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a lump in the salivary gland region often presents as a diagnostic challenge with regards to its site of origin, benign or malignant. most of these occur in the parotid glands ; small percentages occur in the submandibular, sublingual, and minor salivary glands. are a histologically diverse group. tumors of this region comprise 3% of all head and neck and 0.6% of all tumors of human body. the history of fine needle aspiration cytology (fnac) goes back to the 1920s where it came into use simultaneously in europe and the united states [2, 3 ]. fnac is a cytodiagnostic method based on the morphological findings of individual cells, group of cells, and microparticles of tissue, acquired using a needle. the traditional open biopsy is no longer justified because of the risk of tumor spillage and damage to the facial nerve. the method has a high degree of sensitivity in distinguishing the tumors from nonneoplastic lesions of salivary gland [5, 6 ]. fnac is an easily done procedure with minimal incidence of complication and no risk of implantation of tumor cells (< 1%). the complications are rare and bleeding or inflammatory reaction in the region of the puncture. the impairment of the involved nerves has been reported as a very rare complication [7, 8 ]. it is regarded as a diagnostic procedure to be superior to the combination of physical examination and radiological evaluation [9, 10 ]. many authors claimed that it is accurate, safe and cost effective [1113 ]. however, batsakis and colleagues were of the opinion that preoperative fnac has little influence on the clinical management since most of the parotid masses ultimately require surgery. this point of view ignores a considerable number of benign salivary tumors that do not necessitate surgery. because of the distinct morphology of parotid gland, the effectiveness of fnac in its interpretation is still considered controversial. however, the use of fnac helps to distinguish between reactive inflammatory process, which may not require surgery, and neoplastic lesions, between benign and malignant thus allowing proper planning before embarking any treatment. surgical planning is dependent on clinical and radiological investigations ; however, if the pathology is known preoperatively it is easy to counsel the patient and plan surgery. it is here where fnac is of help specially for malignant lesions. in this article a retrospective review was carried out of medical records of patients that underwent parotidectomy for various pathologies between 2002 to 2010. a total of 205 parotid surgeries were performed during this period. of these, only 129 patients were included in this study in which preoperative fnac was done at our institution exclusively. rest of the patients, in which we only performed surgery but fnac was not carried out at our centre, were excluded. fnac was performed at our pathology department using a 22-gauge needle attached to a 10 ml syringe holder by a free hand technique. the specimens were expelled onto two or three slides, and thin smears were prepared between two slides and immediately fixed. the slides were generally stained with papanicolaou and occasionally with may - grunwald giemsa methods. we classified our fnac results into the following categories : true - negative (absence of malignancy correctly diagnosed), true - positive (presence of malignancy correctly diagnosed), false - negative (cytological specimen failed to diagnose a malignancy), and false - positive (cytological specimen was incorrectly considered or suspect for malignancy). we also compared the histopathology of the surgical specimens with the preoperative cytology of the fnac specimens and evaluated the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and overall accuracy of fnac to differentiate between benign and malignant disease. the age range was 15 to 78 years with a mean age of 44 years. there were 98 benign lesions (89 neoplastic and 9 nonneoplastic) while 31 (24%) were malignant tumors (table 2). pleomorphic adenoma was the most common in benign tumor group (60%) while mucoepidermoid carcinoma (12%) in the malignant group. the fnac smears were nondiagnostic in 5 (3.8%) cases, of which 4 were reported benign (2 neoplastic and 2 nonneoplastic) and 1 malignant lesion on final histopathology. an overall of eighty - five percent (110/129) concordance between fnac and final histological diagnosis was established, 78% with malignant and 88% with benign cases on break up (table 2). the cytological diagnosis was true - positive in 26 (20%) cases and true - negative in 96 (74%) cases. twenty - four of the 26 (92%) true - positive neoplasms and 86 of the 96 (90%) true - negative benign lesions were determined as an accurate results (tables 3 and 4). there were 2 (1.5%) false positive and 5 (3.8%) false negative results (table 5). a statistical analysis of 129 cases was carried out to assess the diagnostic accuracy of parotid fnac compared with that of histological results (table 6). a sensitivity of 84% local inflammation was observed in only two cases after performing fnac ; however, no complication such as hematoma, infection, or facial nerve damage was observed. the fnac has been widely used as a diagnostic tool for the management of various head and neck lesions. many authors considered fnac a superior modality and claimed it accurate and safe [6, 1113 ]. in contrast, others argued that it has little influence on clinical management because of its high rates of false - positives and false - negatives and also ultimately patients have to undergo surgery. however, the preoperative cytology helps to the differential diagnosis between benign and malignant lesions of parotid gland, and thus the extent of the surgery can be planned and modified accordingly. this result implicates that if fnac is used as a screening tool in this patient group, 12% of the malignant lesions would have been missed. these values fall within wide range of sensitivity reported in various studies, from as low as 27% up to 97% [9, 1621 ]. the reason often cited for this wide range is the dependence on skills of the cytotechnologist performing fnac and the expertise of the pathologist to assess adequacy and accurate examination of the specimen. specificity reported in the literature has been similarly high, in the range of 84% to 100% [9, 16 ]. a common reason for false negative fnac findings is sampling error. in the recent literature, the accuracy has ranges from 84% to 97% [9, 16, 22 ]. in our study the major drawback of the fnac is the occurrence of nondiagnostic aspirations, which occur in 5 to 15% of cases in literature. in our study, only 5 (3.8%) aspirates were found to be non - diagnostic results. failure to obtain a representative smear could be the result of needle positioning outside the target tissue or of necrosis, hemorrhage, or cystic areas in the tumor. in order to decrease chances of these errors and to increase the diagnostic accuracy various authors [23, 24 ] have utilized ultrasonography to assist fnac but in our series it was not used. because of the varied, complex and overlapping morphological features, the diagnosis of pleomorphic, monomorphic adenoma, and adenoid cystic carcinoma can be sometimes problematic. similarly a case of carcinosarcoma was labelled as warthin 's tumor by fnac (table 5). the rate of false negative results was 3.8% in our review which is significantly less than what zurrida. and this could have happened because of lack of typical features and the presence of atypical squamous cells on fnac. we correctly typed pleomorphic adenoma in 73 of 77 (95%) cases, better than the reported ranges from 82 to 94% in the literature [9, 27 ]. the two acinic cell and 4 adenoid cystic carcinoma were also correctly recognized in our series. according to the experience of shaha. reported correct identification of only 2 of 7 cases of parotid lymphoma by fnac while in a series of al - khafaji. the fact that it was not detected as malignant on fnac is consistent with other reports that lymphoma is difficult to diagnose by fnac and if a lymphoma is suspected on clinical grounds, flow cytometry of fnac can be an adjunct in diagnosing it. still, to some authors, the only relative contraindication of the performing the fnac could be the hemorrhagic disease. many authors exclude the possibility of the implantation of the malignant cells or its recurrence caused by fnab. the phenomenon of tumor cells seeding has become a rare complication with the current use of small - bored needles. no other complications such as hematoma, nerve damage, or infection was observed in our series. the most important questions to be answered by this study is whether results gained from fnac can be useful in the clinical management of patient with parotid lesions. our experience has demonstrated a variety of circumstances in which such data may be valuable. the usual recommendation for the neoplastic lesions regardless of the preoperative cytological diagnosis is excision but recognition of benign lesion, beforehand like in case of warthin 's tumor, in poor risk patients may be of benefit in avoiding inappropriate surgery. our study shows that preoperative fnac plays an important role in the accurate diagnosis of parotid tumors. it is a safe and effective modality for the treatment of patients with parotid lesion. this office based procedure is reliable, well tolerated, easy to perform and cost effective. moreover preoperative differentiation of tumors may help prepare both the surgeon and patient for an appropriate surgical procedure. | objective. histopathology of parotid gland tumors is extremely varied and complex due to heterogeneous cellular composition. preoperative diagnostic tools include fine needle aspiration cytology, the role of which remains controversial. the aim of this paper is to evaluate the usefulness and accuracy of fine needle aspiration cytology (fnac) in the diagnosis of parotid gland tumors. methods. we retrospectively reviewed charts of 129 patients who underwent parotidectomy for parotid lesions at aga khan university hospital from 2002 to 2010. we compared the results of preoperative fnac with final histopathological diagnosis. results. concordance with histological results was observed in 86%, specificity was 98%, sensitivity was 84%, and diagnostic accuracy was 94%. conclusion. our results demonstrate that preoperative cytology in parotid lesions is fairly accurate and useful in diagnosing benign from malignant and in planning appropriate approach for treatment. |
variceal bleeding is the most common lethal complication of cirrhosis, with a mortality rate of 20%. gastroesophageal varices (govs) are present in 50% of cirrhotics, and approximately 25% of patients with portal hypertension have gastric varices. gastric varices are commonly classified as govs (gastric varices in continuity with esophageal varices) and isolated gastric varices (igvs). these are further divided into gov1 (govs along the lesser curve of the stomach), gov2 (govs along the fundus), igv1 (igvs in the fundus), and igv2 (igvs in the gastric body, pylorus, or antrum). although the prevalence and risk of bleeding of gastric varices are lower than those of esophageal varices, gastric variceal bleeding tends to be more severe, requires more transfusions, and is associated with higher mortality (~45%). gastric varices tend to be larger and more tortuous compared with esophageal varices, characteristics that, combined with their anatomical location (particularly fundic varices), make endoscopic management more challenging. current management strategies for gastric varices include pharmacotherapy (-blockers and vasoactive agents), endoscopic therapy (band ligation, thrombin, and tissue adhesives), transjugular intrahepatic portosystemic shunt (tips) placement, and surgical intervention. therefore, recommendations for management of gastric varices are based on recent prospective and retrospective studies, expert consensus opinion, and experience with esophageal varices. the pathophysiology of gastric varices is not as well understood as that of esophageal varices ; however, the former are mostly seen in patients with cirrhotic portal hypertension. no studies to evaluate pharmacologic therapy or endoscopic therapy for primary prophylaxis of gastric varices have been performed, and recommendations are based primarily on the guidelines for managing esophageal varices. large gastric varices with red color signs, especially in patients with advanced liver disease (child 's class c), are most likely to bleed. endoscopic therapy and tips are both considered first - line treatments for gastric variceal bleeding ; however, current guidelines favor endoscopy as the preferred initial intervention. endoscopic therapeutic options for gastric variceal bleeding include band ligation, tissue adhesives, thrombin, and sclerotherapy. the greatest evidence exists for tissue adhesives such as cyanoacrylate (n - butyl-2-cyanoacrylate, cyanoacrylate undergoes rapid polymerization on contact with living tissues and obliteration of varices occurs when the injected cyanoacrylate adhesive hardens on contact with blood. two randomized controlled trials have compared cyanoacrylate injection with variceal band ligation, showing mixed results for initial hemostasis but reduced rebleeding rates with cyanoacrylate (22% versus 44% and 31% versus 54%). another study compared cyanoacrylate injection with sclerotherapy and found better control of initial hemostasis as well as lower rebleeding rates with cyanoacrylate. meticulous adherence to technique is crucial when using tissue adhesives to prevent irreversible damage to endoscopes caused by polymerization. a recent study reported the use of endoscopic ultrasound to guide injection of feeding veins with excellent results ; however, the risk of damaging expensive equipment may prevent its widespread usage. complications of tissue adhesives include embolization, with case reports of portal vein embolization, coronary emboli, splenic infarction, pulmonary emboli, and cerebral stroke [13 - 16 ]. thrombin is a promising agent that does not carry the risk of embolization and damage to equipment. a recent study using bovine thrombin to control gastric variceal bleeding demonstrated a hemostasis rate of 92% and no episodes of rebleeding. several other studies with thrombin have shown promising results [17 - 19 ]. however, controlled trials comparing thrombin with other agents are required before it is universally accepted. the role of tips in the management of gastric varices alone or in comparison with esophageal varices has been investigated in multiple studies. chau. reported a 96% initial hemostasis rate and 29% rebleeding rate with tips in patients with fundal varices, and these were similar to the rates for cirrhotics with esophageal varices. in another retrospective series comparing tips in 40 cirrhotics with gastric variceal bleeding and 232 patients with esophageal variceal bleeding, the rates of rebleeding were similar (20% versus 15%). clearly, with hemostasis rates over 90%, tissue adhesives and tips are both highly effective management options for gastric variceal bleeding. lo. carried out a randomized controlled trial comparing tips (n = 35) and cyanoacrylate glue (n = 37) for secondary prophylaxis of gastric variceal bleeding. however, the rebleeding rate was significantly higher in the cyanoacrylate arm compared with the tips arm (38% versus 11%). in a uk - based retrospective study, the two therapies were equally effective for initial hemostasis but patients with tips had a lower rebleeding rate (20% versus 35%). however, tips was considerably more expensive than cyanoacrylate therapy. finally, in a recent retrospective study comparing tips (n = 44) and cyanoacrylate therapy (n = 61) for gastric variceal bleeding, rebleeding rates and mortality rates were similar between the two groups. however, the tips group had increased morbidity requiring hospitalization and had a higher incidence of encephalopathy (11 versus 1). the pathophysiology of gastric varices is not as well understood as that of esophageal varices ; however, the former are mostly seen in patients with cirrhotic portal hypertension. no studies to evaluate pharmacologic therapy or endoscopic therapy for primary prophylaxis of gastric varices have been performed, and recommendations are based primarily on the guidelines for managing esophageal varices. large gastric varices with red color signs, especially in patients with advanced liver disease (child 's class c), are most likely to bleed. endoscopic therapy and tips are both considered first - line treatments for gastric variceal bleeding ; however, current guidelines favor endoscopy as the preferred initial intervention. endoscopic therapeutic options for gastric variceal bleeding include band ligation, tissue adhesives, thrombin, and sclerotherapy. the greatest evidence exists for tissue adhesives such as cyanoacrylate (n - butyl-2-cyanoacrylate, cyanoacrylate undergoes rapid polymerization on contact with living tissues and obliteration of varices occurs when the injected cyanoacrylate adhesive hardens on contact with blood. two randomized controlled trials have compared cyanoacrylate injection with variceal band ligation, showing mixed results for initial hemostasis but reduced rebleeding rates with cyanoacrylate (22% versus 44% and 31% versus 54%). another study compared cyanoacrylate injection with sclerotherapy and found better control of initial hemostasis as well as lower rebleeding rates with cyanoacrylate. meticulous adherence to technique is crucial when using tissue adhesives to prevent irreversible damage to endoscopes caused by polymerization. a recent study reported the use of endoscopic ultrasound to guide injection of feeding veins with excellent results ; however, the risk of damaging expensive equipment may prevent its widespread usage. complications of tissue adhesives include embolization, with case reports of portal vein embolization, coronary emboli, splenic infarction, pulmonary emboli, and cerebral stroke [13 - 16 ]. thrombin is a promising agent that does not carry the risk of embolization and damage to equipment. a recent study using bovine thrombin to control gastric variceal bleeding demonstrated a hemostasis rate of 92% and no episodes of rebleeding. several other studies with thrombin have shown promising results [17 - 19 ]. however, controlled trials comparing thrombin with other agents are required before it is universally accepted. the role of tips in the management of gastric varices alone or in comparison with esophageal varices has been investigated in multiple studies. chau. reported a 96% initial hemostasis rate and 29% rebleeding rate with tips in patients with fundal varices, and these were similar to the rates for cirrhotics with esophageal varices. in another retrospective series comparing tips in 40 cirrhotics with gastric variceal bleeding and 232 patients with esophageal variceal bleeding, the rates of rebleeding were similar (20% versus 15%). clearly, with hemostasis rates over 90%, tissue adhesives and tips are both highly effective management options for gastric variceal bleeding.. carried out a randomized controlled trial comparing tips (n = 35) and cyanoacrylate glue (n = 37) for secondary prophylaxis of gastric variceal bleeding. however, the rebleeding rate was significantly higher in the cyanoacrylate arm compared with the tips arm (38% versus 11%). in a uk - based retrospective study, the two therapies were equally effective for initial hemostasis but patients with tips had a lower rebleeding rate (20% versus 35%). however, tips was considerably more expensive than cyanoacrylate therapy. finally, in a recent retrospective study comparing tips (n = 44) and cyanoacrylate therapy (n = 61) for gastric variceal bleeding, rebleeding rates and mortality rates were similar between the two groups. however, the tips group had increased morbidity requiring hospitalization and had a higher incidence of encephalopathy (11 versus 1). the initial steps in the management of gastric variceal bleeding are similar to esophageal varices and include attention to airway and obtaining intravenous access with prompt hemodynamic resuscitation, institution of vasoactive medications (somatostatin analogues, octreotide, vasopressin, and terlipressin) to reduce portal pressure by splanchnic vasoconstriction and prophylactic antibiotics followed by diagnostic and potentially therapeutic upper endoscopy (figure 1). the next step in the management may be endoscopic injection of tissue adhesives or tips placement. with a lower complication rate, comparable hemostasis and survival rates, and lower cost, endoscopic therapy with tissue adhesives is likely to be the treatment of choice for acute bleeding and secondary prophylaxis in most patients. on the other hand, tips will remain an important second - line modality in patients who fail endoscopic therapy. however, additional controlled studies are needed before valid conclusions can be made, and individual patient characteristics must be considered in management decisions. primary prophylaxis may be instituted with -blockers in patients with large varices with red color signs and advanced liver disease. further research on management of acute bleeding, primary prophylaxis, and comparisons between tissue adhesives and thrombin would be welcome. | gastric variceal bleeding is a common problem in patients with cirrhosis and is associated with increased morbidity and mortality. management is complex and includes pharmacotherapy, endoscopic therapy, and shunt placement. recent studies indicate that endoscopic therapy with tissue adhesives has similar hemostasis rates and outcomes in terms of mortality as shunt placement but has a lower complication rate and therefore could be considered the first line therapy for acute bleeding and secondary prophylaxis of gastric varices. |
health economics is a branch of economics that studies and evaluates the quantity, quality, and value of the limited resources available to healthcare systems, as well as determining how these factors best combine to produce certain services, with the aim of maximizing effectiveness and efficiency. us health costs are currently 17% of the gross domestic product (gdp) and continue to rise. many other countries also have the same trend toward increasing costs, although they spend less of their gdp on healthcare. hospital costs constitute the largest single component of healthcare expenditure, and in developing countries, 50 - 80% of total annual health expenditure is allocated to healthcare facilities. in iran, 40% of the total health budget is consumed by hospital services. therefore, hospitals must be considered as the primary target of efforts to inhibit rising healthcare costs. nurses are the main users of resources in the hospital setting, by the virtue of their direct links with the patients, and because they constitute the largest group of healthcare professionals. thus, nurses play an important role in the economic management of nursing services, and they must be capable of meeting the demands of healthcare organizations, which have been increasingly concerned with growing costs and wasted resources. their awareness of, and participation in, logical justification of hospital costs, suitable use of resources, waste control, and providing high quality services is of vital importance. although great emphasis has been placed on the importance of the control of hospital costs and productivity, as well as on positive attitudes toward nurses participation in management and cost control, many nurses unfortunately do not understand how their work affects healthcare costs, or how nurses contribute to healthcare revenues. several studies have confirmed that nurse have the insufficient knowledge of budget control and cost management. for example, caroselli found that only 30% of nurses had any basic knowledge of health economics, and a qualitative study showed that nurses do not use cost management in their practice, because they focus only on the care issues. we were unable to find any evidence that a study of this type had previously been conducted in iran. therefore, given the importance of nurses participation in cost efficiency activities and subsequent cost savings, it is necessary to determine their awareness and performance with regard to health economics, as a first step. the present study was conducted to evaluate the awareness and performance of nursing staff with regard to health economics in teaching hospitals. a descriptive cross - sectional study was performed at three teaching hospitals affiliated to the mashhad university of medical sciences, mashhad, iran, between may and august 2013. the study population consisted of all nurses who worked in the medical, surgical, and critical units of these teaching hospitals. the sample size was estimated to be 170 participants, on the basis of a previous study. and using the following parameters - s = 2.2, z = 1.96, d = 0.15 s. in consideration of the sample size and the number of nurses in each hospital, 175 clinical nurses were selected, via a random sampling method. all the nurse managers, including the head nurse, the supervisors, and the matron from the three hospitals were asked to complete the questionnaire. inclusion criteria were nurses employed in the hospitals of the mashhad university of medical sciences, and with at least 2 years of clinical experience. the study protocol was approved by the ethics committee of the mashhad university of medical sciences (grant number was 900582). the purpose of the study was initially explained to respondents, who were assured that participation was voluntary. return of the completed questionnaire was considered as a provision of informed consent to participate. the first of these concerned demographic characteristics, including age, gender, educational level, hospital units of service, job position, years of experience, management, and budgeting experience. part two consisted of 16 multiple choice questions to assess the nurses awareness of economic issues related to healthcare. this was divided into three subscales : nurses awareness of national and regional budgeting (this comprised three questions, with a score ranging from 0 to 3), (this consisted of four questions, with a score ranging from 0 to 4), and nurses awareness of factors that affect hospitals costs (this was composed of nine questions, with a score ranging from 0 to 9). wrong and do nt know answers were given a value of zero (to a maximum value of 16 points). the passing score was determined by using the angoff method, which is one of the most common methods used for setting cutoff scores. each question is rated by a group of five subject matter experts, using criterion - referenced procedures. the expert estimates the percentage of minimally competent persons that will answer each question correctly, after which the judges totals are summed and averaged (8.75 + 7.25 + 8.5 + 7.75 + 6.75 divided by 5) to obtain a cutoff score of 7.8, or 8. therefore, in the present study, following collaboration with an expert panel, nurses that answered at least eight out of 16 questions correctly (50%) were considered to have good awareness of the field of health economics. part 3 of the questionnaire contained 11 questions, and a 5-point likert scale was used to measure economic performance. this section investigated the nurses performance with regard to nursing documentation, as well as consumption and use of drugs, medical supplies, and equipment. the never option was the equivalent of zero and meant that the related performance was never observed in the nurses clinical activities. always option was set at four points and meant that the related performance was constantly observed in the clinical activities carried out. for each question, mean scores above 3, 2 - 3, and below 2 were considered as desirable, moderate, and unfavorable, respectively. the psychometric properties of the questionnaire were examined, and content validity was determined by the means of expert review. four individuals with a ph.d. in nursing, three nursing administrators, and three health economists critiqued the questionnaire. to assess questionnaire reliability, we examined the internal consistency coefficient and achieved a cronbach 's of 0.74 ; values of 0.7 or higher are appropriate and acceptable. data were collected using a combination of the questionnaire, interviews, and observation, as well as an assessment of the available evidence. the first and second parts of the questionnaire were hand delivered to individual units by researchers, and then distributed to staff nurses by the unit manager during different working shifts. two days after distribution, the researchers picked up the questionnaires from the three hospitals. a total of 125 clinical nurses and 50 of 68 nurse managers completed the questionnaire. the third part of the questionnaire was completed by two co - researchers after observing and interviewing the head nurse and nurses working in different shifts, as well as checking the available evidence. a multiple regression analysis was used to determine the predictive power of economics awareness and other variables on the economic performance of the participants. an analysis of variance and pearson 's correlation test were used to evaluate the relationship between variables, and the level of statistical significance was considered to be p 0.05). multiple regression analysis economics awareness, clinical work experience, job position, and age on mean of economic performance of nurses one of the most important findings of this study was that iranian nurses had an insufficient awareness of health economics, such that only 22% of those studied and showed good economic awareness. potential reasons for this knowledge deficit may be a lack of health economics education during nursing training courses, no nurse participation in decision - making regarding economic issues, an absence of continuing education courses and workshops, and little attention to nurses capacity to impact on health system costs. de oliveira. argued that the nurses lack of commitment toward economic issues is related to either a lack of time or to a lack of interest regarding management issues, with the justification being that they have received no academic education in relation to economic management and had no understanding of these concepts. a higher level of awareness with regard to factors that affect hospitals costs, compared to the other two subscales was another notable finding. since the factors that were evaluated in this subscale were more tangible for nurses, and nurses were more involved with them in clinical practice, they had greater awareness in this area. nurses in clinical practice understood the positive effects of various factors, such as standards of care, proper use and maintenance of supplies and equipment, and recording of medication and diagnostic tests. in another study, although 94% of nurses were not involved in the hospital budgeting process, their perception of cost containment and control of supplies and equipment was positive, and over 90% were focused on the importance of procedure standardization for nurses to keep costs down. in the present study, nurse managers had a greater awareness of health economics, but most of them did not have any role in the hospital economic decision - making process. it appears that several factors, such as the nature of the task, a greater number of administrative responsibilities, participating in related meetings and committees, and previous experience are the reasons behind this. nurse managers are in an ideal position to not only affect unit - based economics but also to increase economic awareness and the behavior of staff nurses. however, the centralized organizational structure of almost all of iran 's general governmental hospitals makes it difficult for members within the hospital to contribute to decision - making and organizational policy. the findings showed that there was a significant relationship between the awareness and performance regarding health economics. however, this relationship was very weak, and awareness scores could explain only 5% of the variance in performance scores. this means that although nurses do not have sufficient economic awareness, they pay attention to clinical care matters, such as documentation of consumed drugs and supplies, and careful operation and maintenance of equipment and devices in practice, which have an effect on adjustment of hospital costs. these conditions are primarily the result of formal written clinical guidelines, a shortage of equipment and materials, and close control by the hospital management of the use of expensive drugs and equipment. moreover, the results showed that the lowest score was associated with the recording of routine nursing care activities in patients medical records, which may be a result of its the lack of effect on patients final billing and hospital income. in the present study, 97% of nurses stated that they do not participate in decision - making regarding hospital economy, while the majority (70%) had a positive opinion with respect to the necessity of nurses contribution to economic programs in hospital and cost control activities. it is necessary to make structural changes in the process of hospital budgeting to allow for a greater contribution from the nursing unit. showed that only 6.6% of nurses played a role in hospital budgeting ; however, 69.9% of these indicated that nurse managers should be involved in the planning of a hospital budget. a qualitative study showed that most managers believe that the contribution of nurses to the cost efficiency of interventions would further boost clinical activities and job development, and, subsequently, would have a positive effect on patient care activities. it is clear that nurses should receive health economics training prior to entrance into the profession, with the aim of enhancing productivity, reducing costs, and improving the quality of care. in the present study, 81% of nurses indicated that educational programs can help to reduce hospital costs. in another study, 96% of nurses stated that in - service education sessions on cost containment should be held for all the categories of nurses. since less attention is paid to managerial and economic issues in nursing courses, it is important that nursing students become familiar with the management and economic aspects of the profession, so that in future, they are capable of properly meeting new challenges in this field. the main limitation was the broad nature of the health economics field, which prompted the researchers to implement a broader view in their approach, as well as the design of the questionnaire. taking a more detailed approach, and thus evaluating all of the factors influencing nursing care, such as work environment conditions, communications, and educational and administrative plans that have an effect on costs and outcomes, would severely have affected the size of the questionnaire, which, in turn, would have contradicted the research tool designing principles, and would also have reduced the accuracy of the answers. therefore, in order to increase the validity of the questionnaire, in addition to a preliminary study, the views of several healthcare and economics experts were also considered to maximize the healthcare economics relevance and coverage of the final questionnaire. the small sample size and inclusion of only government teaching hospitals mean that the results may only be cautiously generalized to other settings. nevertheless, this study is a critical and preliminary step that allowed a detailed assessment of economic awareness and performance in nursing clinical practice. an evaluation of the effects of the educational courses and participation of nurses in the cost management of both unit and hospital is suggested for future studies. considering the importance of health economics and priority in healthcare systems in the near future, managers, and staff nurses should upgrade their basic knowledge of economics, cost saving, and optimal usage of equipment and resources. | background : health costs have risen everywhere, worldwide, and nurses play a pivotal role in cost savings and in contributing to the financial stability of hospitals.aim:this study evaluated the awareness and performance of iranian nursing staff, with regard to health economics.materials and methods : a total of 175 nurses who worked in three teaching hospitals in mashhad (iran) were selected for this descriptive cross - sectional study, and data were gathered via a 27-item questionnaire. statistical analysis was performed using one - way analysis of variance, multiple regression analysis, and pearson 's correlation coefficient.results:a total of 78% (n = 39) of nurses did not have a good awareness of health economics. the overall mean score for economic awareness was 5.9 2.1 (possible range, 0 - 16), and for economic performance was 26.6 4 (possible range, 0 - 44). there was a significant relationship between the economic awareness and performance of nurses, and nurses in higher positions had a greater awareness of health economics.conclusions:considering the inadequacy of the health economics awareness and performance of nurses, it is essential that efforts are made to enhance their knowledge and behavior with regard to economic issues and cost saving in all the fields of nursing, through the use of continuing education courses and workshops. |
the construction of long dna oligonucleotides, such as synthetic genes, is useful for a number of reasons. for protein expression, it is possible to improve the yield of a desired protein by optimizing codon usage to reflect the relative codon abundance in the host organism used for protein production. long oligodeoxynucleotides can also be used to produce large amounts of rna for structural or biochemical studies by in vitro transcription methods (1). many rna molecules studied to - date are under 100 nt in length and the dna oligonucleotides needed for their production are readily synthesized and are commercially available (up to 100150 nt long). as larger rna molecules are studied, or if methods such as the use of cis-3 and/or 5 ribozymes to facilitate rna production (24) or if rna - based affinity tags to aid in rna purification (5) are used, it is not possible to purchase dna oligonucleotides of sufficient length to encode the desired rna product. to produce these long dna molecules (over 120150 nt) it is necessary to use techniques originally developed for gene synthesis, such as assembly pcr. pcr methods can be used to construct a synthetic gene through the use of a two - step pcr method referred to as assembly pcr (figure 1) (6,7). in the first step of assembly pcr, multiple oligodeoxynucleotides that contain overlapping regions anneal, and the dna polymerase extends the primers and fills in the regions between the primers. a range of products of different lengths results from the different possible combinations of annealing that involve less than all the oligodeoxynucleotides. in the second pcr step a pair of primers is introduced that is specific for the full - length oligodeoxynucleotide and the full - length product is selectively amplified from the mixture. the code for assembly pcr oligo maker is written in java version 1.4.1 available from. we have attempted to make this program compatible with most browsers, however, if there is any difficulty running the program we recommend downloading the latest java runtime environment available from sun microsystems. the program was tested on the windows (98, 2000, xp), linux (fedora core 3), sgi (irix 6.5) and mac (osx 10.3) operating systems and is functional using virtually any web browser. the default method employed by the program for calculating dna melting temperatures uses the nearest neighbor method employing the unified dna dna hybridization thermodynamic parameters of allawi and santalucia (8) with a monovalent cation correction to the entropy term as outlined by santalucia (9). the default sodium ion (monovalent cation) concentration is 50 mm, while the default dna concentration is 0.5 m. alternately, the user can choose to calculate melt temperatures using the method of wallace. (10) where melting temperature (tm) = 2(a + t) + 4(g + c). vent dna polymerase was purchased from new england biolabs (neb). upon receipt, the oligodeoxynucleotides for the first step of assembly pcr were diluted to 7 m (0.125 g/l) with double distilled water, while the oligodeoxynucleotides for the second pcr step were diluted to 42 m (0.25 g/l). for the first pcr reaction 4 l of each oligo, 4 l of 5 mm dntps, 10 l of 10 thermopol buffer (neb), 1.5 l of vent dna polymerase (2000 u / ml) and 68.5 l of double distilled water were combined. this mixture was then subjected to 8 cycles of amplification at 94c (1.5 min), 54c (2 min) and 72c (3 min). during the first cycle after the last cycle was completed an additional 5 min 72c elongation step was performed. for the second pcr reaction, 1 l of the crude mixture from the first pcr reaction was mixed with 4 l of each primer, 4 l of 5 mm dntps, 10 l of 10 thermopol buffer (neb), 1.5 l of vent dna polymerase and 75.5 l of double distilled water. this mixture each cycle consisted of a 30 s 94c step, a 2 min 54c step and a 1.5 min 72c step. before the first cycle the pcr amplified dna and a sample of the puc18 vector were then digested with the restriction enzymes hindiii and ecori (neb). the pcr amplified dna was then ligated into puc18 using t4 dna ligase (neb) and transformed into dh5 escherichia coli cells. the assembly pcr oligo maker program is designed to be flexible and easy to use. the user can input either an rna sequence or a dna sequence and can alter a number of settings used by the program during the determination of the oligodeoxynucleotide sequences. the user can input the target oligonucleotide sequence in either the 5 to 3 or 3 to 5 directions. for either rna or dna input, the user is queried as to whether the top17 sequence (1) for t7 rna polymerase priming should be added or not. we will note that for highest rna production the sequence following the t7 promoter should code for two or three guanosine nucleotides (1). if rna is used as input, the corresponding dna sequence to be produced is determined and provided as output. the simplest use of the program therefore is to determine the dna sequence needed to produce a particular rna molecule. at this point, the target dna sequence to be produced is known and the program computes the necessary oligodeoxynucleotides for both steps of the two - step assembly pcr synthesis method. by default, the program works by dividing the target dna into an even number of segments with a user - defined maximum length. the user can define the maximum length of the calculated oligodeoxynucleotides to be 40, 50, 60 or 70 nt long. the length of overlap between the different oligodeoxynucleotides is optimized so that the overlapping region matches a target tm. the target tm value employed is a user defined variable with choices available between 50 and 60c, the default value is 55c. a consistent tm value among the different oligodeoxynucleotides helps contributes to successful pcr reactions. a key feature to ensure the success of assembly pcr is the necessity that the oligodeoxynucleotides only anneal where desired. if an oligodeoxynucleotides can anneal in multiple ways, in either an intramolecular or intermolecular fashion, the subsequent pcr reactions would produce a host of undesired products. the assembly pcr oligo maker program tests the oligodeoxynucleotides calculated for both pcr reactions for regions of overlap outside of the desired location. if a stretch of nucleotides is found to overlap at a region outside of the desired site, the program calculates a tm value for this region of overlap. if the tm value of the overlapping region detected is greater than a user defined tm threshold a warning appears notifying the user that an undesired overlapping region was detected the user can then make changes to the sequence of the desired pcr product or ignore this warning and proceed to attempt the pcr reaction despite the presence of an unwanted overlap region. alternately, upon detection of an undesired region of overlap the user can choose to calculate oligodeoxynucleotides with a smaller or greater maximum length in order to try and break up the region of unwanted overlap. the default tm value for triggering a warning for the presence of an undesired overlap is 40c and is user variable between 30 and 45c. the output from the program is available in the main window (figure 2) from which the user can cut and paste text in order to save the output. the output consists of all the necessary oligodeoxynucleotides to perform both of the pcr reactions. alternately, the user can view a summary of the input and output in a single page available from it is also possible to cut and paste text from this page to a file. also present in the results window is a view of how the different oligodeoxynucleotides in the assembly pcr reaction align with each other. initially, a dna oligonucleotide target was used as input, and the oligodeoxynucleotides determined by the program were analyzed to check that the correct dna product would result from the two - step pcr dna synthesis method. next, the ability of the program to find regions of overlap outside of the desired region was confirmed. a target dna oligonucleotide containing a repeat region was used as input. depending on where the repeat region was in relation to where the target dna product was divided, the program was able to detect it, and the calculated tm of the unwanted overlap region was reported. finally, the program was experimentally verified by using the oligodeoxynucleotides determined by the program for the two - step assembly pcr construction of a dna molecule that is to be used to produce an rna molecule. the desired rna product is a 191-nt molecule consisting of 5 and 3 cis hammerhead ribzoymes and a core 20-nt region that forms a hairpin structure and is having its structure studied in our lab by nuclear magnetic resonance methods. when using the default value of 70 for the maximum oligonucleotide length, the program broke this 191-nt dna molecule into four segments for the first pcr reaction and produced the two oligodeoxynucleotide molecules for the second pcr reaction (figure 3b). as shown by gel analyses (figure 3d) the first pcr reaction produces a diffuse band or smear, while the desired full length product results from the second pcr reaction. this behavior is consistent with previous reports of assembly pcr gene construction (6,11,12). the product of the second pcr reaction was cloned into the puc18 plasmid and the correctness of its sequence was verified by dna sequencing. the program we present here for determining the oligodeoxynucleotides needed for the assembly pcr construction of artificial genes has several advantages over similar applications that are currently available. these advantages are in addition to the ability of assembly pcr oligo maker to handle rna as an input and add the top17 sequence to determine the dna sequence needed for rna production. while this is a trivial process, the use of assembly pcr oligo maker does remove the human element from this task that can inadvertently introduce sequence errors into the final product. the other programs available that aid in oligodeoxynucleotide design for the assembly pcr construction of long dna molecules include dna works (11), gene2oligo (12) and dnabuilder (). the major advantage of using assembly pcr oligo maker is that the user has the option of determining the maximum length of oligodeoxynucleotides for assembly pcr that are created by the program. the user thus has the option of choosing to create fewer but longer oligodeoxynucleotides than determined by other programs. working with fewer oligodeoxynucleotides means that the process of dissolving and diluting them and setting up the assembly pcr reaction is simplified and that fewer opportunities for human error are available during the experiment. this function is shown in the example in figure 3a where the 191-nt dna target is broken into four oligodeoxynucleotides of length 56, 64, 64 and 59 nt for a total of 243 nt. for this calculation, a maximum length of 70 (the default value) was chosen as the maximum oligonucleotide length calculated. when the same target is used as input for dnabuilder the output was 6 oligodeoxynucleotides with a total of 306 nt ; dnaworks had an output of 10 oligodeoxynucleotides comprising a total of 371 nt ; and using this sequence as input gene2oligo produced an output of 13 oligodeoxynucleotides made up of a total of 440 nt. this is almost twice the number of nucleotides as obtained from assembly pcr oligo maker. in assembly pcr oligo maker the user has the option to choose to calculate more and shorter oligodeoxynucleotides if desired. typically, the use of shorter oligodeoxynucleotides may be desired due to concerns that the user may have about using the longer oligodeoxynucleotides in a pcr reaction without performing additional purification steps, such as page purification of the longer oligodeoxynucleotides. we will note that we have had no trouble using oligodeoxynucleotides 6065 nt in length in the assembly pcr reaction using them as provided by the manufacturer, without employing any additional purification steps. an additional advantage of our assembly pcr oligo maker program is its flexibility in handling the input sequence. assembly pcr oligo maker is able to correctly handle input containing spaces in the sequence, as you would include if working with the sequence in units of three nucleotides. the program also allows input in both the 3 to 5 direction as well as the 5 to 3 direction. our program is readily accessible on the web and can be accessed with any web browser and does not depend on the computer operating system used. finally, the output of assembly pcr oligo maker is very clear, it lists the oligodeoxynucleotides needed for the assembly pcr reaction and the two oligodeoxynucleotides needed for the second pcr reaction as well as provide the predicted tm values. all the oligodeoxynucleotides in the output are listed 5 to 3 which is in a form that is suitable for cutting and pasting the sequences into an email or web form for ordering oligodeoxynucleotides from vendors of users ' choice. in summary, we have created a program that is freely available on the internet to aid in the design of oligodeoxynucleotides for the construction of long dna molecules. the program can assist in constructing any dna molecule, but has a focus on creating dna templates for rna production. the sequences reported by assembly pcr oligo maker were experimentally verified by the successful construction of a 191-nt dna molecule. the assembly pcr method for constructing long dna molecules. (a) in the first pcr step a pool of oligodeoxynucleotides anneal and are (b) elongated to produce a full - length dna molecule. in addition to the full - length product, a host of shorter molecules also results. (c) in the second pcr step the desired full - length molecule is selectively amplified from the mixture using primers specific for the desired full - length product. the oligodeoxynucleotide sequences for the first pcr step are found in the assembly oligo box, and the two oligodeoxynucleotide sequences for the second pcr step are found in the two flanking primers boxes. user controlled settings and information about the program are accessible under the file and help menus. the construction of a 191-nt dna molecule using the oligodeoxynucleotides determined by the assembly pcr oligo maker program. (c) diagram showing how the four oligodeoxynucleotides anneal to produce the full - length dsdna product (d) agarose gel showing the results of the first (lane 2) and second (lane 3) pcr steps. | we describe a computer program, assembly pcr oligo maker, created to automate the design of oligodeoxynucleotides for the pcr - based construction of long dna molecules. this program is freely available at and has been specifically designed to aid in the construction of dna molecules that are to be used for the production of rna molecules by in vitro synthesis with t7 rna polymerase. the input for assembly pcr oligo maker is either the desired dna sequence to be made or an rna sequence. if rna is the input, the program first determines the dna sequence necessary to produce the desired rna molecule. the program then determines the sequences of all the oligodeoxynucleotides necessary for a two - step assembly pcr - based synthesis of the desired dna molecule. the oligodeoxynucleotide sequences outputted are designed to have a uniform melt temperature and are checked for regions of overlap outside of the desired priming regions necessary for the pcr reaction. the validity of the program was verified experimentally by synthesizing a 191-nt long dna molecule using the dna sequences suggested by the program. |
pilonidal sinus is a well recognized chronic inflammatory condition which affects the natal cleft, finger web space, and it is rare in the penis. we report on a case of penile pilonidal sinus in a 24-year - old man who has severe mental illness, autism, and developmental delay. the literature was reviewed and there were a few cases reported worldwide (val - bernal 1999 ; chikkamuniyappa 2004 ; kalsi 2004 ; okane 2004). a 24-year - old man who is known to have severe mental illness, autism, and a learning disability, presented with more than a one year history of recurrent ulcerative lesion on the middle of the dorsum of his penile shaft, intermittent erythema, and swelling. despite several courses of antibiotics prescribed by his local general practitioner, the lesion did not resolve and the swelling was observed to grow bigger between these episodes. clinical examination revealed an indurated, erythematous 89 mm ulcerative lesion in the middle of the dorsal aspect of the penile shaft. the patient had not been circumcised and had never reported a similar complaint from anywhere else in his body. excisional biopsy was performed, and histology showed the classical findings of pilonidal sinus with hair shaft within. it is essential to highlight the fact that diagnosis was made post - operatively based on histopathological findings rather than on preoperative clinical assessment. in some cases diagnosis pilonidal sinus was first described by anderson (1847). it is a condition that affects hairy areas such as the natal cleft, axillae, perineum, finger web space in the hands of barbers, and amputation stumps (saharay 1997). the pathogenesis of pilonidal sinus is controversial although it is agreed that it is acquired. the present view is that the large majority of pilonidal sinuses have an acquired pathogenesis. the initiating event appears to be follicular hyperkeratosis with plugging, leading to retention of follicular products (lucas 1997). the rupture of a follicular unit or a cyst with secondary infection results in abscess formation. in some instances, sinuses formed to drain the abundant suppuration. the sinuses are lined by stratified squamous epithelium and inflammatory granulation tissue. very often, numerous hair shafts penetrate into dermis and elicit foreign body giant cell reaction (figure 13). the support for the acquired theory of the pilonidal sinus comes from their occurrence in situations such as the finger web space in the hands of barbers, and in amputation stumps. therefore many cases are acquired as a result of trauma from jeep seat, rough riding syndrome, and occupational physical injury (val - bernal 1999). the literature was reviewed and a few cases were reported worldwide (val - bernal 1999 ; chikkamuniyappa 2004 ; kalsi 2004 ; okane 2004). the literature referred to penile pilonidal sinus with some other pathologies such as actinomycosis infection (val - bernal 1999 ; chikkamuniyappa 2004) or erectile dysfunction (kalsi 2004). therefore, a good clinical assessment and bacteriological examination is essential to obtain better results. in our case, we reported on a pilonidal sinus where diagnosis was made on histopathological findings with no evidence of associated bacterial infection or malignancy (rao 2006). our patient had full recovery and no symptoms since, his post - operative follow - up involved two out - patient visits at up to 12 weeks post - operation. the patient had no symptoms or recurrence up to that time, interval circumcision was deferred due to the patient s general health status and his mental disability. | pilonidal sinus is a well recognised chronic inflammatory condition which commonly affects the natal cleft and finger web space. pilonidal sinus of the penis is a rare clinical entity ; few cases have been reported previously. the treatment strategy is almost the same as treating any pilonidal sinus and involves total excision of the sinus. our diagnosis was established on the histopathological findings rather than a clinical - based diagnosis, follow - up is advisable since the recurrence rate is high. our case report is one of few cases reported on pilonidal sinus all over the world. our patient had total excision of the sinus with complete recovery. |
three different types of aiha can be studied based on the behavior of the autoantibodies involved in the pathogenesis:1 (1) warm aiha, which is most commonly mediated through igg autoantiobodies (polygenic ; commonly against the antigens of rh system) that are most potent at temperatures 37 c, (2) cold aiha, which is generally mediated through c3 componant of the complement that binds to and hemolyzes the red blood cells (rbcs) coated with igm antibodies (commonly against i and also against i and pr antigens of the rbcs) typically at temperatures much lower than body temperatures (commonly around 25 c),2 and (3) paroxysmal cold hemmoglobinuria (pch), which is mediated through igg or donath landsteiner antibodies (commonly against p antigen) that fix the complement to the rbcs partially at low temperatures of 30 c3 (commonly 4 c18 c as per in vitro testing)1 and then complete the process at temperatures 37 c. most of the cases of aiha have no identifiable etiology and hence are termed idiopathic or primary aiha. the few cases where an underlying cause is identified are termed secondary aiha and could be associated with lympho - proliferative diseases (lymphoma, chronic lymphocytic lymphoma (cll)), infections (mycoplasma4 as in cold aiha or syphilis5 as in pch and various other viral infections, such as the herpes family,6,7 retrovirus,8 and hepatitis9), drugs (penecillin and methyl dopa),1 and other miscellaneous causes (connective tissue diseases, thyroid disease, ulcerative colitis, pernicious anemia, etc).1 pch occurs much more commonly in young children with infections (where a cold temperature is not involved in vivo). the temperature - dependent chronic primary pch (usually in adults) is extremely rare. lab abnormalities noted among all the aiha in general include anemia, unconjugated hyperbilirubinemia, and elevated lactate dehydrogenase (ldh) levels.1 spherocytosis is often seen in waiha.1 severity of the clinical features and labs depend on the extent and rapidity of onset of the anemia and the ability of bone marrow to compensate for the anemia.1 waiha with a direct anti - globulin test (dat) positive only for c3 is relatively rare. a 79-year - old woman with a history of dementia was referred to our tertiary care center after she was found to have severe anemia with hemoglobin (hgb) of 3.6, hyperbilirubinemia, and a positive coombs test at a small hospital a day previously, where she had presented after being found unresponsive by her family. the patient was transfused 4 units at the initial hospital with appropriate rise in the hemoglobin. however, she was noted to be severely anemic with hgb of 2.6 g / dl upon presentation to our hospital and so was admitted to the intensive care unig for blood transfusion and close monitoring of vitals and hgb. the patient was also started on solu - mederol 1 mg / kg intravenous (iv) and folic acid 1 mg orally once daily. a dat was repeated and was positive for c3 alone and was negative for igg, iga, and igm. a cold agglutinin screen and eluate were negative. her hgb improved to 10.2 g / dl after packed red blood cells (prbc) transfusion, and she was transferred to the step - down unit. she continued to require prbc transfusions on an as needed basis for hgb 8 g / dl. after discussion with the patient s family, the patient was admitted to the palliative care unit with discontinuation of as needed prbc transfusions and any lab testing. a written consent was obtained from the patient s daughter for the purpose of using patient information for this study. a 79-year - old woman with a history of dementia was referred to our tertiary care center after she was found to have severe anemia with hemoglobin (hgb) of 3.6, hyperbilirubinemia, and a positive coombs test at a small hospital a day previously, where she had presented after being found unresponsive by her family. the patient was transfused 4 units at the initial hospital with appropriate rise in the hemoglobin. however, she was noted to be severely anemic with hgb of 2.6 g / dl upon presentation to our hospital and so was admitted to the intensive care unig for blood transfusion and close monitoring of vitals and hgb. the patient was also started on solu - mederol 1 mg / kg intravenous (iv) and folic acid 1 mg orally once daily. a dat was repeated and was positive for c3 alone and was negative for igg, iga, and igm. a cold agglutinin screen and eluate were negative. her hgb improved to 10.2 g / dl after packed red blood cells (prbc) transfusion, and she was transferred to the step - down unit. she continued to require prbc transfusions on an as needed basis for hgb 8 g / dl. after discussion with the patient s family, the patient was admitted to the palliative care unit with discontinuation of as needed prbc transfusions and any lab testing. a written consent was obtained from the patient s daughter for the purpose of using patient information for this study. waiha commonly has a dat positive for igg with or without c3, igm, and iga. however, cases of waiha with a dat positive only for c3, igm, or iga have been reported. though the exact cause of c3 only waiha has not definitively been proved, the following hypotheses have been proposed:10 complement components may get bound to the rbc during adherence of antibody - antigen complexes that later dissociate, leaving the complement still attached to the rbc. when natural antibodies associate with rbc damaged by the in vivo action of microbial enzymes, the complement may also be fixed to the rbc. complement activated in fluid phase by antigen - antibody complexes unrelated to the rbc the complement may fix to the rbc by igm that is present in serologically undetectable quantities. in general, c3 only waiha is a relatively rare entity. also since c3 alone coated rbc are not cleared efficiently by the macrophages11 and since such complement coating of the rbc is seen in normal subjects also,12 theoretically the hemolysis in c3 alone coated waiha should be mild. for the same reason, sparse igg coating13 of the rbc, not detectable by conventional lab testing, has been speculated as a mechanism for increased hemolysis in the rare cases of c3 alone waiha with severe hemolysis. however, different authors have mentioned varying incidences and clinical severity of this disease. for this reason, we sought to perform an analytical literature review of these parameters for this disease. c3 only warm autoimmune hemolytic anemia yielded only one pertinent article.14 further pertinent articles were derived through the references of the initial article and articles further down. some references were also obtained through textbooks of hematology.1,11 through this method, we obtained a total of 13 references of which 6 mentioned an incidence range for this pathology and 3 studies that commented on the presence and severity of hemolysis due to c3 only waiha. most of the authors reported an incidence for c3 only waiha as being less than 25%. commonly it seems to have an incidence ranging between 6% and 13%.1, 10,11,14,15,16 the majority of the patients with dat positive for c3 only do not seem to have any hemolysis. among the patients who do have hemolysis, the clinical picture can range from mild asymptomatic anemia that is self - resolving or responding well to simple conservative measures such as oral steroids, to life threatening anemia requiring multiple blood transfusions and refractory to various conservative therapies including prednisone, ivig, cyclosporine, azathioprine, and danazol and requiring splenectomy. however, in our study, we did not come across any reports of deaths due to this clinical entity.14,16,17 a study17 involving 72 patients and comparing coexisting underlying diseases, severity of hemolysis, and response to therapy in patients with dat positive for igg versus c3 (either alone or in conjunction with igg), mentioned that coexisting chronic diseases such as lymphatic neoplasms and connective tissue disorders were more common in patients with c3 positivity than the other groups. the strength of the dat has not shown a definite correlation to the occurrence or severity of hemolysis despite multiple studies over many years.15 treatment of c3 only waiha is not well - documented given the paucity of these cases. however, in the study mentioned above,17 the most satisfactory initial response to steroids was noted in patients with complement coating and normal cold agglutinins. most of the authors reported an incidence for c3 only waiha as being less than 25%. the majority of the patients with dat positive for c3 only do not seem to have any hemolysis. among the patients who do have hemolysis, the clinical picture can range from mild asymptomatic anemia that is self - resolving or responding well to simple conservative measures such as oral steroids, to life threatening anemia requiring multiple blood transfusions and refractory to various conservative therapies including prednisone, ivig, cyclosporine, azathioprine, and danazol and requiring splenectomy. however, in our study, we did not come across any reports of deaths due to this clinical entity.14,16,17 a study17 involving 72 patients and comparing coexisting underlying diseases, severity of hemolysis, and response to therapy in patients with dat positive for igg versus c3 (either alone or in conjunction with igg), mentioned that coexisting chronic diseases such as lymphatic neoplasms and connective tissue disorders were more common in patients with c3 positivity than the other groups. the strength of the dat has not shown a definite correlation to the occurrence or severity of hemolysis despite multiple studies over many years.15 treatment of c3 only waiha is not well - documented given the paucity of these cases. however, in the study mentioned above,17 the most satisfactory initial response to steroids was noted in patients with complement coating and normal cold agglutinins. waiha with a dat positive for c3 only is relatively rare with an incidence ranging between 6% and 13% and can have a clinical picture ranging from mild to severe anemia. in general, steroids should be used as a first - line therapy in these cases. | polygenic igg autoantibodies are implicated in majority of the cases of warm autoimmune hemolytic anemia (waiha). in some of these cases, complement (c3) proteins accompany the igg antibodies. waiha mediated by c3 alone is relatively rare. we present an interesting case of waiha with a direct antiglobulin test (dat) positive for c3 but negative for igg in a 79-year - old woman and perform an analytical literature review of the incidence and severity of this clinical entity. |
participants came from the twins early development study (teds), a population - based longitudinal study of all twins born in england and wales from 1994 to 1996. the present paper is based on data collected from parents when the twins were approximately 8 (mean sd = 7.89 0.53), 9 (9.01 0.29), and 12 (11.28 0.70) years old, and from teachers when the twins were approximately 9 (9.04 0.29) and 12 (11.54 0.66) years old. zygosity in same - sex twins was established using a parent - rated twin similarity questionnaire (60.5% of twins) and dna genotyping (39.5% of twins). twin pairs were excluded from the analyses if severe pre- or postnatal complications were reported or if either twin had a severe medical condition. to retain the full range of variability in autistic traits, children who had suspected or confirmed asd (n = 238) based on the development and well - being assessment (dawba) were included in the analyses when data were available. after exclusions, parent - reported data on both twins were available from 6,280 families at age 8 years, 3,126 families at age 9 years (the 9-year sample was a subsample of families with twins born between january 1994 and august 1995), and 5,339 families at age 12 years. teacher data were available from 2,663 families at age 9 and 4,405 families at age 12. autistic traits were assessed using the childhood autism spectrum test (cast, formerly the childhood asperger syndrome test). the cast was developed as a screening questionnaire for use in non - clinical settings. the published measure consists of 37 items, 31 of which relate to autistic traits (raters are asked to rate the child on specific behaviors associated with autistic traits), the remaining 6 being control questions on general development. at age 12 years, 1 item (regarding pretend play) was removed for not being age appropriate. each question was rated yes (1)/no (0) at ages 8 and 12, and as not true (0), somewhat true (1), or certainly true (2) at age 9. questionnaires with half or more of the items rated were included in the analyses. only between 0.19% and 2.38% of questionnaires were excluded based on this criterion at any individual age. cast subscale scores were calculated using the method described by ronald. the cast has good internal consistency : in the present study cronbach 's ranged from 0.71 to 0.81, depending on age and rater. it has high sensitivity and specificity for asd (both > 95%) using a cut - point of 15, as well as high test recently, the cast has been used successfully in epidemiological research to screen for previously undetected cases of asd. at ages 8 and 12 years, the full cast was used. at age 9, the 20-item abbreviated version of the cast was used. between ages 8 and 9, the full and abbreviated versions of the cast correlated at 0.63, which for the same measure would be considered acceptable test retest reliability across a 1-year interval (see, e.g., test retest reliability of the strengths and difficulties questionnaire), supporting the abbreviated version as an equivalent but shorter version of the cast. correlations between teacher and parent ratings were modest at age 9 (r =.32, p 95%) using a cut - point of 15, as well as high test recently, the cast has been used successfully in epidemiological research to screen for previously undetected cases of asd. at ages 8 and 12 years, the full cast was used. at age 9, the 20-item abbreviated version of the cast was used. between ages 8 and 9, the full and abbreviated versions of the cast correlated at 0.63, which for the same measure would be considered acceptable test retest reliability across a 1-year interval (see, e.g., test retest reliability of the strengths and difficulties questionnaire), supporting the abbreviated version as an equivalent but shorter version of the cast. correlations between teacher and parent ratings were modest at age 9 (r =.32, p.05), indicating minimal sibling interaction. the multivariate twin models described in the data analyses section were implemented in mx and are presented in table 3. the parameter estimates for parent - rated data indicated a high contribution of additive genetic factors (a) to individual differences in autistic traits at each age for both sexes (6876%). furthermore, the overlap in these additive genetic influences across ages (ra) was substantial : all genetic correlations were > 0.70. there was a small, but significant, contribution of shared environmental influences to parent - rated autistic traits at all ages for girls and at age 9 for boys (717%). in boys, there was a small significant contribution of dominant genetic influences to parent - rated autistic traits at ages 8 and 12 ; the overlap in these influences was very high (rd = 1). there was a consistent contribution of non - shared environmental influences in both sexes at all ages to parent - rated autistic traits (1324%). the overlap in these influences across age (re) was modest (0.370.47). the teacher data showed a more modest contribution of additive genetic factors to autistic traits at individual ages (3565%). the overlap in these influences between 9 and 12 years was considerable (ra = 0.58). the effect of shared environmental factors was small and inconsistent, only reaching significance at 9 years in males and 12 years in females on teacher - rated autistic traits. because the effect of the shared environment was only present at 1 age for each sex, a shared environment correlation could not be estimated. finally, the teacher data suggested a moderate effect of non - shared environmental factors (3149%) at individual ages. however, these factors did not overlap across age (re close to 0), indicating that different non - shared environmental influences were at play at each age. figure 1 presents, separately for boys and girls, the contribution of each variance component to the longitudinal stability in autistic traits according to the multivariate twin models. between all ages, and for both raters and sexes, additive genetic factors accounted for most of the longitudinal stability in autistic traits. in the parent data, there was also a small effect of the non - shared environment on longitudinal stability, as well as a small contribution of dominant genetic effects to the longitudinal stability between 8 and 12 years in boys. the effect of the shared environment on longitudinal stability in autistic traits was non - significant in boys and minimal in girls. in the teacher data, the longitudinal stability in autistic traits the same set of analyses as described for the total cast were carried out on each of the 3 cast subscales (sis, cis, and rrbis). for simplicity, and because the teacher data provided a minimum estimate of longitudinal stability (see discussion), subscale analyses were carried out only on the parent data. the results of these analyses are presented in the supplementary material to this article : table s2 presents the longitudinal phenotypic correlations for each subscale ; tables s3s5 present the twin correlations, and tables s6s8 present the modeling results. longitudinal phenotypic correlations were slightly lower for the subscales (r = 0.330.62), particularly for sis and rrbis. additive genetic correlations tended to be moderate to high (ras > 0.5), the exception being the sis subscale, which demonstrated only modest additive genetic correlations (ras = 0.250.31). dominant genetic correlations, where present, were very high (rds 0.85). non - shared environment correlations were modest for all subscales (res = 0.270.39). because of the inconsistent presence of the c component, it was not possible to estimate a shared environment correlation in the subscale models. figures s1 to s3 present the contribution of each variance component to the longitudinal stability in sis, cis, and rrbis respectively. for all 3 subscales, genetic factors accounted for most of the longitudinal stability, although subscales differed in whether these genetic factors were primarily additive (cis) or non - additive (sis). finally, in accordance with findings from the total scale, non - shared environmental influences played a modest but significant role in longitudinal stability. means and sds for each sex zygosity group can be found in supplement 2. females scored significantly lower than males on the cast and had significantly smaller sds at all ages (p.05), indicating minimal sibling interaction. the multivariate twin models described in the data analyses section were implemented in mx and are presented in table 3. the parameter estimates for parent - rated data indicated a high contribution of additive genetic factors (a) to individual differences in autistic traits at each age for both sexes (6876%). furthermore, the overlap in these additive genetic influences across ages (ra) was substantial : all genetic correlations were > 0.70. there was a small, but significant, contribution of shared environmental influences to parent - rated autistic traits at all ages for girls and at age 9 for boys (717%). the overlap in these influences across age (rc) for girls was minimal. in boys, there was a small significant contribution of dominant genetic influences to parent - rated autistic traits at ages 8 and 12 ; the overlap in these influences was very high (rd = 1). there was a consistent contribution of non - shared environmental influences in both sexes at all ages to parent - rated autistic traits (1324%). the overlap in these influences across age (re) was modest (0.370.47). the teacher data showed a more modest contribution of additive genetic factors to autistic traits at individual ages (3565%). the overlap in these influences between 9 and 12 years was considerable (ra = 0.58). the effect of shared environmental factors was small and inconsistent, only reaching significance at 9 years in males and 12 years in females on teacher - rated autistic traits. because the effect of the shared environment was only present at 1 age for each sex, a shared environment correlation could not be estimated. finally, the teacher data suggested a moderate effect of non - shared environmental factors (3149%) at individual ages. however, these factors did not overlap across age (re close to 0), indicating that different non - shared environmental influences were at play at each age. figure 1 presents, separately for boys and girls, the contribution of each variance component to the longitudinal stability in autistic traits according to the multivariate twin models. between all ages, and for both raters and sexes, additive genetic factors accounted for most of the longitudinal stability in autistic traits. in the parent data, there was also a small effect of the non - shared environment on longitudinal stability, as well as a small contribution of dominant genetic effects to the longitudinal stability between 8 and 12 years in boys. the effect of the shared environment on longitudinal stability in autistic traits was non - significant in boys and minimal in girls. in the teacher data, the longitudinal stability in autistic traits the same set of analyses as described for the total cast were carried out on each of the 3 cast subscales (sis, cis, and rrbis). for simplicity, and because the teacher data provided a minimum estimate of longitudinal stability (see discussion), subscale analyses were carried out only on the parent data. the results of these analyses are presented in the supplementary material to this article : table s2 presents the longitudinal phenotypic correlations for each subscale ; tables s3s5 present the twin correlations, and tables s6s8 present the modeling results. longitudinal phenotypic correlations were slightly lower for the subscales (r = 0.330.62), particularly for sis and rrbis. additive genetic correlations tended to be moderate to high (ras > 0.5), the exception being the sis subscale, which demonstrated only modest additive genetic correlations (ras = 0.250.31). dominant genetic correlations, where present, were very high (rds 0.85). non - shared environment correlations were modest for all subscales (res = 0.270.39). because of the inconsistent presence of the c component, it was not possible to estimate a shared environment correlation in the subscale models. figures s1 to s3 present the contribution of each variance component to the longitudinal stability in sis, cis, and rrbis respectively. for all 3 subscales, genetic factors accounted for most of the longitudinal stability, although subscales differed in whether these genetic factors were primarily additive (cis) or non - additive (sis). finally, in accordance with findings from the total scale, non - shared environmental influences played a modest but significant role in longitudinal stability. the present study is the first large - scale, population - based twin study of genetic and environmental influences on autistic traits longitudinally. consistent with previous evidence, cast scores were significantly and substantially correlated between 8 and 12 years (table 1). the parent - rated scores showed the highest longitudinal correlations (r = 0.590.67). the longitudinal correlations in teacher - rated scores were lower (r = 0.260.35). a plausible explanation for the lower stability in teacher ratings is the fact that twins were usually rated by different teachers (but the same parent) at each age. nevertheless, it is noteworthy that even with different raters for the same individuals, autistic traits showed a significant degree of stability across age. also replicating previous findings, we found that autistic traits as a whole were moderately to highly heritable at each age. approximately 70% of the variance could be accounted for by additive genetic factors when children were rated by their parents. the equivalent figures for the teacher data were around 55% to 65%, the only exception being the relatively modest contribution of additive genetic effects (35%) to teacher - rated autistic traits in females at age 12. the smaller contribution of additive genetic influences to variation in teacher - rated autistic traits was matched by somewhat higher contributions of the non - shared environment (3149%). it is worth noting that measurement error (included in the e parameter) is likely to have been larger in the teacher data because twins were often rated by different teachers, with different levels of knowledge about them. an increase in e puts an upper cap on the a parameter, perhaps explaining some of the discrepancy in heritability estimates between teacher - rated and parent - rated scores. the present study found that the overlap in genetic influences across middle childhood is substantial. in the parent - rated data on total autistic traits, the additive genetic correlation between ages was consistently higher than 0.70, indicating that most of the additive genetic variance was shared across age. a similar pattern emerged in the teacher data : although it was slightly lower than the parent estimates, the additive genetic correlation of 0.58 between 9 and 12 years suggested considerable overlap in genetic influences across age. furthermore, the bivariate heritability and bivariate environmental estimates revealed that longitudinal stability in autistic traits was largely due to genetic effects (figure 1). again, longitudinal stability in each trait could be accounted for primarily by genetic effects. they suggest that autistic traits are not just highly stable across middle childhood, but that this stability is caused largely by genetic influences in both boys and girls. this implies that, if these findings generalize to the clinical extreme, stability is a biological characteristic of autistic symptoms, as opposed to, for example, being due to shared environmental factors. however, the results do not mean that all variance in the stability of autistic traits is caused by genetic influences, nor do they suggest that autistic traits can not be changed. even though the correlation in genetic influences was generally around 0.60 to 0.80 between ages, in no instances did confidence intervals reach 1, suggesting the presence of age - specific genetic influences as well as genetic influences common across age. notably, the environment shared by the twins, often thought of as the family environment, had virtually no influence on the stability of autistic traits. in contrast, the environment unique to each twin, such as being in different classes at school or receiving differential parental treatment, did seem to play a modest role in the stability of autistic traits. overall, the findings suggest that the development of autistic traits in middle childhood is characterized mainly by a set of stable genetic influences, but that, in addition to these influences, new genetic influences as well as modest - to - moderate environmental influences emerge at each age. the specific nature of both these stable and temporary causal influences on autistic traits is an important topic for future research. the strengths of the present study include its large sample size and the corroboration of findings across raters. however, the study also has limitations. an abbreviated version of the cast was used at this age, and items were rated on a 02 scale instead of a 01 scale. this might explain why for boys a dominant genetic effect at 8 and 12 years was replaced by a shared environment effect at age 9. we also can not rule out that this could partly be due to small differences between the full and abbreviated cast. there are also more general limitations of the present study. despite evidence for a continuous distribution of autistic traits in the general population with no clear diagnostic cut - point, the twin design involves important assumptions that need to be met to ensure valid findings, such as the equal environments assumption and the absence of assortative mating (for discussion, see rijsdijk and sham and plomin.). furthermore, gene environment interaction (g e) can not be estimated in the classical twin design, where any such interaction is included in the a parameter. finally, the present study relied on parent and teacher ratings. even though it is a strength of the study to have multiple raters, results would benefit from being corroborated using direct observation. future research should endeavor to explore the molecular genetic substrates of the stability in autistic traits, potential g e interaction, and the causes of stability across the lifespan, which were beyond the scope of this study. | objectivedisorders on the autism spectrum, as well as autistic traits in the general population, have been found to be both highly stable across age and highly heritable at individual ages. however, little is known about the overlap in genetic and environmental influences on autistic traits across age and the contribution of such influences to trait stability itself. the present study investigated these questions in a general population sample of twins.methodmore than 6,000 twin pairs were rated on an established scale of autistic traits by their parents at 8, 9, and 12 years of age and by their teachers at 9 and 12 years of age. data were analyzed using structural equation modeling.resultsthe results indicated that, consistently across raters, not only were autistic traits stable, and moderately to highly heritable at individual ages, but there was also a high degree of overlap in genetic influences across age. furthermore, autistic trait stability could largely be accounted for by genetic factors, with the environment unique to each twin playing a minor role. the environment shared by twins had virtually no effect on the longitudinal stability in autistic traits.conclusionsautistic traits are highly stable across middle childhood. and this stability is caused primarily by genetic factors. |
tum or consisted of a firm, well - circumscribed, ovoid piece of soft pink tissue measuring 21.51 cm in the supraglottic area of the larynx. histologically, the mass showed a fibrous and chondromyxoid stroma containing epithelial and myoepithelial cells. epithelial component consisted of relatively uniform - appearing cells arranged in tubules and small, nested aggregates (figure 1) myoepithelial component was positive for p63, s100 protein (figure 2a), and glial fibrillary acidic protein (gfap) (figure 2b) ; and focally positive smooth muscle actin (sma). (a - b) hematoxylin and eosin (h&e) stained section showing sheets of myoepithelial cells and ductular structures lined with epithelial cells set in chondromyxoid stroma (h&e, a : 40 ; b : 100). (c) adjacent to the lesion, normal laryngeal cartilage can be seen (h&e, 100). immunohistochemistry indicates that the myoepithelial cells cytoplasm is focally positive for s100 protein (a) and glial fibrillary acidic protein (gfap) (b) (a : 100 ; b : 100). pa is a commonly diagnosed benign tumor in the salivary glands that may also occur in a variety of other sites ; however, it is rarely seen in the larynx. the diagnosis of pa is simple, but when it grows in an unusual site like the larynx, it can be mistaken for a malignant neoplasm. pleomorphic adenomas are characterized by epithelial tissue mixed with tissues of myxoid, mucoid or chondroid appearance. histologically, pleomorphic adenoma of the larynx may resemble aggressive epithelial tumors because of the high cellularity and lack of a stromal component. importantly, this feature is not in keeping with that of major salivary glands, which demonstrate relatively reduced myoepithelial cellularity. occasionally, pleomorphic adenomas are composed almost entirely of epithelial cells with few or no stromata myoepithelial cells are variably positive for s100 protein, sma, gfap, calponin, and cd10. although pa is a benign tumor, it can cause problems in clinical management due to its tendency to recur and risk of malignant transformation. histological features indicative of malignant transformation include cytological atypia, increased mitotic figures, satellite tumor nodules, tumor necrosis, and infiltrative margins7. pas have tendency to recur when not widely excised, particularly if they are predominantly mucoid, and have variability in the thickness of the capsule and the tumor invading the capsule. in addition, due to low biological requirements, the neoplastic cells can survive when spilt into the operative sites. pas should be differentially diagnosed from other tumors such as carcinoma ex pleomorphic adenoma, adenoid cystic carcinoma, basal cell carcinoma, basal cell adenoma, myoepithelioma, polymorphous low - grade adenocarcinoma, angiofibroma, hamartoma, epidermoid cyst, hemangioma, vascular malformations, nasopharyngeal carcinoma, and nonepithelial tumors. carcinoma ex pleomorphic adenoma exhibits extensively infiltrative malignancy with necrosis, perineurial invasion, frequent mitotic figures, marked nuclear atypia. adenoid cystic carcinoma usually shows cribriform, solid or tubular pattern similar to cylindromas of the skin. it is composed of small, bland myoepithelial cells with scant cytoplasm and dark, compact, angular nuclei that surround pseudoglandular spaces with periodic - acid schiff (pas)-positive excess basement membrane material and mucin. peripheral perineurial invasion and small, true, glandular lumina are sometimes seen, but no squamous differentiation, and extensive necrosis is usually absent. adenoid cystic carcinoma has high proliferative index, high p53 immunoreactivity, and intense staining for b - cell lymphoma 2 (bcl 2), but negative reactivity for glial fibrillary acidic protein. in contrast, pleomorphic adenoma is not invasive, shows no perineurial invasion and has squamous metaplasia and mesenchyme - like areas. it is an infiltrative tumor with perineurial invasion and vascular invasion as well as variable cytologic atypia and mitotic activity. it is composed of solid, trabecular, tubular or membranous patterns, but no myxoid matrix or cartilagenous areas. basal cell adenoma is composed of basaloid cells sharply delineated from the stroma by basement membrane. polymorphous low - grade adenocarcinoma is usually a nonencapsulated tumor with diverse (polymorphous) growth patterns, infiltrative borders, perineurial invasion and tumor necrosis is rare. differential diagnosis of pa consists of myoepithelioma, a benign epithelial salivary gland tumor, and presence of plasmacytoid or spindled myoepithelial cells. additionally, basal cell adenoma may also be involved in the differential diagnosis. to conclude, pleomorphic adenoma of the larynx is a rare neoplasm and therefore its diagnosis requires a high index of suspicion. recurrence many years after surgical excision as well as malignant transformation should be a concern and therefore long - term follow - up is necessary. | pleomorphic adenoma (pa) is the most common benign neoplasm of the salivary glands. it usually occurs in major salivary glands, such as the parotid and submandibular glands. occasionally, however, it occurs in the larynx. these lesions generally present as a slow - growing, painless mass. malignant transformation is very rare, but it increases with time. the present report is the case of a 59-year - old male who presented with a complaint of hoarseness. right vertical partial hemilaryngectomy revealed an intact, mucosa - covered, fleshy 21.51 cm mass in the supraglottic area of the larynx. lesion had histological characteristics of a pa, and this was confirmed by immunohistochemical expression of cytokeratin, s100 protein, glial fibrillary acidic protein (gfap) and vimentin. their histopathological identification is, however, not always straightforward ; immunohistochemistry can contribute significantly to formulation of a definitive diagnosis and to the realization of appropriate follow - up. |
secondary hyperparathyroidism (shpt) is often underdiagnosed or insufficiently treated in patients not yet receiving dialysis, when therapy would have greater short- and long - term benefits. hyperparathyroidism also frequently persists after successful renal transplantation and is present in up to 50% of patients 1 year after renal transplant surgery. thus, early diagnosis and management of shpt in chronic kidney disease (ckd) patients and recognition and treatment of persistent hyperparathyroidism in post - transplant patients is important and might reduce morbidity from cardiovascular and bone disease. the efficacy of the calcimimetic cinacalcet was recently explored in these two distinct patient populations. in this article, the manifestations of hyperparathyroidism in stage 3 and 4 ckd and in kidney transplant patients will be discussed, and data regarding the efficacy of cinacalcet in these two groups of patients will be presented. patients with stage 3 and 4 ckd are more likely to die than to progress to end - stage renal disease. among patients with stage 3 ckd, 24.3% of patients died within 5 years versus 1.3% who progressed to renal replacement therapy. among patients with stage 4 ckd, 45.7% died within 5 years, compared with 19.9% who progressed to dialysis or transplantation (figure 1). accordingly, the national kidney foundation kidney disease outcomes quality initiative guidelines recommend treatment of shpt in stage 3 and 4 ckd patients to reduce disease severity and associated morbidity. the underrecognition and undertreatment of shpt in these patients has serious consequences, including vascular calcifications and bone disease. thus, in the context of shpt, elevated serum phosphorus has been associated with increased mortality risk and with acute myocardial infarction in ckd patients not receiving dialysis. the hypercalcaemia, hyperphosphataemia and increased calcium phosphorus product (ca p) associated with shpt contribute to the development of vascular calcifications that are commonly observed in chronic dialysis patients. the process of vascular calcification begins early in the progression of ckd and continues as kidney function declines. the incidence of vascular calcifications among ckd patients not receiving dialysis is 40% ; this value increases to 64% in patients who are starting dialysis and to 83% in patients established on dialysis. shpt also contributes to renal bone disease, which may cause additional morbidity in haemodialysis patients. once again, the development of renal bone disease occurs early in the progression of ckd. manipulation of the calcium - sensing receptor (car) impacts the synthesis and secretion of parathyroid hormone (pth) and parathyroid gland hyperplasia. calcimimetics, by modulating the car, can directly address the pathophysiology of shpt early in ckd. charytan. reported the results of a double - blind, placebo - controlled trial to determine the safety and efficacy of cinacalcet in 54 patients with stage 3 and 4 ckd. all patients included in the study had a serum intact pth (ipth) concentration of > 130 pg / ml and a serum calcium concentration of > 9.0 mg / dl. doses of vitamin d could be adjusted appropriately : decreased if hypercalcaemia (calcium > 11 mg / dl) or hyperphosphataemia (phosphorus > 5.5 mg / dl) occurred, or increased if serum calcium was 4-year period in 52 patients with intact graft function after renal transplantation. one year after transplantation, ipth remained elevated (65 pg / ml) in more than 50% of patients. in a similar study, ipth values were measured in the month preceding kidney transplant and over a mean 69-month follow - up period after transplant in 62 patients with stable graft function. after > 2.5 years following transplantation, only 23% of transplant patients with good renal function had normal ipth levels, and 27% of patients had ipth values more than twice the upper normal limit (> 130 pg / ml). persistent hyperparathyroidism causes hypercalcaemia, which is commonly observed following renal transplantation. in a study of 129 transplant patients, reinhardt. found that 52% were hypercalcaemic (> 10 mg / dl) 3 months after kidney transplantation, and 15% were still hypercalcaemic at 24 months. post - transplant hypercalcaemia may pose a significant risk for renal and vascular calcifications following transplantation and a subsequent increased risk of cardiovascular death [relative risk (rr) = 2.6 ; p = 0.033 ] and overall mortality (rr = 1.8 ; p = 0.015). in the study by gwinner., calcifications in the kidneys were present in 26% of renal transplant patients, and those patients with calcifications had both elevated ipth and calcium. interestingly, even though there was no association between serum phosphorus and vascular calcification, post - transplant phosphorus supplementation occurred more frequently in those with vascular calcification. persistent hyperparathyroidism is also associated with high bone turnover and may contribute to bone disease following transplantation. of interest pretransplant ipth level was significantly correlated (r = 0.58 ; p = 0.0001) with post - transplant ipth level in patients with normal transplant function > 2.5 years after transplantation. a similar relationship between pre- and post - transplant ipth levels correlations between pretransplant and post - transplant ipth levels have been shown to persist for up to 4 years after transplantation. these studies suggest that treatment of hyperparathyroidism and hypercalcaemia before transplant surgery might help reduce the severity of hyperparathyroidism after transplantation. the current treatment strategies for hyperparathyroidism after renal transplantation are limited and have important side effects. as mentioned above, phosphorus supplementation has been associated with the occurrence of interstitial calcifications in the graft. bisphosphonates generally do not reduce pth levels significantly and may promote low turnover bone disease. parathyroidectomy is effective to lower pth and to correct hypercalcaemia ; however, several studies have suggested that renal allograft function may deteriorate, and cases of graft loss have been described after parathyroidectomy [2932 ]. based on these data, more suitable and safer therapies that correct persistent hyperparathyroidism and hypercalcaemia and the consequent morbidity would be beneficial to this group of patients. given that hyperparathyroidism remains a problem for many patients after successful renal transplantation [24 ], and because there are few effective therapeutic options, there has been interest in novel treatments for the control of hyperparathyroidism in this group of patients. the effect of cinacalcet in patients with persistent hyperparathyroidism after renal transplant has been reported in a number of small prospective and retrospective open - label studies in transplant patients with stable allograft function [3441 ]. in general, patients in these studies had stable renal function with mean creatinine clearances in the range of 30 75 ml / min. the primary objective of cinacalcet therapy was control of post - transplant hypercalcaemia, and cinacalcet was titrated to achieve this endpoint. avoided the use of vitamin d, whereas apostolou. allowed its use once calcium levels were normalized in general, the cinacalcet dosage needed to control hypercalcaemia was relatively low (average, 3040 mg / day). baseline characteristics of the studies are summarized in table 3. overall, efficacy results across the studies were consistent (table 4). within 2 to 4 weeks of cinacalcet treatment, serum ipth was significantly reduced and maintained over the treatment duration (10 weeks to 14 months). in the study by kruse., the sustained reduction in ipth levels after cinacalcet treatment was not significant because of large ipth differences among patients and the small sample size. reported that cinacalcet reduced serum ipth from baseline by an average of 40% during the first month of treatment ; ipth levels remained 4050% lower than baseline for the duration of the 6-month follow - up. similarly, serra. observed a maximal reduction in ipth following 8 weeks of cinacalcet treatment, with reduced ipth being maintained for 26 weeks (figure 4). (n = 12)(n = 14)(n = 9)(n = 10)(n = 11)(n = 7)(n = 2)(n = 18)age (years)meannrnr52nrnrnr6245range49702365nrnr2264387252711966sex (n)men679nr6428women670nr53010dialysis duration before study entry (months)mean32nrnr65nrnrnr84time from renal transplant to study entry (months)mean28nr59nrnrnrnrnrrange63847168nrnr26043510161276pth at study entry (pg / ml)mean190nr171605nr424nr627rangenr801295nrnr99723nr256407nrnr, not reported. table 4.summary of results from cinacalcet studies in post - transplant patientsgraft functionstudycinacalcetimmuno-(creatinine clearance orstudyreferencedurationpatients (n)pthcapca psuppressantsserum creatinine)serra. 6 months12kruse.. 6 months10nsnrnrnrsrinivas.18 months11nsnrapostolou.18 months 7nrapostolou.14 months 2nsnsnsnrel - amm. 4.reduction in ipth during treatment with cinacalcet in renal transplant recipients (n = 12). baseline demographics in renal transplant patients with persistent hyperparathyroidism summary of results from cinacalcet studies in post - transplant patients nr, not reported ; ns, not significant. reduction in ipth during treatment with cinacalcet in renal transplant recipients (n = 12). cinacalcet significantly reduced calcium levels within days of treatment, and calcium levels overall were maintained in the normal range over the treatment periods [3341 ]. serra. reported that serum calcium was maintained within normal limits in all patients for 6 months, with serum - ionized calcium being reduced by 17.7%. kruse. reported normalization of serum calcium in 12 of 14 patients. in the case report by apostolou., calcium levels declined rapidly after just 1 day of cinacalcet treatment. in general, serum phosphorus remained unchanged or was increased towards the normal range [3441 ]. in one study, after 6 months of treatment with cinacalcet, 90% of patients had serum phosphorus levels within the normal range. when it was measured, serum ca p remained unchanged (table 4). graft function was measured in seven studies of renal transplant patients treated with cinacalcet, and renal function remained stable in five of the studies (table 4) [3336,3841 ]. in one study, the glomerular filtration rate, measured by serum creatinine prediction equations, declined over the observation period of cinacalcet treatment ; however, the observation period was only 3 months, precluding conclusions regarding long - term graft function. in another study, the decline in the glomerular filtration rate after cinacalcet therapy was consistent with the rate of decline before treatment, and therefore, this may not have been a result of cinacalcet therapy. there was no reported interference of cinacalcet with immunosuppressants, and no increase in rejection episodes was reported in these studies. in summary, the results of these small studies show that treatment with cinacalcet effectively normalized hypercalcaemia and significantly reduced ipth levels in renal transplant recipients, without adversely affecting graft function. larger studies are required to confirm these findings and to obtain long - term data regarding safety and efficacy. in patients undergoing chronic dialysis, cinacalcet has been demonstrated to substantially improve the control of shpt compared with standard care [1719,42,43 ]. the role of calcimimetics in the treatment of shpt in stage 3 and 4 ckd patients and in the treatment of persistent hyperparathyroidism in post - transplant patients is less well characterized. nonetheless, the car is a promising target for these two distinct patient populations. in the early studies reported here, cinacalcet significantly reduced ipth concentrations by more than 30% in most patients with stage 3 and 4 ckd. in kidney transplant patients, cinacalcet therapy significantly reduced calcium and ipth levels and placed phosphorus into the normal range. these early clinical studies suggest that cinacalcet is effective in controlling mineral metabolism in patients with stage 3 and 4 ckd and post - renal transplantation. because these studies were of short duration and included only small numbers of patients, long - term studies in larger groups of patients | cinacalcet has proven effective in the treatment of secondary hyperparathyroidism (shpt) in dialysis patients, and it may also have benefits in stage 3 and 4 chronic kidney disease (ckd). the efficacy of cinacalcet in the treatment of shpt was investigated in a study of 54 patients with stage 3 and 4 ckd not receiving dialysis. a significant number of these patients achieved at least a 30% reduction in parathyroid hormone (pth) from baseline with cinacalcet therapy compared with placebo (56% versus 19% ; p = 0.006). another potential use of cinacalcet is in the treatment of persistent hyperparathyroidism (hpt) after kidney transplantation. the pathophysiologic considerations for persistent hpt in patients who have undergone renal transplantation are different from those in stage 3 and 4 ckd. post - transplant patients with normal graft function often present with hypercalcaemia, low serum phosphorus and persistently elevated levels of pth. in eight small open - label studies including a total of 83 patients with persistent hpt after successful kidney transplantation, cinacalcet treatment effectively corrected hypercalcaemia and significantly reduced elevated pth levels. these studies suggest that cinacalcet therapy is an effective therapy in controlling hyperparathyroidism in patients with stage 3 and 4 ckd and in post - transplant patients with persistent hyperparathyroidism. |
p. vivax is the most prevalent cause of human malaria in the americas, accounting for more than 70% of total cases reported. chloroquine (cq) at a dose of 25 mg / kg over three days remains the first line therapy for vivax malaria. to prevent relapse, pq is given to all nonpregnant patients greater than 6 months of age with normal activity of glucose-6-phosphate dehydrogenase (g-6-pd). the peruvian national malaria control program changed from the standard 14-day course of pq at 0.25 mg / kg / day to a 7-day regimen of 0.5 mg / kg / day in 1998. both regimens achieve a total adult pq dose of 210 mg which is assumed to be widely effective against the hypnozoite stage of p. vivax strains acquired outside of southeast asia and the indo - pacific islands. widespread chloroquine resistance (cqr) has been reported in some areas of the world, although, up until now, only two confirmed cases have been reported in peru [2, 3 ]. recurrence of p. vivax following treatment can be classified as recrudescence, which is the recurrence of parasitemia originating from subpatent trophozoites that survived in the blood following treatment, relapse which is the recurrence of parasitemia originating from latent hypnozoites in the liver, or reinfection with a new strain. recrudescence occurs when the blood stage antimalarial drug such as chloroquine fails, either due to inadequate dosing or resistance of the blood stage parasites to the drug. relapse can occur in the absence of a specific treatment against the hypnozoite stage or when the liver stage antimalarial drug such as primaquine fails to completely eradicate the parasite from the liver. in general, reappearance of parasitemia between 17 and 35 days after treatment is attributed to either recrudescence, early relapse, or reinfection, whereas reappearance after 35 days is attributed to a relapse or reinfection. based on the treatment sensitivity demonstrated by p. vivax to blood levels of total cq (parent compound cq plus the active desethylchloroquine metabolite, dcq), a minimally effective concentration (mec) of 100 ng / ml was determined as the criteria for separating between cq - sensitive and cq - resistant strains of p. vivax, whether they arise from recrudescence, relapse, or reinfection. the present protocol studied the efficacy of three different regimens of primaquine for the prevention of relapses of p. vivax at three different sites : padre cocha (pc), san juan (sj), and santa clara (sc), located in or around the city of iquitos in the department of loreto in the amazon basin of peru (figure 1). between 2005 and 2008, a total of 540 informed and consenting subjects between 1 to 77 years of age, with symptomatic microscopy - confirmed p. vivax monoinfection, were enrolled. institutional review boards in peru and the united states had reviewed and approved the study plan and procedures prior to any enrollment (796 - 2004-j - opd / ins and nmrc.2005.0005). study participants were divided into three arms, all of whom received the same standard treatment dose of chloroquine (25 mg base / kg body weight divided into single daily doses over 3 days) but were randomized to receive one of three regimens of pq : (1) 0.5 mg / kg daily for 5 days, (2) 0.5 mg / kg daily for 7 days, or (3) 0.25 mg / kg daily for 14 days. both cq and pq regimens were started on the same day, and consumption of each dose was directly observed. as part of the study protocol, blood smears were collected and evaluated on days 1, 2, 3, 7, 14, 21, and 28, later every 2 weeks until 6 months, and on each occasion when an enrolled study participant returned to the clinic with malaria - like symptoms. patients who developed a recurrence of parasitemia during the 6-month follow - up period were treated as recommended by the peruvian ministry of health (cq : 25 mg / kg base over 3 days : 10 mg / kg on days 1 and 2, and 5 mg / kg on day 3, plus pq : 0.5 a blood smear was performed on day 0 and follow - up days. in the case of recurrence of parasitemia within 1735 days of starting treatment, 2 ml of edta blood were collected for determination of cq blood levels and p. vivax genotyping. a third slide was read in the case of discrepancy between the first two reads. three aliquots of 100 l of whole blood from day - of - recurrence (d - r) samples were spotted onto filter paper for later analysis by high performance liquid chromatography (hplc) to determine the levels of cq and its major metabolite, desethylchloroquine (dcq) as previously described. dna was isolated from 200 l of whole blood - edta samples from d-0 and d - r by using a qiaamp dna blood mini kit (qiagen, nl), following the manufacturer 's instructions. all initial (d-0) and recurrent (d - r) parasitemias were confirmed as p. vivax monoinfections by pcr. genomic dna of p. vivax was amplified at the pvmdr1 and pvcrt loci and sequenced using bigdye terminator sequencing kit (applied biosystems, usa) on an abi 3130 automated dna sequencer (applied biosystems, usa) [5, 6 ]. from the total of 540 patients enrolled in the study at the three sites, four subjects had a recurrence of parasitemia within the 35-day follow - up period. the four patients had fever or history of fever, were between 4 and 11 years old, and had a geometric mean (gm) parasite density of 13,536 parasites/l on d-0. parasitemias recurred with symptoms on day 28 (2 patients), day 30, and day 32. however, the re - reading of the routine, scheduled slides showed that the recurrence of p. vivax parasitemia actually occurred earlier, but without symptoms, on day 28 for sc105 (table 1). initial reading of the day-32 slide for sc105 showed a mixed infection of p. falciparum and p. vivax, but upon review of this slide and reading of the duplicate and triplicate slides for this patient, a monoinfection with p. vivax was determined. p. vivax monoinfection, that is, the absence of p. falciparum, was confirmed in the day-32 whole blood sample of sc105 by pcr. this patient was treated with mefloquine and artesunate for the presumed p. falciparum infection that only later was determined to be a recurrent p. vivax infection. in three patients, the levels of cq plus dcq were subtherapeutic (ccc). none of the mutations in either gene were reported to be associated with cq resistance in p. vivax. according to published criteria for classifying a p. vivax treatment failure as cq resistance, one of four patients in our study would be considered a probable case of cqrpv. multiple criteria that underlie this determination are the following : (1) gmp quality cq, (2) proper weight - adjusted dose determination, (3) complete and directly observed consumption of all doses, (4) normal enteric absorption of the drug, and (5) a cq + dcq blood level at the time of recrudescence 100 ng / ml. in this study, one patient, sc105, had a total cq level of 95 ng / ml in whole blood on day 32 (initially defined as the day of recurrence). however, a second reading of the slides later showed that there was actually reappearance of parasitemia four days earlier on day 28. although a blood sample was not collected on day 28 for this patient, it is likely that the total chloroquine concentration would have been higher on day 28 than on the day it was actually measured, day 32. it seems likely, although it can not be said with complete certainty, that this patient indeed was infected with a chloroquine - resistant strain of p. vivax. we can not say with certainty whether the recurrent parasitemia was a late recrudescence or an early relapse. recrudescence is unlikely, given the fact that cq resistance in p. vivax in peru is quite low, and the combination of cq + pq is very effective against cq - resistant malaria strains ; pq is reported to be even more potent than verapamil in reversing cq resistance [7, 8 ]. early relapse due to failure of the pq to eliminate the hypnozoites seems more likely to have occurred, but if so, this relapse occurred against an impressive block of pq. the recurrent parasites in the blood would have been exposed to the otherwise efficacious level of cq remaining in the blood on day 28, but now in the absence of pq due to its shorter half life, the remaining cq was unable to eliminate the recurrent cq - resistant parasites. upon the reappearance of parasitemia, subject sc105 was treated with mefloquine and artesunate for presumed falciparum malaria on day 32. after that, the patient was lost to followup, but it is likely that the accidental treatment for falciparum malaria successfully treated the recurrence of a cq - resistant p. vivax strain. the other three patients, pc46, sj4, and sc58, were successfully re - treated with cq (25 mg over 3 days) plus pq (0.5 mg / kg daily for 7 days) according to the peruvian national malaria control program policy with no further reappearance of parasitemia. it is understood that pq total dose, more than schedule or duration, is the determinant underlying effective relapse prevention [9, 10 ], and it is also recognized that the curative action of pq in eradicating the hypnozoite stage of p. vivax is potentiated by concurrent cq or quinine therapy [11, 12 ]. primaquine works synergistically with a variety of drugs [1316 ], and the combination of chloroquine plus primaquine was demonstrated to overcome chloroquine resistance in both p. yoelii and p. vivax. more recently, primaquine is also shown to work synergistically with chloroquine against chloroquine - resistant p. falciparum [7, 8 ]. acting on numerous cases of p. vivax recrudescence and/or relapse by the standard 14-day pq regimen of 15 mg / day from locations throughout the old and new world, the us centers for disease control (cdc) recently revised its dosing recommendation for pq antirelapse therapy and radical cure [12, 19, 20 ]. under this new advisement, the adult dose of pq is increased from 15 to 30 mg daily for 14 days, and the pediatric dose has been comparably increased from 0.25 to 0.5 mg / kg / day. under the new cdc recommendation, which has not yet been approved by the us food and drug administration (fda), the four - year - old peruvian subject, sc105, would have received a total pediatric pq dose of 119 mg, considered sufficient to eliminate the pq tolerant chesson - type forms of p. vivax found in southeast asia and the western pacific [10, 21, 22 ]. the patient was in the 14-day arm of this study that received 0.25 mg / kg daily of pq for 14 days, which in this 17 kg child would be a total indicated dose of 59.5 mg pq. in actuality, this subject received a half tablet (7.5 mg) of pq daily which amounted to a 14-day total dose of 105 mg. this total dose, although slightly less than the new cdc recommendation, was considerably higher than the total pediatric dose of 59.5 mg achieved under the old cdc recommendation. the patient was scheduled to receive 25 mg / kg cq base over 3 days, which per her weight of 17 kg should have been a total dose of 425 mg cq base. in actuality, she received a 150 mg base tablet per day, for a total dose of 450 mg cq base. on the day that her recurrent parasitemia was detected, the level of total cq in her blood was at least 95 ng / ml and possibly greater than 100 ng / ml, the minimum therapeutic level. the association between mutations in pvmdr1 and pvcrt and cq resistance in p. vivax is not the same as the strong association between the orthologous genes in p. falciparum and cq resistance in this organism. no obvious mutations in pvmdr1 and pvcrt tentatively linked to chloroquine resistance were found in these samples. codon 976 in pvmdr1, studied for its feasible association to cqr, was wild - type in all eight d-0 and d - r samples. although we used the pvmdr1 and pvcrt gene sequences to distinguish between whether the recurrent strains were the same or different from the original strains, this analysis is limited due to the low level of heterogeneity in their sequences in peruvian strains. as such, while any difference in sequences of d-0 and d - r represents a clear difference in the two strains, the same sequence detected on d-0 and d - r may simply happen by chance and may not indicate that the two strains are identical. while this report confirms previously reported low - level cq resistance by p. vivax in the peruvian amazon region, it is significant in reporting the first occurrence in peru of a failure of the standard cq + pq combination therapy to achieve radical cure. importantly, this treatment failure occurred in the context of a clinical trial in which (1) all drugs used were produced under usfda regulations, (2) dose was carefully determined according to the subject 's weight, (3) pq treatment was administered with food to reduce gastric upset / improve drug absorption, and (4) consumption of each treatment dose of drug was fully supervised and directly observed. the recurrence of p. vivax following combined therapy with chloroquine and high - dose primaquine is unexpected in south america, where in vivo testing has demonstrated relatively few valid cases of cq resistance [3, 23 ]. guyana has been the only other nation in south america that has reported failure of cq - pq combination therapy, but in multiple adult cases, against both low (15 mg / day) and high (30 mg / day) doses of pq, and with parasitemias appearing six weeks following treatment. health providers and travelers should be aware that such difficult cases occur even when there is little evidence of resistance in the region. as in the earlier study of ruebush., it is interesting that all four current cases of cq failure were in children, less than 16 years old. three of the patients in the current study and one of the patients in the previous study had suboptimal levels of chloroquine in the blood on the day of failure. it may be that children fail to absorb the full dose of chloroquine or else eliminate the drug from their systems more rapidly than adults. only a few studies have looked at cq absorption in children but never in conjunction with pq. future studies should address the absorption of cq and pq in children as compared to adults. | the widespread use of primaquine (pq) and chloroquine (cq), together, may be responsible for the relatively few, isolated cases of chloroquine - resistant p. vivax (cqrpv) that have been reported from south america. we report here a case of p. vivax from the amazon basin of peru that recurred against normally therapeutic blood levels of cq. four out of 540 patients treated with combination cq and pq had a symptomatic recurrence of p. vivax parasitemia within 35 days of treatment initiation, possibly indicating cq failure. whole blood total cq level for one of these four subjects was 95 ng / ml on the day of recurrence. based on published criteria that delineate cqrpv as a p. vivax parasitemia, either recrudescence or relapse, that appears against cq blood levels > 100 ng / ml, we document the occurrence of a p. vivax strain in peru that had unusually high tolerance to the synergistic combination therapy of cq + pq that normally works quite well. |
vibrio parahaemolyticus and vibrio vulnificus are important foodborne pathogens associated with the consumption of fish and shellfish, especially oysters, which have long been known to bioconcentrate vibrios within their edible tissues [1, 2 ]. vibrio vulnificus also causes life - threatening illness from wound infections acquired in the marine environment. pathogenic vibrios show seasonal predilection in seawater and shellfish, with high counts during warmer months and low to negligible counts during the colder months [2, 4, 5 ]. recently, we showed that naturally occurring bdellovibrio and like organisms (balos) from coastal seawater significantly reduced the levels of v. parahaemolyticus and v. vulnificus in seawater and v. parahaemolyticus in seawater and oysters. among the balos are marine and terrestrial forms, with the marine forms associated with bacteriovorax, which are exclusively saltwater predators [7, 8 ]. bacteriovorax have shown preferential predation toward v. parahaemolyticus when compared to a broad range of potential host bacteria [912 ]. this suggests that bacteriovorax may invade and kill v. parahaemolyticus in seawater more efficiently than other bacterial pathogens. the life cycle of bacteriovorax and other balos usually involve intracellular invasion of and replication within a host cell, although some are known to grow host - independently [1316 ]. during the attack phase, balos propel themselves with a single polar flagellum to find a susceptible gram - negative bacterium to serve as its host. the balo enzymatically digests a hole in the host membrane, enters the periplasmic space, and, utilizing nutrients from the host, grows in a worm - like fashion in a structure known as a bdelloplast. when mature, the bdelloplast septates into multiple immature cells and are subsequently released from the host as it lyses. the immature cells develop into mature, attack phase cells to repeat the cycle all over again. attack phase balos are small, with a diameter of only 0.20.4 m, making them filterable through 0.45 m pore size membranes, which facilitates their separation from host bacteria. immature balos are about the same length but appear narrower and smoother than those in the attack phase, making them easily filterable as well. in natural unfiltered seawater spiked with > 1 10 cfu / ml v. parahaemolyticus, v. parahaemolyticus counts decreased by 3-logs to nearly nondetectable levels over 72 h, while naturally occurring balos (bacteriovorax) increased by 3-logs from nearly nondetectable initial levels over this same period. some studies have evaluated the seasonality of bacteriovorax in marine systems including estuarine sediment and seawater [18, 19 ]. the present study further evaluates the seasonality of bacteriovorax among estuarine species. in this study, we evaluated natural seawater monthly for 1 year for total culturable bacteriovorax that were capable of infecting vibrio parahaemolyticus o3:k6 host cells (so called vibrio predatory bacteria) from four sites along the delaware bay (figure 1(a)), one site from the gulf coast of alabama (figure 1(b)) and one site in kailua - kona, hawaii (figure 1(c)). three of the collection sites along the delaware bay and the gulf site were estuarine. we also identify seasonal patterns for bacteriovorax abundance, evaluate the effects of temperature and salinity on bacteriovorax levels, identify some of the bacteriovorax phylotypes, and suggest environmental factors that may influence bacteriovorax levels. a clinical strain of v. parahaemolyticus o3:k6 known as rimd2210633 was used as host for the assay of bacteriovorax from seawater. this pandemic strain was originally isolated from an airport quarantine station in japan in 1996 and caused travelers ' diarrhea [20, 21 ]. stock cultures of this isolate were routinely grown in luria bertani (lb) broth (beckton, dickinson and co., sparks, md) supplemented with 2% nacl (3% nacl total) or were streaked on lb agar (beckton, dickinson and co.) with 2% added nacl (3% nacl total). atlantic seawater was surface water collected along the shoreline or rivers ' edges during high tide and analyzed from four delaware bay sites as previously described. in essence, the delaware sites were site 1 : the cape may - lewes ferry terminal in lewes, de (384657.85n ; 750704.73w) ; site 2 : the broadkill river, outside the university of delaware marine laboratory in lewes, de, 0.6 km upstream from the mouth of the river (384726.37n ; 750951.36w) ; site 3 : oyster rocks road boat landing on the broadkill river in milton, de (384808.01n ; 751211.57w) ; and site 4 : scotton landing on the saint jones river in frederica, de (390505.94n ; 752739.99w) (figure 1(a)). food and drug administration laboratory at dauphin island, al (301525.57n ; 88621.73w) (figure 1(b)), and shipped to delaware overnight in an insulated cooler for testing. seawater temperature and salinity were recorded for the delaware and gulf sites at time of water collection. surface seawater was also provided by kona coast shellfish co., kailua - kona, hi (194342.9n ; 156346.2w) (figure 1(c)), and was express shipped in an insulated cooler to the laboratory where it was received within 2 days of collection. temperature of the hawaiian water at time of collection was 24 - 25c and approximately 35 ppt salinity year round. the hawaiian and gulf water samples were shipped with ice packs during the summer ; however, the ice and seawater were physically separated in order to prevent direct contact. after delivery to the laboratory, all seawater samples were analyzed within 4 h. counts of bacteriovorax in 7.5 ml portions of unfiltered or 0.45 m filtered seawater were quantified on lawns of v. parahaemolyticus o3:k6 host cells grown in pp20 agar (polypeptone peptone supplemented with bacto agar), as recently described. preliminary studies showed that seawater filtration was necessary to reduce the levels of non - bacteriovorax microbial contaminants that could otherwise overgrow the lawns of v. parahaemolyticus host cells, thus affecting the quantification of immature or attack - phase bacteriovorax. potential bacteriophage plaques would be distinguished from bacteriovorax plaques by their rapid, overnight formation ; whereas, bacteriovorax plaques require several days to form. although bacteriovorax produce plaques on pp20 agar, bacteriophages generally require more nutritionally complex media for plaque formation. was analyzed per assay, total culturable bacteriovorax counts are listed as counts/7.5 ml seawater throughout this paper. scanning electron microscopy was performed on select isolates as described previously using a quanta 200feg microscope (fei co., hillsboro, or) after glutaraldehyde fixation, rinsing with 0.1 m imidazole buffer (ph 7.0), dehydration in graded ethanol rinses, critical point drying by vacuum (denton, cherry hill, nj), and sputter coating with gold in an argon atmosphere using an edwards scancoat six sputter coater (west sussex, united kingdom). correlation coefficients were obtained between bacteriovorax counts and salinity or temperature, while significant differences in counts, temperatures, and salinities were determined by student 's t test. p values 0.05 were considered statistically significant while values 0.0001 were considered extremely statistically significant. for 16s rrna sequencing, pcr was performed on 13 randomly drawn isolates using 0.2 m final concentration of forward primer (bac676f) and reverse primer (bac1442r), as described by davidov. conditions were 94c denaturation for 1 min followed by 45 cycles at 94c for 1 min, 56c for 1 min, and 72c for 1 min. the cdnas were electrophoretically purified on an 0.8% agarose gel and ethidium bromide - stained bands of ~700 bp were excised and purified using a qiaquick gel extraction kit (qiagen # 28704) following the manufacturer 's instructions. data was trimmed at the terminal ends and sequences ~700 bp long were identified by genbank blast search and compared to phylotypes and clones described for similar sampling sites, as described by pineiro.. one site (site 1) was at the cape may - lewes ferry terminal near the mouth of the delaware bay, while the other three sites were on tidal rivers in close proximity to the delaware bay (figure 1(a)). mean monthly counts of bacteriovorax per 7.5 ml of 0.45 m - filtered seawater were determined for 1 year and water collection scheduled for late october 2012 was postponed because of hurricane sandy which impacted this area with strong tidal surge, hurricane - force winds, and extremely heavy rains during late october. the hurricane caused seawater collection for the october sampling to be delayed until november 5. bacteriovorax counts were at their highest levels at three of the four collection sites one week after the hurricane (figure 2(a), asterisk). the most inland site (site 4) showed no increase after the hurricane but rather decreased from the previous month 's mean count. unlike the other sites, site 4 experienced a dramatic increase in bacteriovorax levels over the summer months, with a peak count of 33 pfu/7.5 ml seawater in early september. the bacteriovorax also showed a seasonal predilection with significantly higher counts in the summer than in the winter (p 0.0001). bacteriovorax were detected from at least one site in the delaware bay every month except in february 2013 when none (25 ppt throughout the year for three of the four sites (figure 2(c)). site 4 showed the lowest salinities throughout the study, ranging from a low of 4.8 ppt in february to a high of 23.4 ppt in late july (figures 2(c) and 3(d)). in addition, there was little to no correlation between salinity and bacteriovorax counts for sites 13 with r values of 0.067, 0.119, and 0.183, respectively ; however, there was a strong correlation between salinity and bacteriovorax counts for site 4 (r = 0.792). spikes in counts were occasionally seen at each site (figures 3(a)3(d)) but were not consistently associated with temperature or salinity, except in the case of sites 13, immediately after hurricane sandy where peaks in counts occurred as water temperatures dipped from > 20c the month before the storm to approximately 10c after the storm (figures 3(a)3(c)). site 1 was the southernmost site directly along a rock - fortified shoreline near the mouth of the delaware bay (figure 1(a)). in contrast, the other three sites were tidal rivers with marsh grasses growing along the banks of the rivers. the presence or absence of marshes in the vicinity of the collection sites could not be shown to influence total bacteriovorax levels over the course of this study. site 4 showed a different trend in total bacteriovorax counts from those of sites 13, which were similar (figure 2(a)). this may be due more to the fact that site 4 was the greatest distance inland from the delaware bay (and had lower salinity) rather than to the presence or absence of marshes in the area. gulf coast seawater ranged in salinities (4.828.8 ppt) and temperatures (12.231.1c) (figure 4(a)). in contrast to delaware seawater, the gulf coast seawater showed low levels of bacteriovorax (5 pfu/7.5 ml of seawater) throughout the summer months and significantly higher (p 0.0001) levels during the winter months (figure 4(a)). this is opposite to the findings in delaware, where significantly higher counts (p 0.0001) were obtained throughout the summer months. in fact, there was a relatively strong negative correlation between temperature and bacteriovorax levels (r = 0.585) for gulf seawater. the highest mean bacteriovorax reading in the gulf was obtained in december, concurrent with the highest salinity reading. as salinity levels dropped from january to february, so did the levels of total bacteriovorax (figure 4(a)). nevertheless, correlation was low between salinity and bacteriovorax counts over the course of the year (r = 0.211). hawaiian seawater, like the delaware bay and gulf coast seawater, was surface water, but it was not subjected to seasonal temperature changes or shifts in salinities. it had nearly constant temperature and salinity (24 - 25c and 35 ppt) throughout the year. results showed two spikes in bacteriovorax counts, one in august followed by one in february, when mean counts increased from levels generally < 5 pfu/7.5 ml of seawater to 23 and 121.8 pfu/7.5 ml of seawater, respectively (figure 4(b)). bacteriovorax counts for both gulf coast and hawaiian seawater may have been affected by shipping, which took 1 and 2 days, respectively. we can not preclude the possibility that counts may have changed during transit, especially if potential host cells were present in the seawater. predation on natural bacteria in the seawater could account for the two spikes in monthly counts that were observed for the hawaiian seawater (figure 4(b)). sequencing of isolates showed 99.9 - 100% identity (maximum of 1 base difference out of 700 bp per isolate) to comparable sequence submitted to genbank by pineiro.. phylotypes and clones are shown in table 1 and indicate that the isolates were within four different phylotype clusters (ix, x, xi, and xii). none of the isolates were of the lower numbered clusters, like iv and v, which are more commonly believed to be low - salt, estuarine species [9, 19, 23 ]. cluster ix isolates were from two sites in the delaware bay near lewes, de, and from one site along the gulf coast (table 1). previously, pineiro. obtained this same isolate from the delaware bay in lewes and named it as clone lewes11. the two delaware isolates were obtained from a medium salinity site (site 3) and a low salinity site (site 4). high levels of microbial contaminants were present in unfiltered atlantic and gulf coast seawater, but hawaiian seawater was cleaner, thus allowing a comparison of bacteriovorax counts in unfiltered and 0.45 m filtered samples (table 2). in unfiltered seawater, bacteriovorax were higher for most of the months (table 2). only in august were the levels in unfiltered and 0.45 m filtered seawater essentially the same (23.5 versus 23.3 pfu/7.5 ml of seawater, resp.). in february 2013, counts in the unfiltered hawaiian seawater were too numerous to count and far exceeded the 121.8 pfu/7.5 ml seawater that was observed for the filtered sample. these spikes in bacteriovorax counts can not be attributed to changes in temperature or salinity because temperature and salinity of the hawaiian seawater remained nearly constant. mean counts, excluding the february 2013 data, indicate the presence of 6.2 times more bacteriovorax in unfiltered seawater than in filtered seawater. representative plaques on pp20 agar plates were picked to further evaluate some of the isolates. plaque sizes often varied from one isolate to another and plaques were generally colorless (figure 5). bacteriovorax were usually isolated by picking from the center of large, well - isolated plaques ; enriching in host cells suspended in sterile seawater ; and imaging by scanning electron microscopy, as previously described. a range of morphologies were observed, as shown in figure 6, and included small, filterable forms (immature and attack - phase stages) and larger forms within the host (bdelloplast stage). therefore, their ability to pass through 0.45 m filters depended on their stage in the life cycle. areas of clearing around colonies were occasionally observed and were associated with the diffusion of inhibitory substances produced by the colonies. over the course of a year, bacteriovorax were detected at the highest levels in the delaware bay during the summer and along the gulf coast during the winter. this latter finding may be surprising at first, since vibrio levels are typically very low during the winter months. however, bacteriovorax are known to have broad host specificities against a wide variety of gram - negative bacteria [912 ]. although this study concentrated on culturable bacteriovorax that infected v. parahaemolyticus and hence may be referred to as vibrio predatory bacteria, it would be expected that the bacteriovorax likely preyed on many other gram - negative bacteria outside the vibrionaceae family, particularly when vibrio levels were low. bacteriovorax were isolated nearly year round from small volumes (7.5 ml) of seawater without the need for enrichment. it should be recognized that the above counts represent the number of culturable bacteriovorax in 0.45 m - filtered seawater and may underrepresent the total number of bacteriovorax within the water. although the immature and attack - phase bacteriovorax are small enough to pass through a 0.45 m filter, an appreciable number of bacteriovorax might not ; thus counts expressed throughout this paper should be considered as minimal counts derived principally from immature and attack phase bacteriovorax. larger bacteriovorax, that is, those maturing as bdelloplasts within the host cell or the so - called host independent strains that occasionally grow as elongated chains, would readily be filtered out. by comparing levels of bacteriovorax in filtered and unfiltered hawaiian seawater (table 2), we estimate that 84% of the bacteriovorax in seawater were unable to pass through a 0.45 m filter. this highlights a shortcoming of using filtered seawater for the quantification of bacteriovorax in culture - dependent methods. it is uncertain if season has any influence on the prevalence of different life forms or sizes of bacteriovorax. the presence of a variety of bacteriovorax phylotypes having different preferences for both salinity and temperature was reported in a study of the cheaspeake bay where some phylotypes (clusters iv and v) were low to medium salinity (estuarine) species, whereas others (clusters ix and xii) were recovered from medium or high salinity sites [9, 23 ]. sequence analysis of 13 samples in this present study showed that all the isolates were in clusters ix, x, xi, or xii (table 1), regardless of the isolates ' origin. as mentioned previously, four of the sequenced isolates obtained from the lowest salinity site in delaware (site 4) were clusters ix, x, or xii clusters more commonly associated with higher salinity seawater. in the present study, salinities did not deviate greatly for three of the delaware bay sites or for seawater obtained from hawaii ; however, seawater from the gulf coast and from delaware bay site 4 varied appreciably. when the salinities were the highest at these two sites, the bacteriovorax levels peaked (figures 3(d) and 4(a)). we showed that salinity and total bacteriovorax counts correlated well (r = 0.792) at the lowest salinity delaware bay site (site 4) but not at the higher salinity delaware sites. compared to the other sites, site 4 had relatively low and widely varying salinities ; therefore, the strong correlation between salinity and bacteriovorax counts at this site must be tempered by the fact that this riverine location never reached the higher salinity levels found in the other delaware bay sites. detected clusters iv and v in low to moderate salinity areas of the delaware bay and in the nearby chesapeake bay. we did not identify any cluster iv or v isolates in delaware or elsewhere, even though these clusters are considered more estuarine species [9, 23 ]. part of the reason may be that our sampling was very limited and was not intended to be a comprehensive analysis of phylotypes, since the dynamic mixing of various proportions of fresh or low - salinity water and seawater in these areas during high tide would be expected to continually alter the phylotype profiles. the identification of only phylotypes ix, x, xi, and xii in the delaware bay does not preclude the presence of other undetected phylotypes from among the many unsequenced bacteriovorax that were enumerated over the year. studies have demonstrated that relaying of oysters to higher salinity areas reduces vibrio levels faster than when oysters are maintained in lower salinity waters [24, 25 ]. high salinity relay has been proposed as a postharvest processing strategy to reduce v. vulnificus in oysters and was also shown effective in eliminating v. parahaemolyticus as well. we recently hypothesized that high salinity supports the proliferation of predatory bacteria and that drought conditions in north carolina from 2007 to 2009 may have been responsible for the apparent disappearance of v. vulnificus from seawater and oysters, as reported by froelich.. other factors beyond salinity and temperature that may well affect bacteriovorax levels and phylotypes within rapidly changing estuarine systems include dramatic fluctuations in flow rates due to storm events, currents and winds, runoff, physical and chemical parameters of the seawater, and perhaps proximity to marshlands, beaches, farmlands, forested or residential areas, or rocky shorelines. the amount of nutrients and suspended solids in the water varied and resuspended sediment was commonly observed. during the winter, bacteriovorax could not be found in top or bottom water samples from the chesapeake bay but were isolated during this period from sediment. this may indicate that greater numbers of bacteriovorax are present in the sediment in the winter and perhaps at other times of the year ; therefore, resuspension of sediment into the water column might influence the bacteriovorax levels detected in the water. our finding that seawater taken from the delaware bay one week after hurricane sandy contained the highest levels of bacteriovorax detected in the delaware waters may be due to the resuspension of large amounts of sediment into the water column by the hurricane. these are all factors that affect shellfish growing areas and the levels of bacteriovorax within these areas. the combination of these highly variable factors will complicate full discernment of the intricacies of predatory interactions with vibrios and other bacteria in the environment. oysters and other molluscan bivalve shellfish are well known for filtering pathogens and other contaminants from seawater making shellfish potential sources for illness. bacteriovorax that may be suspended in seawater or attached to suspended matter are likely sources of food for bivalve shellfish and could concentrate within the shellfish similar to that of pathogens. tests for bacteriovorax quantification within shellfish tissues have not been developed to date ; however, we hypothesize that bacteriovorax within shellfish would continue to parasitize vibrios and other potential pathogens much as they do in seawater, thus rendering shellfish safer to consume when they are obtained from areas where bacteriovorax are present in higher numbers. our previous work indicates that v. parahaemolyticus levels drop significantly in oysters and seawater as vibrio predatory bacteria increase in the surrounding water. our objectives over the next few years are to (a) develop methods to accurately quantify bacteriovorax levels within oyster tissues, (b) determine the relationships between bacteriovorax and vibrio counts in seawater and in shellfish, and (c) develop potential commercial processing strategies using bacteriovorax to reduce vibrios and other potential pathogens in shellfish. | bacteriovorax were quantified in us atlantic, gulf, and pacific seawater to determine baseline levels of these predatory bacteria and possible seasonal fluctuations in levels. surface seawater was analyzed monthly for 1 year from kailua - kona, hawaii ; the gulf coast of alabama ; and four sites along the delaware bay. screening for bacteriovorax was performed on lawns of v. parahaemolyticus host cells. direct testing of 7.5 ml portions of seawater from the atlantic, pacific, and gulf coasts gave mean annual counts 12.2 pfu. spikes in counts were observed at 3 out of 4 sites along the delaware bay 1 week after hurricane sandy. a comparison of summer versus winter counts showed significantly more bacteriovorax (p 0.0001) in the delaware bay during the summer and significantly more (p 0.0001) in the gulf during the winter, but no significant seasonal differences (p > 0.05) for hawaiian seawater. bacteriovorax counts only correlated with seawater salinity and temperature at one delaware site (r = 0.79 and r = 0.65, resp.). there was a relatively strong negative correlation between temperature and bacteriovorax levels (r = 0.585) for gulf seawater. selected isolates were sequenced and identified by phylogenetic analysis as bacteriovorax clusters ix, x, xi, and xii. |
the millennium cohort is the largest prospective study using primary data collection ever undertaken by the department of defense (dod). the study began in 2001 with the objective to collect and evaluate data on health, behavioral risk factors, and occupational characteristics related to military service that may be associated with adverse health outcomes (14). this report includes participants from the first enrollment cycle that spanned 20012003 and comprised 77,047 participants (36% of those able to be contacted). approximately 71% of those participants enrolled at baseline responded to follow - up surveys conducted in 2004 and 2007. the surveys were administered via paper and the internet and included questions on self - reported provider - diagnosed medical conditions, mental health symptoms, physical and functional status, alcohol and tobacco use, occupational status, military exposures, sleep patterns, and demographic information. this study was approved by the naval health research center institutional review board, and informed consent was obtained from all study participants. this research has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research (protocol nhrc.2000.0007). of the 77,047 participants enrolled at baseline, follow - up surveys were completed by 55,021 participants at year 3 and 54,790 at year 6. since incident diabetes was the outcome of interest, eligible participants included those who were followed until either the outcome was reported to have occurred at year 3 or 6, or until their last completed follow - up if the outcome was not reported. exclusions included participants reporting provider - diagnosed diabetes at baseline, missing outcome or exposure data, or if dod outpatient or inpatient medical encounter data contained codes for type 1 diabetes (n = 72) (icd-9 codes 250.x1 and 250.x3). the primary data sources were the self - administered millennium cohort surveys and dod electronic personnel files, managed by the defense manpower data center (dmdc, seaside, ca). for these analyses, electronic records from dmdc provided information on age, sex, race / ethnicity, education, pay grade, service branch, service component, military occupation, dates of separation, and deployment dates. dmdc information was supplemented with self - reported data to reduce missing values when necessary. three sleep variables were included in the analyses : 1) trouble sleeping, 2) sleep duration, and 3) sleep apnea. trouble sleeping was assessed using the sleep items on the prime - md patient health questionnaire (phq) and the ptsd checklist civilian version (pcl - c) (1720). the phq asks, over the last 4 weeks, how often have you experienced trouble falling asleep or staying asleep ? the pcl - c asks, in the past month, have you had trouble falling asleep or staying asleep ? participants with trouble sleeping were defined as those who responded moderately or above on the pcl - c sleep item or several days or longer on the phq sleep item. sleep duration was reported as whole number of hours slept in an average 24-h period over the past month. participants with sleep apnea were identified by self - report as being told by a doctor or other health professional they had sleep apnea. ptsd was assessed using the pcl - c (17), a 17-item self - report screening tool for ptsd symptoms during the past 30 days on a 5-point likert scale, ranging from 1 (not at all) to 5 (extremely). a positive screen for ptsd at baseline was defined as a report of a moderate or higher level of at least one intrusion symptom, three avoidance symptoms, and two hyperarousal symptoms (criteria established by dsm - iv) (21). major depressive disorder was assessed using nine items from the phq (sensitivity = 0.93 ; specificity = 0.89) corresponding to the depression diagnosis based on the dsm - iv (19). a positive screen for depression at baseline was defined as 1) endorsement of depressed mood or anhedonia and 2) response of more than half the days or nearly every day to at least five items. other anxiety (6 items) and panic (15 items) symptoms problem drinking was defined using items from the phq on risky behaviors related to alcohol use, such as driving under the influence more than one time over the past year, where endorsement of at least one item indicated problem drinking. binge drinking was defined as consuming five or more drinks (men) or four or more drinks (women) in 1 day during the past year. smokers were identified as those ever smoking 100 cigarettes in their lifetime, with those persisting in this habit defined as current smokers and those having quit as past smokers. in longitudinal models, service branch, service component, pay grade, military occupation, birth year, race / ethnicity, and education were assessed at baseline. smoking status, alcohol use, bmi, combat deployment, ptsd, depression, anxiety, panic, and all sleep variables were assessed at all available time points. the outcome of interest was incident diagnosed diabetes self - reported on a follow - up survey among people without diabetes at the baseline assessment. at baseline, participants reported if they had ever been told by a doctor or health professional that they had diabetes or at follow - up assessments, participants responded to the same question in regard to the previous 3 years. univariate analyses assessed associations of incident diabetes with sleep, military, demographic, behavioral, and mental health characteristics while accounting for multiple periods of observation per subject. since repeated measurements were available with up to two follow - up assessments per person, generalized estimating equations (gees) were used to account for these multiple assessments while adjusting for fixed and time - varying covariates, to estimate adjusted odds of reporting incident diabetes (22). incident diabetes was determined at each follow - up assessment, and all covariates were evaluated using data from the previous assessment (22). in all multivariable models, adjustments were performed for known type 2 diabetes risk factors captured in the study : age, sex, education, bmi, and race / ethnicity. the final adjusted models were built using a stepwise backward reduction algorithm that retained all significant variables (p 8 h was reported by similar proportions of participants with and without newly self - reported diabetes. participants with new - onset diabetes were on average older, had a higher bmi, and were more likely of nonwhite race. characteristics associated with new - onset diabetes included no deployment, having separated from the military since baseline, army or navy / coast guard service branch, reserves / national guard service component, enlisted pay grade, and administration / supply occupation. binge or problem drinking was less likely to have been reported in people reporting new - onset diabetes, and current and former smoking frequency was similar in groups with and without new - onset diabetes. all mental health symptoms occurred significantly more frequently among those who newly reported diabetes during follow - up. factors associated with a higher odds of incident diabetes in models adjusted for sex and known type 2 diabetes risk factors (age, bmi, education, and race / ethnicity) included trouble sleeping, sleep duration 5 h compared with 7 h, and sleep apnea (table 2). several military service characteristics remained significantly related to the outcome, including no deployment, enlisted pay grade, administration / supply occupation, and having separated from the military since baseline (table 2). binge or problem drinking remained significantly associated, and current smoking emerged as a risk factor for new - onset diabetes after adjustment. screening positive for depression, panic, other anxiety disorder, and ptsd were all significantly associated with higher odds of new - onset diabetes in these adjusted models (table 2). relative odds of incident self - reported diabetes by each baseline characteristic of interest in the multivariable model (table 3), sleep characteristics remained significantly and independently associated with higher diabetes odds. the sleep variables that entered the final model included reported trouble sleeping and sleep apnea, which were associated with significant increases in the odds of diabetes of 1.21 to 1.78, respectively. these associations were not only independent of each other but also the other covariates adjusted for in the model (table 3). military service characteristics significantly associated with diabetes odds included deployment without combat experience, administration or supply occupations, pay grade, and having separated from the military since baseline. a positive screen for ptsd was significantly associated with higher diabetes odds, independent of sleep characteristics and other covariates in the model, and panic disorder was of borderline significance. tests for interactions between the sleep variables included in the models in table 3 and age, sex, and bmi were all nonsignificant (p > 0.10). no variable had a variance inflation factor > 4, indicating that collinearity was unlikely. the model in table 3 was rerun with sleep duration substituted for trouble sleeping but with the other included covariates unchanged. the quasilikelihood information criterion for the model containing trouble sleeping was slightly lower (8,640.9) than when sleep duration was included in the model (8,646.3). the sleep duration variable was insignificant overall in this model (p = 0.1585), although two sleep categories were significantly related to diabetes odds (< 5-h odds ratio [or ] 1.52 [95% ci 1.092.14 ] ; 5-h or 1.28 [1.011.62 ]). several additional modifications of the multivariable model shown in table 3 were conducted. in the first modification, symptoms of depression and anxiety disorder were included in the model to assess whether adjustment for these additional factors altered the association between sleep and diabetes odds. in the second modification, all subjects reporting sleep apnea were removed from the model shown in table 3. in each of these modifications, the statistical significance of the associations between the sleep variables and diabetes odds remained unchanged, and the associations were of similar magnitude to those seen in table 3 (data not shown). longitudinal multivariable regression model of sleep characteristics and mental health symptoms as risk factors for incident self - reported diabetes in the millennium cohort our study findings confirm that sleep characteristics are associated with subsequent new - onset self - reported diabetes. these associations persisted after adjustment for multiple mental health conditions known to affect sleep duration and quality, including ptsd and depression (1113). therefore, it is unlikely that sleep simply serves as a surrogate marker for associated mental health conditions previously shown to predict higher diabetes risk (9,10). additionally, participants reporting provider - diagnosed sleep apnea were at higher risk of developing self - reported diabetes during follow - up. although sleep apnea is associated with higher bmi and both altered sleep quality and quantity, adjustment for these variables did not alter the significance of the higher diabetes risk associated with this condition (23). overall, these results suggest that the associations between type 2 diabetes risk and mental health conditions and sleep characteristics, if causal, may involve different pathways. whereas trouble sleeping and shorter sleep duration were associated with higher risk of diabetes in a model adjusted for known diabetes risk factors, longer sleep duration was not, in contrast to findings that others have reported (4). the reason for this discrepancy is unclear, but it may be due to the relatively younger age of our cohort compared with investigations reporting higher diabetes risk with longer sleep duration (24,25). the independent role of mental health conditions associated with poor quality and quantity of sleep in predicting risk of diabetes has only been addressed in a limited manner in previous research. the nurses health study investigators examined sleep as a predictor of incident diabetes in this cohort from 19861996 and adjusted for an assessment of depression based on the short form 36 health survey that was obtained in 1992, approximately midway through the follow - up period (24). thus, it is unclear whether depression preceded or followed diabetes onset during the first 6 years of follow - up. an analysis using the national health and nutrition examination survey i epidemiologic follow - up study adjusted for depression while examining the association between sleep and diabetes incidence, although no association was seen between depression and diabetes incidence in this cohort (25). lastly, a prospective study of swedish men assessed the presence of depression with a simple yes / no question, do you feel depressed ? and did not provide information on the accuracy of this question in capturing this condition (26). to our knowledge, our study is the first to consider mental health conditions other than depression as potential confounding factors and to measure their occurrence prior to the onset of diabetes. previous prospective research on sleep characteristics and type 2 diabetes risk has not considered whether sleep duration or quality is related to this outcome independent from sleep apnea, a known cause of sleep disturbance. in our fully adjusted analysis, both sleep apnea and trouble sleeping had independent associations with diabetes risk, suggesting that the associations between sleep characteristics and diabetes risk do not merely serve as markers for the presence of sleep - disordered breathing. in addition to the known higher risk of obesity associated with sleep apnea, other factors associated with sleep - disordered breathing may be involved in higher diabetes risk and include abnormal sympathetic activity and release of proinflammatory mediators such as tumor necrosis factor- and interleukin-6 (27). in support of the latter, we observed an independent association between sleep apnea and diabetes risk after adjustment for bmi. this suggests that manifestations of sleep apnea other than the impact of greater general adiposity may be associated with diabetes risk, although it is possible that incomplete adjustment due to residual confounding occurred due to the inaccuracy of bmi in characterizing body composition. the association between trouble sleeping or shorter sleep duration and diabetes risk independent of associated weight gain and sleep apnea is not well understood, but it may be related to both changes in insulin sensitivity, secretion, and glucose effectiveness. one investigation reported significant or borderline - significant reductions in insulin sensitivity, insulin secretion, glucose effectiveness, and the disposition index immediately after a 5-day period of sleep deprivation (4 h of sleep per night) among 11 healthy lean males in a controlled setting (28). all these changes have been associated with higher risk of the development of type 2 diabetes (29). several characteristics of military service were significantly associated with lower diabetes risk in multivariable models, including deployment, officer pay grade, and remaining in military service. for most comparisons, military deployment with or without combat exposure was related to a lower risk for diabetes, possibly reflecting selection of people medically fit for deployment who are at lower risk for diabetes. the mental health conditions independently associated with diabetes risk included those characterized by excessive stress, including ptsd and panic disorder. a review of the literature on stress and diabetes risk concluded that both general emotional and job stress were repeatedly associated with a higher risk of developing diabetes in adults (30). overactivation of the hypothalamic - pituitary axis and abnormal stimulation of the sympathetic nervous system are thought to accompany excessive levels of stress, with accumulation of visceral fat and promotion of an inflammatory state with greater insulin resistance (31). a previous analysis of the members of this cohort recruited in 2001 and followed for 3 years similarly demonstrated a higher diabetes risk associated with ptsd symptoms. first, type 2 diabetes was determined using self - reported data, and it was not confirmed by medical record review. however, because self - reported diabetes compared with physician diagnosis or medical record data have been shown to have a high sensitivity (7099% ; median 81%) and specificity (9299%), there is less likelihood for misclassification (32). also, it is possible that individuals self - reporting type 1 diabetes were included in this analysis. however, individuals with a type 1 diabetes diagnosis in their dod medical records were excluded, although this elimination process would miss those participants no longer receiving care from military treatment facilities after discharge or as reservists. in addition, positive screens for mental disorders were determined from self - report. also, we were unable to adjust for diet or family history, which are known risk factors for diabetes. we expect these findings to generalize to younger populations who are sufficiently healthy to enter into the u.s. armed forces, but it is unclear whether the results would apply to dissimilar populations. self - report of clinician - diagnosed sleep apnea was obtained, and the sensitivity and specificity of this report compared with the medical record are not known. medical encounter data were not used to confirm diagnoses of diabetes or sleep apnea in this cohort due to the potential for some of its members, particularly those in the reserves or national guard, to have received care in non - dod facilities. inaccurate classification of sleep apnea and other covariates potentially may have limited our ability to adjust for confounding. finally, self - reported sleep data were assessed during the prior 4 weeks, and sleep duration was rounded to the nearest whole hour, potentially biasing the estimate of actual sleep time (3335). this is the first study, to our knowledge, to examine baseline sleep characteristics in relation to incident diabetes while adjusting for several mental health conditions known to impair sleep and be associated with type 2 diabetes. furthermore, this research was conducted in a large population - based study of active duty and reserve / national guard u.s. military personnel from all service branches. additional strengths include the large sample size and up to 6-year duration of follow - up. moreover, no association was found between reporting type 2 diabetes at baseline and nonresponse to the first follow - up questionnaire among the first panel of enrollees, suggesting that the development of diabetes was unlikely to have influenced the probability of responding to the follow - up survey (36). finally, previous evaluations of possible biases suggest the cohort is reasonably representative of military personnel in terms of baseline demographic and mental health characteristics, that participants report health and exposure data reliably, and that enrollment in the study is not influenced by health status (14,3740). we conclude that trouble sleeping and sleep apnea predict higher diabetes risk, independent of multiple diabetes risk factors and several mental health conditions. of note, self - reported sleep apnea was also associated with a significantly higher risk for diabetes even after adjustment for other sleep characteristics, bmi, and mental health conditions. substitution of sleep duration for trouble sleeping in multivariable modeling yielded nearly identical results, with significantly higher risk seen with < 6 h of sleep. confirmation of these findings through further analyses may advance our understanding of diabetes pathophysiology and create new opportunities for prevention. | objectiveresearch has suggested that a higher risk of type 2 diabetes associated with sleep characteristics exists. however, studies have not thoroughly assessed the potential confounding effects of mental health conditions associated with alterations in sleep.research design and methodswe prospectively assessed the association between sleep characteristics and self - reported incident diabetes among millennium cohort study participants prospectively followed over a 6-year time period. surveys are administered approximately every 3 years and collect self - reported data on demographics, height, weight, lifestyle, features of military service, sleep, clinician - diagnosed diabetes, and mental health conditions assessed by the prime - md patient health questionnaire and the ptsd checklist civilian version. statistical methods for longitudinal data were used for data analysis.resultswe studied 47,093 participants (mean 34.9 years of age ; mean bmi 26.0 kg / m2 ; 25.6% female). during 6 years of follow - up, 871 incident diabetes cases occurred (annual incidence 3.6/1,000 person - years). in univariate analyses, incident diabetes was significantly more likely among participants with self - reported trouble sleeping, sleep duration < 6 h, and sleep apnea. participants reporting incident diabetes were also significantly older, of nonwhite race, of higher bmi, less likely to have been deployed, and more likely to have reported baseline symptoms of panic, anxiety, posttraumatic stress disorder, and depression. after adjusting for covariates, trouble sleeping (odds ratio 1.21 [95% ci 1.031.42 ]) and sleep apnea (1.78 [1.392.28 ]) were significantly and independently related to incident diabetes.conclusionstrouble sleeping and sleep apnea predict diabetes risk independent of mental health conditions and other diabetes risk factors. |
pathologically, ad is characterized by extracellular aggregation of amyloid - beta into senile plaques and hyper - phosphorylated tau protein accumulation in intraneuronal neurofibrillary tangles (beach., clinically, ad is characterized by progressive cognitive decline that typically presents with memory loss as the initial and primary concern (alzheimer 's association, 2014). worldwide, approximately 47.5 million individuals are living with dementia, the majority of which suffer from alzheimer 's disease (world health organization, 2016). emerging research has focused on the identification of biomarkers that will assist with early diagnosis of ad and allow for the evaluation of potential disease modifying treatments (dubois., considerable effort has been devoted to the development of pet molecular neuroimaging biomarkers of amyloid and tau in ad (e.g. wang., 2016), however, magnetic resonance imaging (mri) continues to offer a non - invasive and easily repeatable method of examining neuropathological changes associated with ad. to date, the majority of mri - based research in ad has focused on tissue loss in grey matter structures (cash., these findings indicate widespread whole brain grey matter atrophy in individuals with ad, including enlarged ventricles and decreased hippocampal volume (gold, 2012, hampel., 2014, the amyloid cascade hypothesis (hardy and higgins, 1992) suggests that axonal degeneration arises as a result of wallerian degeneration. however, the close association of tau with both axonal integrity and with the cognitive symptoms of ad suggests that white matter changes may occur independently and perhaps prior to changes in grey matter. (2007) hypothesizes that white matter degeneration in ad follows a reverse pattern to that observed in early myelination. these ideas lend support to the notion of white matter changes in ad as biomarkers that may be particularly helpful in earlier identification of ad (see amlien and fjell, 2014 for a review). one promising tool to detect early white matter alterations in the brain is diffusion tensor imaging (dti ; alexander.. currently, fractional anisotropy (fa) and mean diffusivity (md) are the most frequently reported dti metrics (amlien and fjell, 2014). fa is a measure of the degree of directionality of water diffusion, thought to be driven by microstructure such as cellular boundaries and myelin (alexander., 2007, amlien and fjell, 2014, soares., 2013), while md is a measure of the mean water diffusion rate (soares., these metrics can provide information regarding changes or differences in restriction to diffusion that may reflect myelination and axonal integrity. specifically, decreases in fa and increases in md are indicative of decreased myelination and loss of axons, as a consequence of neurodegeneration (bosch., 2012, the majority of published ad research has used a cross sectional design and consistently revealed low fa and high md in widespread white matter regions including the frontal, parietal, and temporal lobes (including hippocampal regions), as well as the corpus callosum and longitudinal association tracts (acosta - cabronero and nestor, 2014, sexton., 2011, stebbins and murphy, 2009, stoub., 2014). microstructural water diffusion changes are not unique to ad, however. in fact, some of these changes occur during the healthy aging process. (2010) examined dti indices from young and older adults and found an age - related reduction in fa within a number of white matter structures. these findings are consistent with recent reviews that discuss degeneration of white matter tracts with age, which may result from cerebrovascular changes or other common underlying health conditions (e.g. lockhart and decarli, 2014). recent studies that have utilized a longitudinal design to investigate microstructural changes in ad via dti have revealed decreased fa in the uncinate fasciculus (kitamura., 2013) as well as fa and md changes in the fornix, corpus callosum, and inferior cingulum, over time in individuals with ad (genc., 2016, norwrangi., 2013). the current study is the first to use the alzheimer 's disease neuroimaging initiative (adni) database to investigate changes in diffusion tensor imaging (dti) metrics over time. the adni database has collected and made available dti data from individuals with ad and healthy controls at multiple sites across north america. the overarching aim of the current study was to determine if dti, as a measure of microstructural white matter integrity, has potential as a biomarker of ad. the study had two specific objectives : 1) to examine within - group microstructural white matter changes in individuals with ad and healthy controls at baseline and year one ; and 2) to evaluate between - group differences in individuals with ad and healthy controls at both time points. it was hypothesized that 1) individuals with ad would have decreased fa and increased md across time and that 2) individuals with ad would have lower fa and higher md as compared to healthy controls at both time points. a whole brain exploratory approach was taken to capture differences between groups in any region, however, it was predicted that medial temporal regions would be more greatly affected in ad, compared to healthy controls, given that this is one of the first affected regions in the progression of ad grey matter pathology (braak and braak, 1991, gold, 2012, hampel., 2014, all data were obtained from the alzheimer 's disease neuroimaging initiative 2 (adni2) database (http://adni.loni.usc.edu). michael w. weiner, was launched in 2003 with the goal of testing whether longitudinal brain imaging, biological markers, and neuropsychological assessment can be used together to measure the progression of ad. for more information full eligibility criteria for the adni are described in the adni2 procedures manual (alzheimer 's disease neuroimaging initiative, 2008). briefly, individuals with ad met nincds / adrda criteria for probable ad (mckhann., 1984), demonstrated abnormal memory function on the wechsler memory scale (wms) ii (8 for 16 years education and above), had a mmse score between 20 and 26, and had a clinical dementia rating of 0.5 (very mild) or 1.0 (mild). healthy controls were required to be free of subjective memory concerns, to have a score within the normal range on the wms logical memory ii (9 for 16 years of education and above), have a mmse score between 24 and 30, and a clinical dementia rating of 0 (none). individuals from the adni database were included in the present study if data was available at both baseline and year one. data were retrieved from 34 individuals with ad (mean age = 75.8 7.6 ; 10 females ; mmse = 23.59 1.74 ; logical memory ii = 1.65 1.94) and 33 healthy age - matched controls (mean age = 73.0 6.6 ; 16 females ; mmse = 29.03 1.26, logical memory ii = 11.70 2.84). there were no significant differences between the two groups with respect to age, gender, or education (table 1). the mean number of days from baseline to year one was not significantly different between groups (394 25 for ad, and 403 54 for healthy controls). all adni participants provided informed written consent approved by each sites ' institutional review board. secondary data use for the current study was approved by the human research ethics board at the university of victoria, in british columbia, canada. images were acquired from 3 t mri scanners (ge medical systems) from seven north american sites. scan parameters are as follows : acquisition matrix = 256 256, voxel size = 1.4 1.4 2.7 mm, flip angle = 90, number of slices = 59. there were 46 images acquired for each scan : 41 diffusion - weighted images (b = 1000 s / mm) and 5 non - diffusion - weighted images (b = 0 s / mm). repetition time varied across scanning sites, but was approximately 12,500 to 13,000 ms. all data analyses were performed in functional mri of the brain software library (fsl) version 5.0 (analysis group, fmrib, oxford, uk, http://fsl.fmrib.ox.ac.uk ; smith., 2004). diffusion weighted images were corrected for eddy current distortions and head movement using eddy correct and non - brain tissue was removed using brain extraction tool (smith, 2002). fa maps were created using dtifit and input into tract - based spatial statistics (tbss) to obtain a projection of all participants ' fa data onto a mean fa skeleton (smith., 2006). first, all participants ' fa data were non - linearly aligned to a common space (fmrib58_fa). then, the mean fa image was created and thresholded (fa > 0.2) to create the mean fa skeleton. next, each participant 's fa data was projected onto the thresholded mean fa skeleton. voxelwise statistical analysis of the white matter skeleton was performed using randomise, fsl 's nonparametric permutation inference tool. threshold free cluster enhancement was used to correct for multiple comparisons (p 0.2) to create the mean fa skeleton. next, each participant 's fa data was projected onto the thresholded mean fa skeleton. voxelwise statistical analysis of the white matter skeleton was performed using randomise, fsl 's nonparametric permutation inference tool. threshold free cluster enhancement was used to correct for multiple comparisons (p 0.2) to create the mean fa skeleton. next, each participant 's fa data was projected onto the thresholded mean fa skeleton. voxelwise statistical analysis of the white matter skeleton was performed using randomise, fsl 's nonparametric permutation inference tool. threshold free cluster enhancement was used to correct for multiple comparisons (p < 0.05). tbss was also performed for md ; non - linear registration estimated from the fa images was applied to md data and each participant 's md image was projected onto the mean fa skeleton before applying voxelwise statistics. within - group comparisons were made for individuals with ad from baseline to year one, and for healthy controls from baseline to year one. additionally, between - group contrast comparisons were made between individuals with ad and healthy controls at both baseline and at year one. white matter regions were identified with johns hopkins university 's white matter atlas available in fsl (mori., 2008, the within - group fa analysis showed that individuals with ad had reductions in fa in multiple regions, including the hippocampal cingulum, at year one compared to baseline (table 2 ; fig. healthy controls also had reduced fa in similar regions across time, but these alterations were less extensive (as is visible in fig. there were no significant increases in fa at year one compared to baseline in either group. the within - group md analysis showed that individuals with ad also had increased md in multiple regions including the hippocampal cingulum at year one compared to baseline (table 2 ; fig. healthy controls also had increased md at year one compared to baseline, but once again, these alterations were less extensive than in ad, and did not include the hippocampal cingulum (fig. there were no significant decreases in md at year one compared to baseline in either group. at baseline, between - group tbss revealed that individuals with ad had lower fa relative to healthy controls (table 3 ; fig. 3a). at year one, individuals with ad also had lower fa relative to healthy controls, and these alterations appeared more widespread than at baseline (fig. the between - group tbss also revealed that individuals with ad had higher md relative to healthy controls at baseline (table 3 ; fig. 4a). at year one, individuals with ad had higher md relative to healthy controls in similar regions as baseline, but there were also patterns of higher md in the hippocampal cingulum, not seen at baseline (fig. 4b). no regions demonstrated greater fa or lower md in hc compared to ad for either time point. the within - group fa analysis showed that individuals with ad had reductions in fa in multiple regions, including the hippocampal cingulum, at year one compared to baseline (table 2 ; fig. healthy controls also had reduced fa in similar regions across time, but these alterations were less extensive (as is visible in fig. there were no significant increases in fa at year one compared to baseline in either group. the within - group md analysis showed that individuals with ad also had increased md in multiple regions including the hippocampal cingulum at year one compared to baseline (table 2 ; fig. healthy controls also had increased md at year one compared to baseline, but once again, these alterations were less extensive than in ad, and did not include the hippocampal cingulum (fig. there were no significant decreases in md at year one compared to baseline in either group. at baseline, between - group tbss revealed that individuals with ad had lower fa relative to healthy controls (table 3 ; fig. 3a). at year one, individuals with ad also had lower fa relative to healthy controls, and these alterations appeared more widespread than at baseline (fig. the between - group tbss also revealed that individuals with ad had higher md relative to healthy controls at baseline (table 3 ; fig. 4a). at year one, individuals with ad had higher md relative to healthy controls in similar regions as baseline, but there were also patterns of higher md in the hippocampal cingulum, not seen at baseline (fig. 4b). no regions demonstrated greater fa or lower md in hc compared to ad for either time point. the current study is the first to examine longitudinal white matter changes using dti data from the adni2 cohort. dti indices of fa and md were focused on in the current study, given that these are the most commonly reported metrics and can be interpreted in the context of recent literature (amlien and fjell, 2014). the primary objective of the current study was to examine longitudinal white matter changes in individuals with ad and healthy aging. significant changes in fa and md were observed both in individuals with ad as well as in healthy controls at one year follow up. as expected, individuals with ad demonstrated decreased fa and increased md in widespread white matter tracts (see table 2 and fig. 1, fig. 2), including the hippocampal cingulum at year one compared to their baseline assessment. while healthy controls also exhibited decreased fa and increased md in widespread white matter tracts (see table 2 and fig. 1, fig. furthermore, healthy controls did not demonstrate fa or md changes in the hippocampal cingulum as were observed in those with ad. thus, the relatively widespread changes in larger white matter tracts seen in healthy controls may reflect age - related health factors such as vascular risk factors and the accumulation of other co - morbid health conditions (e.g., vassilaki., 2016). however, changes in microstructural integrity for the hippocampal cingulum over short time intervals (i.e. one year) may more specifically reflect ongoing degenerative process due to ad. the second objective of the current study was to investigate between - group differences in individuals with ad and healthy controls at both baseline and year one. as predicted, significant between - group differences in fa and md were observed in multiple regions at both time points (table 3, fig. 4), including regions of the medial temporal lobe (e.g., hippocampal cingulum). such between - group white matter differences are in line with previous cross - sectional studies that have observed dti alterations in the same regions (i.e. corpus callosum, superior or inferior longitudinal fasciculus ; cingulate ; cingulum ; fornix ; and uncinate fasciculus) when comparing individuals with ad to age - matched healthy controls (agosta., 2011, bosch., 2012, douaud., 2011,, 2012, liu., 2011, parente., 2008, salat., 2010, serra., 2010, shu., 2011, lower fa and higher md in ad relative to controls at each time point likely reflects the more extensive and pathological neurodegeneration observed in ad. in particular, lower fa and higher md in the hippocampal region is consistent with the early pathological progression of ad (braak and braak, 1991, hampel., 2014) as well as memory loss as an initial and primary concern (alzheimer 's association, 2014). although there have been few longitudinal dti studies on individuals with ad, our findings are largely consistent with those published to date (genc. norwrangi., 2013), demonstrating that such findings may be generalizable across ad populations especially, given adni 's multi - site collection. in particular, the observed decreases in fa and increases in md likely reflect the progressive loss of the water diffusion - restricting barriers in white matter (e.g., decreased level of myelination, loss of axons) as a consequence of neurodegeneration (bosch., 2012, kantarci, 2014, serra., 2010). currently, the mechanisms underlying white matter pathology in ad are not well understood. it is hypothesized that some damage to white matter may occur secondarily to grey matter pathology via wallerian degeneration, but additional white matter alterations may also occur independent from grey matter pathology, as put forth by the retrogenesis hypothesis, for example (amlien and fjell, 2014, bartzokis., 2007). in summary, the current whole brain dti study found evidence of a higher rate of decline in fa and increase in md over approximately one year in individuals with ad versus matched healthy controls. these differential changes were seen in a number of white matter regions that correspond to regions know to be affected in ad. importantly however, these dti changes were evident in the hippocampal cingulum only in those with ad and not healthy controls, a finding that is consistent with the known pattern of progression of ad pathology in the grey matter of medial temporal regions (braak and braak, 1991). volume reduction in the hippocampal grey matter is well documented in previous ad research (hampel., 2014). thus, future work should focus on both hippocampal grey and white matter, given that the observed hippocampal changes appear specific to ad relative to aging. there are varying definitions of ad, and the inclusion / exclusion criteria may differ across studies, making cross - study comparisons and generalizations to all individuals with ad challenging. furthermore, there is heterogeneity in the adni ad sample ; disease stage may not be equivalent in the baseline scans and there is no pathological verification of diagnosis for all members of the ad cohort. white matter hyperintensities (wmh) of presumed vascular origin were not accounted for in the current analysis. this represents a potential limitation as wmh are common in older adults and have been shown to be related to lower fa and higher md relative to normal appearing white matter (e.g., munoz maniega., 2015). however, the adni database has previously been shown to have participants with lower levels wmh relative to other large - scale data sources (ramirez., 2016). additionally, although the adni2 database includes neuroimaging data from individuals diagnosed with mild cognitive impairment (mci), the current study did not examine this group, as the primary objective was to characterize white matter in ad, specifically. approximately 10 to 15 percent of individuals with mci progress to ad annually (gong., 2013), in contrast to the 1 to 2 percent of healthy older adults who progress to ad per year (petersen, 2004). thus, future studies of longitudinal dti changes in those with mci may be helpful in determining whether the dti changes noted in our ad sample represent sensitive and specific neuroimaging biomarkers of ad pathophysiologic processes in individuals in the prodromal stages of this disease. it is important to recognize that mci can not be considered synonymous with early ad (balthazar., 2009). as noted by dubois (2000), mci applies to a heterogeneous group of aging adults with cognitive concerns, regardless of the underlying etiology or symptom progression. future studies are likely to require relatively large and well - characterized study samples, longer follow up periods to adequately observe conversion from mci to ad, as well as multimodal imaging protocols in order to adequately address this issue. as new data are added to the database, the current findings should provide support for additional analyses of dti white matter changes to assist in the development of potential biomarkers of ad. future studies that draw from the adni database will benefit from these multi - year longitudinal data (up to five years). not only will the number of participants in each study group grow as additional participants are recruited, the trajectory of the disease progression can be better tracked across longer time periods as data collection with the current cohort continues. a major focus of research on ad centres on the investigation of biomarkers. to date, most studies have focused on changes in grey matter and taken a cross sectional approach. the current study is the first to examine longitudinal white matter changes using the adni2 cohort. the results revealed that changes in fa and md occurred over a one year period in both patients with ad and healthy controls, although the changes were more extensive in ad and more specific to the medial temporal lobe. dti holds potential as an ad biomarker though multi - year tracking of brain imaging and ad clinical signs at different diagnostic stages are needed to fully evaluate its clinical utility. ultimately, better characterization of longitudinal microstructural white matter changes may lead to pre - symptomatic detection and better outcomes for individuals with ad. data collection and sharing for this project was funded by the alzheimer 's disease neuroimaging initiative (national institutes of health grant u01 ag024904) and dod adni (department of defense award number w81xwh-12 - 2 - 0012). adni is funded by the national institute on aging, the national institute of biomedical imaging and bioengineering, and through generous contributions from the following : abbvie, alzheimer 's association ; alzheimer 's drug discovery foundation ; araclon biotech ; bioclinica, inc. eli lilly and company ; euroimmun ; f. hoffmann - la roche ltd. and its affiliated company genentech, inc. ; fujirebio ; ge healthcare ; ixico ltd. ; janssen alzheimer immunotherapy research & development, llc. ; johnson & johnson pharmaceutical research & development llc. ; lumosity ; lundbeck ; merck & co., inc. ; meso scale diagnostics, llc. ; neurorx research ; neurotrack technologies ; novartis pharmaceuticals corporation ; pfizer inc. ; piramal imaging ; servier ; takeda pharmaceutical company ; and transition therapeutics. the canadian institutes of health research is providing funds to support adni clinical sites in canada. private sector contributions are facilitated by the foundation for the national institutes of health (www.fnih.org). the grantee organization is the northern california institute for research and education, and the study is coordinated by the alzheimer 's disease cooperative study at the university of california, san diego. adni data are disseminated by the laboratory for neuro imaging at the university of southern california. the current study was also supported by the canadian institutes of health research (competition : 201310) : catalyst grant for the secondary analyses of neuroimaging databases and the canada graduate scholarships [grant number cse 133352 ] ; natural sciences and engineering research council of canada [grant number pdf-454132 - 2014 ] and create ; alberta innovates - health solutions [grant number 201300567 ]. | backgroundalzheimer 's disease (ad) is a progressive neurodegenerative disorder. current avenues of ad research focus on pre - symptomatic biomarkers that will assist with early diagnosis of ad. the majority of magnetic resonance imaging (mri) based biomarker research to date has focused on neuronal loss in grey matter and there is a paucity of research on white matter.methodslongitudinal dti data from the alzheimer 's disease neuroimaging initiative 2 database were used to examine 1) the within - group microstructural white matter changes in individuals with ad and healthy controls at baseline and year one ; and 2) the between - group microstructural differences in individuals with ad and healthy controls at both time points.results1) within - group : longitudinal tract - based spatial statistics revealed that individuals with ad and healthy controls both had widespread reduced fractional anisotropy (fa) and increased mean diffusivity (md) with changes in the hippocampal cingulum exclusive to the ad group. 2) between - group : relative to healthy controls, individuals with ad had lower fa and higher md in the hippocampal cingulum, as well as the corpus callosum, internal and external capsule ; corona radiata ; posterior thalamic radiation ; superior and inferior longitudinal fasciculus ; fronto - occipital fasciculus ; cingulate gyri ; fornix ; uncinate fasciculus ; and tapetum.conclusionthe current results indicate that sensitivity to white matter microstructure is a promising avenue for ad biomarker research. additional longitudinal studies on both white and grey matter are warranted to further evaluate potential clinical utility. |
herbal ingredients are commonly found in many products, including cosmetics, drugs, foods, and oral hygiene products, such as mouth rinse, toothpaste, and lozenges. for example, the biological properties of herbal substances should be evaluated to exclude the possibility of adverse effects on human tissue. the toxic effects of an herbal substance are of particular concern because the damage caused can not only be acute, such as a degeneration of tissue, but can also be chronic, such as a delay in wound healing. clinacanthus nutans is widely grown in tropical asian countries and constitutes an important herbal medicine in thailand. this plant exerts a potential beneficial effect on wound healing based on evidence provided by practitioners of folk medicine and detailed several scientific reports from thailand. c. nutans or phaya - yor in thai [figure 1 ] has been traditionally used in thailand for the treatment of herpes infections. in addition, this herb also exhibits excellent and rapid - acting anti - inflammatory activity, which makes a topical product of c. nutans, a valuable natural product for the relief of minor skin inflammation. however, scientists in thailand found that dysentery and fever can be treated by this plant. due to its anti - cell lysis property, the plant has been used as an anti - venom agent for snake and scorpion bites and to remove nettle rashes. in addition, this plant has recently received much attention because of its potential application in cancer treatment. clinacanthus nutans (www.madoorayong.com) three levels of biological testing are available for dental materials. most of the new dental materials such as dental adhesives were introduced in the clinical practice to investigate their biocompatibility by in vitro screening test before in vivo experiment. the second level of testing is performed on animals to evaluate the tissue or bone response of the host. preclinical evaluation is necessary to establish the biocompatibility of medical devices and materials used in dentistry. international organization for standardization (iso) 7405 recommends that high priority has to be given to minimize the use of animals in the biological testing of materials. as scientific knowledge advances the understanding of basic mechanisms, an in vitro model that simulates the in vivo test or clinical use, which may yield equally relevant information, is advocated. the dimethylthiazol diphenyltetrazolium bromide (mtt) assay and the neutral red uptake (nru) assay are well - known and widely used methods. a mitochondrial dehydrogenase enzyme in living cells reduces the yellow tetrazolium salt mtt to blue mtt formazan, which is precipitated in uninjured cells. the nru assay is based on the incorporation of the nr dye into lysosomes of viable cells. both assays are based on colorimetric measurements of viable cells after incubation with test materials. the aim of this study was to compare the relevance of the mtt and nru assays for cytotoxicity screening of a thai herb. we used a powder formulation of the thai herb phaya - yor, also known as c. nutans (twin lotus co., ltd., bangkok, thailand). the color of the thai herb powder is brown, with a maximum of moisture 8% and ph range of 6.008.00. 80 sieve with 75% efficiency. c. nutans was dried in a hot air oven at 60c for 72 h, ground into a fine powder using a blender panasonic mx-898 nc (sripiboon electric co., ltd., bangkok, thailand), and then filtered through a no. 80 sieve. c. nutans was weighed at 0.2 g and mixed into 1 ml of (dulbecco 's modified eagle 's medium [dmem ], invitrogen, ca then, the solution was incubated at 37c under 5% co2 for 24 h. the stock solution of c. nutans was centrifuged at 3500 rpm for 10 min, and the supernatant was diluted into 0.5 g%, 0.25 g%, 0.1 g%, and 0.01 g% (w / v) solutions. as a positive control, the iso 10993 - 5 recommended using polyvinyl chloride (hatano research institute, food and drug safety center, kanagawa, japan) with the size of 3 cm/2 ml of media. the films were sterilized by soaking in 70% alcohol for 1 min, washed in normal saline solution for 1 min, and left dry. then, the films were inserted into dmem and incubated at 37c under 5% co2 for 24 h before testing. as a negative control, the iso 10993 - 5 recommended using thermanox plastics cover slips (nunc naperville, il, usa) with a size of 6 cm/2 ml of media. thermanox plastics cover slips were cut into small pieces, soaked in dmem, and incubated in 5% co2 at 37c for 24 h before testing. the target cells used in this experiment were a continuous line of mouse fibroblast l929 cells (atcc, cell line, ecacc no. 2869501, nctc clone 929). cell were maintained at 37c under 5% co2 and 100% humidity in dmem and supplement with 10% fetal calf serum and antibiotics (200 g / ml penicillin g, 200 g / ml streptomycin, 2 g / ml fungizone). when cells reached confluency, they were detached using 0.2% (w / v) trypsin and transferred to new culture flasks. after sufficient growth for experimentation, the cells were trypsinized and plated in 96-cluster - well - culture plates at a concentration of 1 10 cells / well. each well contained 100 l of cell suspension and the plates were incubated for 24 h at 37c under 5% co2 to obtain a monolayer culture. then, 100 l of elute from the c. nutans solution at concentrations of 0.01%, 0.1%, 0.25%, and 0.5% ; the positive control or negative control was inserted into 96-cluster - well - culture plates (8 wells / test material). two 96-cluster - well - culture plates were separately prepared to evaluate cell viability using the mtt and nru assays. the experiments were repeated in triplicate. following a 24-h incubation period at 37c under 5% co2, cell viability of both plates the test materials were removed from each well of the first plate. then, 50 l of mtt reagent (5 mg / ml) was added and incubated for 2 h at 37c in the co2 incubator. the plates were swayed on a shaker to solubilize the formations of purple crystal formazan. the results were used to construct a graph of the cell viability percentage against extract concentrations. then, 150 l of neutral red dye (100 g / ml) dissolved in the serum of free medium (ph 6.4) was added to the culture medium and incubated for 3 h at 37c. cells were washed with phosphate - buffered saline, and 150 l of elution medium (etoh / accooh, 50%/1%) was added followed by gentle shaking for 60 min for complete dissolution. cell viability was expressed as a percentage of the control values. the intra - class correlation coefficient (icc) and the limits of agreement statistic were used to compare the scores. we used a powder formulation of the thai herb phaya - yor, also known as c. nutans (twin lotus co., ltd., bangkok, thailand). the color of the thai herb powder is brown, with a maximum of moisture 8% and ph range of 6.008.00. 80 sieve with 75% efficiency. c. nutans was dried in a hot air oven at 60c for 72 h, ground into a fine powder using a blender panasonic mx-898 nc (sripiboon electric co., ltd., bangkok, thailand), and then filtered through a no. 80 sieve. c. nutans was weighed at 0.2 g and mixed into 1 ml of (dulbecco 's modified eagle 's medium [dmem ], invitrogen, ca then, the solution was incubated at 37c under 5% co2 for 24 h. the stock solution of c. nutans was centrifuged at 3500 rpm for 10 min, and the supernatant was diluted into 0.5 g%, 0.25 g%, 0.1 g%, and 0.01 g% (w / v) solutions. as a positive control, the iso 10993 - 5 recommended using polyvinyl chloride (hatano research institute, food and drug safety center, kanagawa, japan) with the size of 3 cm/2 ml of media. the films were sterilized by soaking in 70% alcohol for 1 min, washed in normal saline solution for 1 min, and left dry. then, the films were inserted into dmem and incubated at 37c under 5% co2 for 24 h before testing. as a negative control, the iso 10993 - 5 recommended using thermanox plastics cover slips (nunc naperville, il, usa) with a size of 6 cm/2 ml of media. thermanox plastics cover slips were cut into small pieces, soaked in dmem, and incubated in 5% co2 at 37c for 24 h before testing. the target cells used in this experiment were a continuous line of mouse fibroblast l929 cells (atcc, cell line, ecacc no. 2869501, nctc clone 929). cell were maintained at 37c under 5% co2 and 100% humidity in dmem and supplement with 10% fetal calf serum and antibiotics (200 g / ml penicillin g, 200 g / ml streptomycin, 2 g / ml fungizone). the medium was changed every other day. when cells reached confluency, they were detached using 0.2% (w / v) trypsin and transferred to new culture flasks. after sufficient growth for experimentation, the cells were trypsinized and plated in 96-cluster - well - culture plates at a concentration of 1 10 cells / well. each well contained 100 l of cell suspension and the plates were incubated for 24 h at 37c under 5% co2 to obtain a monolayer culture. after 24 h of incubation, the old media was removed from each well. then, 100 l of elute from the c. nutans solution at concentrations of 0.01%, 0.1%, 0.25%, and 0.5% ; the positive control or negative control was inserted into 96-cluster - well - culture plates (8 wells / test material). two 96-cluster - well - culture plates were separately prepared to evaluate cell viability using the mtt and nru assays. the experiments were repeated in triplicate. following a 24-h incubation period at 37c under 5% co2, cell viability of both plates was assessed. the test materials were removed from each well of the first plate. then, 50 l of mtt reagent (5 mg / ml) was added and incubated for 2 h at 37c in the co2 incubator. the plates were swayed on a shaker to solubilize the formations of purple crystal formazan. the results were used to construct a graph of the cell viability percentage against extract concentrations. then, 150 l of neutral red dye (100 g / ml) dissolved in the serum of free medium (ph 6.4) was added to the culture medium and incubated for 3 h at 37c. cells were washed with phosphate - buffered saline, and 150 l of elution medium (etoh / accooh, 50%/1%) was added followed by gentle shaking for 60 min for complete dissolution. cell viability was expressed as a percentage of the control values. the intra - class correlation coefficient (icc) and the limits of agreement statistic were used to compare the scores. the cytotoxic effect of the plant extracts on the viability of the l929 cell lines are presented as percent cell viability in figure 2. extracts from the c. nutans were noncytotoxic, whereas the positive control exhibited the highest toxicity. cell viability of l929 cells after exposure to the negative control, positive control, or different concentrations of a thai herb the icc values for agreement of the negative and positive control reactions of the mtt and nru assay were 0.84 and 0.83, respectively. the icc values for agreement of extracts at varying w / v concentrations of 0.01, 0.1, 0.25, and 0.5% were 0.77, 0.68, 0.74, and 0.71, respectively. the results revealed that the strength of agreement ranged from moderate to good [tables 1 and 2, figure 3 ]. criteria for grading the strength of assay agreement intra - class correlation coefficient of the negative and positive controls and various concentrations of clinacanthus nutans for the comparative analysis of dimethylthiazol diphenyltetrazolium bromide and neutral red uptake assays intra - class correlation coefficient values for agreement between the mtt and nru assays the aim of this study was to assess whether the mtt and nru assays reveal comparable cytotoxicity levels for an herbal plant. this study revealed that the correlations between the mtt and nru assays in the cytotoxicity screening of c. nutans at the concentrations of 0.01%, 0.1%, 0.25%, and 0.5% ranged between good and moderate agreement. the study of the effect of c. nutans on a human gingival fibroblast cell line revealed no toxicity. moreover, the extracts at concentrations of 0.01, 0.05, and 0.01% promoted cell proliferation and cell migration, suggesting an alternate adjunctive regimen for oral wound healing. thus, the biocompatibility of c. nutans in the new regimen product should be evaluated before the product is launched into the market. for example, olivier. reported that polyol substances, which are normally found in drugs, food, and cosmetic products, may increase the osmotic pressure within lysosome. this increase promotes the entry of water and the subsequent swelling and fusion of small lysosomes to form a lysosome that is significantly larger than the original size. rorig. also revealed that weakly basic substances introduced into the cytoplasm also contribute to lysosome swelling, resulting in increased nru values. therefore, substances that cause lysosome swelling may result in an underestimation of cytotoxicity when the nru assay is used for cell viability measurements. therefore, the nru assay should be used cautiously with compounds that cause lysosome swelling. vian. compared the correlation of three in vitro cytotoxicity assays (nru, mtt, and [total protein cell content ]) for surfactant evaluation using three different cell lines (sirc, balb / c 3t3, and l929 cells). a kappa analysis of the results revealed moderate agreement among the three assays. lnnroth and dahl evaluated the effects of both light - cured and chemical - cured dental glass ionomer on cell function and viability using both mtt and nru assays. these researchers found that the mtt and the nru assay results were similar for most of the tested materials in l929 cells, but the parallel measurements of the nru assay varied more widely. the present study revealed that mtt and nru assays exhibited good correlation at a 0.01% concentration of c. nutans, but moderate correlations were noted for higher concentrations. both assays can be used to evaluate herbal plant cytotoxicity, but each assay has some limitations. for the nru assay, precipitation of nr occurs at the bottom of the tissue culture plate, thus light may be exposed to this area. although the mtt assay is one of the most sensitive assays for cytotoxicity screening, mtt is categorized as a carcinogen. thus, mtt waste after testing should be eliminated appropriately by the appropriate environmental pollution control agencies. two in vitro cytotoxicity assays, mtt and nru, were compared to determine their correlation in thai herbal plant evaluation. thus, both mtt and nru assays can be used for cytotoxic screening of this herbal species. | objectives : the aim of this study was to compare the results of dimethylthiazol diphenyltetrazolium bromide (mtt) and neutral red uptake (nru) assays of clinacanthus nutans cytotoxicity.materials and methods : mouse fibroblast (l929) cells were exposed to 0.01%, 0.1%, 0.25%, and 0.5% (w / v) c. nutans in a 96-cluster - well - culture plate for 24 h. the cell viability after exposure to c. nutans was determined by mtt and nru assays in separate tissue culture plates. the two assays were compared through an intra - class correlation coefficient (icc) analysis.results:no significant differences in cytotoxicity were noted between the two assays (p > 0.05). the icc values for agreement between two assays for the negative and positive control groups and c. nutans concentrations of 0.01%, 0.1%, 0.25%, and 0.5% were 0.84, 0.83, 0.77, 0.68, 0.74, and 0.71, respectively.conclusion:in general, the mtt and nru assays performed similarly, exhibiting moderate to good correlation for the evaluation of the cytotoxicity of c. nutans. |
cystoid macular edema (cme) following cataract surgery, also known as irvine - gass syndrome, is an important differential diagnosis of painless decrease in vision following uneventful cataract extraction. the pathogenesis is likely multifactorial, ultimately leading to the breakdown of the blood - retinal barrier and cystic accumulation of extracellular intraretinal fluid. the introduction of modern surgical techniques including phacoemulsification and small incision cataract surgery has contributed to a reduction in the incidence of cme since it was initially reported by irvine in 1953. angiographic cme is detected by fluorescein angiography and is significantly more common than clinical cme. clinical and optical coherence tomography (oct) evidence of cme after uncomplicated cataract surgery is present in around 0.12.35 and 5%, respectively [1, 4, 5 ]. the diagnosis of cme was previously made with fluorescein angiography after the patient became symptomatic ; however, the emergence of oct has provided a quick and noninvasive means of diagnosis. patient factors predisposing to cme include comorbidities with high vasoactive profile (diabetic retinopathy, uveitis), while surgical factors include inflammation - inducing complications such as retained lens material, vitreous loss and vitreomacular traction. however, many otherwise healthy patients who undergo routine surgery continue to develop the condition. the most frequently applied treatment paradigms include aggressive topical nonsteroidal anti - inflammatory drugs or steroids aimed at minimizing inflammation [6, 7 ]. while the condition is usually self - limiting and the resolution of symptoms is likely to occur within 312 months, it may occasionally persist and lead to permanent vision loss. the prevention of irvine - gass syndrome continues to pose challenges for ophthalmologists, due to its ability to affect individuals without any obvious predisposing factors at variable times of onset postoperatively. an 82-year - old male with no significant past ocular history was referred for evaluation and treatment of progressive blurred vision secondary to bilateral cataract (1 + cortical, 2 + nuclear sclerotic) by his optometrist. his best - corrected visual acuity (bcva) was 6/9 in both eyes, with no evidence of any other ocular disease. his past medical history was significant only for hypertension, which was well controlled with oral amiodarone and hydrochlorothiazide. he underwent routine phacoemulsification surgery in the left eye with implantation of an intraocular lens [single - piece acrylic toric lens (sn6at3) ; alcon, fort worth, tex. the patient received preoperative topical diclofenac 0.1% hourly for 4 h and a postoperative weaning regimen of chloramphenicol 0.5% and diclofenac 0.1% eye drops, which were ceased on day 28. postoperatively at day 9, bcva decreased to 6/18, with minor fluorescein staining of the cornea. consequently, oct was not performed. at day 22, bcva was reduced to 6/24. ketorolac 0.4% eye drops 6 times per day were commenced, and the patient was referred for medical retina opinion, at which time 0.1% dexamethasone drops 6 times per day was commenced. 1d) and was successfully weaned off all drops 3 months postoperatively. visual acuity in the left eye once the patient 's macular edema had resolved in the left eye, and following extra risk counseling, routine cataract surgery was performed in the right eye [single piece acrylic toric lens (sn6at4) ; alcon ]. the patient was prophylactically prescribed ketorolac 0.4% eye drops q.i.d. and oral indomethacin 25 mg t.d.s. both of these therapies were continued postoperatively in addition to chloramphenicol 0.5% and dexamethasone 0.1% eye drops both q.i.d. unfortunately, at day 32 postoperatively, bcva remained poor at 6/18 in the right eye. no recurrence of cme was seen in the left eye at this time (fig. topical dexamethasone 0.1% and prednisolone acetate 1% drops 6 times daily was used and gradually weaned over 4 months. 2c, d). while the patient continued to experience occasional flashes in both eyes, bcva recovered to od 6/6 + 4 and os 6/6 + 1 five months after his second surgery. improvement in his os bcva vision was noted up to 8 months after his initial operation. bilateral cases of cme have been reported in association with systemic diseases including crohn 's disease and infections. limited cases of bilateral cme after cataract surgery have been reported in the literature [10, 11 ]. irvine - gass syndrome following uncomplicated cataract surgery on this patient 's first eye is not uncommon. 1b), in the presence of symptoms, serves as a valuable reminder that evidence of cme on oct examination may not appear until some weeks after cataract surgery. as such, the possibility of fresh macular pathology must be considered in the assessment of blur postoperatively, even in the light of recently performed normal oct. the occurrence of irvine - gass syndrome in this patient 's second eye, despite topical and systemic prophylactic measures, was unexpected. while it is probable that those who develop cme after their first cataract operation are at increased risk of developing it following surgery on their contralateral eye, there is a paucity of evidence for determining the specific level of risk in these individuals. several studies have reported significant reductions in the incidence of clinical cme with prophylactic use of topical nonsteroidal anti - inflammatory drops, including ketorolac 0.4%, and nepafenac 0.1%, when used in conjunction with topical steroids or oral indomethacin, as was the case with this man 's second eye [12, 13, 14, 15 ]. perhaps higher prophylactic therapy doses or the addition of preoperative topical indomethacin or steroids could be considered in similar cases. irvine - gass syndrome remains an important differential diagnosis in the evaluation of blurred vision after cataract surgery. a normal oct scan 1 week after extraction does not preclude further cme, and repeated oct studies may be warranted where unexplained blurred vision persists. patients who develop cme following their first cataract operation should be counseled about the risks of developing the condition following surgery on the contralateral eye, irrespective of prophylactic measures. the patient 's right and confidentiality have been well protected in all aspects, and he provided informed consent to publish prior to the authors submitting the article. | purposeto report a case of acute bilateral irvine - gass syndrome.methodsthis is an observational case report.resultsan 82-year - old man with no significant ocular history developed postsurgical pseudophakic cystoid macular edema (cme ; irvine - gass syndrome) on consecutive phacoemulsification cataract surgeries. his initial first - eye (left) cme developed 25 days after surgery and was managed with topical preparations of dexamethasone 0.1% and ketorolac 0.4%, in addition to a routine post - cataract surgery drop regime. his left cme resolved completely on optical coherence tomography (oct) by day 100, and he subsequently (after extensive discussion of cme risks) underwent cataract surgery on his right eye. he was commenced prophylactically on dexamethasone, ketorolac and oral indomethacin 25 mg t.d.s. immediately after surgery ; however, he later developed cme (od) on day 32 postoperatively. within 6 months, he achieved complete resolution of his cme in both eyes. his clinical course was documented with serial oct studies.conclusionirvine-gass syndrome remains an important differential diagnosis in the evaluation of blurred vision after cataract surgery, despite decreasing incidence. those who experience cme following their first cataract operation should be counseled about the risks of developing the condition in the contralateral eye, despite prophylactic measures. |
a pigment is a coloured organic or inorganic solid that is usually insoluble in its application media. pigments are used when extreme resistance to light, weather or temperature is needed, e.g. in automotive paints and other outdoor applications. due to its insolubility, a pigment will usually retain its crystal structure throughout the preparation and application process. various physico - chemical properties of pigments, including colour, thermal stability, particle shape, light fastness and fluorescence, are dependent on the crystal packing of the pigment molecules. despite the commercial significance of non - azo organic pigments, only few full crystal structures of these materials have been reported, because single crystals are hard to obtain. the crystallites are often so small that they lead to considerable line broadening in x - ray powder diffraction. single - crystal growth by sublimation methods, on the other hand, rarely produces crystals of suitable quality for conventional x - ray single - crystal structure determination. as a result, the important crystallographic features, controlling the crystal chemistry of such materials, have remained unknown for many pigments. the purpose of this research is to study the feasibility of obtaining the crystal structures of the polymorphs of pigments with the aid of crystal structure prediction (csp) tools in combination with limited experimental data. a similar approach was reported by erk (2001) and schmidt. one can obtain a good impression of the state - of - the - art in crystal structure prediction by looking at the results of csp tests organized in recent years by the cambridge crystallographic data centre (see day. blind tests in 1999, 2001 and 2004 indicated that certain progress has been made in the ab initio prediction of structures of small (not more than 25 atoms) rigid molecules containing only c, h, n and o atoms. as an example of an insoluble pigment with poor crystal quality, unsubstituted linear trans - quinacridone, often called quinacridone (qa), a parent compound of various related pigments (fig. 1), was chosen. a review on various quinacridone - related compounds has been given by lincke (2000). in patent reports, at least six polymorphic forms of quinacridone have been reported on the basis of x - ray powder data (reidinger & struve, 1955 ; struve, 1955 ; manger & struve, 1957 ; filho & oliveira, 1992)., 2002) is available from the cambridge structural database (csd), while for the polymorph only the cell parameters, space group and a powder pattern have been published (paulus., 1989 ; struve, 1955). the structure of the polymorph, derived through calculations and reported in the csd (lincke & finzel, 1996), is likely to be incorrect, as discussed later. crystal structure prediction of quinacridone polymorphs has been mentioned previously (leusen, 1996) by accelrys (formerly molecular simulations ltd) as an example of the use of the cerius polymorph predictor, but the details of the prediction and the resulting structures remained unpublished. the pure and polymorphs of qa were provided by agfa - gevaert nv and used as received. the powder of -qa was obtained by slow water dilution of a concentrated h2so4 solution of qa. for that, a solution of -qa in concentrated sulfuric acid was placed in one test tube and water in a second one. the tubes were connected with each other and the water tube was heated to increase the water vapour pressure in the system. after the precipitation of a dark violet powder in the sulfuric acid solution, the mixture was filtered and the powder of -qa was collected and dried. x - ray powder diffraction (xrpd) patterns of the samples were obtained using a philips pw 1710 diffractometer by scanning the samples over 2 angles ranging from 2 to 35, using cu k radiation with wavelength 1.5418. crystal structure prediction was conducted using the accelrys polymorph predictor (pp) module in the cerius package (accelrys, 2002). in short, this program generates randomly a number of crystal packings in a chosen space group. the structures that are highest in rank are expected to have the highest probability of being found experimentally. for more details on the theory and background of the prediction procedures implemented in the pp, refer to the work of gdanitz. the molecule of quinacridone was first drawn in the three - dimensional sketcher and then energetically minimized in the dreiding 2.21 force field. electrostatic potential derived (espd) point charges were assigned to the atoms in the molecule. fock calculations at the given molecular geometry using the quantum mechanics program gaussian94 (frisch., 1998) with the 6 - 31 g basis set. before starting the prediction procedure, the geometry of the molecule was optimized again in the force field, but this did not contribute any significant changes to the geometry. the dreiding 2.21 force field was also used to minimize the energy of the crystal structures in the final step of the pp procedure. the calculation of the van der waals and coulomb interactions was performed using the ewald summation method ; the dielectric constant r was kept constant (unlike the default cerius setting of distance proportionality). rietveld refinement was performed using the dbws-9006 program (wiles & young, 1981), implemented in cerius. the refinement was performed in the range of 2 from 2 to 35. only the scale factor, background, cell parameters and the peak profile parameters were varied ; the fractional atomic coordinates were kept fixed at this stage. the atomic coordinates were optimized by lattice energy minimization with fixed unit - cell parameters between the rietveld steps. ab initio structure prediction is usually conducted in the space groups most frequently found among organic solids. 18 space groups together account for more than 93% of the molecular crystals reported (baur & kassner, 1992). however, since the space groups of quinacridone polymorphs are already known from the literature [for -qa (although questionable) and for and -qa ], we used this knowledge to limit the choice of the space groups. is not recognized during the pp prediction process, the prediction should be done in a space group that is a subgroup of the desired space group ; the inversion centre is expected to be part of the symmetry elements of the space group. for the present case, the subgroup should contain all symmetry elements of the desired group, excluding the inversion centre. therefore, we conducted the search in the space groups, and only. it is not necessary to search in all three subgroups : with a sufficient number of trials all structures will be found among the structures of each of these subgroups. for practical reasons (to reduce the calculation time), the search was conducted in all three subgroups and the results were subsequently merged. then, the clustering algorithm was applied to remove duplicate structures which resulted in total of 497 structures. minimization of the total potential energy was then performed on the remaining structures, now also relaxing the rigid - body constraints. after the prediction process, an additional step was performed to find the actual symmetry (with the find symmetry tool) followed by a further energy minimization of the crystal structures in the space group found if it differed from the initial space group. the latter step is necessary to reach the optimal structure in the space group found, as the atomic coordinates obtained by imposing the new symmetry may not correspond exactly to an energy minimum, in particular in the case of intramolecular symmetry, where small deviations in bond lengths lead to a substantial increase in energy. as the prediction procedure contains a monte carlo stage, it can give different results for different runs. three runs were enough to obtain reproducible results. in order to evaluate the polymorph prediction results, x - ray powder diffraction patterns of the predicted structures were calculated and compared with the experimental ones. due to the limited amount of predicted structures, it was possible to conduct the comparison manually for every structure. the quality of the experimental data was not good enough for indexing the patterns and deducing the structure, but sufficient to evaluate the pp results. the structures with powder patterns closest to the experimental ones were subjected to the rietveld refinement. the prediction, as a result of three consecutive runs, ended with 103 structures ranging in lattice energy from 53 to 28 kcal mol (1 cal = 4.184 j). we used density as a parameter to present the results in a clearer way and to distinguish structures with the same energy. a higher density might suggest a more stable structure, but this indicator is not as strong as low lattice energies. table 1 gives the information of the first 15 low - energy structures sorted with respect to the lattice energy. the last column shows the attribution of the structures to known polymorphs on the basis of the xrpd pattern. structures in bold are considered to be close enough to the experimental structures to serve as input for the rietveld refinement.. 2. table 2 gives the crystallographic data of the refined structures and published experimental data on - and -qa for comparison. we only consider the three widely accepted polymorphs (,,), which have distinct powder patterns. additional crystal phases mentioned in patents were reported on the basis of powder patterns of poor quality, giving rise to quite some uncertainty concerning their existence. moreover, one of these phases, designated as (deuschel., 1963), was proven to be -qa. variations in the crystallite size and shape caused, in this case, the differences in the powder patterns (potts., 1994). the structure of the polymorph, determined by single - crystal x - ray measurements, is known from the csd (reference code qnacrd04). the prediction of this structure served as a test to evaluate the quality of the force field and polymorph prediction in general. structures 1 and 2 in table 1 have powder patterns similar to the experimental one (fig. the difference between the structures is so small that they can be considered to represent the same structure. 3. each quinacridone molecule forms hydrogen bonds with four neighbours and they are packed in a criss - cross manner in ribbons along. rietveld refinement performed on both structures resulted in r factors of 18.5 and 14.1% (structures 1 and 2, respectively) and nearly the same final structure. in table 2, we cite data for the second structure, as it showed a better rietveld fit. as a test, the experimental structure qnacrd04 was refined against our experimental powder data in the same way as it was for the predicted structure. the r factor of the fit was 10.8%, a value which is an indication for the order of the accuracy of fits for the other predicted structures, given the limited quality of the experimental powder diffraction pattern. in spite of the commercial significance of -qa and many attempts to grow single crystals, its structure is still unknown in the open literature. at present, only the cell parameters, the space group and a sketchy powder pattern are available (paulus., 1989 ; struve, 1955). according to these data, -qa has a monoclinic structure, space group and cell parameters as listed in table 2. the polymorph prediction resulted in several structures for -qa ; all fit the experimental powder pattern well (fig. 5). however, none of the predicted structures reproduces the small peak at 14 in the experimental pattern, which can be attributed to an impurity. structure 4, designated here as 1, is monoclinic, space group, and has a long c axis (31). each molecule forms hydrogen bonds with two neighbours, forming ribbons that are arranged in two directions, along and (fig. the cell parameters of this structure fit well to the data reported in the literature (table 2). structure 5, which is labelled as 2, has the same molecular packing and a similar unit cell as structure 4, though their cell lengths do not correspond. 7. the distances between the planes of the molecules in the stacks in these two structures, 1 and 2, are 2.86 and 2.89, and the angles between molecules in different stacks are 60.2 and 60.3, respectively. this may give the impression that the structures are defined in different settings of the space group and that they can be adjusted to have the same cell parameters. however, the unique axis 3.988 in the 1 structure clearly differs from its value of 5.720 in the 2 structure. structure 14 has nearly the same arrangement of molecules and cell parameters as structure 13, but in space group, which is quite uncommon for a molecule with inversion symmetry. the unit - cell axes lengths of 3 are comparable with the two previously mentioned structures, but the unique axes differ (table 1). the crystal structures of 3 resemble that of 1 and 2 with the difference that the stacks of hydrogen - bonded molecules are arranged in a nearly parallel way, along (fig. the hydrogen - bond patterns are similar for all the structures ; each molecule has four hydrogen bonds to two neighbouring molecules and the resulting ribbons are packed in stacks. rietveld refinement of the 1 and 2 structures against the experimental powder pattern resulted in both cases in a reasonable fit with r values of 13.0 and 12.6%, respectively. the corresponding peak in the experimental pattern is broad and makes it impossible to distinguish between these two structures. it is notable that both refined structures are very close in the energy density plot (fig. both structures of 3 undergo cell compression during the rietveld refinement due to the shortening of the long axis. the energy minimization performed between the rietveld steps forces molecules to leave the nearly parallel arrangement of stacks (see fig. 9). the refinement converges at r values of 19.7% and 20.6% for the structures 13 and 14, respectively, and leads to nearly the same structure in both cases. therefore, only the crystallographic data for the refined structure 13 are listed in table 2. as a result of the rietveld refinement, 1 and 2 show better similarity with the experimental powder pattern, and the published data favour the 1 structure. however, considering that r exp of the structure reported by paulus. (1989) is not known and that the energies of all three structures mentioned here are within the error of the prediction (a few kcal mol), each of them remains a candidate for the experimental structure. it has been derived from the -qa structure by systematic variation of atomic coordinates (considering chemical bonding constraints) in order to fit the experimental powder pattern of -qa (lincke & finzel, 1996). the only difference with the structure is the angle between the molecules in the criss - cross arrangement. using the dreiding force field, we found that this structure converts during minimization readily to the structure of the polymorph ; this makes the qnacrd03 structure questionable. several structures, with numbers 812 in table 1, are predicted by the pp to have powder patterns similar to the experimental one (fig. structures 811 are triclinic, space group. each quinacridone molecule shares hydrogen bonds with two neighbours, forming ribbons similar to those in -qa, along. these are packed in such a manner that all molecules in the structure lie parallel to one another (fig. structure 12 is monoclinic, space group, and it has a different packing motif. crystallization of centrosymmetric molecules in a non - centrosymmetric space group is, however, very rare. the quality of the experimental powder pattern was not high enough to obtain an acceptable fit of the patterns. structures 10 and 11, as being closest to the experimental powder pattern, were used further for the rietveld refinement. the refinement converged at r values of 19.6% and 21.4%, respectively, and produced the structures at substantially much higher energy as compared with the initial values (fig. rietveld refinement of the structure 12 resulted in a relatively low r value of 14.3%. visual inspection of the resulting xrpd pattern showed, however, that the structure 12 fitted the experimental pattern badly, mainly due to the mismatch of the peaks in the area 2530. the low r value is misleading in this case. structure 10, showing the best rietveld fit among all five structures for the polymorph, can be regarded as the closest to the experimental structure. it is debatable if such a high r value is acceptable to confirm the similarity of the predicted structure and the experimental one. a higher quality powder pattern, at least, is necessary to arrive at a better fit. what are the other structures in table 1 ? showing no similarity with the known experimental powder patterns, they have molecular packing and hydrogen - bond patterns that are similar to the polymorphs discussed above. structure 3 has a criss - cross molecular motif, similar to that of -qa, but its powder pattern is very different from the experimental one (fig. structures 6 and 7, as 1-qa, consist of hydrogen - bonded ribbons running in two directions. structure 15 contains squeezed layers of molecular ribbons which resemble the packing of the 2 structure after the rietveld refinement. an important question is how certain one can be of the results of the polymorph prediction approach outlined in this paper. the confidence in the results heavily depends on the quality of the force field and the atomic charges used. the dreiding force field (mayo., 1990), a generic force field, can be applied to a wide range of heteroatomic organic molecules with reasonable accuracy. it shows the best results when used with the charges derived from high - level quantum mechanics calculations (espd charges). it models hydrogen bonds in crystal structures reasonably well. in certain cases, for example hydrogen bonds in acids, the force - field description of the structures in the present case appears to be quite adequate, since all three polymorphs were found within 3 kcal mol. moreover, -qa, usually mentioned in the literature as a stable polymorph at room temperature, is found to have the lowest lattice energy. if calculated lattice energies are close to the sublimation enthalpies, this can serve as another validation of the force field used. lattice energy for the structures listed in table 1 is 5053 kcal mol. the sublimation enthalpy of quinacridone, as for many other pigments, is hard to measure due to the difficulties in precise measurements at high sublimation temperature. a number of promising developments have been reported in the area of force - field development, such as a better description of the electrostatics by means of a multipole expansion (van eijck., 2001), using a large number of charged cells to describe the charge density (gavezzotti, 2002, 2003), or developing a tailor - made potential for the compound under investigation from quantum chemical calculations (day & price, 2003). as current progress in csp showed, the problem in predicting is not the generation of all the possible structures (for that, present algorithms are good enough), but rather the ranking and choosing the right structures from the list. in the case of pure ab initio prediction, we combine ab initio csp with experimental xrpd patterns to increase the chance of arriving at the right structure. the differences arise from the limited prediction power of the force field and from the bad quality of the experimental diffraction pattern. an absolute match of the powder patterns in such cases is mostly regarded as a pure coincidence and is rather rare. thus, the effectiveness of powder pattern comparison, in order to choose the right structures from the list of predicted ones, varies from case to case. to get an impression of the deviation from the experimental pattern, it is interesting to compare powder patterns for -qa. 12 shows the experimental powder pattern together with four calculated ones, namely calculated from the csd structure qnacrd04 before and after energy minimization in the force field, the powder pattern of structure 2 in the list of predicted structures (minimized), and that of structure 2 after rietveld refinement against the experimental data. in this figure it is remarkable that the patterns (c) and (d) are identical. this means that the structure predicted by the pp is the same as the experimental structure minimized using the same force field and, therefore, it is probable that these two structures are also identical. 12 also demonstrates how the effect of the force field leading to the difference between patterns (b) and (c) is eliminated by rietveld refinement. three peaks at 1215 are overlapping in the experimental pattern (a) and distinct in the one calculated from the csd structure (b). due to the inaccuracy of the force field, minimization of the energy results in changes in the structure (fig. 13) and the three peaks merge into two in pattern (c). after the rietveld refinement three peaks are resolved again in the pattern (e). these five structures are assumed to be the same, but their powder patterns differ to a certain extent. this difference should be kept in mind when comparing other predicted structures with the experimental powder patterns. rietveld refinement is often used to complete the prediction procedure and to refine the trial structures against powder data. full rietveld refinement is, however, only worthwhile if the experimental data are of excellent quality (e.g. collected using synchrotron radiation) and the input structure is close enough to the experimental one. in the case of small particle size or poor crystallinity, like for many pigments, the low quality of the powder diffraction patterns can not be improved by using more powerful x - ray sources. moreover, rietveld refinement is not efficient if the candidate crystal structure is not close enough to the experimental one and too few peaks in the simulated and experimental powder patterns exactly match (which happens very often). an example of the intrinsic ambiguity of powder data can be found in the structure of -qa that was deposited in the csd under the reference code qnacrd03 (lincke & finzel, 1996). this structure has a molecular packing very close to the criss - cross arrangement of molecules in -qa (and far from -qa, as predicted by us) ; only the angle between the molecules is different. however, this criss - cross -qa structure produces a powder pattern very close to the experimental pattern of -qa, which leads to confusion. the reason to have more confidence in our variant of -qa is that it represents a real local minimum in a reliable force field. in this case and in general, similarity of powder patterns of different polymorphs is not uncommon [for example, two polymorphs of terephthalic acid (bailey & brown, 1967 ; brown, 1984) ]. thus, the methods that use the powder patterns or other types of experimental information solely to distinguish the polymorphs contain by default some degree of uncertainty. for more accuracy, a combination of different physico - chemical characterization methods should be applied. another indication of the quality of the present prediction is the stability order of the polymorphs. according to the calculated lattice energy of the structures, the order of descending stability for the predicted polymorphs is or (table 1), depending on which structure to accept for -qa. as was mentioned before, experimental data about the stability of these polymorphs are not available. -qa is often mentioned in the literature as the most stable form and -qa as the least stable form (jaffe, 2001) (-qa is therefore not commercially important)., 2007), we observed that -qa sublimed faster and at a lower temperature than -qa. this leads us to the conclusion that, at least at the temperature of sublimation (673 k), the polymorph is less stable than the polymorph. possibly, -qa is less stable than -qa for the whole range of temperatures (monotropically related polymorphs), since no polymorphic transition could be observed till 773 k (jones. assuming such a monotropic relation, our experimental data confirm the predicted stability order for - and -qa. experimentally observed metastable polymorphs, in general, are not necessarily next in this ranking, even in the case of perfect prediction results, as kinetics determine the circumstances under which they are formed and kinetics are not part of the prediction method. the structures of three polymorphs of quinacridone were predicted using polymorph predictor in combination with xrpd patterns of limited quality. the known structure was predicted correctly as first in the ranking. three different structures were proposed for the unknown -qa that closely match the experimental powder pattern. for -qa, the present work demonstrates a method to obtain crystal structures using polymorph predictor in combination with experimental xrpd patterns. ab initio prediction (without any help of additional information, like xrpd) is still not very reliable, even for simple molecules, mainly due to the limited quality of the force field. the use of x - ray powder data helps to improve the reliability of the prediction. the effectiveness of the latter step is limited by the flexibility of the molecule and/or the quality of the xrpd pattern. in the present prediction method, the xrpd pattern need not be indexable. we expect this method to be useful in the case of rigid organic molecules, if a corresponding non - indexable powder pattern of reasonable quality is available. pigments are a good example of the application of this method, since pigment molecules are often rigid and xrpd patterns of pigments are usually difficult to index because of the peak broadening as a result of small crystal sizes. while the paper was in press, another paper on the single - crystal x - ray structure determination of -quinacridone was published (nishimura. the structure reported in this paper fits very nicely our 1-structure, which is the highest in ranking for the predicted -structures. | the structures of the, and polymorphs of quinacridone were predicted using polymorph predictor software in combination with x - ray powder diffraction patterns of limited quality. the present work demonstrates a method to obtain crystal structures of industrially important pigments when only a low - quality powder pattern is available. |
nowadays, post - operative nausea and vomiting (ponv) sound to be the leading cause of morbidity and discomfort among pediatric surgical patients. ponv may at least direct patients to have longer stay at hospital after surgery but some more serious consequences such as bleeding, wound dehiscence, dehydration, electrolyte disturbance and/or pulmonary aspiration of gastric contents can be resulted by severe types of ponv[2, 3 ]. the incidence of ponv is higher in pediatric patients than in adults[2, 4 ]. it has been reported that up to 40% of children undergoing appendicectomy who have not received any antiemetic drug may have ponv. clonidine, an 2-adrenoceptor agonist, is an effective premedicant which has been shown to decrease the incidence of ponv in children undergoing strabismus surgery[6, 7 ], but its efficacy for prevention of ponv after appendectomy in pediatric patients has not been studied yet. we therefore decided to evaluate the effect of oral clonidine given preoperatively on the incidence of ponv in children after appendectomy. following approval of institutional review board of medical faculty of tehran university of medical sciences and written parental consent, 60 children, with american society of anesthesiologists physical status i, ii, aged 5 - 12 years, who were scheduled for appendectomy were enrolled in this randomized double blinded clinical trial. this study was carried out at bahrami children 's hospital, tehran from february to november 2009. we excluded patients with perforated appendicitis, history of gastrointestinal diseases, motion sickness, and unexpected cardiac or respiratory events during or after the surgery and also those patients who had received antiemetic within 24 hours before surgery. patients in clonidine group were given 4 g.kg -1 clonidine in 20 cc of apple juice and patients in control group were given only 20 cc of apple juice 1 hour before transporting to operating room. standard monitoring included electrocardiography, noninvasive blood pressure, and pulse oximetry and capnography. heart rate and systolic and diastolic blood pressures were measured before and after anesthesia induction and after tracheal intubation in patients of the two groups. anesthesia was induced with 2 g.kg -1 fentanyl, 5 mg.kg -1 sodium thiopental, and 2 mg.kg -1 succynyl choline and maintained with isoflorane, fentanyl, atracurium and nitrous oxide. lactate ringer was administered to all of patients before induction of anesthesia (5ml.kg -1) and during the surgery with regard to hemorrhage intensity and body fluid loss. twenty minutes before the end of the procedure, all patients were given paracetamol 30 mg.kg -1 rectally. at the end of surgery muscle relaxation the children were extubated at the end of surgery when awake and then transferred to the post anesthesia care unit. at the surgical ward patients the episodes of vomiting as well as antiemetic usage were observed and recorded for 24 hours after the surgery. when two or more episodes of emetic symptoms occurred, intravenous metoclopramide 0.2 mg.kg-1 was administered slowly. all intra and postoperative observations were made by trained technicians and nursing staff who were blinded to which study group the patients had been allocated. data were expressed as simple count or mean [sd ]. statistical analysis of patients data from the two study groups was conducted using analysis of variance for repeated measurements (anova), fisher 's exact test, or chi square test where appropriate. a power analysis based on the result of a similar study and our pilot study under the present protocol showed that sample size of 30 patients in each group deemed to have a power of 80% to detect a difference in ponv between groups with a significance value of p<0.05. sixty patients were recruited in this study and no patient was excluded or lost from the study. there was no statistically significant difference between the groups with respect to sex, age and weight. also there was no significant difference in the mean values of systolic and diastolic blood pressures and heart rate and total time of surgery between the two study groups (table 1). patients who had received clonidine had significantly less episodes of ponv than patients in control group (p<0.001)(table 2). difference between patients in the two study groups with respect to number of patients who were given rescue antiemetic drug was also statistically significant (table 2). no clinically significant adverse effect such as bradycardia and hypotension was seen during perioperative period. flow diagram of patients characteristics of the patients in two groups sd : standard deviation number of patients with nausea and vomiting and requiring rescue antiemetic medication during 24 h after anesthesia to the best of our knowledge this is the first study aimed to evaluate the effect of premedication with clonidine to prevent ponv in patients undergoing appendectomy. we showed that oral clonidine with the dose of 4 mg.kg-1 in children undergoing appendectomy can significantly decrease the incidence of ponv as compared with control group (70% vs. 16.7%). the incidence of post operative vomiting in control group of our study was 70% which is higher than the result of a similar study. ponv is a multifactorial complication of anesthesia and surgery which accounted for 13 - 42% of all post - operative complications in pediatric surgical patients. many drugs (antihistamines, butyrophenones, dopamine receptor antagonists) have been attempted to prevent and treat ponv, but undesirable adverse effects, such as excessive sedation, hypotension, dry mouth, dysphoria, hallucinations and extrapyramidal signs have occasionally been noted. clonidine is an 2-agonist which is used as a sedative premedication in children. due to the lower costs of clonidine in comparison with other drugs such as opioid and benzodiazepines, as well as less complications of clonidine it has been shown that this drug can provide preoperative sedation, suppress cardiovascular responses to laryngoscopy and tracheal intubation and reduces anesthetic requirement. moreover, clonidine has been reported to reduce shivering both after general and spinal anesthesia and the incidence of ponv has been shown to be reduced after both systemic and epidurally administered clonidine[10, 11 ]. however, there are conflicting results in the published literature about the effect of clonidine on the incidence of ponv. some investigations in adults concluded that oral clonidine can reduce the incidence of ponv[1215 ], but in children the results are somewhat different. our results are consistent with the two earlier studies who examined the effect of clonidine in children undergoing strabismus surgery and showed that preoperative oral clonidine at a dose of 4 g.kg-1 is associated with a reduced incidence of postoperative vomiting in this group of patients [6, 7 ]. in contrast, some other investigators reported no significant antiemetic effect of clonidine given orally to children[16, 17 ]. the difference between the above mentioned results may be related to the nature and origin of vomiting during perioperative period. in addition to age, sex, a history of ponv and motion sickness and also type of surgery which accounted as uncontrollable risk factors for ponv, there are anesthesia - related factors under control of anesthetists that have impact on the incidence and severity of ponv. it seems that the variety number of factors affecting the incidence of ponv in adults and children can be accounted for the different results of investigations evaluating the effect of clonidine on ponv. the mechanism of antiemetic effect of clonidine has not been yet clearly specified but some investigators believe that antiemetic effect of clonidine can result from its sedative and anxiolytic action which can directly reduce anesthetic and analgesic requirement of patients in perioperative period. it also has been suggested that clonidine can decrease ponv through its inhibitory action on sympathetic outflow and catecholamine release which can then block triggering of nausea and vomiting. one of the most important systemic side effects of clonidine is hypotension along with bradycardia in adults which have not been considered as main post - operative complications among children[17, 18 ]. however, none of the patients in our study experienced such episodes of severe hemodynamic change. in our study all patients were comparable regarding demographic data, anesthetic technique, surgical procedure and postoperative medications, therefore the difference between the study groups with respect to incidence of ponv can be attributed to premedication with oral clonidine. in this study we used oral clonidine with the dose of 4 g.kg-1 which has been shown that it can reduce the incidence of ponv[6, 7 ]. because of slow onset (0.5-l h) and a long duration (12 h) of action, we administered oral clonidine to patients in intervention group one hour before transporting patients to the operating room. our study had a few limitations. in the present study the level of sedation and discharge time because of sedative effect of clonidine, further studies are required to elucidate if clonidine prolongs hospital stay in patients receiving clonidine in the perioperative period. however, to verify the antiemetic effect of clonidine in postoperative period, we need to design similar studies with larger sample size in appendectomy patients and also other pediatric surgical populations. we have shown that oral clonidine at a dose of 4 g / kg-1 administered preoperatively is associated with a reduced incidence of postoperative vomiting in children who have undergone appendectomy. | objectiveclonidine is an 2-agonist which is used as a sedative premedication in children. there are conflicting results in the published literature about the effect of clonidine on the incidence of post operative nausea and vomiting (ponv). we therefore decided to evaluate the effect of oral clonidine given preoperatively on the incidence of ponv in children after appendectomy.methodssixty children, 5 - 12 years old, classified as american society of anesthesiologists physical status i and ii, who were scheduled for appendecectomy were enrolled in this randomized double blinded clinical trial. patients were randomly assigned into two groups of 30 patients. patients in clonidine group were given 4 g.kg -1 clonidine in 20 cc of apple juice and patients in control group were given only 20 cc of apple juice 1 hour before transporting to operating room. the protocol of general anesthesia and postoperative analgesia was the same for two groups. incidence of ponv and antiemetic usage of patients were assessed during 0 - 24 hours after anesthesia.findingsthe patients characteristics were similar in two groups. patients who had received clonidine had significantly less episodes of ponv and also less rescue antiemetic usage than patients in control group.conclusionwe showed that oral clonidine at a dose of 4 g.kg -1 administered preoperatively is associated with a reduced incidence of postoperative vomiting in children who have undergone appendectomy. |
the ecology of tularemia is represented by a literature that reflects the diversity of this complex zoonosis. like the literature of many other infections, the sheer volume of observations makes it difficult to organize and synthesize sets of working hypotheses for how the causative agent exists in nature. an organized understanding of tularemia ecology serves as the basis for developing public health interventions and to predict or explain changes in incidence or distribution. in addition, knowing how the agent is currently maintained in nature provides information that helps us to reconstruct its evolutionary history. we review herein features of tularemia ecology that are particularly critical and suggest lacunae that hinder us from distinguishing major themes from variations on themes. the reader is referred to excellent reviews of the subject by hopla (1974), friend (2006), and petersen. (2009) for significantly more detail on the breadth and diversity of the ecological literature. we use some basic terms and concepts in the population biology of infectious agents that help to organize our interpretation of the existing literature. maintenance refers to the life cycle of the agent : how one infection gives rise to at least one other infection. perpetuation of zoonotic infections may involve a vector, an intermediary in the life cycle that imparts directionality to the agent. hematophagous arthropods are vectors because they require blood and thus directly transport an infectious agent to a relevant host. in contrast, scalars may also maintain an agent, but there is no directionality ; copepods containing dracunculus medinensis nematodes, for example, are passively imbibed with water. vectors may support biological transmission of an agent, a process that involves replication or developmental changes. vectorial capacity (spielman and rossignol, 1984) refers to the sum of vector traits that ensures that the basic reproduction number (brn) of an infection exceeds unity and comprises competence (ability to support replication and effectively deliver the agent) as well as factors such as abundance, longevity, and narrowness of host range. a mosquito that requires a large dose of pathogen and rarely passes it during feeding has poor vector competence and thus might not contribute much to brn, but even a highly competent vector (agent replicates well and is readily ejected during feeding) can have poor vectorial capacity if it feeds only on an animal that is a the central question in the ecology of infectious agents is to describe how an agent ensures that brn > 1 (anderson and may, 1981) ; brn 90% of the > 6000 tularemia case reports that he had compiled from 1924 to 1935 were associated with exposure to cottontail rabbits or hares, and this analysis surely helped to formally develop tularemia 's reputation as rabbit fever. it is possible that the strong rabbit association was due to an active market for rabbit meat in the north central states where there was a tradition of rabbit hunting (yeatter and thompson, 1952). this rabbit association, interestingly, obscured the fact that in the south central us, tick exposure accounted for 70% of all cases (brooks and buchanan, 1970) during the 1960s. tularemia incidence in the us started to diminish in the 1960s (boyce, 1975), perhaps as a result of a loss in popularity of rabbits as food and of hunting in general. it seems unlikely that the force of transmission of the agent diminished in nature during this time. the tick vectors (d. andersoni, d. variabilis, and a. americanum) for tick - transmitted tularemia in the us are the same as those for rmsf, which increased in incidence during the 1960s and 1970s (childs and paddock, 2002). russia and japan had concurrently discovered tularemia (francis, 1934 ; pollitzer, 1967). episodes of morbidity and mortality in hares were associated with an increase in the number of human cases of yato - byo and the disease could be acquired by skin contact with hare tissues (ohara, 1926). apparently, 90% of all japanese tularemia cases were associated with exposure to the hare lepus brachyurus (toyoshima and ohara, 1967). thus, in north america and japan, during the very first decades of epidemiologic investigations of the disease, lagomorphs (rabbits and hares) were the main focus of attention. researchers in the former soviet union were extremely active in investigations of tularemia, producing 1300 publications from 1928 to 1960 (pavlovsky, 1966), and were the first to describe at least six perpetuation scenarios centered around habitat types (floodplain swamp ; meadow field ; forest ; steppe ; piedmont river ; and desert floodplain) ; lagomorphs were requisites for none. paradoxically, a hypothesis for the evolution of f. tularensis by russia 's most prominent tularemia researcher (olsufiev, 1963) focused on associations with lagomorphs, mainly based on their great degree of susceptibility and sensitivity to infection as well as a scenario for the zoogeography of the steppes, which were thought by the former ussr workers to have been a pivotal habitat for tularemia. differences in distribution, ecology, biochemistry, and virulence led to the seminal classification of tularemia into distinct types (olsufiev., 1959). type a organisms (now known as f. tularensis tularensis) are prevalent in north america but not in eurasia, are frequently transmitted by ticks, and may cause severe disease. type b (f. tularensis holarctica) causes episodic outbreaks (epizootics) in beavers, muskrats, and arvicoline rodents in either north america or eurasia, may be isolated from water or soil, and may cause a milder disease (jellison. these eco - epidemiological paradigms retain tremendous utility and argue for modeling brn separately. the perception that tularemia was due to lagomorphs was largely the influence of francis himself and also of william jellison of the rocky mountain laboratories, who compiled and interpreted the existing literature on tularemia biology in a seminal monograph (jellison, 1974). jellison argued that human risk and geographic distribution of north american tularemia was strongly associated with cottontail rabbits (jellison and parker, 1945 ; jellison., 1961). cottontail rabbits are very susceptible to infection by type a, dying within 7 days, and are large enough animals to attract attention when there is an epizootic, making them good sentinels for transmission activity. furthermore, because of their value as food, their populations were a focus of attention by local residents and by state game management divisions : 25,000,000 rabbits were killed annually with a value of $ 5,000,000 during the 1920s (henderson and craig, 1932). whether cottontail rabbits are required for type a brn > 1 remains unproven and requires further study. it may be that the question has been considered resolved due to conflating the requirements for maintenance with the proximal determinants for human exposure. of course, human exposure (the subject of epidemiology) may provide clues to the mode of perpetuation (ecology) but this is not axiomatic. zoonotic infections may exist in sites with no implied human risk in the absence of an effective epidemiological bridge. the classical theory of natural nidality (pavlovsky, 1966) posits that most zoonotic agents exist in longstanding foci that comprise optimal physical (weather, geology) and biological (fauna, flora) associations and that humans only become aware of their existence when they intrude. accordingly, rabbits and hares may only be the epidemiological bridge and are not necessarily an element of natural focality. the concept of rabbits as central to type a ecology was bolstered by the identification of the rabbit tick, haemaphysalis leporispalustris, as an effective enzootic vector (parker 1934). these ticks are widely distributed across north america and their feeding is focused primarily on lagomorphs. narrowness of host range (bites focused on relevant hosts) greatly contributes to vectorial capacity and brn (spielman and rossignol, 1984). these ticks transmit f. tularensis and pass the agent by inheritance (transovarial transmission ; parker 1934) ; presumed type a isolates were made from field collected h. leporispalustris (philip and parker, 1938). rabbits may be infested by hundreds of these ticks and all stages may infest a rabbit simultaneously, thereby providing an opportunity for non - systemic (co - feeding) transmission (randolph., 1996). thus, rabbits and rabbit ticks could serve to powerfully maintain type a. in addition, because subadults (larvae and nymphs) of this tick will infest ground - inhabiting birds, including those that migrate, the agent could be readily transported. this fact begs the question : why has f. tularensis not been detected in latin america south of mexico, particularly given the presence of rabbits throughout south america ? the central importance of rabbits and their ticks in type a ecology is not supported by recent studies on martha 's vineyard (mv). this is the only site in the us which has endemic primary (inhalational) pneumonic tularemia (matyas., 2007) ; of more than 100 tularemia cases reported from mv from 2000 to 2010, case control studies demonstrate that landscapers are the major risk group and use of lawnmowers or leaf blowers are the main risk factors, suggesting environmental contamination. landscapers there insist that they rarely mow over animal carcasses because they visually inspect properties prior to their activities to reduce hazard due to rocks and other debris. the nature of the fomites that served to infect these case - patients remains undescribed but unseen remnants of animal carcasses, animal feces, urine - soaked soil, ticks, tick feces, fleas, and contaminated water are possible sources. why mv alone reports numerous pneumonic tularemia cases when tularemia is more prevalent in the south central us where lawnmowers are certainly used remains enigmatic. it is possible that the heavily salt - spray influenced landscape of the ocean facing southern edge of mv is more conducive to the agent remaining viable for a longer duration than elsewhere in the us (berrada and telford, in press). ecological studies, extended from lyme disease surveillance starting in 1994, quickly suggested that dog ticks (d. variabilis) were important to type a perpetuation (goethert., 2004 ; matyas., 2007 ; goethert and telford, 2009). infection has been found in mv dog ticks every year to date, comprising a large degree of genetic heterogeneity (goethert., 2009). rabbits were indeed infected but virtually disappeared on mv, probably due to tularemia mortality ; their disappearance did not influence the force of transmission of type a, which continued to remain prevalent in dog ticks. intensive studies of cottontail rabbits had been undertaken on nantucket island, which is visible from the eastern portion of mv. rabbits attained densities of 1520 per hectare and were heavily infested by h. leporispalustris (telford and spielman, 1989 ; goethert and telford, 2003). evidence of tularemia had never been detected in more than 200 rabbits sampled on nantucket, even though five human cases had been identified from 1990 to 2005. one of these cases was definitively associated with rabbits : a worker who had helped his colleague move a rabbit that had been mutilated by a lawnmower developed pneumonic tularemia (goethert and telford, 2005). this event demonstrated that even though type a had been introduced at least once to nantucket, despite the presence of dense rabbits and heavy h. leporispalustris infestations, the agent did not perpetuate. no mass die - offs of rabbits were noted during the year when the lawn mowing incident occurred, nor did active surveillance for rabbit carcasses by landscapers yield any evidence of mortality due to tularemia. there is one important difference in the ecology of nantucket relative to mv : nantucket lacks appreciable numbers of dog ticks (indeed, the tick may now be extirpated from that island due to the recent widespread use of topical anti - ectoparasiticides on dogs) because it does not have their reproductive hosts, skunks, raccoons, foxes, or coyotes. although one exception to the rule of rabbits maintain tularemia does not necessarily invalidate the rule, we note that mv is one of few sites where longitudinal ecological studies have been undertaken and thus where incidental findings can be distinguished from general findings. we suspect that rabbits are not necessarily critical to the brn of type a, or if they are, it is a function of local conditions. this dependence on local conditions, in fact, is the challenge of developing a quantitative model for the brn of tularemia : should there be a general model, or should we approach the subject as did the researchers of the former ussr, focusing on independent natural foci ? at one extreme, the 4 genotypes / subclades of type a and 11 of type b (kugeler., 2009 ; vogler., 2009) might each require a specific brn model. but, a general model would have to assume that all elements for the ecology of type a would apply to type b and vice versa, not necessarily a good assumption. although our hypothesis is that dog ticks are critical to brn on mv, experimentally infected as well as naturally infected ticks die more quickly than do those that are not (reese. these findings stand in contrast to our empirical observation of infected ticks each year, and suggest that there may be factors that mitigate the negative fitness of infection in nature. tularemia in eurasia and non - rabbit or tick associated infection in north america seem to have a strong environmental basis, acquired from agricultural activities such as hay threshing ; from water contaminated by muskrats or water voles ; or during the trapping of furbearers (pavlovsky, 1966 ; syrjala., 1985 ; reintjes., 2002 ; allue., 2008). in addition, transmission of the pathogen could occur via contamination of foodstuffs by urine or fecal material from infectious rodents (karpoff and antonoff, 1936 ; parker., 1951). experimental studies with voles suggested the possibility that some f. tularensis - infected animals developed a chronic nephritis and bacteriuria that could serve as a protracted source of environmental contamination (bell and stewart, 1975). of particular interest was the suggestion that voles became partially immune due to low level infection resulting from cannibalism of tularemic carcasses and that this immunity allowed survival of the vole during subsequent infection, increasing the probability for shedding in the excreta. (cannibalism of moribund cagemates is well known as a mode of transmission for type a in the laboratory, owen and buker, 1956, and could be a complementary factor in perpetuation.) this suggestion of orally induced immunity has not been explored further but if confirmed could be a critical factor for the brn of type b, particularly in the context of environmental persistence. ticks may be infected by type b and are said to be the reservoir (pavlovsky, 1966). in the former soviet union, 17 species of ixodid ticks have been found to be naturally infected (balashov, 1972), presumably by type b inasmuch as type a is virtually restricted to north america. in addition, type b is well known to be tick - transmitted in north america and both types may be present in ticks in the same site (markowitz., 1985). human cases certainly result from tick exposure but this mode of transmission is less common than exposure to furbearers or contaminated water (pavlovsky, 1966). dermacentor marginatus and dermacentor reticulatus appear to be the main vectors there as well as into central europe. as with type a, type b - infected d. marginatus or d. variabilis die more rapidly than do uninfected ticks (petrov, 1960 ; reese., 2010), which again raises the question of whether a non - adaptive trait may be associated with stable brn. mosquitoes are strongly implicated as vectors in sweden, given that tularemic ulcers are most frequently found on the upper back, neck, and ears of case - patients (eliasson and back, 2007), where mosquitoes are more likely to feed. in addition, the agent has been isolated from mosquitoes there (olin, 1942) and recent studies provide evidence for mosquito larvae acquiring infection from water, perhaps by the ingestion of predatory protozoa (mathisen., gis studies of the orebro endemic area in sweden demonstrate that there is temporal spatial association of incidence with mosquito breeding (svensson., 2009). one recent study reported a third of mosquito pools to be infected in alaska (triebenbach., 2010) when tested by pcr, but this finding was at odds with the epidemiological evidence as well as with the difficulty of finding infection in animals. older studies in alaska failed to isolate f. tularensis from mosquitoes (hopla, 1974) and thus it is not clear what the pcr findings represent ; it should be noted that the assay that was used might also detect francisella novicida. future mosquito surveys should always attempt to confirm pcr findings with a complementary assay such as culture or even indirect immunofluorescence using monoclonal antibodies. at the very least definitive evidence for biological transmission by naturally infected mosquitoes might be provided by the use of deliberately placed sentinel mice but given the difficulties of animal experimentation in sweden, not a likely approach. the recent suggestion that infectious agents may be detected by assaying sugar sources probed by mosquitoes (hall - mendelin., 2010) may be an effective alternative approach to demonstrating that naturally infected mosquitoes can transmit. given that mechanical transmission causes infection and thus brn > 1, it might be considered academic to determine whether biological transmission occurs. but, the duration of mosquito infectivity would differ depending on whether it was mechanical or biological transmission, thereby impacting the magnitude of brn. in addition, the possibility that mosquito larvae may acquire infection from water might greatly enhance brn if those larvae became adult females that transmitted. whether there is a main mode of perpetuation (e.g., ticks and rodents) with ancillary cycles (spillover into a water cycle, mechanical transmission by mosquitoes), whether it is the other way around (perpetuation within water and spillover into rodents and their ectoparasites) or whether there may be multiple parallel cycles in sites where there are ticks would be difficult to answer with field studies but might be facilitated by mathematical modeling of brn. such questions have more than just academic interest : if ticks and rodents drive the ecology of type b, then intervention could be considered to reduce risk, for example, by rodent or tick control. if water drives the ecology, then risk reduction would have to focus on personal protection (e.g., with vaccination) given the difficulty of manipulating water ecosystems. both type a and type b are highly infectious and may be transmitted mechanically on the mouthparts of various hematophagous arthropods, or by contact with body or tissue fluids through abraded and even intact skin, and by ingestion, in addition to true biological (vector) transmission. such a wide spectrum of modes of exposure and great infectivity helps to explain the wide range of kinds of animals known to be exposed or infected (burroughs., 1945 ; friend, 2006). it is likely that most of these animals do not serve as amplifying hosts (reservoirs) that increase the brn of the agent of tularemia, but are incidental many of the reports of an animal serving as host simply document exposure (seroreactivity) in a limited sample which does not allow inference about whether the exposure might be common over many sampling periods or among many sites. if an animal contributes significantly to brn, it should do so for successive generations and in more than one site. this is the rationale for undertaking longitudinal ecologic studies : to determine whether an observation is incidental or is a generality. the reason it is important to determine whether there is a main theme for perpetuation (one important reservoir host such as a lagomorph) as opposed to many themes (almost anything can serve as a reservoir) is that an ecological paradox otherwise exists : if virtually any hematophagous arthropod and vertebrate could maintain infection, then tularemia should be readily perpetuated and extremely common across the holarctic. thorough search of any site should document the presence of f. tularensis. from an epidemiological standpoint, tularemia is only moderately common, with global incidence in the range of 1001000 cases annually (paddock and telford, in press). tick surveys in known endemic sites generally record prevalence of f. tularensis infection in the range of 0.15% (e.g., green, 1931 ; hopla, 1960 ; hubalek and halouzka, 1997 ; goethert., 2004) which is similar to that for tick borne encephalitis (tbe) virus in i. persulcatus complex ticks (gresikova and nosek, 1967 ; korenberg, 1994 ; schafer., 1999). tbe is considered to be a common tick borne infection, with 100010,000 new cases each year (paddock and telford, in press). f. tularensis has been detected in i. ricinus, the main european vector of tbe. with the potential for transmission by mosquitoes, in addition to tick transmission and environmental exposure, tularemia risk (human incidence) over the palearctic should approach or exceed that of tbe. becomes even more vexing given the possibility of environmental persistence, that is, perpetuation that may be independent of vertebrate hosts. surface water and sediment yielded indisputable dna sequence evidence of contamination with type b in swedish endemic sites, even in years with little epidemiological activity (broman., infected carcasses contaminated water for 10 days and contaminated water stored in the cold infected animals after 2 weeks. naturally contaminated mud remained infectious as long as 10 weeks (parker., 1951). experimental microcosm experiments demonstrated that f. tularensis in silt could infect animals for 2 months. about half of rodents immersed in contaminated water became infected with exposure to as few as 1001000 cfu / ml (pavlovsky, 1966), which appears to be large dose but the spleen alone of a mouse dying of tularemia may have 10 cfu (molins., 2010) and it would not take many carcasses to contaminate a contained body of water. indeed, many have speculated that environmental persistence depends on continual contamination by dead animals. however, water invertebrates such as shrimp or snails could retain viable organisms for 20 days (mironchuk and mazepa, 2002), and indeed, invertebrates were first described as contributing to f. tularensis survival within water by former ussr research (pavlovsky, 1966). more recent reports (anda., 2001) of crayfish infection suggest that additional surveys using modern methods are warranted. even more interesting is the hypothesis that free - living amebae serve as hosts (berdal., 1996 ; abd., 2003 ; el - etr., 2009), which would allow for even greater duration of persistence and even the possibility of amplification in the absence of vertebrates. in terms of brn, environmental persistence within aquatic invertebrates or protozoa would have implications for ensuring that brn > 1 when vertebrates are scarce but water contamination could serve to greatly increase brn and trigger an epizootic or outbreak. the ecology of tularemia is sufficiently complex that limited resources for research should be targeted to the most relevant questions. developing quantitative models of the brn for tularemia, probably separately for type a and type b, would appear to be a priority because such models provide structure for observations. critical life cycle breakpoints and influential variables can be hypothesized a priori and tested by field observations. surveys should be undertaken to identify useful study sites where the agent is reliably detected and longitudinal observations (on a scale of years) may be undertaken. cooperative agreements should be developed among researchers and long term funding jointly sought to support such studies. we have outlined some of the more interesting lacunae in our knowledge of tularemia ecology, and additional information for all of these would greatly enhance iterative refinements of brn quantitative models in the future. 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. | the epidemiology of tularemia has influenced, perhaps incorrectly skewed, our views on the ecology of the agent of tularemia. in particular, the central role of lagomorphs needs to be reexamined. diverse observations, some incidental, and some that are more generally reproducible, have not been synthesized so that the critical elements of the perpetuation of francisella tularensis can be identified. developing a quantitative model of the basic reproduction number of f. tularensis may require separate treatments for type a and type b given the fundamental differences in their ecology. |
assessment of left ventricular (lv) function is the most common and clinically relevant task in the congenital echocardiography laboratory. historically, those measurements have been estimated using m - mode (mm) and two - dimensional (2d) echocardiography, with several limitations. strain echocardiography, on the other hand, permits the quantification of lv global and regional function, which provides new insights in different experimental and clinical scenarios. recently, lv strain derived from 3d speckle - tracking echocardiography (3d - ste) has been validated against cardiac magnetic resonance imaging in children and adults. this new technique has the potential to better evaluate the lv function in neonates with congenital heart disease and to be of help in the surgical decision - making. however, to the best of our knowledge, there are no data on 3d - ste in full - term neonates to assess lv mechanics. in this study, we sought to establish the feasibility of the acquisition and analysis of 3d data sets, and to establish using 3d - ste, normal values for lv volumes, and ejection fraction (ef) as well as the 3d global strain (gs) parameters in a group of healthy neonates. all the subjects were born in our institution and were either awaiting the discharge of their mothers or presenting for their first newborn baby regular checkup. the inclusion criteria were : no history of congenital oracquired heart disease, andcompletely normal results on 2d and doppler echocardiography, with no structural defects and with normal chamber size and systolic function. no history of congenital or acquired heart disease, and completely normal results on 2d and doppler echocardiography, with no structural defects and with normal chamber size and systolic function. exclusion criteria were : structural congenital or acquired heart disease, abnormal cardiac rhythms, andhypertension and/or other acute or chronic illnesses. structural congenital or acquired heart disease, abnormal cardiac rhythms, and hypertension and/or other acute or chronic illnesses. all echocardiography studies were performed by an experienced cardiac sonographer using ie 33 platform scanner (philips medical systems, andover, ma). matrix - array transducer (s8) was used for the routine mm, 2d, and doppler study, and a dedicated 3d full sampling transducer (x7) was used for the 3d full volume data sets acquisition. studies were performed with the babies asleep or quite suckling on a bottle of formula ; attention was made to acquire the data sets during the resting phase of the periodic breathing or shallow breathing to minimize heart translation and the effect of breathing. the echocardiograms were transferred to xcelera r3.3 cardiology pacs system (philips medical systems, andover, ma) and were analyzed offline. the routine echo study was reported in a separate session than that of the 3d analysis. the same experienced observer, analyzed and reported the 3d study, blinded to the finding of the routine examination. adequate 3d data sets (without stitching artifacts and containing the whole of the lv) were analyzed using 4d lv analysis software v3.1, (tomtec imaging systems). the software allows a semi - automated detection of the lv border following the identification of the mitral valve annulus and lv apex by the operator [figure 1 ]. the others resemble the two and four chamber views of the lv, used in the biplane method of lv volumetric measurement. in addition, following manual adjustments of the endocardial surface was performed as necessary to include the papillary muscles within the lv cavity ; the 3d endocardial surface is automatically tracked using 3d speckle - tracking technology throughout the cardiac cycle. the software eventually provides longitudinal, circumferential, radial, and tangential time curves, from which peak gs and averaged peak strain at the basal, mid - ventricular, and apical levels are determined [figure 2 ]. initial step of the analysis of the three - dimensional dataset : an automated tri - plane construction of the left ventricle automated border detection, both at end diastole and systole with the resultant volume and deformation calculations principal tangential strain in 4d lv analysis describes the shortening of a surface element along the main contraction direction. it is, thus, a measure of regional myocardial contraction ability, and may represent the vector summation in longitudinal and circumferential direction, and the resulting direction of the contraction of the muscle fibers that are aligned tangentially to the myocardial surface. in addition to track the endocardial border, the software allowed the tracking and tracing the epicardial border as well. in this way, the system calculated the volume of the endocardial cavity and estimated myocardial mass. as a result, the volume of the lv all through the cardiac cycle was measured and the myocardial mass was calculated. to assess intraobserver variability, the same observer (z.b.) measured the 3d strain analysis of 10 randomly selected newborns twice at an interval of 2 months to avoid recall bias. to assess interobserver variability, 3d strain measurements were performed by a second observer (g.s.), who was blinded to the results of the first observer (z.b.). all demographic, conventional, echocardiographic and 3d strain data are presented as numbers or percentages (mean standard deviation). linear correlation analysis was performed to assess the relationship between 3d strain variables (global longitudinal strain [gls ], global circumferential strain [gcs ], global circumferential strain [grs ], global transverse strain [gts ], and twist and torsion) and age, heart rate, weight, height, and 3d derived lv volumes, lv ef, lv stroke volume (sv), and lv mass indexed for body surface area (bsa). multiple linear regression analysis was performed to determine the additional effect of anthropometrics (age, heart rate, weight, and height) and conventional echocardiographic parameters on the 3d - studied strain variables. the anthropometric and echocardiographic parameters were considered as potential dependent variables, and all variables were simultaneously entered in the regression. statistical analyses were performed using spss for windows version 16.0 (spss, inc., intraobserver and interobserver agreements were calculated using the coefficient of variation (i.e., the percentage absolute difference between the measurements divided by their mean value). all demographic, conventional, echocardiographic and 3d strain data are presented as numbers or percentages (mean standard deviation). linear correlation analysis was performed to assess the relationship between 3d strain variables (global longitudinal strain [gls ], global circumferential strain [gcs ], global circumferential strain [grs ], global transverse strain [gts ], and twist and torsion) and age, heart rate, weight, height, and 3d derived lv volumes, lv ef, lv stroke volume (sv), and lv mass indexed for body surface area (bsa). multiple linear regression analysis was performed to determine the additional effect of anthropometrics (age, heart rate, weight, and height) and conventional echocardiographic parameters on the 3d - studied strain variables. the anthropometric and echocardiographic parameters were considered as potential dependent variables, and all variables were simultaneously entered in the regression. statistical analyses were performed using spss for windows version 16.0 (spss, inc., chicago, il). intraobserver and interobserver agreements were calculated using the coefficient of variation (i.e., the percentage absolute difference between the measurements divided by their mean value). of the 50 healthy full - term newborns, 10 were excluded because their parents refused to give consent for this study. two babies had more than mild shunting lesions (atrial septal defect type ii and a large patent ductus arteriosus) ; both were not included in our data analysis. thus, the final study group consisted of 38 healthy newborns (mean age was 3.7 4.7 days, range : 1 - 15 days, and gestational age of 38.1 1.1 weeks). mean weight and bsa were 3.1 0.5 kg and 0.19 0.02 m, respectively. general characteristics of the studied sample bsa = body surface area, hr = heart rate conventional echocardiographic parameters and 3d lv global myocardial strain variables of the studied subjects are presented in table 2. measured echocardiographic parameters of the studied sample 3d lv edv = three - dimensional left ventricular end diastolic volume, 3d lv esv = three - dimensional left ventricle end systolic volume, 3d lv ef = threedimensional left ventricular ejection fraction, 3d lv sv = three - dimensional left ventricular stroke volume, 3d gls = three - dimensional global longitudinal strain, 3d gcs = three - dimensional global circumferential strain, 3d grs = threedimensional global circumferential strain, 3d gts = three - dimensional global transverse strain linear regression analysis was performed to assess the relationship among 3d strain variables, age, weight, lv volumes, lv mass lv sv, and lv ef. gcs was significantly correlated with age (r = 0.41, p = 0.01), sv (r = 0.55, p = 0.0003), lv mass index (r = 0.36, p = 0.027), and ef (r = 0.90, p < 0.0001). grs was significantly correlated with age (r = 0.43, p < 0.0001) and ef (r = 0.79, p < 0.0001), while gts was significantly correlated only with ef (r = 0.61, p = 0.0002). multiple linear regression analyses among the anthropometric and standard echocardiographic parameters were performed to determine the independent effect of these parameters on the 3d gs variables. only age was significantly associated with gls (p = 0.046, r = 0.20), while gcs (p < 0.0001, r = 0.90), grs (p < 0.0001, r = 0.79), and gts (p = 0.003, r = 0.61) were significantly associated only with ef. the coefficient of variation was very good for gls (intraobserver 0.09% and interobserver 1%) and gcs (intraobserver 2.5% and interobserver 2.8%), but poor for grs and gts (intraobserver 10% and interobserver 15%). the coefficient of variation was very good for gls (intraobserver 0.09% and interobserver 1%) and gcs (intraobserver 2.5% and interobserver 2.8%), but poor for grs and gts (intraobserver 10% and interobserver 15%). our data show that 3d - ste analysis is feasible on the consecutive healthy newborns, and we presented reference data for this population. the most recent ase\eacvi guidelines consider ste - derived gls as reproducible and feasible for clinical use offering incremental prognostic data over lv ef in a variety of cardiac conditions. thus, gls may become a sensitive tool to assess lv mechanics and function in newborns with various congenital heart diseases. unfortunately, only few data are available in the pediatric and newborn population on ste - derived strain variables and 3d - ste data are available only for preterm ones [table 3 ]. however, differences in gls values across different studies also reflect the use of different machines, software, and different algorithms. comparison with previous studies na = not available, 2d = two - dimensional, 3d = three - dimensional, cpa = cardiac performance analysis in agreement with the previous studies, gls is a very reproducible parameter and this could facilitate its routine clinical application. only a very weak association (r = 0.20) between age and gls was demonstrated. while at multivariate analysis, gcs, gts, and grs were significantly associated only with 3d lv ef. this finding is consistent with the previous observations that lv size and mass continue to grow from birth to puberty but that lv systolic function, as shown by lv ef and now by strain variables, is relatively consistent from infancy. in this regard, the use of normative values for 3d - derived gls will be greatly facilitated by its substantial independence of anthropometric, echocardiographic, and demographic factors that constitute confounding effects in other techniques. the determination of reproducibility of 3d lv strain and torsion measurements is also important when applying this new technology to daily clinical practice. inter and intraobserver variability of 3d gls and gcs was very good, suggesting these parameters are ready for the clinical use. on the other hand, reproducibility of gts, grs twist, and torsion is still high, underscoring the need for more technical advancement before is ready for clinical practice. there is a learning curve for operators new to the technique to optimize imaging for the entire endocardial wall throughout the cardiac cycle and provide reproducible point selection for tracing. fundamental limitations of ultrasound, such as reverberation, shadowing, dropouts, chamber foreshortening, and movement artifacts, can limit feasibility. in children and newborns, most of these limitations are overcome because of the usual, very good image quality as confirmed by our findings and previous studies on infants or children with over 90% feasibility. the relatively low frame rate of rt3de imaging compared with a high heart rate could potentially lead during the analysis to underestimating strain values. because of this limitation, strain rate and lv twisting and untwisting velocities can not be examined. our radial strain values are significantly lower than in other studies, this may be due to different algorithms and formulas, which are proprietary and hidden from end users, and probably because in previous studies, radial strain calculations were derived from the endocardium. it should be noted that the published literature has suggested that there is variability in lv volume, mass, and function among certain ethnic groups. the determination of reproducibility of 3d lv strain and torsion measurements is also important when applying this new technology to daily clinical practice. inter and intraobserver variability of 3d gls and gcs was very good, suggesting these parameters are ready for the clinical use. on the other hand, reproducibility of gts, grs twist, and torsion is still high, underscoring the need for more technical advancement before is ready for clinical practice. there is a learning curve for operators new to the technique to optimize imaging for the entire endocardial wall throughout the cardiac cycle and provide reproducible point selection for tracing. fundamental limitations of ultrasound, such as reverberation, shadowing, dropouts, chamber foreshortening, and movement artifacts, can limit feasibility. in children and newborns, most of these limitations are overcome because of the usual, very good image quality as confirmed by our findings and previous studies on infants or children with over 90% feasibility. the relatively low frame rate of rt3de imaging compared with a high heart rate could potentially lead during the analysis to underestimating strain values. because of this limitation, strain rate and lv twisting and untwisting velocities can not be examined. our radial strain values are significantly lower than in other studies, this may be due to different algorithms and formulas, which are proprietary and hidden from end users, and probably because in previous studies, radial strain calculations were derived from the endocardium. it should be noted that the published literature has suggested that there is variability in lv volume, mass, and function among certain ethnic groups. 3d gls analysis using the new 3d - ste is feasible and reproducible in the newborns. further investigation is warranted for potential clinical application of this new technology in a pediatric population. | aims : three - dimensional speckle - tracking echocardiography (3d - ste) is a promising new technique to evaluate left ventricular (lv) mechanics. the feasibility and normal values of lv strain using 3d - ste have recently been established in adults and children. unfortunately, no data are available in neonates. the aims of this study were to evaluate the feasibility and establish normal values of 3d lv volumes, ejection fraction (ef), and the 4 normal strains in healthy neonates.materials and methods : of 50 consecutive newborns who were delivered at our hospital or returned to their first newborn follow - up within the first 3 weeks of life, 38 babies underwent full echocardiographic evaluation, including the acquisition of at least 3 full volume data sets from the apical window, while naturally sleeping. data sets were analyzed offline. global lv longitudinal, circumferential, and radial strain, as well as 3d lv volumes and ef, were measured using 3d-ste.results:of the 50 newborns, 2 patients were excluded because of significant intra - cardiac shunts, and in another 10 subjects, parents did not give consent. at least one data set was adequate for analysis in all the remaining subjects. mean indexed lv diastolic, systolic volumes, and ef were 24.7 3.6 ml / m2, 9.2 1.3 ml / m2, and 63% 3.7%, respectively. normal global longitudinal, circumferential, radial, and tangential 4d strain were 20.9% 2.8%, 32.4% 3.1%, 44.3% 3.4%, and 39.7% 3.4%, respectively.conclusions:3d-ste is feasible in newborns without the needed for sedation. reference values of normal, regional, and global lv 4d strain and volumes were obtained. |
a. pereda, c. perfumo, and d. r. perez contributed to the concept and design of the study ; a. rimondi, m. angel, j. ye, t. sutton, m. dibrbora, v. olivera, and m. i. craig analyzed and interpreted the virological and molecular data ; j. cappuccio, m. quiroga, and m. machuca performed necropsies, analyzed, and interpreted the histopathological data ; a ferrero provided technical support ; a. pereda, c. perfumo, and d. r. perez revised and approved the final version of the manuscript. phylogenetic tree of hemagglutinin (1a), na (1b), and mp (1c) genes. unrooted trees were generated by the neighborjoining method in 11 the paup program. viruses characterized in this study are highlighted in bold, and swine influenza clusters are identified in colored boxes. h1n1pdm corresponds to h1n1 2009 pandemic virus, ch1n1a to classical swine h1n1, rh1n1b to triple reassortant swine h1n1, h1n2c to triple reassortant swine h1n2, huh1d to humanlike swine h1, hun1 or hun2 to humanlike swine n1 and n2, respectively, and swn1 to swine n1. | please cite this paper as : pereda. (2011) evidence of reassortment of pandemic h1n1 influenza virus in swine in argentina : are we facing the expansion of potential epicenters of influenza emergence ? influenza and other respiratory viruses 5(6), 409412.in this report, we describe the occurrence of two novel swine influenza viruses (sivs) in pigs in argentina. these viruses are the result of two independent reassortment events between the h1n1 pandemic influenza virus (h1n1pdm) and humanlike sivs, showing the constant evolution of influenza viruses at the human swine interface and the potential health risk of h1n1pdm as it appears to be maintained in the swine population. it must be noted that because of the lack of information regarding the circulation of sivs in south america, we can not discard the possibility that ancestors of the h1n1pdm or other sivs have been present in this part of the world. more importantly, these findings suggest an everexpanding geographic range of potential epicenters of influenza emergence with public health risks. |
the older population is increasing in sweden,1 as it is in the rest of europe.2 elderly people are and will be affected by multiple chronic conditions that is, multimorbidity3 and several mental disorders,4 even when dementia disorders are not taken into account.5 multimorbidity is associated with mental health problems among elderly people6 and is often treated with polypharmacy.7 it seems likely that future home health care will increase among older people8 and that a high number of community - dwelling seniors with multimorbidity will become a challenge in swedish elder care9 and among those who provide home help services and home health care.10 in the present study, seniors with multimorbidity are referred to as clients or seniors because they are not defined as patients in this context. mental health problems among seniors have become a major public health issue in sweden11,12 and in other high - income countries.13 the prevalence of mental disorders in seniors who live in communities is high, and may affect 30%50% of this population.14,15 multiple factors associated with psychogeriatric problems have been studied among older community patients,16 and for instance, depression has been related to being female, being unmarried, and to having somatic disease, functional disability, and a smaller social network.17,18 further, anxiety symptoms have been associated with the female sex, chronic disease, and functional limitations.18 seniors with multimorbidity are at risk of developing a generally low quality of life,19 psychological distress,3 and depressive disorders.20,21 nevertheless, seniors with multimorbidity may lack access to health - promoting dialogues that could facilitate mental health.22 the increase in the senior population implies changing needs, indicating that many health care systems will face challenges regarding caring for seniors with mental disorders.23 the organizations that provide care and services to the very old vary between countries, depending on the structure of each one s health care system and its finances.24 in sweden, the county councils are responsible for health care, and the municipalities handle all other care efforts, while the government bears responsibility for legislation.25 swedish seniors (> 65 years of age) who remain in their own homes may be provided support through the municipal home help service, according to the social service act (sfs 2001:453).26 this publicly funded support varies27 according to the client s needs and facilitates daily life in areas such as grocery purchases, food distribution, cleaning, and personal care. before a senior can receive such support from a municipality, an assessment of his or her individual requirements must be made by a public home care officer,28 also known as care managers (cms). the granted support is then provided in ordinary housing by nursing aides (nas) or enrolled nurses10,29,30 or trained nursing assistants working as home care assistants (hcas). these unlicensed personnel often work alone in the seniors homes, where they provide personal care, services, and housekeeping, and they often relieve district nurses (dns) in the administration of prescribed medication.31,32 despite increasing numbers of seniors receiving home help services, there are fewer and fewer staff members who can provide this granted support.33 this implies that the workload for hcas has increased, and they often face situations that demand more knowledge on a broader range of subjects.2 although most hcas have completed a high - school education (or equivalent),33 this staff may need increased knowledge in matters of mental illness among senior adults.34 in swedish primary care, general practitioners (gps) and dns have the primary responsibility for health promotion activities.35 dns work on health promotion and disease prevention among homebound patients, in addition to providing care to patients of all ages and medical diagnoses.36 the job of these registered nurses also involves prescribing certain medicines, technical materials, and equipment for patients with diabetes and incontinence. it is within a dn s prerogative to delegate the administration of medication to the hcas providing social services ; these nurses, further, can not fulfill their responsibilities without delegating to hcas the administration of chronically ill seniors prescription medication.37 therefore, dns and hcas are both present in a given senior s home, and the administration of medicine has become a matter that involves both health care and social service care.38 furthermore, caring for elderly persons with mental disorders presents a complex situation for the hcas,24 and despite a high incidence of mental disorders among older adults, direct care personnel may be insufficiently prepared and trained in mental illness and geriatrics.39,40 in addition, previous research has concluded that training of nas may promote quality of care for older adults with mental illness as well as improve personnel job satisfaction.41,42 the swedish national board of health and welfare states that older people with comprehensive and complex needs for care and support mostly remain in their homes, receiving that care and support from various providers and through intermittent hospitalizations.33 even though seniors with multimorbidity are at risk of developing mental health problems such as depression, the current situation among seniors with mental disorders has been very little studied. earlier studies have sought to increase the understanding of mental health care delivery in the primary care setting,4345 or to determine whether seniors with mental disorders are provided with adequate home help services,24 or to report staff members views of caring for patients experiencing late - life depression.46 hcas have rarely been the subject of investigation on their own. one reason for this may be that the idea that hcas may play an active part in health promotion work for their clients is a new one.47 as hcas often encounter homebound seniors, they may be pivotal in identifying those clients who are at risk of or are already experiencing mental health problems, and they are in a position to initiate actions that facilitate the early detection of mental health problems such as depression. even so, limited research has been found targeting hcas involvement and specific training in the area of mental health. in order to increase the quality of care provided to homebound seniors with multimorbidity, it is important to understand hcas experiences of the ways in which mental health problems may be detected and mental health may be promoted within this population. in order to make progress in this area, the aim of this study was to describe hcas perspectives on detecting mental health problems and promoting mental health among community - dwelling seniors with multimorbidity in order to meet this study s aim, we chose a descriptive and qualitative design in which focus group interviews were used in the process of data collection.48 qualitative research is designed to describe, interpret, and understand human experience and to describe the meaning that this experience has for the participant.49 a focus group interview gives the researcher an opportunity to understand several informants differing points of view.50 the data from the focus group interviews were analyzed using a content analysis technique inspired by graneheim and lundman.51 the study examined the municipal home help services in one urban district in sweden. encounters with the older persons varied from single and multiple daily visits to a single visit with one older person that lasted for almost an entire day. participants were selected according to a convenience sample technique,52 and the inclusion criterion was any experience caring for seniors with multimorbidity. the first author (g) contacted the medically responsible nurses in 13 municipalities, who were sent written information about the study and its purpose. four of those medically responsible nurses responded and referred us to the operations managers of five municipal home help services. the operations managers were then contacted by the first author to confirm their participation and to agree upon a place and time for an interview and on the nature of written content about the hcas participation in the study. ultimately, five focus group interviews took place, with four to six participants in each group. the participants were all swedish - speaking hcas from four different municipalities : 23 women and three men between 21 and 65 years of age. the hcas were all unlicensed assistive personnel : 21 informants had an upper secondary education as assistant nurses three were nas who had completed shorter education programs, and two lacked education in either health care or social services. their professional experience as hcas ranged between 6 months and 32 years. the hcas worked part or full time, and all had experience providing home help services to seniors with multimorbidity. all focus group interviews began with the question : how may mental health problems be detected among elderly people with multimorbidity ? the initial question was followed by : how may mental health be promoted among these seniors ? follow - up and probing questions were sometimes used in order to learn more about informants experiences in this area and to facilitate dialogue and interaction in each group. at the end of each interview, the interviewer repeated the two main questions and asked whether the hcas had anything to add. the interviews were transcribed verbatim and were subsequently reread several times to assess the material. the resulting text was examined using an inductive, manifest, and latent content analysis inspired by graneheim and lundman,51 as described in a recent study.22 according to sandelowski,53 important insights can emerge from the different ways in which different persons look at data in qualitative research. in this study, four authors in a research team read through the material separately to obtain a sense of the complete text. the research team then reread all interview transcripts to ensure consistency, keeping in mind the aim of the study and the content analysis. units of text (eg, words, sentences, paragraphs) that answered the two research questions were marked, condensed into a description of their manifest content, and coded. the components of each focus group interview were lumped together and designated as a single unit. the codes were compared based on differences and similarities and organized into three categories, each with two subcategories which constituted the manifest content. as a final step, the underlying meaning (ie, the latent content) of the text was formulated into an overarching theme that encompassed all the categories (table 1). the first author was responsible for all data collection and transcription, the main part of the analysis, and manuscript preparation. to increase the trustworthiness of the present study, all authors were involved in the entire research process and discussed the findings until consensus was reached. this study was approved by the regional ethical review board in stockholm, sweden (nos. both verbal and written information was provided about the study, and written consent was obtained from every participant. participants were informed that their participation was voluntary and could be terminated at any time. in order to meet this study s aim, we chose a descriptive and qualitative design in which focus group interviews were used in the process of data collection.48 qualitative research is designed to describe, interpret, and understand human experience and to describe the meaning that this experience has for the participant.49 a focus group interview gives the researcher an opportunity to understand several informants differing points of view.50 the data from the focus group interviews were analyzed using a content analysis technique inspired by graneheim and lundman.51 encounters with the older persons varied from single and multiple daily visits to a single visit with one older person that lasted for almost an entire day. participants were selected according to a convenience sample technique,52 and the inclusion criterion was any experience caring for seniors with multimorbidity. the first author (g) contacted the medically responsible nurses in 13 municipalities, who were sent written information about the study and its purpose. four of those medically responsible nurses responded and referred us to the operations managers of five municipal home help services. the operations managers were then contacted by the first author to confirm their participation and to agree upon a place and time for an interview and on the nature of written content about the hcas participation in the study. ultimately, five focus group interviews took place, with four to six participants in each group. the participants were all swedish - speaking hcas from four different municipalities : 23 women and three men between 21 and 65 years of age. the hcas were all unlicensed assistive personnel : 21 informants had an upper secondary education as assistant nurses three were nas who had completed shorter education programs, and two lacked education in either health care or social services. their professional experience as hcas ranged between 6 months and 32 years. the hcas worked part or full time, and all had experience providing home help services to seniors with multimorbidity. all focus group interviews began with the question : how may mental health problems be detected among elderly people with multimorbidity ? the initial question was followed by : how may mental health be promoted among these seniors ? follow - up and probing questions were sometimes used in order to learn more about informants experiences in this area and to facilitate dialogue and interaction in each group. at the end of each interview, the interviewer repeated the two main questions and asked whether the hcas had anything to add. the interviews were transcribed verbatim and were subsequently reread several times to assess the material. the resulting text was examined using an inductive, manifest, and latent content analysis inspired by graneheim and lundman,51 as described in a recent study.22 according to sandelowski,53 important insights can emerge from the different ways in which different persons look at data in qualitative research. in this study, four authors in a research team read through the material separately to obtain a sense of the complete text. the research team then reread all interview transcripts to ensure consistency, keeping in mind the aim of the study and the content analysis. units of text (eg, words, sentences, paragraphs) that answered the two research questions were marked, condensed into a description of their manifest content, and coded. the components of each focus group interview were lumped together and designated as a single unit. the codes were compared based on differences and similarities and organized into three categories, each with two subcategories which constituted the manifest content. as a final step, the underlying meaning (ie, the latent content) of the text was formulated into an overarching theme that encompassed all the categories (table 1). the first author was responsible for all data collection and transcription, the main part of the analysis, and manuscript preparation. to increase the trustworthiness of the present study, all authors were involved in the entire research process and discussed the findings until consensus was reached. this study was approved by the regional ethical review board in stockholm, sweden (nos. both verbal and written information was provided about the study, and written consent was obtained from every participant. participants were informed that their participation was voluntary and could be terminated at any time. most hcas stated that they were experienced in caring for clients with mental health problems such as anxiety, depression, sleep problems, and high alcohol consumption. the participants engaged in detailed discussions according to their experiences both negative and positive. analysis of the data revealed an overarching theme that described how mental health problems may be detected and how to improve mental health : continuity provided by an accessible and competent team may increase social and physical activity. findings are presented collectively in the three categories observation, collaboration, and social support in order to describe ways of detecting mental health problems as well as factors that may promote mental health (table 1) : activities in these categories emerged as ways of detecting mental health problems and promoting mental health. several hcas articulated that good personal knowledge of the client was crucial to detecting mental health problems. most participants explicitly indicated that continuity and continuous home visits were essential to noticing any changes in behavior or expressed emotions. observing and evaluating a client s behavior and emotions constituted important sources of information in determining whether the hcas should act to promote mental health. knowledge about a senior s medical history was especially pivotal if hcas were to detect mental health problems such as anxiety and depression. several participants argued that mental health problems among seniors were to be expected, and that depression could be explained by various well - known causes, such as multiple chronic conditions, physical impairments, dementia, stroke, pharmacological treatment, constipation, social loneliness, high alcohol consumption, and being in need of care and support. the challenge was to evaluate whether the seniors became depressed because of their increased alcohol consumption or started to abuse alcohol after signs of depression and anxiety had already appeared. one participant described poor mental health as synonymous with depression, asserting that such illness could be explained in terms of interacting causes : the more diseases and the more medications [] the greater the risk that you end up there with depression [] losing zest for life [] having difficulty sleeping [] and their appetite disappears []. the psyche suffers most because one suffers so many obstacles [] if you become multi - morbid and experience dizziness and take a lot of medicines that also have a lot of side effects, it affects the psyche very deeply. you may even take medications that make you constipated, and you ca nt eat as you normally do. it s also hard to get around, so they do nt do things they used to do before [] because of all these barriers. the more diseases and the more medications [] the greater the risk that you end up there with depression [] losing zest for life [] having difficulty sleeping [] and their appetite disappears []. the psyche suffers most because one suffers so many obstacles [] if you become multi - morbid and experience dizziness and take a lot of medicines that also have a lot of side effects, it affects the psyche very deeply. you may even take medications that make you constipated, and you ca nt eat as you normally do. it s also hard to get around, so they do nt do things they used to do before [] because of all these barriers. an ocular observation of the client s personal appearance and home environment was performed in order to evaluate his or her behavior and to identify changed emotions that might reflect the development of poor mental health. observing behavior involved identifying poor appetite, malnutrition, physical and social inactivity, and low inter est in personal hygiene or in the tidiness of their home. in these observations, several hcas reported that they might find unopened boxes of food that hcas had left on an earlier visit and empty boxes and bottles of wine ; these were interpreted as signs of worsening mental health. a senior s consumption of alcohol might also be detected by hcas during an ambulatory home visit responding to a senior who had fallen and was unable to rise from the floor. some participants indicated that there were other not - so - obvious signs of mental health problems. for example, a client might seem depressed or stressed during a personal meeting with a well - known hca : then there are signs that you see when you talk to them. they become easily stressed and feel unbalanced when [] someone comes to them [] and there you see at once [] that there s something wrong. then there are signs that you see when you talk to them. they become easily stressed and feel unbalanced when [] someone comes to them [] and there you see at once [] that there s something wrong. other hcas described additional signs of worsening mental health : a senior may seem especially tired, have difficulty sleeping during the night, or want to stay in bed during the day. further, several clients had themselves perceived that they had a decreased sparkle and interest in life and told hcas so ; they might even state that they no longer wished to live. several hcas identified as signs of declining mental health a client s increased suspicion or confusion, an apparently weakened short - term memory, and indications that they saw things or people that were not present. these clients seemed to encounter particular difficulty when interacting with new people, according to the participants : we have many women here who may [] find it difficult to meet people [] in the beginning. but as soon as they begin to get to know us, it will be a little better [] it is also a sign that there is something mentally [amiss ]. we have many women here who may [] find it difficult to meet people [] in the beginning. but as soon as they begin to get to know us, it will be a little better [] it is also a sign that there is something mentally [amiss ]. observing a client s emotions entailed evaluating identified or expressed emotions that were both expected and unexpected among seniors with multimorbidity and seniors with mental health problems. the emotions the hcas reported observing among the seniors in focus were a general sadness, sadness combined with loneliness, fear of loneliness, insecurity, and verbal aggressiveness among those with cognitive impairments. several hcas argued that these emotions were difficult to evaluate ; anxiety was especially difficult to separate from symptoms that the hcas could also relate to the seniors chronic conditions : and that anxiety can, of course, also prove that there is some problem with the heart, or that it hurts []. it might not really [] be the heart that hurts, but instead it hurts in the soul [] this can show up as physical [] symptoms. and that anxiety can, of course, also prove that there is some problem with the heart, or that it hurts []. it might not really [] be the heart that hurts, but instead it hurts in the soul [] this can show up as physical [] symptoms. even though a client s emotions and state of mind seemed quite easy to evaluate, several hcas stated that they had experienced difficulty dealing with a senior s expression of sadness or the wish that his or her life would end. there was generally a lack of time to initiate a dialogue about these seniors mental health conditions, since the time allotted for each hca s home visit was calculated based on the time that each granted task should take. the hcas reported not knowing what to do or how to behave if a client became more emotional and cried, for instance, when a well - known hca arrived in his or her home environment. home visits are often planned to provide a specific form of support ; this problem is related to the cm s decision making and to the priority of practical tasks : so we feel the pressure of time because we cost money [] so then you have to almost just put on the vacuum cleaner [] even though this is someone who is crying [] because the task is to vacuum. so we feel the pressure of time because we cost money [] so then you have to almost just put on the vacuum cleaner [] even though this is someone who is crying [] because the task is to vacuum. several hcas viewed collaboration with colleagues and other health care professionals as important when they themselves lacked knowledge about how to detect and handle mental health problems or how to improve mental health. such collaboration involved the transmission of information about a client s mental health status or about how to promote mental health. in fact, hcas seemed dependent on effective cooperation with other actors in these matters. the hcas reported that it was quite difficult for them to detect potential problems or to interact with seniors with various mental health problems : they depended on other actors to transmit information about changed behavior to inform them, for instance, if a senior had become more emotional. study participants seemed especially dependent on information from the responsible gp or dn about a senior s chronic conditions and current mental health status. even though dns often seemed to transmit information about a senior s mental health problems, several hcas lacked guidance in how to respond to a client with mental health problems or in providing support for those with known anxiety or diagnosed depression : it s really important that we get the right information, i think [] from a doctor [] before we go to them []. we must know exactly what the disease is [] and how to treat that person. it s really important that we get the right information, i think [] from a doctor [] before we go to them []. we must know exactly what the disease is [] and how to treat that person. participants also seemed dependent on their collaboration with the responsible cm, with whom they apparently communicated only rarely, although hcas wanted to transmit information about a client s changed behavior, alcohol consumption, and their own evaluation of the granted support. if a senior could not transmit information about his or her own mental health status, the hcas usually were contacted by relatives or neighbors, who might report changed behavior and the impression that the client s mental health had declined. a few hcas mentioned that swedish legislation about transmitting information could pose a barrier when hcas wish to inform health care professionals or talk to relatives or neighbors about a client : so even if you want to sound the alarm [] and you want to try to circumvent potential problems, you must always pursue each person s best interests, as well []. for example, i might decide that now i have to [] break a little confidentiality here because it serves the person s best interests. so even if you want to sound the alarm [] and you want to try to circumvent potential problems, you must always pursue each person s best interests, as well []. for example, i might decide that now i have to [] break a little confidentiality here because it serves the person s best interests. although hcas viewed their collaboration with colleagues as essential, there seemed to be no planned time for mutual information sharing about clients receiving granted support. the hcas especially highlighted the importance of a forum, currently nonexistent, through which to transmit information about a senior s state of mind and to share suggestions about how colleagues could provide support during the next home visit : we ought to all just sit down and talk a little bit about some of our joint clients. and if i knew you were going over there in the morning, i could tell you, she was sad today [] can you help her by going out with her [] or making food for her [] so she wo nt need to cry again ? we ought to all just sit down and talk a little bit about some of our joint clients. and if i knew you were going over there in the morning, i could tell you, she was sad today [] can you help her by going out with her [] or making food for her [] so she wo nt need to cry again ? cooperation was another aspect of collaboration, since most participants reported that they did not have practical support or contact with other actors close to the senior in focus. the improvement they wanted in their cooperation with health care professionals such as dns was crucial ; hcas seemed to want an agreement that would clarify each actor s responsibility in matters of mental health. closer and more pragmatic cooperation with dns constituted one way to improve hcas knowledge of mental health, according to most participants. further, they seemed to think that working more closely with dns would provide opportunities for questions related to a senior s mental health and for additional intermittent supervision regarding how multimorbidity may affect mental health and the best ways to deal with it : supervision in how to handle [] our mentally ill [] people, clients, patients with multiple diagnoses []. supervision in how to handle [] our mentally ill [] people, clients, patients with multiple diagnoses []. that would be great ! several participants stated that they had no access to a forum for cooperation with their colleagues, even though they seemed to consult with them by phone between home visits and during lunch breaks. other participants gave examples of intermittent and planned forums for learning about their colleagues experiences in the field : then one can get some advice too [] based on other people s experiences []. then we can try this, maybe, and [] see if it works [] so maybe we can come up with a solution together. then one can get some advice too [] based on other people s experiences []. then we can try this, maybe, and [] see if it works [] so maybe we can come up with a solution together. some participating hcas were unable to name specific actors whom they could contact when they needed support or wanted to discuss a client they were concerned about. the participants considered cooperation with mental health care professionals especially important if they were to increase their own knowledge about mental health issues : well, then it must surely be a psych team that you have access to []. this would provide more knowledge []. so we need to be connected to a psychiatric team that could sort of supervise us. well, then it must surely be a psych team that you have access to []. this would provide more knowledge []. so we need to be connected to a psychiatric team that could sort of supervise us. according to several participants, seniors with multimorbidity often live alone and need different kinds of social support, including both instrumental and emotional support. most hcas cited living alone and feeling lonely as reasons why seniors might develop mental health problems like depression. accordingly, some hcas understood that their own presence and work made a difference because they could break up periods of loneliness simply by visiting a client and providing support with practical everyday tasks. other participants stated that it was difficult to provide support that could improve mental health and that they needed greater knowledge in this area, as well as more time for dialogue with clients, for conversations that might help improve mental health : i mean, okay, if they the clients now live at home [] and are healthy and have no mental problems [] then we need to have more time with them so that we can help them []. i mean, okay, if they the clients now live at home [] and are healthy and have no mental problems [] then we need to have more time with them so that we can help them []. instrumental support entails practical actions that promote mental health, such as sitting down to chat over a cup of coffee. it seemed especially important to many hcas that homebound seniors have an opportunity to leave their home environment in order to increase their social contacts or physical activity : thus, most people are alone [] and it s loneliness that affects mental health, actually ! they need this social part. through this social interaction [] we can actually make sure that they get to walk in the yard [] to actually talk [] or we can drink coffee with them. thus, most people are alone [] and it s loneliness that affects mental health, actually ! we can actually make sure that they get to walk in the yard [] to actually talk [] or we can drink coffee with them. another component of instrumental support was body massage ; still another was helping a client to contact a mental health care provider when a senior expressed suicidal thoughts or to contact a physician or nurse in primary health care to evaluate a senior s pharmacological treatment. some hcas argued that polypharmacy was itself a reason why a client s mental health might be poor : it s rare that anyone looks at the patient and asks, do you need all these drugs ? [] or asks, can we stop some medications ? []. it could also be, among those who do nt feel so good or eat so well [] that they do nt feel good mentally just [] because there is so much happening in the body. it s rare that anyone looks at the patient and asks, do you need all these drugs ? [] or asks, can we stop some medications ? []. it could also be, among those who do nt feel so good or eat so well [] that they do nt feel good mentally just [] because there is so much happening in the body. contacting a library to request a book and reading to the senior from a newspaper were other actions that hcas reported could help avert loneliness and thus promote mental health. the hcas repeatedly mentioned that they had no time for such social support because clients were rarely granted support specifically meant to disrupt loneliness or promote mental health. even so, few hcas said that they could contact the responsible chief or cm to highlight the senior s need for social support or to suggest that the granted support should be evaluated or that the senior needed other assistance aimed at promoting mental health. being granted time for company during meals or for physical activities such as walks and social daycare was a form of (rarely granted) support that the hcas said could improve mental health and prevent depression. one participant indicated that it was often difficult (though not impossible) to convince a client of the importance of social activities : they need to have confidence in the person [hca ] [] so we can help get them on the right path. and it s the same thing [] the client goes to social daycare now : i tried to convince her that it would be good for her, and it s worked out great. they need to have confidence in the person [hca ] [] so we can help get them on the right path. and it s the same thing [] the client goes to social daycare now : i tried to convince her that it would be good for her, and it s worked out great. emotional support is another kind of social support that most participants highlighted as a means of promoting mental health. several hcas repeatedly stated that it was crucial to cultivate a trustful relationship with the seniors and that this required time, in combination with interpersonal skills such as perception and the ability to listen actively and ask questions about what might help the senior feel better more generally. hcas indicated that it was especially important to have the goal of decreasing clients feelings of loneliness and increasing their feelings of security about home help services and their overall purpose : the client can know that they re not alone [] there is someone who cares about them [] someone who will help and whom they can turn to. the client can know that they re not alone [] there is someone who cares about them [] someone who will help and whom they can turn to. several participants repeatedly stated that dialogue was the most important way to improve mental health : the seniors in focus needed professional partners in dialogue, such as psychologists and social workers. some hcas highlighted their own role as dialogue partners as a means of both detecting mental health problems and promoting mental health : so after you start talking to them [] when they feel a little bit calmer [] you can see whether there s a question mark, something amiss that has to do with mental health. so after you start talking to them [] when they feel a little bit calmer [] you can see whether there s a question mark, something amiss that has to do with mental health. the dialogues hcas engaged in with their clients aimed to identify a client s state of mind so that the hca could establish trust and provide hope for the future. even though the hcas seem to lack time to sit down for a conversation that could promote mental health, some reported that such dialogues could take place while hcas were preparing food, cleaning, or walking outside with the client. the content of some such dialogues seemed to center on joyful matters in the seniors lives, aiming to distract them from anxiety or to decrease any sadness they had expressed ; others focused on helping the senior verbalize current emotions. with this in mind, it seemed crucial that an hca talk in a relaxed way and that dialogue involve active listening and probing questions that show personal interest in the senior s expression of matters that troubled them. such dialogue could also lead to the detection of mental health issues : and then other things may emerge, too [] that they are keeping within themselves. because when you start talking and start a conversation [] one thing leads to another. yes, it could be [] anything and that may be annoying, as they carry on [] that s hard. but then maybe it all comes out : they may need to talk, speak about themselves [] and feel that there is somebody who listens. and then other things may emerge, too [] that they are keeping within themselves. because when you start talking and start a conversation [] one thing leads to another. yes, it could be [] anything and that may be annoying, as they carry on [] that s hard. but then maybe it all comes out : they may need to talk, speak about themselves [] and feel that there is somebody who listens. several hcas articulated that good personal knowledge of the client was crucial to detecting mental health problems. most participants explicitly indicated that continuity and continuous home visits were essential to noticing any changes in behavior or expressed emotions. observing and evaluating a client s behavior and emotions constituted important sources of information in determining whether the hcas should act to promote mental health. knowledge about a senior s medical history was especially pivotal if hcas were to detect mental health problems such as anxiety and depression. several participants argued that mental health problems among seniors were to be expected, and that depression could be explained by various well - known causes, such as multiple chronic conditions, physical impairments, dementia, stroke, pharmacological treatment, constipation, social loneliness, high alcohol consumption, and being in need of care and support. the challenge was to evaluate whether the seniors became depressed because of their increased alcohol consumption or started to abuse alcohol after signs of depression and anxiety had already appeared. one participant described poor mental health as synonymous with depression, asserting that such illness could be explained in terms of interacting causes : the more diseases and the more medications [] the greater the risk that you end up there with depression [] losing zest for life [] having difficulty sleeping [] and their appetite disappears []. the psyche suffers most because one suffers so many obstacles [] if you become multi - morbid and experience dizziness and take a lot of medicines that also have a lot of side effects, it affects the psyche very deeply. you may even take medications that make you constipated, and you ca nt eat as you normally do. it s also hard to get around, so they do nt do things they used to do before [] because of all these barriers. the more diseases and the more medications [] the greater the risk that you end up there with depression [] losing zest for life [] having difficulty sleeping [] and their appetite disappears []. the psyche suffers most because one suffers so many obstacles [] if you become multi - morbid and experience dizziness and take a lot of medicines that also have a lot of side effects, it affects the psyche very deeply. you may even take medications that make you constipated, and you ca nt eat as you normally do. it s also hard to get around, so they do nt do things they used to do before [] because of all these barriers. an ocular observation of the client s personal appearance and home environment was performed in order to evaluate his or her behavior and to identify changed emotions that might reflect the development of poor mental health. observing behavior involved identifying poor appetite, malnutrition, physical and social inactivity, and low inter est in personal hygiene or in the tidiness of their home. in these observations, several hcas reported that they might find unopened boxes of food that hcas had left on an earlier visit and empty boxes and bottles of wine ; these were interpreted as signs of worsening mental health. a senior s consumption of alcohol might also be detected by hcas during an ambulatory home visit responding to a senior who had fallen and was unable to rise from the floor. some participants indicated that there were other not - so - obvious signs of mental health problems. for example, a client might seem depressed or stressed during a personal meeting with a well - known hca : then there are signs that you see when you talk to them. they become easily stressed and feel unbalanced when [] someone comes to them [] and there you see at once [] that there s something wrong. then there are signs that you see when you talk to them. they become easily stressed and feel unbalanced when [] someone comes to them [] and there you see at once [] that there s something wrong. other hcas described additional signs of worsening mental health : a senior may seem especially tired, have difficulty sleeping during the night, or want to stay in bed during the day. further, several clients had themselves perceived that they had a decreased sparkle and interest in life and told hcas so ; they might even state that they no longer wished to live. several hcas identified as signs of declining mental health a client s increased suspicion or confusion, an apparently weakened short - term memory, and indications that they saw things or people that were not present. these clients seemed to encounter particular difficulty when interacting with new people, according to the participants : we have many women here who may [] find it difficult to meet people [] in the beginning. but as soon as they begin to get to know us, it will be a little better [] it is also a sign that there is something mentally [amiss ]. we have many women here who may [] find it difficult to meet people [] in the beginning. but as soon as they begin to get to know us, it will be a little better [] it is also a sign that there is something mentally [amiss ]. observing a client s emotions entailed evaluating identified or expressed emotions that were both expected and unexpected among seniors with multimorbidity and seniors with mental health problems. the emotions the hcas reported observing among the seniors in focus were a general sadness, sadness combined with loneliness, fear of loneliness, insecurity, and verbal aggressiveness among those with cognitive impairments. several hcas argued that these emotions were difficult to evaluate ; anxiety was especially difficult to separate from symptoms that the hcas could also relate to the seniors chronic conditions : and that anxiety can, of course, also prove that there is some problem with the heart, or that it hurts []. it might not really [] be the heart that hurts, but instead it hurts in the soul [] this can show up as physical [] symptoms. and that anxiety can, of course, also prove that there is some problem with the heart, or that it hurts []. it might not really [] be the heart that hurts, but instead it hurts in the soul [] this can show up as physical [] symptoms. even though a client s emotions and state of mind seemed quite easy to evaluate, several hcas stated that they had experienced difficulty dealing with a senior s expression of sadness or the wish that his or her life would end. there was generally a lack of time to initiate a dialogue about these seniors mental health conditions, since the time allotted for each hca s home visit was calculated based on the time that each granted task should take. the hcas reported not knowing what to do or how to behave if a client became more emotional and cried, for instance, when a well - known hca arrived in his or her home environment. home visits are often planned to provide a specific form of support ; this problem is related to the cm s decision making and to the priority of practical tasks : so we feel the pressure of time because we cost money [] so then you have to almost just put on the vacuum cleaner [] even though this is someone who is crying [] because the task is to vacuum. so we feel the pressure of time because we cost money [] so then you have to almost just put on the vacuum cleaner [] even though this is someone who is crying [] because the task is to vacuum. several hcas viewed collaboration with colleagues and other health care professionals as important when they themselves lacked knowledge about how to detect and handle mental health problems or how to improve mental health. such collaboration involved the transmission of information about a client s mental health status or about how to promote mental health. in fact, hcas seemed dependent on effective cooperation with other actors in these matters. the hcas reported that it was quite difficult for them to detect potential problems or to interact with seniors with various mental health problems : they depended on other actors to transmit information about changed behavior to inform them, for instance, if a senior had become more emotional. study participants seemed especially dependent on information from the responsible gp or dn about a senior s chronic conditions and current mental health status. even though dns often seemed to transmit information about a senior s mental health problems, several hcas lacked guidance in how to respond to a client with mental health problems or in providing support for those with known anxiety or diagnosed depression : it s really important that we get the right information, i think [] from a doctor [] before we go to them []. we must know exactly what the disease is [] and how to treat that person. it s really important that we get the right information, i think [] from a doctor [] before we go to them []. we must know exactly what the disease is [] and how to treat that person. participants also seemed dependent on their collaboration with the responsible cm, with whom they apparently communicated only rarely, although hcas wanted to transmit information about a client s changed behavior, alcohol consumption, and their own evaluation of the granted support. if a senior could not transmit information about his or her own mental health status, the hcas usually were contacted by relatives or neighbors, who might report changed behavior and the impression that the client s mental health had declined. a few hcas mentioned that swedish legislation about transmitting information could pose a barrier when hcas wish to inform health care professionals or talk to relatives or neighbors about a client : so even if you want to sound the alarm [] and you want to try to circumvent potential problems, you must always pursue each person s best interests, as well []. for example, i might decide that now i have to [] break a little confidentiality here because it serves the person s best interests. so even if you want to sound the alarm [] and you want to try to circumvent potential problems, you must always pursue each person s best interests, as well []. for example, i might decide that now i have to [] break a little confidentiality here because it serves the person s best interests. although hcas viewed their collaboration with colleagues as essential, there seemed to be no planned time for mutual information sharing about clients receiving granted support. the hcas especially highlighted the importance of a forum, currently nonexistent, through which to transmit information about a senior s state of mind and to share suggestions about how colleagues could provide support during the next home visit : we ought to all just sit down and talk a little bit about some of our joint clients. and if i knew you were going over there in the morning, i could tell you, she was sad today [] can you help her by going out with her [] or making food for her [] so she wo nt need to cry again ? we ought to all just sit down and talk a little bit about some of our joint clients. and if i knew you were going over there in the morning, i could tell you, she was sad today [] can you help her by going out with her [] or making food for her [] so she wo nt need to cry again ? cooperation was another aspect of collaboration, since most participants reported that they did not have practical support or contact with other actors close to the senior in focus. the improvement they wanted in their cooperation with health care professionals such as dns was crucial ; hcas seemed to want an agreement that would clarify each actor s responsibility in matters of mental health. closer and more pragmatic cooperation with dns constituted one way to improve hcas knowledge of mental health, according to most participants. further, they seemed to think that working more closely with dns would provide opportunities for questions related to a senior s mental health and for additional intermittent supervision regarding how multimorbidity may affect mental health and the best ways to deal with it : supervision in how to handle [] our mentally ill [] people, clients, patients with multiple diagnoses []. supervision in how to handle [] our mentally ill [] people, clients, patients with multiple diagnoses []. that would be great ! several participants stated that they had no access to a forum for cooperation with their colleagues, even though they seemed to consult with them by phone between home visits and during lunch breaks. other participants gave examples of intermittent and planned forums for learning about their colleagues experiences in the field : then one can get some advice too [] based on other people s experiences []. then we can try this, maybe, and [] see if it works [] so maybe we can come up with a solution together. then one can get some advice too [] based on other people s experiences []. then we can try this, maybe, and [] see if it works [] so maybe we can come up with a solution together. some participating hcas were unable to name specific actors whom they could contact when they needed support or wanted to discuss a client they were concerned about. the participants considered cooperation with mental health care professionals especially important if they were to increase their own knowledge about mental health issues : well, then it must surely be a psych team that you have access to []. this would provide more knowledge []. so we need to be connected to a psychiatric team that could sort of supervise us. well, then it must surely be a psych team that you have access to []. this would provide more knowledge []. so we need to be connected to a psychiatric team that could sort of supervise us. according to several participants, seniors with multimorbidity often live alone and need different kinds of social support, including both instrumental and emotional support. most hcas cited living alone and feeling lonely as reasons why seniors might develop mental health problems like depression. accordingly, some hcas understood that their own presence and work made a difference because they could break up periods of loneliness simply by visiting a client and providing support with practical everyday tasks. other participants stated that it was difficult to provide support that could improve mental health and that they needed greater knowledge in this area, as well as more time for dialogue with clients, for conversations that might help improve mental health : i mean, okay, if they the clients now live at home [] and are healthy and have no mental problems [] then we need to have more time with them so that we can help them []. i mean, okay, if they the clients now live at home [] and are healthy and have no mental problems [] then we need to have more time with them so that we can help them []. instrumental support entails practical actions that promote mental health, such as sitting down to chat over a cup of coffee. it seemed especially important to many hcas that homebound seniors have an opportunity to leave their home environment in order to increase their social contacts or physical activity : thus, most people are alone [] and it s loneliness that affects mental health, actually ! they need this social part. through this social interaction [] we can actually make sure that they get to walk in the yard [] to actually talk [] or we can drink coffee with them. thus, most people are alone [] and it s loneliness that affects mental health, actually ! we can actually make sure that they get to walk in the yard [] to actually talk [] or we can drink coffee with them. another component of instrumental support was body massage ; still another was helping a client to contact a mental health care provider when a senior expressed suicidal thoughts or to contact a physician or nurse in primary health care to evaluate a senior s pharmacological treatment. some hcas argued that polypharmacy was itself a reason why a client s mental health might be poor : it s rare that anyone looks at the patient and asks, do you need all these drugs ? [] or asks, can we stop some medications ? []. it could also be, among those who do nt feel so good or eat so well [] that they do nt feel good mentally just [] because there is so much happening in the body. it s rare that anyone looks at the patient and asks, do you need all these drugs ? [] or asks, can we stop some medications ? []. it could also be, among those who do nt feel so good or eat so well [] that they do nt feel good mentally just [] because there is so much happening in the body. contacting a library to request a book and reading to the senior from a newspaper were other actions that hcas reported could help avert loneliness and thus promote mental health. the hcas repeatedly mentioned that they had no time for such social support because clients were rarely granted support specifically meant to disrupt loneliness or promote mental health. even so, few hcas said that they could contact the responsible chief or cm to highlight the senior s need for social support or to suggest that the granted support should be evaluated or that the senior needed other assistance aimed at promoting mental health. being granted time for company during meals or for physical activities such as walks and social daycare was a form of (rarely granted) support that the hcas said could improve mental health and prevent depression. one participant indicated that it was often difficult (though not impossible) to convince a client of the importance of social activities : they need to have confidence in the person [hca ] [] so we can help get them on the right path. and it s the same thing [] the client goes to social daycare now : i tried to convince her that it would be good for her, and it s worked out great. they need to have confidence in the person [hca ] [] so we can help get them on the right path. and it s the same thing [] the client goes to social daycare now : i tried to convince her that it would be good for her, and it s worked out great. emotional support is another kind of social support that most participants highlighted as a means of promoting mental health. several hcas repeatedly stated that it was crucial to cultivate a trustful relationship with the seniors and that this required time, in combination with interpersonal skills such as perception and the ability to listen actively and ask questions about what might help the senior feel better more generally. hcas indicated that it was especially important to have the goal of decreasing clients feelings of loneliness and increasing their feelings of security about home help services and their overall purpose : the client can know that they re not alone [] there is someone who cares about them [] someone who will help and whom they can turn to. the client can know that they re not alone [] there is someone who cares about them [] someone who will help and whom they can turn to. several participants repeatedly stated that dialogue was the most important way to improve mental health : the seniors in focus needed professional partners in dialogue, such as psychologists and social workers. some hcas highlighted their own role as dialogue partners as a means of both detecting mental health problems and promoting mental health : so after you start talking to them [] when they feel a little bit calmer [] you can see whether there s a question mark, something amiss that has to do with mental health. so after you start talking to them [] when they feel a little bit calmer [] you can see whether there s a question mark, something amiss that has to do with mental health. the dialogues hcas engaged in with their clients aimed to identify a client s state of mind so that the hca could establish trust and provide hope for the future. even though the hcas seem to lack time to sit down for a conversation that could promote mental health, some reported that such dialogues could take place while hcas were preparing food, cleaning, or walking outside with the client. the content of some such dialogues seemed to center on joyful matters in the seniors lives, aiming to distract them from anxiety or to decrease any sadness they had expressed ; others focused on helping the senior verbalize current emotions. with this in mind, it seemed crucial that an hca talk in a relaxed way and that dialogue involve active listening and probing questions that show personal interest in the senior s expression of matters that troubled them. such dialogue could also lead to the detection of mental health issues : and then other things may emerge, too [] that they are keeping within themselves. because when you start talking and start a conversation [] one thing leads to another. yes, it could be [] anything and that may be annoying, as they carry on [] that s hard. but then maybe it all comes out : they may need to talk, speak about themselves [] and feel that there is somebody who listens. and then other things may emerge, too [] that they are keeping within themselves. because when you start talking and start a conversation [] one thing leads to another. yes, it could be [] anything and that may be annoying, as they carry on [] that s hard. but then maybe it all comes out : they may need to talk, speak about themselves [] and feel that there is somebody who listens. this study illuminated hcas experiences caring for seniors with multimorbidity and these care providers perspectives on detecting mental health problems and promoting mental health among these seniors. participants related both positive and negative experiences of addressing mental health among homebound seniors, and their discussions indicate that they were engaged in these matters. the primary results show that the participants had encountered mental health problems such as depression, anxiety, and difficulty sleeping among seniors with multiple chronic conditions. several participants seemed to think that such mental health problems were to be expected among most of their clients, especially those who were living alone with somatic diseases and polypharmacy. this perception may stem from participants experiences caring for elderly people or from stereotyping beliefs about aging and illnesses.54 the hcas mentioned causes, or risk factors, that related first and foremost to multiple chronic conditions, feelings of loneliness, and social isolation. concerning mental health and aging, a previous study reported that community - dwelling seniors with multimorbidity stated that mental health problems were considered a normal part of aging.55 such a belief about the correlation between aging and mental health problems can not be considered acceptable, for negative stereotypes of aging may translate into age discrimination and further social exclusion and hence into worsening mental and physical health.54 on the other hand, people s positive attitudes toward and affirmative beliefs about senior adults and aging appear to bolster seniors mental health.56 above all, participants indicated that good personal knowledge of a senior (and his or her medical history) was the most important factor in hcas ability to observe changes in client behavior, including altered emotions that could be warning signs of worsening mental health. such signs included negative feelings, alcohol abuse, not eating, and not maintaining good personal hygiene. it was especially important to participants that they have the ability to observe the client s home environment in order to evaluate the client s level of interest in keeping it tidy. continuity among hcas home visits was crucial if an hca was to get to know a person well and to fulfill the important prerequisite of evaluating and comparing a senior s condition from one visit to the next. the hcas especially highlighted that continuity of care was essential to detecting mental health problems and to addressing seniors condition if they stated that they no longer had a desire to live. these findings should be compared with those of a previous study showing that continuity is important not only in detecting mental health issues but continuity of care, knowledge, and staff competence are also crucial to client satisfaction with the care older people receive in their homes.57 the hcas were unsure whether the seniors in focus used alcohol because they were depressed or became depressed because of their alcohol consumption. hcas apparently needed to interpret and explain the cause - and - effect relation in situations of alcohol abuse or self - medicating among this group of seniors. these discussions align with a previous study s finding of a direct relationship between alcohol abuse or dependence and major depression.58 further, problems with alcohol may lead to an increased risk of major depression, which can be explained according to a self - medication model and by the fact that major depression may lead to an increased risk of alcohol abuse or dependency.58 it is therefore important that hcas be provided with support and supervision in managing clients with alcohol abuse or dependency, since these clients may suffer from undetected major depression. it should also be noted that many seniors with multimorbidity are prescribed several drugs that may interact with alcohol, so recognized alcohol abuse should therefore be considered by the responsible physician and dn in home health care. the evaluation of medical treatment seems especially important among gps, since these physicians are the main prescribers of psychotropic drugs to older people.59 it appears that the hcas compared various signs of cognitive impairments and symptoms of dementia, depression, or psychosis. hcas further reported that it was difficult to separate cognitive impairments from the physical symptoms of some somatic diseases. previous studies have shown similar findings : numerous factors relate to psychogeriatric symptomatology in elderly people with physical illnesses,16 and mental health in old age is particularly related to several psychological, sociodemographic, and somatic factors, which may explain the reason for the low rate of detection of mental problems.60 even though it is not hcas primary responsibility to detect mental health problems among their clients, it is essential that these staffers day - to - day observations of current symptoms, changed behavior, and suicidal thoughts be taken into account when responsible health care professionals evaluate prescribed medication ; doing so will provide a broader perspective that will aid the responsible physician in diagnosing dementia or mental health problems such as depression. however, continuous quality improvement in home care can take place only if all the actors involved learn from each other. hcas personal knowledge about their clients is vital because hcas are the care providers closest to the action taking place in a client s home.47 according to participants in this study, social support constitutes an important mental health - promoting initiative because social isolation and feelings of loneliness are well - known causes of or risk factors for mental health problems among these clients, who mostly live alone. in the data analysis, instrumental support related to practical tasks that the client once performed but was now unable to without assistance (eg, the administration of medicine). the hcas suggestions regarding instrumental support indicated that they thought it was crucial to break clients social isolation and to increase their physical activity, mainly through oversight, home visits, dialogue, social daycare, and walking outdoors. however, some participants reported that it was sometimes difficult to motivate clients to participate in such social activities. according to the participants, clients with mental health problems were rarely granted assistance that included social support with the goal of alleviating social isolation or of increasing physical activity. these statements align with the findings of previous studies that have highlighted the importance of existential belonging, as well as the fact that seniors over age 65 with mental disorders generally have no access to daily activity centers in the community61 ; people with mental health issues struggle for social integration in the community.62 other researchers stress that older adults who receive home care do not receive support for regular physical activity,63 even though it clearly benefits senior adults mentally and physically.47 the emotional support that participants advocated in the present study related primarily to dialogues that could distract clients from negative and unwanted emotions or change their mood ; important components in such dialogues are the hcas active listening, asking questions to confirm understanding, and demonstrating personal interest in the clients. these dialogues, further, should have an unstructured form without an explicit or conscious aim or objective. our finding should be compared to a previous systematic review and synthesis of qualitative studies, indicating that to experience care as something positive, elderly persons need to be acknowledged for who they are and feel appreciated, as well as given the opportunity to contribute to their own decision - making and maintain their relationships.64 ultimately, even though the hcas offered various suggestions on mental health - promoting initiatives related to social support, it is important that care providers understand what each client values in life and how that client makes sense of changes in his or her life situation.65 further, staff members working with mental health care in the community need to ensure that clients with mental health issues also develop a sense of belonging in the community by helping these clients grow their social networks and achieve social integration.62 in contrast, although hcas indicated that their tasks were associated with cms decision - making to organize the delivery of granted home help services, participants rarely contacted their cms about needs assessment in situations of declining mental health or about the need for social support to improve a client s mental health. according to chapter 5, 5 of the social services act (sfs 2001:453) [sol ], the social board should ensure that older people receive good housing and provide those who need it with support and help in their homes, along with other easily accessible services.26 arguably, hcas should be able to provide feedback to the cms who would assist them in evaluating the granted support when a client s personal needs have changed. also valuable would be scheduled time for hcas to perform tasks that constitute social support with the purpose of improving mental health. another finding of the present study was that collaboration between dns and cms was crucial. according to the participants, a dn could transfer and respond to information about a client s behavior and emotions, as well as supervise hcas in managing clients with mental health problems. from the hcas perspective, a cm was in a position to grant assignments in a way that increases the time hcas have for activities that promote mental health and facilitate dialogue, such as increasing social and physical activities. the participants expressed a need for collegiality and stressed the importance of sharing the responsibility and decision - making about client tasks based on the hcas needs assessment. collaboration and teamwork with health care professionals such as dns and social services personnel emerged as especially crucial to transferring information about a client s changed behavior and emotions. according to several hcas, there was no forum through which hcas, dns, and cms could discuss issues or transfer and respond to information about a client. in addition, communication among health care professionals is important to improving health care among homebound seniors with mental and behavioral conditions.66 participants in the present study were uncertain whose responsibility it was to promote mental health or to diagnose and treat mental health problems when they arise. this may explain why participants in this study seemed uncertain about whom they should contact first if a client s mental health deteriorated. these findings may also reflect the difficulties the hcas perceived in providing care and support to a heterogeneous population of elderly people with multiple chronic conditions ; perhaps, that was why hcas expressed a desire for care coordination with actors such as dns. this finding aligns with previous research, highlighting the importance of care coordination and health promotion among persons with multimorbidity.47 other researchers have stated that multidisciplinary care is essential and should include the development of a comprehensive education system for all those involved in caring for an aging population.46,67 several researchers have suggested that interprofessional collaboration constitutes a method of improving health because it involves a comprehensive team of experts (both health care professionals and social workers) who work collaboratively toward the shared goal of treating a person with complex needs in the community.6771 personal meetings with dns may be useful when clients are in poor health,72 and such arenas for collaboration increase the opportunity for hcas to receive supervision and to ask questions about medical32 and mental health issues. according to further findings in the present study, personal resources appeared to be an important prerequisite for both detecting mental health problems and promoting mental health. several hcas repeatedly stated that they lacked time for such assignments because they were responsible for tasks related to household and personal care, and they were constrained by the limited time allotted for these tasks : those organizing the assistance in clients homes planned such assignments to fit into specific time slots without allowing for addition interpersonal interaction. specifically, participants lacked time for dialogue with clients through whom they could assess how a client was feeling or adequately comfort a sad client. this lack of time and the tight schedule may also be the reason why some hcas seemed to have distanced themselves from clients in need of comfort or conversation about their mental health. in these cases, hcas may decide to focus on the specific type of granted support they are supposed to provide, such as cleaning, rather than becoming attached to clients with whom they had little time to converse and truly engage.24 this finding about time aligns with the findings in our previous study indicating that seniors with multimorbidity preferred accessible interlocutors who had time for conversations that could promote mental health.22 the perceived lack of time could also reflect that hcas must perform time - consuming tasks related to their delegated responsibility for administering medication and other care to several clients.32 however, since relationships with clients were important to personal knowledge and were crucial in detecting worsening mental health, hcas may be caught between different interests related to ethical challenges in their granted assignments, including efforts to provide quality care to several clients in one work shift. in order to obtain a varied sample, the study initially applied a convenience and purposive sampling technique. however, we encountered challenges in recruiting enough hcas who had time for a focus group interview to assign a moderate number of participants to each group. this resulted in a convenience sample52 of mostly women of various ages and levels of experience in home help services and in caring for clients with multimorbidity. one limitation consists of the voluntary nature of study participation73 and the further risk that the most experienced hcas in this area were recruited. focus group interviews were considered a suitable method of data collection ; a distinct feature of this method is its group dynamics through social interaction, which may facilitate participants elucidation and further exploration of their perspectives on a specific topic.48 given the limitations in accessibility to more hcas of both sexes, along with the final sample size, the data include a fair amount of variation in experience ; thus, the generalizability increases. the interview guide was designed specifically to give participants the opportunity to discuss and share both negative and positive experiences with their colleagues. this approach in data collection was successful in eliciting both depth and breadth of data. however, the findings can not be generalized to other types of staff in elder care, and conclusions must therefore be drawn carefully, considering the participants context. the results indicate that the hcas had knowledge about certain risk factors for mental health problems even though they seemed insecure about which health professionals had primary responsibility for their clients mental health. they were often able to detect early signs of mental health problems even though good personal knowledge and continuity in home visits were needed in order to do so. when it came to mental health promotion, hcas suggestions were related to the goal of alleviating social isolation, decreasing feelings of loneliness, and increasing physical activity. the study also indicates that the hcas seemed dependent on supervision from dns and on cms decision - making about the granted support ; hcas relied on these professionals to schedule assignments in a way that would facilitate the detection of mental health problems and allow mental health promotion among this population. the results reported here present new knowledge about the opportunities and difficulties in the work of promoting mental health faced by staffers who meet clients on a regular basis. we suggest multidisciplinary and interprofessional approaches as means of facilitating the early detection of mental health problems and of increasing hcas competence in mental health promotion among their clients. hcas experience and competence vis - - vis a client s behavior and emotions is important because their observations may reflect worsening mental health over time. investments should therefore be made to provide hcas with the tools and skills they need to support clients with mental health problems and to help them remain integrated in their communities. | introductionelderly people with multiple chronic conditions, or multimorbidity, are at risk of developing poor mental health. these seniors often remain in their homes with support from home care assistants (hcas). mental health promotion by hcas needs to be studied further because they may be among the first to observe changes in clients mental health status.aimto describe hcas perspectives on detecting mental health problems and promoting mental health among homebound seniors with multimorbidity.methodswe applied a descriptive qualitative study design using semi - structured interviews. content analyses were performed on five focus group interviews conducted in 2014 with 26 hcas.resultsmost hcas stated that they were experienced in caring for clients with mental health problems such as anxiety, depression, sleep problems, and high alcohol consumption. the hcas mentioned as causes, or risk factors, multiple chronic conditions, feelings of loneliness, and social isolation. the findings reveal that continuity of care and seniors own thoughts and perceptions were essential to detecting mental health problems. observation, collaboration, and social support emerged as important means of detecting mental health problems and promoting mental health.conclusionthe hcas had knowledge of risk factors, but they seemed insecure about which health professionals had the primary responsibility for mental health. they also seemed to have detected early signs of mental health problems, even though good personal knowledge of the client and continuity in home visits were crucial to do so. when it came to mental health promotion, the suggestions related to the aim of ending social isolation, decreasing feelings of loneliness, and increasing physical activity. the results indicate that the hcas seemed dependent on supervision by district nurses and on care managers decisions to support the needed care, to schedule assignments related to the detection of mental health problems, and to promote mental health. |
since earliest times, food and nutrition have always remained one of the darkest areas and the world has failed to tackle the situation completely. the 1996 world food summit brought back to center stage the issue of hunger and food insecurity as both cause and effect of poverty and slow growth. in the wake of this new push, reducing hunger and food insecurity also became one of the millennium development goals, bringing with it the necessity by individual countries to measure progress in achieving the proposed targets. a well - nourished population is important to a country 's long - term development and is a desirable outcome objective in itself. unfortunately, the levels of child malnutrition in india are very high - 36.4% of urban indian children are underweight, according to the national family health survey 3 (nfhs3). chronic energy deficiency in a similar proportion of adults suggests perpetuation of childhood malnutrition into adulthood. the nutritional status of an individual is the outcome of a complex interaction of a broad range of host and environmental factors, the latter encompassing physical, biological, and especially sociocultural ones. it is thought that if people have access to sufficient quantity and variety of foods, they will meet their nutritional needs. an inadequate diet, poor in both quality and quantity has been one of the reasons for high levels of malnutrition in children. in most of the settings, household is the basic unit for food consumption, and if there is sufficient food, individual members of the household can consume a diet with the recommended nutrient densities and meet their specific requirements. assessing dietary adequacy in terms of quality and quantity at the household level is, therefore, important. adequacy implies that that the food must satisfy the dietary needs of every individual, taking into account age, gender, body size, and physical activity level. to define quantitatively, nutrient adequacy is the level of intake of an essential nutrient in relation to the nutrient requirement for adequate health, which is expressed as the percentage of recommended dietary allowance (rda). the quantitative characterization of nutrient needs and its expression as nutrient adequacy have been considered as significant instruments of food and nutrition strategy in numerous countries and have engrossed the attention of global bodies. to develop an effective nationwide preventive program to combat malnutrition, it is necessary not only to assess the nature and magnitude of the problem but also to identify factors affecting it especially at the household level. the present study aims to estimate the prevalence of nutrient adequacy in a sample of households in a rural area of darjeeling district and to find out the factors, particularly those socioeconomic factors known to influence nutrition, associated with nutrient adequacy. the decision to study only the effects of socioeconomic factors on nutrient intake was dictated by the consideration that the others are modifiable and can be readily manipulated to improve nutritional status. the factors included in the study were per capita income, education of both the head of the family and his wife, family size, and per capita expenditure on food. a community - based cross - sectional study was carried out in siliguri subdivision of darjeeling district of west bengal, india, from january 2014 to december 2014. the data employed in this study were collected from households in five villages under matigara ii gram panchayat, the villages under the field practice area of the department of community medicine, north bengal medical college, west bengal, india. before starting the study, ethical approval was obtained from the institutional ethics committee. since published data on nutrient adequacy in this part of the country were not available, the prevalence was assumed as 50% to yield maximum sample size. sample size was calculated as 384 using the formula for single proportion n = z(1/2)p (1 p)/d, where z(1/2) is the standard normal deviation at 95% confidence interval (1.96), p is the anticipated prevalence of nutrient adequacy (50%), d is absolute precision (5%). considering the design effect of 2 and 10% nonresponse rate, the final sample size was 845. first, all the 4280 households of the five villages in the rural field practice area were listed. sampling interval was calculated by dividing the number of households by the sample size, which came out to be 5.1, rounded off to 5. on reaching the household, the family members were explained about the purpose of the study and their informed consent was taken. data were collected from the head of the family or any other responsible member by the investigators with the help of a validated, uniform questionnaire in the local vernacular. the questionnaire comprised of three different parts : sociodemographic details, a dietary survey of the food consumed in the preceding 24 h and clinical exanimation, including anthropometric measurements. sociodemographic variables included age and sex of individual family members, religion, family income, and literacy status of the head of the family and his wife. dietary survey included food intake by the family members in the 24 h preceding the survey. average calorie and protein requirements were estimated for every member of each household using the indian council of medical research (icmr) standards. the major dependent variable used in this study was the nutrient adequacy ratio (nar) of a household. to estimate the nutrient adequacy of the diet, nar was calculated for 10 nutrients (energy, protein, fat, vitamin a, riboflavin, thiamine, vitamin c, calcium, and iron). nar was calculated for each nutrient as the ratio of daily individual intakes to standard recommended amounts for subject 's sex and age category. mar = nar (each truncated at 1)/number of nutrients, where nar = daily nutrient intake / recommended amount of nutrient. the mean nar measures whether a household is consuming more or less than, or the equivalent of, its need. a value of 100 specifies that the intake of nutrient is equal to the recommended intakes, and a value below 100 specifies lower than the recommended intake for one or more nutrients. per capita income of the family was calculated by dividing the total family income by the number of the family members. body mass index (bmi) was calculated by dividing weight in kg by square of height in meters. for children under 5 years, the bmi was plotted against age in months in the new who growth chart and was interpreted accordingly. undernutrition was considered if any adult member of the family had their bmi < 18.5 or the bmi for any under - five child fell below -2sd of the who growth standards. completed questionnaires were collated and entered in ms excel datasheet after proper data cleaning activities (validation, range, and consistency checks). the categorical data were expressed in frequencies and percentages in tabular form ; chi - square test was applied for analysis. nar was used as the dependent variables, the predictor variables included number of family members, number of under - five children in the family, literacy of head of the family and his wife, per capita income of the family, and percentage expenditure on food. out of the 845 households approached for the study, 24 participants refused to participate in the study (response rate 97.2%). thus, the sample population included 821 households with 4708 individuals, who were investigated for 1-day oral (24 h recall) diet survey. the sociodemographic profiles of the families show that 89.6% of families followed hinduism in 60.2% of the families. there were more than five family members, and 639 families had a per capita income less than rs. 1500, where literacy was concerned ; the heads of families were illiterate in 242 families whereas in 60.7% families, the wives were illiterate. it is evident from table 1 that the mean nar of a family was significantly associated with presence of literacy status of both head of the family and his wife, number of family members, per capita income, and percentage expenditure on food. the prevalence of nutrient adequate households was found to be 35.3% among the study households. relation between mean nar and characteristics of the study population (n=821) table 2 shows that mean nar was positively associated with per capita income, expenditure on food, literacy status of head of the family and his wife, and negatively associated with family size and number of under - five children in the family. linear regression analysis between mean nar as dependent variable and other factors as predictor variables (n=821) from the regression results, it was observed that the percentage expenditure on food had the highest contribution toward nutrient adequacy, followed by number of under - five children in the family, literacy of the wife of the head of the family.. the regression model for this purpose can be best expressed as follows : nar = 93.313 1.380 (number of family members) 3.0 (number of under - five children in the family) + 0.309 (literacy of head of the family) + 2.719 (literacy of the wife of the head of the family) + 3.901 (percentage expenditure on food). thus, applying the above model, approximately 13.2% of the total variation in nutrient adequacy can be best explained. undernutrition was found to be prevalent in 56.6% of the households [figure 1 ]. undernutrition in the family was associated significantly with mean nar, percentage expenditure on food, family size, and literacy of the wife of the head of the family. the relation between undernutrition and per capita income of the family and educational status of the head of the family was not found to be statistically significant [table 3 ]. prevalence of undernutrition among the study households n = 821 factors associated with undernutrition in the households (n=821) nutritional status can be seen as the output of a health production function in which nutrient intake is one input, but in which other individual, household, and community variables also feature. dietary intake in terms of caloric intake is often considered to be a strong proxy for nutritional status, even though it is one of the immediate causes of malnutrition. the rdas are standards for nutrient intake designed to meet the nutrient needs of virtually all healthy individuals and are often used as the basis for determining whether nutrient intake is adequate. in india, these guidelines have been developed by the icmr. income is an important factor in determining nutrition and provides an index of the purchasing capacity of the family. in 1980, the world bank stated in its report that the serious and extensive nutritional deficiencies that exist in almost all of these countries are largely a reflection of poverty, people do not have enough income for food. with the improvement of household income, absolute expenditure on food is likely to go up, as is the calorie and protein intake of the household. in the present study, both per capita income of the family and percentage expenditure of income on food likewise, in a study done among seniors in ontario, keller. concluded that income was a major contributing factor of nutrient inadequacy. the association between parental education and the dietary intake of a household has been dealt in many studies around the world. it is expected that better - educated parents are able to provide more wholesome diets in their family due to their ability to identify the nutritious value of food. a study done by galobardes. in geneva showed that lower education contributed to determining differences in dietary habits and dietary intake. just as in the present study, better literacy status of the head of the household and his wife were associated significantly with a higher mean nar. in two different studies done among spanish and indian children, educational status of the mother family size affects the dietary intake of the family in such a way that the allocation of food per member is likely to decrease with the increase in the number of household members, which, in turn, may have a negative effect on per capita nutrient intake. in a study done in rural bangladesh, family size was shown to have a negative effect on nutrient adequacy. in the present study also, family size was adversely associated with mean nar. however, in a study done in a marginal community of el salvador, no association was found between caloric sufficiency and family size. undernutrition was present in more than half of the households which can be corroborated with nfhs3 results which states undernutrition was more common in rural households. studies done among children and adults of north bengal have reported very high prevalence of undernutrition. the underlying reasons of malnutrition are multidimensional, including economic, social, and political factors. poverty has been widely acknowledged as both a cause and consequence of malnutrition, but has been obscured and bypassed as the main determinant to tackle when choosing solutions to combat malnutrition in the developing countries. a study done in philippines by garcia and pinstrup - andersen found that in households with severely malnourished members, insufficient income was the most limiting factor. quite contrarily, in the present study undernutrition was not significantly associated with per capita income of the family. this can be due to the fact that majority of the study households had a lower per capita income. according to anand and harris, food expenditure per capita is a better and more dependable indicator of well - being than income. surprisingly, in the present study, malnutrition was significantly more prevalent in the households with higher percentage expenditure on food. maternal education plays an important role in determining nutritional status of children and the family, as a whole. the present study reveals that malnutrition was significantly higher in the households with an illiterate mother. in another study done in iran, stunting in children was found to be significantly associated with lower maternal education. in studying the correlates of undernutrition in children under 3 years of age by mahgoub., a negative relationship between the number of under three children in the family, family income, maternal education, and the nutritional status (underweight) of children was found. a larger family size was seen to be associated with an increased risk of severe acute malnutrition among under - five children in a case control study done in ethiopia by amsalu and tigabu. corroborating with these findings, our study points out the fact that in families with 5 or more members, undernutrition was significantly associated. in a study in bangladesh by das., chronic malnutrition in 1223 months of age was found to be associated with households having large family size, poor wealth index, and maternal illiteracy. the limitation of the study was that intrafamilial distribution of food could not be measured which may have contributed to undernutrition. in addition, the issue of food security was not addressed, which may have played a role in the underweight status. in addition, 24 h recall method of dietary survey, though widely accepted, has the inherent limitation that it depends on the reliability of household consumption estimates. the present study has brought forth important and useful information regarding the pervasiveness of nutrient inadequacy and expectedly, undernutrition in the study area. socioeconomic status and illiteracy improved access to food and better nutrition for the rural population can only be achieved on a sustainable basis if these underlying causes are addressed. all these issues and challenges need to be addressed in a pragmatic and intersectoral manner to achieve better health for the nation and the community at the large. | introduction : nutrient adequacy is the level of intake of an essential nutrient in relation to the nutrient requirement for adequate health, which is expressed as the percentage of recommended dietary allowance. to develop an effective nationwide preventive program to combat malnutrition, it is necessary not only to assess the nature and magnitude of the problem of nutrient inadequacy but to identify factors affecting it especially at the household level.objective:to estimate the prevalence of nutrient adequacy in a sample of households in a rural area of darjeeling district and to find out the factors associated with nutrient adequacy.materials and methods : a community - based cross - sectional study was carried out from january 2014 to december 2014 in 821 households of darjeeling district, india. the major dependent variable used in this study was the mean nutrient adequacy ratio of a household and the independent variables were number of family members, number of under - five children in the family, literacy of head of the family, literacy of the wife of the head of the family, income of the family, and percentage expenditure on food.results:the prevalence of nutrient adequate households was found to be 35.3% among the study households. it was observed that the percentage expenditure on food had the highest contribution toward nutrient adequacy, followed by number of under - five children in the family and literacy of the wife of the head of the family. undernutrition was found to be prevalent in 56.6% of the households.conclusion:majority of the study population had a diet less than the required amount and expectedly, undernutrition was also present in huge proportions. |
using exome sequencing and a variant prioritisation strategy that focuses on loss - of - function variants, we identified biallelic, loss - of - function cep57 mutations as a cause of constitutional mosaic aneuploidies. cep57 is a centrosomal protein and is involved in nucleating and stabilizing microtubules. our data indicate that these and/or additional functions of cep57 are crucial in maintaining correct chromosomal number during cell division. |
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the protocol was approved by the institutional review boards for human studies of baylor college of medicine, the harris county hospital district, and the university of washington. all kpd patients were identified by acute presentation with an episode of dka, defined by the presence of all of the following : anion gap 15, blood ph 200 mg / dl, serum ketones 5.2 the a kpd patients were identified by the absence of gad65, islet antigen-2, or znt8 autoantibodies, measured in sera by quantitative radioligand binding assays with recent modifications (8,9). as previously described (1), kpd patients are classified as a if the autoantibody index for all three autoantibodies are below the ethnic - specific 98th percentile. patients were classified as + or based on the presence or absence of -cell functional reserve, measured by fasting serum c - peptide concentration and c - peptide response to glucagon 14 weeks after resolution of ketoacidosis. cutoffs defining -cell functional reserve were as previously established and longitudinally validated using receiver - operator characteristic curve analysis (1) ; briefly, + status is defined by fasting c - peptide > 1 ng / ml and peak c - peptide after glucagon stimulation > 1.5 ng / ml. provoked a+ kpd patients were defined by the presence of a clinically defined precipitating factor associated with or immediately preceding the index dka episode (4). we have previously shown that the phenotype of provoked a+ kpd is characterized by progressive loss of -cell function after recovery from the index dka episode, no sex predominance, inability to discontinue insulin or relapsing to insulin requirement after a brief period off insulin, and an increased frequency of type 1 diabetes - associated hla class ii alleles dqb1 0302 and drb1 04 (4). unprovoked a+ kpd patients were defined by the absence of a clinically defined precipitating factor associated with or immediately preceding the index dka episode. we have previously shown that the phenotype of unprovoked a+ kpd is characterized by marked improvement and sustained preservation of -cell function after recovery from the index dka episode, male predominance, insulin independence, and an increased frequency of the protective allele dqb1 0602 (4). a kpd patients were defined exclusively by a and status, without regard to dka precipitant, because the latter does not affect the natural history or phenotypic characteristics of this kpd subgroup. blood was drawn for autoantibody, phenotyping, and t - cell responses during routine follow - up visits of the subjects to the kpd clinic at ben taub general hospital. all subjects were clinically stable, without evidence of acute illness or inflammation at the time of the blood draw. blinded whole - blood samples were transported, using a validated shipping protocol, overnight in a blinded fashion to the university of washington, seattle, wa, for t - cell analysis (10,11). serum samples were shipped frozen to the university of washington for measurement of islet autoantibodies. gad65, islet cell antigen (ia-2), and zinc transporter isoform 8 (znt8) autoantibodies were determined by radioligand binding assay, as previously described (8,9). recombinant s - antigen was produced in an in vitro coupled transcription and translation system with sp6 rna polymerase and nuclease - treated rabbit reticulocyte lysate (promega, madison, wi). sera (2.5 l) were incubated with s - antigen (25,000 of trichloroacetic acid precipitable radioactivity). after an overnight incubation at 4c, antibody - bound s - antigen was separated from unbound antigen by precipitation with pas (invitrogen, carlsbad, ca). the immunoprecipitated radioactivity was counted on a wallac microbeta liquid scintillation counter (perkinelmer life and analytical sciences, inc., antibody - positive control sera were included as a standard and used to express immunoglobulin - binding levels as a relative unit. samples with a gad65 antibody titer > 65 units / ml were considered positive for gad65-antibody. ia2-antibody positivity was established as exceeding the binding of the 98th percentile for healthy controls (30 units / ml). autoantibody positivity for znt8r and znt8 w were set at 15 units / ml and 26 units / ml, respectively, based on the 98th percentile observed in 50 healthy human control sera. the autoantibody laboratory (c.s.h.) participated in the diabetes autoantibody standardization program (dasp) workshop (12) ; the gad65 autoantibody assay showed a sensitivity of 86% and specificity of 93%, and the ica512/ia-2 autoantibody assay showed a sensitivity of 66% and a specificity of 98%. cellular immunoblotting was performed on blinded freshly isolated peripheral blood mononuclear cells (pbmcs) to test for the presence of islet - reactive t cells, as previously described (13). briefly, normal human islet cell preparations were subjected to preparative one - dimensional 10% sds - page and electroblotted onto nitrocellulose. the nitrocellulose was cut into molecular weight regions (blots) and then solubilized to form nitrocellulose particles. the nitrocellulose particles containing islet proteins were used to stimulate pbmcs, at a concentration of 3.5 10 pbmcs per well (5,6,10,11,13,14). type 1 diabetic patients have been shown to respond to 418 molecular weight proteins and normal control subjects (without diabetes) to 03 molecular weight regions (14). human pancreatic islets were obtained from the national institutes of health supported islet cell resource centers (icr - abcc). the specificity of the t - cell responses from diabetic patients to islet proteins has been demonstrated previously (13). cellular immunoblotting has been validated to have excellent specificity and sensitivity for distinguishing t - cell responses to islet proteins between t1d patients and control subjects (10,11,15). the specificity and sensitivity for the cellular immunoblotting assay was similar to those for the islet autoantibody assays performed in the individual workshops and exceeded the specificity and sensitivity of other t - cell assays validated at this time. pbmcs were stained with antibodies specific to human cd14 and human cd16 (bd pharmingen, san jose, ca). data acquisition was performed using bd cellquest 5.2.1 software on a bd facscalibur (bd biosciences). hla genotyping for class ii alleles associated with susceptibility to or protection from autoimmune type 1 diabetes was performed at benaroya research institute, seattle, wa. briefly, hla genotyping for dr1, dr3, dr4, dr13, dr15, dq2, dq6, and dq8 was performed by real - time pcr using taqman specific assays, as described previously (16,17). real - time pcr was done using taqman gtxpress genotyping master mix (applied biosystems, life technologies, inc.) on an abi steponeplus real - time pcr system (applied biosystems), as described (17). anovas, the wilcoxon signed rank test, and mann - whitney tests were used to compare groups. multivariate regression analyses were performed to evaluate positivity for monocytes and islet - reactive t - cell responses to bmi, age, sex, and duration of diabetes. the protocol was approved by the institutional review boards for human studies of baylor college of medicine, the harris county hospital district, and the university of washington. all kpd patients were identified by acute presentation with an episode of dka, defined by the presence of all of the following : anion gap 15, blood ph 200 mg / dl, serum ketones 5.2 the a kpd patients were identified by the absence of gad65, islet antigen-2, or znt8 autoantibodies, measured in sera by quantitative radioligand binding assays with recent modifications (8,9). as previously described (1), kpd patients are classified as a if the autoantibody index for all three autoantibodies are below the ethnic - specific 98th percentile. patients were classified as + or based on the presence or absence of -cell functional reserve, measured by fasting serum c - peptide concentration and c - peptide response to glucagon 14 weeks after resolution of ketoacidosis. cutoffs defining -cell functional reserve were as previously established and longitudinally validated using receiver - operator characteristic curve analysis (1) ; briefly, + status is defined by fasting c - peptide > 1 ng / ml and peak c - peptide after glucagon stimulation > 1.5 ng / ml. provoked a+ kpd patients were defined by the presence of a clinically defined precipitating factor associated with or immediately preceding the index dka episode (4). we have previously shown that the phenotype of provoked a+ kpd is characterized by progressive loss of -cell function after recovery from the index dka episode, no sex predominance, inability to discontinue insulin or relapsing to insulin requirement after a brief period off insulin, and an increased frequency of type 1 diabetes - associated hla class ii alleles dqb1 0302 and drb1 04 (4). unprovoked a+ kpd patients were defined by the absence of a clinically defined precipitating factor associated with or immediately preceding the index dka episode. we have previously shown that the phenotype of unprovoked a+ kpd is characterized by marked improvement and sustained preservation of -cell function after recovery from the index dka episode, male predominance, insulin independence, and an increased frequency of the protective allele dqb1 0602 (4). a kpd patients were defined exclusively by a and status, without regard to dka precipitant, because the latter does not affect the natural history or phenotypic characteristics of this kpd subgroup. blood was drawn for autoantibody, phenotyping, and t - cell responses during routine follow - up visits of the subjects to the kpd clinic at ben taub general hospital. all subjects were clinically stable, without evidence of acute illness or inflammation at the time of the blood draw. blinded whole - blood samples were transported, using a validated shipping protocol, overnight in a blinded fashion to the university of washington, seattle, wa, for t - cell analysis (10,11). serum samples were shipped frozen to the university of washington for measurement of islet autoantibodies. gad65, islet cell antigen (ia-2), and zinc transporter isoform 8 (znt8) autoantibodies were determined by radioligand binding assay, as previously described (8,9). recombinant s - antigen was produced in an in vitro coupled transcription and translation system with sp6 rna polymerase and nuclease - treated rabbit reticulocyte lysate (promega, madison, wi). sera (2.5 l) were incubated with s - antigen (25,000 of trichloroacetic acid precipitable radioactivity). after an overnight incubation at 4c, antibody - bound s - antigen was separated from unbound antigen by precipitation with pas (invitrogen, carlsbad, ca). the immunoprecipitated radioactivity was counted on a wallac microbeta liquid scintillation counter (perkinelmer life and analytical sciences, inc., antibody - positive control sera were included as a standard and used to express immunoglobulin - binding levels as a relative unit. samples with a gad65 antibody titer > 65 units / ml were considered positive for gad65-antibody. ia2-antibody positivity was established as exceeding the binding of the 98th percentile for healthy controls (30 units / ml). autoantibody positivity for znt8r and znt8 w were set at 15 units / ml and 26 units / ml, respectively, based on the 98th percentile observed in 50 healthy human control sera. the autoantibody laboratory (c.s.h.) participated in the diabetes autoantibody standardization program (dasp) workshop (12) ; the gad65 autoantibody assay showed a sensitivity of 86% and specificity of 93%, and the ica512/ia-2 autoantibody assay showed a sensitivity of 66% and a specificity of 98%. gad65, islet cell antigen (ia-2), and zinc transporter isoform 8 (znt8) autoantibodies were determined by radioligand binding assay, as previously described (8,9). recombinant s - antigen was produced in an in vitro coupled transcription and translation system with sp6 rna polymerase and nuclease - treated rabbit reticulocyte lysate (promega, madison, wi). sera (2.5 l) were incubated with s - antigen (25,000 of trichloroacetic acid precipitable radioactivity). after an overnight incubation at 4c, antibody - bound s - antigen was separated from unbound antigen by precipitation with pas (invitrogen, carlsbad, ca). the immunoprecipitated radioactivity was counted on a wallac microbeta liquid scintillation counter (perkinelmer life and analytical sciences, inc., antibody - positive control sera were included as a standard and used to express immunoglobulin - binding levels as a relative unit. samples with a gad65 antibody titer > 65 units / ml were considered positive for gad65-antibody. ia2-antibody positivity was established as exceeding the binding of the 98th percentile for healthy controls (30 units / ml). autoantibody positivity for znt8r and znt8 w were set at 15 units / ml and 26 units / ml, respectively, based on the 98th percentile observed in 50 healthy human control sera. the autoantibody laboratory (c.s.h.) participated in the diabetes autoantibody standardization program (dasp) workshop (12) ; the gad65 autoantibody assay showed a sensitivity of 86% and specificity of 93%, and the ica512/ia-2 autoantibody assay showed a sensitivity of 66% and a specificity of 98%. cellular immunoblotting was performed on blinded freshly isolated peripheral blood mononuclear cells (pbmcs) to test for the presence of islet - reactive t cells, as previously described (13). briefly, normal human islet cell preparations were subjected to preparative one - dimensional 10% sds - page and electroblotted onto nitrocellulose. the nitrocellulose was cut into molecular weight regions (blots) and then solubilized to form nitrocellulose particles. the nitrocellulose particles containing islet proteins were used to stimulate pbmcs, at a concentration of 3.5 10 pbmcs per well (5,6,10,11,13,14). type 1 diabetic patients have been shown to respond to 418 molecular weight proteins and normal control subjects (without diabetes) to 03 molecular weight regions (14). human pancreatic islets were obtained from the national institutes of health supported islet cell resource centers (icr - abcc). the specificity of the t - cell responses from diabetic patients to islet proteins has been demonstrated previously (13). cellular immunoblotting has been validated to have excellent specificity and sensitivity for distinguishing t - cell responses to islet proteins between t1d patients and control subjects (10,11,15). the specificity and sensitivity for the cellular immunoblotting assay was similar to those for the islet autoantibody assays performed in the individual workshops and exceeded the specificity and sensitivity of other t - cell assays validated at this time. cellular immunoblotting was performed on blinded freshly isolated peripheral blood mononuclear cells (pbmcs) to test for the presence of islet - reactive t cells, as previously described (13). briefly, normal human islet cell preparations were subjected to preparative one - dimensional 10% sds - page and electroblotted onto nitrocellulose. the nitrocellulose was cut into molecular weight regions (blots) and then solubilized to form nitrocellulose particles. the nitrocellulose particles containing islet proteins were used to stimulate pbmcs, at a concentration of 3.5 10 pbmcs per well (5,6,10,11,13,14). type 1 diabetic patients have been shown to respond to 418 molecular weight proteins and normal control subjects (without diabetes) to 03 molecular weight regions (14). human pancreatic islets were obtained from the national institutes of health supported islet cell resource centers (icr - abcc). the specificity of the t - cell responses from diabetic patients to islet proteins has been demonstrated previously (13). cellular immunoblotting has been validated to have excellent specificity and sensitivity for distinguishing t - cell responses to islet proteins between t1d patients and control subjects (10,11,15). the specificity and sensitivity for the cellular immunoblotting assay was similar to those for the islet autoantibody assays performed in the individual workshops and exceeded the specificity and sensitivity of other t - cell assays validated at this time. pbmcs were stained with antibodies specific to human cd14 and human cd16 (bd pharmingen, san jose, ca). data acquisition was performed using bd cellquest 5.2.1 software on a bd facscalibur (bd biosciences). hla genotyping for class ii alleles associated with susceptibility to or protection from autoimmune type 1 diabetes was performed at benaroya research institute, seattle, wa. briefly, hla genotyping for dr1, dr3, dr4, dr13, dr15, dq2, dq6, and dq8 was performed by real - time pcr using taqman specific assays, as described previously (16,17). real - time pcr was done using taqman gtxpress genotyping master mix (applied biosystems, life technologies, inc.) on an abi steponeplus real - time pcr system (applied biosystems), as described (17). anovas, the wilcoxon signed rank test, and mann - whitney tests were used to compare groups. multivariate regression analyses were performed to evaluate positivity for monocytes and islet - reactive t - cell responses to bmi, age, sex, and duration of diabetes. patient demographic, clinical, and phenotypic data are reported in tables 1 and 2. there were no significant differences among the three groups in age at the time of t - cell testing, sex, or ethnicity (table 1). the a kpd patients had been diagnosed with diabetes at a younger age than the two a+ kpd groups (p 4 years). moreover, provoked a+ kpd patients have a high frequency of hla class ii alleles linked to susceptibility to autoimmune type 1 diabetes (dqb1 0302 and drb1 04), suggesting that their pathophysiology includes a component of islet autoimmunity, whereas unprovoked a+ kpd patients lack the increased frequency of susceptibility alleles (4). hence, in the current study, we used cellular immunoblotting and flow cytometry to determine whether the differences observed in the clinical characteristics and natural histories of these three a kpd subgroups are related to the presence or absence of islet - specific t cells and/or proinflammatory monocytes (cd14+cd16 +). proinflammatory monocytes (cd14+cd16 +) are increased in a variety of acute infections and severe inflammatory states such as sepsis. therefore, all samples from subjects were obtained when they were clinically stable, without evidence of acute inflection or inflammation, to avoid the potential increases of inflammatory monocyte populations associated with inflammation or infection. we discovered an inverse relationship between bmi and the presence of proinflammatory monocytes. because an association of obesity with increased numbers of circulating proinflammatory monocytes has been reported (20), this finding suggests that some factors unique to kpd may drive the systemic inflammatory response and override the effects of obesity per se. frequencies of hla class ii alleles associated with susceptibility to or protection against autoimmune type 1 diabetes were measured in all of the patients in this study to assess genetic contributions to cell - mediated islet autoimmunity in the a kpd patients. the results, in conjunction with those previously obtained in a separate, larger cohort of kpd patients, indicate that 1) in the a kpd population, having a protective hla class ii allele is associated with absence of both humoral (4) and t - cell autoreactivity to islet antigens and that 2) compared with the other a kpd subgroups, unprovoked a+ kpd, previously shown to have a lower frequency of susceptibility hla class ii alleles (4), also has a higher frequency of protective alleles. together with the present results indicating that unprovoked a+ kpd has a low frequency of t - cell positivity and that having a protective allele is strongly associated with absence of t - cell positivity, these findings strengthen the notion that the cause of -cell dysfunction in unprovoked a+ kpd is not related to islet autoimmunity. in summary, our results lead to the conclusion that provoked a+ kpd is associated with a high frequency of islet autoimmunity and proinflammatory monocyte populations, which could contribute to the propensity to ketoacidosis and the subsequent progressive loss of -cell function in these patients. on the other hand, unprovoked a+ kpd is a distinct syndrome of reversible -cell dysfunction and sustained -cell functional reserve comprising patients who lack evidence for humoral and cellular islet autoimmunity and whose proneness to ketosis may be explained by specific defects in oxidation of ketones and fatty acids and in the metabolism of the ketogenic amino acid leucine (21). patients in the a subgroup are demographically different from the other two a kpd subgroups, being lean, with earlier age of onset of diabetes, and completely insulinopenic. in our earlier analyses, etiology of severe -cell dysfunction on the basis of absent autoantibodies and a frequency of hla susceptibility alleles that is lower than that of a+ kpd patients (who have classic, autoimmune type 1 diabetes) (1). it appears, therefore, that a kpd subsumes multiple etiologies of severe -cell dysfunction, including cellular islet autoimmunity (t - cell positive patients) and monogenic causes of defective islet development or function, as we have previously reported (22). thus, these findings demonstrate that cellular islet autoimmunity is present in some but not all a forms of kpd and expand the autoimmune basis of -cell dysfunction beyond the boundaries of classic, autoimmune type 1 diabetes. | objectiveketosis - prone diabetes (kpd) is characterized by diabetic ketoacidosis (dka) in patients lacking typical features of type 1 diabetes. a validated classification scheme for kpd includes two autoantibody - negative (a) phenotypic forms : a (lean, early onset, lacking -cell functional reserve) and a+ (obese, late onset, with substantial -cell functional reserve after the index episode of dka). recent longitudinal analysis of a large kpd cohort revealed that the a+ phenotype includes two distinct subtypes distinguished by the index dka episode having a defined precipitant (provoked, with progressive -cell function loss over time) or no precipitant (unprovoked, with sustained -cell functional reserve). these three a kpd subtypes are characterized by absence of humoral islet autoimmune markers, but a role for cellular islet autoimmunity is unknown.research design and methodsislet - specific t - cell responses and the percentage of proinflammatory (cd14+cd16 +) blood monocytes were measured in a (n = 7), provoked a+ (n = 15), and unprovoked a+ (n = 13) kpd patients. genotyping was performed for type 1 diabetes associated hla class ii alleles.resultsprovoked a+ and a kpd patients manifested stronger islet - specific t - cell responses (p < 0.03) and higher percentages of proinflammatory cd14+cd16 + monocytes (p < 0.01) than unprovoked a+ kpd patients. a significant relationship between type 1 diabetes hla class ii protective alleles and negative t - cell responses was observed.conclusionsprovoked a+ kpd and a kpd are associated with a high frequency of cellular islet autoimmunity and proinflammatory monocyte populations. in contrast, unprovoked a+ kpd lacks both humoral and cellular islet autoimmunity. |
lung cancer is the leading cause of cancer - related deaths in the united states. even after 30 years of improved therapeutic approaches, the 5-year mortality of lung cancer remains alarmingly high at 86%.1 the emerging application of pharmacogenomics offers the potential to identify drugs and drug combinations in the treatment of lung cancer according to an individual s unique genetic makeup. a series of randomized clinical trials have demonstrated that survival of patients with stage iii or iv non - small - cell lung cancer (nsclc) is improved with the use of chemotherapy compared to best supportive care.2,3 among the initial chemotherapeutic agents that demonstrated reproducible activity in nsclc was the non - classical alkylating agent cisplatin. more recently, newer agents such as gemcitabine and taxanes have been combined with cisplatin ; however, the ability to predict which patients are likely to benefit from chemotherapy remains a challenging dilemma with no uniformly reliable clinical predictors other than performance status (ps) and weight loss. several studies have linked cisplatin resistance to high expression of the dna repair gene excision repair cross - complementation group 1 (ercc1).47 ercc1 is 1 of 16 genes that encodes the nucleotide excision repair complex. ercc1 is a nucleotide excision repair enzyme playing an important role in dna repair mechanism that removes the therapeutic platinum dna adducts from the tumor dna. nsclc patients with surgically removed early stage tumors, who received no further therapy, had a better survival if their tumors were ercc1 positive.8 these studies also have emphasized that patients with lowest levels of mrna expression of ercc1 tend to have better response to cisplatin - based therapy. this indicates the importance of considering ercc1 mrna expression as a predictive marker for the effectiveness of cisplatin - based chemotherapy. in addition to ercc1, there are several other pharmacogenomic markers that could have prognostic impact, although they act through different mechanisms. angiogenesis plays an important role in tumor growth and has attracted interest as a potential therapeutic target.9 studies have shown that 14 - 3 - 3 sigma (14 - 3 - 3) protein levels are more highly expressed in human lung cancers compared to normal tissues. the aim of this study was to correlate clinical factors and pharmacogenomic markers, such as ercc1 and 14 - 3 - 3, with overall survival in patients with advanced stage nsclc and also to determine the feasibility of utilizing pharmacogenomic markers as prognostic markers and to determine their value for incorporation into future treatment protocols. fifty patients with advanced stage nsclc were included in the study in the period between january 2007 and december 2008. we included patients with advanced stage nsclc (recurrent nsclc or stage iii or iv nsclc ; squamous, adenocarcinoma, or large cell undifferentiated nsclc) with adequate tissue to undergo further analysis. most of the patients with advanced stage were only diagnosed with fine needle aspiration or small biopsies. three patients had surgical resection, followed by adjuvant systemic chemotherapy, however, were included in the study as they became progressive with recurrent advanced nsclc. pharmacogenomic testing was carried out in the majority of the 50 patients using the biopsy samples of the advanced disease, but for patients who had prior surgical resection, the available surgically resected tumor was tested. all patients had chest x - ray and a computed tomography (ct) scan of the chest and upper abdomen before entry into the study and underwent repeated evaluation at least every 6 weeks. all patients gave signed informed consent, and the study was approved by the institutional ethics review boards. archival tumor specimens from each patient were retrieved from the pathology core facility after review of the h&e (hematoxylin and eosin) stained slides. this was a retrospective study, approved by the university of cincinnati institutional review board, in which 50 patients with stages iii and iv nsclc from the university medical center and the va medical center were included between 1998 until 2008. the patients medical records were reviewed and their gender, race, ps, chemotherapy regimens, and pharmacogenomic marker levels were documented and evaluated for correlation with survival. the evaluation was blinded and they used immunohistochemical (ihc) staining and included : ercc1, vascular endothelial growth factors (vegf) and vegf receptor, nuclear factor kappa beta (nf - kb [p65 ]), 14 - 3 - 3, and phosphorylated akt (pakt) and pten. ihc staining was performed using vectastain elite abc universal staining kit (vector laboratories, ca, usa) according to the manufacturer s protocol. paraffin blocks of stage iii and iv nsclc were sectioned at 4 microseconds and de - waxed for 2 hours at 65 c. sections were boiled at 95 c in antigen demasking buffer (10 mm sodium citrate at ph 6.0) for 15 minutes. these sections were further incubated with 3% h2o2 in 100% methanol to quench the endogenous peroxidase activity and blocked with 10% goat or horse serum for 2 hours. the treated sections were incubated overnight at 4 c with anti - vegf (a-20) rabbit polyclonal at 1:100 dilution ; anti - p65 (c-20) rabbit polyclonal at 1:100 dilution ; anti-14 - 3 - 3 (n-14) goat polyclonal at 1:500 dilution (santa cruz biotechnology, ca, usa) ; anti - vegfr2 (kdr-55b11) rabbit monoclonal at 1:250 dilution ; anti - pakt (ser 473) rabbit monoclonal at 1:100 dilution (cell signaling, ma, usa) ; anti - pten rabbit polyclonal at 1:100 dilution (abcam, ma, usa) ; and anti - ercc1 (8f1) rabbit polyclonal at 1:200 dilution (thermo fisher scientific, ca, usa). peroxidase staining was revealed using a dab - enhanced liquid substrate system (3,3-diaminobenzidine tetrahydrochloride) (sigma, mo, usa). results were interpreted independently by two pathologists and graded by the following criteria : grade 0 (negative or non - reactive expression), grade 1 (weak or focal expression), grade 2 (moderate expression), and grade 3 (strong or diffused expression). ihc staining was performed using vectastain elite abc universal staining kit (vector laboratories, ca, usa) according to the manufacturer s protocol. paraffin blocks of stage iii and iv nsclc were sectioned at 4 microseconds and de - waxed for 2 hours at 65 c. sections were boiled at 95 c in antigen demasking buffer (10 mm sodium citrate at ph 6.0) for 15 minutes. these sections were further incubated with 3% h2o2 in 100% methanol to quench the endogenous peroxidase activity and blocked with 10% goat or horse serum for 2 hours. the treated sections were incubated overnight at 4 c with anti - vegf (a-20) rabbit polyclonal at 1:100 dilution ; anti - p65 (c-20) rabbit polyclonal at 1:100 dilution ; anti-14 - 3 - 3 (n-14) goat polyclonal at 1:500 dilution (santa cruz biotechnology, ca, usa) ; anti - vegfr2 (kdr-55b11) rabbit monoclonal at 1:250 dilution ; anti - pakt (ser 473) rabbit monoclonal at 1:100 dilution (cell signaling, ma, usa) ; anti - pten rabbit polyclonal at 1:100 dilution (abcam, ma, usa) ; and anti - ercc1 (8f1) rabbit polyclonal at 1:200 dilution (thermo fisher scientific, ca, usa). peroxidase staining was revealed using a dab - enhanced liquid substrate system (3,3-diaminobenzidine tetrahydrochloride) (sigma, mo, usa). results were interpreted independently by two pathologists and graded by the following criteria : grade 0 (negative or non - reactive expression), grade 1 (weak or focal expression), grade 2 (moderate expression), and grade 3 (strong or diffused expression). the main objective of this study was to identify molecular determinants of clinical outcomes related to cancer prognosis and survival. the expression rates of the protein markers vegf receptor and 14 - 3 - 3, as determined by ihc labeling, were treated as continuous variables, while the remaining markers (vegf, p65, pten, pakt, and ercc1) were treated as ordinal categorical variables. cells with protein expression rates above 0 (vegf receptor and 14 - 3 - 3) or stained as 1 + or 2 + (vegf, p65, pten, pakt, and ercc1) were considered positive. cells with 0 expression or stained as 0 + were considered negative for the marker. overall survival was defined as the time from the date of diagnosis to the time of death from any cause or the time at the last follow - up. progression - free survival was defined as the duration between the date of diagnosis and disease progression or the last follow - up. the kaplan meier method was used to estimate the distribution of survival time of the patients, overall and after stratifying by race and gender. univariate analysis was conducted to examine the survival curves across the protein expression levels of the molecular markers (classified as positive and negative) using the log rank test. in order to determine the joint effect of the predictors (age, gender, race, type of chemotherapy, and protein expression of each marker) on the overall survival and progression - free survival of the patients, comparisons of categorical data were performed using the chi - square test or fisher s exact test. the wilcoxon two - sample test with t approximation continuous variables, including the overall survival and progression - free survival, were correlated with expression levels of the markers using spearman correlation coefficients. fifty patients with histologically proven nsclc of advanced stage (stage iii or iv) were included in our study. forty patients received a platinum - based chemotherapy regimen, while 10 received non - platinum based regimens. median age was 65 years (range 4284) ; age did not correlate with os (overall survival) (p = 0.24). ecog ps was 0 in 8 patients (16%), 1 in 29 patients (58%), 2 in 7 patients (14%), and 3 in 6 patients (14%). ecog ps of 0 and 1 were significantly associated with higher overall survival (p = 0.004). females had significantly longer survival compared to males (31.7 months versus 12.4 months, p = 0.04) as shown in figure 2 and table 1. caucasians had better overall survival in comparison to african americans with median survival of 14.8 months versus 10.4 months, although this did not reach statistical significance (p = 0.1) as shown in table 1. patients treated with gemcitabine therapy had a median survival of 19 months versus 13 months in the non - gemcitabine therapy group (p = 0.003) ; patients treated with platinum - based therapy had a median survival of 15 months versus 8 months in the non - platinum based therapy group, but this did not reach statistical significance. however, in patients treated with platinum, ercc1 negativity was strongly associated with longer survival (p = 0.007) as shown in figure 3. higher expression of the vegf receptor was associated with improved overall survival of 14 months versus 9.7 months with lower expression, but the difference did not reach statistical significance. 14 - 3 - 3, pakt, and pten tissue expression did not correlate with overall survival. nf - kb (p65) expression was positive in 4 out of 46 patients lung cancer tissue. the positivity of nf - kb (p65) was associated with shorter survival of 9.9 months versus 13 months in patients with negative expression of the same receptor, with only a statistical trend of p = 0.1 (see table 1). since cisplatin and carboplatin are the most commonly used base agents for combination chemotherapy for the treatment of advanced nsclc, many studies have focused on the benefit of platinum doublets. older randomized trials repeatedly failed to identify differences in survival between any of these doublets, which gave way to the new era of personalized chemotherapy and the selection of patients with ercc1 negative expression for receiving platinum - based chemotherapy. adjuvant therapy with platinum doublet showed benefit in the ercc1-negative and the ercc1-positive population of patients.10 in 1995, the non - small - cell lung cancer collaborative group (nsclccg) published a meta - analysis showing a strong trend in favor of adjuvant cisplatin - based chemotherapy (hazard ratio[hr ], 0.87 ; 95% confidence interval [ci ], 0.741.02 ; p = 0.08), with an absolute improvement in the 5-year survival rate of 5%. although the benefit was not statistically significant, this observation prompted additional clinical trials to address the appropriate role of adjuvant chemotherapy in patients with resected nsclc (table 2).11 the standard of care for stage ii iiia nsclc patients is adjuvant cisplatin - based doublet chemotherapy after appropriate surgical resection to improve os. the benefit for patients with stage ib nsclc is less apparent, likely because of the heterogeneity of this population. the latest revisions to the tnm staging criteria should assist in risk stratification.11 our data confirm the benefits of ercc1 selectivity in improving the outcome of patients with nsclc. among the patients who received platinum - based therapy, those who were negative for ercc1 had significantly longer overall survival than those who were positive as shown in table 1. the degree of ercc1-positive expression also correlated well with the overall survival, but this could also be attributed to the fact that most of our patient population (80%) received platinum - based therapy as per the standard guidelines. we were unable to test for ribonucleotide reductase messenger rna (rrm1) expression, the marker of resistance to gemcitabine therapy.12,13 in spite of that, our patient population with advanced nsclc showed better survival when treated with gemcitabine, whether first or second line, compared to other regimens (19 versus 13 months ; p = 0.003). this contrasts with the group of patients receiving platinum therapy, in whom platinum therapy was only significantly associated with improved overall survival in ercc1-negative patients. this is the first study, however, that shows that gemcitabine therapy would influence the overall survival of patients with advanced nsclc if we do not have the tools to test for the specific rrm1 unlike ercc1 and platinum therapy. the vegf (vegf - a, -c, -d) and the vegf receptors (vegfr-1, -2, and -3) are important molecular markers in angiogenesis and lymphangiogenesis. a study by donnem.14 elucidated the prognostic significance of these molecular markers in tumor cells, as well as in the tumor stroma of resected nsclc tumors. in that study, high tumor cell expression of vegf - a, vegfr-1, vegfr-2, and vegfr-3 was a significant negative prognostic indicator of disease - specific survival (dss).14 in tumor stroma, however, high expression of vegf - a, vegf - c, vegf - d, vegfr-1, and vegfr-2 correlated with good prognosis. in multivariate analyses, high expression of vegfr-3 in tumor cells was an independent negative prognostic factor for dss, whereas in stromal cells, high vegf - c had an independent positive association with survival.14 in our study, however, the tumor tissue was obtained at biopsy for advanced stage disease. the abundance of tumor tissue made the discrimination between the tumor stromal tissue and the tumor cells very challenging. our data regarding vegf and vegf - r tumor expression were not statistically significant in relation to overall survival ; this could be attributed not only to the limited patient population, but also to the limited volume of tumor tissue obtained during biopsy rather than surgical resection. this might lead to the conclusion that vegf and vegf - r expression data should be reviewed with caution especially given data on stromal or tumor tissue expression as well as available information on the amount of available tumor tissue examined prior to any conclusions for prognosis or selection of therapy. in another study on 115 patients, serum levels of 14 - 3 - 3 were associated with a significantly longer survival in the methylation - positive group (15.1 versus 9.8 months ; p = 0.004).15 in our study, however, we tested 14 - 3 - 3 in paraffin - embedded tumor tissue, and both the positive and negative groups had similar survival. this suggests that the expression of 14 - 3 - 3 may be of more value for prognostic stratification of patients with nsclc if measured in the serum rather than by ihc analysis of paraffin - embedded tissues. over - expression of pakt and loss of pten expression in nsclc have been previously correlated with poor differentiation, lymph node involvement, distant metastasis, late stages as well as worse overall survival.16 unfortunately, our study did not confirm these findings. in fact, both negative and positive expressions of both receptors had almost equal survival. nf - kb, also known as p65, is a key transcription factor thought to play a major role in carcinogenesis, and its expression was shown to be significantly higher in advanced tnm stages (iii iv) than in earlier stages (i ii).16 in that study, 394 formalin - fixed and paraffin - embedded specimens from surgically resected lung tumors were examined. the overall survival analysis in patients with nsclc (n = 298) showed that nf - kb did not significantly influence the overall survival or disease - free survival.16 our results, however, are only relevant to the advanced stages of nsclc (stage iii, iv). in our study, patients with positive expression of nf - kb had a median survival of 9.9 months compared to 13 months in those with negative expression. however, only 4 of 46 patients had positive expression, making this a limited sample to draw broader conclusions. a larger scale study of nf - kb expression is warranted, especially with the ongoing research in targeted therapy utilizing nf - kb inhibitors in patients with lung cancer.17 for the future implications of our study, we are currently investigating the role of pharmacogenomics in relation to the k - ras mutation and have presented an abstract at iaslc / aacr (abstract) and the potential use of these markers if used in combination with molecular profiling. our study is retrospective, limiting the measurement of some key statistics. as a one - center study, most of the patients with advanced stage were only diagnosed with fine needle aspiration or small biopsies, and thus limited tissue was available for further studies. gemcitabine therapy, ercc1 negativity and treatment with platinum therapy, ecog ps 0 or 1, and female gender correlated with improved overall survival in patients with advanced nsclc. | introductionperformance status (ps) is the only known clinical predictor of outcome in patients with advanced non - small - cell lung cancer (nsclc), although pharmacogenomic markers may also correlate with outcome. the aim of our study was to correlate clinical and pharmacogenomic measures with overall survival.methodsthis was an irb approved, retrospective study in which the medical records of 50 patients with advanced nsclc from 19982008 were reviewed, and gender, race, ps, and chemotherapy regimens were documented. stromal expression of pharmacogenomic markers (vegfr, ercc1, 14 - 3 - 3, pakt, and pten) was measured. clinical factors and pharmacogenomics markers were compared to overall survival using a cox proportional hazards model.resultsforty patients received platinum - based therapy. median age was 65 years. improved ps, female gender, and gemcitabine therapy were significantly associated with longer overall survival (p = 0.004, p = 0.04, and p = 0.003, respectively). age was not associated with survival. caucasians had better overall survival in comparison to african americans with median survival of 14.8 months versus 10.4 months (p = 0.1). patients treated with platinum - based therapy had better survival of 15 months versus 8 months for non - platinum based therapy (p = 0.01). there was no significant association between any of the pharmacogenomics markers and overall survival other than in patients treated with platinum, in whom ercc1 negativity was strongly associated with longer survival (p = 0.007).conclusionercc1 negativity with platinum therapy, gemcitabine therapy, good ps, and female gender all correlated with improved overall survival in patients with advanced nsclc. |
indeed, patients with dementia often have neuropsychiatric symptoms collectively termed behavioral and psychological symptoms of dementia (bpsd).1 depression is quite common in elderly patients with dementia.2,3 aggressive behaviors that cause great difficulties in the social functioning of patients are also common, significantly increasing rates of nursing home placement.4,5 verbal and physical aggression could be strictly connected with depressive symptoms,68 but the relationship between depression, aggressive behavior, and dementia is not well defined. the objective of this study was to evaluate the relationship between neuropsychiatric symptoms and progressive cognitive decline by assessing cognitive impairment, depressive symptoms, and aggressive behavior in a sample of elderly nursing home residents. increased knowledge of the interaction between aggression, cognitive impairment, and depressive symptoms could lead to the identification of specific strategies in the management of health care for elderly patients. the study sample consisted of 201 men and women over the age of 65 years who were residing in one of three different care homes for the elderly. informed consent to take part in the study was provided by each subject or, in some cases, by a subject s legal guardian. the authors collected the subjects sociodemographic and health status data from medical records and through physical examination, with a particular focus on medical comorbidity. the authors also reported the functional status and psychopathologic symptoms using the activities of daily living scale and the neuropsychiatric inventory. for the purpose of the present study, each subject was evaluated using the mini - mental state examination (mmse),9 the geriatric depression scale (gds),10 and the modified overt aggression scale (moas).11 the mmse was developed as a brief screening tool to provide a quantitative assessment of cognitive impairment and to record cognitive changes over time. typically, these are grouped into seven cognitive domains : (1) orientation to time, (2) orientation to place, (3) registration of three words, (4) attention and calculation, (5) recall of three words, (6) language, and (7) visual construction. the test yields a total score of 30 and provides a picture of the subject s present cognitive performance based on direct observation of completion of test items or tasks. levels of impairment have been classified as mild (mmse score 2429), slight (mmse score 1623), moderate (mmse score 1115), and severe (mmse score 010). the gds is a 30-item self - report assessment designed specifically to identify depression in the elderly. the assessment requires yes or no answers this is thought to be simpler than scales that use a five - category response set and is generally recommended as a routine part of a comprehensive geriatric assessment. a score of 10 or lower is the usual threshold to separate depressed from nondepressed patients. levels of depression have been classified as borderline (gds score 1014) and present (gds score > 14). the moas, developed by kay and colleagues11 is based on the overt aggression scale.12 the moas is divided in four domains : (1) verbal aggression, (2) aggression against objects, (3) aggression against self, and (4) aggression against other people. a score from 0 to 4 is assigned to each act : 0 indicates no aggressive behavior and higher scores indicate increasing severity. the score in each category is multiplied by a factor assigned to that category : 1 for verbal aggression, 2 for aggression against objects, 3 for aggression against self, and 4 for aggression against other people. thus, the total moas score ranges from 0 (no aggression) to 40 (maximum grade of aggression). margari.13 validated the psychometric properties of the moas in italy by assessing its inter - rater reliability (p > 0.90) and predictive power. descriptive statistics were used to summarize the variables studied and the characteristics of the subjects. the authors analyzed correspondence between mean gds scores and score subgroups of the mmse and between mean moas scores and score subgroups of the mmse and the gds. the pearson s correlation coefficient (r), which can only take on values between 1 and 1, was used ; this could indicate a positive (as one variable increases, so too does the other) or a negative correlation (as one variable increases, the other decreases). furthermore, student s t - test was used to compare the means of two samples (male and female). with this test the authors evaluated the role of gender on depressive symptoms, aggressive behavior, and cognitive impairment. the authors used the data processing program statistical package for social science (v 19.0 ; spss inc, chicago, il) for statistical processing. the study sample consisted of 201 men and women over the age of 65 years who were residing in one of three different care homes for the elderly. informed consent to take part in the study was provided by each subject or, in some cases, by a subject s legal guardian. the authors collected the subjects sociodemographic and health status data from medical records and through physical examination, with a particular focus on medical comorbidity. the authors also reported the functional status and psychopathologic symptoms using the activities of daily living scale and the neuropsychiatric inventory. for the purpose of the present study, each subject was evaluated using the mini - mental state examination (mmse),9 the geriatric depression scale (gds),10 and the modified overt aggression scale (moas).11 the mmse was developed as a brief screening tool to provide a quantitative assessment of cognitive impairment and to record cognitive changes over time. typically, these are grouped into seven cognitive domains : (1) orientation to time, (2) orientation to place, (3) registration of three words, (4) attention and calculation, (5) recall of three words, (6) language, and (7) visual construction. the test yields a total score of 30 and provides a picture of the subject s present cognitive performance based on direct observation of completion of test items or tasks. levels of impairment have been classified as mild (mmse score 2429), slight (mmse score 1623), moderate (mmse score 1115), and severe (mmse score 010). the gds is a 30-item self - report assessment designed specifically to identify depression in the elderly. the assessment requires yes or no answers this is thought to be simpler than scales that use a five - category response set and is generally recommended as a routine part of a comprehensive geriatric assessment. a score of 10 or lower is the usual threshold to separate depressed from nondepressed patients. levels of depression have been classified as borderline (gds score 1014) and present (gds score > 14). the moas, developed by kay and colleagues11 is based on the overt aggression scale.12 the moas is divided in four domains : (1) verbal aggression, (2) aggression against objects, (3) aggression against self, and (4) aggression against other people. a score from 0 to 4 is assigned to each act : 0 indicates no aggressive behavior and higher scores indicate increasing severity. the score in each category is multiplied by a factor assigned to that category : 1 for verbal aggression, 2 for aggression against objects, 3 for aggression against self, and 4 for aggression against other people. thus, the total moas score ranges from 0 (no aggression) to 40 (maximum grade of aggression). margari.13 validated the psychometric properties of the moas in italy by assessing its inter - rater reliability (p > 0.90) and predictive power. descriptive statistics were used to summarize the variables studied and the characteristics of the subjects. the authors analyzed correspondence between mean gds scores and score subgroups of the mmse and between mean moas scores and score subgroups of the mmse and the gds. the pearson s correlation coefficient (r), which can only take on values between 1 and 1, was used ; this could indicate a positive (as one variable increases, so too does the other) or a negative correlation (as one variable increases, the other decreases). furthermore, student s t - test was used to compare the means of two samples (male and female). with this test the authors evaluated the role of gender on depressive symptoms, aggressive behavior, and cognitive impairment. a p - value of 14, indicating the presence of depressive symptoms ; the evaluation was not possible in 19.90% of the subjects because of the severity of clinical impairment. females showed more depressive symptoms than males (f = 13.21 ; p = 0.008). for the moas test, 20.89% of subjects showed self- or hetero - directed aggressive behavior (moas score > 0). in these subjects, the mean moas score was 5.19, corresponding to expressions of verbal aggression and aggression against objects. no gender differences were found for aggressive behavior (f = 10.18 ; p = 0.1). cognitive impairment, depressive symptoms, and aggressive behavior in elderly subjects are summarized in table 2. figure 1 presents the distribution of the mean gds score across the different ranges of mmse score. subjects with mmse scores at either end of the range reported lower moas scores than subjects with mmse scores in the central range. the borderline gds score (1014) was associated with mild cognitive impairment (mmse score 2429) and slight dementia (mmse score 1623) ; a higher mean score on the gds (> 14) corresponded to moderate dementia (mmse score 1115). the positive correlation between mmse and gds scores was not statistically significant (r = 0.087 ; p = 0.217). the range of distribution of mean scores between the mmse and the moas showed major impairment of cognitive functioning corresponded to an increase in aggressive behavior (figure 2). the subgroup without cognitive impairment (mmse score 30) corresponded to an absence of aggressive behavior (moas score 0), the subgroup with mild cognitive impairment (mmse score 2429) corresponded to a mean moas score of 1.1, the subgroup with slight dementia (mmse score 1623) corresponded to a mean moas score of 1.2, the subgroup with moderate dementia (mmse score 1115) corresponded to a mean moas score of 1.5, and the subgroup with severe dementia (mmse score 010) corresponded to a mean moas score of 1.7. analysis of the relationship between mmse and moas scores showed a statistically significant negative correlation (r = 0.440 ; p = 0.000). the range of distribution of mean scores between the gds and the moas showed an increase in moas score corresponded to an increase in gds score (figure 3) : the subgroup that showed no depressive symptoms (gds score 010) corresponded to the mean moas score of 0.4, the subgroup with a borderline score for depressive symptoms (gds score 1014) corresponded to the mean moas score of 0.8, and the subgroup with depressive symptoms present corresponded to the mean moas score of 1.3. in addition, the authors analyzed the relationship between age and the results of each rating scale test. the findings showed a statistically significant positive correlation between age and cognitive impairment (r = 0.150 ; p = 0.033). on the contrary, depressive symptoms and aggressive behavior did not have a significant correlation with age (depressive symptoms, r = 0.092, p = 0.195 ; aggressive behavior, r = 0.108, p = 0.126). the relationships between mmse score, gds score, moas score, and patient age are summarized in table 3. bpsd may affect up to 90% of patients with dementia during the course of their illness and includes verbal and physical aggression, psychotic symptoms, agitation, anxiety, and depression ; these are the major factors of institutionalization and of the associated health care costs.1417 depression is one of the most frequent comorbid psychiatric disorders in alzheimer disease (ad) and other dementias.3,18 in addition, patients with ad seem to have more depressive symptoms than patients with subcortical dementias. the relationship between depression and dementia is still not clear, as dementia and depressive syndromes demonstrate a substantial symptom overlap19 indeed, there are two types of depressive syndrome : (1) depression related to the development of dementia and (2) a reactive depression. the mmse detected cognitive impairment in 167 (83%), confirming that cognitive impairment is one of the most common reasons for admission to nursing home care.2022 the gds detected symptoms of depression in 105 (52%) of the 201 subjects, revealing a greater frequency than clinical examination. the distribution of the mean gds scores across the mmse score range might suggest that depressive symptoms have a tendency to increase with the deterioration of cognitive functioning ; this correlation is not statistically significant. when the cognitive decline is severe, the low gds scores in those subjects with severe cognitive impairment indicated by the mmse might be related to the difficulty of recording depressive symptoms in patients with severe dementia who are unable to communicate their feelings and thoughts and who may experience depressive symptoms linked to agitation and aggression. in the severe stages of dementia, the symptoms can only be indirectly observed as overt behavior, such as physically or verbal aggressive behavior. furthermore, verbal and physical aggression among patients with dementia may be related to a higher frequency of comorbid depressive symptoms.7,8 in addition, cognitive impairment was more common in male subjects. depression symptoms were more frequent in female subjects.23 in an epidemiologic study of community dwellers and nursing home residents, lyketsos al24 found that 23.7% of patients with dementia were rated as being agitated or aggressive. other studies involving older nursing home residents with dementia found a higher rate of verbal aggression than physical aggression17,25,26 and observed that physical aggression is usually preceded by verbal aggression.25 in agreement with these findings, results of the present study show that 20.9% of elderly subjects manifested aggressive behavior with a mean moas score of 5.19, corresponding to expressions of verbal aggression and aggression against objects. according to the literature,15,23,27 analysis of the data of 201 subjects admitted to nursing homes suggests a statistically significant correlation between a greater impairment of cognitive functioning and an increase of aggressive behavior. this means that subjects with severe dementia tend to exhibit more frequent verbal and physical aggression. furthermore, analysis of data suggests there is a relationship between depression and aggressive behavior. this finding suggests that aggressive behavior is correlated with depressive symptoms and the severity of cognitive impairment. in contrast, no significant correlation was found between age and symptoms of depression or aggressive behavior. therefore, aggressive behavior and depressive symptoms may be part of the clinical picture of senile dementia. for these reasons garcia - alloza supposed that cortical cholinergic acetylcholinesterase could be a putative marker of accelerated cognitive decline in ad and may also be implicated in bpsd such as aggressive behavior, supporting the use of cholinesterase inhibitors in alleviating noncognitive behavioral symptoms in patients with ad. lanctt found that the final stages of ad are associated with gamma - aminobutyric acidergic changes, which may contribute to depression and apathy in ad.29 a recent study by mitchell suggests that neuropsychiatric behaviors in ad share certain neurochemical features with psychiatric disorders such as major depression, and that distinct or overlapping disease processes leading to similar neurochemical alterations (eg, 5-ht1a receptor deficits) may manifest clinically as behavioral symptoms in both conditions. in a review study, borroni,32 reported that the involvement of dopamine- or serotonin - related pathways and the associated genetic variability have been demonstrated as being interesting candidates for the neuropsychiatry manifestations of dementia. moreover, genetic variations of neurotrophins, such as brain - derived neurotrophic factor, have been related to depression susceptibility in ad. these studies on neurobiological mechanisms may explain the relationship observed in the present study between cognitive impairment, aggressive behavior, and depression symptoms. the use of self - reports for assessing depression in elderly subjects with cognitive impairments was a limitation of this study. measurement of depression in dementia is difficult because symptoms such as apathy, indecisiveness, insomnia, and weight loss may be undetected. the gds test is known to show an adequate diagnostic accuracy in elderly people living in the community but does not show the same accuracy for elderly people in institutions. therefore, the gds should be used cautiously as a screening instrument in a population in which dementia is prevalent or in people known to have dementia, because the gds is not sensitive in detecting depression in demented subjects.33 to resolve these study limitations in any future research, self - reports for the elderly should be specific to elderly with cognitive impairments. these self - reports should be short, easily understood, and appropriate in terms of the size of letters in the items and in terms of the elderly subject s level of education ; they should include relevant age - related items ; and they should provide normative data on the elderly population with dementia. the use of self - reports for assessing depression in elderly subjects with cognitive impairments was a limitation of this study. measurement of depression in dementia is difficult because symptoms such as apathy, indecisiveness, insomnia, and weight loss may be undetected. the gds test is known to show an adequate diagnostic accuracy in elderly people living in the community but does not show the same accuracy for elderly people in institutions. therefore, the gds should be used cautiously as a screening instrument in a population in which dementia is prevalent or in people known to have dementia, because the gds is not sensitive in detecting depression in demented subjects.33 to resolve these study limitations in any future research, self - reports for the elderly should be specific to elderly with cognitive impairments. these self - reports should be short, easily understood, and appropriate in terms of the size of letters in the items and in terms of the elderly subject s level of education ; they should include relevant age - related items ; and they should provide normative data on the elderly population with dementia. in conclusion, the results of this study show that aggressive behavior and depressive symptoms are associated with progressive cognitive decline in elderly subjects. early assessment of these conditions can promote rational therapeutic strategies that may improve the quality of life and delay institutionalization for elderly patients. | patients with dementia often have neuropsychiatric symptoms. the objective of this study was to evaluate the relationship between neuropsychiatric symptoms and progressive cognitive decline by assessing cognitive impairment, depressive symptoms, and aggressive behavior in a sample of elderly subjects. the study sample consisted of 201 subjects admitted to nursing homes. for the purpose of the present study each subject was evaluated using the mini - mental state examination, the geriatric depression scale, and the modified overt aggression scale. the results show that aggressive behavior and depressive symptoms are associated with progressive cognitive decline in elderly subjects. early assessment of these conditions can promote rational therapeutic strategies that may improve the quality of life and delay institutionalization for elderly patients. |
during july 2011april 2014, a total of 45 blood samples were collected in edta - containing collection tubes from wild japanese macaques in aomori (n = 25), yamagata (n = 5), and wakayama (n = 15) prefectures in japan. the animals were captured by licensed trappers, in accordance with the wildlife protection and proper hunting act, by using large hand - made cage traps and commercial cage traps (no. the physical conditions of each animal were recorded before they were euthanized, according to the guidelines of the japanese veterinary medical association. freeze - thawed blood samples were spread onto chocolate agar plates (8) for isolation of bartonella spp. and incubated at 35c under 5% co2 for up to 4 weeks. bartonella - specific pcrs that targeted the glta (9) and rpob (10) genes and the 16s23s rdna intergenic transcribed spacer (its) regions (11) were used for identification of bartonella isolates ; genomic dna of b. alsatica strain ibs 382 and nuclease - free distilled water were used as positive and negative controls for the pcrs, respectively. bartonella isolates were obtained from 6 (13.3%) of 45 japanese macaques ; 1 (4.0%) of 25 macaques in aomori, 1 (20.0%) of 5 in yamagata, and 4 (26.7%) of 15 in wakayama prefectures. the bacteremia levels in the macaques ranged from 5.0 10 to 3.7 10 cfu / ml. the dna sequences of all 30 isolates were identical in the glta (338 bp), rpob (825 bp), and its (1,297 bp) regions ; the sequences were registered in genbank / european nucleotide archive in embl, and ddbj under accession nos. lc031777 (glta), lc031778 (rpob), and lc031779 (its). blast searches (http://blast.ncbi.nlm.nih.gov/blast.cgi) indicated that the dna sequences of the isolates had the highest degree of similarity (100% for glta and rpob, 99.5% for its) with those of b. quintana rm11 strain from rhesus macaques. subsequently, mlst analysis with 9 loci (12) revealed that 6 representative strains (mf11, mf31, mf101, mf111, mf191, and mf341 strains) from each culture - positive macaque were identical and belonged to a new sequence type (st), st22. the allelic profiles of st22 and other sts are shown in the technical appendix table. a phylogenetic relationship between st22 and other known sts was analyzed by using eburst version 3 (http://eburst.mlst.net/default.asp) in combination with the mlst data. a clonal complex was defined as the group of sts that had identical alleles at 8 of 9 loci, and the lineage was defined as the group of sts that had identical alleles at 7 of the 9 loci. as previously reported (6,12), sts 14, sts 6 and 7, sts 810 and 14, and sts 1521 formed clonal complexes 1, 2, 3, and 4, respectively, whereas st22 remained a singleton (figure 1). in terms of lineage classification, all sts, except st22, were divided into 3 individual lineages by the host animal species : sts 17 for human strains, sts 814 for cynomolgus macaque strains, and sts 1521 for rhesus macaque strains. in contrast, st22 from japanese macaque strains belonged to a singleton lineage. phylogenetic relationship among 1 to 22 sequence types (sts) of bartonella quintana strains based on eburst analysis (http://eburst.mlst.net/default.asp). clonal complexes 1, 2, 3, and 4 consist of sts 14, sts 67, sts 810 and 14, and sts 1521, respectively. a lineage was defined as a group of sts that had 7 identical alleles. we constructed a phylogenetic tree with the concatenated sequences (4,270 bp) of the 9 loci in each st using the maximum - likelihood method in mega6 (13). the sts 17 from human strains, sts 814 from cynomolgus macaque strains, and sts 1521 from rhesus macaque strains were classified into groups 1, 2, and 3, respectively, as with the lineage classification by eburst analysis. all strains of st22 formed a monophyletic clade defined as group 4 (figure 2). the tree was constructed from the concatenated sequences (4,270 bp) of the 9 loci used for multilocus sequence typing by using the maximum - likelihood method based on the tamura 3-parameter model in mega6 (13). the 22 sequence types (sts) of b. quintana strains from humans (sts 17), cynomolgus macaques (sts 84), rhesus macaques (sts 1521), and japanese macaques (st22) were included in the tree. our study shows that natural infection with b. quintana can occur in free - ranging nonhuman primates in japan. the japanese macaques harboring b. quintana showed no clinical abnormalities, although bacteremia levels were considerably high (> 10 cfu / ml) in 3 animals that tested positive. these data suggest that japanese macaques are one of the natural reservoirs of b. quintana. all isolates from japanese macaques were more closely related to rhesus macaque strains than to human strains in the glta, rpob, and its regions. by mlst analysis with 9 loci, all 6 representative strains from japanese macaques were identified as st22 (6). thus, st22 is likely a new genotype of b. quintana specific to japanese macaques. because wild - caught japanese macaques from only 3 prefectures were examined for b. quintana bacteremia, a large - scale surveillance study would help elucidate the genetic diversity of japanese macaque strains. according to eburst analysis, human, cynomolgus macaque, and rhesus macaque lineages as reported previously (6,12), the primary founders in clonal complexes 1, 3, and 4 were reconfirmed as sts 2, 9, and 15, respectively. however, st22 from wild - caught japanese macaque strains was not found in any other clonal complex and formed an independent lineage. through phylogenetic analysis with concatenated mlst sequences, li. (6) showed that 3 groups were formed by each host species ; this finding was confirmed in our study. are known to have an adaptive strategy of causing asymptomatic and prolonged bacteremia in their specific reservoirs (14). our data support the idea that b. quintana may have separately co - evolved with the macaque species and humans. notably, bartonella dna was recently detected in gorillas from west africa, suggesting that nonhuman primates, including apes, could be naturally infected with bartonella spp. further studies are necessary to clarify the prevalence of b. quintana and the vector of the organism in other nonhuman primates, and the potential of these primates to serve as a source of infection to humans. | bartonella quintana bacteremia was detected in 6 (13.3%) of 45 wild - caught japanese macaques (macaca fuscata). multilocus sequence typing of the isolates revealed that japanese macaques were infected with a new and specific b. quintana sequence type. free - ranging japanese macaques thus represent another natural reservoir of b. quintana. |
as described in detail below, we first estimated the exposure - response relationship between observed daily mortality and temperature data in manhattan. we then obtained downscaled future temperature projections from sixteen climate models and two greenhouse gas emissions scenarios for manhattan. these two inputs were combined to estimate future mortality related to future temperatures, which were compared to temperature - related mortality in a climatalogical baseline period. we separately accounted for cold - season and warm - season mortality, and also computed net annual changes. historical data on daily deaths covering the 1982 1999 period for manhattan were obtained from the us national center for health statistics (nchs). daily death counts for all internal causes (icd-9 codes 0799.9 for 19821998 and icd-10 codes a00-r99 for 1999) were pooled, excluding accidental causes. we chose this definition for consistency with previous studies ; however this would tend to underestimate heat - related mortality since heat stroke is an daily tmax data were obtained from the national climatic data center for 1982 to 1999 at the central park station. a statistical model (see supplemental equation 1) was developed using poisson general linear regression with log daily non - accidental death counts as the outcome variable and the following predictors : a spline function of daily tmax with 3 degrees of freedom, a natural spline of time with 7 degrees of freedom per year, and a day of week indicator variable. this approach was similar to that used to study temperature for 11 eastern us communities. sensitivity analyses investigated potential confounding by ozone, pm10, dewpoint temperature and influenza epidemics. future temperature projections were developed using downscaled outputs from 16 global scale general circulation models (gcms) used in the intergovernmental panel on climate change fourth assessment report, in conjunction with two future greenhouse gas emissions scenarios. the approach uses monthly bias - corrected and spatially disaggregated (bcsd) climate projections at 1/8 degree resolution derived from the wcrp cmip3 multi - model dataset. the output from the land - based grid box corresponding to central park, ny was used to create change factors for each calendar month based on the difference between each 30-year future time slice and the same gcms 30-year baseline time slice. these change factors are then applied to the daily central park weather data to create a future projection with the same statistical characteristics and sequence as the observations. the approach described here does not explore how changes in intra - annual and inter - annual temperature variability may change, for several reasons. additionally, the new york city weather station used in this study does not show a significant trend in the variance of either daily summer maximum temperatures or winter minimum temperatures. further, in an analysis of daily projections for the nyc gridbox from three gcms, horton. showed that neither summer tmax nor winter tmin showed significant changes in variance through 2080. by not considering sub - monthly changes in variability, we were able to use fine spatial resolution projections (as the 1/8 degree bcsd product is monthly, not daily) and analyze the entire 21 century (whereas using daily data would have constrained the analysis to the 2046 - 2065 and 20812100 timeslices for which only a subset (9) of the gcms are available from the pcmdi data portal). note that by applying the delta method separately for each calendar month, we do capture one component of possible changes in intra - annual variance, changes in the annual temperature cycle. this methodology yielded a set of 32 synthetic future temperature projections for daily tmax from 2010 to 2100 based on the three 30 year time slices, and for a baseline period 19701999. greenhouse gas emissions scenarios represent specific blends of demographic, social, economic, technological and environmental assumptions. we selected 2 scenarios, a2 and b1, which represent relatively high and low greenhouse gas growth projections, respectively. the a2 scenario assumes relatively rapid population growth and limited sharing of technological change, which combine to produce high greenhouse gas levels by the end of this century, with emissions growing throughout the entire century. the b1 scenario assumes a high level of environmental and social consciousness, which leads to sustainable development, low population growth, high economic and technological advancement, and low energy use. projected mortality impacts were estimated using modeled daily tmax. for any day with tmax greater than 71f, the change in mortality was calculated relative to the minimum mortality temperature for the heat effect, i.e. 59 f. for any day with tmax less than 63f, the change in mortality was calculated relative to the minimum mortality temperature for the cold effect, i.e. 72 f. for days with tmax from 63f to 71f daily additional deaths were computed as (1)mortality = y0ercpop where : mortality is daily temperature - related additional deathsy0 is baseline daily mortality rate (per 100,000 population)pop is county populationerc is percentage change in mortality for a specified change in temperature, derived from the statistical analysis of observed data as described above. mortality is daily temperature - related additional deaths y0 is baseline daily mortality rate (per 100,000 population) pop is county population erc is percentage change in mortality for a specified change in temperature, derived from the statistical analysis of observed data as described above. we computed temperature - related daily deaths in this way for each time period (1980s, 2020s, 2050s, 2080s), and then computed the average number of temperature - related deaths per year (figure 2). we also computed percent changes in annual average deaths from the 1980s to future time periods (table 1). the population of manhattan was based on data obtained from the us census 2000 survey, and was held constant throughout the projection period. baseline mortality rates for all ages, which excluded deaths attributable to external causes, were obtained from the u.s. centers for disease control and prevention. | global average temperatures have been rising for the past half - century, and the warming trend has accelerated in recent decades1. further warming is expected over the next few decades, with significant regional variations. these warming trends will likely result in more frequent, intense and persistent periods of hot temperatures in summer, and generally higher temperatures in winter. daily death counts in cities increase markedly when temperatures reach levels that are very high relative to what is normal in a given location24. relatively cold temperatures also appear to carry risk2,4. rising temperatures may result in more heat - related mortality but may also reduce cold - related mortality, and the net impact on annual mortality remains uncertain. here we use 16 downscaled global climate models and two emissions scenarios to estimate current and future seasonal patterns in temperature - related mortality in manhattan, new york. all 32 projections yielded warm season increases and cold season decreases in temperature - related mortality, with positive net annual temperature - related deaths in all cases. monthly analyses showed that the largest percentage increases may occur in may and september. these results suggest that, over a range of models and scenarios of future greenhouse gas emissions, increases in heat - related mortality could outweigh reductions in cold - related mortality, with shifting seasonal patterns. |
polyamines (pas) are small positively charged aliphatic molecules ubiquitous in almost all life forms. in plants, spermine (spm), spermidine (spd), and their precursor putrescine (put) are the major pas present in cells at micromolar - to - millimolar concentrations. pas have been implicated in a wide range of life processes in plants including seed germination, growth, floral initiation, floral development, pathogen defenses, and environmental stress responses [24 ]. following the classic report of richards and coleman on pa accumulation in potassium starved leaves, many investigations have demonstrated the physiological relevance of pas in response to diverse environmental stresses, including heavy metal stress, so2 pollution, osmotic stress, chilling stress, drought stress, ph stress, nutritional stress, biotic stress, and heat stress (reviewed by [4, 68 ]). although the explicit physiological role of the increase in pa remains obscure, exogenous addition of pas to plants under stress conditions has been reported to alleviate stress damage or to increase tolerance to adverse environments [6, 8 ]. a recent study suggested crosstalk with or direct involvement of the similarly multifunctional molecule nitric oxide (no) in a pa - mediated response. regardless of the fact that underlying mechanism of its action remains unclear, it is evident that pas are an integral part of plant stress responses. plants, owing to their sessile nature, are compelled to endure stress due to perpetual environmental changes. they do so through morphological, biochemical, or physiological adjustments. under stressful conditions, the tiny globally - distributed water ferns of the genus azolla respond through a unique rapid loss of their roots, a phenomenon termed rapid root abscission [1113 ]. such shedding sets its fronds free from root - entangled mats and facilitates their dispersion to a potentially better environment. the phenomenon is thus considered to be an important survival strategy for azolla [12, 13 ]. uehda and coworkers have demonstrated that rapid root abscission is primarily due to rapid osmotic expansion of cells at the base of the roots with presumptive activation of hydrolytic enzymes in the cell wall to hasten the root separation [12, 14 ]. unlike typical abscission events in other plants, programmed cell death previous studies revealed that rapid abscission in azolla can artificially be induced by nutrient stress, a high concentration of nitrite, chemical stress (i.e., treatment with the inhibitors of oxidative phosphorylation sodium azide, 2, 4-nitrophenol and carbonyl cyanide m - chlorophenyl - hydrazone), and by transient exposure to high temperature. an array of stimuli culminating to the same response in azolla leave open the possibility of a common inducer or internal mediator involved in the root abscission process. since higher plants exposed to a broad spectrum of abiotic stresses exhibit alteration in pa metabolism, we sought to investigate the potential role of pas in rapid root abscission of azolla. alterations of pa content have been observed during high - density induced sporulation in azolla and in response to exposure to ozone and nitrogen dioxide. to the best of our knowledge, no study has yet specifically examined the effect of polyamines on rapid root abscission. the aim of this study was to assess the effect of exogenously applied pas on rapid root abscission phenomenon. fronds of the water fern were originally collected from a local taro field in okinawa, japan. fronds were treated with a solution containing 0.12% (v / v) sodium hypochlorite and 0.01% (v / v) triton x-100 for 3 min and then repeatedly washed in a large volume of distilled water before transferring to the nutrient medium. a two - fifth strength nitrogen - source - free hoagland 's e - medium was used for the culture of a. pinnata. it contained 200 m potassium dihydrogenphosphate, 400 m magnesium sulphate heptahydrate, 0.31 m zinc sulphate heptahydrate, 0.14 m copper sulphate pentahydrate, 14.32 m ferric chloride hexahydrate, 920 m calcium chloride, 3.68 m manganese chloride tetrahydrate, 18.5 m boric acid, 0.15 m disodium molybdate (vi) dehydrate, and 41.26 m ethylene diamine - n, n, n, n-tetraacetic acid, disodium salt, dehydrate. in addition, 0.12 m cobalt chloride hexahydrate was also supplied into the medium, and ph was adjusted to 5.8 with potassium hydroxide. fronds were grown in a plant growth chamber (type fli-2000h, eyla, japan) maintained at 27 1c, 80% humidity, 16 : 8 h light : dark photoperiod and 50 mol m s (at plant level) provided by fluorescent lamps (type fl 40 sbr - a, nec, japan). for experiments, 1520 fronds were randomly selected from the cultured a. pinnata stock and derooted manually using forceps. rootless fronds were then washed at least twice in distilled water and cultured in the nutrient medium. abscission experiments were carried out with fronds which had produced new roots of the same age. in order to assess the potential of pas to induce root abscission, 2 - 3 fronds (2030 roots) of a. pinnata were first placed on a 10 mm potassium phosphate buffer solution, ph 7 (control set) at a room temperature (25c). then pa putrescine dihydrochloride, spermidine or spermine (0.55 mm) was subsequently added. the number of roots (> 10 mm in length) abscised was recorded every 10 min for 2.5 hr. total abscission rate (%) was determined as the ratio of the detached to the initial number of roots. exogenous addition of 2 mm spm and spd resulted in change in the ph of the phosphate buffer rising from neutral to 9.0 and 8.3, respectively. in order to determine whether the ph rise influenced pa - induced abscission in a. pinnata, experiments were conducted with or without readjusting the ph of the buffer to 7.0 after addition of 2 mm spm or spd using hydrochloric acid. we also assessed root abscission in response to 2 mm spermine tetrahydrochloride (spm4hcl) ; the acidic form of spm which does not increase the ph to alkaline. as a control, a. pinnata fronds were placed in 10 mm potassium phosphate buffer with ph ranging from 4 to 10 and root loss monitored for 2.5 hr. to study the effect of temperature, a. pinnata fronds were transferred to beakers containing neutral phosphate buffers, and then 2.0 mm spm the beakers were then placed in a water bath maintained at 25c, 27c, 32c, and 38c. abscission rate was recorded every 10 min for 2 h. in order to examine the effect of light and light intensity, a. pinnata fronds suspended in phosphate buffer containing 2 mm spm were subjected to varying light environment, that is, dark condition (0.01 mol m s), ambient light (5 mol m s), and high light (250 mol m s). abscission was monitored every 10 min for 2 h. in order to gain some insight into the identity of the molecular inducer in pas - induced abscission, experiments were performed using the catabolic products of higher pas. polyamine oxidase (pao) degrades spd and spm and produces a stress signaling molecule hydrogen peroxide (h2o2) and 1, 3-diaminopropane (dap) ; the latter can be further converted to -alanine (ala). a. pinnata fronds were treated with 2 mm ala, dap, or 210 mm h2o2 dissolved in the neutral buffer (ph 7.0), and the abscission induced by each treatment was monitored. pas are reported to stimulate no production in plants via an unknown pathway [23, 24 ] as well as modulate the activity of nitrate reductase (nr), the key enzyme in the nitrite - dependent no production in plants. no emission was detected with a sievers nitric oxide analyzer (noa) 280i, which employs ozone - chemiluminescence technology, and the data was collected by noanalysis software. a confluent layer of a. pinnata covering the surface area of a 10 cm diameter petridish containing 20 ml of neutral phosphate buffer was used for no measurement at room temperature (25c). the petridish contained two holes, one at the side for the inserting the tip of noa and the other at the top cover to supply the chemicals. to ensure proper mixing, basal no production of a. pinnata in neutral phosphate buffer was measured for 10 min. then 0.1 mm sodium nitrite, a well - known no source in plants, was supplied to initiate no production. after 10 min, 2 mm of spm or spd was added to examine the effect of pas on nitrite - dependent no production. at 25 min, the same concentration of pa was added and the no measurement carried out until 50 min. spermidine trihydrochloride (spd3hcl) and spermine tetrahydrochloride (spm4hcl), the acidified form of spd and spm, respectively, were used to prevent the medium ph from increasing to highly alkaline. throughout the experimental period, photos of entire fronds were taken with a digital camera (eos kiss digital, canon, japan). for close - up observations, a zoom stereomicroscope (model sz 61, olympus, japan) with a digital camera (model c7070wz, olympus, japan) was used. the proximal portion of an abscised root was observed under a light microscope (model eclipse 80i, nikon, japan). figure 1 presents a typical sequence of events of root abscission induced by pas. exogenous addition of spd or spm to the free - floating a. pinnata fronds caused significant shedding of roots within 2.5 hours after the treatment (figures 1(a) and 1(b)). we observed that pa - induced abscission was accompanied by rounding off of the cells at the proximal end of the detached roots (figure 1(f)), similar to that observed in response to inhibitors of oxidative phosphorylation. figure 2 shows time courses of root abscission in a. pinnata in response to 0.55 mm pas. spd or spm induced significant rapid root abscission of a. pinnata. both the extent of the response and the onset of abscission were dependent on the concentration of the pa. an increase in spd or spm concentration from 0.5 mm to 5 mm reduced the time for the onset of abscission from approximately 80 min to 30 min, respectively. however, with a longer incubation period (48 hrs), put was found to have the same effect as spd and spm, consistent with its conversion (albeit slow) to spd and spm (data not shown). abscission rates in response to spd and spm showed similar concentration dependency within the 2.5 h experimental period (figure 2). neutral phosphate buffer alone without any added pas failed to induce abscission (data not shown). the total abscission rate after 2.5 hr of 0.55 mm put treatments was negligible (0.52%) (figure 2(a)). there was no significant difference between the total abscission rates by 0.5 or 2 mm spd and spm. moreover, an increase in their concentration to 3 or 5 mm resulted in only a 9 - 10% rise in the abscission rate (figures 2(b) and 2(c)). since spm was the most effective pa, 2 mm spm was used for further experiments. it was suggested that enzyme(s) involved in the cell wall dissolution process during rapid root abscission in azolla may be activated by neutral or weakly alkaline ph. in the present study, we did observe substantial ph increases in the phosphate buffer solution upon addition of pas and found that this ph increase did enhance the abscission response (figure 3). however, an increase in ph could not solely be responsible for the pa - induced root abscission based on our observations of effective abscission in response to pas even after readjustment of ph to 7 or acidified for spm and an almost negligible occurrence of abscission in potassium phosphate buffers at various phs ranging from 4 to 10 (figure 3). we further verified ph effects with good buffers (mes, mops, hepes, tricine) from ph 6 to ph 9 and confirmed that ph changes alone were ineffective in triggering the response (data not shown). exposure of a. pinnata to a temperature gradient did not affect the total abscission rate (7580%) in response to spm, but higher temperatures resulted in a significantly shorter lag time for the onset of abscission and total time required to reach 50% abscission rate (figure 4). the time lag was 40 min at 25c, 30 min at 27c, and 32c, 20 min at 38c. similarly to increasing temperature, the time required to shed 50% of the roots also decreased : 75 min at 25c and 27c, 60 min at 32c, and 40 min at 38c. the availability of light as well as light intensity affected pas - induced abscission rate, time required for the onset of abscission, and time to reach 50% root abscission in a. pinnata (figure 5). exposure to 2 mm spm under ambient light in the laboratory showed a time lag of 50 min a total abscission rate of 80% and took 65 min for 50% root abscission. when the light intensity was increased by 50 times to 250 mol m s, the time lag did not change but abscission rate decreased to 60%, and it took longer (100 min) to reach 50% root abscission. in contrast, dark conditions (0.01 mol m s) stimulated the response ; the time lag reduced to 40 min, 50% abscission was attained within 60 min, and final abscission rate at 2 h was 88%. previous studies have revealed an important role for pa catabolism via polyamine oxidase (pao) in plant stress tolerance. thus, it was essential to check the effect of the pa oxidation products on the rapid root abscission phenomenon. the pa oxidation products dap, ala and h2o2 showed no significant abscission - triggering effect (figures 6(a) and 6(b)). the abscission rates in the presence of ala were only 12.5% and were negligible (< 5%) in response to treatments with h2o2 and dap. furthermore, no change was observed in the total abscission rate by spm in presence of hydroxyethyl hydrazine (heh), an inhibitor of pao (data not shown). these results suggest that spm and spd do not induce root abscission by supplying their degradation products ; the parent compound spm or spd is required for the induction. pa metabolism has been suggested to be associated with plant no synthesis [23, 24, 27 ]. no is a signaling molecule that is involved in diverse plant environmental responses [28, 29 ]. possible crosstalk between pas and no in stress responses led us to examine the involvement of no in the pa - induced root abscission. no released from a. pinnata was measured directly in the gas phase using a sievers nitric oxide analyzer (noa) 280i (figure 7). no detectable amount of no signal was observed when the three major pas were added (data not shown). however, when nitrite was supplied, the signal increased rapidly and reached to a range of 80100 ppb. when dh2o or pas were supplied, a slight decrease in the signal was observed for couple of seconds and the signal bounced back to its original pace of no production. these results suggest that a. pinnata does not stimulate detectable no emission in response to pas and has no effect on nitrite - dependent no production. the presence of pas in a. pinnata was found more than a decade ago but their physiological roles have remained unclear. the present study has clearly shown that the pas spd and spm are strong inducers of rapid root abscission whereas put is ineffective in the short term (figures 1 and 2). a smaller effect of put relative to the higher pas spd and spm has been found in many previous studies that investigated effects of pas in various physiological processes, including stress responses [4, 79, 30 ]. those different lines of observations may underlie an unknown common mechanism which involves spd and spm as inducers or signals for plant cells. assessment of the transport / uptake and metabolism of the exogenously added pas could clarify the observed discrepancy among individual pas. in higher plants, cellular uptake of pas is very rapid (within a few minutes) and is known to be accelerated by auxin. however, the identity of transporters for each polyamine remains unknown [23, 31 ]. further studies on uptake, transport as well as measurement of cellular pas on exposure to exogenous pas are essential to address the abscission - inducing efficiency of pas. in the time courses of spd or spm - induced root abscission, a one - hour time lag should be beyond that required for the uptake of chemicals into cells (figure 2). since pas are rapidly taken up by higher plants and stored in vacuoles, it is logical then to assume that the time lag reflects transport or their metabolism in the cells. plants, including a. pinnata, contain the enzyme polyamine oxidase which oxidizes higher pas to yield h2o2, dap, and ala. h2o2 is known to function as a signaling molecule in biotic and abiotic stress responses while ala derived from dap functions as an osmoprotectant. however, h2o2 could not stimulate abscission even at a very high concentration (figure 4(b)). furthermore, the other degradation products, dap and ala, also failed to trigger root abscission in a. pinnata (figure 4(a)) which led us to rule them out as the candidate for the abscission inducer. tun and coworkers and silveira and coworkers demonstrated that treatment with the pas spm and spd results in the production of no in higher plants, suggesting that no may be involved in pa - mediated physiological response in plants. pas are now considered as one of several candidate substrates for no production in plants via an unknown pathway [23, 24 ]. laboratory cultures of a. pinnata cultivated under favorable conditions produced very low amounts of no (figure 7). interestingly, nitrite, which stimulates no production in a. pinnata (figure 7), initiated rapid root abscission in a. pinnata with a shorter time lag compared with spm or spd. moreover, detectable amounts of no emission from a. pinnata could not be measured in response to these pas. thus, our results do not point to an obvious role for no in the polyamine - induced root abscission process in a. pinnata. in conclusion our results have clearly shown that higher pas spd and spm are efficient inducers of rapid root abscission in a. pinnata, and the subsequent rise in ph is not the only factor responsible for the onset of the abscission. to the best of our knowledge it is the first study to scrutinize the effect of pas on the unique adaptive response of a. pinnata. although pas are omnipresent stress markers in plants exposed to virtually all stresses, their explicit mode of action remains largely unknown. the rapid root abscission phenomenon in a. pinnata provides a unique clue for understanding the function of pas in stressed plant cells. | floating ferns of the genus azolla detach their roots under stress conditions, a unique adaptive response termed rapid root abscission. we found that azolla pinnata plants exhibited dose - dependent rapid root abscission in response to the polyamines spermidine and spermine after a substantial time lag (> 20 min). the duration of the time lag decreased in response to high ph and high temperature whereas high light intensity increased the time lag and markedly lowered the rate of abscission. the oxidation products of polyamines, 1,3-diaminopropane, -alanine and hydrogen peroxide all failed to initiate root abscission, and hydroxyethyl hydrazine, an inhibitor of polyamine oxidase, did not inhibit spermine - induced root abscission. exposure of a. pinnata to the polyamines did not result in detectable release of no and did not affect nitrite - dependent no production. the finding of polyamine - induced rapid root abscission provides a facile assay for further study of the mode of action of polyamines in plant stress responses. |
they include low dietary intake, conditions associated with decreased absorption or increased urinary phosphate excretion, and conditions associated with shifts in phosphate from the extracellular to intracellular fluid. the latter include various cellular uptake syndromes, in which hypophosphatemia develops as a result of phosphate consumption by rapidly proliferating cells.1,2 the association between hypophosphatemia and cytokines has recently been investigated, and some authors have reported that hypophosphatemia can be induced directly by certain cytokines.3,4 the eshap (etoposide, methylprednisolone, cytarabine, and cisplatin) protocol is an effective treatment for hodgkin s lymphoma,5,6 and the effect of this protocol on serum electrolytes has been reported previously in some individual observational studies. other electrolyte imbalances are seen mainly in patients receiving cisplatin. in a study published in 2012, there was a significant decrease in serum electrolytes including magnesium, potassium, phosphorus, and calcium, following use of this agent.7 hypophosphatemia induced by eshap is not well described, and is rarely considered as a side effect of the chemotherapeutic agents in this protocol. herein, we report a case of severe hypophosphatemia concomitant with administration of the first cycle of the eshap protocol to a patient with hodgkin s lymphoma. a 51-year - old woman (weight 55 kg, height 160 cm) with a history of hypertension, hepatitis c infection, chronic liver disease with ascites, esophageal varices, and mild renal impairment, was admitted electively to the national centre for cancer care and research, doha, qatar, on april 22, 2012 to receive her first cycle of salvage eshap protocol for treatment of recently relapsed hodgkin s lymphoma. her medications consisted of entecavir 1 mg once daily, propranolol 20 mg twice daily, spironolactone 50 mg once daily, allopurinol 100 mg once daily, furosemide 40 mg once daily, and lactulose 30 ml twice daily. on april 23, 2012 she was started on the first dose of cycle 1 of the intravenous eshap protocol as follows : etoposide 40 mg / m / day on days 14 ; methylprednisolone 500 mg / day on days 14 ; cytarabine 2000 mg / m on day 5 ; and cisplatin 25 mg / m / day as a continuous infusion on days 14. her laboratory values prior to, throughout, and 24 hours after finishing this cycle of chemotherapy were essentially normal (table 1), and she was discharged home on april 28, 2012 (day 6 of chemotherapy). two days later (day 8 of chemotherapy) she was readmitted with a temperature of 38.9c. at this time she had a normal complete blood count and electrolyte levels (table 1), and was started empirically on intravenous vancomycin 1 g every 12 hours and piperacillin - tazobactam 4.5 g every 8 hours. on day 10 (5 days after completion of treatment), she developed leukopenia and neutropenia (white cell count 900/l and absolute neutrophil count 800/l), and her phosphorus level was 0.73 she was started on granulocyte colony - stimulating factor (filgrastim 300 g subcutaneously once a day for 8 days), at which time her white cell count continued to drop and reached 200/l on day 13 (8 days after completion of treatment), and then started to recover, as shown in table 1. the phosphorus level continued to drop, and on day 17 (12 days after completion of treatment) reached a critical level of 0.34 mmol / l (asymptomatic hypophosphatemia) and potassium dropped to 2.4 mmol / l, although renal function and serum calcium were normal. she was given a 20 mmol intravenous potassium phosphate infusion on two successive days and was started on neutral phosphate 250 mg orally three times daily. her serum phosphate returned to normal after 4 days, and was maintained at 1.35 mmol / l after discontinuing supplementation. this report describes a patient with severe hypophosphatemia after administration of the first cycle of the eshap protocol and granulocyte colony - stimulating factor started on day 10 (5 days after completion of treatment) to treat the chemotherapy - induced leukopenia and neutropenia. eshap is an effective salvage strategy for hodgkin s lymphoma, and is associated with an overall response rate of approximately 70%80%.8,9 the effect of this protocol on serum electrolytes has been reported previously by some individual observational studies, and magnesium has been the electrolyte most commonly mentioned. further, hypophosphatemia has been reported as a complication of cisplatin therapy, although has been usually asymptomatic.10 being infrequent complications, the exact cause of cisplatin - induced hypophosphatemia and hypobicarbonatemia is not known, but significant hypophosphatemia was observed in 67% of patients in one study, particularly in female patients.11 another phase ii study examined the effect of fludarabine, cytarabine, cyclophosphamide, cisplatin, and granulocyte macrophage colony - stimulating factor in patients with richter s syndrome and refractory lymphoproliferative disorders, and hypophosphatemia was reported in 10% of cases.11 the association between hypophosphatemia and cytokines has recently been investigated, and some authors have reported that hypophosphatemia can be induced directly by certain cytokines.3,4 cytokines are low molecular weight proteins and glycoproteins that are secreted principally by activated lymphocytes and macrophages, and have a wide range of functions in the hematopoietic and immune systems. it has been postulated that characteristic clinical and histopathological features of malignant lymphomas may be due to activation of cytokines. however, there are some conflicting and unreliable trends in interleukin-6 levels after chemotherapy, with persistently elevated levels seen in patients in clinical remission.12 various cytokines are associated with the development of hypophosphatemia. recent research has evaluated the interrelationship between cytokine release, rise in white cell count, and development of hypophosphatemia.4 the median time taken to reach the minimum phosphate level was 8 days. there was a significant correlation between day of the minimum phosphate level and day of maximum white cell count, and a significant correlation between the decrease in phosphate level and the increase in white blood cells.3,4 in the present case report, our patient developed asymptomatic hypophosphatemia starting on day 10 (ie, 8 days after receiving chemotherapy and granulocyte colony - stimulating factor, which is known to be associated with release of cytokines and an increase in white cell count) and showed a significant correlation between day of minimum phosphate level and day of maximum white cell count, along with a significant correlation between the decrease in phosphate level and increase in white cell count (table 1). cytokine levels were not measured, but the clinical findings in our patient were consistent with previously reported data. serious manifestations of hypophosphatemia were prevented by phosphate infusion, and recurrence of hypophosphatemia was prevented by oral supplementation, and addressing other factors that might have contributed to development of hypophosphatemia (vitamin d, urinary phosphate excretion, parathyroid hormone, arterial blood gas analysis, or any other drug which might have aggravated the effect of cisplatin). using the naranjo scale for adverse drug reactions, we obtained a score of 7 (probable adverse drug reaction, see table 2).13 based on this score and our clinical and investigation findings, we considered the possibility of severe hypophosphatemia being induced after the first cycle of the eshap protocol. in view of the critical level of hypophosphatemia seen in our patient, close monitoring of phosphate levels and consideration of supplementation is warranted with the eshap protocol, especially when it is used in combination with granulocyte colony - stimulating factor and diuretics. this is a novel finding in the sense that, to the authors knowledge, this side effect has not been reported previously. health care providers should be aware of this potential toxicity, and appropriate monitoring of patients should be implemented. patients receiving this protocol should have their serum phosphate levels monitored throughout treatment, because some would require phosphate supplementation in the second and third weeks after chemotherapy, taking into consideration the need to evaluate other potentially contributing factors, including concomitant medications, vitamin d levels, urinary phosphate excretion, parathyroid hormone, and arterial blood gas analysis. | the effect of the eshap (etoposide, methylprednisolone, cytarabine, cisplatin) salvage protocol on serum electrolytes has been previously reported by individual observational studies. the most commonly described electrolyte affected by the eshap protocol is magnesium. in addition, hypophosphatemia has been studied and reported as a complication of cisplatin therapy, although it is usually asymptomatic. this is a case report of a 51-year - old woman with relapsed hodgkin s lymphoma who developed severe hypophosphatemia following administration of the first cycle of the eshap protocol. the patient started to develop gradually decreasing phosphate levels 2 weeks after receiving chemotherapy, which needed to be corrected by phosphate supplementation. this case report raises concern regarding hypophosphatemia as a possible side effect of the eshap protocol and points to a need for close monitoring, taking into consideration vitamin d levels, urinary phosphate excretion, parathyroid hormone levels, and arterial blood gas analysis to rule out other contributing factors. health care providers should be made aware of this possible toxicity. critical monitoring of phosphate levels and considering supplementation is warranted with the eshap protocol, especially when it is used in combination with granulocyte colony - stimulating factor and diuretics, to prevent such possible hypophosphatemia. further investigations may be required to confirm and evaluate the significance of this type of toxicity. |
osteoporosis is a metabolic bone disease characterized by loss of bone mass and disruption of bone microarchitecture, and consequently increases the risk of fracture3,5,11,14). it is well known that estrogen plays a fundamental role in skeletal growth and bone homeostasis1 - 3,6). this is of particular importance in postmenopausal women, whose estrogen levels are naturally lowered, and in whom the incidence of osteoporosis is high, because a rapid decrease in estrogens in this population is the predominant cause of the imbalance between bone formation and bone resorption12). although no single animal model precisely mimics the human condition of osteoporosis, appropriate animal models can provide information on bone quality and structure not obtainable during clinical trials. ovariectomy (ovx) provides the most popular model for studying events associated with postmenopausal osteoporosis with estrogen deficiency3,5,12,16), and it has been well established that ovariectomy elicits bone loss and increased bone turnover in rats3,5,7 - 9,11,12,16). thus, ovx rat models of osteoporosis can mimic conditions in postmenopausal women, and are suitable for the evaluations of potential therapeutics designed to prevent or treat osteoporosis7 - 9,16). in the present study, in an effort to devise a reliable and reproducible ovx rat model of osteoporosis, we performed histomorphometric analyses of the lumbar spine using micro - computed tomography (ct) scans and evaluated changes in serum bone turnover markers after ovariectomy in rats. all animal experiments were performed in accordance with the animal care guidelines issued by the national institute health, and were approved by the institutional animal care committee at kyungpook national university. twelve female sprague - dawley rats (11 weeks old, body weight 190 - 210 g) were purchased from samtako bio inc. (osan, korea) and acclimated to conditions for one week before the experiment. animals were housed in an air - conditioned room (relative humidity 45 - 65%) under a 12-h light / dark cycle at 222 and given free access to food and tap water. acclimated rats were assigned to one of two groups, either a sham - operated group (sham, controls ; n=6) or a surgically ovariectomized group (ovx ; n=6). at surgery, rats were initially anesthetized intraperitoneally with a mixture of xylazine (10 mg / kg) and ketamine (60 mg / kg). a 2 cm skin incision was then made just medial to the most bulging part of the back. after pulling away periovarian fat with the ovaries, complete bilateral ovariectomy was performed, and then muscle, fascia, and skin were sutured using 4 - 0 silk. eight weeks after surgery, all animals were sacrificed and blood samples were collected by cardiac puncture for serum isolation. serum was separated by centrifugation (at 1500g) and then stored at -80 until required for bone metabolic marker assays. serum estrogen (estradiol, e2) was determined using an estradiol elisa kit (elisa, drg instruments gmbh, germany). in addition, 4th lumbar vertebrae were removed, fixed in a 3.7% formaldehyde in phosphate - buffered saline solution (ph 7.4) for 16 h and then stored (4) in 80% ethanol for bone mass measurements. serum osteocalcin levels and alkaline phosphatase (alp) activities (both sensitive biochemical markers of bone formation) were determined using osteocalcin eia kits (nordic bioscience diagnostics, herlev, denmark) and quantichrome alp assay kits (dalp-250, bioassay systems, ca, usa), respectively. serum levels of c - terminal telopeptide fragment of type i collagen c - terminus (ctx), which is generated by the osteoclast and is a marker of bone resorption, were determined using ratlaps elisa kits (nordic bioscience diagnostics, herlev, denmark)3,6). bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system (explore locus sp, ge healthcare) with x - ray energy settings of 80 kv and 80 a. samples were scanned over one entire 3600 rotation at an exposure time of 3000 ms / frame. an isotopic resolution of 15 - 40 m voxel size that displayed the microstructure of rat lumbar vertebra was selected, and the angle of increment around the sample was set to 0.4, which resulted in the acquisition of 900 2d images. a modified feldkamp cone - beam algorithm was used for 3d reconstruction. for bone analysis, bone mineral contents (bmc), bone mineral densities (bmd), trabecular bone volume fractions (bv / tv, %), trabecular thicknesses (tb.th.), trabecular numbers (tb.n.), trabecular separations (tb.sp.), and cortical bone mineral densities (cr.bmd) were used for the quantitative analysis, which was performed using 2.0 + aba microview software provided with the micro - ct system13,14,17). each bone was positioned on the two lower supports of the anvil of a universal testing machine (instron 4202 ; instron, canton, ma, usa). load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests. the load versus displacement data were recorded automatically by the instron software (instron series ix automated materials tester, version 8.04.00), which subsequently calculates the mechanical parameters from the load - displacement curves. repeated measure anova was used to compare body weights in the ovx and sham groups. the two - sample t test was used to identify significant differences between the groups, and p values of < 0.05 were considered significant. all animal experiments were performed in accordance with the animal care guidelines issued by the national institute health, and were approved by the institutional animal care committee at kyungpook national university. twelve female sprague - dawley rats (11 weeks old, body weight 190 - 210 g) were purchased from samtako bio inc. (osan, korea) and acclimated to conditions for one week before the experiment. animals were housed in an air - conditioned room (relative humidity 45 - 65%) under a 12-h light / dark cycle at 222 and given free access to food and tap water. acclimated rats were assigned to one of two groups, either a sham - operated group (sham, controls ; n=6) or a surgically ovariectomized group (ovx ; n=6). at surgery, rats were initially anesthetized intraperitoneally with a mixture of xylazine (10 mg / kg) and ketamine (60 mg / kg). a 2 cm skin incision was then made just medial to the most bulging part of the back. after pulling away periovarian fat with the ovaries, complete bilateral ovariectomy was performed, and then muscle, fascia, and skin were sutured using 4 - 0 silk. eight weeks after surgery, all animals were sacrificed and blood samples were collected by cardiac puncture for serum isolation. serum was separated by centrifugation (at 1500g) and then stored at -80 until required for bone metabolic marker assays. serum estrogen (estradiol, e2) was determined using an estradiol elisa kit (elisa, drg instruments gmbh, germany). in addition, 4th lumbar vertebrae were removed, fixed in a 3.7% formaldehyde in phosphate - buffered saline solution (ph 7.4) for 16 h and then stored (4) in 80% ethanol for bone mass measurements. serum osteocalcin levels and alkaline phosphatase (alp) activities (both sensitive biochemical markers of bone formation) were determined using osteocalcin eia kits (nordic bioscience diagnostics, herlev, denmark) and quantichrome alp assay kits (dalp-250, bioassay systems, ca, usa), respectively. serum levels of c - terminal telopeptide fragment of type i collagen c - terminus (ctx), which is generated by the osteoclast and is a marker of bone resorption, were determined using ratlaps elisa kits (nordic bioscience diagnostics, herlev, denmark)3,6). bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system (explore locus sp, ge healthcare) with x - ray energy settings of 80 kv and 80 a. samples were scanned over one entire 3600 rotation at an exposure time of 3000 ms / frame. an isotopic resolution of 15 - 40 m voxel size that displayed the microstructure of rat lumbar vertebra was selected, and the angle of increment around the sample was set to 0.4, which resulted in the acquisition of 900 2d images. a modified feldkamp cone - beam algorithm was used for 3d reconstruction. for bone analysis, bone mineral contents (bmc), bone mineral densities (bmd), trabecular bone volume fractions (bv / tv, %), trabecular thicknesses (tb.th.), trabecular numbers (tb.n.), trabecular separations (tb.sp.), and cortical bone mineral densities (cr.bmd) were used for the quantitative analysis, which was performed using 2.0 + aba microview software provided with the micro - ct system13,14,17). each bone was positioned on the two lower supports of the anvil of a universal testing machine (instron 4202 ; instron, canton, ma, usa). load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests. the load versus displacement data were recorded automatically by the instron software (instron series ix automated materials tester, version 8.04.00), which subsequently calculates the mechanical parameters from the load - displacement curves. repeated measure anova was used to compare body weights in the ovx and sham groups. the two - sample t test was used to identify significant differences between the groups, and p values of < 0.05 were considered significant. 1 shows changes in mean body weights in the two groups with different time. at the start of the experiment, however, at 4 weeks after surgery, mean body weight was significantly greater in the ovx group compared with the sham group, and this significance was maintained throughout the period (p=0.015, 0.037, and 0.011 at 4, 6, and 8 weeks, respectively). as shown in fig. 2, 8 weeks after surgery, serum estradiol levels were significantly lower in the ovx group (1.610.62 pg / ml vs. 19.953.47 pg / ml ; p=0.001). 3a, b summarize the serum levels of the biochemical markers of bone formation, osteocalcin, and alp, and fig. the ovx group had on average a 75.4% higher osteocalcin level than the sham group, which was statistically significant (p=0.002). iu / l in the ovx group, they were not significantly different (p=0.136). on the other hand, serum ctx levels in the ovx group were 72.5% higher than in the sham group, which was significant (p=0.018). as shown in the representative micro - ct images, ovx rats had fewer trabecular bone structures than the sham controls (fig. 4). in the analyses of micro - ct scans of 4th lumbar vertebrae, the ovx group showed a significant 12% reduction in bmd, and a significant 9.1% reduction in trabecular bone volume fraction (bv / tv) (p=0.005 and p=0.002, respectively). furthermore, mean bmc in the ovx group was lower than in the sham group (22.384.12 mg vs. 25.524.37 mg, respectively), though this was not significantly different (p=0.252). however, tb.n. in the ovx group were lower than that of the sham group (4.20.6 in the sham group and 3.80.5 in the ovx group), which was not significantly different (p=0.351). in the histomorphometric analysis of cortical bone, the ovx group also showed a significant 6.2% reduction in cr.bmd versus the sham group (p=0.021) (fig. the three point bending test showed that the 4th lumbar vertebrae of ovx group have on average 39% lower maximal load value compared to that of the sham group (p=0.001) (fig. 1 shows changes in mean body weights in the two groups with different time. at the start of the experiment, however, at 4 weeks after surgery, mean body weight was significantly greater in the ovx group compared with the sham group, and this significance was maintained throughout the period (p=0.015, 0.037, and 0.011 at 4, 6, and 8 weeks, respectively). as shown in fig. 2, 8 weeks after surgery, serum estradiol levels were significantly lower in the ovx group (1.610.62 pg / ml vs. 19.953.47 pg / ml ; p=0.001). fig. 3a, b summarize the serum levels of the biochemical markers of bone formation, osteocalcin, and alp, and fig. the ovx group had on average a 75.4% higher osteocalcin level than the sham group, which was statistically significant (p=0.002). iu / l in the ovx group, they were not significantly different (p=0.136). on the other hand, serum ctx levels in the ovx group were 72.5% higher than in the sham group, which was significant (p=0.018). as shown in the representative micro - ct images, ovx rats had fewer trabecular bone structures than the sham controls (fig. 4). in the analyses of micro - ct scans of 4th lumbar vertebrae, the ovx group showed a significant 12% reduction in bmd, and a significant 9.1% reduction in trabecular bone volume fraction (bv / tv) (p=0.005 and p=0.002, respectively). furthermore, mean bmc in the ovx group was lower than in the sham group (22.384.12 mg vs. 25.524.37 mg, respectively), though this was not significantly different (p=0.252). however, tb.n. in the ovx group were lower than that of the sham group (4.20.6 in the sham group and 3.80.5 in the ovx group), which was not significantly different (p=0.351). in the histomorphometric analysis of cortical bone, the ovx group also showed a significant 6.2% reduction in cr.bmd versus the sham group (p=0.021) (fig. the three point bending test showed that the 4th lumbar vertebrae of ovx group have on average 39% lower maximal load value compared to that of the sham group (p=0.001) (fig. the causes of postmenopausal osteoporosis are accelerated bone resorption and systemic calcium imbalance due to menopause - induced estrogen deficiency3,5,11,12,14). the animal models of osteoporosis are essential for anticipating treatment efficacy and safety2,10), and of the various animal models used, rodents have numerous advantages, for example, they are inexpensive, easy to house, the rapid generation time, and their skeletons are sensitive to the loss of ovarian hormones18). there is extensive literature studying ovx rat including the histomorphometric changes and the biochemical markers of bone turnover2 - 6,10,11,13,14,17). in the present study, we investigated changes in bone turnover and bone loss after ovariectomy in spraque - dawley rats. at 8 weeks after ovariectomy, serum estradiol levels in the ovx group were significantly lower than in the sham group (1.640.62 pg / ml vs. 19.943.46 pg / ml), which supports the presence of ovarian deficiency in the ovx model. furthermore, as was expected, mean body weights were greater in the ovx group than in the sham group (p=0.011). omi.9) described that weight gain efficiency (body weight gain / food intake) in their ovx group was significantly greater than in the sham group. turner.18) also found that ovariectomy increases body weight more in ovx group than in sham group. accordingly, our results are comparable to those of previous studies that have used an ovx rat model3,7 - 9,12,16,18). biochemical markers of bone turnover have been widely used as measures of the status of bone remodeling. the bone remodeling cycle involves the achievement of balance between the bone resorbing activities of osteoclasts and the bone forming activities of osteoblasts. after menopause, bone resorption markers increase due to the activation of bone resorption by estrogen deficiency, and bone formation increases to fill the higher number of resorption cavities, and as a result, increase in their serum levels. the currently accepted mechanism of skeletal bone loss in estrogen deficient rats is that of an imbalance in bone turnover, that is, where bone resorption exceeds bone formation1,16). the biochemical markers of bone turnover used in clinical and experimental studies include osteocalcin and alp (a sensitive marker of bone formation) and ctx (a sensitive marker of bone resorption). in a study of ovariectomized rats, kim.3) found higher plasma osteocalcin and ctx concentrations in their ovx group than in sham operated animals, and similarly, it has been reported that estrogen deficiency increases serum alp, osteocalcin, and ctx levels, which indicated the increased bone turnover2,9,12,15). in the present study, ovariectomized animals were found to have higher osteocalcin, alp and ctx level than sham controls, indicating increased bone turnover due to menopause - induced estrogen deficiency, which is entirely consistent with previous studies2,9,12,15). in ovariectomized animals, as in postmenopausal women, bone loss induced by ovarian deficiency mainly results from trabecular bone loss3). although bmd measurements could be performed by dual - energy x - ray absorptiometry, micro - ct can also be used for monitoring bone microarchitecture and bmd in small animals. scanned micro - ct images can separate cancellous and cortical bone, and therefore, they can be used to produce realistic depictions of apparent and tissue bmds, which in turn reflect degrees of bone mineralization. in the present study, we analyzed bone histomorphometric parameters and microarchitectural properties, bmd, bv / tv, %, tb.th. lei.5) reported a significant decrease in femoral bmd in 8-week ovx rats as compared 8-week sham rats. in a recent study of histological observa - tions of bone resections of vertebrae and femurs, bone mass was found to be obviously reduced in ovariectomized rats than in sham group10). similarly, in the present study, we also observed that bmd and bv / tv in 4th lumbar vertebrae and cortical bmd were significantly lower in the ovx group. these findings support the notion that our model successfully mimics the effects of osteoporosis. when considering animal models of osteoporosis, it is important that bone sites should be taken into account. it is well known that, in ovariectomized animals, cortical bone loss does not match that of trabecular bone2,9). in a study of ovariectomized rats, ferretti.2) reported that bone mass started to decrease earlier and more extensively in trabecular than in cortical bone. in particular, mean bmd of 4th lumbar vertebra at 8 weeks after surgery, had reduced by 12.0%, but mean cortical bmd had only reduced by 6.2%. the present study demonstrates that ovariectomy in rats significantly increases bone turnover, and this was confirmed by elevated levels of bone turnover markers. furthermore, it shows a significant loss of trabecular bone in lumbar vertebrae 8 weeks after ovariectomy. finally, our study demonstrates that the ovariectomized rat model offers a reproducible and reliable model of osteoporosis. | objectivethe purpose of this study was to verify the appropriateness of ovariectomized rats as the osteoporosis animal model.methodstwelve female sprague - dawley rats underwent a sham operation (the sham group) or bilateral ovariectomy [the ovariectomy (ovx) group ]. eight weeks after operations, serum biochemical markers of bone turnover were analyzed ; osteocalcin and alkaline phosphatase, which are sensitive biochemical markers of bone formation, and c - terminal telopeptide fragment of type i collagen c - terminus (ctx), which is a sensitive biochemical marker of bone resorption. bone histomorphometric parameters and microarchitectural properties of 4th lumbar vertebrae were determined by micro - computed tomographic (ct) scan.resultsthe ovx group showed on average 75.4% higher osteocalcin and 72.5% higher ctx levels than the sham group, indicating increased bone turnover. micro - ct analysis showed significantly lower bone mineral density (bmd) (p=0.005) and cortical bmd (p=0.021) in the ovx group. furthermore, the ovx group was found to have a significantly lower trabecular bone volume fraction (p=0.002).conclusionour results showed that bone turnover was significantly increased and bone mass was significantly decreased 8 weeks after ovariectomy in rats. thus, we propose that the ovariectomized rat model be considered a reproducible and reliable model of osteoporosis. |
dacron arterial grafts are widely used in thoracic aortic replacement, but few cases of non - anastomotic aneurysmal dilatation or rupture have been reported. in most cases that have been reported, knitted, rather than woven, we report the clinically successful result of thoracic endovascular repair (tevar) of the late rupture of a woven dacron graft. the dacron graft had been implanted in the patient s descending thoracic and upper abdominal aorta 20 years previously. a 56-year - old man with a descending thoracoabdominal aortic aneurysm underwent descending thoracic and upper abdominal aorta replacement with a hemashield 20-mm woven dacron graft (meadox medicals inc., he was followed up as an outpatient, and no adverse events were noted until 2009, when he presented with sudden - onset chest pain and severe dyspnea. at that time, chest computed tomography (ct) revealed a massive right hemothorax, a large pseudoaneurysm around the implanted graft just above the diaphragm level, and marked contrast medium extravasation from the graft (fig. 1). the patient underwent an emergency open thoracotomy, in which a disruption was found at the site of the anastomosis between the dacron graft and lumbar intercostal arteries. the disrupted site was repaired with 2 layers of running sutures, and the graft was wrapped with the calcified aneurysmal sac. in 2012, the patient presented with back pain, abdominal pain, and indigestion. a follow - up chest ct revealed that the size of the aneurysm in the descending aorta had increased in comparison with the findings of the previous ct examination. dye collection in the space between the graft and native aneurysmal sac was also observed, and was suspected to be indicative of a late rupture of the dacron graft itself (fig. the rupture was unrelated to the previous anastomosis or disrupted site. due to the high morbidity and mortality associated with reoperation, we decided to perform thoracic endovascular stent therapy as an alternative to open thoracotomy for the late graft failure. the primary goal of using tevar was to cover the entire length of the dacron graft in the descending aorta with a stent graft. the right common femoral artery was exposed under general anesthesia, and a guidewire and angiographic catheter were introduced through it. aortic angiography revealed multiple dye leakages from the graft that were caused by late failure of the dacron graft (fig. 3). after obtaining an aortogram, two stent grafts (3030150 mm and 3636100 mm ; s&g biotech inc., seongnam, korea) were also introduced through the right femoral artery and placed in the dacron graft. the proximal end of the stent graft was deployed distally to the left subclavian artery, and the distal end was placed just above the celiac trunk. the total length of the thoracoabdominal aorta covered by the stent graft was approximately 220 mm. a postoperative ct scan showed that the disruption of the dacron graft had been successfully treated (fig. the patient was discharged with no procedure - related complications and, during the 1-year follow - up period, was free of any symptoms. dacron vascular grafts are known to be durable and effective as aortic vascular prostheses. however, a few cases of non - anastomotic aneurysmal dilatation or rupture of these grafts after implantation in the descending thoracic aorta have been reported. the probable cause was degeneration over time of the graft fibers or the round black yarn used as the guideline. intrinsic graft failure of dacron grafts is a rare complication (0.5%3%) ; and in most reported cases, knitted, not woven, dacron grafts had been used. in our case, woven dacron graft rupture occurred 20 years after the initial operation, probably caused by the degeneration of graft fibers over a long time. although woven dacron graft rupture is extremely rare, reoperation is inevitable when it does occur. however, the hospital mortality after aortic reoperation remains high (8%15% in many reports), and is even higher (25%50%) among patients receiving urgent or emergency care. tevar is increasingly being used for descending aortic diseases as a safe and feasible alternative to open graft repair. in light of the high morbidity and mortality associated with reoperation, we decided to perform endovascular therapy as an alternative management strategy for late graft failure in our patient. to the best of our knowledge, the endovascular repair of primary graft rupture in a woven dacron graft has not been reported. the case presented here demonstrates the effectiveness and feasibility of tevar implantation to treat the non - anastomotic rupture of a dacron graft after descending aortic replacement. | the intrinsic structural failure of a dacron graft resulting from the loss of structural integrity of the graft fabric can cause late graft complications. late non - anastomotic rupture has traditionally been treated surgically via open thoracotomy. we report a case of the successful use of thoracic endovascular repair to treat a dacron graft rupture in the descending aorta. the rupture occurred 20 years after the graft had been placed. two stent grafts were placed at the proximal portion of the surgical graft, covering almost its entire length. |
it was not until the year 1900, when karl landsteiner at the university of vienna, discovered why some blood transfusions were successful while others could be deadly. landsteiner discovered the abo blood group system by mixing the red cells and serum of each of his staff. he demonstrated that the serum of some people agglutinated the red cells of other. from these early experiments, he identified three types, called a, b and c (c was later to be re - named o for the german the fourth less frequent blood group ab, was discovered a year later. in 1930, landsteiner received the nobel prize in physiology and medicine for his work (1). the gene that determines human abo blood type is located on chromosome 9 (9q34.1) and is called abo glycosyltransferase. the abo locus has three main allelic forms : a, b, and o, as mentioned above and each of them is responsible for the production of its glycoprotein. it is therefore the combination of alleles that are inherited from parents that determines which glycoproteins (antigens) are found on persons blood cells and thereby their abo blood type (1). as investigations have demonstrated on monkeys (table 1), human blood groups are very old genetic indicators which have evolved during several million years (2). based on the primary races hypothesis, it was thought that in the three major races of man, blood groups a in europe, b in asian, and finally o in south america have been emerged and gradually due to the migration and mixing of the races, became the present situation. but we know that in each continent, the isolated populations are seen that have completely different blood groups. for example, there is relatively high prevalence of blood group o in siberian inhabitants ; also this blood group is very common in some areas of switzerland (3). according to another hypothesis, the emergence of all blood groups a and b and their subgroups, are resulted from successive mutations, from a basic and common blood group, which is the o group, and have been branched over millions of years (fig. the old races have o blood group, such as red indians of south america, and eskimos that among them the frequency of o blood group is between 75100%. while in most of recent ethnic groups a and b blood groups are dominant. in another hypothesis, the first blood group had been ab blood group, which gradually and over the time due to genetic mutations was resulted in a and b and finally o blood groups (fig. 2). base on this theory, perhaps a few million years ago all people have had type o blood only, which is more resistant against many infectious diseases. geographic distribution and racial blood groups a and b and o in the world (according to the mourant design 1958) are shown in figures 3 to 5(4). the geographical spread not only is a result of the above assumptions, but the current process of natural selection against environmental factors such as diseases, climate, humidity, altitude and etc after discovery of the first human blood groups (abo) by karl landsteiner in 1901 (5), gradually from 1927, other blood groups were also discovered and reported which its collection is given in table 2. it is important to mention that landsteiner together with his american colleague alexander wiener discovered the rh blood group and reported it in 1940, 1941. karl landsteiner was born on 14 june 1868, in vienna, austria ; he died on 26 june 1943 ad, at 75 years old, in the united states. landsteiner in his 17 scientific paper in 1901 reported blood group abo which was displayed at the beginning with the letters abc. in 1930 in addition to the known blood groups (table 2), nearly twenty public antigens and also sixty - specific antigen or family antigen (private antigens) have been reported (3). moreover, the main blood groups abo, gradually discovered and reported (3) which the most notably of them are as follows : a subgroups, including a1, a2, a3, and also rare types a4, a5, a6, z, x, end, boutu, g, i.b subgroups, including b1, b2, b3, and rare types w, x, v, m.subgroups, including o1, o2, o3, and other types such as yy, hh, xx, and bombay. a subgroups, including a1, a2, a3, and also rare types a4, a5, a6, z, x, end, boutu, g, i. b subgroups, including b1, b2, b3, and rare types w, x, v, m. subgroups, including o1, o2, o3, and other types such as yy, hh, xx, and bombay. in a compilation by mourant in 1958 (4), referring to a limited and small sampling from iran (tehran) by a.ajir was seen, but there was no systematic and comprehensive research about types and frequencies of blood groups, serum proteins, and red blood cell enzymes, found in iran. the first report about the frequency of lutheran blood group in iran was published in 1979 (23). after a long study and targeted collection, detailed reports of the frequency of abo blood groups in different iranian ethnic groups was released (24). in another study, the frequency of blood groups, serum proteins and red cells enzymes in various iranian populations furthermore, a collection of valuable and extensive cooperation with iran blood transfusion organization, different types of blood groups in various population of iran, was reported. this report included the study of abo and rh blood groups phenotype and genotype frequencies among 291857 individuals and their geographical spread in different provinces of iran (26). in this report, in addition to the abo blood groups and rh, genotype and phenotype frequencies of rare blood groups, including kell (n=5522), daffy (n=3764), kidd (n=3650), lutheran (n=3199), kp (n=1489), xg (n=3227), were also presented (26). since over 2030 years have passed from that sampling in different provinces of iran, population displacement, and various environmental factors, diseases, immigration, exogamous marriages within different ethnic groups, no doubt that provincial prevalence of blood groups distribution, at this time has changed, too. however, over time, case reports and local frequencies of blood groups in different regions of iran, were prepared and published, including abo and rh blood groups report in population of larestan and lamerd, fars (27). ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors. | the evolution of human blood groups, without doubt, has a history as old as man himself. there are at least three hypotheses about the emergence and mutation of human blood groups. global distribution pattern of blood groups depends on various environmental factors, such as disease, climate, altitude, humidity etc. in this survey, the collection of main blood groups abo and rh, along with some minor groups, are presented. several investigations of blood groups from iran, particularly a large sampling on 291857 individuals from iran, including the main blood groups abo and rh, as well as minor blood groups such as duffy, lutheran, kell, kp, kidd, and xg, have been reviewed. |
homogeneous gold catalysis is a relatively new field of research that became popular in the early years of this century. although there were a few scattered papers describing reactions of aromatic compounds with gold salts, the first signals of the synthetic potential of gold in homogeneous catalysis came with the work of teles and tanaka on the addition of alcohols and water to alkynes. we focus on the developments of synthetic methods that were inspired on our initial studies of platinum(ii)-catalyzed reactions and, in particular, on the multifaceted nature of cyclopropyl metal carbenes, key electrophilic intermediates formed by the activation of alkynes in functionalized 1,n - enynes and related substrates. inspired by the seminal work of the group of trost on the electrophilic activation of enynes using palladium(ii) complexes as catalysts, which was followed by related work by murai and chatani using ruthenium(ii) and by the synthesis of phenols by intramolecular reactions of furans with alkynes developed by hashmi, we initially examined the intramolecular reaction of allylsilanes and allylstannanes with alkynes, discovering that ptcl2 was the best catalyst, although palladium(ii), ruthenium(ii), and silver(i) salts and complexes could also be used. several other groups had almost concurrently found that platinum(ii) also catalyzed the skeletal rearrangement of 1,6-enynes and other cycloisomerizations under relatively mild conditions. we also found that ptcl2 was an excellent catalyst for the addition of water and alcohols (hydroxy- and alkoxycyclization) to 1,6-enynes such as 1a d, a process that proceeds stereospecifically (scheme 1). although these reactions proceed by an exo - dig pathway, the alternative endo - dig cyclization mode was also observed in other cases. the formation of five- or six - membered ring compounds 2a c or 3, respectively, was rationalized by the competitive opening of a common intermediate 4 by attack of the nucleophile at the most electrophilic site, which was dictated by the substitution of the alkene following the markovnikov rule, a hypothesis that was supported by dft calculations (scheme 2). intermediates 4 can be described as cyclopropyl platinum(ii) carbenes, although the contribution of a charge - separated resonance form corresponding to metal stabilized homoallyl carbocations is also significant. it is important to note that the involvement of very similar reactive intermediates was already suggested in the palladium(ii)- and ruthenium(ii)-catalyzed reactions of 1,6-enynes. in a parallel work, we also examined the synthesis of phenols by cyclization of furans with alkynes using platinum(ii) catalysts instead of gold(iii), as originally reported by hashmi, concluding that this transformation is mechanistically related to metal - catalyzed cycloisomerization of enynes in which the furan ring acts as an electron - rich alkene (scheme 3). according to the dft studies performed for the platinum(ii)-catalyzed reaction, the attack of the furan to the -alkyne metal complex 5 leads to a cyclopropyl metal carbene 6, which opens up to form 7. cyclization to 8, followed by elimination of the metal generates oxepine 9, in tautomeric equilibrium with arene oxide 10, which leads to phenols 11. although almost all the examples reported concerned with the intramolecular reaction of furanynes, we recently developed an intermolecular version using cationic gold(i) catalysts. during our initial studies focused on the use of platinum(ii) complexes as catalysts, we also found that highly lewis acidic aucl3 could be used as a catalyst in some cases. however, we soon realized that cationic phosphine gold(i) complexes were more convenient catalysts, actually being the most active and selective for reactions of alkynes, outperforming platinum(ii) and all the other metal catalysts that we had examined for the addition of heteronucleophiles, cycloisomerizations of 1,6-enynes, and similar transformations. first, we generated in situ the reactive cationic gold(i) complexes by protonolysis of neutral complex ph3paume, which leads to the formation of methane. however, this method introduces a strong brnsted acid into the reaction medium, which may promote unwanted side reactions. more convenient proved to be the use of ph3paucl and a silver(i) salt with a noncoordinating anion such as agsbf6. recently, we found that the mode of activation of the laucl precatalysts with silver(i) salts has an important effect in catalysis. thus, if complexes laucl are allowed to react with agx in poorly coordinating solvents and in the absence of the substrate, dinuclear chloride - bridged species [lauclaul]x are readily formed, regardless of the counterion. these very robust complexes are poor catalysts, so that their formation significantly reduces the reaction rate. the formation of [lauclaul]x can be minimized by premixing laucl with the substrate, followed by addition of silver salts, allowing formation of complexes [lau(substrate)]x, which can directly enter the catalytic cycles. of course, the formation of dinuclear chloride - bridged complexes can be entirely circumvented by the use of silver - free, cationic [laul]x, where l is a weakly coordinating ligand, which are often the alternative of choice in gold(i) catalysis. along with our initial study in 2004 on the cycloisomerization of 1,6-enynes such as 1e to form 1,3-diene 12a by skeletal rearrangement, the groups of frstner and toste reported related examples of cycloisomerization of 1,5-enynes of type 13a, b to give bicyclo[3.1.0]hex-2-ene derivatives 14a, b, respectively, under almost identical conditions using ph3paucl and a silver salt (scheme 4). a gold(i)-catalyzed conia ene reaction of -ketoesters with alkynes was also reported by toste. in general, cationic gold(i) complexes activate selectively alkynes even in the presence of other potentially coordinating functional groups as alkenes. it is important to remark that, unlike other transition metals, gold(i) does not promote alder ene cycloisomerizations of 1,n - enynes, which require the unfavorable coordination of gold(i) to both the alkyne and the alkene. the oxidative cyclometalation of gold(i) to form a gold(iii) metallacycle, is also a very unlikely process. in the case of 1,6-enynes, gold(i) forms (-alkyne)metal complexes 15(40) that react intramolecularly with the alkene to form cyclopropyl gold(i) carbene - like intermediates 16 and/or 17 by 5-exo - dig or 6-endo - dig cyclization, respectively (scheme 5). similar pathways are followed by 1,5-, 1,7-, and higher enynes in the presence of gold(i) catalysts. according to dft calculations, intermediates 16 and 17 are even more delocalized cationic structures than their pt(ii) counterparts, although for convenience, we prefer to represent these complexes as cyclopropyl gold(i) carbenes to highlight their propensity to undergo cyclopropanation reactions. however, although the back - donation from gold(i) to the cationic centers in these metal carbene - like structures is low, it still provides sufficient stabilization as has recently been shown in a few well - characterized gold carbenes [lau = cr2 ] and [l2au = cr2 ]. the bond between au and c in these gold(i) carbenes has been described as a half - double bond. terminologically, we recommend using the term gold carbenes rather than gold carbenoids for these species since genuine gold(i) carbenoids lau ch2x (x = cl, sph) have been structurally characterized. these neutral complexes can be considered as formal precursors of gold methylidene complex [lau = ch2 ] by an -elimination of the x group. intermediates 17 usually lead to bicyclo[4.1.0]hept-2-ene derivatives 18 by -proton elimination (scheme 5). on the other hand, intermediates 16 of 5-exo - dig cyclization afford 1,3-dienes 19 by a skeletal rearrangement (single - cleavage) in an interesting process that gives rise directly the -coordinated gold(i) diene complex and involves a 1,3-suprafacial migration of the terminal carbon of the alkene via transition state ts1619. intermediates 16 can also undergo a slightly different migration (double - cleavage rearrangement) by the formal insertion of the terminal alkene carbon into the alkyne carbons that forms a new gold(i) carbene 20. compounds 21 (r = h) with z configuration usually predominate, although both e- and z - configured products have been observed. the scenario shown in scheme 5 is a simplification, as other similar pathways may also be possible depending on the substitution pattern of the substrate. thus, intermediates 17 of endo - dig cyclization can undergo ring expansion to give (-cyclobutene)gold(i) complexes 22, which can evolve to form bicyclo[3.2.0]hept-2-ene derivatives 23 (scheme 6). intermediates 22 can also open up to form 24 and then dienes 19 in an overall single - cleavage process. isomeric 1,6-enynes 1f and 1 g, which differ in the substitution pattern of the alkyne, react with gold(i) to form the same 1,3-diene 12b by two different mechanisms, single- and double - cleavage rearrangement, respectively (scheme 7). other 1,6-enynes bearing terminal alkynes such as 1d and 1h give 1,3-dienes 12c, d by single - cleavage rearrangement. l, with strongly electron - donating substituents at the terminal alkene carbon, react with cationic gold(i) catalyst 25a to give z - configured dienes 12f h (scheme 7). this z - selectivity was also found using different electrophilic gold(i) or platinum(ii) catalysts. this is rather mechanistically puzzling, since both the platinum(ii)- and the gold(i)-catalyzed single - cleavage rearrangements are in the vast majority of cases stereospecific processes in which the configuration of the alkene is retained. complexes of gold(i) bearing commercially available bulky dialkyl biphenyl phosphines such as 25a, proved to be particularly useful in homogeneous gold(i) catalysis. experimental and theoretical studies confirmed that gold(i) does not interact significantly with the closest arene ring of the ligand. we examined the intermolecular reaction between alkynes and electron - rich alkenes with many different gold(i) catalysts, although only complex mixtures of oligomers were obtained. in addition, electron - rich alkenes are very good ligands for gold(i), thus competing with the alkyne for the coordination to the metal. however, by using cationic gold(i) complex 25b with a very bulky phosphine, cyclobutenes 26 were finally obtained regioselectively for a variety of electron - rich alkenes (scheme 8). interestingly, the isolated yields were improved in most cases by changing the anion from sbf6 to softer bar4, presumably by minimizing the formation of unproductive, -digold(i) alkyne digold(i) complexes, an undesired side reaction in many gold - catalyzed reactions of terminal alkynes. the same improvement was observed in the intramolecular [2 + 2 ] cycloaddition of alkynes with alkynes in substrates of type 27 to form macrocycle 28 (scheme 9). the regioselective formation of cyclobutenes by [2 + 2 ] cycloaddition probably proceeds by electrophilic addition of the (-alkyne)gold(i) complex to the alkene via ts2930 to form a cyclopropyl gold(i) carbene 30 very similar to intermediate 16 involved in the cyclization of enynes (scheme 10). intermediate 30 was also trapped intramolecularly with an alkene to form the corresponding cyclopropane. the intermolecular reaction of propiolic acid and related alkynes with strongly electron - withdrawing groups gives dihydropyrones 32 or 1,3-dienes 33 via a similar mechanism, although the initial attack of the alkene to the (-alkyne)gold(i) complex occurs with the opposite regiochemistry (scheme 10). thus, cyclopropyl gold(i) carbene intermediate 34, with gold bonded to the internal carbon of the alkyne, evolves by intramolecular opening to form 32 by attack of the carboxylic acid to the most substituted carbon of the alkene. on the other hand, in the reaction of alkenes with two very similar substituents, intermediate 34 undergoes a 1,3-migration similar to that found in the single - cleavage rearrangement to form stereospecifically 1,3-dienes 33. a transition state similar to ts3433 has been proposed in the reaction of cyclopropyl gold(i) carbenes generated by retro - buchner reaction of 7-cyclopropyl 1,3,5-cycloheptatrienes. gold(i) complexes are able to catalyze the addition of amines, alcohols, or water to 1,n - enynes affording products of amino-, alkoxy-, or hydroxycyclization under much milder conditions than other metal catalysts. these additions are stereospecific, as illustrated by the alkoxycyclization of diastereomeric enynes 1 m and 1h, and proceed via opening of cyclopropyl gold(i) carbene intermediates 35 by attack of the nucleophile to the cyclopropane ring (scheme 11). gold(i) complex and the heteronucleophile to an alkene following the markovnikov regiochemistry, which is further demonstrated by the reaction of enyne 1d with gold(i) in the presence of meoh to form six - membered ring 3 via intermediate 36. 1,6-enynes such as 1n bearing an aryl substituent at the alkyne terminus undergo an analogous 5-exo methoxycyclization to afford 2f in the presence of gold(i) and meoh (scheme 12). however, similar 1,6-enyne 1o tethered by a benzene ring undergoes gold(i)-catalyzed hydroxy- or alkoxycyclizations preferentially via 6-endo - dig pathway through intermediates 37 giving rise to dihydronaphthalenes 38. amino- and alkoxycyclizations can also take place intramolecularly starting from amino- or hydroxy-1,n - enynes. thus, 1,6-enyne 1p containing a propargylic alcohol gave 40 quantitatively upon treatment with gold(i) as a result of an intramolecular attack of the hydroxyl group to cyclopropyl gold carbene intermediate 39 (scheme 13). in a mechanistically related transformation, amino- or hydroxy-1,5-enynes 41 afforded in the presence of gold(i) spirofused heterobicyclic compounds 43 through intermediates of type 42. in a similar vein, 1,n - enynes bearing nucleophilic moieties such as carboxylic acids, carbamates, or carbonates are also prone to undergo gold - catalyzed tandem cyclizations, giving rise to a variety of complex molecular architectures. oxo-1,6-enynes 44 also react in the presence of gold(i) by a formal [2 + 2 + 2 ] alkyne / alkene / carbonyl cycloaddition to afford predominantly oxatricyclic compounds 48, together with minor amounts of dienes 49 (scheme 14). these reactions proceed via intramolecular attack of the carbonyl to cyclopropyl gold carbenes 45 to form oxonium cations 46, which undergo a prins - type cyclization to give intermediates 47. demetalation from 47 forms oxatricycles 48, whereas an alternative elimination with fragmentation of the seven - membered ring leads to dienes 49. we exploited gold(i)-catalyzed intramolecular [2 + 2 + 2 ] alkyne / alkene / carbonyl cycloadditions in the synthesis of several oxygen - bridged sesquiterpenoids. thus, ketoenynes 44d and 44e were respectively converted into oxatricycles 48d and 48e, which are key intermediates in the total syntheses of (+) -orientalol f (50) and pubinernoid b (51) (scheme 15). oxo-1,6-enyne 44f was analogously cyclized to form 48f, which was subsequently converted into ()-englerin a (52). another total synthesis of 52 included a very similar gold(i)-catalyzed cyclization as the key step. remarkably, an unprotected aldol subunit could be used in both syntheses as the substrate for the gold - catalyzed reaction. 1,n - enynes also react intermolecularly with carbonyl compounds giving rise to a wide range of products depending on the substitution pattern of the alkene and the nature of the carbonyl compound. 1,6-enynes such as 1q react with aromatic aldehydes in a formal [2 + 2 + 2 ] cycloaddition to give oxabicyclic adducts of type 55 together with dienes 56, which result from a metathesis - type reaction of the enyne with the aldehyde (scheme 16). the formation of these products can be explained by attack of the aldehyde to the cyclopropyl gold carbene intermediate to form 53a, which undergoes a prins - type cyclization followed by either demetalation to form 55 or fragmentation to give 56. this mechanism is analogous to that proposed for the intramolecular [2 + 2 + 2 ] cycloaddition of oxo-1,6-enynes (scheme 14). 1,7-enynes also undergo [2 + 2 + 2 ] cycloadditions with carbonyl compounds to give analogous products. on the other hand, following a related mechanism, 1,6-enyne 1q reacts with cyclopropenones in a ring - expanding spiroannulation to afford spirocyclic cyclopentenones 58 after incorporation of a molecule of water to rearranged intermediate 57. 1,6-enynes 1r bearing a monosubstituted alkene react with aldehydes and ketones in a different way to form tricyclic compounds 61. this transformation presumably proceeds via trapping of the rearranged carbene that results from the enyne to form oxonium cation 59, followed by prins cyclization and demetalation. the intermolecular reaction of terminal alkynes with oxoalkenes 62 leads to 8-oxabicyclo[3.2.1]oct-3-enes 64 as a result of a formal [2 + 2 + 2 ] alkyne / alkene / carbonyl cycloaddition via cyclopropyl gold carbene intermediates 63 (scheme 17). electron - rich aromatic and heteroaromatic compounds can act as nucleophiles reacting with 1,6-enynes. cyclopropyl gold(i) carbenes that result as intermediates in the cyclization of 1,6-enynes can act as bifunctional electrophiles, reacting with arenes either at the carbene or at the cyclopropane. hence, indole reacted with 1,6-enyne 1s in the presence of different gold(i) complexes to give two different adducts 66 and 67 (scheme 18). we found that the more electrophilic gold(i) complexes with less donating phosphite or phosphine ligands favored the attack to the cyclopropane ring to give 66, whereas complexes with more donating nhc ligands favored the formation of 67 by attack at the metal carbene. an analogous reaction in the presence of a gold(i) catalyst generated in situ from ph3paucl / agsbf6 was reported to give exclusive formation of products of type 66 by attack to the cyclopropane ring. 1,3-dicarbonyl compounds can also add to 1,6-enynes such as 1s acting as carbonucleophiles through their enol tautomer to give compounds 68 (scheme 19). however, some dicarbonyl compounds such as 1,3-cyclohexadione may act as o - nucleophiles to form adduct 69. as in the addition of heteronucleophiles to 1,6-enynes, products derived from the attack at the carbene carbon are favored with more donating ligands, whereas more electrophilic complexes favor the attack to the cyclopropane. gold(i) also catalyzes the addition of allyl silanes to 1,6-enynes, as well as the addition of a variety of carbonucleophiles to 1,5-enynes. intramolecular attack of aryl groups in 1,6-enynes of type 1 t leads stereospecifically to tricyclic products such as 74 of intramolecular formal [4 + 2 ] cycloaddition (scheme 20). this is a very general reaction that proceeds via initial exo - cyclization of the enyne followed by opening of the cyclopropyl gold(i) carbene 70 by a friedel crafts - type reaction. these cycloadditions can be performed enantioselectively in the presence of chiral phosphine or phosphite gold(i) complexes. the endo - cyclization of the 1,6-enyne also takes place in certain cases, being the preferred pathway for arylalkynes bearing enesulfonamides or enamines such as 72 to afford tricycles 74. benzyl - substituted 1,5-enynes such as 13c also undergo a formal [4 + 2 ] cycloaddition via 5-endo - dig cyclization to afford tricyclic product 75. in contrast, 1,5-enynes 13d with an aryl substituent at the alkyne react with gold(i) through a different mechanism to form dihydrobenzofluorenes 78 in a formal [3 + 3 ] cycloaddition. this transformation can be explained by a 1,2-h shift in intermediate 77, followed by a friedel crafts alkylation (scheme 21). the carbene - like character of the intermediates formed in metal - catalyzed cycloisomerizations of enynes is more clearly exhibited in intra- and intermolecular cyclopropanations of alkenes. formation of tetracyclic compounds 81a, b by cyclization of dienynes 1u, v at room temperature nicely illustrates the high reactivity and stereoselectivity usually exerted by gold catalysts (scheme 22). this reaction, which was first reported using ruthenium(ii) complexes under more forcing conditions (80 c, toluene), proceeds by a 5-exo - dig cyclization followed by cyclopropanation of the resulting cyclopropyl gold(i) carbene 80. when the starting dienynes are cyclic substrates these intramolecular cyclopropanations lead to very complex ring systems. 1,5-dienynes such as 13e react similarly to afford pentacyclic product 83 through endo - carbene 82. dienynes bearing a methoxy or other or group at the propargylic position such as 87a undergo an intramolecular 1,5-or migration in the presence of gold(i) to form tricyclic compounds 88a (scheme 23). this reaction presumably takes place via cyclopropyl gold(i) carbene intermediate 85, which leads to bridged system 86 by attack of the or group at the cyclopropane. opening of 86 results in the formation of,-unsaturated gold(i) carbene 87, which is prone to undergo intramolecular cyclopropanation with the alkene at the side chain to form 88a. this cascade cyclization process is ideally suited for the total syntheses of sesquiterpenes ()-4,7-aromadendranediol (92), ()-4,7-aromadendranediol (93), and ()-epiglobulol (94) from a single dienyne 84b by using a stereodivergent gold(i)-catalyzed reaction as the key step (scheme 24). in the absence of any external nucleophile, cyclopropyl gold(i) carbene intermediate 89 undergoes a 1,5-obn migration followed by intramolecular cyclopropanation to form tricyclic compound 88b, which is the precursor of 93 and 94. on the other hand, by adding an external nucleophile such as allyl alcohol, intermediate 89 leads to 91 and then to 88c after intramolecular cyclopropanation. intramolecular cyclopropanation of a gold(i) carbene can lead to very complex structures starting from 7-alkynylcyclohepta-1,3,5-trienes 95 (scheme 25). this reaction generates highly fluxional barbaralyl gold(i) cations 96 that can be trapped by the pending alkene to form 97a, b, which are in equilibrium by cope rearrangement both in the solid state and in solution. intermediate cyclopropyl gold(i) carbenes such as 65 resulting from the cyclization of 1,6-enynes can also be trapped intermolecularly by cyclic or acyclic alkenes to afford adducts 98 stereoselectively (scheme 26). 1,6-enynes such as 1d bearing a terminal alkene react differently yielding 100, probably by rearrangement of cyclopropyl gold(i) carbene 36 to give carbene 99. the involvement of carbene 99 in the trapping by the alkene also supports the involvement of this type of species as intermediates in the double - cleavage rearrangement (see scheme 5). in the presence of gold(i), 1,6-enyne 84c undergoes a cyclization followed by 1,5-acetoxy migration to give,-unsaturated carbene 103, which reacts intermolecularly with alkene 104 giving rise to 105 with only 5% loss of enantiomeric excess (scheme 27). this transformation has been used as the key step in the total synthesis of antiviral sesquiterpene (+) -schisanwilsonene a (106). it is remarkable that the cyclization/1,5-acetoxy migration is faster than the alternative 1,2-acyloxy migration, which would lead to racemization. gold(i) catalysts trigger complex reactions of substituted 1,n - enynes by stabilizing the key reactive cationic intermediates, which can be viewed in a simplified form as cyclopropyl gold(i) carbenes. these intermediates react with diverse nucleophiles inter- or intramolecularly and cyclopropanate alkenes leading to complex structures with total atom economy. this work, along with that of many other groups, has promoted gold from a mere curiosity to the metal of choice for the activation of alkynes in complex molecular settings under homogeneous conditions. the same basic principles uncovered during the mechanistic study of gold(i)-catalyzed reactions of 1,n - enynes can also been extended for reactions of more complex systems as well as for the development of more challenging intermolecular transformations between alkynes and alkenes. in this regard, in order to fulfill the synthetic potential of intermolecular transformations, new types of gold(i) catalysts that selectively activate alkynes in reactions with highly functionalized alkenes are still required, particularly in the arena of enantioselective synthesis. | cycloisomerizations of 1,n - enynes catalyzed by gold(i) proceed via electrophilic species with a highly distorted cyclopropyl gold(i) carbene - like structure, which can react with different nucleophiles to form a wide variety of products by attack at the cyclopropane or the carbene carbons. particularly important are reactions in which the gold(i) carbene reacts with alkenes to form cyclopropanes either intra- or intermolecularly. in the absence of nucleophiles, 1,n - enynes lead to a variety of cycloisomerized products including those resulting from skeletal rearrangements. reactions proceeding through cyclopropyl gold(i) carbene - like intermediates are ideally suited for the bioinspired synthesis of terpenoid natural products by the selective activation of the alkyne in highly functionalized enynes or polyenynes. |
we describe here a method that can rapidly and accurately compute the potentials of mean force (pmfs) between spherical univalent ions in water. the pmf between an ion a and ion b represents the reversible work (as a function of distance between the two ions, r), averaged over all the configurations of the surrounding solvent, needed to bring a and b from infinite separation to a distance r. pmfs are central to many solvation processes. for example, some pairs of ions form a stable contact pair or salt bridge ; others form a stable solvent - separated pair and have intervening bridging water molecules. the pmf is relevant to understanding the solubilities of salts in water, the equilibria and dynamics when a drug molecule approaches and binds to a protein binding site, and how some parts of a protein molecule approach other parts as the protein molecule folds. currently, the gold standard for computing the pmfs between large molecules such as are typical in biology is to use explicit - solvent simulations with semiempirical force fields. however, these simulations are often computationally too expensive for many of the calculations that would be of practical interest. even in the case of various sampling techniques have been introduced to mitigate the computational expense : constraints, umbrella sampling, and weighted histogram methods, for example. on the other hand, treating water explicitly in analytical theories is challenging, because of the need to properly account for the orientation (angular) effects of water. for simple salt solutions, integral - equation theories often predict qualitatively correct trends but not quantitative details. an alternative is to use implicit - solvent models (such as poisson boltzmann (pb) or generalized born (gb)), in which the solvent is approximated as a dielectric continuum. implicit - solvent models have the advantage of computational speed : a pmf costs around 100 h of md simulation time in explicit water but only minutes on a personal computer for pb pmf or fractions of a second for gb. pmfs of a model nacl from explicit - solvent md simulation (symbols) and implicit - solvent theories : pb (continuous blue line) and gb (dashed green line). pb calculations were done with apbs 1.3 software, and gb results were obtained according to the gb model of still. the permittivity of water was set to 78.54 in the pb and gb calculations. parameters from dang. were used for na and cl ions (see table 1). however, implicit - solvent models do not capture the subtle features of pmfs, which require some representation of particulate water (see figure 1). to correct for the energy errors in implicit modeling implicit - solvent models can allow interstitial high dielectrics (e.g., water), but they give incorrect pmfs between nonbonded atoms. salt bridges, for example, treated via gb models are typically too stable compared to explicit - solvent results, which leads to oversampling such conformations in md simulations. much work has been done to overcome these difficulties, such as the following. a method for including a single explicit bridging water between the pairs of the salt bridge charges was proposed by yu., and it was shown that this significantly improves the predictions. geney. introduced an empirical correction to the dielectric radii of hydrogen atoms of charged protein groups, bringing the contact depth of gb pmfs in better agreement with explicit solvent for the tested salt bridges. recently, nguyen. did more systematic fits to improve the gb solvent model parameters for protein simulations. to account for the proper coupling between nonpolar and polar solvation energies in implicit - solvent models, dzubiella. proposed a variational formalism where the gibbs free energy of the system is expressed as a functional of the solvent volume exclusion function. the method captures the sensitivity of solvent expulsion in cases of simple spherical solutes. proposed an extension of the gb model to correctly describe the solvent - excluded volume of each pair of atoms, and that improved the nonbonded pmfs. an empirical model of chen and brooks captures the context dependence of the effective surface tension, and the authors showed that it can resolve deficiencies in pmfs of nonpolar peptide side chain analogues. we first perform md simulations in explicit solvent as our sort of gold standard for what we aim to capture in a fast computational method. we describe here a way which we call interpolation pmf (i - pmf)to determine any ion ion pmf from this set of presimulated md explicit - water pmf simulations. i - pmf gets its speed because we simply capture the explicit results by simple interpolations of the precomputed results that we store in look - up tables. we show here that this simple approach results in fast and accurate pmf determinations. i - pmf is in the spirit of a recent solvation model called sea (semi - explicit assembly) in which solvation free energies of single spheres are computed through an explicit - simulation precompute step, and then assembled into the solute of interest at run - time by summing regional free energies. we describe below the two components of the i - pmf method : first, we performed extensive md simulations of small spherical ions of different charges and radii, and harvested their pmfs. second, we describe our interpolation algorithm, the i - pmf, that allows us to capture the presimulated md results with interpolation fits. next, we show that the i - pmf method gives good agreement with md and experimental results. we performed md simulations, mostly on the titan supercomputer at oak ridge national laboratory, using version 4.6.2 of gromacs. each simulation consisted of two charged solute particles and 635 water molecules in a rhombic dodecahedron box. hoover thermostat (coupling constant of 2 ps) and parrinello rahman barostat (coupling constant of 10 ps). the equations of motion were integrated using the leapfrog algorithm with a time step of 0.2 ps. the smooth version of the particle mesh ewald (pme) was used to treat electrostatics (grid spacing of 0.12, pme order of 4, real - space cutoff of 9, and ewald screening parameter of 0.347), and a 9 cutoff for lennard - jones (lj) interactions was used along with long - ranged dispersion corrections for energy and pressure. the lj size parameter (lj) of the solutes ranged from 2 to 5.5 in increments of 0.5, while the lj energy parameter (lj) was set to 0.1 kcalmol for all solutes. the choice for the lj range and for the lj value was based on the values for the alkali metal (na, k, rb, cs) and halogenide (cl, br, i) ion lj parameters of the dang force field. the solute particles were each given a formal charge of 1 or + 1. we simulated all possible combinations of solute pairs (of different sizes and charges), a total of 136 combinations (+ 1:1, + 1:+1, and 1:1 ion pairs) for a given water model. we obtained the pmfs from md simulations in which constraints were applied to hold the solute pairs at a series of fixed interparticle separations during the course of the simulation. the closest distance between the solute particles was 1.6, and the largest interparticle distance was 12. in the interval from 1.6 to 5, the separation step was 0.1, from 5 to 8 it was 0.2, and 0.4 increments were used for the rest (up to 12). therefore, to construct the pmf for a given solute pair, 60 independent simulations were performed where, after energy minimization and 0.1 ns equilibration, the trajectory over 3 ns was collected (in total, this amounted to 136 60 = 8160 independent md runs). the individual pmf was calculated by integrating the average mean force over the solute separation distance, while the entropic force due to the increase in phase space with solute separation was added to the average mean force prior to integration. uncertainties in the average mean force values were estimated from the limiting value of the block averages, and the errors in pmf were calculated by integrating the variances of the mean force. in short, in the presimulation step of the i - pmf method, we performed explicit - solvent (tip3p water) md simulations to obtain the pmfs for different combinations of solute charges (q1, q2) and lj size parameters (lj,1 and lj,2). we tabulated the values of the presimulated pmfs for given (q1, q2) and (lj,1, lj,2) at the end of the supporting information file. to validate that the i - pmf method is predictive, we performed additional test md runs for selected charge and size combinations of ions with the same protocol. the lj energy parameter was the same for all ions (lj, ion = 0.1 kcal mol). a pmf is the free energy as a function of distance r between the two ions. in addition, here we aim to account for two other variables at the same time, lj,1 and lj,2, the diameters of the two ions, each of which has a given fixed charge. below is the procedure for performing this interpolation from our md simulation data, and we use this process to determine pmfs for any given pair of charges on the ions. here is a short summary of how interpolations are performed on two variables at the same time. suppose that we are given a matrix of functional values, f(xi, yj), that correspond to points on a two - dimensional (n m) grid of points (xi, yj). here, i goes from 1 to n and j goes from 1 to m. in a two - dimensional interpolation, we want to estimate, by interpolation, the function f at some point not tabulated (xa, yb)we want to find f(xa, yb). the point (xa, yb) falls into a grid square ; i.e., four tabulated points surround the desired interior point. say that xi xa xi+1 and xj yb xj+1 ; let us denote these four functional points in the following way1 in the simplest case of interpolation on the grid square t [0, 1 ] and u [0, 1 ] are3 in order to obtain a smoother interpolated surface, higher - order interpolation techniques are applied. for example, in bi - cubic spline interpolation, we enforce the smoothness of some of the derivatives as the interpolating point cross grid square boundaries. to do a bicubic interpolation within a grid square, one needs to know the function f and the derivatives fx = f/x, fy = f/y, fxy = f/xy at each of the four corners of the square. the values of the derivatives at the grid points are determined globally by one - dimensional splines. the interpolating function has the following form4and we need to find 16 coefficients cmn by first solving the equations for the square grid points. the function f was in our case the pmf, and xi and yj were the lj. besides bilinear and cubic spline interpolation, we tested the performance of two additional interpolation techniques (nearest - neighbor and piecewise - cubic - hermite - polynomial interpolations). for a more detailed discussion regarding the two - dimensional interpolation algorithms, see refs (52) and (53). now, we describe the i - pmf interpolation methodology for capturing the pmf for any particular pair of ion sizes and charges and water model. for a given pair of solutes with charges q1 and q2 (qi = + 1 or 1) and for a given interparticle separation distance r, a matrix of pmf values corresponding to different combinations of the solutes lj size parameters (lj,1 and lj,2) was built from the presimulated pmfs. the span of lj,1 and lj,2 was from 2 to 5.5 in increments of 0.5, so the matrix dimension was 8 8. in total, 60 such matrices were constructed, each belonging to a given interparticle separation distance r [1.6,12 ] (see section above). for every matrix, a two - dimensional interpolation fit through the grid points was performed. for that purpose a matrix of interpolated values was constructed and used as a look - up table to determine the value of the pmf at a given r for an arbitrary pair of lj,1, lj,2 [2, 5.5 ] values. lj,1 and lj,2 are different from the size parameters lj,1 and lj,2 of the ions used in the grid. we stored the interpolated values of the pmf for every lj,1 in the range from 2 to 5.5 in increments of lj,1 = lj,2 = 0.01 (this means that the matrix of interpolated values was of dimension 351 351 at each r). although, for example, lj values of ions in dang force field are given to five significant digits (see table 1), no significant differences in predicted pmfs were observed if smaller lj were used to store the interpolated data. we tested different interpolation algorithms. by comparing the pmf obtained via an interpolation scheme and a full explicit - solvent simulation, we concluded that the cubic spline interpolation method worked best in the majority of cases. details of this comparison are shown below. for our initial i - pmf implementation, it takes approximately 8 s on a personal computer (pc) to build tables of presimulated values, interpolating through the grid points, making the look - up tables of interpolated values, and extracting from them the pmf for a desired combination of charges and ion sizes. after the interpolation is performed a single time and the look - up tables are available, the pmf can be extracted in 23 s on a pc. our md pmfs required a cluster of computers with many processors (the value of the pmf at each r is its own simulation), and in total, it takes more than 100 h of cpu time to get a single pmf. presimulating a systematic series of ion size and charge combinations on a supercomputer, oak ridge national lab s titan in this case, provides us with a data set of accurately calculated pmfs. we perform independent two - dimensional interpolations over these solute size combination grids to determine the interaction strength for two arbitrarily sized solutes across the full range of particle separation distances. the estimated pmf is finally assembled from this interpolation data for this specific solute pair. figure 3 shows the results of the i - pmf method utilizing four different interpolation algorithms. a size asymmetric + 1:1 system mimicking potassium iodide (lj,+ = 3.3345, lj, = 5.1705 ; see table 1) at infinite dilution was selected as a representative case. a similar analysis of i - pmf predictions in other cases shown in this paper is provided in the supporting information. the simulated pmf shows a deep first minimum, corresponding to the contact ion pair (rcp 3.6), a desolvation barrier at r 4.6, and further peaks involving the solvent shared / separated pairs (cf. table 2). we see that all of the interpolation algorithms give a pmf that qualitatively agrees with the simulated one. nevertheless, the performance of the nearest - neighbor interpolation is the least accurate of the four interpolation algorithms over a broad range of interparticle separation distances. it overestimates the contact pair (cp) peak and underestimates the desolvation barrier (numerical data are collected in table 2). the bilinear interpolation underestimates the depth of the contact pair minimum, and the values at short r (repulsive wall) are shifted to somewhat larger r. the value of the cp which follows from piecewise cubic hermite polynomial interpolation lies on the border of the experimental error, but the rest of the pmf agrees within the error bars with the md results. the pmf obtained via the cubic spline interpolation through the grid points gives the best agreement with the simulated pmf. the positions of the characteristic peaks are within the error bars (table 2). here, we want to find the best interpolation method. the goodness of the interpolation schemes for the i - pmf method can be expressed in terms of the reduced parameter (see the supporting information file for the definition). the values of red are 22.0 for bilinear interpolation, 8.2 for nearest - neighbor interpolation, 1.8 for piecewise cubic hermite polynomial interpolation, and 0.4 for cubic spline interpolation algorithm. the large value of red in the case of the bilinear interpolation algorithm originates in the differences between the predicted and simulated pmf at small interparticle separation distances (the repulsive wall of the pmf). the values of the pmf on the steep repulsive wall are on the order of 10 kcalmol (and larger), but the error in the case of + 1:1 ion pairs is small (10 kcalmol). even though the differences between the predicted and simulated pmfs are only around 1 kcalmol, the red becomes extremely large due to the small error in the simulated pmf. this region of the pmf is not very important when one discusses the structural aspects of ion ion and ion water interactions (the red was indeed defined in such a way as to avoid points on the repulsive wall ; see the supporting information file). if two points on the steep repulsive wall, lying just before the cp minimum (r 1 indicates that the fit has not fully captured the data (or that the error estimate has been underestimated), while red rcp concentrations of free ions and of the ion pair can be estimated by integrating the gab(r). the ratio of the concentration of the ion pair to the total concentration of the electrolyte (ctot) is equal to5and the concentration of the free ions is [a ] = [b ] = ctot [ab ]. rmax is the largest interparticle separation distance (12 in our case). the total concentration of the electrolyte is ctot = 3/(na4rmax3), where na is the avogadro constant. in table 3, we compare predicted association constants for ion pair formation, obtained from integrating the appropriate parts of the predicted pmfs (eq 5), with the ka from reference md simulation pmfs. we report the values for all combinations of cation anion pairs given in table 1. prior to integration, a cubic spline interpolation was used to smooth the original pmfs. it was demonstrated in previous studies that this does not significantly affect the value of ka. the error in the association constant was determined as ka = |ka ka|/2, where ka and ka correspond to constants obtained from a pmf increased and decreased by its variance, respectively. from table 3, we see that the agreement of the predicted association constants with the ones obtained by integration of the simulated reference pmfs falls in most cases within the limits of the estimated errors. the predicted ka is systematically smaller than the reference one in the cases of kcl, kbr, and rbbr, and larger in the case of rbi and csi. small differences in predicted and simulated pmfs accumulate in the integration process, resulting in larger ka differences than one might expect through visually comparing both pmfs. also, the rmax in eq 5 should in principle be, but we were restricted by the dimensions of the simulation box. differences in pmfs at larger interparticle separation distances amplify by integration and are reflected in estimated ka. in figure s6 of the supporting information, we show predicted association constants for size symmetric + 1:1 ion pairs as a function of ion sizes. we see that the predicted ka agree well with constants determined from reference md simulation pmfs. finally, we explore the law of matching water affinities of k. d. collins. in short, this is the idea that small small ion pairs are insoluble in water (because of strong electrostatic attractions), that large large ion pairs are poorly soluble in water (because they act largely like hydrophobic spheres), but that large small ion pairs are very soluble in water. here since we can consider the cp state as a precipitated form of the salt, the ka correlates with the solubility of the salt in question. figure 7 therefore shows inverse values of the experimentally determined solubilities in panel a and our predicted ka values in panel b. experiments show that nai and cscl (one ion big and the other small) have large solubilities (s is small), whereas csi and nacl (two big and two small ions) have the smallest solubilities (s is large). with respect to a given cation, the predicted association constants show a qualitatively similar pattern as experiments : for cs salts ka diminishes from csi to cscl, for rb and k there is not much difference with respect to the anion, while for na the constants increase going from nai to nacl. for potassium salts, experiments show a more significant increase in s for kcl than is reflected in the predicted ka values. for salts with a common anion, the predicted ka values increase with increasing size of the cation (from na to cs). such a trend is expressed also in experimental data for i and br salts but not for cl. it seems that the predicted ka value for kcl is too small and the one for csi too large. in that respect, the predicted ka for cscl does not follow the law of matching water affinities. it should be noted that these ka results are only for the set of ion parameters listed in table 1. many other sets of ion parameters have been proposed, and alternate sets may show different behavior. figure s7 of the supporting information shows a density map of predicted ka values for a systematic variation in the sizes of anion cation pairs (from 3 to 5.5). we see that ka is the largest in the case of two big ions and the smallest for the combination of one big and one small ion, in agreement with collins proposed behavior. inverse values of the experimentally determined solubilities, s (panel a), and ion - association constants from predicted pmfs, ka (panel b), for selected alkali metal halides. we performed extensive md simulations of the pmfs between ions of different sizes and charge in tip3p water. by applying an interpolation scheme (i - pmf) on this presimulated pmf data set, we can calculate pmfs accurately and quickly for an arbitrary combination of size and charge of the two ions. we tested various interpolation formulas and found that the cubic spline interpolation algorithm gives the most accurate representation of the majority of md results. the accuracy of the prediction is scalable based on the density of the presimulated pmf grid. improved agreement could come from a denser grid of pmfs or an expanded set of sizes. the advantage of the interpolations is about a 5 order of magnitude gain in speed, without loss of predictive accuracy (within the error bars). our interpolated pmfs are consistent with experimentally observed ion - pair solubilities and with collins law of matching affinities. the i - pmf method may be useful where there is a need to calculate accurate pmfs rapidly. | we perform extensive molecular dynamics (md) simulations between pairs of ions of various diameters (25.5 in increments of 0.5) and charge (+ 1 or 1) interacting in explicit water (tip3p) under ambient conditions. we extract their potentials of mean force (pmfs). we develop an interpolation scheme, called i - pmf, that is capable of capturing the full set of pmfs for arbitrary combinations of ion sizes ranging from 2 to 5.5. the advantage of the interpolation process is computational cost. whereas it can take 100 h to simulate each pmf by md, we can compute an equivalently accurate i - pmf in seconds. this process may be useful for rapid and accurate calculation of the strengths of salt bridges and the effects of bridging waters in biomolecular simulations. we also find that our data is consistent with collins law of matching affinities of ion solubilities : small small or large large ion pairs are poorly soluble in water, whereas small large are highly soluble. |
the approval of five new treatments for metastatic castrate - resistant prostate cancer (mcrpc) within the last 5 years has been an unprecedented milestone in prostate cancer treatment [figure 1 ]. not only have these drugs successfully changed the therapeutic landscape for this disease but at the same time these challenges include the identification and integration of novel blood - based biomarkers, sequencing or combination of the androgen - receptor (ar) targeting drugs, use of early chemotherapy intervention, and more recently, the role of genomic profiling in disease prognosis and treatment. while clinical trials remain the backbone of any drug under development, their role is pivotal towards bringing novel approaches earlier into the clinical arena. the introduction of the first autologous immune - based cellular therapy, sipuleucel - t has led to a change in the metastatic treatment paradigm ; despite a survival benefit, the lack of a robust antitumor effect has made this agent less appealing. overall, there are many challenges that immune therapies bring to the treatment tableau, and it remains unclear whether prostate is in fact an ideal target for such approaches. while the prevailing standard of care for patients with metastatic disease remains hormonal therapy with a gnrh agonist or antagonist, with or without an antiandrogen, those patients who become castration resistant have multiple options including the usual attempt at antiandrogen withdrawal. as such, patients the same observations are true for patients currently undergoing treatment with either steroids or the ar targeting drugs. patients who have failed these modalities could be offered immunologic therapy, clinical trials, or chemotherapy with recent data suggesting the benefits of early intervention with chemotherapy at the time of diagnosis of metastatic disease. the united kingdom - led stampede trial confirmed the observations of sweeney. and found that adding docetaxel chemotherapy to standard hormone therapy markedly improved survival for men with newly diagnosed advanced prostate cancer not previously treated with hormone therapy (hormone - naive). men who received docetaxel plus standard hormonal therapy lived on average 10 months longer than those who received only standard therapy. stampede used a novel trial design that was a multiarm, multistage platform to test whether the addition of treatments at the time of long - term hormone therapy initiation improved overall survival. this is the first time that a clinical trial had multiple arms with different agents in which patients could be studied prospectively. the trial included interim activity analyses based on failure - free survival to select groups to continue accrual for fully powered survival analysis. the patient population included men with metastatic (m1), high - risk localized (n0), or node - positive (n+) prostate cancer who were newly diagnosed or had high - risk recurrent disease following prior localized therapy. these were stratified to receive standard of care, i.e., hormonal therapy (n = 1184) or standard of care plus docetaxel at the dose of 75 mg / m (n = 592). the median overall survival was 71 months (interquartile range 32 to not reached) for the hormonal arm - only, 81 months (41 to not reached) for hormones and docetaxel (078, 066093 ; p = 0006). these studies endorse the potential benefits of using docetaxel in conjunction with standard hormonal therapy ; physician choice based on clinical assessment is still paramount to initiating treatment. the standards of care continue to be initiating first and second line antiandrogens i.e., the addition of antiandrogens or ar - directed therapies in the setting of patients who have been on single agent agonist or antagonists or have been on prior antiandrogens. for the latter, it should be noted that following a standard treatment algorithm for all patients may not be reasonable as these patients may have a more aggressive biology and need other means of evaluating the potential of the behavior, i.e., gene profiling of tumor or assessing circulating tumor cells. when to initiate docetaxel in this setting remains the physician 's choice. the rationale for using docetaxel first line after standard hormonal therapies in lieu of its immediate use posthormonal therapy may be based on several factors. patients whose prostate - specific antigen (psa) is rapidly rising and unresponsive to first - line hormonal therapy are more symptomatic, i.e., failure to thrive, poor oral intake, decreased performance status, or having multiple sites of pain whereby significant radiation would be needed are appropriate candidates to start docetaxel. not only was a survival benefit observed but it also improved the quality of life. determining the biology through the disease 's natural history is important in determining how to proceed with any given therapy. the approval of enzalutamide (xtandi) and abiraterone (zytiga) given before or after docetaxel has changed the natural treatment landscape of prostate cancer. however, patients continue to develop resistance to these drugs through a variety of means. as such, new strategies patients are aware of the successes of these agents in providing a survival benefit and the rapid onset of antitumor responses. nevertheless, many patients prefer to avoid the use of toxins which they often think are synonymous with chemotherapy and want a more natural approach, i.e., using their own immune system to fight the cancer. many approaches have been used to date including synthetic mimes of known carbohydrate molecules over expressed on prostate cancer cells and cells lines, dna vaccines, prime - boost virus vector vaccines, irradiated prostate cancer cell lines transduced with the genes for growth factors, as well as combination approaches with chemotherapy. we have learned a considerable amount about vaccine trials in prostate cancer [table 1 ] but still are behind the accomplishments seen in other solid tumors such as melanoma, bladder, renal cell, and nonsmall cell lung cancers. lessons learned : prostate cancer vaccine trials unlike other solid tumors, prostate cancer has been viewed as an inflammatory disease ; the extent of immune dysregulation leading to prostate cancer pathogenesis has been focused largely on inflammatory cytokines. several murine models have been generated in an attempt to understand the transition into malignancy. one novel model of prostatitis showed prostatic mast cell infiltration with subsequent accumulation of neutrophils, t lymphocytes, and macrophages, as well as increased expression of several chemokines. this chronic inflammation preceded the formation of prostatic intraepithelial neoplasia lesions that demonstrated the infiltration by immune cells. many studies have evaluated the association between specific immune cells and prostate cancer ; a majority of these investigated cd3 +, cd4 +, or cd8 + cells in prostatectomy or biopsy tissue of patients and found such cells to be pro - tumorigenic. for example, a cross - sectional study of lymphocyte infiltration in tumor tissue microarrays from patients with biochemical relapse reported that extremely high or low cd3 cell counts correlated with reduced psa recurrence - free survival (rfs). cd3 was the only immune cell marker included in this study, and no t - cell subset analyses were performed. while it may be assumed that the variation in clinical outcomes across cd3 cell quartiles may partially reflect differences in t - cell populations, there are data that suggest that higher numbers of regulatory t - cells (treg) were associated with more advanced tumor stage and psa - rfs. this led to the unsubstantiated assumption that the cd3 cells whose low numbers conferred poor prognoses were protective in nature. sipuleucel - t became the first approved treatment for asymptomatic or minimally symptomatic crpc with a survival benefit. the preparation is comprised of the patient 's own peripheral blood mononuclear cells that have been transduced with a gene for a fusion protein of prostatic acid phosphatase and granulocyte / macrophage colony - stimulating factor. the cells are then returned to the patient as three separate infusions each 2 weeks apart. despite the survival benefit, minimal or the original trials did not follow patients beyond 6 months ; based on data with checkpoint inhibitors in melanoma, the thought is that it would take many months for the treatment to work, preceded initially by a disease flare then a regression. there is evidence to suggest that antigen - specific t and b cell responses can be generated early, i.e., following the first infusion and these could be res - stimulated subsequently in vitro. cytokines were also associated with t - cell activation and could be detected in the cell culture fluids following the second and third stimulations. interleukins (ils) that were detected included il-2, 4, 5, 6, 10, 13, 17, and interferon gamma (ifn-). there was an increase in known t - cell activation markers cd134 and cd136 on ce4 + and cd8 + t cells after culture with the fusion protein. recall responses suggestive of sensitivity to the treatment many urologists have advised its use in the setting of biochemically relapsed prostate cancer with the consideration that this therapy may be changing the immune milieu early on in the disease thereby making other subsequent therapies more effective. attempts to enhance this approach have been made by others including a combination of sipuleucel - t with biologic agents. these efforts continue in an attempt to optimize the use of sipuleucel - t within the prostate cancer treatment continuum. despite the enthusiasm for using immune therapies in prostate cancer, there remains a paucity of agents for testing. prostvac, a dna vaccine comprised a recombinant vaccinia vector as the primary immunotherapy backbone. the vectors contain transgenes for psa and tricom, the latter being 3 co - stimulatory molecules intracellular adhesion molecule-1 (cd54), b7.1 (cd80), and leukocyte function - associated antigen-1 (cd58). unlike sipuleucel - t, this construct was based on the inherent immunogenicity of the pox virus. an anti - psa directed t - cell response is generated but at the same time, other antigens may be exposed that could activate other t cells. results of phase i and phase ii trials have been encouraging with the phase ii trial suggesting a survival benefit comparable to that of sipuleucel - t. however, the results of the completed phase iii trial, are eagerly awaited. the excitement over immunotherapy in solid tumors has occurred as a result of the significant and durable responses obtained in several malignancies using checkpoint inhibitors. the first, a monoclonal antibody directed against the checkpoint molecule, ctla-4 (ipilimumab, yervoy) was approved for melanoma in the setting of improved survival and antitumor effects. ctla-4 is a protein receptor that resides within the t cells and downregulates the immune system [figure 2 ]. upon t - cell engagement with dendritic or antigen presenting cells (apcs), the cells that present cancer or foreign antigens to the t cell, the t cell must have certain costimulatory molecules that tell it to either proliferate or abort its interaction with the apc. engagement of the t - cell receptor must be accompanied by a second signal that results from the binding of receptors on the t cell with either soluble factors, such as il-2, or cell - surface molecules on the antigen - presenting cell. cd28 and ctla-4 are receptors on t cells that play critical roles in the initial activation and subsequent control of cellular immunity. cd28 is expressed constitutively on t cells ; it provides a co - stimulatory signal upon binding to target ligands on antigen - presenting cells. the signal delivered through ctla-4 down regulates t - cell function and inhibits excessive expansion of activated t cells. blockade of cd28 signal with ctla-4 immunoglobulin or b7 antibodies will inhibit t - cell activation. the end result of using antibodies to ctla-4 allows the inhibition to be released and unrestricted proliferation of t cells. reproduced by permission of r and d systems a phase i / ii dose - escalating trial in patients with mcrpc of ipilimumab alone or following radiation to bone lesions, the latter in an attempt to induce antigen release, demonstrated safety and tolerability of the drug alone and in combination with radiation but predictable autoimmune events such as colitis and hypophysitis occurred. these occurred irrespective of the dose and were treated with high doses of steroids and resolved over time. the phase iii trial of ipilimumab with and without prior radiation for patients who failed docetaxel did not confirm an overall survival benefit, but there was a suggestion that patients with visceral metastases had a worse prognosis and poorer survival. how do we strategize the implementation of immune therapies when the dramatic impact seen in other solid tumor far outshines that which is seen in prostate cancer ? among the theories for suboptimal responses in prostate cancer is the concern that prostate cancer is not a hypermutated disease as seen in other diseases [figure 3 ]. other approaches now are focusing on armored or chimeric antigen receptor directed t cells, whereby the patient 's own t cells can be redirected to recognize and kill tumor cells that express a particular antigen on its surface. it has had significant successes in hematologic malignancies such as acute lymphocytic leukemia but has been limited in prostate cancer. the reasons are many such as presence of sclerotic, bone disease may prevent these cells from trafficking to sites of active tumor, many more cells may been needed given that the reticuloendothelial system may engulf these cells and prevent them from reaching their target, and the durability of the cells may not be as long as may be needed to effect antitumor effects, i.e., hours to days or weeks. while immune therapies can be used to treat at any time during the disease progression, there is still a need for immune agents that can be used either alone or in concert with other therapies. prostate as a solid tumor remains a challenge in that we are lacking the robust antitumor responses seen with other agents despite survival benefits. it is eagerly anticipated that as we gain more experience with these agents, we will be able to maximally enhance treatment responses and their durability. | introduction : prostate cancer remains a challenge as a target for immunological approaches. the approval of the first cell - based immune therapy, sipuleucel - t for prostate cancer introduced prostate cancer as a solid tumor with the potential to be influenced by the immune system.methods:we reviewed articles on immunological management of prostate cancer and challenges that lie ahead for such strategies.results:treatments have focused on the identification of novel cell surface antigens thought to be unique to prostate cancer. these include vaccines against carbohydrate and blood group antigens, xenogeneic and naked dna vaccines, and pox viruses used as prime - boost or checkpoint inhibitors. no single vaccine construct to date has resulted in a dramatic antitumor effect. the checkpoint inhibitor, anti - ctla-4 has resulted in several long - term remissions, but phase iii trials have not demonstrated an antitumor effect or survival benefit.conclusions:multiple clinical trials suggest that prostate cancer may not be optimally treated by single agent immune therapies and that combination with biologic agents, chemotherapies, or radiation may offer some enhancement of benefit. |
nppe is an uncommon complication of extubation in the early post - operative period and most commonly caused by laryngospasm. acute laryngeal spasm results in airway obstruction and can cause life threatening pulmonary edema due to negative intra thoracic pressure. the incidence of nppe has been reported to be 0.05 to 0.1% of all anesthesiology practices however it may be underreported due to failure of recognizing or misdiagnosing. a 37-year - old male patient was referred to the emergency department due to developing of severe chest pain, hypotension, and dyspnea following septoplasty operation. on admission, physical examination revealed blood pressure of 70/40 mmhg, heart rate of 75/min, bilateral diffuse crackles at the lung and wheezy breathing. oxygen saturation was 60 - 70% while breathing high - flow oxygen through a face mask. twelve - lead resting electrocardiography (ecg) revealed deep and down - sloping st - segment depressions of 3 mm in the precordial leads [figure 1 ]. due to elevated cardiac biomarkers on admission (troponin i level : 2,1 ng / dl and creatine kinase myocardial band (ck - mb) level : 7,4 mg / dl) he immediately underwent cardiac catheterization with the diagnosis of non - st elevation myocardial infarction, acute pulmonary edema and cardiogenic shock. twelve - lead resting electrocardiography showing deep and down - sloping st segment depressions in the precordial leads bedside transthoracic echocardiography showed. ejection fraction was normal, estimated pulmonary arterial pressure was not elevated and there was no evidence of thrombus in the main pulmonary artery. there was only increase in diffuse density of the lungs supporting congestion : [figure 2 ]. thoracic computerized tomography angiography demonstrating increase in diffuse density of the lungs supporting acute diagnosis acute pulmonary edema after coronary angiography, the patient was followed in the intensive care unit. during the follow - up, his oxygen saturation and blood pressure levels were increased, chest pain, and dyspnea were improved with high - flow oxygen, inotropic support, and diuretics. based on these laboratory parameters, imaging results and clinical course, the patient was diagnosed as hypoxia - induced acute subendocardial myocardial infarction, which is an unexpected result of negative pressure pulmonary edema. nppe is one of the causes of non - cardiogenic pulmonary edema which occurs with increased inspiration effort and upper respiratory tract obstruction following upper respiratory, oropharyngeal, and nasal surgeries. nppe, usually occurs during emergence from general anesthesia in the early post - operative period especially after upper respiratory tract surgery and may less frequently occur in various clinical situations (electroconvulsive therapy, etc.). upper respiratory tract obstruction caused by laryngospasm which occurs especially after extubation has been suggested as the most common cause of the nppe. increased inspiration effort caused by upper respiratory tract obstruction elevates the negative intrathoracic pressure and this condition progresses to the nppe. there are some investigations which reports healthy, young, and well - built adults are more prone to nppe because of their ability to further increase the negative intrathoracic pressure. an entirely different population carries high nppe risk which consist of the patients who are obese and patients who have obstructive sleep apnea syndrome, mediastinal mass, and upper respiratory tract obstruction. depending on the clinical circumstance, if diagnosed early, many patients do not need aggressive therapies but some patients will need invasive or non - invasive mechanical ventilation. patients usually respond to well fluid restriction, diuretics and corticosteroids. to the best of our knowledge, the diagnosis of nppe was missed in the early post - operative period, and therefore, the clinical status of the patient deteriorated with time. the complains of chest pain and dyspnea were misdiagnosed as acute coronary syndrome, acute pulmonary edema and hypotensive shock in the emergency service. in conclusion, other than anesthesiologists, the cardiologists, pulmonologists, and emergency physicians must be aware of the nppe, a diagnosis of exclusion, in the differential diagnosis of acute pulmonary edema. | negative pressure pulmonary edema (nppe) is defined as fluid transudation into the pulmonary interstitium which occurs as a result of elevated negative intrathoracic pressure caused by the upper respiratory tract obstruction and strong inspiratory effort. nppe is usually seen during emergence from general anesthesia in the early post - operative period especially after upper respiratory tract surgery. we present a case of a 37-year - old male patient who underwent septoplasty operation and developed nppe which could not diagnosed and progressed to acute subendocardial myocardial infarction. |
tufted angioma is a rare vascular neoplasm clinically characterized by slowly progressive pink to red macules or plaques on the neck, trunk, or shoulders of children or young adults. these tumors follow a benign course of angiomatous proliferation, without any evidence of malignancy. we report herein a case of multifocal tufted angiomas with annular morphology in a child for the rarity of the condition. an 8-year - old boy was referred to us for the evaluation of multiple reddish elevations on the back, which were first noticed 4 years before presentation. starting as two small reddish asymptomatic patches, the lesions gradually became elevated and fresh lesions appeared in the vicinity of the older ones. the lesions have remained stable for the preceding several months. increased sweating over the patches was noted by the parents. family history and past medical history was unremarkable. on examination, two brightly erythematous annular plaques of about 6 cm and 3 cm diameter in addition, several erythematous papules and plaques of varying sizes were seen around the lesions as well as on the right side of the back. routine laboratory tests, blood coagulation profile, and serum biochemistry panel were within normal limits. two annular erythematous plaques and several papules and plaques of varying sizes on the back we considered the differential diagnoses of tufted angioma, sudoriferous angioma (eccrine angiomatous hamartoma), and capillary hemangioma. crescent - shaped cleft like spaces were seen around the lobules [figures 2 and 3 ]. based on the clinical findings and characteristic histology, a diagnosis of tufted angioma was made. after discussion about the possible modalities of treatment and the chance of spontaneous regression, the parents preferred regular follow - up without any active intervention and the child was kept under observation. histopathology showing well - circumscribed poorly - canalized capillary lobules in the dermis with crescent - shaped spaces around them. h and e, 100 showing well - defined capillary tufts surrounded by crescent - shaped clefts. ta was described for the first time by wilson jones in 1976 as an acquired vascular proliferation of peculiar histological characteristics and reports of additional cases had helped better characterization of this clinicopathologic entity. it commonly presents in infancy or early childhood, though adult - onset cases have been reported also. ta usually presents as a macule, papule or plaque over the upper trunk, neck and proximal part of the limbs ; involvement of other locations like face, oral mucosa and lip is also known. ta is a sporadic disease with more than half of the cases occurring during the first 5 years of life. hyperhidrosis, as in our case, and hypertrichosis can also occur over the lesion. eccrine sweat gland elements however were not seen to be increased in the histopathology of our patient. ta should be differentiated from kaposiform hemangioendothelioma, juvenile capillary, or strawberry angioma, haemangiopericytoma, glomeruloid haemangioma, or pyogenic granuloma. ta can however be diagnosed by the distinctive histopathological pattern characterized by the vascular tufts of densely packed capillaries, randomly scattered throughout the dermis in a so called cannonball configuration. there are crescentic spaces surrounding the vascular tufts and lymphatic - like spaces within the tumor stroma. immunohistochemical stains show strong positivity for ulex uropaeus i lectin and en4 and unlike infantile hemangioma, negative staining for glut 1. ta, like kaposiform hemangioendothelioma, may be associated with the kasabach - merritt syndrome (kms). physicians should look for the appearance of petechiae or ecchymotic patches and if they occur, a complete hemogram (including a platelet count), prothrombin time and/or activated partial thromboplastin time and full screening for disseminated intravascular coagulation should be ordered. common association with kms, morphologic similarity, association in the same patient, and common expression of lymphatic markers had led to the suggestion that ta may be a minor form of kaposiform hemangioendothelioma or part of a spectrum of the same condition. ta usually pursue a persistent course, but spontaneous regression may occur in a proportion of cases. different modalities of treatment including complete surgical excision, cryosurgery, and radiotherapy had been tried for ta. pulsed dye laser has also been reported as an effective option for treatment of ta. embolization and vincristine are found to be effective in patients having kms. in conclusion, we have reported a rare case of multiple tufted angiomas with annular morphology in a child. the possibility of this tumor should be kept in mind while dealing with patients having lesions of similar morphology. we described a boy who had multifocal lesions with large annular plaques as well as papules and nodules. | tufted angioma (ta) is a localized benign hamartomatous vascular proliferation usually presenting in the childhood as an erythematous plaque. we report here a rare case of multifocal ta in an 8-year - old boy who presented which two large annular lesions as well as multiple papules and nodules on the back for the duration of 4 years. histology showed typical well circumscribed poorly canalized vascular lobules with cannon ball configuration. |
upper gastrointestinal bleeding is one of the most common emergencies managed by gastroenterologists that results in high patient morbidity and medical care costs (1). the main sources of upper gastrointestinal bleeding are peptic ulcers, esophagitis, drug - induced mucosal damage, sequelae of portal hypertension (esophageal varices, varices of the gastric fundus, portal hypertensive gastropathy), vascular anomalies, traumatic and postoperative lesions, and tumors (3). more recent data suggest that the proportion of cases caused by peptic ulcer disease has declined (4, 5, 7). peptic ulcers were responsible for only 21 percent of episodes of upper gastrointestinal bleeding among 7822 patients included in a national, united states database between 1999 and 2001. patients with variceal bleeding were excluded from the analysis (6). in the present study all kinds of duodenal and gastric ulcers were treated as a single entity - peptic ulcers. every patient with peptic ulcer should already be tested for h.pylori at the initial endoscopy with rapid urease test and pathologic exam (giemsa staining for helicobacter pylori). peptic ulcer as one of the most important reasons leading to upper gastrointestinal bleeding are mainly resulting from h.pylori infection or inappropriate and progressive use of nonsteroidal antiinflammatory drugs (nsaids), stress in hospitalized patients and gastric acid and pepsin (9, 10). although helicobacter pylori infection is prevalent in developing countries, recent reports from different parts of asia have shown a declining trend for h. pylori - associated ulcers (11, 12). since h. pylori - negative ulcers have been shown to have a higher incidence of mortality and recurrent bleeding, documenting the proportion of such cases is important (13). therefore more new studies are required to show recent changes in the incidence of various causes resulting in upper gastrointestinal bleeding. the number and the proportion of patients with non- h.pylori, non - nsaids peptic ulcer disease is important, given the increase in use of eradication therapy for h. pylori and widespread use of acid suppressor agents accompanied by nsaids (26). in response to recent changes in the incidence of various causes of upper gastrointestinal bleeding and peptic ulcer diseases, the present study aimed to determine the proportion and actual numbers of patients with various causes of upper gastrointestinal bleeding, referred to the emergency room of taleghani hospital from january 2010 till december 2010 with a diagnosis of upper gastrointestinal bleeding and also the proportion and actual numbers of non - nsaid, non - helicobacter pylori peptic ulcers seen in endoscopy of these patients. this was a descriptive study conducted retrospectively on 157 medical records of patients admitted with a diagnosis of upper gastrointestinal bleeding referred to taleghani hospital. medical records of patients referred to the emergency room of taleghani hospital from january the first 2010 till december the 29th 2010 with a diagnosis of upper gastrointestinal bleeding in the form of hematemesis (regurgitation or vomiting of blood), coffee ground material in nasogastric tube or melena (black tarry stool with positive occult blood test) were included in this study. patients with hematochezia (passage of bright red blood per rectum) with evidence of a source of bleeding from upper gastrointestinal tract in endoscopy were also included in this study. occasionally, the only finding pointing to a gastrointestinal hemorrhage is a laboratory result, such as iron - deficiency anemia or a positive test for occult blood in the stool with or without subjective signs such as fatigue, pallor, dyspepsia and etc. these patients were not enrolled in our study because our study focused on patients referred to emergency room. 21 patients were excluded from study ; 15 patients were referred with melena but with evidence of proximal lower gastrointestinal bleeding and no evidence of upper gastrointestinal bleeding in endoscopy, 4 patients did not consent to follow up with endoscopy and 2 patients had a diagnosis of hemoptysis and blood swallowing due to epistaxis respectively. therefore, care was taken not to enter patients with bleeding from upper respiratory tract or paranasal sinuses and lower gastrointestinal tract in this study. the mean age of patients was 55.9 years old with range of 15 - 89 years. after enrolment in the study, a questionnaire including demographic data, important points in the history, history of acid peptic disease, nsaid use, regular ingestion of aspirin, anticoagulants and other drugs can cause gastrointestinal bleeding eg, bisphosphonates, previous history of upper gastrointestinal bleeding and the cause of it, co - morbidities, physical exam such as ongoing vital signs, pallor, organomegaly, ascites, jaundice, laboratory tests such as ongoing bleeding, admission hemoglobin (hgb), activated prothrombin time (pt), platelet count (plt), partial thromboplastin time (ptt), and the results of endoscopy and etc was filled. also, mortality rate and re - bleeding in hospital and within 3 days after admission, blood transfusion need and surgery were registered. a hundred and thirty six patients entered the study, of which 51 (37.5%) were female and 85 (62.5%) were male. the most common signs on presentation among our patients were hematemesis and coffee ground vomiting (35%) and melena (69%). they had shock (blood pressure 20 mmhg decrease in systolic blood pressure or > 10mmhg in diastolic blood pressure from supine to standing position). the incidence of patients with coagulopathy (prolonged prothrombin or thromboplastin time compared with standard rates or low platelet levels < 150000) was 25%. the incidence of patients referred with comorbidities such as cirrhosis, diabetes, kidney stones, chronic renal failure, pancreatitis, chronic obstructive pulmonary disease, coronary artery disease, malignancies of the upper gastrointestinal tract was 39.7%. the frequency and incidence of patients referred with different comorbidities are shown in table 1. comorbidities seen in study population (n = 136) note that it was probable to see more than one comorbidity in one patient. sixty percent of patients gave a history of acid - peptic disease before presentation with upper gastrointestinal bleeding. thirty six percent of patients required emergent endoscopic intervention such as band ligation or sclerotherapy and 6.6% of patients required urgent surgery. during 3-day follow up of patients, 14 patients, (10.29%) died. there was a significant relationship between death and existence of comorbidities (p = 0.02) and unstable hemodynamic on arrival (p = 0.01). frequency and incidence of abnormalities seen in endoscopy of these patients referred to taleghani hospital with the impression of upper gastrointestinal bleeding are shown in table 2. frequency and incidence of abnormalities seen in endoscopy of study population (n = 136) abnormalities responsible for recent upper gastrointestinal bleeding in patients referred to taleghani hospital with upper gi bleeding are shown in table 3. abnormalities responsible for recent upper gastrointestinal bleeding in study population (n = 136) normal endoscopies could be due to subtle abnormalities, such as dieulafoy 's lesions, that can be missed easily during endoscopy. further studies are required to find out the real reason of upper gastrointestinal bleeding in these patients because normal results may be due to a careless endoscopy. in this study, peptic ulcer disease (all kinds of ulcers such as ulcers with simple clot, with oozing from ulcer bed, with adherent clot, with visible vessels, and with spurting artery in stomach and duodenum were calculated as duodenal and gastric ulcers) was seen in 61 patients (5 patients had the both duodenal and gastric ulcers). incidence of different drugs used in patients referred to the emergency room of taleghani hospital with upper gastrointestinal bleeding is shown in table 4. incidence of different drugs used by study population only 47 of these ulcers were source of recent bleeding in these patients. multiple biopsies were taken and be sent to laboratory for rapid urease test and pathologic exam (giemsa staining for h.pylori). 11.47% of patients had ulcers associated with both reasons (h.pylori and consumption of nsaids). the frequency and incidence of peptic ulcers related and not related to h.pylori and nsaids are shown in table 5. different kinds peptic ulcer disease in study population (n = 61) the relationship between different causes of peptic ulcer disease (helicobacter pylori related, nsaid use related, both helicobacter pylori and nsaid use related, no - nsaid no - helicobacter pylori related) and existence of comorbidities is shown in table 6. the relationship between different causes of peptic ulcer disease and existence of comorbidities there was no significant relationship between existence of comorbidities and peptic ulcers related to h.pylori, nsaid use, both h.pylori and nsaid use. (p = 0.734, p = 0.598, and p = 0.536 respectively) but there was a significant relationship between existence of comorbidities and no - nsaid no - helicobacter pylori peptic ulcers. in this study, peptic ulcer disease was seen in 61 patients, 45% of abnormalities seen at the endoscopy of patients referred to taleghani hospital with a diagnosis of acute upper gastrointestinal bleeding. only 47 of these ulcers were source of recent bleeding in these patients. 11.47% of patients had ulcers associated with both reasons (h.pylori and consumption of nsaids). there was no significant relationship between existence of comorbidities and peptic ulcers related to h.pylori, nsaids use, both helicobacter pylori and nsaids use. (p = 0.734, p = 0.598, and p = 0.536 respectively) but there was a significant relationship between existence of comorbidities and non - nsaids non- h.pylori peptic ulcers (p = 0.035). there was no significant relationship between age and different kinds of ulcers. according to the study of chow dk, non - nsaid non-h.pylori ulcer disease, that was believed to account for a minority of bleeding gastroduodenal ulcers, has been increasingly recognized for the past decade. their study suggests that both relative proportion and actual numbers of patients with non - nsaids and non-h.pylori ulcers have increased, whereas the prevalence of h.pylori -positive ulcers have declined. they made evidence to support non - nsaid non- h.pylori ulcers are associated with a higher risk of recurrent ulcer bleeding and a higher overall mortality as compared to h. pylori - positive ulcer disease. patients with non - nsaids non-h.pylori ulcers are often older, sicker and more frequently experience bleeding episodes while in hospital (29). the results are in agreement with chow dk. the proportion of non - nsaids non - h. pylori has been increased recently because of a decline in the proportion of ulcers related to helicobacter pylori and nsaidsthe pathogenesis of non - nsaids non- h.pylori ulcer is largely unknown. more studies are necessary to discuss about causes resulting in non - nsaids non - helicobacter pylori ulcers to justify the increase in the actual number of these ulcers in recent studies. the results of this study supports the results of study done by yakoob j. that suggests the incidence of h.pylori infection related with duodenal ulcer is common, and in the presence of co - morbidities, non-h.pylori and non - nsaids duodenal ulcers are likely to be present (24). in our study there was a significant relationship between comorbidities and non - nsaids, non- h.pylori peptic ulcers (p = 0.035), but there was no significant relationship between non - nsaids, non- h.pylori ulcers and the age of patients. | aimthe purpose of the present study was to evaluate the number and proportion of various causes of upper gastrointestinal bleeding and actual numbers of non - nsaid, non - helicobacter pylori (h.pylori) peptic ulcers seen in endoscopy of these patients.backgroundthe number and the proportion of patients with non- h.pylori, non - nsaids peptic ulcer disease leading to upper gastrointestinal bleeding is believed to be increasing after eradication therapy for h.pylori.patients and methodsmedical records of patients referred to the emergency room of taleghani hospital from 2010 with a clinical diagnosis of upper gastrointestinal bleeding (hematemesis, coffee ground vomiting and melena) were included in this study. patients with hematochezia with evidence of a source of bleeding from upper gastrointestinal tract in endoscopy were also included in this study.resultsin this study, peptic ulcer disease (all kinds of ulcers) was seen in 61 patients which were about 44.85% of abnormalities seen on endoscopy of patients. among these 61 ulcers, 44 were duodenal ulcer, 22 gastric ulcer (5 patients had the both duodenal and gastric ulcers). multiple biopsies were taken and be sent to laboratory for rapid urease test and pathological examination. about 65.53% of patients had ulcers associated with h.pylori, 9.83% had peptic ulcer disease associated with nsaids and 11.47% of patients had ulcers associated with both h.pylori and consumption of nsaids. 13.11% of patients had non - nsaids non- h.pylori peptic ulcer disease.conclusionthe results of this study supports the results of other studies that suggest the incidence of h.pylori infection related with duodenal ulcer is common, and that non - h pylori and non - nsaids duodenal ulcer is also common. |
basal cell carcinoma (bcc) is the most common type of skin cancer in the head and neck region. the incidence of bcc is on the rise, and it represents approximately 65% of all skin carcinomas [13 ]. bcc is not usually life threatening, unlike malignant melanoma, but it is locally invasive and may lead to considerable morbidity and complications. bcc is the most frequent tumor type among the us population and also has a strongly rising incidence in europe. although, the incidence of bcc in turkey is not accurately known, we think that there is a rise in the number of patients who are admitted to medical facilities. with the increase in the incidence of bcc, even though mortality is relatively low, the morbidity and treatment - related costs represent a significant burden to health care systems. recurrence is more common, especially with positive peripheral margins and certain types of bcc, like morpheaform forms. positive surgical margins in primary bcc excisions in the head and neck region were reported to be 320% in the literature. recurrence rates in incompletely excised patients were also reported at 2667%. in our study, we present a case with a series of 320 patients with 331 bccs, involving the head and neck region, and who were treated with complete surgical excision. the aim of this paper is to describe the clinical, histopathological, and epidemiological features of completely excised bcc and also to evaluate clinical features of the recurrent cases with previous histologically clear margins in the head and neck region. we performed a retrospective review of the database of our institution, ataturk training and research hospital, which has a wide acceptance of patients from the capital city ankara and surrounding cities. all patients between january 2008 and january 2013 with head and neck bccs were evaluated. only completely excised primary cases with histopathologically confirmed bccs were included in this study. all 331 lesions of 320 patients were categorized with regard to age, sex, personal and family history, skin type, tumor location, size, clinical and histopathological subtypes, reconstruction method, and recurrence rates. in order to achieve a systematic analysis of the collected data, the head and neck region were divided into the following 8 subunits : scalp, frontotemporal, orbital, nose, cheek, auricula, perioral, and chin - neck area. the diagnosis of bcc was clinically undertaken first, but, in some cases when the clinical diagnose was not clear, the dermatologist performed a pretreatment biopsy. excision margins were planned, depending on the clinical features, size, and location of the lesion. with loupe (5) magnification, the excision margin was marked in the dermatology department. while the 3 mm safe margin excision was used in small and well - defined lesions, where up to 10 mm safe margins were chosen for large tumors and unclear borders, deep margins were systematically located in the subcutaneous tissue or were more deeply situated, according to the tissue characteristics of the region. incomplete excision was defined as a residual tumor at, or within, 1 mm of the lateral or deep incision margins. all incompletely excised lesions, whether or not they were reexcised, were excluded from the study. all excision materials underwent routine histopathological examination by the pathology department and were all confirmed as bcc. methods of reconstruction were primary closure, skin grafts, and local and distant flaps. routine outpatient controls were planned as postoperative one week, one month, three months, six months, and one year. statistical analysis was performed using spss 16.0 software (chicago, il, usa). nonnormally distributed continuous variables were expressed as median, and categorical variables were expressed as numbers and percentages. mann - whitney u test was used in comparison with the continuous variables, while the chi - square test was used in comparison with the categorical variables. a sample of 320 patients, presenting 331 histopathologically confirmed bcc of the head and neck, were identified in the retrospective review. there were 176 (55%) men and 144 (45%) women. in the whole head and neck region, there was no statistically significant difference between the male and female population. when the subunits were evaluated, scalp, frontotemporal, and auricular regions presented male predominance, while the perioral region presented female predominance (p < 0.05). tumors were rare below the age of 20, with only one case in the present study who revealed no history of an underlying disease such as nevoid bcc syndrome or xeroderma pigmentosum. higher prevalence of patients (34.7%) was noted in the 6070 age group which was found to be statistically significant (p < 0.05). the personal history of chronic sun exposure which is defined by spending every working day in open areas due to occupational reasons was reported in 199 (62.2%) cases. none of the patients had a predisposing disease such as gorlin 's syndrome or xeroderma pigmentosum. one hundred and twenty patients (37.5%) were smokers for more than ten years. the fitzpatrick classifications of the patients were type 1 in 9 (2.8%) patients, type 2 in 129 (40.3%) patients, type 3 in 165 (51.6%) patients, and type 4 in 17 (5.3%) patients, respectively. there was no significant difference in the lesions that was related to a patient 's skin type, in regard to subunits (table 1). tumors most commonly occurred on the nose, with 107 (32.3%), followed by the orbital at 63 (19.1%), cheek 60 (18.1%), frontotemporal 42 (12.7%), auricular 23 (6.9%), scalp 21 (6.4%), perioral 9 (2.7%), and the chin - neck 6 (1.8%) regions. the size of the tumors was analyzed in 3 groups ; 185 (55.9%) tumors were smaller than 10 mm in diameter, 143 (43.2%) were between 10 mm and 30 mm, and 3 (0.9%) were larger than 30 mm. in the nose and cheek regions the lesions were under 10 mm and in scalp region lesions between 10 and 30 mm were higher in number (p < 0.05). reports of clinical evaluation showed that all of the examined primary bccs were of the nodular type and were either ulcerated or pigmented. with confirmation to histopathological analyses, 46 (13.8%) were of the mixed type (nodular + micronodular) ; 39 (11.7%) were of the micronodular type ; 37 (11.2%) were of the superficial type ; 31 (9.3%) were of the basosquamous type ; 23 (6.9%) were of the adenoid type ; 14 (4.2%) were of the sclerotic type of bcc. the rest of the lesions 141 (42.6%) were reported as being of the nodular type. regarding the distribution of histopathologic patterns, no statistically significant difference was found in the entire head and neck region. in the scalp, frontotemporal, nose, and cheek regions, two hundred and ninety - four excisions (88.9%) were undertaken under local anesthesia ; 37 (11.1%) were done under general anesthesia. the reconstruction method regarding the size of the defect was comprised of primary closure 84 (25.4%), full thickness skin grafting 54 (16.3%), and local flaps 192 (58%). one defect (0.3%) was reconstructed with the radial forearm free flap procedure (table 2). recurrent lesions were in the scalp (n = 3), orbital (n = 2), and nose (n = 4) regions. the histopathological subtypes of these tumors were sclerotic (n = 4), micronodular (n = 3), mixed (n = 1), and nodular (n = 1). bcc is the most common type of skin cancer in caucasians, which predominantly occurs on the exposed parts of the body, with 7585% of the lesions found in the head and neck regions [1, 2, 69 ]. according to our findings, more than half of the lesions are found in the nose (32.3%), orbital (19.1%), and cheek (18.1%) areas which are the most central and prominent parts of the entire head and neck region. these regions are also more prone to chronic sunlight exposure [2, 5, 10, 11 ]. most of the bcc lesions are reported in the 4079 age group, with the mean age of 62. in tropical regions and in patients with family history, we have had a statistically significant increase in the 6070 age group (34.7%), in all the head and neck regions. but in other mediterranean countries, the predominance is in the 7080 age bracket [8, 1315 ]. regarding the patient 's age and the location of the lesions, no significant difference was found. this can be explained with the siesta culture in these countries, which prevents their population from rush - hour sun exposure. men generally have up to 2 times higher rate of bcc [9, 10, 16 ]. in our study, when the whole head and neck region is encountered, there was no statistically significant difference between men (55%) and women (45%) that was consistent with other studies [10, 13, 17 ]. most of our patients are from central anatolia, where the population is generally comprised of men and women who both work during the daytime as farmers. when the subunits were evaluated, scalp, frontotemporal, and auricular lesions are more common in men, which can be explained by androgenic alopecia or short hair on men, when compared to the long hair or kerchief worn by women. perioral lesions are found less in men, especially on the upper lip, which can be contributed to moustache on men. phenotypic characteristics, such as fair skin type, red hair, and freckling are risk factors for bcc [9, 19 ]. most of our patients were of type 2 (39.4%) and type 3 (51.6%), regarding the fitzpatrick classification. when the subunits were encountered, there was no significant difference in the lesions based on the patients ' skin type. most of the lesions were found to be less than 10 mm in size (55.9%), when the whole head and neck region was evaluated. this might be related to the recent increase in the public cognition of health issues and admission numbers at health facilities in turkey. the ratio of tumors less than 10 mm was found to be higher in cosmetically more recognized regions, such as the nose and cheek (65.4% and 68.3%, resp.). although a universally accepted classification scheme is lacking, commonly accepted clinical types are nodular, superficial, fibroepitelial, and morpheaform [9, 12 ]. there are also histopathologic patterns, such as nodular, micronodular, adenoid, superficial, and sclerotic, which are referred to as subtypes of clinical type of nodular bcc [9, 20 ]. all of our patients were clinically evaluated as the nodular type bcc ; histopathologically most of the lesions were nodular (42.2%). in the scalp, frontotemporal, nose, and cheek regions, the main goal of the bcc treatment is to eradicate the tumor with the safest and most cost - effective method available and to provide an aesthetically and functionally pleasant outcome. even though different treatment modalities for bcc have been described, surgical excision is the most commonly preferred method for tumor removal. in the literature, the 3 mm peripheral surgical margin is adequate for the clearance of 85% of small and well - defined bccs, and the 4 - 5 mm margin will raise this to 95%. it has been shown that loupe magnification can assist in reducing incomplete excision rates [2022 ]. in our practice, with loupe magnification, the 3 to 10 mm excision margin was used, depending on the clinical features, size, and location of the lesion. it has been shown that the recurrence rate for primary bccs after surgical excision varies between 5% and 14% [1, 5 ]. lesions in the head and neck region are at more risk for recurrence, when compared to lesions in trunk and extremities [1, 5, 7, 23 ]. even though some factors, like anatomical localization, histopathological characteristics, and initial treatment strategy, have been proposed, there is lack of accepted understanding in the recurrence of the lesions. incomplete excision was reported as one of the risk factors for recurrence [6, 10, 20, 24 ]. the main difference of our study when compared with the previous studies is the precise evaluation of the factors effective on recurrence in completely excised bccs. in our series, recurrent lesions were in the scalp (n = 3), orbital (n = 2), and nose (n = 4) regions. interestingly, all recurrences were in the median parts of head and neck region ; this may be attributed to high recurrence rates in embryonic fusion planes [9, 2527 ]. histopathological subtype of recurrent tumors was mainly micronodular and sclerotic, which is more difficult to eradicate and has high risk of recurrence. our study revealed that recurrence is related to the localization and histopathologic subtype, whereas they were not related to age, sex, and size of the lesion. the low recurrence rates in our series might be due to the relatively low rates of histopathologically aggressive subtypes, excision with appropriate margin, and no positive surgical margins after surgery. in conclusion, this study presents a relatively large number of series of surgically treated bccs in the head and neck region. we would like to emphasize the importance of the preoperative evaluation of the patient keeping the epidemiology in mind, defining the surgical margins in order to get lower recurrence rates, and by motivating the patient for follow - up visits, in order to evaluate outcomes and diagnose recurrences. | the aim of the study is to analyze all completely excised bccs in the head and neck region with regard to age, sex, personal and familial history, skin type, tumor localization and size, histopathological subtype of tumor, reconstruction method, and recurrence rates. incompletely excised bccs were not included in this study since incomplete excision is the most important preventable risk factor for recurrence. in 320 patients, 331 lesions were retrospectively evaluated by dividing into the following 8 subunits : scalp, frontotemporal, orbital, nose, cheek, auricula, perioral, and chin - neck area. most of the patients were in 6070 age group (34.7%). the nose (32.3%) was the most common site of presentation. clinically, all lesions and, histopathologically, most of the lesions (42.2%) presented were of the nodular type. all cases of recurrence after complete excision (n = 9, 2.7%) were located in the median parts of the head and neck region and were mainly diagnosed histopathologically as sclerotic and micronodular. even though completely excised, head and neck region bccs, especially which are more prone to recurrence due to anatomical and histopathological properties, should be more closely monitored in order to decrease morbidity and health care costs. |
myocardial infarction (mi) is one of the leading causes of morbidity and mortality throughout the world. in china, a newly industrialized country, it is estimated that 3 million patients have suffered from mi and an additional 0.5 million patients experience acute mi yearly. substantial progress has been made in myocardial reperfusion strategies, revascularization procedures and novel pharmacological approaches in the past 30 years. however adult cardiomyocytes have a very limited capacity to regenerate themselves, and therapies for rescuing dead cardiomyocytes remain limited. basic research in this area has progressed rapidly since the early 1990s, and furthermore, the evidence from preclinical studies has also been encouraging. the first clinical trial of ctx for mi menasche in 2002 and since then hundreds of clinical safety and efficacy studies have been performed worldwide. bone marrow (bm)-derived cells have been the most popular cell source in current studies. recently, a meta - analysis was performed to review the outcome of 18 studies involving 999 patients who received bm - derived ctx for cardiac repair. although the administration of bm cells was safe, it only improved heart function by 3% increase in ejection fraction and a 5% reduction in infarct scar size. from a clinical point of view, this moderate improvement is far from satisfactory. in addition to a search for a new more robust cell source, some other potential hurdles must be considered. in the end there is the possibility that cell survival and redistribution after transplantation might drastically improve the outcome and safety of cell therapy. poor survival of engrafted cells has adversely influenced the therapeutic efficacy of ctx for mi. after intracoronary infusion of bm - mononuclear cells, only 5% of transplanted cells could be detected in the myocardium within 2 hrs after infusion ; furthermore, only 1% of infused cells resided in the heart 18 hrs after transplantation. although intramyocardial injection was considered to be the most effective route for precise cell delivery to target areas, the intramyocardially injected cells decreased rapidly from 3480% at 0 hr to 0.33.5% at 6 weeks. besides the delivery route and cell types, several reasons may account for the poor cell survival rate, as illustrated in fig., the transplanted cells face the serious challenge of an unfriendly environment in which they may not survive. the deprivation of oxygen and nutrient supply, the mechanical tension from myocardial motion and various cytotoxic factors in the ischemic myocardium can result in large - scale cell apoptosis and death. the in vivo cell proliferation and differentiation of transplanted cells usually occurs weeks after transplantation. the first week is the crucial time for cell survival and can determine the final therapeutic efficacy. in our previous in vitro study, we demonstrated that serum deprivation, not hypoxia, was the main reason for the apoptotic cell death of bm - derived ischemic mesenchymal stem cells (mscs). mitochondrial dysfunction leading to increased caspase-3 activation could be a major contributor to this process. however, the choice of different oxygen tensions, serum concentrations and various cell types makes it difficult to elucidate the precise mechanisms underlying apoptotic cell death. clearly, with the increasing understanding of the signalling pathways involved in cell survival, dedicated strategies should be capable of creating a friendly milieu of host infarcted myocardium to improve long - term cell viability in vivo. in a non - human primate model, culture - expanded mscs were infused by an intravenous route. the cells were distributed to a wide range of tissues including gastrointestinal tissues, lung, skin, thymus, etc.. our previous study also revealed extensive extracardiac cell distribution after intramyocardial injection into the border zone of an acute mi area. in another preliminary study, we injected male mscs into a sex - mismatched rat chronic mi model (fig. one hour after ctx, 56% of injected cells were untraceable and 8% of cells were harboured by filter organs. interestingly, we found 3% and 4% of cells in venous and arterial blood, respectively. these findings indicate that cell migration is initiated in an ultra - early stage after transplantation and that blood flow is the main thus venous blood could collect escaped cells from the right and left side of the heart and transport them into the lung. similarly, cells washed from the left side of the heart could be pumped into the systemic circulation via the left ventricular space and then captured by reticulo - endothelial systems, mainly located in liver and spleen. we presume that the survived cells had been integrated into the micro - environment at a late stage after ctx. therefore, the myocardial contractile force would not squeeze more retained cells into the blood at a late stage. however, the total amount of escaped cells accounted for 37% of total traceable cells at 2 weeks after transplantation. quantification of implanted male mscs in female rats by real - time polymerase chain reaction. the data are expressed as the average percentage, relative to the initial number of injected cells. at this time, little is known about the impact of escaped progenitor cells on extracardiac organs. a previous study has shown that transplanted bm cells could be a double - edged sword with an unexpected negative impact on the liver. in the clinical experience of angiogenic gene therapy, unwanted angiogenesis, including liver haemangioma and retinopathy, was observed. therefore, a control of the expressed angiogenic factors to the local ischemic myocardium was advocated. recently, more robust cell types, including induced pluripotent stem cells (ips) and cardiac stem cells were introduced into the field of heart cell therapy [14, 15 ]. until now, the purity of ips or embryonic stem cells derived cardiomyocytes has been unsatisfactory. undoubtedly, transplanted ips or embryonic stem cells without a cardiomyocyte phenotype hold more potential for differentiation and teratoma formation. once these cells escape from the heart, they could have a more serious impact on the extracardiac organs. various approaches have attempted to support the survival and engraftment of implanted cells (summarized in fig. 3). in vitro gene modification could be very labour intensive and carry inherent risks for future clinical translation. therefore, some more convenient approaches such as hypoxia, heat shock and low - level laser irradiation (llli) have also been applied for in vitro cell manipulation. these approaches could induce faster cell proliferation, augment cytokine release and increase preconditioning or anti - hypoxia ability. another easy way to enhance cell retention is to increase the size of the injected cells with microencapsulation. the size of freshly isolated progenitor cells is usually 1020 m, so the cells could be packed into microcapsules (150250 m). with the increased size, the cells would not be drained to the myocardial vein, collateral channels or ventricular lumen and thus reside in the injection sites. fabrication of cell - seeded biodegradable scaffolds is a promising tissue - engineering approach to nourish grafted cells and maintain neo - cardiac tissue geometry and structure. however, many issues remain unsolved, including the optimal scaffold biomaterial, cell adhesion and diffusion of nutrients. besides direct pre - treatment with cytokines, recently some small molecules have been identified as novel regulators that are capable of enhancing cell survival rates. lysophosphatidic acid, as an endogenous lipid mediator, could prevent cell apoptotic death and increase cell survival 2-fold within 1 week after cell injection. local injection of tannic acid could cross - link fibrous collagen and inhibit matrix metalloproteinase activity. the consequent stabilization of the extracellular matrix would reduce myocardial mechanical tension and thereby promote cell survival. some associated procedures could be combined with direct cell injection during open - chest revascularization surgery for patients with mi. we found that llli augments the expression of growth factors and superoxide dismutase in the myocardium, and hence markedly enhances early survival of transplanted mscs. beginning in 2009, we initiated a clinical study at our centre to evaluate the efficacy of coronary bypass surgery, combined with pedicled omentum - wrapped heart tissue patch implantation. the omentopexy could significantly stimulate angiogenesis in the locally wrapped area and hence improve cell survival. poor cell survival and extensive cell migration have caused clinicians and scientists to re - evaluate the safety and efficacy of heart cell therapy. to achieve satisfactory cell survival, we need to apply a comprehensive strategy to join in vitro and in vivo approaches that precondition both the cells and the infarcted myocardium. although there are clearly many obstacles to overcome, new understanding and technologies will hopefully move cell - based regenerative therapy closer to the clinic. | abstractcell transplantation has become an attractive option for cardiac regenerative therapy. however, poor cell survival and extensive redistribution throughout the body can drastically affect the outcome and safety of cell therapy. although various approaches have been attempted to support the survival and engraftment of implanted cells, we need to apply a new comprehensive strategy by melding the in vitro and in vivo approaches to recondition the cells and infarcted myocardium. here we summarize our understanding of cell survival and migration after transplantation into the damaged heart. |
the quality of life (qol) assessment in elderly people has been a priority in many developed countries to guide public policy, including social, health, and environmental actions. the world health organization (who) developed an international cross - culturally comparable qol assessment instrument, whoqol. to adapt whoqol to use with elderly people, physical activity has been associated with better qol and fewer chronic diseases. despite the benefits of physical activity however, physical activity programs are needed, especially in countries such as brazil, which is experiencing a rapid demographic transition and a burden of chronic diseases. in 2002, the brazilian government created a public program, the academia da cidade program (acp) to encourage community individuals to have physical and leisure activity at public parks (www2.recife.pe.gov.br/projetos-e-acoes/projetos/academia-da-cidade., accessed 22 january 2012). at present, acps are located in recife in open public spaces in different neighborhoods with approximately 32,000 participants, 15% of whom are elderly. our aim is to assess the qol according to whoqol - bref and whoqol - old instruments on elderly participants of acp in recife. this cross - sectional study evaluated elderly people attending the acp between march and december 2013. this study was previously approved by the research ethics committee of imip and participants signed an informed consent. acp activities are led by physical education teachers and offer free supervised leisure - time physical activity sessions, including stretching, aerobic, and resistance training exercises, walking, dancing classes, and other activities. acp activities are offered every hour from 5 am to 10 am and 5 pm to 10 pm during weekdays. participants aged 60 years or more, who have been participating in the program at least twice a week, were selected. the whoqol - old and whoqol - bref questionnaires were applied, and for illiterate participants, the questionnaire was filled out by the researchers. whoqol - bref explores six domains : environment (8 items), physical (7 items), psychological (6 items), social relationships (3 items), and overall qol (2 items). whoqol - old accesses elderly - specific facets of qol covering : sensory abilities, autonomy, past, present, and future activities, social participation, death and dying, and intimacy. both portuguese version of these questionnaires were validated and showed adequate internal consistency ; cronbach 's coefficients were ranged from 0.71 to 0.88. these questionnaires were administered by two trained interviewers without knowing the goal of the study. a third questionnaire was used to obtain information about the duration of participation in the acp, demographic, socioeconomic, and clinical data of the participants. whoqol - old and whoqol - bref scores were presented in mean (standard deviation). proportions were compared by chi - square test and the distribution of metric variables among tertiles of whoqol scores by kruskal univariate and multivariate analysis, using linear regress models, were performed to identify variables associated with whoqol - bref and whoqol - old. most of them were retired women, had few years of schooling, and had low family income. main characteristics of the 181 participants demographic, socioeconomic, and clinical data according to the whoqol - bref and whoqol - old scores in tertiles (lowest, intermediate, and highest) are shown in table 2. according to the whoqol - bref and whoqol - old scores, only the duration of participation in the apc showed a significant difference of p = 0.030 and p = 0.003, respectively. demographic, socioeconomic, and clinical data of participants according to the whoqol - bref and whoqol - old scores in tertiles (lowest, intermediate, and highest) multivariate analysis linear regression models were adjusted. predictor variables for whoqol - bref were age, gender, duration of participation in the apc, and marital status and for whoqol - old they were gender, age, duration of participation in the apc, retired and blood hypertension ; all showed p < 0.20 in univariate analysis [table 2 ]. after mutual adjustment, only the duration of participation in the apc remained significantly associated with whoqol - bref and whoqol - old scores (p = 0.023 and p = 0.038), respectively [table 3 ]. adjusted effects of the variables age, time, and marital status on the variables whoqol - bref and whoqol - old through the adjustment of the linear regression a longer duration of participation in this community - based physical activity program, acp, was associated with a positive trend in qol of the participants according to both whoqol - bref and whoqol - old scores. acp is a brazilian government program focusing on physical activity and aims to provide the community of recife city with the opportunity to engage in regular physical activity by offering free physical exercise classes. this program is offered in public places (parks and squares), the classes are conducted by physical education teachers, and healthy lifestyles are recommended. this acp results are in agreement with other studies that have shown a positive impact of community - based physical activity program in health. recently, sampaio and ito described in japan that the highest influence on whoqol - bref was physical activity. the scores of qol in elderly people have been linked to other factors, such as age, family income, schooling, and positive perceptions about health and depression. in our study, age, income, and schooling however, we studied a specific elderly age and all the participants had low income and schooling. this reason could justify the finding of no differences among these variables in our study. a significant increase in life expectancy and a decrease in the fertility rate in recent decades in brazil have led to a significant aging population. aging is associated with the loss of independence and autonomy, which may contribute to a decreased qol. however, the elderly people studied seem to be already committed and motivated to this specific physical activity program, acp. this may explain our finding of similar whoqol - old and whoqol - bref scores among the elderly at different ages. the duration of participation in acp may have influenced the variations in response about qol regarding age on the questionnaire, and we observed a significant increase in the values of the whoqol - old and whoqol - bref scores according to the time participated in acp. however, our study had a cross - sectional design, and exposition (acp) and outcome (qol) were assessed simultaneously women in our region have about a 10 years longer lifespan as compared to men. another explanation is that women, especially in our culture, adhere more to preventive health programs than men. our whoqol - old and whoqol - bref scores are similar to elderly women from rural area in brazil, which are greater as compared to residents in the urban area our scores are also similar to those found in elderly brazilian women with good physical and psychosocial health. these comparisons seem to indicate that acp can compete to a better qol for those women participants. possibly because the whoqol - bref emphasizes areas questioning physical aspects and social relations, as well as psychological and environmental aspects that the whoqol - bref questionnaire showed a significant relationship between the higher score and participating time in the acp program. our whoqol - bref scores were comparable with normative data from a randomly selected sample of a general brazilian population. the whoqol - old questionnaire also surveys the aspect of social participation, but rather in the areas of sensory functioning, autonomy, death and dying, intimacy and past, present, and future activities. a subgroup of elders with a greater degree of dependence and fragility might reveal more consistent differences on the whoqol - old questionnaire if they were participating in the acp program. it is a fact, however, that most elderly participants in acp are independent and autonomous. our study design did not allow us to conclude that a longer participation in the program was responsible for an increase in qol scores despite the association with aging.. the sample may be insufficient for a more faithful indication of qol and it was drawn from participants who joined the acp ; therefore, it may not be representative. groups with a similar number of individuals in each period of time in the program (e.g., 1, 2 and 5 years) could specify the relationship between duration of participation in acp and qol scores. acp seems to be an important strategy for favoring the qol of the elderly population in recife. | purpose : to assess the quality of life (qol) of elderly people in the academia da cidade program (acp), a public brazilian government program encourages physical and leisure activity in recife, brazil.materials and methods : this cross - sectional study evaluated 181 elderly people attending the acp at least twice a week. demographic, socioeconomic, and clinical data were presented according to world health organization qol (whoqol)-bref and whoqol - old scores.results:univariate analysis showed that whoqol - bref and whoqol - old scores were positively associated with longer duration of participation in acp, p < 0.030 and p < 0.003, respectively, a multivariate regression analysis showed that duration of participation in acp remained associated with higher whoqol - bref (p = 0.023) and whoqol - old (p = 0.038) scores.conclusion:a longer duration of participation in a community - based physical activity program, acp, may contribute to a better qol for the elderly in brazil. |
adults with type 2 diabetes inadequately controlled (a1c 711%) with maximally tolerated stable doses of metformin, sulfonylurea, or both for 3 months were randomized (1:1) to 1.8 mg liraglutide once daily or 10 g exenatide twice daily. after 26 weeks, patients continued into a nonrandomized 14-week extension : all exenatide patients were switched to 0.6 mg liraglutide once daily for 1 week, then escalated to 1.2 mg for another week, and then given a final maintenance dose of 1.8 mg. background oral antidiabetes drugs remained unchanged, although sulfonylurea doses could be decreased by 50% if unacceptable hypoglycemia occurred. visits occurred at weeks 2 (screening), 0 (randomization), 4, 8, 12, 20, 26, 34, and 40 for both groups. efficacy and safety assessments during the extension phase (weeks 2640) were identical to those previously described (3). extension intention - to - treat (itt) (all randomized patients exposed to trial product who entered the extension) and extension safety (all patients exposed to trial product who entered the extension) populations were used for efficacy and safety analyses, respectively. changes from baseline (last available observation up to 26 weeks) to week 40 within each treatment group were analyzed by paired t tests. post - baseline missing values were imputed using last observation carried forward. unless noted, mean (se) values are presented. demographics were well matched between groups and similar to those previously reported (3). overall, 376 of 389 patients (97%) completed the extension : 10 of 187 (5.3%) with exenatideliraglutide and 3 of 202 (1.5%) continuing liraglutide withdrew. withdrawals (n [% ]) in the exenatideliraglutide and liraglutide groups, respectively, were due to either adverse events (6 [3.2% ] and 0), ineffective therapy (0 and 2 [1.0% ]), protocol noncompliance (0 and 1 [0.5% ]), meeting withdrawal criteria (1 [0.5% ] and 0), or other reasons (3 [1.6% ] and 0). demographic and screening characteristics were similar between patients who withdrew during the extension and those who completed the extension, with the exception of mean duration of diabetes, which was longer for those withdrawing (12.2 years) than completers (7.9 years). mean a1c further decreased from 7.2% at week 26 to 6.9% at week 40 (0.32 0.043% ; p < 0.0001) after switching from exenatide to liraglutide, but remained similar with continued liraglutide (7.0 to 6.9% ; 0.06 0.041%, p = 0.1222) (fig. effect of switching from exenatide to liraglutide or continuing liraglutide on various measures of efficacy (extension itt population). b : percentage of patients reaching a1c targets at week 26 (after the main part of the trial) and week 40 (after the exenatide group switched to liraglutide for 14 weeks). all patients originally in the exenatide group switched to liraglutide at week 26, whereas the liraglutide group continued liraglutide. the white line within each bar indicates the percentage of patients reaching a1c targets at week 26 and the top of the bar indicates the percentage reaching a1c targets at week 40. in a and c e, post - baseline timepoints were imputed using last observation carried forward. after switching from exenatide to liraglutide, further reductions in fpg (fig. 1d ; 0.9 0.15 kg, p < 0.0001), and sbp (fig. 1e ; 3.8 0.84 mmhg, p < 0.0001) occurred while the homeostasis model of -cell function (homa - b) assessment increased (14.5 4.4%, p = 0.001), consistent with the fpg reductions. in patients continuing liraglutide, reductions in fpg (fig 1d, 0.4 0.15 kg, p = 0.0089), and sbp (fig. 1e ; 2.2 0.88 mmhg, p = 0.0128) occurred. no significant changes in postprandial glucose (except after lunch with exenatideliraglutide [0.64 0.21 mmol / l, p = 0.0032 ], diastolic blood pressure, fasting insulin, fasting c - peptide, proinsulin - to - insulin ratio, or homeostasis model assessment similar numbers of patients reported one or more adverse events during the extension (exenatideliraglutide : 70 [37.4% ] and liraglutide : 76 [37.6% ]). most adverse events were mild in severity (exenatideliraglutide : 79/117 events, liraglutide : 81/120 events) ; investigators assessed most as unrelated to trial drug (exenatideliraglutide : 72/117 events, liraglutide : 94/120 events). nausea and diarrhea occurred in 3.2% of patients switching from exenatideliraglutide and 1.5% in those continuing liraglutide, whereas vomiting occurred in 0.5% switching from exenatideliraglutide and 2.0% in those continuing liraglutide. one major hypoglycemic episode occurred in a patient continuing liraglutide, while extension rates (episodes / patient - year) of minor hypoglycemia were 1.30 (exenatideliraglutide), down from 2.60 with exenatide at week 26 (3), and 0.74 (liraglutide). thirteen adverse events associated with withdrawal from the extension of six patients in the exenatideliraglutide group were myocardial infarction, diarrhea (three events), impaired gastric emptying, eructation, nausea, lethargy, paraesthesia, anxiety, depressed mood, depression, and dyspnea. for two of these six patients, the adverse events associated with their withdrawal from the extension (nausea and diarrhea) had been previously reported as separate events during the 26-week exenatide treatment period. four patients in the exenatideliraglutide group had seven severe adverse events (cardiac failure, myocardial infarction, cataract, chest discomfort, chronic obstructive pulmonary disease [two events ], dyspnea). five patients continuing liraglutide had eight severe adverse events (cerebral infarction, cerebrovascular accident, transient ischemic attack, acute coronary syndrome, coronary artery occlusion, portal vein thrombosis, rectal cancer, and depression). two deaths occurred (exenatideliraglutide : myocardial infarction after 198 days of treatment ; liraglutide : cerebral infarction [patient completed the study but died shortly after ]). calcitonin levels remained at the lower level of the normal range (< 1 pg / ml) and did not differ between groups. mean a1c further decreased from 7.2% at week 26 to 6.9% at week 40 (0.32 0.043% ; p < 0.0001) after switching from exenatide to liraglutide, but remained similar with continued liraglutide (7.0 to 6.9% ; 0.06 0.041%, p = 0.1222) (fig. 1a). effect of switching from exenatide to liraglutide or continuing liraglutide on various measures of efficacy (extension itt population). b : percentage of patients reaching a1c targets at week 26 (after the main part of the trial) and week 40 (after the exenatide group switched to liraglutide for 14 weeks). all patients originally in the exenatide group switched to liraglutide at week 26, whereas the liraglutide group continued liraglutide. the white line within each bar indicates the percentage of patients reaching a1c targets at week 26 and the top of the bar indicates the percentage reaching a1c targets at week 40. in a and c e, post - baseline timepoints were imputed using last observation carried forward. after switching from exenatide to liraglutide, further reductions in fpg (fig. mmol / l, p < 0.0001), body weight (fig. 1d ; 0.9 0.15 kg, p < 0.0001), and sbp (fig. 1e ; 3.8 0.84 mmhg, p < 0.0001) occurred while the homeostasis model of -cell function (homa - b) assessment increased (14.5 4.4%, p = 0.001), consistent with the fpg reductions. in patients continuing liraglutide, reductions in fpg (fig. 1d, 0.4 0.15 kg, p = 0.0089), and sbp (fig. 1e ; 2.2 0.88 mmhg, no significant changes in postprandial glucose (except after lunch with exenatideliraglutide [0.64 0.21 mmol / l, p = 0.0032 ], diastolic blood pressure, fasting insulin, fasting c - peptide, proinsulin - to - insulin ratio, or homeostasis model assessment insulin resistance [homa - ir ]) occurred in either group. similar numbers of patients reported one or more adverse events during the extension (exenatideliraglutide : 70 [37.4% ] and liraglutide : 76 [37.6% ]). most adverse events were mild in severity (exenatideliraglutide : 79/117 events, liraglutide : 81/120 events) ; investigators assessed most as unrelated to trial drug (exenatideliraglutide : 72/117 events, liraglutide : 94/120 events). nausea and diarrhea occurred in 3.2% of patients switching from exenatideliraglutide and 1.5% in those continuing liraglutide, whereas vomiting occurred in 0.5% switching from exenatideliraglutide and 2.0% in those continuing liraglutide. one major hypoglycemic episode occurred in a patient continuing liraglutide, while extension rates (episodes / patient - year) of minor hypoglycemia were 1.30 (exenatideliraglutide), down from 2.60 with exenatide at week 26 (3), and 0.74 (liraglutide). thirteen adverse events associated with withdrawal from the extension of six patients in the exenatideliraglutide group were myocardial infarction, diarrhea (three events), impaired gastric emptying, eructation, nausea, lethargy, paraesthesia, anxiety, depressed mood, depression, and dyspnea. for two of these six patients, the adverse events associated with their withdrawal from the extension (nausea and diarrhea) had been previously reported as separate events during the 26-week exenatide treatment period. four patients in the exenatideliraglutide group had seven severe adverse events (cardiac failure, myocardial infarction, cataract, chest discomfort, chronic obstructive pulmonary disease [two events ], dyspnea). five patients continuing liraglutide had eight severe adverse events (cerebral infarction, cerebrovascular accident, transient ischemic attack, acute coronary syndrome, coronary artery occlusion, portal vein thrombosis, rectal cancer, and depression). two deaths occurred (exenatideliraglutide : myocardial infarction after 198 days of treatment ; liraglutide : cerebral infarction [patient completed the study but died shortly after ]). calcitonin levels remained at the lower level of the normal range (< 1 pg / ml) and did not differ between groups. no medullary thyroid carcinoma or pancreatitis cases were reported during the extension. this extension shows that patients can be simply and safely switched from twice - daily premeal exenatide to meal - independent once - daily liraglutide using weekly dose escalation from 0.6 to 1.2 to 1.8 mg. additional reductions in body weight and sbp occurred in both groups. over 40 weeks, the magnitude of these changes and differences between liraglutide and exenatide are consistent with results reported in phase 3 lead (liraglutide) (38) and exenatide (911) trials. the greater efficacy of liraglutide may be due to sustained levels achieved over 24 h by once - daily dosing compared with biphasic levels achieved during the 2.4-h half - life of exenatide after dosing within 1 h of breakfast and dinner (12). given the likely preference for once - daily meal - independent dosing, liraglutide appears to be a useful addition to the diabetes treatment armamentarium. | objectiveto evaluate efficacy and safety of switching from twice - daily exenatide to once - daily liraglutide or of 40 weeks of continuous liraglutide therapy.research design and methodswhen added to oral antidiabetes drugs in a 26-week randomized trial (liraglutide effect and action in diabetes [lead]-6), liraglutide more effectively improved a1c, fasting plasma glucose, and the homeostasis model of -cell function (homa - b) than exenatide, with less persistent nausea and hypoglycemia. in this 14-week extension of lead-6, patients switched from 10 g twice - daily exenatide to 1.8 mg once - daily liraglutide or continued liraglutide.resultsswitching from exenatide to liraglutide further and significantly reduced a1c (0.32%), fasting plasma glucose (0.9 mmol / l), body weight (0.9 kg), and systolic blood pressure (3.8 mmhg) with minimal minor hypoglycemia (1.30 episodes / patient - year) or nausea (3.2%). among patients continuing liraglutide, further significant decreases in body weight (0.4 kg) and systolic blood pressure (2.2 mmhg) occurred with 0.74 episodes / patient - year of minor hypoglycemia and 1.5% experiencing nausea.conclusionsconversion from exenatide to liraglutide is well tolerated and provides additional glycemic control and cardiometabolic benefits. |
threshold values are of prime importance in providing a sound basis for public health decisions. a key issue is determining threshold or maximum exposure values for pollutants and assessing their potential health risks. environmental epidemiology could be instrumental in assessing these levels, especially since the assessment of ambient exposures involves relatively low concentrations of pollutants. this paper presents a statistical method that allows the determination of threshold values as well as the assessment of the associated risk using a retrospective, longitudinal study design with a prospective follow - up. morbidity data were analyzed using the fourier method, a time - series analysis that is based on the assumption of a high temporal resolution of the data. this method eliminates time - dependent responses like temporal inhomogeneity and pseudocorrelation. the frequency of calls for respiratory distress conditions to the regional mobile medical emergency service (mmes) in the city of leipzig were investigated. the entire population of leipzig served as a pool for data collection. in addition to the collection of morbidity data, air pollution measurements were taken every 30 min for the entire study period using sulfur dioxide as the regional indicator variable. this approach allowed the calculation of a dose - response curve for respiratory diseases and air pollution indices in children and adults. significantly higher morbidities were observed above a 24-hr mean value of 0.6 mg so2/m3 air for children and 0.8 mg so2/m3 for adults.(abstract truncated at 250 words)imagesfigure 1.figure 2. afigure 2. bfigure 2. cfigure 2. dfigure 3.figure 4.figure 5. |
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the origin of tumors of minor salivary glands account for less than 25% of all salivary neoplasms with 40 kleinsasser and klein introduced the term basal cell adenoma to describe a benign salivary gland tumor comprised of uniform appearing basaloid cells arranged in solid, trabecular, tubular, and membranous patterns, but lacking the myxoid and chondroid mesenchymal - like component as seen in pleomorphic adenoma. it has been recognized as one of the nine subcategories of salivary gland adenomas in the second edition of the salivary gland tumors classification of the world health organization (who), and accounts for approximately 1 2% of all salivary gland epithelial tumors. it is considered as a low - grade malignant tumor with a high recurrence rate. a 32-year - old male patient reported with a chief complaint of painless swelling on the right side of the palate, which was first noted by a local physician two months prior. on intraoral examination an, asymptomatic solitary swelling with an insidious onset and growth was seen on the palate. on inspection it was ovoid in shape, measuring about 3.5 2 cm, with well - circumscribed borders. it extended from the mesial aspect of 14 to the distal aspect of 17, and laterally from the free gingival margin in relation to 14 17 to 0.5 cms away from the midline. on palpation the swelling was nontender, with a smooth surface, and was soft - to - firm in consistency. a well - defined radiolucency with distinct margins was noted in relation to 14 17. with a clinical diagnosis of adenoma of minor salivary glands, the cytosmears stained with rapid papanicolaou stain revealed predominantly basaloid cells arranged in clumps with hyperchromatic nuclei and an increased nuclear - cytoplasmic ratio [figure 1 ]. suspecting it to be an adenoma of minor salivary glands, excisional biopsy was recommended. the biopsy specimen obtained was about 3.0 2.0 1.5 cms in dimension, brownish - white in color [figure 2 ], and soft - to - firm in consistency. the sections revealed an overlying orthokeratinized type of stratified squamous epithelium with an underlying connective tissue. in the deeper layers of the mucosa was a well - circumscribed encapsulated tumor mass enclosing multiple islands and cords of epithelial cells, supported by a small amount of fibrous stroma [figure 3 ]. the basaloid cells making up the bulk of the tumor were found to be isomorphic. the peripheral cells were palisaded with a cuboidal - to - columnar shape, while the central cells were relatively rounded. these peripheral cells were hyperchromatic, while the central cells had pale staining nuclei [figure 4 ]. the sharp demarcation between the neoplastic epithelial cells and the surrounding connective tissue was evident. pas positive membrane - like structures surrounding cellular nests showed intense eosinophilia that clearly delimited the basaloid cells [figure 5 ]. papanicolaou stained cytosmears with basaloid cells arranged in clumps with hyperchromatic nuclei and an increased nuclear - cytoplasmic ratio submitted biopsy specimen, brownish - white in color an overlying orthokeratinized type of stratified squamous epithelium with underlying well circumscribed encapsulated tumor mass in connective tissue enclosing the multiple islands and cords of epithelial cells supported by a small amount of fibrous stroma an island of isomorphic cells with peripheral palisaded cuboidal to columnar shape and central cells relatively rounded pas stain clearly exhibiting intensely eosinophilic basal membrane - like structures surrounding cellular nests clearly delimiting basaloid cells to confirm our diagnosis, we performed an immunohistochemical examination using antibodies against the markers pancytokeratin and alpha smooth muscle actin. the tumor tissue revealed no positivity for alpha smooth muscle actin [figure 6 ], though it was found to be positive in normal salivary gland acini [figure 7 ]. no immunoreactivity for alpha smooth muscle actin alpha smooth muscle actin showing positivity only in areas of normal salivary gland acini focal areas of positivity for pancytokeratin on intraoral examination an, asymptomatic solitary swelling with an insidious onset and growth was seen on the palate. on inspection it was ovoid in shape, measuring about 3.5 2 cm, with well - circumscribed borders. it extended from the mesial aspect of 14 to the distal aspect of 17, and laterally from the free gingival margin in relation to 14 17 to 0.5 cms away from the midline. the swelling was nontender, with a smooth surface, and was soft - to - firm in consistency. a well - defined radiolucency with distinct margins was noted in relation to 14 17. with a clinical diagnosis of adenoma of minor salivary glands, the cytosmears stained with rapid papanicolaou stain revealed predominantly basaloid cells arranged in clumps with hyperchromatic nuclei and an increased nuclear - cytoplasmic ratio [figure 1 ]. suspecting it to be an adenoma of minor salivary glands, excisional biopsy was recommended. the biopsy specimen obtained was about 3.0 2.0 1.5 cms in dimension, brownish - white in color [figure 2 ], and soft - to - firm in consistency. the sections revealed an overlying orthokeratinized type of stratified squamous epithelium with an underlying connective tissue. in the deeper layers of the mucosa was a well - circumscribed encapsulated tumor mass enclosing multiple islands and cords of epithelial cells, supported by a small amount of fibrous stroma [figure 3 ]. the basaloid cells making up the bulk of the tumor were found to be isomorphic. the peripheral cells were palisaded with a cuboidal - to - columnar shape, while the central cells were relatively rounded. these peripheral cells were hyperchromatic, while the central cells had pale staining nuclei [figure 4 ]. the sharp demarcation between the neoplastic epithelial cells and the surrounding connective tissue was evident. pas positive membrane - like structures surrounding cellular nests showed intense eosinophilia that clearly delimited the basaloid cells [figure 5 ]. papanicolaou stained cytosmears with basaloid cells arranged in clumps with hyperchromatic nuclei and an increased nuclear - cytoplasmic ratio submitted biopsy specimen, brownish - white in color an overlying orthokeratinized type of stratified squamous epithelium with underlying well circumscribed encapsulated tumor mass in connective tissue enclosing the multiple islands and cords of epithelial cells supported by a small amount of fibrous stroma an island of isomorphic cells with peripheral palisaded cuboidal to columnar shape and central cells relatively rounded pas stain clearly exhibiting intensely eosinophilic basal membrane - like structures surrounding cellular nests clearly delimiting basaloid cells to confirm our diagnosis, we performed an immunohistochemical examination using antibodies against the markers pancytokeratin and alpha smooth muscle actin. the tumor tissue revealed no positivity for alpha smooth muscle actin [figure 6 ], though it was found to be positive in normal salivary gland acini [figure 7 ]. no immunoreactivity for alpha smooth muscle actin alpha smooth muscle actin showing positivity only in areas of normal salivary gland acini focal areas of positivity for pancytokeratin basal cell adenoma represents 54% of monomorphous adenomas and 1 3% of major salivary glands tumors with an incidence of 7.5% among primary epithelial parotid gland tumors. it usually affects older persons over 50 years (mean - 57.7 years) with female predilection (1:1.02). our case was in the minor salivary glands of the palate, a rarer site. most bca of the parotid gland are unilateral, round or oval in shape with well - circumscribed borders. described a rare case of bilateral basal cell adenomas in the parotid glands of a 65-year - old woman. usually there is no regional lymph node involvement, and no calcification or cystic component within the tumor. the resected mass is usually yellowish - white in color, soft - to - firm in consistency, well - circumscribed, encapsulated and highly vascular. the color of the cut surface is usually uniform and gray - white or tan - white. aspirates consist of two populations of basaloid cells : a group of small oval cells with bland hyperchromatic nuclei, scant cytoplasm, and indistinct nucleoli, and a group of larger oval - to - polygonal cells, with moderate amounts of delicate pale cytoplasm. the basaloid cells are uniform and haphazardly arranged in clusters of variable size or in trabeculae, often with peripheral palisading of the smaller population of cells. squamous morules, a characteristic feature of basal cell tumors, are sometimes present in well - sampled cases. histopathologically, these neoplasms feature as relatively uniform, small, dark basaloid epithelial cells in the stroma, and are surrounded by a fibrous connective tissue capsule. one group consists of small cells with little cytoplasm and intensive basaloid rounded nuclei that are usually located in the periphery of the tumoral nests or islands. the other group is formed by large cells with abundant cytoplasm and pale nuclei that are located in the center of the tumoral nests. a basal membrane - like structure surrounds these tumoral nests and clearly separates them from the surrounding nonmucoid stroma. globally, as it has been referred to in classic texts, the tumor adopts an ameloblastoma - like pattern. the basaloid cells are arranged in solid, trabecular, tubular, and membranous pattern. ultrastructurally, the tumor appears to be derived from the intercalated portion of the duct, with minor participation of the myoepithelial cells. basal cell adenocarcinoma is a rare malignant counterpart of bca and may be present as a solid, tubulotrabecular, or a membranous subtype. ultrastructural analysis of a case of basal cell adenoma reported by hemachandran. showed characteristic features including reduplicated basal laminae around the tumor cells, presence of intermediary filaments, and rough endoplasmic reticulum in the cytoplasm of the tumor cells. basaloid cells in trabecular and solid areas express vimentin, actin, subunit of s-100 protein, and cytokeratins. in our case the tumor cells in the islands were focally positive for cytokeratins and negative for alpha smooth muscle actin. hemachandran. reported the first case of bca of the minor salivary gland, which showed selective positivity for pancytokeratin, s-100, and smooth muscle actin in the tumor, which highlighted the participation of myoepithelial cells in histogenesis. clinical differential diagnosis includes mucocele, which usually appears in the lower lip of young people, whereas, bca appears in the upper lip of the elderly. warthin 's tumor and membranous bca are usually unilateral with pleomorphic adenoma and tubular basal cell adenomas exhibiting multicentricity or bilaterality. among the radiographic differential diagnoses, pleomorphic adenoma and malignant salivary gland tumors mostly show increased enhancement in delayed phases on computed tomography (ct). the age of the patient and the attenuation on unenhanced and contrast - enhanced ct may help in differentiating bca from pleomorphic adenoma. histopathological differential diagnosis includes salivary gland basaloid lesions such as basal cell adenocarcinoma (solid form), benign mixed tumor, adenoid cystic carcinoma, cellular pleomorphic adenoma, chronic sialadenitis, cutaneous basal cell carcinoma, metastatic basaloid squamous carcinoma, and sialoblastoma. basaloid cells in trabecular and solid areas express vimentin, actin, subunit of s-100 protein, and cytokeratins. in our case the tumor cells in the islands were focally positive for cytokeratins and negative for alpha smooth muscle actin. hemachandran. reported the first case of bca of the minor salivary gland, which showed selective positivity for pancytokeratin, s-100, and smooth muscle actin in the tumor, which highlighted the participation of myoepithelial cells in histogenesis. clinical differential diagnosis includes mucocele, which usually appears in the lower lip of young people, whereas, bca appears in the upper lip of the elderly. warthin 's tumor and membranous bca are usually unilateral with pleomorphic adenoma and tubular basal cell adenomas exhibiting multicentricity or bilaterality. among the radiographic differential diagnoses, pleomorphic adenoma and malignant salivary gland tumors mostly show increased enhancement in delayed phases on computed tomography (ct). the age of the patient and the attenuation on unenhanced and contrast - enhanced ct may help in differentiating bca from pleomorphic adenoma. histopathological differential diagnosis includes salivary gland basaloid lesions such as basal cell adenocarcinoma (solid form), benign mixed tumor, adenoid cystic carcinoma, cellular pleomorphic adenoma, chronic sialadenitis, cutaneous basal cell carcinoma, metastatic basaloid squamous carcinoma, and sialoblastoma. the final diagnosis requires histopathological analysis, which can be inverterated by immunohistochemical examination. in any suspected neoplastic salivary gland lesion, due to prognostic implications, this case report describes a case of basal cell adenoma in a relatively rare site, that is, the hard palate, which had been immunohistochemically confirmed. | basal cell adenoma (bca) of the salivary glands is an uncommon type of monomorphic adenoma. its most frequent location is the parotid gland. it usually appears as a firm and mobile slow - growing mass. histologically, it is seen as nests of isomorphic cells and interlaced trabeculae with a prominent basal membrane. there is also slack, hyaline stroma with absence of a myxoid or chondroid component. we describe a case of bca of palatal minor salivary glands, a rare site for its occurrence. we also briefly review the literature on the same. |
autistic spectrum disorders (asds) are developmental disorders estimated to affect 1 in 150 children. increasing professional awareness has led to higher levels of diagnosis and support, possibly explaining the recorded rise in prevalence over the last 30 years. asd is commoner in males (m : f autism 4 : 1, aspergers 10 : 1). genetic factors are complex, and no single gene mutation or chromosome abnormality has been linked, although the condition has the highest heritability of any psychiatric disorder (approximately 90%) ; the risk of having a second child with autism is increased by 2030. the national autism plan for children (napc) emphasises the importance of early intervention and close collaboration between parents and professionals and provides a gold standard for services. benefits for parents include increased capacity to seek information from external sources and make use of available health services. this service involved sessions from child and adolescent psychiatrists (cap), community paediatricians (cp), camhs practitioners, specialist speech and language therapists (salt), and educational psychologists (ep). in addition to the core symptoms, there are other common features, such as abnormalities in sleep and mood. for a diagnosis of asds to be made if there are concerns during developmental screens, specific screening for autism is strongly encouraged. most commonly used is the autism diagnostic observation schedule (ados) ; however not all characteristics may be demonstrated during the test. difficulties can become more apparent when changes in routine occur, for example, starting a new school, and this is often when children are referred. the diagnosis of autism, however, is often not made until 2 to 3 years after symptoms are recognized. there are considerable demands on parents as they accept and adjust to their child 's communication and social interaction impairments. many people seek information on their own, from other parents, websites, books, and autism groups and newsletters. children and families want accessible mental health services which provide support when needed and involve them as users. they also want to know what services are available to help them support their child. the napc guidelines address identification, assessment, diagnosis, and access to early interventions for children with asds. stage 1 is a general multidisciplinary developmental assessment for any child with possible developmental problems. the assessment should be completed and fed back to the family within 17 weeks of referral. the napc states that feedback to parents should include information about services, for example, the national autistic society (nas). in manchester, all scait professionals receive training in assessing asds. referrals from educational psychologists, health visitors, and school nurses are accepted if the general practitioner or a paediatrician is informed. parent intervention programmes can enhance interaction with children, promote development, and increase parental satisfaction and mental health. scait offers five 2-hour sessions : understanding asd, understanding and managing behaviour, understanding and working with the education system, enabling your child 's communication and understanding, and further resources for you and your child. to assess parental satisfaction with scait assessments and compare the service with the napc guidelines. parents and carers of the 35 children diagnosed with asds from december 2008 to may 2010 were invited to participate. participants were invited to have a telephone or face - to - face alternative to complete the questionnaire. a previous manchester audit tool was enhanced to provide further information about the postdiagnostic group (appendix 1). questionnaires were scored according to total satisfaction, and satisfaction in each of the 4 areas of the assessment process. thirty - five questionnaires were posted, and 20 (57%) parents participated, with 1 father responding. of the 20 participants, 1 parent completed the interview via telephone and the other 19 returned the questionnaire by post. the child 's age at diagnosis and the present time showed 2 peaks (figure 1), consistent with transition to primary and secondary school. most parents (86%) were especially satisfied with the assessment process (table 1), but less happy with the information received before and after the assessment. most parents felt they were well informed about the assessment before the appointment, with 65% receiving information. however, 14 would have liked to have known the different parts of the process, and 8 wanted to know the questions they would be asked. about half of parents would have liked to have known the name and professional background of the clinician (table 2). nineteen parents felt they were definitely listened to carefully, with the final patient choosing yes to some extent. additionally, 18 parents felt they were able to discuss their concerns and give feedback at the time of assessment, and 80% felt the assessment could not have been communicated differently. the family who needed an interpreter reported that the information they were provided with before the appointment was very poor, although they believed the actual process was good. notably, 85% of parents would have liked a letter with a plan for further assessment and appointment dates. most parents had received a report which 65% claimed to definitely understand, with 20% understanding to some extent. eighteen parents had the opportunity to give feedback at the time of the assessment. however, 59% stated they either did not have any say or only to some extent. all of the parents said they were able to ask questions at following meetings. with regards to the postdiagnostic workshops, 8 parents received information regarding the aims. when parents are invited to attend the sessions, written information about the course is also sent with the letter. every respondent was happy with the assessment process, with 60% reporting it to be very good, 100% of parents said they were definitely treated with trust and dignity. after the assessment, 100% of patients were happy with the level of information they had regarding the process. although 18 parents received additional information in some form, 6 felt they had not received enough information about the condition and future interventions. with regards to the postdiagnostic workshops, only 15 parents said they were invited to attend. the reasons for this were not explored, although one patient did report moving house. when asked why they were unable to attend, childcare was a key theme as was timing. looking at the time interval from referral to diagnosis, great variance was apparent (figure 2). the average time was 46 weeks with only 6 diagnoses reported in the recommended time frame of 17 weeks. of note, the child with the shortest timeframe from referral to diagnosis was the least satisfied, while the longest timeframe elicited the highest satisfaction score. the results show 2 peaks, with satisfaction higher at the times of school transition (35 and 1012 year olds). one weakness was size, although sampling the past 18 months allowed the collection to be achieved within the allocated timescale. if the study was increased to the past 3 years, the results may show a more significant trend, although parents might find the process harder to recall. parents wanted more information regarding diagnosis and management. a longer follow - up appointment after diagnostic feedback could allow needs to be individually assessed. during this appointment, professionals could discuss the purpose of the postdiagnostic workshops. the information provided during these workshops is obviously most beneficial when attending the group sessions, as they are interactive and parents have the opportunity to share experiences and strategies. however, if all parents received the written information provided within these sessions, it would be helpful to those who could not attend. as childcare and timing were the commonest reasons for nonattendance, this could be addressed by combining all 5 sessions into 2 days within school hours. written communication could be enhanced by a standardised response letter from the scait team. this could include the role of scait, the purpose of the assessment, the appointment time, the assessment structure and timescale, the name and profession of all clinicians, and possible question areas. this should help ease parent 's anxiety and ensure that scarce appointments achieve maximum benefit. the initial page should contain a standardised format with the outcome clearly stated, the individuals and professional roles of those involved, and the date of the diagnosis. although, most parents were happy with the assessment service, satisfaction could be increased by providing more information about the assessment as well as the condition. it is important to actively involve parents in the sessions, allowing them to build a trusting relationship with the health professionals involved as children may need support from the team for many years. giving each family the time to discuss their concerns will also help tailor individual needs and further increase parental satisfaction. the fact that parents are considerably more likely to have a second child with asds emphasises the importance of engaging families. the study was fed back to scait, and standardized letters and workshop information sheets have already been implemented. furthermore, the team will complete the audit loop by undertaking the audit again in 18 months time. this study has provided a key opportunity to offer a value - added service with service - user feedback. | background. user feedback is now an integral part of both clinical governance and service development, and it also provides a key route to engaging parents and children. autistic spectrum disorders (asds) can impact on all members of a family, and close working between parents and professionals is essential. aim. to explore parental satisfaction rates and identify areas in need of improvement. method. a postal survey was completed by parents whose children had been diagnosed with an asd in the past 18 months in a manchester child and adolescent mental health service. the national autism plan for children was used as a gold standard. results. parents were particularly satisfied with the way team members dealt with them and their children during appointments. however, the standard of written information provided about the condition, diagnosis, and support available could be improved. the findings show the benefits of receiving a diagnosis in the recommended timeframe. discussion. we discuss ways of effectively using scarce resources. |
ecology is generally described as the study of the relationships between organisms and the (a)biotic environment. such relationships are best understood as interactions because the environment is not only dictating the fate of organisms, but organisms also affect the environment. organisms make use of their phenotype (behaviour, morphology, physiology and life - history traits) to deal with their environment (both opportunities and constraints). because phenotypic traits are shaped by evolutionary processes, evolutionary history has the potential to affect ecological interactions and, hence, the prosperity of species in rapidly changing environments under human impact (e.g., purvis. 2000 ; jones. 2003). there is a growing recognition of the significance of evolutionary thinking for conservation biology (e.g., ashley. although there is still a long way to go to fully develop eco - evolutionary conservation strategies in biodiversity management and policymaking, several recent papers provide stimulating perspectives (e.g., kinnison and hairston 2007 ; mace and purvis 2008 ; lankau. this new wave of interest is not independent of climate change and the need to better understand (limits of) evolutionary adaptation in non - model organisms in the field (hoffmann and sgro 2011). the recent appreciation that evolution is not necessarily a slow process is another factor of significance in this context ; there is evidence for rapid evolutionary responses of organisms, particularly in human - dominated environments (hairston. 2011), although the relative contribution of rapid evolution on ecological dynamics may vary considerably (ellner. i will discuss the significance of an evolutionary way of thinking about what actually represents a species habitat. the current vision on the concept of habitat in ecology and conservation, and surprisingly even in several evolutionary studies, appears often as a remarkable example of typological, static thinking. structural habitat (e.g., vegetation or land cover types) as it is perceived by humans and used on maps for different conservation applications as habitat units may not (closely) reflect the functional habitat of an organism. organisms are able to pay attention to specific relevant information of the environment and screen or ignore many other signals. therefore, habitat use is not a fixed species - specific trait, but a trait that may show significant intra - specific variation and deserves a more functional, mechanistic approach. although there is rich literature on the adaptive significance of animal communication since the pioneer work of tinbergen (1963), the way habitat and habitat use have been adopted in several ecological and conservation biology studies largely ignores this aspect of integrating sensory inputs and how it may deviate from human perception. an appropriate functional definition and conception of the habitat of an animal should integrate this behavioural viewpoint on the sensory interactions between the organism and its environment. therefore, i will emphasize in this paper the conceptual link with the umwelt - concept from ethology and psychology and explore its significance for animal conservation. first, i will briefly discuss how human activities create anthropogenic niches that affect ecological and evolutionary responses of organisms, and i will also illustrate how such environments may interfere with information processing of organisms. secondly, i introduce the umwelt concept as it may help us avoid a far too human perception on how animals perceive, and hence deal with, their environment. there is evidence of adaptive changes in perceptual ability relative to changes in landscape structures. to integrate these aspects into a conservation context, i will discuss the resource - based habitat concept as a functional, organism - centred view of the interactions between the organism and its relevant environment. organisms are influenced by the environment, but they may also modify their environment, which in turn may have consequences for their own ecology and evolution, as well as for other organisms. 2006), and there is a recent synthesis on how human activities and different types of land use can be viewed as forms of stable or unstable niche construction (rowley - conwy and layton 2011). niches created by humans (e.g., several urban, industrial and agricultural land - use types) have been shown to have negative impacts on the population dynamics of many species. human activity can have a direct impact on animals (e.g., by persecution and harvesting) or an indirect impact via effects on the quantity, quality or configuration of their habitat. however, particular human - made environments can be favourable to some organisms as surrogates of natural environments (e.g., benes. 2003), or as novel environments that result from novel physical or biotic conditions (e.g., hobbs. it has, for example, been hypothesized that a considerable number of british butterflies have taken advantage of warmer man - made, early successional vegetations like grasslands, heathlands and forest clearings (thomas 1993). a recent analysis by mller (2010) showed elevated success of birds breeding inside human buildings (like barns) compared with outdoors as in human buildings they can easily escape nest predation. human activities may even create opportunities for diversification of species, with the food resources provided by human activities and livestock farming suggested to provide an opportunity for an island raptor species to colonize the canary islands (agudo. 2010). anthropogenic environments provide interesting opportunities for studying population differentiation, adaptation and rapid evolution (lankau. anthropogenic environments have a strong capacity to alter phenotypic traits of wild organisms by rapid evolution (darimont. 2009) ; however, it is more widely acknowledged that anthropogenic niches typically constrain most wild organisms. adaptive behaviour relies upon accurate information use of relevant ecological parameters (dall. 2005). in environments under strong human impact, physical and biotic environmental changes can be of significance to several organisms, but the way these organisms are behaviourally operating may be affected as well. humans are interfering with information processing and with the use of environmental cues by wild organisms to a much larger extent than is often realized. with industrialization, noise pollution from machinery and other human activities continues to expand in space and intensity (barber. the problem is not limited to terrestrial organisms as freshwater and marine species can also be affected by noise either from terrestrial sources or from traffic and other sources on or in the water (slabbekoorn. recent work has mainly focused on the impact of noise on intraspecific animal communication (e.g., slabbekoorn and ripmeester 2008), but siemers and schaub (2011) provided experimental evidence of an impact of traffic noise on foraging efficiency in acoustic predators and hence on predator prey interactions. many ecologists have neglected to consider artificial night lighting as a relevant environmental factor, while conservationists have largely neglected to include the nighttime environment in conservation strategies (longcore and rich 2004). we are at the very beginning of understanding evolutionary effects of light pollution and how they may in turn feedback on ecological interactions. the case of light pollution illustrates that human - dominated environments do not only alter the presence and quality of habitats, but also the environmental cues organisms use to deal with their environment. for example, artificial lighting has been shown to disrupt the nocturnal movements of sea turtle hatchlings to the ocean, as artificial light will attract them landward rather than seaward (tuxbury and salmon 2005). interestingly, some minor details or even unnoticed sources of information to the human observer can be essential for habitat selection in other organisms. this observation has received much attention in dairy management practices and with respect to the well - being of farm animals (grandin and johnson 2005). in the same vein, we should recognize the importance for wild animals and hence several of their conservation issues. take the case of polarized light that is used by several insects to locate water surfaces. several human creations (including glass buildings, asphalt roads and even particular cars) appear to offer similar cues to these insects (kriska. hence, they are attracted to wrongly interpret anthropogenic substrates and defend territories and lay eggs. interestingly, some predators take advantage of such trapped insect prey in urban areas (e.g., robertson. 2010), illustrating potential positive feedbacks for other species and their urban life styles. the polarized light examples bring us to the concept of ecological and evolutionary traps that have attracted much attention in anthropogenic environments (e.g., schlaepfer. ecological traps occur when, by various mechanisms, low - quality habitat is more attractive and hence preferred over available good habitat (e.g., hollander. patten and kelly (2010) argue that also the converse problem may occur : the avoidance of high - quality habitat because it is less attractive. perceptual trap in such a case and showed evidence with a field experiment in the lesser prairie - chicken tympanuchus pallidicinctus (patten and kelly 2010). however, the label perceptual trap is somewhat confusing as perception is also much of an issue in the other cases of trapping. ecological and evolutionary trapping is anyway a significant issue for conservation in anthropogenic landscapes, even if it often remains difficult to show empirically and in an unequivocal way that reduced breeding success in preferred, low - quality habitat is related to population decline. sensory systems have evolved to respond to relevant functional subsamples of information from the environment. moreover, the performance and sensitivity of visual systems vary considerably among organisms as illustrated earlier with the example of light pollution. for defining and recognizing the habitat of a species, therefore, it is relevant to connect the habitat issue to the umwelt-concept, a longstanding concept from ethology and psychology. from his discussion of the animal 's relationship with its environment, von uexkll (1909) argued already at that time that different organisms live in different perceptual worlds and that we should be more sensitive to the environmental carriers of significance that differ among species and individuals. the analysis of information use by animals is considered a central field to organismal biology, and hence to evolutionary and behavioural ecology (dall. 2005). a behavioural viewpoint on habitat recognition and selection is also of significance for conservation biology, including conservation action on the ground (e.g., species action plans). manning. (2004) discussed the relevance of the umwelt concept for improving realism of landscape fragmentation models, as common simplifications that ignore the organism 's perspective can be problematic for conservation and land management. here, i want to emphasize and even widen this conclusion or warning for several, if not most aspects in conservation and land management that explicitly or implicitly deal with functional habitat and movements of animals. what is perceived and used as habitat by organisms is driven by their perception and their responses to environmental cues. dispersal is widely recognized as a key process for ecology, evolution and conservation (ronce 2007). the issue of available information and sensitivity to cues would also need careful treatment during dispersal. habitat use and dispersal behaviour may alter with changing landscapes, stressing the evolutionary dimension that has often been ignored in landscape ecology (baguette and van dyck 2007). the spatial scale of interaction between landscape structure or more precisely resource distributions and the perception of the organism is referred to as the functional grain of a landscape (baguette and van dyck 2007). (2009), for example, demonstrated modification of the search strategies of red flour beetles (tribolium castaneum) in response to the scale of habitat structure, emphasizing the significance of functional grain. they concluded that spatially explicit, organism - centred studies focusing on behavioural responses to different habitat configurations are much needed to improve our ability to accurately predict space use of organisms in landscapes. our biased and incomplete view on how organisms perceive and use their environment can be illustrated with manipulative field experiments adding or removing particular ecological resources and conditions within and among vegetation types at the landscape level. a simple field experiment i conducted with orange tip butterflies (anthocharis cardamines) is illustrative in this context (h. van dyck, unpublished data). potted host plants of this butterfly were placed for short periods during the adult flight period inside and outside the local patchwork of known (in the sense of vegetation type) in a local network population in a human - dominated agricultural landscape in ne - belgium where its host plant (cardamine pratensis) typically occurs in damp meadows this experiment allowed examination of the extent this butterfly would scan the landscape matrix outside the patches of damp meadows. host plants were even placed at places where it was physically unable to grow and at distances up to > 1300 m from the nearest occupied meadow. after a timeframe of 2 days only, i noticed that > 70% of the experimental plants outside habitat patches carried eggs. moreover, the average number of eggs per used host plant was 3.6 times higher on the host plants placed across the landscape matrix compared with plants placed in meadows (maximum number of eggs per host plant was nine outside meadows and two only within meadows). this is remarkable as it has been demonstrated that a. cardamines females tend to avoid plants that already carry eggs because of the females response to an oviposition - deterring pheromone that is deposited with each egg (dempster 1992). this experiment illustrates that females were readily able to find and use host plants at different places across the landscape matrix even without any suitable habitat from the vegetation - type - based habitat view. the landscape matrix was more intensively scanned for resources than is generally appreciated in species with a patchy host plant distribution. high egg loads outside habitat patches also suggest that movement through a resource - poor matrix may affect reproductive decisions (i.e., acceptance of already used host plants), which may signify deferred search costs of dispersal in fragmented landscape (stamps. 2005). anthocharis cardamines is often considered to be a grassland species, but most grasslands in nw - europe have been created by human activity for agriculture. so, the butterfly most likely has a longer evolutionary history as an organism using resources in forest clearings, likely explaining why it still makes use of forest edges and hedgerows in agricultural landscapes. it would be interesting to understand how search behaviour, movements and perception of such an insect may have been altered from forested to agricultural landscapes. comparing phenotypes and behavioural performances among different landscape types would hence be promising in this context. a behavioural viewpoint on the interactions between an organism and its environment is essential to get a better mechanistic understanding of habitat selection and dispersal, including the costs of dispersal across anthropogenic landscapes (baguette and van dyck 2007 ; bonte. a particular species in rapidly changing landscapes may deviate from commonly made assumptions like in least cost path analyses that have become popular in conservation and applied landscape ecology (e.g., fahrig 2007). more mobile species are considered more resilient to the dynamics of anthropogenic landscapes than less mobile species. however, if more mobile organisms experience increased mortality rates as they move into more hostile anthropogenic parts of the landscape matrix, it may compromise their survival in such landscapes even more than less mobile organisms (thomas 2000 ; fahrig 2007). individuals of the same species may show different behavioural searching strategies rather than one species - specific strategy (heinz and strand 2006). despite the growing interest for studying the eco - evolutionary dynamics of dispersal, current studies rarely assess to what extent, and under which conditions, naturally selected dispersal behaviour has failed to promote population persistence (delgado. individuals can be selected to have traits that diminish population - level performance ; individuals can disperse less (or more) than would be ideal for a population. delgado. (2011) argue that as conservation managers typically need to take a population - level view of performance, these novel insights may necessitate their intervention if it differs from what is selected for. the potential discrepancy between individual and population level for conservation in an eco - evolutionary context will need more attention in the future as it has not been fully appreciated yet. movements other than those that aim for displacement (e.g., foraging movements) may also be altered in anthropogenic landscapes and should not be ignored. from a behavioural point of view, real displacement movements and routine foraging movements can be very different, and may evolve independently in changing landscapes (van dyck and baguette 2005 ; bonte. the umwelt of organisms that decided to emigrate from a site may be different from the umwelt of conspecifics that make local, routine movements to forage ; dispersing animals may need to ignore particular cues from resources, conspecifics and other species (e.g., predators), or at least alter their responses to them compared with individuals in a different behavioural mood. if dispersers are not a random sample of the population based on their perceptual abilities (together with other phenotypic traits), they may have a different umwelt compared with the average resident individual. this may have consequences for choosing relevant individuals for experimental studies on dispersal (e.g., release experiments). studies on dispersal often interpret vegetation or land - use types in terms of barriers and corridors based on a human perspective or interpretation of the landscape. for example, a recent genetic study by leidner and haddad (2010) could not find any indication that existing levels of urbanization were barriers to dispersal in a coastal endemic butterfly. as the degree to which anthropogenic environments creates barriers to animal dispersal remains poorly understood, our understanding would benefit from integrating knowledge about landscape perception. it is well appreciated that species that live in different environments may need different sensory abilities. the same can be true at the intraspecific level, if populations of the same species have to contend with, for example, different levels of habitat fragmentation (lima and zollner 1996). field release experiments of varying distances to a target habitat have been successful in showing variation in perceptual range (e.g., in mammals ; zollner 2000). our work on the speckled wood butterfly (pararge aegeria l.) in different landscapes offers an interesting case in this context. although many butterflies in nw europe have shown negative population trends over the last decades, p. aegeria is among the few successful butterfly species that have increased considerably in distribution and abundance (van dyck. it has recently expanded its habitat use in nw europe from mainly forests to much more open environments, including agricultural land with some hedgerows and small woodlots. for a flying heliotherm, forested and agricultural landscapes represent very different operational worlds in terms of the biotic and abiotic environmental interactions (karlsson and van dyck 2005). our comparative work on forest and agricultural populations has already pointed to several phenotypic differences in functional morphology, behaviour and life history (e.g., gibbs and van dyck 2010 ; vandewoestijne and van dyck 2010). pararge aegeria has not shifted but expanded its habitat use, with evidence of ecotypic, and hence phenotypic, differentiation. interestingly, there is also evidence for evolutionary effects on their perceptual ability relative to landscape type. field experiments with wild caught p. aegeria individuals showed that butterflies from an agricultural landscape were able to detect a target habitat from a greater distance than forest butterflies (merckx and van dyck 2007). more recently, we confirmed this effect with laboratory reared males and females of the two types of landscape and replicate populations (e. ckinger and h. van dyck, submitted manuscript). this is a highly relevant result within a framework of functional landscape connectivity as the same fragmented landscape will be perceived as more fragmented (less functionally connected) by speckled woods with a recent evolutionary history in forested landscapes than by conspecifics with a history in fragmented agricultural landscapes. based on the case of p. aegeria, we have recently hypothesized that the success in dynamic anthropogenic landscapes might be related to the existence of seasonal plasticity in morphology and life history in this multivoltine species (van dyck. this idea builds upon the baldwin effect that plasticity can be significant in the successful colonization of novel environments (yeh and price 2004). the idea of a link between phenotypic plasticity and conservation status now warrants a well - designed test with multiple species within a phylogenetically controlled, analytical framework. using simulation models, olden. (2004) have explored the potential for plasticity in perceptual ranges and argued that context - dependent perceptual ranges need to be carefully considered and further explored in future landscape ecological studies. conservationists may wonder about the practical relevance and feasibility of detailed behavioural studies inspired by the umwelt - concept for application in conservation the habitat is one of our basic concepts in ecology and evolutionary biology as it captures where an organism lives. however, there is quite some confusion about the definition of a habitat (hall. different habitat concepts have been applied, but often without carefully considering the underlying assumptions and simplifications (dennis. the way we consider the habitat of an organism is strongly biased by our own, largely visual perception of the environment. most ecologists, evolutionary biologists and conservationists treat habitat as being synonymous with a particular vegetation or land - cover category without questioning the underlying assumptions of the adopted habitat concept. structural units as perceived by humans do not necessarily reflect the functional units of habitat for other organisms. this point has been appreciated within the context of dispersal across the landscape matrix because structural and functional connectivity related to structural and functional landscape heterogeneity have been distinguished in landscape ecology (e.g., fahrig. however, the same philosophy should also apply to an organism 's habitat (dennis. structural variation like vegetation types (or biotopes) can at most be proxies of the functional habitat of a species in particular cases, but in several other cases such a habitat approach is unable to realistically recognize what is the habitat of a species and to predict the presence of a species (e.g., vanreusel and van dyck 2007). a functional habitat model based on resource distributions and individual movements is the resource - based habitat concept (dennis. 2003, 2006) and has attracted much recent interest (e.g., fattorini 2010 ; bates. hence, a habitat from this viewpoint is the intersection and union of the necessary complementary resources (i.e., consumables and conditions) for an organism at all its life stages, where resources are connected by daily movements to give functional habitats by combination and overlap (dennis. conditions will include relevant physical, but also biotic parameters (e.g., relative to predation). so, the concept reconnects to the ecological niche by being the multi - dimensional, functional space of an organism (hutchinson 's hypervolume whittaker. the resource - based habitat is then the spatial projection on the ground of this functional space (fig 1). the umwelt - concept refers to the observation from ethology that different organisms may perceive their environment different than do human observers. the organism 's unique sensory world explains why different organisms can have different umwelten, even though they share the same structural environment. conservation biology often assumes that structural habitats reflect functional habitat. the resource - based habitat concept (seedennis. 2003) offers a model that takes into account specific resources and conditions, as well as movements at the appropriate spatial scale and hence provides a means to translate human observed structural habitats into organism - centred functional habitat. there are a growing number of papers stressing the importance of considering animal behaviour and the scale of habitat - use decisions when assessing landscape conditions (e.g., a koala 's eye view of spatial variation in habitat qualitymoore. ; popescu and hunter 2011). the key point of the resource - based habitat approach is that it is an organism - centred approach that focuses on the behavioural interactions between the organism and the relevant part of its environment. this is of particular significance in rapidly changing anthropogenic landscapes because resource distributions and relevant conditions may change without obvious alteration in the general vegetation structures. these changes can be the result of (unintentional) interventions including conservation management, but also of several other human activities. changes in resources may also occur in an indirect way through, for example, pollution effects. the combination of regional warming and atmospheric nitrogen deposition may, for example, make herbaceous vegetation grow more densely which in turn makes them too cold for larval development under spring conditions in a number of declining butterfly species (wallisdevries and van swaay 2006). in this final section, i will discuss three issues to illustrate the practical significance for conservation of a functional habitat approach that takes into account the organism 's perspective to the environment : (i) detecting additional conservation opportunities, (ii) avoiding failures of habitat restoration and (iii) implementing the functional habitat approach with geographic information systems (gis). the fact that habitat is often perceived in a simplistic binary way as discrete patches of a particular vegetation type in a hostile landscape matrix may limit conservation opportunities (dennis. davison and fitzpatrick (2010) provide an instructive example with the florida scrub - jay (aphelocoma coerulescens), a highly endangered habitat specialist bird that was known to be confined to remnant native scrub patches surrounded by agricultural and suburban landscapes. but against common belief among conservationists, they showed that regenerated pastures could also serve as suitable habitat (with equal breeding success), at least when adjacent to native scrub. the authors defended additional conservation strategies involving the relaxing of accepted definitions of suitable habitat for this species (davison and fitzpatrick 2010). the bigger issue here is that habitat definitions may constrain the opportunities for conservation and restoration in landscapes under intense human use. restoration actions may also deteriorate the habitat quality for threatened species that are unable to respond to altered conditions (e.g., predation risks) as a result of restoration. in other words, the structural changes in the environment do not necessarily alter the umwelt of the threatened species. i will illustrate this with a recent study on the critically endangered desert lizard acanthodactylus beershebensis (hawlena. the strong decline of the species occurred in parallel with a large landscape restoration project including the planting of trees. as the species disappeared from both natural and altered sites in the area, land managers argued that the decline could not have been caused by the afforestation. (2010) showed, however, experimental evidence with artificial trees that the increased structural complexity in planted patches favoured avian predation. it is argued that the lizards have not evolved to assess structural cues to evaluate elevated predation risks in this system. (2010) demonstrates that local anthropogenic changes in habitat structure may have a considerable negative effect, even beyond the immediate area of action because it may induce ecological trapping affecting the entire network of local populations. conservation managers are accustomed to working with spatial information like vegetation units and land - use types in different gis applications (i.e., geographic information system). however, by now it should be clear that making use of information on structural habitat may not be the same as functional habitat as it is perceived and used by species as we applied for heathland butterflies of conservation interest in a national park in ne - belgium, one can make use of gis - based data and modelling techniques to create resource - based functional habitat maps that more closely match the organism - specific environment than methods directly applying general vegetation types from land cover maps (vanreusel and van dyck 2007). moreover, the approach was found to be fruitful for ecological niche modelling as models based on a quantitative resource - based habitat approach were successfully transferred among nature reserves in the same region (vanreusel. so, although it would require additional layers of information and some gis - based calculations to combine the resource - based information and spatial scale of interaction with the environment, the final output of such an exercise will be a map with different functional habitat areas and indications of zones that lack particular resources or conditions (see vanreusel and van dyck 2007 for an example). autecology behind such a final map, the end product is not that different from what is currently often used by conservation practitioners. but the delineated zones based on a functional habitat approach may differ significantly from the generally adopted vegetation type or biotope approach (e.g., turlure. evolutionary biologists rarely use the extensive environmental data and tools available from gis and spatial land cover databases for dealing with environmental variation. for a detailed discussion and review on how evolutionary studies can integrate gis - based data and approaches, evolutionary studies along environmental gradients and mosaics that adopt such a functional, resource - based habitat approach provide a very interesting scope for a better mechanistic understanding of the match and mismatch of different phenotypes and their environment in anthropogenic landscapes. in summary, the way several ecological, conservation and evolutionary studies perceive and treat the habitat of organisms is a clear example of simplistic, typological thinking. moreover, there is a strong bias towards human perception in terms of vegetation or land - use types. such an approach can be ignorant to the specific sensory information different organisms will use (i.e., their umwelt). a functional habitat concept based on the ecological resources and conditions a population (or species) will use offers a better perspective for the recognition of functional habitat compared with the use of vegetation or land - use types (i.e., general structural habitats) as a proxy. such an approach may detect conservation opportunities and help avoid failures of habitat restoration for endangered species. i suggest that functional habitat approaches and the umwelt concept need to be essential ingredients of evolutionary conservation programs, as they will help creating better conditions for this single living planet with its multiple perceptual worlds. | there is a growing recognition for the significance of evolutionary thinking in ecology and conservation biology. however, ecology and conservation studies often work with species - specific, fixed traits that ignore intraspecific variation. the way the habitat of a species is considered is an example of typological thinking biased by human perception. structural habitat units (e.g., land cover types) as perceived by humans may not represent functional habitat units for other organisms. human activity may also interfere with the environmental information used by organisms. therefore, the umwelt - concept from ethology needs to be integrated in the way we think about habitat and habitat selection. it states that different organisms live in different perceptual worlds dealing with specific subsamples of the environment as a result of their evolutionary and developmental history. the resource - based habitat concept is a functional habitat model based on resource distributions (consumables and conditions) and individual movements. this behavioural approach takes into account aspects that relate to the perceptual world of organisms. this approach may offer new opportunities for conservation and may help avoid failures with habitat restoration. perceptual ability may be subject to adaptive change, but it may also constrain organisms from showing adaptive behaviours in rapidly changing environments. |
laparoscopic sleeve gastrectomy (sg) was introduced by gagner and colleagues as a first - step procedure to minimize surgical risk for super - super obese (sso) or high - risk patients, followed by either laparoscopic biliopancreatic diversion with duodenal switch or laparoscopic roux - en - y gastric bypass (rygb). sg is now established as a primary bariatric procedure apart from being a first - stage procedure in a planned bariatric approach for high - risk patients. super obese (so) patients with a body mass index (bmi) 50 kg / m are a difficult - to - manage population because of the huge liver, leading to limited working space ; excessive torque on the instruments because of the thick abdominal wall ; comorbidities ; and high - risk anesthesia. the management of patients with super - super obesity (bmi 60 kg / m) also remains a challenge. robotic surgery may be a suitable alternative to the laparoscopic approach in so and sso patients. robotic surgery allows for more precise manipulations and increased dexterity by downscaling the surgeon 's movements and filtering out physiological tremor. it liberates the surgeon from torque on the instruments because of the thick abdominal wall and decreases the port - site trauma because of remote - center technology, thereby conferring improved ergonomic positioning. furthermore, a true 3-dimensional view of the intra - abdominal anatomy with robotic surgery facilitates fine tissue dissection. the main disadvantages associated with robotic surgery remain its high cost and longer setup time compared with the laparoscopic equivalent for many procedures. with increased experience, it may be assumed that this setup time will be reduced, and in the future, operative costs may decrease as material prices are reduced. this study evaluates our initial experience with and technique for robot - assisted sleeve gastrectomy (rasg) in morbidly obese (mo) and so patients. a robotic platform was used for bariatric surgery starting in march 2012 at our well - established center of laparoscopic bariatric surgery. the patients met the national institutes of health consensus criteria, as well as the institutional policies, for undergoing a bariatric procedure. after the required training in robotic surgery, the surgeons initially performed a few simple cases such as cholecystectomy, hernia repair, and hiatal surgery before proceeding with bariatric procedures. preoperative evaluation included bmi, anthropometry, routine blood parameters, nutritional markers (vitamin b12, folic acid, iron, ferritin, vitamin d3), diabetes profile (fasting blood glucose, hemoglobin a1c, fasting insulin, and c - peptide levels), lipid and thyroid profile, sleep study, upper gastrointestinal endoscopy, pulmonary function test, dobutamine stress echocardiogram, chest radiography, and ultrasonography of abdomen. the following parameters were evaluated : patient demographic characteristics, body mass index (bmi), comorbidities, total operative time (further split into docking time and imbrication time), mean blood loss, conversions, perioperative complications and deaths, length of hospital stay, and excess body weight loss at 6 months after the procedure. the operative time was defined as the time from skin incision to skin closure, including any associated procedures. the so (bmi 50 kg / m) subset of patients was compared with the mo patient group based on the previously mentioned parameters. statistical analysis was performed using spss software, version 19.0 (ibm, armonk, new york). the patient is placed in the supine position with his or her legs by the side. a 38f gastric calibration tube is used after induction to empty out the gastric contents and is kept in place to guide the sleeve formation. pneumoperitoneum is created with a veress needle at palmer 's point up to a pressure of 15 mm hg. all distances for port placement are measured after insufflation because measurements obtained before pneumoperitoneum is achieved may be misleading. pre - emptive infiltration of 0.5% bupivacaine is performed before incision at all port sites. the camera port (12-mm - diameter, 150-mm - long endopath xcel trocar ; ethicon endo - surgery, cincinnati, ohio) is placed 20 cm below the xiphisternum slightly to the left of the midline under vision using a 0 10-mm laparoscope. the ports for the robotic arms (r1, r2, and r3) are established next. r1 (8-mm da vinci cannula ; intuitive surgical, sunnyvale, california) is placed at the left midclavicular line approximately 20 cm from the xiphisternum. r2 (5-mm da vinci cannula) is placed in the right hypochondrium at the midclavicular line, with care taken to ensure that the entry of the port is below the margin of the liver. r3 (5-mm da vinci cannula) is placed in the left flank at the level of the camera port. the assistant port (12-mm - diameter, 100-mm - long endopath xcel trocar) is placed between the camera port and r2 with a distance of at least 10 cm from both of them. a 5-mm epigastric port is made and used to place a nathanson liver retractor for retraction of the left lobe of the liver (cook inc., a1 = assistant port ; n = nathanson retractor ; r1/r2/r3 = ports for robotic arms ; mcl = mid clavicular line ; sul = spino - umbilical line. all 3 arms of the da vinci surgical system (intuitive surgical) are used. the patient cart is brought from the head end of the patient, and docking is performed. the instruments used for rasg are as follows : 8-mm da vinci harmonic ace curved shears (ethicon endo - surgery, intuitive surgical, sunnyvale, ca, usa) (r1, for gastrolysis), 5-mm bowel graspers (r2 and r3), and 5-mm needle driver (r1, for over - sewing staple line). a 5-cm umbilical tape is used to measure the distance from the pylorus, and gastrolysis is performed along the greater curvature of the stomach using a harmonic scalpel (ethicon endo - surgery, intuitive surgical, sunnyvale, ca, usa). the short gastric vessels from the gastrosplenic ligament and posterior gastric adhesions with the pancreas are divided. one can appreciate a bilaminar parchment like membrane if one remains close to the stomach on the greater curvature. care is taken to ensure that the bougie is withdrawn in the esophagus during this step ; otherwise, it can lead to difficulty in dissection around the hiatus. the gastric calibration tube is pushed forward by the anesthetist and guided by the surgeon into the first part of the duodenum using bowel graspers. rotation of the bougie by the anesthetist and correct amount of traction by the surgeon help in negotiating the pylorus. this leads to the bougie nicely resting along the lesser curve of the stomach and helps in formation of an even sleeve. a sleeve of stomach is created using an articulating stapler from the assistant port while the console surgeon applies traction on the stomach so that it lies in the right orientation. sequential stapling first with a green load and subsequently with blue loads is performed while not being very snug to the bougie. after the sleeve is formed, the staple line is imbricated using no. 2 - 0 polydioxanone running suture with a sliding loop at one end. gastroscopy with saline solution immersion is performed in all cases to rule out any leak, bleed, or obstruction. the patient is placed in the supine position with his or her legs by the side. a 38f gastric calibration tube is used after induction to empty out the gastric contents and is kept in place to guide the sleeve formation. pneumoperitoneum is created with a veress needle at palmer 's point up to a pressure of 15 mm hg. all distances for port placement are measured after insufflation because measurements obtained before pneumoperitoneum is achieved may be misleading. pre - emptive infiltration of 0.5% bupivacaine is performed before incision at all port sites. the camera port (12-mm - diameter, 150-mm - long endopath xcel trocar ; ethicon endo - surgery, cincinnati, ohio) is placed 20 cm below the xiphisternum slightly to the left of the midline under vision using a 0 10-mm laparoscope. the ports for the robotic arms (r1, r2, and r3) are established next. r1 (8-mm da vinci cannula ; intuitive surgical, sunnyvale, california) is placed at the left midclavicular line approximately 20 cm from the xiphisternum. r2 (5-mm da vinci cannula) is placed in the right hypochondrium at the midclavicular line, with care taken to ensure that the entry of the port is below the margin of the liver. r3 (5-mm da vinci cannula) is placed in the left flank at the level of the camera port. the assistant port (12-mm - diameter, 100-mm - long endopath xcel trocar) is placed between the camera port and r2 with a distance of at least 10 cm from both of them. a 5-mm epigastric port is made and used to place a nathanson liver retractor for retraction of the left lobe of the liver (cook inc., a1 = assistant port ; n = nathanson retractor ; r1/r2/r3 = ports for robotic arms ; mcl = mid clavicular line ; sul = spino - umbilical line. all 3 arms of the da vinci surgical system (intuitive surgical) are used. the patient cart is brought from the head end of the patient, and docking is performed. the instruments used for rasg are as follows : 8-mm da vinci harmonic ace curved shears (ethicon endo - surgery, intuitive surgical, sunnyvale, ca, usa) (r1, for gastrolysis), 5-mm bowel graspers (r2 and r3), and 5-mm needle driver (r1, for over - sewing staple line). a 5-cm umbilical tape is used to measure the distance from the pylorus, and gastrolysis is performed along the greater curvature of the stomach using a harmonic scalpel (ethicon endo - surgery, intuitive surgical, sunnyvale, ca, usa). the short gastric vessels from the gastrosplenic ligament and posterior gastric adhesions with the pancreas are divided. one can appreciate a bilaminar parchment like membrane if one remains close to the stomach on the greater curvature. care is taken to ensure that the bougie is withdrawn in the esophagus during this step ; otherwise, it can lead to difficulty in dissection around the hiatus. the gastric calibration tube is pushed forward by the anesthetist and guided by the surgeon into the first part of the duodenum using bowel graspers. rotation of the bougie by the anesthetist and correct amount of traction by the surgeon help in negotiating the pylorus. this leads to the bougie nicely resting along the lesser curve of the stomach and helps in formation of an even sleeve. a sleeve of stomach is created using an articulating stapler from the assistant port while the console surgeon applies traction on the stomach so that it lies in the right orientation. sequential stapling first with a green load and subsequently with blue loads is performed while not being very snug to the bougie. after the sleeve is formed, the staple line is imbricated using no. 2 - 0 polydioxanone running suture with a sliding loop at one end. gastroscopy with saline solution immersion is performed in all cases to rule out any leak, bleed, or obstruction. a total of 35 patients (15 female and 20 male patients) with a mean bmi of 48.17 11.7 the bmi ranged from 33 to 82.6 kg / m, with 3 patients having a bmi > 80 kg / m. the mean operative time was 116.3 24.7 minutes, and the mean docking time was 8.9 5.4 minutes. the mean time taken to imbricate the staple line was 21.4 6.4 minutes. mean blood loss was 19.36 4.62 ml, and there were no complications, conversions, or perioperative deaths in the series. a steep decline in operative time and docking time was observed after the initial 10 cases. we believe that the operating room team surpassed the learning curve for robotic bariatric surgery by the 10th case. the team was already well - versed in laparoscopic bariatric procedures and could adjust to the new system fairly quickly. the so group was compared with the mo group regarding patient demographic characteristics, bmi, and comorbidities (table 1), as well as perioperative outcomes, complications, and weight loss (table 2). the comparison showed no significant difference in operative time (p =.145), blood loss (p =.523), and length of hospital stay (p =.213). the excess body weight loss at 6 months ' follow - up was also not significantly different between the 2 groups. perioperative outcomes, complications, and ebw loss in morbidly obese versus super obese patients ebw = excess body weight. laparoscopic bariatric surgery, especially in so patients, presents a complex situation in which the surgeon has to deliver the best results despite compromised technology. with the advent of robotic technology, its application to bariatric procedures may eliminate certain limitations such as torque on rigid instruments because of the thickness of the abdominal wall and difficulty in dissection or suturing in a limited working space because of a heavy liver and excess omental fat. it also provides for a stable and ergonomically comfortable platform with 3-dimensional vision and precise movements without any tremor. sg, on the other hand, is considered a relatively simple procedure compared with rygb. however, there are certain peculiarities in sg ; for example, it uses a long staple line with a potential to leak, and precise and safe dissection is required in the area of the left crus and hiatus to entirely mobilize the fundus. compared with laparoscopic surgery, robotic surgery offers the possibility of endowrist application (intuitive surgical), and this action facilitates the over - sewing of the staple line. over - sewing of the staple line was accepted by most of the surgeons (95%) participating in the international sleeve gastrectomy expert panel. any leak with sg is a high - pressure leak and is difficult to manage compared with an rygb leak. these difficulties become more pronounced when operating in an so patient because of limited working space. so and sso patients are high - risk candidates, and sg is considered the first step in a 2-stage procedure or, more and more often, is performed as a single - stage procedure. from a surgical point of view, so patients remain a difficult population to manage, usually with outcomes slightly less favorable than those for mo patients. there have been comparisons of robot assistance in rygb in so patients versus mo patients, and there are studies that state that rasg is a safe and feasible operation. however, there are no or very few studies comparing the perioperative outcomes of using a robot for sg in so patients versus mo patients. the results of rasg for the so group versus the mo group were compared to look for any advantages provided by the robotic system in the so group. in our experience, the outcomes in both the so and mo groups were the same, with no complications in either group. this occurred despite the fact that there were a few very high - risk sso patients in this series, including 3 patients with a bmi > 80 kg / m. in addition, a robotic system helps in precisely over - sewing the staple line (as is the routine practice in our center) even in sso patients with a severely limited working space. thus, using a robotic system may help overcome many difficulties in so patients and enables the surgeon to perform a similar procedure to that in mo patients with equal precision, similar time requirement, and little or no extra effort. we believed that during introduction of robotic surgery to a bariatric program, sg was a relatively simple and safer procedure to start with. it enables the whole team to become accustomed to the setup of the robotic system, docking, and troubleshooting. once the team is comfortable with rasg, it can proceed to robotic rygb and revision surgical procedures. in this series the learning curve for rasg was 10 procedures for a team well - versed in laparoscopic bariatric surgery and well - trained in handling the da vinci surgical system. after 10 cases, our docking time, setup time, and console time plateaued with a < 5% change in the operative time. first, the study had a small sample size, and patients were not randomized. yet, it described our initial experience with rasg and tried to address issues such as safety, outcomes, and the learning curve. second, the study included the learning curve of the team, which may have increased the mean operating room times. third, the study did not analyze the additional cost of using a robot, but we believe that clinical outcomes are much more important and that costs are bound to decrease with time. in addition, the study did not compare rasg with laparoscopic sg, and such a comparison is required to ascertain the real benefit of using a robotic system. in addition, the ease and precision with which the staple line is imbricated are definite advantages. rasg can act as a good procedure to introduce robotics in a bariatric surgery center before going on to perform rygb and revision procedures. the learning curve for rasg is about 10 cases for an established bariatric surgery center. the real advantages in comparison with the laparoscopic approach are still being debated and require further studies for the subpopulation of so patients. | background and objectives : this study evaluates our technique for robot - assisted sleeve gastrectomy for morbidly obese and super obese patients and our outcomes.methods:a retrospective analysis of patients who underwent robot - assisted sleeve gastrectomy at a single center was performed. the procedure was performed with the da vinci si hd surgical system (intuitive surgical, sunnyvale, california). the staple line was imbricated with no. 2 - 0 polydioxanone in all cases. the super obese (body mass index 50 kg / m2) subset of patients was compared with the morbidly obese group in terms of demographic characteristics, comorbidities, operative times, perioperative complications, and excess body weight loss.results:a total of 35 patients (15 female and 20 male patients) with a mean body mass index of 48.17 11.7 kg / m2 underwent robot - assisted sleeve gastrectomy. of these patients, 11 were super obese and 24 were morbidly obese. the mean operative time was 116.3 24.7 minutes, and the mean docking time was 8.9 5.4 minutes. mean blood loss was 19.36 4.62 ml, and there were no complications, conversions, or perioperative deaths. when compared with the morbidly obese patients, the super obese patients showed no significant difference in operative time, blood loss, and length of hospital stay. there was a steep decline in operating room times after 10 cases of robot - assisted sleeve gastrectomy.conclusion:this study shows the feasibility and safety of robot - assisted sleeve gastrectomy. robotic assistance might help overcome the operative difficulties encountered in super obese patients. it shows a rapid reduction in operative times with the growing experience of the entire operative team. robot - assisted sleeve gastrectomy can be a good procedure by which to introduce robotics in a bariatric surgery center before going on to perform roux - en - y gastric bypass and revision procedures. |
transposons employ intricate mechanisms of excision and reinsertion, and they appear to play an important role in the evolution of their hosts, often accounting for a substantial part of the genomes [1 - 5 ]. more recently, however, they have primarily attracted attention as tools for the manipulation of the genomes of their natural hosts and, more importantly, the genomes of other species that are not closely related. transposons have even been found to be active in organisms that do not belong to the kingdom of their natural host. this review summarizes the advances that have been made with one of these versatile genomic tools, namely the transposon minos. minos is a dna (class ii) transposon that was fortuitously identified as a repetitive element in the genome of the fruit fly drosophila hydei. the natural element, approximately 1.8 kilobases in length, is characterized by 254 base pair (bp) perfect inverted terminal repeats flanking a two - exon transposase gene (figure 1). the minos element carries a single gene that is interrupted by a 60 bp long intron and encodes a transposase. the amino - terminus of the transposase contains a putative dna - binding domain resembling the paired domain, a conserved feature of the pax protein family. the carboxyl - terminus of the minos transposase contains a d, d34e catalytic triad, which is also found in transposases of related elements such as tc1 and bari1. the presence of an unmapped nuclear localization signal has been inferred based on the nuclear localization of a minos transposase - enhanced green fluorescent protein (egfp) fusion. idr, inner direct repeat ; itr, inverted terminal repeat ; odr, outer direct repeat. surveys among drosophila spp. have revealed widespread occurrence of minos - like elements ; 21 out of 26 analyzed species of the repleta group and 5 out of 7 analyzed species of the saltans group carry minos - like transposons. evolutionary analysis suggests that the distribution of minos in the genus drosophila is best explained by horizontal transfer of the element across species. like most dna transposons, minos moves in a host genome with a cut - and - paste mechanism, whereby the transposase excises the element from the original site of insertion and reinserts it into a new locus in a nonreplicative manner. the transposition of minos, like that of the other tc1/mariner - like elements, occurs into a ta dinucleotide that is duplicated upon insertion ; this implies that a staggered cut of the target dna leads to 2 bp single strand ta overhangs as the first step in the insertion reaction. analysis of the sequences that flank insertion sites in the genome of drosophila melanogaster revealed that minos transposase has little sequence preference beyond the ta target dinucleotide. this is in contrast to most other transposable elements studied thus far, which exhibit variable degrees of flanking sequence preference, resulting in biased insertion and consequently the existence of ' hot ' and ' cold ' spots along the genome. although much of the recent work on transposons is concerned with the transgenesis of vertebrates, simpler model organisms and cell lines are more suitable for the analysis of the transposition mechanism. the introduction of a non - autonomous minos element and of a transgene expressing transposase into the d. melanogaster genome allowed the study of the minos transposition mechanism by mobilization of the non - autonomous element and subsequent molecular analysis of the excision and transposition events. two types of chromosomal sites were recovered after excision : sites precisely restored and sites containing leftovers (footprints) of the mobilized element. precise excision (restoration of the original site of insertion) was detected only in flies heterozygous for the insertion, presumably resulting from gene conversion. most footprints were 6 bp long and consisted of the four terminal nucleotides of either one of the inverted repeats plus the duplicated ta dinucleotide. the structure of the footprints suggested a possible mechanism of excision involving staggered cuts by the transposase at the ends of the inverted repeats, creating single strand overhangs of the four terminal bases of each end. during reinsertion, the overhangs of the mobilized element are joined to the ta overhangs of the target site, followed by polymerase - mediated fill - in of the single stranded dna. this model predicts that a heteroduplex flanked by the ta duplication is formed at the site of excision. indeed, the existence of such heteroduplexes in the genome of d. melanogaster flies in which minos is actively excising was directly demonstrated. several excision sites were also cloned from vertebrate cells, in which a single non - autonomous minos element was mobilized by trans acting transposase. these footprints, however, were much more heterogeneous in their size and structure, reflecting differences in the post - excision processes compared with drosophila. an analysis of minos excisions from a plasmid in the simple chordate ciona intestinalis identified the typical 6 bp footprints as well as variants thereof with short insertions or deletions. sequence - specific interaction of transposase and the transposable element at or near the inverted repeats is required for transposition. each of the minos inverted repeats contains two 18 bp direct repeats near the ends of the inverted repeat (figure 1). dnaasei protection with purified recombinant minos transposase protein shows that these direct repeats are bound by transposase (figure 2a). binding of transposase to the minos inverted terminal repeat and internal minos transcription initiation sites. g+a reaction ' indicates g and a specific sequence reaction ; the dnaasei protection reactions were done in duplicate. ' + transposase ' indicates twofold molar excess of transposase over target dna, and ' + + transposase ' indicates 20-fold molar excess of transposase. the positions of the protected sequences (open boxes), the hypersensitive positions (filled box), as well as the positions of the inner (bottom arrow) and outer direct repeats (top arrow) are indicated. a ', ' c ', ' g ', and ' t ' indicate base specific sequencing reactions. also shown are primer extension reactions with a minos - specific primer on total rna isolated from d. melanogaster populations with (+) or without (-) a minos insertion. the lower horizontal arrow indicates a transcription start about 30 base pairs downstream from the inner direct repeat. an additional transcription start site is detected between the two direct repeats (upper horizontal arrow). although the binding site close to the end of the element (the ' outer ' direct repeat) is expected to play a role in the excision of the element, the function of the second, ' inner ' repeat is not known. in addition to minos, two other well studied transposons of the tc1/mariner family carry two direct repeats per inverted terminal repeat : tc3 and sleeping beauty. transposon mobility assays suggest that both the outer and the inner pairs of binding sites are required for full activity of sleeping beauty and minos (klinakis a and savakis c, unpublished data) but not that of tc3. the mechanism underlying the involvement of the inner direct repeat in minos and sleeping beauty transposition remains to be elucidated. it is intriguing that the inner direct repeat overlaps a putative tata box in the 5 ' inverted repeat of minos, and primer extension analysis revealed a transcriptional initiation site for the minos transposase approximately 30 bp downstream of this repeat (figure 2b). these observations suggest a possible role for the inner transposase binding site in autoregulation of transposase expression. negative feedback mechanisms that regulate transposition have been suggested for other transposable elements too [23 - 25 ]. in vitro activity assays established for other members of the tc1/mariner transposon superfamily although direct biochemical evidence is not yet available for minos, the ability of minos - based vectors to transpose in cells of a wide range of metazoan species [19,28 - 40 ] is indicative of the element 's host independence (figure 3). assays for three types of minos activity are distinguished and indicated (extrachromosomal, chromosomal, and germline). transposon mediated transgenesis mimics nature ; many transposons found in genomes are non - autonomous derivatives. non - autonomous elements also carry functional inverted repeats but no functional transposase gene, because of the accumulation of point mutations and deletions. elements that lack an intact transposase gene are unable to catalyze their own transposition, but they can be mobilized with transposase supplied in trans. in a typical engineered transposon, the transposase gene is inactivated through the insertion of a selective marker or deleted and replaced by a marker gene. transposase is supplied in trans from a copy of the transposase gene introduced into the genome, or (more often) expressed from co - injected plasmid or transposase mrna. the transient nature of the supply of transposase is the key to the generation of stable insertions. a construct carrying a non - autonomous transposon is usually termed a ' donor ' and a construct carrying an active transposase gene is termed a ' helper '. the minos transgenesis system is supported by an extensive toolkit that has allowed experimentation in a wide spectrum of species. first, extra - chromosomal assays have been devised to facilitate and speed up the process of assessing the effectiveness of the minos components in diverse organisms of interest. second, an ever - increasing number of minos - based transformation vectors has been created, containing either fluorescent proteins such as gfp or more traditional marker genes for the easy identification of integration events and transgenic individuals [14,17,30 - 32,35 - 37,39,40,42 - 44 ]. third, the availability of different transposase sources, varying from a chromosomal copy of the transposase gene to a helper plasmid or in vitro synthesized mrna, can fit most purposes and experimental settings. the development of minos - based gene manipulation for mammals was based on a first phase of evaluation in insects. as in the case of all other insect transposons, d. melanogaster has been the favored testing ground for most components and applications of the minos system. the original use of minos as a germline transformation vector in d. melanogaster was followed by the transformation of the medfly ceratitis capitata, in the first report of a transposon system applied in a genus other than the one in which it was originally identified. the validation of improved minos tools in activity assays conducted in mosquito and lepidopteran embryos and cell lines opened the way to the stable germline transformation of a species of great medical importance, namely the human malaria vector anopheles stephensi. more recently, the development of even more efficient minos transposase sources and of versatile fluorescent marker systems facilitated the genetic transformation of diverse arthropod species, ranging from diptera (the olive fly bactrocera oleae) to coleoptera (the red flour beetle tribolium castaneum), lepidoptera (the silkworm bombyx mori), and crustacea (the amphipod parhyale hawaiensis). the genetic transformation of d. melanogaster using the p - element over 25 years ago triggered the development of innovative experimental approaches that revolutionized molecular genetic analysis in this model organism. however, the use of the p - element is limited to species closely related to d. melanogaster. the establishment of minos and other transposon systems allowed a number of these forward and reverse genetic methodologies to be transferred to several non - drosophilid insects of experimental and practical interest, as well as to a crustacean. for instance, analysis of cis regulatory elements with reporter constructs has been demonstrated in ceratitis and parhyale, enhancer trapping has been undertaken in tribolium and parhyale (pavlopoulos a, averof m, unpublished data), conditional mis - expression systems have been developed in anopheles and parhyale (pavlopoulos a, averof m, unpublished data), and post - transcriptional gene silencing via rna interference constructs has been employed in anopheles. minos activity is not limited to arthropod species ; it has also been used for germline transformation and enhancer trapping in two species of ascidians. finally, minos - based transposons have been mobilized successfully in cultured human cells and in the mouse soma and germline. in addition to minos, there is a handful of other dna transposons used for vertebrate transgenesis, the most widely employed of which is sleeping beauty. the relative evaluation of these different transposons is difficult unless experiments are performed under comparable conditions, which is generally not the case. however, one such comparison has been done with regard to transposition efficiency of the transposons sleeping beauty, tol2, mos1, and piggybac. in all four different vertebrate cell lines one should, however, be careful not to take the transposition rate as the only criterion of the usefulness of a transposable element as a genomic tool. depending on the intended application, insertional preference, the capacity to carry large inserts, mutagenicity, the ability to create deletions upon excision, and other properties can be at least equally important as high transposition frequency. the availability of sequenced genomes for several model and nonmodel organisms signals the next challenge in genomics research, namely the functional annotation of all predicted genes. classic genetic analysis relies on the use of mutations to study the function of the affected gene. the most common experimental approach is inactivation of the gene by chemical mutagenesis or insertional mutagenesis using transposable elements or viruses. insertional mutagenesis has the advantage that the targeted gene is at the same time tagged with the inserted dna, and so it is relatively easy to identify and retrieve. in principle, the short target sites of most transposable elements would allow a single transposon to saturate the whole genome with mutagenic insertions. in practice, however, every transposable element exhibits insertional biases for certain genomic loci, rendering mutagenesis of the entire genome problematic. for example, the ability of the p - element to target new genes in d. melanogaster appears to have reached its limits. in accordance with the distinct target preferences between minos and p, a pilot study indicated that more than half of minos generated insertions were in genes not previously hit by the p - element. although the p - element tends to insert in 5 ' untranslated regions, minos insertions in drosophila genes occur preferentially into introns. these insertions appear to be spliced out with the targeted introns and thus do not interfere with normal gene function. however, re - mobilizing such insertions by providing transposase in trans leads at reasonable frequency to deletions of flanking dna, so that induced excision can be used to ' knockout ' the targeted gene. because of the ability of minos to transpose at high frequencies and the fact that 60% of all minos insertions were in or close to genes, the minos system has become an integral component of the drosophila gene disruption project, which is a systematic effort to mutate all genes in the drosophila genome (bellen h, personal communication). the efficiency of large - scale mutagenesis screens is enhanced in genetic schemes that utilize two different transposons, say a and b. the ' jump starter ' element a (with inverted repeats a) encodes transposase b that, when expressed, mobilizes the ' mutator ' element b (with inverted repeats b) into new genomic loci. the independent development of both minos and piggybac based gene transfer systems in the beetle tribolium castaneum allowed the initiation of a large - scale insertional mutagenesis screen by the geku consortium (research groups based in goettingen, erlangen, kansas, and the us department of agriculture grain marketing and production research center). in such screens, minos might be more useful as the mutator element because, unlike piggybac, it can excise imprecisely, offering the possibility to generate mutant alleles secondarily. other transposon based approaches to genomic analysis include the various ' trapping ' schemes, in which expression of a reporter or selection gene carried by the inserted transposon depends on the targeted gene. in enhancer trapping the expression of a transposon encoded marker gene is activated or modulated by transcription enhancer elements near to the transposon insertion site. in such traps, expression of the marker gene follows partly or fully the spatial and temporal expression of the gene normally controlled by the ' trapped ' enhancer elements. minos based enhancer trapping has been demonstrated in the ascidian ciona, in tribolium, in parhyale (pavlopoulos a, averof m, unpublished), and in d. melanogaster (metaxakis t, savakis c, unpublished data). in exon trapping, expression of a transposon - encoded marker gene depends on appropriate splicing of the marker gene upon insertion of the element into an intron. the preference of minos to insert into introns in the fly and the mouse (zagoraiou l, unpublished data) suggests that minos may be particularly useful for this type of analysis. indeed, the choice of an exon trapping minos based transposon as mutagen in human cells in culture was justified by the outcome of the experiment. this transposon carried an intronic splice acceptor followed by a selection gene that would be expressed only upon insertion into an intron of an active gene and then spliced in the correct frame to the upstream exon. using this transposon mediated mutagenesis (tramm) method, it was demonstrated that the minos system can potentially trap / tag all genes in a mammalian genome. co - transfection of a minos transposon construct with a helper plasmid expressing transposase into hela cells resulted in 4% of the cells being stably transformed, with on average two minos insertions per transformed cell. in later studies, efficient inter - chromosomal transposition of minos was demonstrated in double transgenic mice, carrying tandem copies of non - autonomous minos elements and the minos transposase gene under the control of tissue specific promoters. the observed transposition rate was much greater in oocytes than in lymphocytes (8% versus 0.6%). its high germline activity makes minos a powerful tool for genome - wide functional studies in whole mice and probably in other vertebrates. minos has attracted interest as a tool for basic research, for biotechnological purposes, and for developing strategies to control arthropod disease vectors and agricultural pests [30 - 32,51 ]. minos has been successfully evaluated as a tool for the transgenesis of diverse animal species, both vertebrates and invertebrates, whereas its functionality in plants has yet to be tested. in most species tested, minos transposition occurs with high frequency, transposons can carry relatively large transgenes, insertions are stable in the absence of transposase, and the only obvious target site preference is the ta dinucleotide. overall, these features demonstrate the usefulness of minos as a tool for functional genetic and genomic approaches in model and nonmodel organisms, including vertebrates. cs is a founding member and nonexecutive director of a company, minos biosystems ltd, which owns intellectual property related to the minos transposable element. this article has been published as part of genome biology volume 8, supplement 1, 2007 : transposons in vertebrate functional genomics. | transposons are powerful tools for conducting genetic manipulation and functional studies in organisms that are of scientific, economic, or medical interest. minos, a member of the tc1/mariner family of dna transposons, exhibits a low insertional bias and transposes with high frequency in vertebrates and invertebrates. its use as a tool for transgenesis and genome analysis of rather different animal species is described. |
sunitinib is a small molecule multitargeted tyrosine kinase inhibitor currently used for the treatment of cancer. this class of drugs has also been reported to have antidiabetic effects [1 - 3 ], but the mechanism has not been elucidated. here we describe a patient with a nonfunctioning pancreatic neuroendocrine carcinoma with liver metastasis, who developed life - threatening hypoglycemia after treatment with sunitinib. histological examination of the metastatic mass in the liver suggested that the nonfunctioning neuroendocrine cells were converted into insulin - producing cells, causing hyperinsulinemia and severe hypoglycemia. a 46-year - old woman was carried to the emergency room with sudden loss of consciousness before breakfast. during transfer in the ambulance, 1a), with metastases to retroperitoneal lymph nodes, left supraclavicular lymph nodes and liver (fig. the patient had received this diagnosis 4 months prior, after presenting with right flank pain. needle biopsy and immunohistochemical (ihc) staining of the supraclavicular lymph node had been positive for cd56, chromogranin, and synaptophysin, and negative for glucagon and insulin. one month later, she started taking sunitinib 37.5 mg per day because the disease progressed and the right flank pain increased. after 2 months of sunitinib treatment she felt severe fatigue from which she was diagnosed with hypothyroidism, a common adverse event of sunitinib. she began levothyroxine, but even after normalization of thyroid hormone levels, she experienced intermittent weakness, dizziness, and hunger. to relieve fatigue and hunger, she increased oral intake and experienced weight gain of 10 kg over a month. she reported having no personal or family history of thyroid disease or diabetes mellitus. on physical examination her vital signs were within the normal range, height 156.5 cm and body weight 53 kg. there were two palpable, hard, and nontender lymph nodes of less than 1 cm each in the left supraclavicular area. she had tenderness in the right flank on percussion, but hepatosplenomegaly was not noted. grade 1 hand - foot syndrome (mild erythema), a skin - related side effect of sunitinib was observed. the blood cell count, urinalysis, and serum chemistry and electrolytes were within normal range. an electrocardiogram revealed a normal sinus rhythm and chest x - ray showed no abnormality. computed tomography of the abdomen demonstrated an increase in the size of the liver mass from 1.8 to 2.8 cm. overnight fasting plasma glucose was 16 mg / dl with c - peptide and insulin levels of 6.1 ng / ml and 27.2 iu / ml, respectively. these inappropriately elevated fasting insulin and c - peptide levels compared to the glucose level confirmed that the patient 's hypoglycemia resulted from endogenous hyperinsulinemia. differential diagnosis of endogenous hyperinsulinemia was based on negative titers for insulin antibody and insulin receptor antibody. we performed a liver biopsy to obtain metastatic tissue, and the histologic examination revealed diffuse infiltration of the cancer cells in the liver. in addition, the metastatic lesion in the liver which had been negative at the initial diagnosis (fig. we concluded that the nonfunctional neuroendocrine carcinoma that metastasized to the liver changed into an insulin - producing tumor after 2 months of administration of sunitinib. however, even with frequent dietary intake, the patient required more than 500 g of glucose per day via the central vein to prevent hypoglycemia. high - dose glucocorticoid and glucagon administration were not effective in relieving the severe hypoglycemia. eighteen days after discontinuation of sunitinib, severe hypoglycemia persisted and she underwent transarterial chemoembolization (tace) for the metastatic lesions in the left lobe of her liver. intravenous glucose infusion was slowly tapered to 200 g per day during the 2 weeks after tace. then a -cell toxin, streptozotocin (500 mg / m), and adriamycin (50 mg / m) were administrated intravenously the patient underwent another round of tace for the right lobe of the liver and intravenous streptozotocin / adriamycin, and she was successfully discharged. one month after the final treatment, her fasting blood glucose was 101 mg / dl. local control of metastatic carcinoma and systemic administration of -cell toxin had reversed her severe hypoglycemia. generally, tumorous conditions that induce endogenous hyperinsulinism such as insulinoma and nesidioblastosis cause clinical hypoglycemia even for small tumors. therefore, it is unlikely that the slight increase in tumor burden in the liver in the current case had caused conversion of " preclinical " insulinoma into clinical insulinoma. the patient 's liver metastases did not impair liver function, so liver function did not contribute to the hypoglycemia. she developed severe hypoglycemia from endogenous hyperinsulinemia after treatment with sunitinib without initial evidence of insulinoma or impaired liver function. ihc staining for insulin in tumor specimens before and after sunitinib treatment suggests that neuroendocrine carcinoma cells were transformed from nonfunctioning to insulin - producing cells after treatment with sunitinib. this hypothesis is further supported by the fact that she recovered from severe hypoglycemia after controlling metastatic tumors with tace and streptozotocin. however, we should note that her supraclavicular lymph node was biopsied at initial diagnosis of pancreatic neuroendocrine carcinoma because it was easily accessible. in contrast, liver tissue was obtained after use of sunitinib due to significant progression of liver metastases. our findings suggested that the metastatic liver mass turned to producing insulin upon sunitinib treatment, but we could not rule out production of insulin by the same liver metastatic tissue before sunitinib treatment because pretreatment liver metastatic tissue was not available. we previously published a case of sunitinib - induced hypoglycemia occurring in nonfunctioning pancreatic neuroendocrine carcinoma with liver metastasis. in that case, we identified endogenous hyperinsulinism but did not attempt to examine insulin production by tumor cells because the hypoglycemia resolved after treatment with a small dose of prednisolone. recently reported a similar rare case of transformation from nonfunctioning neuroendocrine tumor into insulin - producing tumor, where the patient had liver metastases. however, hypoglycemia was not ascribed to sunitinib treatment as various chemotherapeutic regimens had been used. in our case, the patient developed severe hypoglycemia after treatment with only sunitinib, with the exception of thyroid hormone which has no previous report of causing hypoglycemia. the similar characteristics of nonfunctioning pancreatic neuroendocrine carcinoma with liver metastases in all these cases suggest that a common mechanism might exist, such as the milieu of liver tissue in which the neuroendocrine carcinoma is embedded. the molecular mechanism of the glucose - lowering effect of sunitinib has not been elucidated. several in vitro and in vivo experiments have suggested that imatinib, another kind of small molecule tyrosine kinase inhibitor, is involved in the autoimmune process, -cell protection, and insulin sensitivity [6 - 10 ]. the platelet - derived growth factor signaling pathway, through which sunitinib works, was recently shown to control age - dependent -cell proliferation in mouse and human pancreatic islets. further studies are warranted to understand the molecular mechanism of neuroendocrine cell fate to produce insulin by sunitinib. in conclusion, this case and a review of the literature suggest that the use of sunitinib in a patient with pancreatic neuroendocrine carcinoma with liver metastases can bring about hypoglycemia, which could be lethal. considering the possibility of such a serious adverse effect, we recommend that oncologists carefully monitor hypoglycemic symptoms and blood glucose levels of patients treated with sunitinib. the possibility of transformation of neuroendocrine cells into insulin - producing cells by sunitinib would provide a novel way of manipulating -cell fate. | we report a rare case of severe hypoglycemia after sunitinib treatment for pancreatic neuroendocrine carcinoma. we describe the initial clinical presentation, laboratory results, pathologic findings, and managment in a patient with a nonfunctioning pancreatic neuroendocrine carcinoma with liver metastases who developed life threatening hypoglycemia after 2 months of sunitinib therapy. a 46-year - old woman presented to the emergency department with loss of consciousness from hypoglycemia. serum c - peptide and insulin levels at fasting state revealed that the hypoglycemia resulted from endogenous hyperinsulinemia. she had been diagnosed with nonfunctioning pancreatic neuroendocrine carcinoma based on a biopsy of metastatic cervical lymph node and was being treated with sunitinib, a small molecule tyrosine kinase inhibitor. immunohistochemical stain of the metastatic liver mass demonstrated that the initially nonfunctioning neuroendocrine carcinoma cells had changed into insulin - producing cells after sunitinib therapy. transarterial chemoembolization of the liver masses and systemic chemotherapy with streptozotocin / adriamycin relieved the hypoglycemia. a nonfunctioning pancreatic neuroendocrine carcinoma was transformed into an insulin - producing tumor after treatment with sunitinib, causing endogenous hyperinsulinemia and severe hypoglycemia. |
a new distribution for analysing time - to - event data was introduced by, known as generalised exponential distribution. generalised exponential distribution can be used as an alternative to the well - known and used weibull distribution in lifetime data analysis and reliability engineering according to. the generalised exponential distribution has the distribution, density, and survival functions, respectively, as (1)ft;,p=1exptp,,p, t>0,ft;,p = p1exptp1expt, st;,p=11exptp, where p is the shape parameter and the scale parameter. let the ge distribution with the shape parameter p and the scale parameter be denoted by ge(, p). according to, the two - parameter ge(, p) can be used quite effectively in analysing many lifetime data and can assume the place of the two - parameter gamma and two - parameter weibull distributions. the two - parameter ge(, p) can have increasing and decreasing failure rates depending on the shape parameter. studies that involve time - to - event or survival data analysis are focussed on measuring time - to - event of an outcome. time - to - event could vary from time to either death or the occurrence of a clinical endpoint such as disease or the attainment of a biochemical marker. a special course of difficulty in the analysis of time - to - event data is the possibility that some individuals or units may not be observed for the full time to failure. in some circumstances, some individuals or units do not fail but are lost - to - followup during the observed period. instead of knowing the failure time t, all we know about these individuals is that their time - to - failure exceeds some value, say x, where x is the follow - up time of these individuals in the study, which is referred to as censoring. under random or noninformative censoring, a sample of, say, n, elements are followed for some time, say t. an instance of this type of censoring occurs when the termination date for a medical trial is not fixed before the study starts but is rather chosen later, where the choice is influenced by the results of the study up to the termination time. in a straightforward overview of this scheme, which can be considered as time censoring, each element has a maximum inspection time of, say, ti, for i = 1,, consider an experiment where we start with an observation of 50 cancer patients and terminate the experiment after a certain amount of time irrespective of the number of patients that have died or survived at the specified time. the survival of the patients may be due to withdrawal, inadequate monitoring mechanism, or deaths, which is not related to the purpose of the study. maximum likelihood estimator (mle) is very popular both in the literature and in practice. some researches have been done to compare mle and the bayesian approach in estimating the two parameters of the generalised distribution using hybrid and complete failure time data. amongst them reference determined the bayes estimates of the reliability function and the hazard rate of the weibull failure time distribution by employing squared error loss function. reference studied bayesian parameter and reliability estimate of weibull failure time distribution ; reference studied the approximate bayesian estimates for the weibull reliability function and hazard rate from censored data by employing a new method that has the potential of reducing the number of terms in lindley procedure. the main aim of this paper is to compare the classical maximum likelihood estimator to the proposed bayesian estimators with two loss functions for the unknown parameters of the generalised exponential distribution for different sample sizes and parameter values. tn) be the set of n random lifetimes with respect to the generalised exponential distribution with p and as the parameters, where is the scale parameter and p the shape parameter. in random censoring as stated by, we assume ti = min(ti, ci), = 1 if ti ci and = 0 if ti > ci. the observed data from n individuals is assumed to consist of the pair (ti, i), i = 1,, n, so that the final result obtained will be the same provided ci is available for all i. it is therefore assumed that tc ; that is, t and c are independent of each other, which implies that the censoring time c is noninformative in analysing the failure time t. in order for this assumption to be valid, one has to ensure that the loss to follow - up of individuals is not as a result of the failure time defined. the likelihood function with respect to random censored data is (2)l,ti;,p=i=1nftiisti1i, where s() is the survival function. calculation of the maximum likelihood estimator often requires that some iterative (e.g., newton - raphson) procedures be implemented to obtain the parameters estimates. in this section we consider the bayes estimation of the two unknown parameters. since both parameters are assumed to be greater than zero (0), we let both take on the following gamma prior distributions : (3)1b1expa, >0,2ppd1exppc, p>0. assume that the hyperparameters a, b, c, and d are known and > 0. the joint density function of the data, and p, can be obtained as (4),p, t,ldata,p12p. bayesian inference is based on the posterior distribution which is given as (5),p ti,i=,p, t,0,p, t,d dp. the ratio of the two integrals given in (5) can not be obtained in a closed form. we can apply a numerical integration technique, which may be computationally intensive especially in high - dimensional parameter space. it is also possible to make use of numerical approximation methods such as and/or. in this paper, we shall consider both methods for this type of censoring scheme and for this distribution, since we are unaware of any study employing both methods for this distribution and with this type of censored data apart from the former by with uncensored data. this approach is considered under two loss functions, namely, linex and squared error loss. this approach has been used by several authors like [3, 6 ] to obtain the approximate bayes estimators. reference shows the approximate procedure for evaluating ratio of integrals of the form (6)eu() x=expldvexpld, where = (1, 2,, m) and () is the logarithm of the likelihood function and (), v() are arbitrary functions of. assume that v() is the prior distribution for and () = u() v() with u() being some function of interest. the posterior expectation of q() is given as (7)eq t=uexpl+dexpl+d, where () = logv(). an outline of the procedure can be obtained from and a recent paper by. lindley 's procedure can be approximated asymptotically by (8)eu x = u+12ijuij+2uijij + 12ijkllijkijklul. considering the bayesian estimator under the squared error loss function, which is the posterior mean, the following can be obtained where u1 and u11 are the first and second derivatives of the scale parameter () while u2 and u22 are also the first and second derivatives of (p) : (9)u=, u1=1, u11=0,up = p, u2=1, u22=0,=ln1+ln2p,1=b1a, 2=d1pc.11=l201, 22=l021. unlike the symmetric loss function (squared error), this loss function measures the degree of underestimation and overestimation of the estimated parameter. the bayes estimator of, say,, which is denoted by ^bl under linex loss function, is (10)^bl=1klneexpk, provided that e[exp(k) ] exists and is finite. the bayes estimator u^bl of a function ubl = u[exp(k), exp(kp) ] under linex is given as (11)u^bl=ubl12pl,ti;,pd dp12pl,ti;,pd dp, where (12)^=expk, u1=u=kexpk,u11=2u2=k2expk, u2=u22=0,p^=expkp, u2=up=kexpkp, u22=2up2=k2expkp, u1=u11=0. observing from section 3.1, it is clear that the lindley approach demands or requires that we evaluate the third derivatives of the likelihood function. depending on the distribution and the number of parameters involved, this approach can be very difficult to achieve. tierney and kadane through laplace approximation procedure gave an alternative to the lindley approach which only requires the first and second derivatives of the likelihood function. let l(; t) be the likelihood function of based on n number of observations. () represents the prior distribution defined over the parameter space, v() represents the loss function, and q(t) represents the posterior distribution of. the bayes estimate of a function q() under the squared error loss function is the posterior mean and is given as (13)q^=eq t=expnldexpnld, with (14)l=log+loglt n, l=logv+log+loglt n. equation (13) can be approximated in the form (15)q^bs=1/2expnl^l^. this can similarly be expressed as (16)q^bs=1/2q^q^ tq^ t, where ^ and ^ maximize () and (), respectively, and and are the negatives of the inverse hessians of and, respectively. the matrix takes the form (17)=2l22l2p2lp2lp. we can similarly obtain the expression for the matrix, which involves the partial derivatives of. in applying the method the following need to be maximised : (18)l=1nl,ti;,pa1lnb+c1lnp pd+logl,ti;,p(19)l=1nl,ti;,pln+lnp+a1lnb+c1lnp pd+logl,ti;,p. setting / and /p to zero produces the following system of equations : (20)l=1na1b+logl,p;ti,i,lp=1nc1pd+logl,p;ti,ip, where logl(, p ; ti, i)/ and logl(, p ; ti, i)/p are easy to obtain the bayesian estimator u^bl of a function ubl = u[exp(k), exp(kp) ] under linex with respect to tierney and kadane procedure is given as (21)u^bl=ubl12pl,ti;,pd dp12pl,ti;,pd dp, where (22)l=1na1lnb+c1lnppd + logl,ti;,p, l=1nlnexpk+lnexpkp+a1ln b+c1lnppd+logl,ti;,p. the same approach is also adopted with the squared error loss function to obtain the bayes estimates of the unknown parameters. the data for this example are on survival of patients with cervical cancer, recruited to a randomised trial aimed at analysing the effect of addition of a radio sensitiser to radiotherapy (new therapytreatment b) compared to using only radiotherapy (controltreatment a). the data are in days since the start of the study ; the event of interest is death caused by this cancer. our main interest is on the patients under treatment a, which is fairly small to illustrate the proposed methods in this paper. starred observations are censored : 90, 890, 142, 1037, 150, 1090, 269, 1113, 291, 1153, 468, 1297, 680, 1429, 837, 1577. the data represent survival times for 121 breast cancer patients who were treated over the period 19291938. times are in months and asterisks denote censoring times : 0.3, 0.3, 4.0, 5.0, 5.6, 6.2, 6.3, 6.6, 6.8, 7.4, 7.5, 8.4, 8.4, 10.3, 11.0, 11.8, 12.2, 12.3, 13.5, 14.4, 14.4, 14.8, 15.5, 15.7, 16.2, 16.3, 16.5, 16.8, 17.2, 17.3, 17.5, 17.9, 19.8, 20.4, 20.9, 21.0, 21.0, 21.1, 23.0, 23.4, 23.6, 24.0, 24.0, 27.9, 28.2, 29.1, 30, 31, 31, 32, 35, 35, 37, 37, 37, 38, 38, 38, 39, 39, 40, 40, 40, 41, 41, 41, 42, 43, 43, 43, 44, 45, 45, 46, 46, 47, 48, 49, 51, 51, 51, 52, 54, 55, 56, 57, 58, 59, 60, 60, 60, 61, 62, 65, 65, 67, 67, 68, 69, 78, 80, 83, 88, 89, 90, 93, 96, 103, 105, 109, 109, 111, 115, 117, 125, 126, 127, 129, 129, 139, 154. since it is difficult to compare the performance of the proposed methods theoretically, we have performed an extensive simulation to compare the estimators through mean squared errors and absolute biases by employing different sample sizes with different parameter values. if u follows a uniform distribution in the interval, then y = (ln(1 u)/) follows ge(, p). consequently, with a very good uniform random number generator, the generation of ge(, p) random deviate is immediate. a lifetime t is generated from the sample sizes indicated above from the ge(, p) distribution which represent failure of the product. the values of the assumed actual shape parameter (p) of the ge(, p) distribution were taken to be 0.8, 1.2 and 2.0. the scale parameter () was considered throughout to be 1 without loss of generality. the same sample size is generated from the uniform distribution for the censored time c with (0, b), where the value of b depends solely on the proportion of the observations that are censored. in our study, we considered the percentage of censoring to be 25. ti = min(ti, ci) is taken as the minimum of the failure time and that of the censored time of the observed time t. to compute the bayes estimates, an assumption is made such that and p take, respectively, gamma(a, b) and gamma(c, d) priors. we set the hyperparameters to 0 ; that is, a = b = c = d = 0 ; this makes the priors noninformative. the values of the loss parameter for the linex loss function are k = 0.7. the mean squared errors and the absolute biases are determined and presented for the purpose of comparison. the main objective of this study is to obtain the estimates of the generalised exponential distribution parameters and compare the proposed methods applied in this paper. in order to examine the estimates of the parameters which can not be obtained analytically, we made use of different numerical approximation procedures and have obtained absolute biases and mean squared errors of the estimated parameters. observing from table 1 and figures 1 and 2, it is evident that the smallest mean squared errors vis - a -- vis the absolute biases for the estimated scale parameter (^) occurred under the bayesian estimator with the linear exponential loss function. the loss parameter from which we obtained the smallest mean squared errors is 0.7, which is above zero, implying this approach is preferred if overestimation is more serious than underestimation. this occurred largely with the lindley numerical approximation procedure, followed by tierney and kadane. as the sample size increases, all the estimators ' mean squared errors correspondingly decrease. another observation made that needs to be mentioned is that lindley approximation method under the squared error loss function performed better than that of the tierney and kadane with respect to the generalised exponential scale parameter. as illustrated clearly in figure 2, considering table 2 alongside figures 3 and 4, which contain the mean squared errors and the absolute biases of the estimated shape parameter (p^), we noticed that the bayesian estimator under the tierney and kadane method performed better than the lindley approach but maximum likelihood estimator overall had the smallest mean squared error followed by tierney and kadane. the bold numbers indicate the smallest and minimum biases of the estimated parameters with their corresponding estimators. the bayes estimator with lindley numerical approximation procedure for the exponential distribution performed better under the squared error loss function for the shape parameter than that of the tierney and kadane numerical method to a very large extent. the bayesian estimator under linear exponential loss function with the positive loss parameter has the smallest standard error as illustrated in table 3. this happened with the approximation procedure suggested by tierney and kadane ; it implies that linear exponential loss function overestimates the scale and shape parameters of the generalised exponential distribution. from this example, where the sample size is considered to be fairly small, we noticed that using the tierney and kadane approach via bayes under squared error loss performs fairly better than that of the lindley method as well as the maximum likelihood estimator. using the iterative procedure suggested in this paper for both mles and bayes with respect to data 2, the mles of ^ and p^ are 0.765027 and 6.277847 with their corresponding standard errors as 0.006323 and 0.010377. since we do not have any prior information on the hyperparameters, we assume a = b = c = d = 0. this makes the priors on ^ and ^ noninformative. for computing the bayes estimators, we considered the squared error loss and linear exponential loss functions and gamma priors on both and same as the approach used in the simulation section. after computing the bayes estimators via lindley approximation procedure under squared error loss for ^ and p^, the following parameters estimates and standard errors were obtained, respectively, 0.765027, 6.277847 and 0.006325, 0.010376. computing the bayes estimates of ^ and p^ and their corresponding standard errors under the linear exponential loss function with a loss parameter of 0.7, we have 0.765029, 6.277860 and 0.006323, 0.010377. with the loss parameter being 0.7, we have 0.765025, 6.277840 and 0.006323, 0.010376, respectively. considering 95% confidence interval of mle, we have ^ = (0.752634, 0.777419) and p^ = (6.257508, 6.298185). bayes credible intervals under squared error loss function of ^ and p^ are 0.752634, 0.777419 and 6.257508, 6.298185, respectively. the bayes credible intervals with respect to the linex loss function with the loss parameter 0.7 for ^ and p^ are 0.752637, 0.777421 and 6.257521, 6.298198 and those of the 0.7 are 0.752633, 0.777417 and 6.257501, 6.298178, respectively. computing the bayes estimators using tierney and kadane (t & k) approximation procedure under squared error loss function for ^ and p^, we have, respectively, the following parameters estimates and standard errors : 0.764725, 6.275374 and 0.006320, 0.010373. calculating the bayes estimates via tierney and kadane of ^ and p^ with their corresponding standard errors under the linear exponential loss function with a loss parameter of + 0.7, we have 0.765633, 6.282807 and 0.006328, 0.010385. with the loss parameter of 0.7, we have 0.763671, 6.277809 and 0.006311, 0.010358, respectively. bayes credible intervals using tierney and kadane under squared error loss function of ^ and p^ are 0.752338, 0.777113 and 6.255044, 6.295704. the bayes credible intervals with respect to the linex loss function with the loss parameter + 0.7 for ^ and p^ are 0.753231, 0.778035 and 6.262453, 6.303161 and those of 0.7 are 0.751301, 0.776041 and 6.246441, 6.287045, respectively. as clearly stipulated above, the estimator with the smallest standard error is bayesian under the linear exponential loss function for both the scale and shape parameters. this is followed by bayes estimator using the squared error loss function, again with the tierney and kadane method. we observed that the linear exponential loss function had the narrowest credible intervals with respect to the tierney and kadane approach as compared to the credible intervals of bayes using lindley and the confidence intervals obtained from maximum likelihood estimator. this happened with a negative loss parameter, an indication of underestimation of the generalised exponential distribution parameters. from the results and discussions above it is evident that the bayesian estimator under linear exponential loss function performed quite better well than bayes under squared error loss function and maximum likelihood estimator for estimating both the scale parameter (^) and shape parameter p^, with both mse and absolute bias. lindley method performed better than t & k for the scale parameter with regard to mean squared errors while t & k performed better for the shape parameter with both the mean squared errors and the absolute bias. considering the standard errors obtained for the real data analysis, we can state that the t & k method outperformed the lindley numerical approximation and the maximum likelihood estimator. | a unit is said to be randomly censored when the information on time occurrence of an event is not available due to either loss to followup, withdrawal, or nonoccurrence of the outcome event before the end of the study. it is assumed in independent random / noninformative censoring that each individual has his / her own failure time t and censoring time c ; however, one can only observe the random vector, say, (x ;). the classical approach is considered for analysing the generalised exponential distribution with random or noninformative censored samples which occur most often in biological or medical studies. the bayes methods are also considered via a numerical approximation suggested by lindley in 1980 and that of the laplace approximation procedure developed by tierney and kadane in 1986 with assumed informative priors alongside linear exponential loss function and squared error loss function. a simulation study is carried out to compare the estimators proposed in this paper. two datasets have also been illustrated. |
supernumerary teeth can occur in primary or permanent dentition, but are more frequently reported in permanent dentition. they can occur in different forms, unilaterally or bilaterally and either in the maxilla or mandible. supernumerary teeth may be an incidental finding on a radiograph or the cause for failure of eruption of permanent teeth. the most common supernumerary tooth is the mesiodens that occurs between the maxillary central incisors. heredity may also play a role in the occurrence of this anomaly, as supernumeraries are more common in the relatives of affected children than in the general population. occurrence of supernumeraries is reported to be 0.2 to 0.9 % of the general population and is seen more frequently in permanent dentition. approximately 90% of cases present in the maxilla with a strong predilection for the incisor region (mesiodens), followed by maxillary and mandibular fourth molars (distomolars or distodens), premolars, canines and lateral incisors. on the basis of morphology, they are classified as supplemental (where the tooth has a normal shape for the teeth in that series)tuberculate (also called peg shaped)compound odontome (multiple small tooth - like forms)complex odontome (a disorganized mass of dental tissue). supplemental (where the tooth has a normal shape for the teeth in that series) tuberculate (also called barrel shaped) conical (also called peg shaped) compound odontome (multiple small tooth - like forms) complex odontome (a disorganized mass of dental tissue). on the basis of position, a supernumerary tooth may be referred to as a mesiodens, para premolar, para molar, or a distomolar. multiple supernumerary teeth are usually associated with conditions such as cleft - lip and palate or syndromes like cleidocranial dysplasia and gardners syndrome. however, it is rare to find multiple supernumeraries in individuals with no other associated disease or syndrome. a 35-year - old man came to the department of conservative dentistry and endodontics, bhabha college of dental sciences, bhopal with a chief complaint of pain in relation to the lower left first molar. the patient was healthy with no mental retardation.the facial appearance was normal and presented no skeletal or other abnormalities suggestive of any syndrome (fig 1). intra oral examination revealed presence of full complement of permanent teeth except for the lower third molars, and supernumerary teeth were present in all four quadrants. another finding was a grossly decayed 18. in the upper arch, supernumerary teeth resembling conical or triangular shaped crown were present in between normally appearing 14 and 15, 24 and 25 without any displacement of the permanent teeth. in the lower arch, two were located lingual to 34 and 35 and subsequently displacing same. on the right side, two supplemental teeth were present ; of which one was lingual to 44 and 45 and the other was between 45 and 46 (figs 2 and 3). a panoramic radiograph was taken to rule out any underlying pathology or unerupted supernumerary teeth that showed unerupted 13 present at the floor of the right maxillary sinus and apex of 14 and 15. supplemental supernumerary teeth showed a small crown - root proportion with a conical - shaped root and no bone or root resorption. panoramic radiograph showed impacted 38 and 48 with over - retained deciduous canine in relation to 13 (fig 4). 36 was treated endodontically, followed by a full metal crown.18 was extracted under local anesthesia. supernumerary teeth were not causing any kind of discomfort for the patient and patient was informed about the position of his teeth and importance of regular follow - ups. though a number of theories have been put forth, most commonly accepted explanations are dichotomy of the tooth bud. another theory suggests that supernumeraries are formed as a result of local independent condition, hyperactivity of the dental lamina. on the other hand although until recently the precise mechanism of transmission was not known, the underlying hereditary pattern has gradually been elucidated, thanks to the publication of different cases in which several members of one family presented hyperdontia. although multiple supernumerary teeth appear to develop more often in patients with some relative with at least one supernumerary tooth, the hereditary trait does not exhibit a simple mendelian pattern. there are few cases of multiple supernumerary teeth published in the literature not associated with complex syndromes. different studies have reported a 0.15% to 3.8% prevalence of supernumerary teeth in the permanent dentition. single supernumerary teeth are found in 76 - 86% of the cases, while two supernumerary teeth are found in 1223% of the cases, and three or more such teeth in the same individual are only found in 28% of cases. however, based on a study conducted by rajab and hamdan, this percentage was less than 1% when hyperdontia comprises five or more supernumerary teeth, while aikgz. reported a 0.06% prevalence of multiple supernumerary teeth [11, 12 ]. according to a study performed by hyun. the conical type of supernumerary teeth is relatively common in the anterior maxilla (75%), while supplemental supernumerary teeth can occur in any location of the dental arch, with predilection to the maxillary lateral incisors (7%). however, presence of multiple supernumerary teeth is associated with problems of displacement, rotation, impaction, ectopic eruption of the adjacent teeth, resorption of the teeth and even formation of cysts. in the present documented case, full complement of permanent dentition was in normal occlusion and the patient was completely asymptomatic and presence of the supernumerary teeth was an accidental finding. there are no fast set rules for management of supernumerary teeth ; the management of supernumerary teeth depends on its location and presence of associated complications. the various treatment modalities for impacted teeth include observation, intervention, relocation of the supernumerary tooth and extraction. if the supernumerary tooth is unerupted, it is generally better to wait till the root formation of the teeth in its vicinity is completed before its extraction. the extraction of supernumerary teeth should be carried out carefully, without causing damage to the roots of the adjacent teeth and adjacent anatomical structures. extraction is generally considered when there is failure of eruption of permanent teeth or potential complications such as crowding, root resorption, impaction and cyst formation. if the supernumerary teeth are not causing any complications and also do not interfere with orthodontic treatment, they can be monitored periodically. describe the surgical and orthodontic management of impacted teeth and identify their position and angulation, length of treatment, available space and the presence of keratinized gingival tissue as critical factors that can affect the prognosis and treatment outcomes. each case has to be evaluated on an individual basis and early detection greatly helps the treatment outcome. | the presence of supernumerary teeth is not uncommon in the general population. they occur more frequently in patients with a family history of such teeth. it is rare to find multiple supernumeraries in individuals with no other associated disease or syndrome. there have been very few documented cases of bilateral maxillary and mandibular supernumeraries in the premolar region. an unusual case of a 35-year - old man with six para premolars and complete dentition is presented. |
laboratory bacterial strains used are derived from e. coli k12 (bw25113 obtained from the coli genetic stock center or mg1655 obtained from atcc). the uropathogenic e. coli strain uti89 was kindly provided to us from matt conover and scott hultgren. the ciprofloxacin - resistant uropathogenic e. coli isolate (upec cipro) was collected from the brigham and women s hospital specimen bank (sup. table 3). deletion mutants on the bw25113 background were derived from the keio collection following kan cassette removal. deletion mutants on the mg1655 background were constructed by allelic transduction from keio collection strains using classical p1 phage transduction, followed by kan cassette excision. the dam null phenotype was confirmed by pcr alone or with electrophoresis of genomic dna digested with dpnii, which cleaves unmethylated gatc sites only. for construction of the dam uti89 and dam upec cipo strains, the parent strain bearing a km208 plasmid - based red - recombinase system was electroporated with a pcr amplicon encoding the dam::kan allele. the genotype of each deletion strain was verified by colony pcr. the plasmid used in the dam complementation studies, namely pzs31 (fig. pzs31 has a psc101 origin of replication (which yields a low copy number of 35 plasmids per cell) and a chloramphenicol - resistance marker. genes encoding either dam (with 500bp upstream flanking region) or gfp were inserted into the multiple cloning site. for complementation experiments using mutated versions of dam, the plasmid containing the dam insert was engineered using either gibson cloning or site - directed mutagenesis (neb, q5 site - directed mutagenesis kit). quinolone resistance - conferring mutations in the cipo upec clinical isolate were identified though whole - genome illumina sequencing of genomic dna (purelink pro-96 genomic purification kit ; life technologies). raw sequencing reads were processed by trimming adapter sequences and discarding reads shorter then 28bp. the genome alignments were searched for known quinolone resistance - conferring mutations in acra, acrr, beas, cpxa, cpxb, envz, gyra, gyrb, mara, marr, mdta, mdtb, mdtc, ompc, ompf, ompr, parc, pare, soxr, soxs and tolc genes and their regulatory regions. for timecourse kill curves and mbc assays, stationary - phase bacterial cultures were diluted at 1:1,000 in 25mls of lb medium in 250ml baffled flasks. cultures were transferred to 24-well plates at 500 l per well, or to 96-well plates at a final volume of 150ul per well, and either left untreated or treated with the indicated drugs at specified doses. plates were sealed using breathable membranes (breatheeasy, cat # : bem-1) and incubated at 37c and 900rpm for the remainder of the experiment. cfus were enumerated at desired time points (4 hours for mbc determination) by spot plating 5 l of ten - fold serially diluted culture onto lb agar and counting colonies after overnight growth at 37c. percent survival at each timepoint was calculated in relation to the cfu immediately before treatment (0h). for mic determination, antibiotics were serially diluted in a 96-well plate and mixed with stationary - phase bacterial cultures diluted at 1:10,000 in a final volume of 150 l lb per well. genomic dna (gdna) was extracted from e. coli k12 mg1655 lb cultures grown in the presence or absence of ampicillin using the genelute bacterial genomic dna extraction kit (sigma). to assess genomic methylation status, gdna extracted from stationary - phase cultures was quantified, digested using dpnii (neb) and run on an 0.8% agarose gel containing ethidium bromide. for methylome analyses, samples were sent to umass medical school deep sequencing core, where methylome data were obtained by pacbio core enterprise instrument smrt. smrtbell dna template libraries for smrt sequencing were prepared according to the instructions described in the procedure & checklist for 10 kb template preparation and sequencing document (pacific biosciences). briefly, genomic dna samples were first sheared to a target shear size of 10 kb using g - tube devices (covaris, inc.), treated with dna damage repair mix, end - repaired and ligated to hairpin adapters. the smrtbell libraries were prepared using the dna template prep kit 2.0 (310 kb) fro pacific biosciences. incompletely formed smrtbell templates were digested using exonuclease iii (new england biolabs) and exonuclease vii (affymetrix). the prepared smrtbell libraries were sequenced using a 120-min movie acquisition time and p4 polyerase - c2 dna sequencing reagent kits following standard instructions for a pacbio rs ii instrument (pacific biosciences). each e. coli sample was sequenced on four or more smrt cells yielding a total of approximately 200-fold double - stranded coverage of the bacterial genome, and two or three biological replicates were sequenced for each antibiotic treatment condition (sup. sequencing coverage was comparable between methylated and non - methylated sites (sup. table 1, sup. data set 2), ruling out coverage loss as an explanation for the absence of methylation. genome - wide detection of base modification and the affected motifs was performed using the standard (default) settings in the the fasta reference genome sequence (e. coli k12 mg1655, ncbi nc_000913.2) used for the base modification detection analyses was obtained from pacific biosciences. for motif identification, the base modification quality value (qv) threshold setting was left at the default value of 30. interpulse duration (ipd) values were measured for all nucleotide positions in the genome and compared with expected durations in an in silico kinetic model of the polymerase for significant associations. frac values were calculated in smrt analysis using a standard mixture model analysis of the pooled kinetic data for a given sample. the frac output value provides information about the fraction of individual molecules displaying a methylation signal at each identified motif site within the genome (sup. methylation frac values were derived from ipd data within the smrt pipeline using the single site mixture model. the values from two or three experimental replicates were compared by student s t - test and fdr adjusted p - values were obtained by the method of benjamini and hochberg (sup. circular graphs were generated using the circos software package e. coli log - phase cultures were transferred to a 96-well plate (200 l / well) and treated with ampicillin (2.5 g / ml) or hydrogen peroxide (100 mm) for 30 minutes to 2 hours at 37c and 900rpm. cell pellets were resuspended vigorously in 200 l of cold 4% paraformaldehyde / pbs and incubated at room temperature for 30 minutes to allow fixation. bacteria were centrifuged again, then resuspended in 200 l of cold permeabilization buffer (0.1%tritonx-100 in 0.1% sodium citrate). after pelleting the cells and discarding the supernatant, cells were resuspended in 50 l of tunel labeling mix (dutp - fitc and tdt enzyme) or 50 l tunel labeling reagent (dutp - fitc) according to manufacturer s instructions (roche ; in situ cell death detection kit, fluorescein). cells were then washed twice with pbs, resuspended in 1 g / ml pi / pbs and analyzed by flow cytometry (bd lsr fortessa). pi negative cells, which lack genomic material, were excluded from the analysis. 3d and statistical analysis, background staining with labeling reagent only was subtracted for each sample to account for treatment dependent shifts in auto - fluorescence or stain retention. statistical analysis performed on log10-transformed data (for survival experiments) or on untransformed data (for tunel assay) using a two - way anova followed by a post - hoc t - test using sidak s multiple comparison test correction. in all cases, laboratory bacterial strains used are derived from e. coli k12 (bw25113 obtained from the coli genetic stock center or mg1655 obtained from atcc). the uropathogenic e. coli strain uti89 was kindly provided to us from matt conover and scott hultgren. the ciprofloxacin - resistant uropathogenic e. coli isolate (upec cipro) was collected from the brigham and women s hospital specimen bank (sup. table 3). deletion mutants on the bw25113 background were derived from the keio collection following kan cassette removal. deletion mutants on the mg1655 background were constructed by allelic transduction from keio collection strains using classical p1 phage transduction, followed by kan cassette excision. the dam null phenotype was confirmed by pcr alone or with electrophoresis of genomic dna digested with dpnii, which cleaves unmethylated gatc sites only. for construction of the dam uti89 and dam upec cipo strains, the parent strain bearing a km208 plasmid - based red - recombinase system was electroporated with a pcr amplicon encoding the dam::kan allele. the genotype of each deletion strain was verified by colony pcr. the plasmid used in the dam complementation studies, namely pzs31 (fig. pzs31 has a psc101 origin of replication (which yields a low copy number of 35 plasmids per cell) and a chloramphenicol - resistance marker. genes encoding either dam (with 500bp upstream flanking region) or gfp were inserted into the multiple cloning site. for complementation experiments using mutated versions of dam, the plasmid containing the dam insert was engineered using either gibson cloning or site - directed mutagenesis (neb, q5 site - directed mutagenesis kit). quinolone resistance - conferring mutations in the cipo upec clinical isolate were identified though whole - genome illumina sequencing of genomic dna (purelink pro-96 genomic purification kit ; life technologies). raw sequencing reads were processed by trimming adapter sequences and discarding reads shorter then 28bp. the genome alignments were searched for known quinolone resistance - conferring mutations in acra, acrr, beas, cpxa, cpxb, envz, gyra, gyrb, mara, marr, mdta, mdtb, mdtc, ompc, ompf, ompr, parc, pare, soxr, soxs and tolc genes and their regulatory regions. for timecourse kill curves and mbc assays, stationary - phase bacterial cultures were diluted at 1:1,000 in 25mls of lb medium in 250ml baffled flasks. cultures were transferred to 24-well plates at 500 l per well, or to 96-well plates at a final volume of 150ul per well, and either left untreated or treated with the indicated drugs at specified doses. plates were sealed using breathable membranes (breatheeasy, cat # : bem-1) and incubated at 37c and 900rpm for the remainder of the experiment. cfus were enumerated at desired time points (4 hours for mbc determination) by spot plating 5 l of ten - fold serially diluted culture onto lb agar and counting colonies after overnight growth at 37c. percent survival at each timepoint was calculated in relation to the cfu immediately before treatment (0h). for mic determination, antibiotics were serially diluted in a 96-well plate and mixed with stationary - phase bacterial cultures diluted at 1:10,000 in a final volume of 150 l lb per well. genomic dna (gdna) was extracted from e. coli k12 mg1655 lb cultures grown in the presence or absence of ampicillin using the genelute bacterial genomic dna extraction kit (sigma). to assess genomic methylation status, gdna extracted from stationary - phase cultures was quantified, digested using dpnii (neb) and run on an 0.8% agarose gel containing ethidium bromide. for methylome analyses, samples were sent to umass medical school deep sequencing core, where methylome data were obtained by pacbio core enterprise instrument smrt. smrtbell dna template libraries for smrt sequencing were prepared according to the instructions described in the procedure & checklist for 10 kb template preparation and sequencing document (pacific biosciences). briefly, genomic dna samples were first sheared to a target shear size of 10 kb using g - tube devices (covaris, inc.), treated with dna damage repair mix, end - repaired and ligated to hairpin adapters. the smrtbell libraries were prepared using the dna template prep kit 2.0 (310 kb) fro pacific biosciences. incompletely formed smrtbell templates were digested using exonuclease iii (new england biolabs) and exonuclease vii (affymetrix). the prepared smrtbell libraries were sequenced using a 120-min movie acquisition time and p4 polyerase - c2 dna sequencing reagent kits following standard instructions for a pacbio rs ii instrument (pacific biosciences). each e. coli sample was sequenced on four or more smrt cells yielding a total of approximately 200-fold double - stranded coverage of the bacterial genome, and two or three biological replicates were sequenced for each antibiotic treatment condition (sup. sequencing coverage was comparable between methylated and non - methylated sites (sup. table 1, sup. data set 2), ruling out coverage loss as an explanation for the absence of methylation. genome - wide detection of base modification and the affected motifs was performed using the standard (default) settings in the rs_modification_and_motif_analysis.1 protocol included in smrt analysis version 2.3.0 patch 5. the fasta reference genome sequence (e. coli k12 mg1655, ncbi nc_000913.2) used for the base modification detection analyses was obtained from pacific biosciences. for motif identification, the base modification quality value (qv) threshold setting was left at the default value of 30. interpulse duration (ipd) values were measured for all nucleotide positions in the genome and compared with expected durations in an in silico kinetic model of the polymerase for significant associations. frac values were calculated in smrt analysis using a standard mixture model analysis of the pooled kinetic data for a given sample. the frac output value provides information about the fraction of individual molecules displaying a methylation signal at each identified motif site within the genome (sup. methylation frac values were derived from ipd data within the smrt pipeline using the single site mixture model. the values from two or three experimental replicates were compared by student s t - test and fdr adjusted p - values were obtained by the method of benjamini and hochberg (sup. e. coli log - phase cultures were transferred to a 96-well plate (200 l / well) and treated with ampicillin (2.5 g / ml) or hydrogen peroxide (100 mm) for 30 minutes to 2 hours at 37c and 900rpm. cell pellets were resuspended vigorously in 200 l of cold 4% paraformaldehyde / pbs and incubated at room temperature for 30 minutes to allow fixation. bacteria were centrifuged again, then resuspended in 200 l of cold permeabilization buffer (0.1%tritonx-100 in 0.1% sodium citrate). after 2 minutes at room temperature after pelleting the cells and discarding the supernatant, cells were resuspended in 50 l of tunel labeling mix (dutp - fitc and tdt enzyme) or 50 l tunel labeling reagent (dutp - fitc) according to manufacturer s instructions (roche ; in situ cell death detection kit, fluorescein). cells were then washed twice with pbs, resuspended in 1 g / ml pi / pbs and analyzed by flow cytometry (bd lsr fortessa). pi negative cells, which lack genomic material, were excluded from the analysis. 3d and statistical analysis, background staining with labeling reagent only was subtracted for each sample to account for treatment dependent shifts in auto - fluorescence or stain retention. statistical analysis performed on log10-transformed data (for survival experiments) or on untransformed data (for tunel assay) using a two - way anova followed by a post - hoc t - test using sidak s multiple comparison test correction. in all cases, | antibiotic resistance is an increasingly serious public health threat1. understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets28. we explore the role of the bacterial epigenome in antibiotic stress survival using classical genetic tools and single - molecule real - time sequencing to characterize genomic methylation kinetics. we find that escherichia coli survival under antibiotic pressure is severely compromised without adenine methylation at gatc sites. while the adenine methylome remains stable during drug stress, without gatc methylation, methyl - dependent mismatch repair (mmr) is deleterious, and fueled by the drug - induced error - prone polymerase poliv, overwhelms cells with toxic dna breaks. in multiple e. coli strains, including pathogenic and drug - resistant clinical isolates, dna adenine methyltransferase deficiency potentiates antibiotics from the -lactam and quinolone classes. this work indicates that the gatc methylome provides structural support for bacterial survival during antibiotics stress and suggests targeting bacterial dna methylation as a viable approach to enhancing antibiotic activity. |
the transmission risk of foot - and - mouth disease (fmd) in japan was evaluated using a mathematical fmd transmission model. the distance - based transmission rate between farms, which was parameterized using the fmd epidemic data in 2010 in japan, was used to calculate the local - level reproduction numbers expected numbers of secondary infections caused by one infected farm for all cattle and pig farms in the country, which were then visualized as a risk map. the risk map demonstrated the spatial heterogeneity of transmission risk in the country and identified risk areas with higher possibility of disease spread. this result suggests that, particularly in high - risk areas, it is important to prepare for the smooth and efficient implementation of control measures against fmd outbreaks. |
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the population of old people is rising exponentially in thailand. according to the foundation of thai gerontology research and development institute, tgri (2013) it is projected that the aged population in thailand will significantly rise to approximately 22 million by 2040. in 25 years, 33.5% of the entire thailand population will be old people. people in this age group usually have the highest rate of disability and are often the most dependent. in spite of the efforts made by the thai government to enhance the quality of healthcare services for the older population, the prevalence of disparate access to services, inadequate quantity of healthcare resources (such as equipment, personnel, and finance), and lack of interinstitutional and intrainstitutional coordination among healthcare institutions still persists. in a bid to counter these problems, the thai government commissioned a working group to make changes to the country 's national plan on older persons in 2009. the working group drew inspiration from the findings of an evaluation of this plan that had been previously conducted and recommended for the establishment of community - based long - term care institutions for the elderly. these institutions would offer medical and social care for the elderly in the comfort of their homes. in the past, adl scale was basically constructed for individuals suffering from stroke and has two items pertaining to climbing stairs and continence. a combination scale that assesses adl, social involvement, and communication an earlier step in disability intensity may be recognized as a limitation in instrumental activities of daily living (iadl). such activities essentially involve cognitive functions and other basic actions such as shopping, telephoning, shifting from one place to another, and dealing with medicines and funds. these days adl and iadl are often used for measuring health in both clinical studies and community - based surveys of older group [2, 3 ]. the calculations regarding iadls are asymmetrical across diverse cultures, even if the evaluations of adls have been normally regular among various countries and in varying time frame. all of those activities that are auxiliary in everyday life are different for every country as seen in the national surveys, due to cultural, geographical, and temporal deviations [48 ]. so, the barthel index has been modified for assessment of activity in daily living in thai older people. this adl instrument comprises eating, grooming, transferring in house, toileting, walking, climbing stairs, dressing, bathing, control of urination, and control of defecation. in addition, chula adl index (cai) has been modified for assessment of instrumental activity in daily living, which includes walking outdoors, cleaning house, public transportation, cooking, and money exchange. a study conducted by village health communicators in 1989 in a rural northern thai community found that the overall prevalence rate of the disabled aged population was 6.3/1000. a study conducted in 1997 reported that 19% of participants aged 60 years and over have a long - term disability. later, the study in 2001 found that the prevalence rates of disability in females were higher than that of males among the elderly living in central region. with respect to international comparison, the occurrence of the adl disability in a population aged 60 years and above was as much as 25.7% in lebanon. however, more recent studies carried out after 2008 showed that the adl disability is not as widely prevalent, with a population comprising 65-year - old and older population in china showing a prevalence of around 14.9%, whereas in the united states it was about 18.4%. much effort has been invested in identifying the risk factors associated with how disability sets in, and the corresponding model proposed by nagi (1976) is a major source of input in this regard, constituting components related to pathology, functional impairments, functional limitations, and disability. beyond any doubt factors like age and gender are linked with the functional limitation and complexity with adls and iadls. people who are over 70 years of age are more prone to these problems in contrast to the individuals in their 60s. moreover, it was clearly expressed that females tend to show more difficulties than men when they are asked to perform a similar task [1820 ]. area of residence does not play a predominant role in alteration of function but it is generally believed that older people or rural areas show less functional restrictions than the residents of urban areas. therefore, it is demonstrated in the literature review that there are several elderly people suffering from functional disability, with an annual increase in this dependency rate. down the line, disabled individuals could be expected to require a certain amount of ltc services in formal or informal settings, within an institution, in the community or at home. it is important to understand that the elderly have lots of different needs which are not adequately met, thereby forcing many of them to go about their daily lives with these additional challenges. likewise, it is expected that the risks involving disability and long - term illnesses on the part of the elderly will see a steady and sharp rise because of the increase in people 's longevity. nonetheless, no current studies have been conducted to look into the disability risk factors throughout the country beginning from the year 2002 to 2011. in view of this, the study sought to determine the disability risk factors of six disabilities (eating, dressing, squatting, lifting a 5 kg object, ease of stair use, and being able to travel by bus) among thai aged population in 2002, 2007, and 2011. for each of the disability items the question was phrased as can you perform these activities by yourself ? with response categories no, not all, in so doing, the disability items were transformed prior to merging three datasets ; then we defined disability as older people who could not do the task or older people who could do it only with help. in addition, we excluded the urination and defecation continence from our analysis, since it was considered as physical health in the survey. the national statistical office (nso) had conducted four cross - sectional surveys in 1994, 2002, 2007, and 2011. unfortunately, the nso does not allow using 1994 dataset, so there are three datasets. the prime goal of this survey is to form a database of demographic, socioeconomic, and health characteristics and living arrangements of people aged 50 years and over in thailand can be represented nationally. to obtain this goal, a stratified multistage sampling technique was done by the national statistical office in all 76 provinces of thailand which were further classified based on administered classification into urban and rural areas. the blocks for municipal areas and villages for nonmunicipal areas were the main sampling units. the probability of selection depends on the number of households existing in a block or village. a total of 5,796 blocks / villages were chosen in 2002 and 2011, while 5,793 blocks / villages were chosen in 2007. there were 15 households including a person aged 50 years or older chosen systematically from each chosen block and for the nonmunicipal area ; 12 households were chosen systematically from each village. in 2002 and 2011, 79,560 households were chosen for final sample and, in 2007, 79,542 households were chosen for final sample. in this study we selected only people aged 60 and above so the interviews were conducted with 24,835, 30,427, and 34,173 people in the 2002, 2007, and 2011 surveys of elderly thai people, respectively. it was covered by the survey of the elderly in thailand with a variety of demographic, socioeconomic, and health characteristics and management of people. disability indicator in old age was the capability of doing activities on daily basis which is associated with the need for personal assistance. since the surveys have almost the identical questions on doing activities, then the availability of three cross - sectional datasets potentially presents the capability to quantify the differences of disability between three points of time while caution will require to be exercised. this part deals with detecting and removing errors and inconsistencies from data in order to improve the quality of data. also, missing data were coded as system - missing in spss. this part was employed in order to determine whether the formatting of the variables needs to be changed. consistency in setting up response category values such as yes and no is one way to make the dataset more user friendly. the original response category values of variables of each study are shown in table 1. the variables were recoded to make the data fit with the purpose of the study. to do so we identified variables that were present in three datasets and recoded them. the identifier variable assures that the values of the same individual are on the same line. at this step, the dependent variables were recoded to dummy variable (see the new variables list in table 2). persons who could do the task without help were considered not to have the disability. persons who could not do the task or could do it only with help were considered to have the disability. the purpose is to have a longitudinal file which allows the understanding of changes, so we combined files from different years in order to run a regression utilizing year indicators. in the 2002 survey, eight items related to self - care were questioned (feeding, dressing, bathing plus toileting, squatting, lifting 5 kg objects, walking 1 kilometer, stair climbing : 2 - 3 steps, and transferring by bus or boat alone). in 2007 survey, nine items related to self - ability were questioned including eating, dressing, bathing / toileting / toothbrushing, squatting, lifting 5 kg object, walking 200300 meters, stair climbing : 2 - 3 steps, getting out by bus or boat alone, and money management. in 2011 survey, fourteen items were queried (feeding, putting on clothes, taking a bath, washing face / brushing teeth, toileting / cleaning oneself after urinating / defecating, shaving / combing, putting on shoes, squatting, lifting 5 kg object, walking 200300 meters, stair climbing : 2 - 3 steps, taking a bus or boat alone, money management, and taking medicine correctly). since three surveys have six identical questions on doing activities, there are six activities in this study that determine disability status : eating, dressing, squatting, lifting a 5 kg object, ease of stair use, and being able to travel by bus or boat. survey participants in all three years (2002, 2007, and 2011) specified for each task whether theycould not do the task at all;could do the task with help;could do the task without help.before merging the data, we recoded these 3 responses into a dummy variable. persons who could do the task without help were considered not to have the disability. persons who could not do the task or could do it only with help were considered to have the disability. could not do the task at all ; could do the task with help ; could do the task without help. the following sixteen independent variables were transformed to dummy variables and considered in multiple logistic regression analysis as follows : (1) age in years, (2) gender, (3) education, (4) income, (5) married but not living as a couple (unmarried is reference), (6) married and living as a couple (unmarried is reference), (7) working status in the past 7 days, (8) any of five chronic diseases, (9) area (0 = urban ; 1 = rural), (10) year 2007 (year 2002 is reference), (11) year 2011 (year 2002 is reference), (12) living in bangkok, (13) the central region (excluding bangkok), (14) the northeast region, (15) the north region (the south region is reference for all regions), and (16) living arrangements were self - reported during an interview according to living alone = 0 and living with others = 1. logistic regression analysis was used to assess the extent to which the selected independent variables explain the disability of the elderly in thailand. confidence intervals (cis) were calculated at the 95% level to estimate statistical significance. in addition to odds ratios, the modeled absolute prevalence of each disability was calculated with and without each independent variable, at the mean values of all other independent variables. for each independent variable, the resulting difference in modeled prevalence this procedure gave a measure of the impact of each independent variable on each disability (e.g., the modeled prevalence of difficulty lifting 5 kg, in persons who had and had not worked during the past 7 days, and at the mean levels of all other independent variables was 32.8% and 15.6%, respectively, for a modeled prevalence difference of 17.2%. this difference constituted a proportion of 0.560 of the weighted lifting disability prevalence of 30.7% (0.172/0.307 = 0.560)). then, for each independent variable, these proportions were averaged over all 6 disabilities. to enable comparison of the relative impacts of the independent variables, proportions for the independent variables were arranged in descending order of this average proportion. (this approach is somewhat similar to calculating and comparing modeled population - attributable risk fractions for the independent variables, except that it does not take their prevalence into account. thus this approach may be viewed as an assessment of absolute impact of the risk factors, regardless of their frequency in the study population.) also, this approach adds the benefit of considering absolute prevalence differences associated with risk factors and not merely odds ratios, which differ substantially at different baseline prevalence rates of studied outcomes and which can therefore be misleading. as indicated above, the analyses reported here employed secondary data, which were originally collected by thailand 's national statistical office (nso) in its nationwide surveys of 2002, 2007, and 2011. the nso obtained written consent from all participants in these surveys. completed consent forms are on file at the nso additionally, the analyses reported here were approved by the ethics review committee for research involving human research subjects, health sciences group, chulalongkorn university, certificate of approval number 031/2015. the total numbers of the elderly with the people aged 60 and above were about 5,969,030 people in 2002, 7,020,959 people in 2007, and 8,266,304 people in 2011. the mean age of all participants was 68.6, 69.0, and 69.2 years in the 2002, 2007, and 2011 surveys, respectively. more than 90% of the elderly in the samples lived with others, decreasing slightly but continually from 93.7 in 2002 to 92.3 in 2007 and to 91.4 in 2011. the percentage of elderly people who had at least one chronic disease increased from 29.6 in 2002 to 40.3 in 2007 and to 43.8 in 2011 (table 3). the number of elderly people who had not attended school decreased from 20.6 in 2002 to 16.4 in 2007 and to 11.8 in 2011. approximately 30% lived in the northeast region, which had the highest proportion of elderly people of all regions in thailand. figure 1 and table 3 show the prevalence of the six limiting activities by years. it was found that the prevalence of difficulty in lifting 5 kg decreased from 37.4% in 2002 to 27.0% in 2007 and increased slightly to 29.2% in 2011. prevalence of difficulty in transportation declined somewhat, from 30.5% in 2002 to 25.8% and 24.0% in 2007 and 2011, respectively. the percentage of the elderly in 2002 that reported difficulty to step up 2 - 3 stairs was 10.1% which was increased to 13.6% in 2007 and it witnessed a bit decrease to 11.9% in 2011. for difficulty in squatting, the prevalence was 6.5%, 12.4%, and 12.7% in 2002, 2007, and 2011, respectively. the prevalence of the elderly who reported being difficulty in dressing was 2.1% in 2002 which increased to 3.0% in 2007 and then slightly decreased to 2.7% in 2011. the prevalence of elderly persons who had eating limitation was 1.2% in 2002, 2.3% in 2007, and 2.2% in 2011. overall, prevalence of lifting - related and travel - related disabilities was high, prevalence of squatting- and stair climbing - related disabilities was intermediate, and prevalence of dressing- and eating - related disabilities was low. over all three surveys, the disability with highest prevalence activity that was most difficult for the sample population was lifting 5 kg (30.7), followed by traveling alone by bus or boat (26.4). twelve percent of elderly people had difficulty climbing 2 - 3 stairs, a proportion slightly higher than that for squatting (10.9). table 4 demonstrated the associations of subject characteristics with disability risk. as expected, age indicated the highest risk for difficulty with dressing (or = 1.09, 95% ci : 1.091.10), eating (or = 1.09, 95% ci : 1.081.10), using stairs (or = 1.10, 95% ci : 1.091.10), traveling alone (or = 1.12, 95% ci : 1.121.13), lifting 5 kg (or = 1.10, 95% ci : 1.091.10), squatting (or = 1.08, 95% ci : 1.081.09), and any disability (or = 1.11, 95% ci : 1.101.11) at p < 0.001. for education level, no education indicated highest risk for difficulty with dressing (or = 1.34, 95% ci : 1.201.49), eating (or = 1.59, 95% ci : 1.411.79), using stairs (or = 1.21, 95% ci : 1.141.28), traveling alone (or = 1.42, 95% ci : 1.361.49), lifting 5 kg (or = 1.27, 95% ci : 1.211.33), squatting (or = 1.13, 95% ci : 1.061.20), and any disability (or = 1.39, 95% ci : 1.321.45) at p < 0.001. income inadequacy showed highest risk for difficulty with dressing (or = 1.14, 95% ci : 1.291.54), eating (or = 1.29, 95% ci : 1.161.43), using stairs (or = 1.35, 95% ci : 1.291.41), traveling alone (or = 1.28, 95% ci : 1.23 - 1.33), lifting 5 kg (or = 1.09, 95% ci : 1.051.13), squatting (or = 1.26, 95% ci : 1.201.32), and any disability (or = 1.20, 95% ci : 1.161.40) at p < 0.001. living with others indicated highest risk for difficulty with dressing (or = 2.79, 95% ci : 2.213.52), eating (or = 1.96, 95% ci : 1.542.51), using stairs (or = 1.38, 95% ci : 1.271.51), traveling alone (or = 1.18, 95% ci : 1.101.26), lifting 5 kg (or = 1.10, 95% ci : 1.031.17), squatting (or = 1.45, 95% ci : 1.321.59), and any disability (or = 1.12, 95% ci : 1.051.19) at p < 0.001. having at least one chronic disease (heart problems, diabetes, hypertension, cancer, or stroke) had a highly significant association with dressing (or = 3.41, 95% ci : 3.103.76), eating (or = 3.19, 95% ci : 2.853.56), using stairs (or = 2.16, 95% ci : 2.062.26), traveling alone (or = 1.85, 95% ci : 1.781.92), lifting 5 kg (or = 1.70, 95% ci : 1.641.76), squatting (or = 2.07, 95% ci : 1.972.17), and any disability (or = 1.77, 95% ci : 1.711.83) at p < 0.001. being female had a negative association with difficulty in eating and dressing and a positive association with squatting, lifting 5 kg, using stairs, traveling by bus, and any disability. in terms of marital status, those who were married and lived with a spouse showed better ability to perform daily activities than their counterparts who were not married (divorced, widowed, and separated). those who lived in a rural residence had a higher likelihood of having difficulty performing with any disability and traveling alone by bus / boat than those in an urban residence, but they were less likely to have difficulty eating, dressing, squatting, lifting 5 kg, and using stairs. interestingly, being unemployed seven days prior to interview indicated the highest risk for difficulty with dressing (or = 6.03, 95% ci : 4.887.45), eating (or = 5.15, 95% ci : 4.106.47), using stairs (or = 4.04, 95% ci : 3.734.37), traveling alone by bus (or = 3.28, 95% ci : 3.123.44), lifting 5 kg (or = 2.64, 95% ci : 2.532.76), having any disability (or = 2.64, 95% ci : 2.542.75), and squatting (or = 2.61, 95% ci : 2.442.80) at p < 0.001. proportional impacts of independent variables on disability prevalence are presented in figure 2 and table 5. on average, the characteristics with the greatest adverse impact on disability prevalence were not working in the past 7 days (average impact : 61.2%), age per 10 years (53.7%), and presence of one or more chronic diseases (46.3%). living with others, lack of education, and lower income were associated with moderate positive proportional impacts on disability prevalence (average impacts : 16.4%, 15.1%, and 12.6%, resp.). female gender was associated with an overall positive impact but exhibited considerable variability across disabilities (average 12.5%, range 37.6% to 12.2%). living in a rural area was generally associated with lower disability risk than living in an urban area. being married was consistently associated with lower risk than being unmarried, especially for married participants who lived together as couples. the levels of difficulty experienced by those surveyed decreased between the 2002 and 2007 surveys. the sole category in which the level of difficulty decreased between 2002 and 2007 and increased between 2007 and 2011 was the ability to travel alone by bus or boat. in one to two disabilities, the level of difficulty was reported to decline in 2007 and increase mildly in 2011. in more than three disabilities, the opposite was the case, with the level of difficulty rising in 2007 and dropping in 2011. across all six activities, as it is not necessarily the case that all surveys were completed by the concerned parties themselves, there may be slight inconsistencies found with regard to the levels of difficulty observed in the results. the reasons for these trends are complex and include shifts in socioeconomic status of the elder population, in the distribution of underlying conditions and limitations in capacity that may be related to use of medical treatments, and in the uptake of assistive and other convenience technologies. this study has shown that the factors most associated with disability are old age, being female, lack of education, low income, cohabitation, unemployment seven days prior to interview, having at least one chronic disease (hypertension, heart problems, diabetes, strokes, or cancer), and living in a rural setting. it has already been demonstrated in past studies that categories such as age, gender, and socioeconomic status may be linked to physical disability in the elderly [23, 24 ]. this study connects the factors of age, income, and marital status to the issue. it is not easy to compare the different studies because a variety of the adl measurements and age groups have been used. in malaysia, the uniqueness of the studies lies in the fact that the sample used in malaysia was younger (mean age : 69.0) than that of the u.s. and chinese population samples (74.5 and 75.1). (2007) show that prevalence of adl and iadl limitations varies across asian countries and trend was increasing. the prevalence of adl disability in singapore was 3.9% (1999), in beijing it was 4.7% (1997), in indonesia it was 6.5% (1997), in taiwan it was 9.2%, and in philippines it was 14.7% (2000). iadl restrictions in singapore were 17.2% (1999), in beijing they were 17.7% (1997), and they were 25.1% in taiwan and 27.7% in philippines (2000). at present, there is a higher burden of dependency in sub - saharan african region, which has the dependency region of almost 20%. it is expected that there will be a high increase in this ratio in sub - saharan africa, latin america, and asia. it was estimated in this study that there will be a higher increase in the dependency ratio in china (16%) and india (14%) in the next few years, along with the population increase. the study ended on the note that several countries are going to face the burden of rising number of dependent people and a lot more significance needs to be awarded to disability prevention. the physical and mental health issues of the elderly population living in a rural region of sepand in malaysia were examined in a cross - sectional study. prestructured questionnaires were used for interviewing the elderly in the villages, including geriatric depression scale, barthel 's index, and elderly cognitive assessment questionnaire. the existence of physical health problems like chronic illness and functional dependence was found to be 60.1% and 17.5%. it was further found that the existence of physical reliance in at least one adl was 17.5% and it was 17.1% for one or more adls. basic adl disability and functional restrictive rates among elderly population in america were examined in a study spanning from 2000 to 2005 so as to ascertain if there was an increase or decrease in the percentages of basic adl disabilities and functional restrictions among the community residents and the institutionalized elderly. the yearly percentages of prevalence of basic adl disabilities and functional restrictions were computed using data from american community survey and national nursing home survey. this data was then used in regression lines to evaluate the changing trends with time. it was observed that there was a 9% increase in the percentages of basic activities of daily living (badl) disabilities among the elderly in the community aged 65 and over. the institutionalized elderly adl disability rates were found to be consistent among males but showed an increase for females in the years 2000 to 2005. it was henceforth deduced in this study that this growth in adl disability with time among the elderly population greatly affects the healthcare mechanism. even if there is a huge range of disparity in the way surveys have questions regarding adl functioning, there is some agreement among all of these surveys related to what activities of everyday life should be incorporated. according to guralnik., the calculations might differ merely due to the reason that the participants have a different understanding of the question seeing as the self - report instruments do not include distinct meaning for the calculated activity or plausible answer categories. as stated by m. w. linn and b. s. linn, the criteria of evaluation of disability by individuals can be affected by language, education, and culture. the factors like time period of disability, the kind of help that was given, and what range of complexity they experience while performing every adl vary in surveys. result of our study can not be straight compared with these results but we can generalize that any disability is elevated along with the thai elderly. in terms of gender, adl disability variations that indicate disability prevalence are more common among females compared to males [25, 32 ]. a study undertaken in lebanon revealed that higher disability existed in elderly women (31.3%) than among elderly men (18.7%). another study in spain on people aged 65 indicated that the adl disability prevalence among females was 15.8% and for males it was 10.6%. such differences may be attributed partially to a higher mortality rate among males and a higher adl disability prevalence in females. few studies have examined differences in health and adl disability based on residence (urban versus rural). the studies have indicated that greater adl disability prevalence and poor health are found among aged rural dwellers [34, 35 ]. another study in australia and canada established that 18-year - old adults from rural areas had poor health. it was discovered that a correlation existed between the health of over 18-year - old adults and living in rural areas, particularly with regard to poor health. additionally, adults living in rural areas exhibited an unhealthy way of life, for instance, smoking, overweight, unhealthy eating habits, and less physical activity. with regard to disability, income, rather than education, appeared related in the current study. some past studies corroborate the finding [24, 38 ], whereas others state that income has no role in the elderly disability. interestingly, this study found that aged individuals living with others had a higher risk of disability, an outcome that is consistent with tsai. it is possible that individuals living alone must complete chores on their own whereas those who live with others have assistance as aged couples assist each other when functional ability wanes. the older people might invite another person to come and stay with them when they become disabled. further research should be undertaken to explain this finding, even though many studies indicate that low economic status and loneliness increase the risk of disability among the older people. the reason for the strong association between the risk of disability and the status of not working in seven days before the interview is not clear. the high probability may be due to the difficulty of defining unemployment, as it may include people who are not working because of a disability, those who are looking for work or are on temporary sick leave, the retired, home makers, and people whose spouse and/or children do not allow them to work. the main strength point of the study is to provide description of aged individuals and to create a framework of forming practices that offer a better insight on thai ageing. different disability limitations and risks in thailand in 2002, 2007, and 2011 are examined thoroughly in the study and the findings can be applied to the broader thai population. thus, the possibility of inaccurate data could not be ruled out, particularly if a family member or friend participated in the reporting. this study did not consider health behaviour variables, such as drinking clean water, smoking, and exercise, because these behaviours were not queried consistently in the 3 survey years. however, evidence from literature reveals that exercise and physical activities positively affect adl and physical functioning among the aged. this may reduce the risk of facing an adl disability. a study in the u.s. found a relationship between lower incidents of disability and higher rates of physical activity. the current study omitted self - rated health as an independent variable because many individuals can rate their health basing on their past health. finally, since the study was cross - sectional with regard to design, it is possible that determining the causality was not achieved. because chronic illnesses relate strongly to disability, health prevention mechanisms of reducing later life disability should be promoted. lack of work had a strong link with a disability, though the causality factor can not be dismissed because an increase in working opportunities can reduce disability among aged individuals. effects of risk factors over time are often influenced by effects of age, period (time of data collection), and/or birth cohort. the modeled effects of putative risk factors presented in this report were adjusted for age and period (year of data collection). strictly speaking, it is not possible to model age, period, and cohort simultaneously, because these three characteristics are a linear combination of each other (cohort + age = period). to address this issue, we chose to construct separate models (not shown), adjusting for age and birth cohort. in these, modeled effects of factors other than age, period, and cohort were similar to those in the models reported here. thus, the effects analyzed were robust with different model specifications. this observation increases confidence that the findings reported here are valid. although overall prevalence has dropped, the absolute number of disabled people continues to increase in thailand. thus, the healthcare system and individuals themselves are faced with an increased trouble of disability and new challenges. since older people who did not work had higher risk for disability in our study, so extending the default retirement age of 60 in thailand and increasing the work opportunity in older people may reduce the risk of disability although the causality is not clear. in view of the fact that people living alone had a lower risk for disability in our study, so we suppose that older people living alone may be at a lower risk for disability in the future compared to those living with others | background. there is an important need to characterize risk factors for disability in thailand, in order to inform effective prevention and control strategies. this study investigated factors associated with risk of 6 types of disability in thailand 's ageing population in 2002, 2007, and 2011. methods. data came from the cross - sectional national surveys of older persons in thailand conducted by the national statistical office (nso) in 2002, 2007, and 2011. stratified two - stage sampling was employed. interviews of 24,835, 30,427, and 34,173 elderly people aged 60 and above were conducted in the respective study years. prevalence of disabilities was measured, and factors associated with disability risk were assessed with probability - weighted multiple logistic regression. results. disability prevalence decreased slightly over the study period. the characteristics with greatest positive impact on disability prevalence were not working over the past week (average impact : 61.2%), age (53.7% per decade), and suffering from one or more chronic illnesses (46.3%). conclusions. the strong observed positive impact of not working on disability prevalence suggests that raising the mandatory retirement age might result in some reduction of disability risk. also, the observed positive impact of living with others (versus alone) on disability risk was somewhat unexpected. |
the most common hospital - acquired infection is catheter - associated urinary tract infection (cauti) accounting for almost 40% of all the nosocomial infections. the single most important predisposing factor for cauti is the insertion of urinary catheter. urinary (foley) catheters are used very frequently in hospitalized patients, and almost 25% of them undergo urinary catheterization during their stay in the hospital. the frequency of urinary catheterization in intensive care unit (icu) can range as high as 100%. catheter - associated urinary tract infection produces substantial morbidity in hospitalized patients including discomfort, fever, malaise and unnecessary antibiotic use, which may become an important source of antibiotic resistant organisms. further, the catheterized urinary tract acts as a reservoir for the dissemination of these drug resistant organisms to other patients. there are increased chances of catheter blockage, urinary tract stones and even increased risk of malignancy of the urinary tract following cauti. if it gets complicated by bacteremia, it increases the cost of care tremendously besides increasing the mortality in these hospitalized patients. most doctors and nurses are unfamiliar with the indication of catheterization and further there is no defined management plan to monitor the presence of unnecessary catheter. since the number of catheter days in icu setting is more, the frequency of cauti is expected to be higher. but many studies have shown similar rates of cauti in the icu and non icu patients. this shows that all hospitalized patients including non icu patients are an important group for surveillance and prevention of cauti. many studies have shown that single most important modifiable risk factor for decreasing the incidence of cauti is reducing unnecessary catheter use. preventive measures such as catheter reminders for removing the catheter as soon as possible restrict the number of catheter days and thus lead to decreased incidence of cauti. training the health care personnel and introducing the prevention of cauti as a high priority in hospitals is strongly associated with decreased incidence of cauti. there are limited studies from india, which have assessed the knowledge of health care personnel regarding indication for catheterization and methods to prevent cauti. the present study was planned to assess the knowledge of various health care personnel regarding indication for catheterization and measures to prevent cauti. the study also assessed the attitude of health care provider (hcp) regarding cauti. the questions regarding the indication for catheterization and measures for prevention of cauti were taken as per centers for disease control (cdc) guidelines. the content of the questionnaire was given independently to all the authors for assessing the simplicity of questions, clarity of language, accuracy and adequacy of questions for the purpose of the study. the survey covered demographic details of the respondents including age, sex, qualification, designation years of experience in health care setup after obtaining graduation and area of posting. the contents of the questionnaire were designed to assess the knowledge of the health care professionals regarding indication for catheter use (10 questions) and the methods of preventing cauti (15 questions) in a catheterized patient. knowledge regarding indication for catheter insertion was assessed by asking the respondents to grade their answers on a 4 point scale (4 : almost always indicated, 3 : sometimes indicated, 2 : rarely indicated, 1 : never indicated) they were given 10 situations in which they had to grade their answers [table 1 ]. demographic details of the study group (n=154 ; 49 : doctors ; 105 : nurses) knowledge regarding the methods to prevent cauti included 15 methods [table 2 ]. the respondents had to grade their answers on a 4 point scale (4 : large effect, 3 : moderate effect, 2 : some effect, 1 : no effect). the answers for both the indication and prevention of cauti were compared with the cdc guidelines. for evaluating the indication for catheterization, the response almost, always and sometimes indicated, was taken as appropriate for the indication and the response of rarely and not indicated was taken as appropriate for nonindication. for evaluating the preventive measures, the response of large effect and moderate effect were taken as effective, and that of some effect or no effect were taken as noneffective. frequency of respondents having identified the indications correctly there were 10 questions to assess the attitude and the answers had to be given as whether they agree or disagree with the statements. this was not compared with any reference, and the format of the questionnaire was designed to be used for this study. the data were compiled and analyzed using spss for windows version 12.0. various statistical test used were simple frequency table, chi - square test, fischer 's exact t - test for significance and univariate analysis for testing the correlation. the data were compiled and analyzed using spss for windows version 12.0. various statistical test used were simple frequency table, chi - square test, fischer 's exact t - test for significance and univariate analysis for testing the correlation. the data were compiled and analyzed using spss for windows version 12.0. various statistical test used were simple frequency table, chi - square test, fischer 's exact t - test for significance and univariate analysis for testing the correlation. in the present study, 154 health care personnel (doctors = 49 and nurses = 105) out of the 180 participants completed a questionnaire and were included in the study. the mean age of the study group was 31.9 years (range : 2359) and the mean years of experience was 7.94 years (range : 136). though doctors had significantly higher knowledge as compared to nurses regarding various indications for catheterization, more than half of the doctors could not identify important indications such as urethral stricture or in patients receiving large volume infusions during surgery [table 2 ]. more than one - third of the respondents identified urine output monitoring in a mobile patient or even obtaining a urine sample for culture sensitivity as a valid indication for catheterization [table 2 ]. most respondents could not correctly identify noneffective measures such as bladder irrigation or twice daily meatal care. in general, the hcp could not differentiate the effective measures from noneffective ones [table 3 ]. number of doctors and nurses correctly identifying methods to prevent cauti doctors had significantly better knowledge than nurses regarding the indications of catheterization and preventive measures for cauti [table 4 ]. hcp posted in high - risk areas such as icus had significantly better knowledge than the hcp posted in wards regarding various indications, but there was no difference in their awareness in respect to preventive measures [table 4 ]. univariate predictors of indications of catheterization most of the hcp felt that including prevention of cauti as a high priority in the hospitals and use of renewal reminders would definitely help in preventing cauti. more than 90% of the hcp also felt that education regarding basic catheter care would also help prevent cauti [table 5 ]. almost 40% of the nurses and 25% of the doctors felt that catheters could be inserted even for nursing convenience. nearly, 10% of the respondents showed hopelessness regarding prevention of cauti [table 5 ]. urinary catheters once inserted in the hospitalized patients tend to remain in place even after the indication for its use ends. this leads to cauti being one of the most common health care associated infection. the most important preventive measure for decreasing the incidence of cauti is limiting catheter use. this is possible only if the hcp has adequate knowledge regarding the appropriate indications for inserting urinary catheters. we assessed the knowledge of doctors and nurses regarding indication for catheterization to help manage the problem of inappropriate catheterization and cauti. overall the knowledge of doctors was significantly better than nurses in identifying the indications for catheterization (p < 0.05). since in our facility doctors decide on the catheterization protocol, they would be more aware regarding the indications. educating nurses regarding indications for catheterization will help prevent cauti as they could remind the doctors on removing catheters as soon as the indication for catheterization ends. this finding is consistent with other observers in that nurse 's daily determination for the need of catheter in a patient helps reduce the incidence of cauti. almost 79% of the nurses and 12% of the doctors justified the use of catheters for nursing care of incontinent patients. almost one - third of the respondents justified the use of catheters for routinely obtaining urine samples for culture and sensitivity testing and for urine output monitoring even in mobile patients. it would be worthwhile educating both doctors and nurses regarding the various indications for catheterization as this can keep a check on unnecessary catheter use. knowledge regarding various preventive measures was also suboptimal in the study group. the health care personnel could not identify the preventive measures, which are less effective or noneffective. being over cautious is better than being ignorant but this lack of knowledge regarding preventive measures may lead to wastage of time on unimportant issues at the cost of truly effective measures. a complete knowledge regarding all effective preventive measures would help them prioritize the care of the urinary catheters. almost 77% (119/154) of the hcp felt that regular bacteriological monitoring is effective for prevention of cauti. this causes increased costs of treatment for the patients in addition to increasing sample load in the laboratories. almost 45% (68/154) of the hcp felt that prophylactic antimicrobials for 3 days can prevent cauti. this is ineffective besides exposing the patients to unnecessary antibiotics and selecting out on antibiotic resistant organisms. in spite of the fact that nurses are involved with routine catheter care in our setup, more than one - third of the nurses did not know that the urinary collection bag should be below the level of the bladder and it should be emptied regularly to allow unobstructed urine flow. these seemingly simple measures can have a major effect in the prevention of cauti. almost 25% (38/154) of the hcp felt that isolation of the patient helps in preventing cauti. if they follow this practice, it will lead to emotional, social stress in the patients. thus, appropriate knowledge regarding all preventive measure would save on cost and time and would help in more efficient patient care. the knowledge regarding the correct preventive measures can be imparted through educational training and reinforcement thereafter. in our study, females were more aware regarding the indications and preventive measures. we also observed that experience had no effect on awareness levels, and knowledge in one domain did not affect the awareness levels in another domain. the study again reiterates the finding that educating the hcp regarding indications and preventive measures will help them do evidence - based practice. more than 95% of the hcp felt that hospitals focusing on prevention of cauti as a high priority and use of urinary catheter reminders would help in preventing cauti. despite this, most of the hospitals in india have not implemented any defined strategy for prevention of cauti. the finding is in agreement with other observers and even in many developed nations many hospitals do not have any protocol to monitor the number of catheterization and duration of catheterization. most of the hcp also felt the need for education regarding basic catheter care for prevention of cauti. thus, despite the need being felt for these measures, the implementation was lacking at various levels including organizational levels. this hopelessness can be improved by educating them and showing them the definite evidences of prevention of cauti following appropriate preventive measures. multifaceted interventions in the form of routine educational trainings to hcp regarding cauti and its prevention, implementation of catheter reminder system to discontinue unnecessary catheters, changes in the hospital policies would definitely help in reducing unnecessary catheter use, decrease the number of catheter days, and thus reduce the incidence of cauti. surveillance of the incidence of cauti on a regular basis in all the hospitals would be desirable to initiate preventive measures as early as required. catheter - associated urinary tract infection is one of the most common health care associated infections. it is largely preventable if indications for catheterization, methods for catheter care and other preventive measures are followed diligently. in the present study, it was seen that though doctors had significantly better awareness than nursing staff regarding reasons for urinary catheter indication and preventive measures, yet it was suboptimal and had a lot of scope of improvement. further, it was observed that cauti was not thought of as a serious problem. all the health care personnel including the doctors should have regular training regarding prevention of cauti, and all efforts should be made by hospital authorities to include prevention of cauti in its high priority list. surveillance of the incidence of cauti would also be helpful in recording the improvement observed as a result of these education based and administrative interventions. | background and aims : catheter - associated urinary tract infection (cauti) is one of the most common health care acquired infection encountered in clinical practice. the present study was planned to assess the knowledge and attitude of health care providers regarding the indications for catheterization and methods of preventing cauti.methods:a prospective questionnaire - based survey was done from march 2011 to august 2011. a structured questionnaire comprising of 41 items related to demographic details of the respondents, their knowledge regarding indications for catheterization and methods of preventing cauti was given to 54 doctors and 105 nurses. the response was evaluated for statistical correlation using a computer software.results:the mean years of experience of the respondents in the health care setup was 6.8 years. only 57% of the respondents could identify all the measures for prevention of cauti. the knowledge regarding the indication for catheterization though suboptimal was significantly better amongst the doctors as compared to nurses.conclusion:the knowledge regarding indication and preventive measures was suboptimal in our study group. there is a tremendous scope of improvement in catheterization practices in our hospital and education induced intervention would be the most appropriate effort toward reducing the incidence of cauti. |
pit displays the characteristic histological features of a thymoma, with the additional characteristic of being surrounded by lung or visceral pleura without evidence of a thymic lesion in the anterosuperior mediastinum. due to its rarity, variety, unclear natural course, and non - specific features on diagnostic imaging, a formal diagnosis can be made only based on a pathological examination. therefore, surgical resection should be considered for an exact diagnosis, unlike is the case for mediastinal thymomas. herein, we report the case of a patient who was initially suspected to have lung cancer on the basis of a bronchoscopic biopsy but was later diagnosed with pit after surgery followed by a pathological examination. written informed consent was obtained from the patient for the publication of this case report and all accompanying images. a 59-year - old asymptomatic female with a history of partial thyroidectomy for papillary thyroid cancer visited for the evaluation of pulmonary nodules observed in a low - dose chest computed tomography (ct) scan. there was a 1.1-cm nodule in the right upper lobe (rul) and a 5-mm small ground glass nodule in the left upper lobe. the 1.1-cm rul nodule was suspected to be primary lung cancer, and a contrast ct scan was performed. in the contrast ct scan, the rul nodule was observed as a 1.5-cm finger - shaped lesion with a smooth margin in the subsegmental bronchus of the posterior segment. in positron emission tomography (pet) with 18-f fluorodeoxyglucose (fdg), the maximum standardized uptake value (suvmax) of the rul endobronchial nodule was 4.1 (fig. lung cancer was strongly suspected from the findings of the radiological studies, and fiber - optic bronchoscopy was performed to confirm the tissue diagnosis. the bronchoscopy revealed a smooth surface mass obstructing a subsegmental bronchus of the posterior segment (fig. however, spindle cell proliferation with moderate cellularity, mild nuclear pleomorphism, and mitosis over 10 per high - power field were identified in the biopsy specimen. the clinical diagnosis was ct1an0m0 primary lung cancer of the salivary gland type or a spindle cell tumor showing epithelial differentiation ; subsequently, thoracoscopic rul lobectomy and mediastinal lymph node dissection were performed. in the macroscopic examination, the white - to - gray tumor measuring 1.4 cm1.0 cm0.8 cm showed no evidence of hemorrhage or necrosis. a microscopic evaluation showed a relatively fascicular spindle cell pattern with round - to - oval nuclei (fig. immunohistochemical studies revealed diffuse immunoreactivity for pan cytokeratin, cytokeratin 5/6, p40, and p63 (fig. 2d), but negative for vimentin, s-100 protein, thyroid transcription factor-1, napsin a, and smooth muscle actin. according to the world health organization (who) classification, these histopathological findings are consistent with type a thymoma. pit is a rare disease, and thus far, only 37 cases of this condition have been reported. according to the epidemiological data reported by myers. in 2007, autoimmune paraneoplastic syndromes, such as myasthenia gravis or good syndrome, can be accompanied and are associated with a poor prognosis. masses were located in the right lung in approximately 65% and in the left lung in approximately 35% of the patients. the size of the masses ranged from 9 to 129 mm in diameter. on the basis of the who classification, there were 7 cases of type a, 8 of type ab, 6 of type b1, 5 of type b2, and 7 of type b3 thymoma. it has been suggested that pit involves thymomas with an ectopic location, tumors arising from an uncommitted germinative cell, and migration of a mediastinal thymoma into the lung. since the theory of ectopic location is considered to have the most support, pit has been classified as a tumor of an ectopic origin in the recently revised who histological classification of lung tumors. the radiological findings of pit are non - specific. in ct, most pits present as well - circumscribed hetero geneous masses that are not diagnostic. in the present case, the nodule was a cylindrical lesion with a smooth margin and showed a homogeneous enhancement pattern on the ct. the homogeneous enhancement pattern in this case may have been related to the small size of the lesion. a thymoma can show an increased fdg uptake in pet, as in the case of lung cancer, and the amount of the fdg uptake has been reported to have a correlation with the who subtype. however, pet is non - specific with respect to the differential diagnosis of pit because it is not possible to make a histological diagnosis by using suvmax. the fdg uptake in the present case was not high, and the nodule was presumed to be a low - grade malignancy. however, it was not possible to suspect pit until a pathological diagnosis was performed using a surgical specimen. most pits are diagnosed postoperatively on the basis of the histopathological features of the surgically obtained specimens. a cytologic diagnosis based on a biopsy with a small amount of the thymoma tissue is extremely challenging. this is in part attributed to the fact that epithelial cells are difficult to recognize in lymphoid - rich aspirate smears and there is an inherent sampling error in a tumor that frequently displays heterogeneous histopathology. therefore, most cases, including ours, are typically clinically diagnosed as primary lung cancer and staged accordingly before surgery. in the fourth edition of the who classification, the diagnostic criteria of thymomas have been revised to include histological features using hematoxylin and eosin - stained specimens, which are justified as obligatory criteria. optional criteria and immunohistochemical features for the diagnosis are also introduced in order to reduce previous ambiguities, improve the unsatisfactory reproducibility of the who classification, and help to classify thymomas with ambiguous histology. immunohistochemical markers such as cytokeratin 19/20, p20, p63, p40, and tdt are routinely used for this purpose. after a pathological examination of the surgical specimen, we confirmed it to be type a thymoma because of its well - circumscribed and spindle - shaped epithelial cell - rich appearance with relatively low mitotic activity and minimal atypia in comparison with its histological findings. further, in the immunohistochemical study of our case, the surgical specimen was positive for ae1/3, indicating the epithelial keratin origin of the tumor. we assumed it to be the epithelium of the salivary gland or a lung - origin malignancy because of the positive result of the cytokeratin (ae1/3) staining of the biopsy specimen, which is also strongly associated with spindle - shaped epithelial - cell neoplasms, such as type a thymoma. moreover, the positive result for p40 staining, which is a p53 marker that is positive in 97% of thymomas, supported this result. additionally, p63, another maker of the spindle - shaped epithelial cells of thymoma, was positive in our case. the antigen cd20 reveals the existence of focal micronodular areas with a lymphoid stroma and is helpful in the diagnosis of type a thymoma. however, 50% of type a thymomas show a negative result for cd20, and our result was also negative. the fourth edition of the who classification states that tumors should be diagnosed on the basis of hematoxylin and eosin staining ; thus, we concluded that our case was type a thymoma, although some immunohistochemical studies did not follow the usual pattern of type a thymoma. pit should be staged according to the lung cancer staging system because pit is classified as a malignant tumor of the lung according to the who histological classification. the masaoka staging system, which is based on the extent of invasion into mediastinal structures, is not appropriate for the staging of pit, because pit exists inside of a lung, and not in the mediastinum, and has an identical anatomical condition to that of lung cancer. further, nodal involvement was described in several cases, which shared an anatomy similar to that of primary lung cancer. surgical resection, lobectomy, and systematic lymph node dissection should be the treatment of choice in the case of pit. a significantly better survival rate was reported in patients who underwent surgical treatment than in those who were conservatively treated in a systematic review by veynovich.. they suggested that adjuvant radiation therapy be considered in cases of incomplete resection or expansion of the tumor tissue beyond a circumscribed capsule. in particular, the need for lymph node dissection has not been adequately discussed because of the rarity of pit. as previously mentioned, terashima. reported a case of pit with lymphatic spread ; the sites of nodal metastases were similar to those of primary lung cancer. they argued that systematic mediastinal lymph node dissection according to the lymph node map for primary lung cancer be considered for better staging and most likely, better prognosis. in our case, video - assisted thoracic surgery lobectomy and mediastinal lymph node dissection were performed, and no adjuvant therapy was conducted subsequently, because the mass was confirmed to be type a thymoma without invasion. in conclusion, pit is a very rare lesion with uncertain pathogenesis. pit should be included in the differential diagnosis when the pathology of a lung tumor shows uncommon features. since the natural history of the disease has not yet been fully elucidated, regular long - term follow - ups should be performed in accordance with primary lung cancer. | primary intrapulmonary thymoma (pit) is a very rare lesion of uncertain pathogenesis. pit should be considered when the histopathological appearance of a lung tumor shows features that are uncommon but similar to those of a thymoma. in this case report, we discuss the case of a 59-year - old female with a solitary pulmonary nodule that was confirmed to be pit on the basis of pathological tests. treatment with complete resection showed good results. |
web- and mobile - phone - based applications are now commonly used to improve coverage and quality of health services. in india, the mother and child tracking system (mcts) is one such web - based applications that is used nation - wide to improve the coverage of maternal, newborn, and child health (mnch) services. similarly, the use of mobile - phone application such as mobile health (mhealth) for improving health services is becoming popular in india. the first step of utilizing any e - health initiative is registration of eligible beneficiaries ; however incomplete registration is a major concern. only 35% of pregnant women were found to be registered through the mcts in india in 2011. the purpose of the current study was to assess an innovative mhealth intervention to ensure completeness of pregnancy, delivery, and death registration by the accredited social health activists (ashas) in the tribal areas of gujarat. this cross sectional study was undertaken by a local voluntary organization, society for education welfare and action rural (sewa rural) among randomly selected five predominant tribal villages of two primary health centers (phcs) of bharuch district for a period of 2 months (april - may 2015). sewa rural and the department of health and family welfare, government of gujarat, india implemented an innovative mhealth application called the innovative mobile - phone technology for community health operations (imtecho) since 2013. the purpose of the imtecho is to improve the coverage and quality of the mnch services through improving support and supervision of ashas and phc staff. some of the strategies used in the imtecho to ensure completeness of registration were as follows : point of care registration using the imtecho mobile - phone application, linkage of ashas performance - based incentives to electronic record in the imtecho, regular feedback to ashas regarding completeness of registration, and demand generation among the community by the use of mobile - based videos. to find the actual number of events (reference) in the sampled villages, trained and independent data collectors conducted house - to - house survey to enumerate every household. using a pretested questionnaire, the members of each household were asked to identify current events after obtaining written informed consent from them. a detailed account of each event was collected including last menstrual period (lmp), date of delivery, native village, etc. information was collected from a key informant as well such as asha and anganwadi workers. after completing information collection in every village, asha 's record was checked in her mobile to identify the women who were not registered through the imtecho application ; subsequently, asha was interviewed to identify reasons behind the nonregistration of the event through the imtecho application. every event found during household survey was compared with the imtecho system to identify those events which were missed by asha using the imtecho application. to detect 10% difference in sensitivity between registration of deliveries in the imtecho and reference, the required sample size calculation was 98 deliveries in both groups considering 90% power and 5% two - sided alpha error. those villages were included where single ashas had been using the imtecho application since january 1st 2014. native currently pregnant women having lmp more than 3 months from the day of survey were included in the study. the data of native women who delivered between january 2014 and april 2015 and all infant who died between january 2014 and april 2015 were studied. the villages were selected through stratified random sampling so that equal numbers of villages are chosen from two study phcs. the study was approved by the scientific committee of sewa rural and written informed consent was taken from participants after explaining the whole procedure of our study. primary outcome of interest for the study was the proportion of pregnancies, deliveries, and infant deaths registered through the imtecho application compared to actual number of events. there were 844 household enumerations, 39 pregnancies, 102 deliveries and 15 infant deaths that were recorded. the median age of the study participants was 23 years (range : 18 to 36 years). table 1 tabulates data on pregnancies (n = 39), deliveries (n = 102) and infant deaths (n = 5) found during the household survey, 38 (97.43%), 101 (99.01%), and 5 (100%) were already registered by the ashas through the imtecho application. the asha missed one case of pregnancy and delivery because she was under the impression that the women did not belong to the study village as they spent majority of time in another village. for the pregnancies, deliveries, and infant deaths found in household survey, the sensitivity of the imtecho application with the household survey was 97% (95% ci 0.85 - 1.00), 99% (95% ci 0.93 - 0.99), and 100% (95% ci 0.46 - 1.00), respectively. number of pregnancies, deliveries, and infants deaths found during the household survey and in the imtecho among sampled villages (n = 5 villages) 95% cis for sensitivity was calculated using the wilson method on vassarstats online statistics software. the study found that the proportion of pregnant women registered through imtecho was similar to the enumeration. a survey done in india in 2011 - 2012 showed 95% of districts reporting data on pregnant women and 93% on children enrolled under the mcts, while in gujarat, india only 51% of pregnant women among estimated gujarat had 7.69% of data on pregnant women and 10.27% of data on children that were not reported appropriately by sub - centres under the mcts. a similar study carried out in rural ahmedabad shows only 79% of the women have a health card with 82.5% women having documentation of treatment and advice for the antenatal details and only 3.9% of women had been given advice regarding postnatal details. a possible explanation for the difference in findings is the registration being made into this innovative mhealth application. near - complete registration is ensured via numerous safeguards incorporated into the program that includes linking performance - based incentives with digital record, demand generation, regular feedback, and point of care registration. as documented in another publication, the ashas found the use of mobile application quite acceptable, feasible, and useful ; therefore, they were more inclined to timely register pregnant women and deliveries. one of the limitations of the study is its small sample size : using a larger population sample would be helpful, as it would demonstrate the effects of the imtecho application in a more appropriate manner. also, sewa rural has been active in the study area for more than a decade, which might have positive influence on the results. however, similar findings were noticed in another new project area that is far from the reach of sewa rural. to conclude, the strategies used to ensure complete registration in the imtecho application could be applied in other e - health initiatives. similar mhealth initiatives might be considered for scaling up in other tribal areas to improve the coverage and quality of the mnch services. | background : the innovative mobile - phone technology for community health operation (imtecho) is a mobile - phone application that helps accredited social health activists (ashas) in complete registration through the strategies employed during implementation that is linking ashas incentives to digital records, regular feedback, onsite data entry, and demand generation among beneficiaries.objective:to determine the proportion of pregnancies, deliveries, and infant deaths (events) being registered through the imtecho application against actual number of events in a random sample of villages.materials and methods : five representative villages were randomly selected from the imtecho project area in the tribal areas of gujarat, india to obtain the required sample of 98 recently delivered women. a household survey was done in the entire villages to enumerate each family and create a line - listing of events since january 2014 ; the line - listing was compared with list of women registered through the imtecho application. the proportion of events being registered through the imtecho application was compared against the actual number of events to find sensitivity of the imtecho application.result:a total of 844 families were found during household enumeration. out of actual line - listing of pregnancies (n = 39), deliveries (n = 102), and infant deaths (n = 5) found during household enumeration, 38 (97.43%), 101 (99.01%), and 5 (100%) were registered by ashas through the imtecho application.conclusion:the use of mobile - phone technology and strategies applied during the imtecho implementation should be upscaled to supplement efforts to improve the completeness of registration. |
recent advances in high - throughput sequencing technologies have greatly increased the scale and scope of genomics research, and this was evident throughout the recent biology of genomes meeting at the cold spring harbor laboratory. here in one of several talks that investigated the causes, dynamics and phenotypic effects of regulatory change, mike snyder (stanford university, stanford, usa) used chromatin immunoprecipitation followed by dna sequencing (chip - seq) to examine the variability in transcription factor binding among individuals in yeast (saccharomyces cerevisiae) and human. in both species, significant variation was observed, and the amount of binding was strongly correlated with gene expression. much of the observed binding variation could be associated with specific single nucleotide polymorphisms (snps) and structural changes to the genome. on the basis of the patterns of variation, snyder suggested that gene regulation may work like a government, with global regulators and local regulators all having strong effects, but with some focused on a more limited set of loci. snyder 's talk introduced two themes that appeared throughout the meeting : the widespread adoption of high - throughput sequencing as an analysis strategy ; and a focus on identifying and understanding regulatory elements. axel visel (lawrence berkeley national laboratory, berkeley, usa) continued these themes in a talk that highlighted the limitations of using comparative genomics to identify enhancers. by performing chip - seq with the enhancer - associated p300 protein on mouse forebrain and embryonic heart tissue, he and colleagues identified a large number of heart enhancers with very low evolutionary conservation compared to forebrain enhancers. this surprising result suggests that deep pathway conservation does not imply regulatory sequence conservation, that enhancer conservation is not predictive of function, and that there are global differences in enhancer conservation between tissues. li ding (washington university, st louis, usa) described how sequencing samples from the same patients at different stages of the same cancer can help track changes that have occurred during cancer progression, and possibly lead to improved drug therapies. thanks to an efficient pipeline, their genome - sequencing center can analyze tumor / pair samples in only 12 days. the analysis of about 150 cancer genomes using this pipeline has enabled comparisons of different cancer genomes from different points of view, including mutation rate, mutation spectrum, copy number variation and structural variation. the results showed by elaine mardis (washington university, st louis, usa) are an example of how powerful these tools are and what they are able to achieve. she presented the analysis of the relapse genome of an acute myelogenous leukemia patient and a comparison with the genome sequenced at initial presentation. interestingly, this study was able to pinpoint relapse - specific mutations most likely involved in disease progression. one of the largest open issues in human genetics deals with the question of ' missing heritability ' : given the generally high estimates of heritability for many complex traits (such as genetic susceptibility to complex diseases), why have genome - wide association studies (gwas) identified variants that explain only a small fraction of the heritable variation we know is out there ? one hypothesis suggests that rare variants of large effect, which will generally be missed by gwas, are a crucial component of this missing variability. richard durbin (wellcome trust sanger institute, cambridge, uk) and others described progress on the 1000 genomes project, which by next year will generate low - coverage (around 4) whole - genome sequence data from more than 2,000 individuals. this dataset, in conjunction with new imputation algorithms for base - calling low - coverage data, will provide a near - complete catalog of rarer variants (for example, minor allele frequency 0.005) across the human genome, which in turn will facilitate efforts to identify rare variants affecting disease susceptibility. jeffrey barrett (wellcome trust sanger institute, cambridge, uk) addressed the subject of ' synthetic associations '. it has been proposed that many gwas hits are not the result of common variants of modest effect, but rather are artifacts caused by linkage to multiple rare (but highly penetrant) variants. barrett 's talk outlined several compelling sources of evidence suggesting that these synthetic associations are likely to be quite rare. so, while rare variants may or may not explain the ' missing heritability ' problem, they are not a probable cause of the associations already discovered by gwas. one alternative approach to understanding the genetic architecture of complex traits is to use a more tractable genetic system. barak cohen (washington university, st louis, usa) presented detailed genetic analyses of sporulation efficiency in the yeast s. cerevisiae. for this phenotype, just four snps (located in three transcription factor genes) combine to explain 87% of the total phenotypic variation, although very little of this (around 25%) could be ascribed to additive effects. cohen also described a thermodynamic model that might explain the strong interactions (epistasis) observed among snps. this and other work raises the possibility that gene - gene interactions may be a large part of the answer to the missing heritability question. next - generation sequencing technologies now enable researchers not affiliated with genome centers to conduct their own genome - sequencing projects. peter donnelly (oxford university, uk) described some preliminary findings from the panmap project, a collaborative effort to sequence and analyze the genomes of ten western chimpanzees (pan troglodytes verus). donnelly and colleagues were especially interested in the evolution of recombination rates, and the recent fusion between chimpanzee chromosomes 2a and 2b can be used as a ' natural experiment ' to estimate if and how chromosome position influences recombination rate. the results suggest that recombination rates are more affected by chromosomal position than they are by local sequence context. another exciting genome project was described by svante pbo (max planck institute for evolutionary anthropology, leipzig, germany). as a follow - up to the recent publication of the neanderthal genome, pbo and colleagues have now generated roughly 2 coverage of the whole genome from an unclassified hominin fossil found in denisova cave in southern siberia. preliminary results suggest that the denisova fossil is more closely related to neanderthals than to modern humans, though the divergence between neanderthal and the denisova fossil is larger than the divergence between any two extant modern humans. further studies will be needed to clarify the precise evolutionary relationships between this fossil and other hominin groups. several talks gave exciting glimpses of how next - generation sequencing technology can enable novel, high - throughput experimental analyses. rob mitra (washington university school of medicine, st louis, usa) introduced a promising new technology for investigating the regulatory networks of development across a cell lineage. by attaching a transposase to a transcription factor, mitra forces the insertion of a transposon ' calling card ' near the site of dna - binding events. these transposons, and thus binding sites, can then be identified by next - generation sequencing. as these transposons survive cell divisions, the binding history of a transcription factor can be traced though a cell lineage. the method has been applied successfully in yeast and tests are in progress in vertebrates. if successful, this approach would yield a powerful tool for decoding how regulation drives tissue - specific development. although the meeting focused primarily on humans and model organisms, considerable attention was given to the large quantities of diverse data being generated by the sequencing of microbial communities through projects such as the global ocean sampling expedition and the human microbiome project (hmp). katherine pollard (gladstone institutes, university of california, san francisco, usa) argued that traditional approaches to genomic analysis must be significantly adapted to take advantage of the new kinds of information in metagenomic data, which is produced by shotgun sequencing the dna extracted from environmental samples. she showed that phylogenies inferred from metagenomic sequence reads allow new ways of defining species, such as operational taxonomic units (otus), of discovering novel otus, of defining and comparing microbial community diversity, and of estimating microbial ranges in geographic and niche spaces. in regard to the human microbiome, jennifer wortman (university of maryland school of medicine, baltimore, usa) emphasized the hmp 's goal of discovering potential correlations between changes in microbial community composition and the health of the human host. referencing studies of the vaginal and gut microbiomes, she showed that different types of communities require bioinformatic tools with different levels of resolution and specialization. by sequencing the complete genomes of several closely related microbes collected from coastal ocean populations, b jesse shapiro (massachusetts institute of technology, cambridge, usa) took a step towards understanding microbial speciation with his presentation of a well - supported sympatric model of speciation in which populations are ecologically differentiated by a set of niche - specific genes. finally, james taylor (emory university, atlanta, usa) offered an integrated vision of how we might aim to do science in the age of next - generation sequencing. he emphasized two fundamental directions : first, increasing access to the ability to perform large - scale computational analyses ; and second, and perhaps more important, ensuring that such analyses are done in a way that supports and encourages the integrity of scientific investigation. taylor demonstrated by an analysis of a mitochondrial genome resequencing experiment that commercial cloud computing platforms can be used in conjunction with galaxy, a web - based genome analysis tool, to facilitate large - scale analyses that potentially involve multiple software programs, while maintaining the transparent provenance of the data and parameters. the resulting record of every step of the workflow guarantees that an analysis is reproducible and can be clearly communicated. taylor also noted that in order to completely guarantee reproducibility, the original data themselves must be stored permanently, which raises another challenge for this new scientific paradigm. new experimental technologies are giving individual labs the opportunity to conduct large - scale genomic studies that were unimaginable just a few years ago. however, the data generated on this scale present new challenges in interpretation, analysis and data management. given the quality of the science presented at this meeting, we are confident that the community will find creative and collaborative solutions for these issues. | a report on the 23rd annual meeting on ' the biology of genomes ', 11 - 15 may 2010, cold spring harbor, usa.meeting reportrecent advances in high - throughput sequencing technologies have greatly increased the scale and scope of genomics research, and this was evident throughout the recent biology of genomes meeting at the cold spring harbor laboratory. here we describe some highlights of the meeting. |
the three goals of radical prostatectomy () are cancer control, preservation of urinary control, and preservation of sexual function. as the techniques of rp have evolved, our understanding of the relevant anatomy has improved and better oncological and functional outcomes have been reported. despite this, there remains no consensus on operative technique and there remain many technical components of the procedure itself where controversy exists. one such contentious area is the role of bladder neck sparing (bns) versus bladder neck reconstruction (bnr) and the impact of both techniques on oncological and functional outcomes. those in favour of bns suggest this technique allows earlier return of continence and a lower incidence of post - operative strictures, without compromising oncological clearance. surgeons who favour bnr state concerns about compromising surgical margins with no definitive improvement in functional outcome. bns has been incorporated into open rp (orp), laparoscopic rp (lrp) and robot - assisted laparoscopic prostatectomy (ralp) techniques. technical aspects of bns vary between the surgical approaches because of the technical difficulties this can present and the prolonged learning curve. circumferential, anterior and lateral approaches to the have all been described in orp, lrp and ralp. improved surgical techniques and appreciation of the local anatomy have seen huge modifications to the technique of rp since the first described procedure by proust in 1901. preservation of the neurovascular bundle was first reported in 1983 by walsh. and since then, sparing of suspensory mechanism of the urethra preservation of urethral length posterior rhabdosphincter sparing, bns and seminal vesicle sparing have all been described as important techniques for optimising functional outcome after rp. bns has evolved from a better understanding of the anatomy and physiology of mechanisms of uc. new research based on embryological, 3d imaging and urodynamic studies have redefined our understanding of the urinary sphincter mechanism. we now understand the contributions of the bn as an integral part of larger, complex sphincter mechanism that consists of both striated and smooth muscle fibres, rather than an independent external (rhabdo) and internal (lisso) sphincter as previously thought. the rhabdosphincter is horseshoe - shaped and overlies the circular and longitudinal smooth muscle of the urethra. during periods of increased intra - abdominal pressures it contracts and coapts the urethra. due to the relatively low volume of slow twitch muscle fibres compression of the rhabdosphinter can not be sustained for longer than about 60 s. during the storage phase, the lissosphincter maintains urethral resistance and is the primary mechanism responsible for maintaining resting and baseline continence. urodynamics studies have demonstrated that an intact lissosphincter maintains continence in the absence of a viable rhabdosphincter. bns technique aims to leave most of the lissosphincter mechanism intact allowing preservation of its function. we performed a search of the pubmed, science direct and wiley library databases to identify original and review articles in english which addressed bns and bn reconstruction techniques, oncological and functional outcomes of orp, lrp and ralp, where bns technique was used. the search terms used were bn, spare, preservation, reconstruction, urinary incontinence, positive margin, orp, lrp and ralp. relevant articles were reviewed, analyzed and summarized in line with the intentions of this review article. our initial online database search returned 345 results ; 133 via science direct, 122 via pubmed and 90 via wiley library. after narrowing the search down to the relevant papers, current review included 12,806 patients in 31 original studies plus further two review articles directly pertinent to the topic of bns and bnr. there are 19 studies describing outcomes of bns in orp included in the review, six studies on bns in lrp and five original articles on outcomes of bns in ralp. the only prospective, randomised study included patients undergoing both ralp and orp (level of evidence 1b). remaining studies are non - randomised prospective and retrospective studies with varying population size sample (level of evidence 2 - 4). the relationship between bns and positive surgical margin (psm) is well - documented in the literature [table 1 ]. however, only one study is a prospective, randomised, blinded study with a control group. gomez. in a prospective study did not find any sole psm at bn in the cohort of 50 men undergoing orp and bns, although there were six patients (12%) who had multiple positive margins involving bn. wood demonstrated that all nine patients (12%) who had psm at the bn had multiple psm 's and t3 disease. reported there was no sole psm at bn after orp and 7.5% patients had psm at the bn, which were multiple psm 's. soloway and neulander found only 1% sole psm at bn. in his study of 676 patients, 4.3% patients had multiple psm also involving bn. in the series of 365 patients undergoing orp shelfo. reported total psm rate at 32%, but only two patients had sole bn psm similarly, poon. in the retrospective cohort study of 220 men concluded no statistical difference between patients undergoing bns versus the control group. as described by bianco. in the larger prospective studies of 555 men undergoing orp, total psm was 32% with 13% psm at bn and 0.36% sole psm at bn., in a series of 619 men undergoing ralp, assessed psm rate at the base of the prostate in the bns versus control group. similarly, friedlander. in a retrospective evaluation of a large cohort of over 1000 men demonstrated identical psm for bns and control group, which was 13.8%. in a new prospective, randomised, controlled and blinded study from the heidelberg group, only 2% of bns group had a sole psm at the bn compared with 12.5% psm rate in the control group. published results of their prospective study, where they ceased recruitment after 70 patients as the psm rate was 10%, all being at the bn irrespective of the group. in lrp, katz. published results of the series, where overall psm was 26% and psm at bn as 9.75%. during the study, they abandoned bns approach and decreased psm at bn to zero. several studies stressed that appropriate patient selection is crucial in order not to compromise the surgical outcome. oncological outcomes of bns high gleason score on the prostatic biopsies or suspicion of extra - prostatic extension increases the risk of psm at the bn. aydin. described the significance of a sole psm at the bn. in his retrospective study of 164 men with psm after orp, 23% had sole psm at the bn. those patients had 69.8% 5-year actuarial risk of biochemical progression compared with 33% who had negative bn margin. the data highlights that the presence of a psm at the bn has a significant impact on long - term outcomes. both marcovich. and aydin. have found that a psm at the bn results in higher rates of biochemical recurrence when compared with single or multiple psm at other sites. however, in 2010, pierorazio. published the results of large review on over 17,000 patients. patients with an isolated psm at the bn had prostate specific antigen (psa) free survival rate of 37% and cancer free survival of 92%, which was comparable to prognosis of patients with a seminal vesicle invasion and an extracapsular extension respectively. they concluded that american joint committee on cancer should reconsider bn involvement staging as t4. assessing long - term oncological outcomes, we were able to identify only four studies where biochemical recurrence - free survival (bcrfs) in bns cohorts was compared with non - bns cohort. friedlander. assessed psa, surgical margin status and pathological stage and grade and demonstrated no difference in bcrfs between bns and control groups with a follow - up period of up to 72 months (hazard ratio 1.20, 95% confidence interval 0.62 - 2.31, p = 0.596). similarly, in their prospective, controlled study, bianco. reported that during 7-year of follow - up, bns did not compromise disease free survival (dfs). univariable and multivariable analyses of psm at bns was not a statistically important factor for dfs. licht. also assessed dfs between bns and non - bns groups and showed no statistical difference. reported that at 5.2-year follow - up, 90% of bns group had a psa of less than 0.2 mcg / l and 80% of the non - bns group had a psa of less than 0.2. post - operative return to uc is similar in the reported orp, lrp and ralp series. where the bn was preserved uc varies between 36% and 100% at 1 month and 84% to 100% at 12 months follow - up. the method of assessment of uc post - rp has always been controversial and there is no consensus definition. in a prospective study lowe defined uc as usage of zero pads per day. in his series, uc at 1 month was 23% in the bns group and 11% in the non - bns group were continent, which was statistically significant. long - term uc was similar, 89% and 86% bns and non - bns cohorts respectively. studied continence in patients undergoing orp and bladder bnr (anterior bladder tube reconstruction [abtr ] vs. tennis racket repair [trr ]). differences in maximal urethral closing pressures were the only statistically significant findings between both groups post - operatively. following analysis, preservation of functional urethral length was significantly longer in patients undergoing abtr. furthermore, poon. compared outcomes of bns with abtr and trr. at intervals of 1 week, 4 weeks, 3 months, 6 months and 12 months there was no statistical difference in uc among those groups., however, due to the high prevalence of psm at the bn recruitment to the study was halted pre - maturely. a significant improvement in uc post - bns was reported by gaker and steel, and deliveliotis. in both prospective studies, early return of uc in bns groups the study of 600 patients undergoing lrp reported a return to uc at 12 months at 84%. bns was associated with a statistically insignificant faster recovery of uc at 3 months and statistically significant difference in uc at 12 months between bns and non - bns groups, 94.9% and 80.1%, respectively using one pad or less as a definition of continence. more recently, studies aiming at evaluation of bns technique in ralp have been published. reported significantly better early uc in bns group versus non - bns group at 65.6% and 26.5% respectively. at 2-year follow - up, there was no statistical difference between bns and non - bns groups and was 100% and 96.1%, respectively. in 2012, in a large retrospective cohort of 1067 men showed a significant improvement in early uc between bns and control groups particularly at 5 and 12 months follow - up. in another study, you. compared functional outcomes of ralp with bns and bns plus posterior urethral reconstruction (pur) ; however, their study was performed retrospectively on a relatively small sample. nevertheless, it demonstrated a significant improvement in return to uc between a control and pur groups and control and bns - pur groups. the authors concluded that pur and bns are related with improved uc post - ralp. the only prospective, randomised, controlled, blinded study comes from the heidelberg group. 84.2% of patients with bns were continent compared with 55.3% in non - bns procedure. similarly at 12 months, the difference in uc was statistically significant and rates were 94.5% and 81.4% for bns and non - bns groups. moreover, in the multiple logistic regression analysis bns was an independent positive predictor of uc. functional outcomes post - bns gomez. were reported that neo vesico - urethral anastomosis stricture rate were lower in bns group compared with the non - bns group. subsequent literature consistently supported these findings and currently there is an agreement that stricture rate post - bns is relatively lower than in patients not undergoing bns. poon. reported bn stricture at 5% for bns and 11 - 18% for bnr techniques. gaker and steel in 2004 reported that 2.9% of bns patient had an anastomotic stricture. other studies present similar findings. in 2004, besarani. demonstrated that strictures occur mostly within 3 months post - surgery (75%) and are less frequent after 12 months from surgery (5%). more recently, the heidelberg group did not observe the occurrence of any bn strictures post - rp in either bns and control groups. available evidence suggests that bns technique is likely to be superior to non - bns for preventing anastomotic strictures. however, most of evidence comes from non - randomised trials hence level of evidence is relatively low. the relationship between bns and positive surgical margin (psm) is well - documented in the literature [table 1 ]. however, only one study is a prospective, randomised, blinded study with a control group. gomez. in a prospective study did not find any sole psm at bn in the cohort of 50 men undergoing orp and bns, although there were six patients (12%) who had multiple positive margins involving bn. wood demonstrated that all nine patients (12%) who had psm at the bn had multiple psm 's and t3 disease. reported there was no sole psm at bn after orp and 7.5% patients had psm at the bn, which were multiple psm 's. soloway and neulander found only 1% sole psm at bn. in his study of 676 patients, 4.3% patients had multiple psm also involving bn. in the series of 365 patients undergoing orp shelfo. reported total psm rate at 32%, but only two patients had sole bn psm. similarly, poon. in the retrospective cohort study of 220 men concluded no statistical difference between patients undergoing bns versus the control group. as described by bianco. in the larger prospective studies of 555 men undergoing orp, total psm was 32% with 13% psm at bn and 0.36% sole psm at bn., in a series of 619 men undergoing ralp, assessed psm rate at the base of the prostate in the bns versus control group. similarly, friedlander. in a retrospective evaluation of a large cohort of over 1000 men demonstrated identical psm for bns and control group, which was 13.8%. in a new prospective, randomised, controlled and blinded study from the heidelberg group, only 2% of bns group had a sole psm at the bn compared with 12.5% psm rate in the control group. published results of their prospective study, where they ceased recruitment after 70 patients as the psm rate was 10%, all being at the bn irrespective of the group. in lrp, katz. published results of the series, where overall psm was 26% and psm at bn as 9.75%. during the study, several studies stressed that appropriate patient selection is crucial in order not to compromise the surgical outcome. oncological outcomes of bns high gleason score on the prostatic biopsies or suspicion of extra - prostatic extension increases the risk of psm at the bn. aydin. described the significance of a sole psm at the bn. in his retrospective study of 164 men with psm after orp, 23% had sole psm at the bn. those patients had 69.8% 5-year actuarial risk of biochemical progression compared with 33% who had negative bn margin. the data highlights that the presence of a psm at the bn has a significant impact on long - term outcomes. both marcovich. and aydin. have found that a psm at the bn results in higher rates of biochemical recurrence when compared with single or multiple psm at other sites. however, in 2010, pierorazio. published the results of large review on over 17,000 patients. patients with an isolated psm at the bn had prostate specific antigen (psa) free survival rate of 37% and cancer free survival of 92%, which was comparable to prognosis of patients with a seminal vesicle invasion and an extracapsular extension respectively. they concluded that american joint committee on cancer should reconsider bn involvement staging as t4. assessing long - term oncological outcomes, we were able to identify only four studies where biochemical recurrence - free survival (bcrfs) in bns cohorts was compared with non - bns cohort. assessed psa, surgical margin status and pathological stage and grade and demonstrated no difference in bcrfs between bns and control groups with a follow - up period of up to 72 months (hazard ratio 1.20, 95% confidence interval 0.62 - 2.31, p = 0.596). reported that during 7-year of follow - up, bns did not compromise disease free survival (dfs). univariable and multivariable analyses of psm at bns was not a statistically important factor for dfs. licht. also assessed dfs between bns and non - bns groups and showed no statistical difference. reported that at 5.2-year follow - up, 90% of bns group had a psa of less than 0.2 mcg / l and 80% of the non - bns group had a psa of less than 0.2 post - operative return to uc is similar in the reported orp, lrp and ralp series. where the bn was preserved uc varies between 36% and 100% at 1 month and 84% to 100% at 12 months follow - up. table 2 summarises the relevant studies. as with the assessment of oncological outcomes, only one study provides 1b level of evidence. the method of assessment of uc post - rp has always been controversial and there is no consensus definition. in a prospective study lowe defined uc as usage of zero pads per day. in his series, uc at 1 month was 23% in the bns group and 11% in the non - bns group were continent, which was statistically significant. long - term uc was similar, 89% and 86% bns and non - bns cohorts respectively. studied continence in patients undergoing orp and bladder bnr (anterior bladder tube reconstruction [abtr ] vs. tennis racket repair [trr ]). pre - and post - operative urodynamics studies were performed to evaluated continence parameters. differences in maximal urethral closing pressures were the only statistically significant findings between both groups post - operatively. following analysis, preservation of functional urethral length was significantly longer in patients undergoing abtr. furthermore, poon. compared outcomes of bns with abtr and trr. at intervals of 1 week, 4 weeks, 3 months, 6 months and 12 months there was no statistical difference in uc among those groups. no significant difference in uc at 6 months was noted by srougi., however, due to the high prevalence of psm at the bn recruitment to the study was halted pre - maturely. a significant improvement in uc post - bns was reported by gaker and steel, and deliveliotis. in both prospective studies, early return of uc in bns groups the study of 600 patients undergoing lrp reported a return to uc at 12 months at 84%. bns was associated with a statistically insignificant faster recovery of uc at 3 months and statistically significant difference in uc at 12 months between bns and non - bns groups, 94.9% and 80.1%, respectively using one pad or less as a definition of continence. more recently, studies aiming at evaluation of bns technique in ralp have been published. reported significantly better early uc in bns group versus non - bns group at 65.6% and 26.5% respectively. at 2-year follow - up, there was no statistical difference between bns and non - bns groups and was 100% and 96.1%, respectively. in 2012 in a large retrospective cohort of 1067 men showed a significant improvement in early uc between bns and control groups particularly at 5 and 12 months follow - up. in another study, you. compared functional outcomes of ralp with bns and bns plus posterior urethral reconstruction (pur) ; however, their study was performed retrospectively on a relatively small sample. nevertheless, it demonstrated a significant improvement in return to uc between a control and pur groups and control and bns - pur groups. the authors concluded that pur and bns are related with improved uc post - ralp. the only prospective, randomised, controlled, blinded study comes from the heidelberg group. 84.2% of patients with bns were continent compared with 55.3% in non - bns procedure. similarly at 12 months, the difference in uc was statistically significant and rates were 94.5% and 81.4% for bns and non - bns groups. moreover, in the multiple logistic regression analysis bns was an independent positive predictor of uc. functional outcomes post - bns gomez. were reported that neo vesico - urethral anastomosis stricture rate were lower in bns group compared with the non - bns group. subsequent literature consistently supported these findings and currently there is an agreement that stricture rate post - bns is relatively lower than in patients not undergoing bns. poon. reported bn stricture at 5% for bns and 11 - 18% for bnr techniques. gaker and steel in 2004 reported that 2.9% of bns patient had an anastomotic stricture. other studies present similar findings. in 2004, besarani. demonstrated that strictures occur mostly within 3 months post - surgery (75%) and are less frequent after 12 months from surgery (5%). more recently, the heidelberg group did not observe the occurrence of any bn strictures post - rp in either bns and control groups. available evidence suggests that bns technique is likely to be superior to non - bns for preventing anastomotic strictures. however, most of evidence comes from non - randomised trials hence level of evidence is relatively low. post - operative return to uc is similar in the reported orp, lrp and ralp series. where the bn was preserved uc varies between 36% and 100% at 1 month and 84% to 100% at 12 months follow - up. table 2 summarises the relevant studies. as with the assessment of oncological outcomes, only one study provides 1b level of evidence. the method of assessment of uc post - rp has always been controversial and there is no consensus definition. in a prospective study lowe defined uc as usage of zero pads per day. in his series, uc at 1 month was 23% in the bns group and 11% in the non - bns group were continent, which was statistically significant. long - term uc was similar, 89% and 86% bns and non - bns cohorts respectively. studied continence in patients undergoing orp and bladder bnr (anterior bladder tube reconstruction [abtr ] vs. tennis racket repair [trr ]). pre - and post - operative urodynamics studies were performed to evaluated continence parameters. differences in maximal urethral closing pressures were the only statistically significant findings between both groups post - operatively. following analysis, preservation of functional urethral length was significantly longer in patients undergoing abtr. furthermore, poon. compared outcomes of bns with abtr and trr. at intervals of 1 week, 4 weeks, 3 months, 6 months and 12 months there was no statistical difference in uc among those groups. no significant difference in uc at 6 months was noted by srougi., however, due to the high prevalence of psm at the bn recruitment to the study was halted pre - maturely. a significant improvement in uc post - bns was reported by gaker and steel, and deliveliotis. in both prospective studies, early return of uc in bns groups the study of 600 patients undergoing lrp reported a return to uc at 12 months at 84%. bns was associated with a statistically insignificant faster recovery of uc at 3 months and statistically significant difference in uc at 12 months between bns and non - bns groups, 94.9% and 80.1%, respectively using one pad or less as a definition of continence. more recently, studies aiming at evaluation of bns technique in ralp have been published. reported significantly better early uc in bns group versus non - bns group at 65.6% and 26.5% respectively. at 2-year follow - up, there was no statistical difference between bns and non - bns groups and was 100% and 96.1%, respectively. in 2012 in a large retrospective cohort of 1067 men showed a significant improvement in early uc between bns and control groups particularly at 5 and 12 months follow - up. in another study, you. compared functional outcomes of ralp with bns and bns plus posterior urethral reconstruction (pur) ; however, their study was performed retrospectively on a relatively small sample. nevertheless, it demonstrated a significant improvement in return to uc between a control and pur groups and control and bns - pur groups. the authors concluded that pur and bns are related with improved uc post - ralp. the only prospective, randomised, controlled, blinded study comes from the heidelberg group. 84.2% of patients with bns were continent compared with 55.3% in non - bns procedure. similarly at 12 months, the difference in uc was statistically significant and rates were 94.5% and 81.4% for bns and non - bns groups. moreover, in the multiple logistic regression analysis bns was an independent positive predictor of uc. gomez. were reported that neo vesico - urethral anastomosis stricture rate were lower in bns group compared with the non - bns group. subsequent literature consistently supported these findings and currently there is an agreement that stricture rate post - bns is relatively lower than in patients not undergoing bns. poon. reported bn stricture at 5% for bns and 11 - 18% for bnr techniques. gaker and steel in 2004 reported that 2.9% of bns patient had an anastomotic stricture. other studies present similar findings. in 2004, besarani. demonstrated that strictures occur mostly within 3 months post - surgery (75%) and are less frequent after 12 months from surgery (5%). more recently, the heidelberg group did not observe the occurrence of any bn strictures post - rp in either bns and control groups. available evidence suggests that bns technique is likely to be superior to non - bns for preventing anastomotic strictures. however, most of evidence comes from non - randomised trials hence level of evidence is relatively low. there is a growing body of literature on the role of bns in rp, although outcome data is still mainly based on non - randomised, prospective and retrospective studies with variable sample size (level 2 - 4 evidence). only one randomised, controlled, blinded study has been performed to date (level 1b). in the studies reviewed, psm rates varied between 6% and 32%. katz. in his prospective series reported that after abandoning the bns technique the psm rate dropped by 9.75%, however, total psm rate was still 26%. those findings were not confirmed by other studies, which documented that psm did not negatively correlate with bns technique. moreover, newer, larger review postulates that sole psm at bn is very rare (1.2%) and if present confers 12-year cancer free survival of 92%. while there is no agreement, it appears that the only absolute contraindication is high risk disease which increases the risk of psm involving bn. a relative contraindication is a previous pelvic or transurethral prostate surgery as this may increase technical intraoperative difficulties. some authors postulate additional investigations pre - operatively such as additional bladder base biopsies, pre - operative magnetic resonance imaging, and biopsies of the spared bn. the only randomised, prospective study on the subject concluded that bns does not negatively impact oncological outcomes. the sole psm rate at bn was only 2% compared with total psm rate being 13.6%. this study also reported objective and subjective assessment of uc and demonstrated that bns enables early return of uc and long - term uc. in the remaining studies included in our review, lowe. defined uc as zero pads per day while srougi. other series used an open interview as a method of assessment of uc without any validated questionnaires. more recently international continence society (ics) male and ics male short - form were utilised by others. available data uniformly shows that uc rates post - rp with bns are at least as good as post non - bns and it is very likely that bns expedites early return of uc and improves long - term uc. with changes in surgical technique, bns has been increasingly incorporated into the rp. evidence summarised in this review demonstrates that bns technique leads to early return of uc and long - term uc without compromising oncological outcomes. bns is superior to non - bns techniques and authors of this review postulate application of the method in feasible cases. | the role of a bladder neck sparing (bns) technique in radical prostatectomy (rp) remains controversial. the potential advantages of improved functional recovery must be weighed against oncological outcomes. we performed a literature review to evaluate the current knowledge regarding oncological and functional outcomes of bns and bladder neck reconstruction (bnr) in rp. a systematic literature review using on - line medical databases was performed. a total of 33 papers were identified evaluating the use of bns in open, laparoscopic and robotic - assisted rp. the majority were retrospective case series, with only one prospective, randomised, blinded study identified. the majority of papers reported no significant difference in oncological outcomes using a bns or bnr technique, regardless of the surgical technique employed. quoted positive surgical margin rates ranged from 6% to 32%. early urinary continence (uc) rates were ranged from 36% to 100% at 1 month, with long - term uc rate reported at 84 - 100% at 12 months if the bladder neck (bn) was spared. bns has been shown to improve early return of uc and long - term uc without compromising oncological outcomes. anastomotic stricture rate is also lower when using a bns technique. |
using a modal matching theory, we demonstrate the generation of short - range, chiral electromagnetic fields via the excitation of arrays of staggered nanoslits that are chiral in two dimensions. the electromagnetic near fields, which exhibit a chiral density greater than that of circularly polarized light, can enhance the chiroptical interactions in the vicinity of the nanoslits. we discuss the features of nanostructure symmetry required to obtain the chiral fields and explicitly show how these structures can give rise to detection and characterization of materials with chiral symmetry. |
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most of us agree that the assumption that nurses care and doctors cure is too simplistic, and that it is not uncommon for many nurses not to care very much at all, and for some doctors to care a great deal. however, in spite of this, there still is a theoretical discrepancy between nursing and medicine in relation to how care is handled ; while care may be said to constitute an important part of professional self - understanding in nursing1 [7, 27, 28, 38, 39 ], it still may be said to be situated in the margins of the ethical thinking in medicine [9, 42, 51, 52 ], and seldom applied to medical education. nursing and the nursing profession may be criticized for monopolizing the concept of care. in light of this theoretical discrepancy and the fact that many patients complain about a lack of care when meeting with their doctors, and experiencing ignorance, insensitivity or degrading behaviour [24, 26, 35 ], this urges a further analysis of the relevance of the care discourse in medicine. implementing nursing care perspectives in medicine is controversial for a number of reasons, and may be why it has not been explored thoroughly. however, this is not a reason to avoid the topic. on the contrary therefore, in this paper i attempt to bring some of this knowledge from nursing into the field of medicine, taking the influential scholarly works on care by norwegian nursing scientist and philosopher kari martinsen as my point of departure.2 kari martinsen s work has influenced the current understanding of care in nursing in scandinavian countries, and she belongs to a different theoretical tradition than the gilligan s anglo - american. investigating martinsen in the context of medicine will be interesting because it will represent an attempt to bridge the gap between related research traditions in medicine and nursing, and it will represent an attempt to relate the scandinavian nursing debate on care inspired by phenomenology to the anglo - american tradition of care ethics. interestingly, sren holm asks a similar question in his article, what should other healthcare professions learn from nursing ethics ?. however, holm and i reach different conclusions : while holm rejects the influence and importance of ethics of care in relation to nursing ethics and health care ethics in general,3 i welcome these perspectives, also in medicine. in this paper i focus my attention on martinsen s analysis of the different clinical gazes, and i will explore how this perspective may represent a prolific approach towards care in medicine., so focusing on the way physicians see may represent an advantageous way of approaching care in medicine. the dominating gaze in medicine, however, is often referred to as powerful, objectifying and dehumanizing [14, 22, 24 ]. therefore, i argue with kari martinsen that medicine needs to encourage and develop a perceiving eye. the fostering of such an eye, or gaze,4 may facilitate care in each medical encounter, as well as refining clinical proficiency. in support of this claim, i first draw on some of the main features in martinsen s care theory, trying to make her thinking comprehensible for readers outside nordic countries as well. subsequently, i analyse martinsen s essay seeing with the heart s eye, focusing on her analysis of what seeing means in health care (ibid.). by using examples, i aim to illustrate how the development of a sensous and perceiving eye in medicine may be of ethical as well as epistemological importance for the clinical encounter. finally, i will discuss different objections to focusing on care in medicine, in relation to the perspective developed above. influenced by phenomenology and the historic roots of caring in nursing, kari martinsen has approached the concept of care through different levels of analysis. i rely for the most part in this paper on her later works in which she is thinking with, to use her own expression, the danish philosopher and theologian knud e. lgstrup by analysing care phenomenologically [3941 ]. she argues that as human beings we are basically social and dependent on other people and relies here on lgstrup s view of human life as a life in interdependence5 [39, 40 ]. the assumption that human beings are interconnected and dependent upon each other thus represents a central ontological feature of martinsen s theory. thus, care constitutes a fundamental precondition of our lives : as human beings, we are basically dependent, and this dependency requires a human response in the form of care. martinsen always interprets a situation of care as being contextual, emotionally laden and particularistic in nature : care is to be concrete and present in a relationship by our senses and our bodies. it is always to be in a movement away from ourselves and towards the other, she writes [36, p. 11 ]. care is to relate to the other in an unconditional and spontaneous way as a response to the concrete situation of the other. nevertheless, in order to accomplish this, we must be able to recognize his or her needs in the first place. therefore, noticing the need to care may be conceived of as a basic element of care.6 similarly, martinsen argues that in order to care for the patient, we must both be able to see and express the [patient s ] appeal for help in order to strengthen the [patient s ] life courage in the suffering [37, p. 9 she thus highlights the importance of noticing the need to care (to see), at the same time as focusing on the importance of practical action in care (to express). she also indicates an important ethical implication : namely, the strengthening of a patient s life courage. the essential element, however, is to be able to recognize the need of the other and to act accordingly. for that reason, practical action represents an important dimension in martinsen s care approach, together with a relational and a moral dimension. care is thus a trinity : relational, practical and moral simultaneously [3, 38 ]. a central feature of the care philosophy of martinsen is her emphasis on perception in our interaction with others, as well as in clinical situations, and she considers perception to constitute an important part of clinical judgment.7 in the essay seeing with the heart s eye martinsen explores different dimensions of the clinician s gaze. she starts by drawing a distinction between perceiving and recording : by recording is meant the putting of oneself in an outside position, classifying, she argues (p. 72). when we record, we systematize and differentiate, albeit within the frameworks of an already existing conceptual system. the recording eye is reductionistic and neutral ; it may reduce living characteristics, such as a laughing face, to clinical signs, characteristics and marks. it is the eye of the disinterested observer, in which the other, as a person, may become indifferent to us. in her description of the recording eye, martinsen is also inspired by and refers to lgstrup s conception of the eye of the epoch and to michel foucault s well - known analyses of the classifying gaze and the examining gaze in medicine. in contrast the perceiving eye is characterized by openness towards the world, where sensation and emotions are working together, says martinsen. the perceiving eye opens for a seeing emotion, in which we are touched and emotionally involved before we understand the needs of the other. martinsen refers in this connection to the story of the good samaritan : the samaritan saw the half - dead stranger and was moved with compassion. he did not record first ; instead, he was struck, pained by the wounded stranger. martinsen refers here to the greek word in the text, esplanknistae, which is a verb made from a noun, meaning something to do with guts, stomach, abdomen, in other words an incredible physical pain he was pained in the gut, meaning that he saw with his whole body, which was touched the samaritan s understanding of the situation was thus led by his senses, by his sensuous and perceiving eye. so, why does martinsen bring the story of the good samaritan into an essay on seeing in health care?8 the story illustrates how the perceiving eye may facilitate care. in the story, the priest, the levite and the samaritan all saw the half - dead man, although they saw him in different ways. significantly, the way they saw him had a major impact on the way they acted : the well - established and theoretically educated men, the priest and the levite, saw the half - dead man and passed him by. did they not trust their eyes ? did they have recording eyes and not perceiving eyes ? was the ailing man recorded as a stranger and therefore not of their concern, based on theory and culture ? the samaritan, on the other hand, got within the perceiving range of life. he did not record first ; instead, he was struck, pained by the wounded stranger. how can we further transfer these theoretical insights to medicine ? what gaze is at work here ? as physicians we are trained to create a thorough medical history and make a clinical examination in order to give a proper diagnosis. the process of interviewing and examining the patient operates within a fixed framework, and even though we are encouraged to ask open - ended questions, the medical interview follows a standard scheme, investigating the patient s heredity, social history, education, employment history, drug history, tobacco and alcohol consumption, home circumstances, and the history of the present complaint(s). likewise, the physical examination of the patient operates within the same framework, leading us towards a possible diagnosis of the patient. this perspective is evident if we go to a recenty updated text book in clinical examination for medical students : the examination really begins from the moments you set eyes on the patient, as the patient approaches you in the consulting or examination room, observe the posture, gait and character of the stride, and when making your initial acquaintance with the patient, the grip of the handshake usually provides some useful information (p. 2021). thus, the first step of the examination is the inspection, during which we initially try to create a general impression of the patient : does she look well, or not ? is there any striking physical abnormality ? then we inspect the patient more specifically in relation to our examination of the different body systems, looking for signs and stigmata before we continue with palpation and auscultation of the heart and lungs. the medical exploring gaze thus operates within a fixed framework by systematizing, differentiating and classifying the information from the patient within a fixed totality. at the same time we usually understand the word professional to mean keeping sufficient distance, not being overly involved (restraining emotions) and being objective and a matter of fact in one s speech. in light of this, i will argue that the dominant gaze of medical practice is a recording gaze, focusing too heavily on diagnoses and diseases alone and leaving out the sensitive openness to the patient s particular situation.9 this is also underscored by recent empirical findings, pointing to how physicians actually essentialise the patients stories by breaking them down into concrete complaints and categorising the symptoms into medical sense, as well as studies pointing to how the professional gaze actually may lead to humiliation and shame on part of the patient. a possible consequence of such a recording gaze in medicine may be that physicians do not always trust their own eyes when meeting with patients who suffer. in an article in thejournal of the norwegian medical association, kristin ribe, who has suffered for several years from self - injury and suicidal thoughts, describes her meeting with health personnel (first and foremost physicians) at the emergency ward, arriving with self - inflicted bleeding wounds again and again. she describes anger or indifference by the doctors who meet her with expressions such as are you here again, pull yourself together ! or one doctor did not even say hello to me, she writes. he just equipped himself and talked over an hour with the nurse about his cottage, while i was only something lying there, a scrap, a nothing it is tempting and may be relevant to draw a parallel here between the doctors behaviour and that of the priest and the levite in the story of the good samaritan. like the priest and the levite a young woman arrives at the emergency unit with bleeding wounds, and they meet her with ignorance, blame or anger. they probably saw her with a recording gaze, founded on their medical knowledge and the medical culture. among many health professionals, there has been a widespread tendency to think and talk about self - injuring patients as manipulative. did the doctors record the patient as manipulative and therefore outside of their moral concern just like the priest and the levite, who recorded the injured man as a stranger and as someone they were not obliged to help, based on their culture and their laws ? the recording gaze is powerful in its objectification of the other. through a recording eye, we may see a person as an object or a completed fact, and this may threaten the person s integrity. integrity means that which must not be hurt, damaged or altered, but which should be respected and protected, and for a person who is seen as an object, it may be both painful and damaging. the self - injuring girl precisely delineates the damaging effects of a recording eye when she says she felt like a scrap, a nothing when treated by the doctor who ignored her. through this type of objectifying behaviour as a consequence, we see how the recording activity of the professionally trained eye may be damaging to the patient. however, this kind of viewing of the patient is also necessary in order to understand the patient s medical condition and to reach a diagnosis. thus, in order to care for the patient in a broader sense than just making a diagnosis and initiating treatment, we must strive to integrate and combine the two modes of seeing the patient : to see with openness and ease, such as seeing with the perceiving eye, while also seeing in an academic, exploring and evaluating way. besides the protection of patient integrity, martinsen points to another important ethical implication of displaying a perceiving gaze in medicine, namely, the strengthening of patients life courage. this aspect of the perceiving gaze is also illustrated in the article by ribe, in which she describes physicians who actually saw her, receiving her appeal for help, rather than abandoning it. she describes the good in a physician who saw her scars and said : i see you have had a difficult time, and she describes the positive potential in such a response from another person, as something that was very good to feel and to take with her when she returned outside again. i need the physician at the emergency ward, whose hands trembled while he was suturing, she writes. for him i was not routine. care may also be epistemologically essential in the clinical encounter, and in the following example i try to illustrate how seeing patients with a perceiving eye also may contribute to refining the clinical abilities of the physician : earwax is the problem for the next patient on the list, and the doctor envisages a short consultation, hoping to catch up with some work that is behind. after examining the patient s ears, the physician confirms the diagnosis and orders the appropriate treatment. the consultation is over with in five minutes, and the patient is supposed to leave. the doctor knows the patient from previous consultations ; he is a middle - aged man, well - ordered and with no medical history. maybe it is something in the way the patient walks, or talks that makes it difficult for the physician to end the consultation as planned. maybe it is something in the patient s eyes that touches the physician, so that instead of turning to his computer, he asks the patient one more time how he is doing. well, for how long a time have you not been sleeping ? the physician asks. since i was twelve years old, answers the patient, who then he loses his well - ordered appearance and tells a history of many years of child abuse in his childhood home, terrorized by a violent father. the patient is upset and starts to cry, and the consultation turns into something quite different from the treatment of bilateral earwax. earwax is the problem for the next patient on the list, and the doctor envisages a short consultation, hoping to catch up with some work that is behind. after examining the patient s ears, the consultation is over with in five minutes, and the patient is supposed to leave. the doctor knows the patient from previous consultations ; he is a middle - aged man, well - ordered and with no medical history. maybe it is something in the way the patient walks, or talks that makes it difficult for the physician to end the consultation as planned. maybe it is something in the patient s eyes that touches the physician, so that instead of turning to his computer, he asks the patient one more time how he is doing. well, for how long a time have you not been sleeping ? the physician asks. since i was twelve years old, answers the patient, who then he loses his well - ordered appearance and tells a history of many years of child abuse in his childhood home, terrorized by a violent father. the patient is upset and starts to cry, and the consultation turns into something quite different from the treatment of bilateral earwax. as i see it, this example illustrates the epistemic potential in holding on to the concreteness in the situation by displaying the sensuous and perceiving eye instead of immediately starting a process of abstraction. the physician could have disciplined the situation by following his plan for an efficient consultation and doing a proper job treating the patient s earwax. and but an extra weight in the patient s steps or something in the patient s eyes moved the physician, and by being aware of his own seeing emotions in meeting with the patient, he captured some important information regarding the patient s clinical situation. this is important both in regard to the medical state of the patient, by initializing further examination and treatment of his insomnia, but also in relation to bringing forward the courage to live, as martinsen expresses it. so far, i have discussed central conceptions in the care philosophy of kari martinsen in relation to medicine, focusing on her discussion of seeing in health care and her conceptions of the perceiving and the recording eye. i have asserted that seeing in medicine is dominated by a recording gaze and that the development of a sensuous and perceiving gaze is important in order to facilitate care and refine clinical proficiency. there are many aspects to discuss here, and i will not discuss martinsen s theoretical perspective in this context more,10 but i will discuss the possible drawbacks of highlighting such a perspective in medicine. in this connection, it is important to point out that even though care has been a marginalized area of inquiry within the philosophy of medicine and medical ethics, there is an ongoing transition in the medical ethical landscape by which the feasibility of ethics of care in relation to medicine is being recognized within philosophy of medicine [10, 11, 42, 58 ], medical education, feminist ethics and medical anthropology. there are, of course, many worries that might be raised for a care orientation in the context of medicine and medical ethics. in the following section i will deal with some of the arguments related to the assertion that a care perspective in medicine will likely bring about bad consequences for patients and physicians. by this, i limit my discussion to exclude critical arguments trying to assert that the notion of care itself is problematic and unnecessary [2, 12, 19 ]. let us look at some of the objections that are particularly germane to the present discussion : first, it might be said that caring interferes with proper medical practice by inhibiting the doctor s organization of information, in addition to the decision - making process. beauchamp and childress, discussing the virtue of compassion in medicine, point to this connection in how compassion can blind reason and impartial reflection. constant contact with suffering can overwhelm and even paralyze a compassionate physician and nurse, they write (p. 40). in response to this worry, it is important to point out that there is nothing intrinsic to the care perspective that excludes appropriately detached forms of concern and compassion, to paraphrase carse. a good health care professional should be able to summon the appropriate degree of emotional detachment, or equanimity, when this is crucial to serving the well - being of the patient, if we relate this objection to martinsen s perspective, we may ask whether the display of a perceiving gaze in medicine may inhibit rather than facilitate medical practice. first, according to martinsen, proper care is not concordant with actions that disrupt the necessary medical work. here, i rely on her emphasis on professional reflection in relation to the clinical encounter. in martinsen s view, clinical judgment depends on professional knowledge to structure and organize the information, as well as an ability to be open and sensitive toward the patient. because of this, comforting the patient instead of initiating proper medical treatment is not consistent with good care, but is more like what martinsen denotes as sentimental care. according to martinsen, sentimental care is care in which the caregiver s participation in the other person s suffering is limited to the caregiver s self - centred emotions. when the caregiver confines him or herself to pitying the patient and refrains from helping the patient in a professional manner, the care becomes sentimental. second, as we experienced in the example with the patient with earwax and insomnia, care may be epistemologically essential in the clinical encounter because caring attitudes and sensitivities may allow access to important knowledge about the patient s subjective experiences of illness, which may be crucial in acquiring a proper understanding of the patient s condition.11 accordingly, from my point of view, the understanding of care as an inhibitor of medical practice may be due to an inadequate conception of care as well as a failure to recognize how care may contribute to refining clinical proficiency by being epistemologically relevant. second, it might be argued that caring for a patient may lead to difficulties for the physician with regard to causing the patient therapeutically necessary pain or breaking bad news to the patient. as i see it, such a worry is based on an inadequate conception of care and i refer to martinsen s conception of sentimental care ; according to martinsen, care is more than feeling sorry for, having a liking for, or being emotionally attached to someone. care also involves a practical participation in the other person s suffering and concrete action based on professional judgement. therefore, to leave out a painful but important procedure for the patient because of one s caring for that patient is not concordant with proper care. third, it might be said that there are important moral issues that may fall outside the reach of an ethics of care. one main worry is that an ethic of care will have nothing to say about certain forms of injustice. will physicians be inclined to favour their patients over other physicians patients or the need of the community ? in this regard it is important to point out that the partialistic feature of an ethic of care does not afflict the caring agent with tunnel vision. this point is also evident in martinsen s perspective that, besides emphasizing a spontaneous care response towards the other, mediated through a perceiving eye, also emphasizes the importance of professional judgment and reflection in the clinical encounter. accordingly, the professional may not have the opportunity to focus his or her attention on the care of one person or group of persons to the complete exclusion of the wants and needs of others. it is also important to point out that what is in question in the care perspective is not necessarily to question the importance of justice, but pointing to additional ways of moral reasoning. therefore, an adequate moral theoretical approach may well involve an integration of the justice and care orientations so as to retain their respective strengths through rehabilitated notions of justice and care. the fourth difficulty relates to the previous objection and concerns the possible tendency of an ethics of care to allow favouritism. it would require a saint to care for some really disgusting patients, says curzer, referring to a claim that it is not possible to care equally for all patients. this is one of the reasons i consider ribes s article (2009) to be both interesting and relevant. there has been an understanding among doctors that self - injuring patients are difficult, demanding and manipulative [6, 43 ]. at the same time, recent research has found that good care from doctors and health personnel is important for these patients to recover. so, how do we manage to care for difficult and demanding patients ? yet, i consider martinsen s understanding of care as being able to see and express the [patient s ] appeal for help, involving an emotional awareness and a relational and a practical competence to represent a more feasible approach to the problem than an understanding of care, meaning to have a liking for, which involves emotional attachment. the latter refers to the understanding of care that curzer refers and criticizes in his article. following martinsen, the important task is not whether we like the patient or whether we feel attached to the patient as a friend, but whether we are able to see, or become aware of the person s suffering and express it independent of his or her sympathetic or unsympathetic traits. burnout on the part of the physician also is identified as a possible drawback to caring in medicine. a usual way of approaching this issue in medicine has been to argue in favour of a detached model of care in which the physician remains professional, i.e., cool and undisturbed by the patient s situation. on the contrary, the american physician and philosopher jodi halpern argues convincingly against such an ideal of detachment in medicine, pointing to empirical studies indicating an increased risk of burnout in relation to a medical practice characterized by detachment, as well as pointing out how an empathic communication with patients makes being a physician more meaningful and satisfying [15, 16 ]. kari martinsen also has been criticized in a similar manner for pursuing an altruistic concept of care, allowing for self - sacrifice on the part of the health care professional [18, 50 ]. i do not agree that martinsen s care perspective is altruistic in the sense that it implies self - sacrifice. as i see it, these critics make their claim without taking into consideration the consciousness of limit inherent in the professional reflection that martinsen emphasizes in relation to practical care work. in this paper i have argued that the development of a sensous and perceiving gaze is important in medicine in order to facilitate care and refine clinical proficiency. relying on the care philosophy of kari martinsen, i suggest that an understanding of care as being able to see and express the [patient s ] appeal for help may represent a fruitful way of dealing with care in medicine. by this, both the importance of attentiveness as conceptualized by the perceiving eye, as well as the relational and practical dimension of care, such an approach may further contribute to protecting the integrity of patients, in addition to contributing to refining the clinical abilities of physicians in the clinical encounter. possible problems have been addressed, while some questions are also left open for further inquiry : for instance, how do we train and educate physicians in this way of seeing in medicine ? | in this paper i introduce a theoretical framework on care developed by the norwegian nurse and philosopher kari martinsen, and i argue that this approach has relevance not only within nursing, but also within clinical medicine. i try to substantiate this claim by analysing some of the key concepts in this approach, and i illustrate the potential clinical relevance of this approach by applying it in relation to two care scenarios. finally, i discuss some of the concerns that have been raised in relation to the aim of highlighting care in medicine. |
one of the major factors that can negatively affect breastfeeding is the lack of breast milk. there are many instructions on the subject of breast milk in iranian traditional medicine resources. this article attempts to investigate causes and reasons for the lack of breast milk from the perspective of the great scholars in this field. this study reviews the literature based on the iranian traditional medicine. the literature review included traditional medicine resources and a survey of reputable databases using keywords such as morzae, sady, pestan, sheer, sheerkhar, and hifzossehhe. according to the viewpoint stated in traditional medicine literature, the primary substance for milk production is blood that transforms to milk after crossing the breast glandular tissue. the main causes of milk shortage can be classified into four categories, namely food - related factors, factors related to blood impaired, factors related to breast tissue and psychological and physical factors. one of the main reasons for milk shortage is the impaired quality and quantity of food. appropriate mizaj of breast tissue is required for adequate milk production as it develops sufficient ability to absorb and transform the substance into milk. on the other hand, the ability of breast tissue is greatly influenced by the main organs of the body, particularly the heart ; being the core of understanding psychological factors. thus, psychological factors can have a significant effect on the process of milk production. due to the importance of breastfeeding, reflection on other viewpoints, such as those mentioned in iranian traditional medicine, could pave the way towards new research areas. | background : support for breastfeeding is a public health priority. one of the major factors that can negatively affect breastfeeding is the lack of breast milk. there are many instructions on the subject of breast milk in iranian traditional medicine resources. this article attempts to investigate causes and reasons for the lack of breast milk from the perspective of the great scholars in this field.methods:this study reviews the literature based on the iranian traditional medicine. the literature review included traditional medicine resources and a survey of reputable databases using keywords such as morzae, sady, pestan, sheer, sheerkhar, and hifzossehhe. the content analysis was used after collecting data.results:according to the viewpoint stated in traditional medicine literature, the primary substance for milk production is blood that transforms to milk after crossing the breast glandular tissue. the main causes of milk shortage can be classified into four categories, namely food - related factors, factors related to blood impaired, factors related to breast tissue and psychological and physical factors. one of the main reasons for milk shortage is the impaired quality and quantity of food. appropriate mizaj of breast tissue is required for adequate milk production as it develops sufficient ability to absorb and transform the substance into milk. on the other hand, the ability of breast tissue is greatly influenced by the main organs of the body, particularly the heart ; being the core of understanding psychological factors. thus, psychological factors can have a significant effect on the process of milk production.conclusion:due to the importance of breastfeeding, reflection on other viewpoints, such as those mentioned in iranian traditional medicine, could pave the way towards new research areas. it could also steer policies towards an extra focus on breastfeeding by mothers. |
over the next 30 years, the elderly population (65 years and older) in the united states is expected to double. by the year 2040, 20% of americans will be 65 years of age and older as compared to 12.97% in 2010. from 2010 to 2050, the total population is expected to increase by 142% ; the age group of 65 years old and over is expected to increase by 220%, the age group of 85 years old and over is expected to increase by 331%, and the age group of 100 years old and over is expected to increase by 761%. although there has been an increase in life expectancy, the later years may not be lived in good health. the burden associated with providing health care to an aging population is likely to increase, with nutrition being one of the most important factors for maintaining quality of life. to maintain health, independence, and well - being, however, many studies indicate that seniors ' diets are low in calories and other nutrients. within the community, efforts have been made to develop nutrition programs targeting older people of high nutritional risk. home delivered meals program, such as those provided by mow, can make a significant contribution to the nutrient intakes of the homebound. despite the sincere intentions of organizations, administrators of charitable programs often over - focus on the nutritional content of meals with little concern for recipients ' food preferences, satisfaction, or more importantly, avoidances. therefore, this study was conducted to evaluate recipients ' perceptions of food quality, volunteer service and responsiveness, and their satisfaction of home delivered meals program. malnutrition among the elderly may be related to illness, cognitive impairment, medication usage, functional decline, and mental illness [6 - 8 ]. previous research showed that 74% to 98% of elderly recipients of mow were at nutrition risk. furthermore, many elderly people have physical problems that make it difficult for them to consume certain foods. used the nutrition screening initiative (nsi) screening tool and reported that 83% of mow participants were at high risk for poor nutritional status compared to the 74% considered at risk by the nutritional assessment criteria of their study. even though the rate of participants at high nutritional risk in this study was lower than those originally assessed from mow programs, the result still indicated that the number of participants at high nutritional risk is high. while certain groups of seniors are at greater - risk of hunger, hunger cuts across the income spectrum. for example, over 50% of all seniors who are at - risk for hunger have incomes above the poverty line ; likewise, it is present in all demographic groups. seniors living in non - metropolitan areas are as likely to experience food insecurity as those living in metropolitan areas, suggesting that food insecurity cuts across the urban - rural continuum. since adequate nutrition is essential for maintaining health, decreasing existing health problems, and maintaining functional independence, improving nutritional status and maintaining functional independence are seriously important to prolong good health status and well - being. weatherspoon. found that enjoyable meal times and appropriate nutrition intake allow the elderly to improve their health and quality of life ; therefore, health status and quality of life might be influenced by recipients ' satisfaction with the meals and services provided from health - care facilities. service quality assessment has received considerable attention in hospitality research [10 - 13 ]. service quality is defined in the marketing literature as a customer 's post - consumption evaluation of service that compares expectations with perceptions of performance. the evaluation of service quality is based on the manner in which the service was delivered and the outcomes that resulted from that service. service quality addresses issues of quality only, while satisfaction may include non - quality components (e.g. price). satisfaction is an overall measure of how happy or content customers are in general with a product or service offered. this model utilizes a 44-item measurement that compares differences between customers ' expectations of service and their assessment of actual performance. respondents ' expectations of excellent service are evaluated first in this instrument by 22 questions (before consumption), followed by another 22 matching questions rating perceived performance of the service provided (after consumption). five dimensions of service quality have been specified in servqual : reliability, responsiveness, empathy, assurance, and tangibles. despite the popularity of the servqual model proposed an instrument called dineserv to assess customers ' perceptions of a restaurant 's service quality. dineserv was adapted and refined from servqual containing 29 statements on a seven - point response scale. they selected a random sample of people from telephone directories of a midsize city in the north central us and used dineserv to conduct telephone interviews with adults who had eaten out six or more times during the previous 6 months in three restaurant segments : quick service, casual / theme, and fine dining. dineserv consists of five dimensions : assurance, empathy, reliability, responsiveness, and tangibles. in order to evaluate customer perceptions of foodservice quality, kim. conducted a study to evaluate the reliability and validity of a modified dineserv instrument to fit the needs of specific establishments : a casual italian restaurant in the us and an american - style casual restaurant in taiwan. another study was conducted to validate five dimensions of the dineserv instrument in korean casual dining restaurants and explored any possible differences in the perceived service quality of those restaurants. one could easily assume that recipients would be satisfied with the meals and services of mow or other congregate meal programs because almost all meals from these programs are free, and most staff of these programs are volunteers. however, some research has indicated that many recipients explicitly expressed their dissatisfaction ; for example, in a study by lirette, some recipients felt that the vegetables were too firm (19%) and the meat was too tough (24%). they also indicated their dissatisfaction about texture, vegetables, salads, and ethnic choices. in roy and payette 's study, nineteen subjects (95%) usually or never consumed their entire meal, and eight (40%) subjects reported they threw away parts of their meal. this study indicated that fifteen (75%) were satisfied with meal quality, time of delivery, variety, choice, and temperatures of foods. these results imply that mow recipients may not eat sufficient amounts of food to meet their nutritional needs. moreover, research has suggested that recipients ' preferences and acceptance should be strictly monitored and evaluated to ensure that meals are going to be eaten. therefore, mow agencies must understand recipients ' perceptions about food quality, volunteer service, and level of satisfaction. in the current study, midwest mow recipients ' perceptions and meal satisfaction were investigated. consequently, this study was designed to evaluate recipients ' satisfaction with meals and services from the mow program in lubbock, texas. the specific objectives of this research were to : evaluate the recipients ' satisfaction with meals from mow.assess the recipients ' satisfaction with services from mow.evaluate the recipients ' overall satisfaction with meals and services from mow.investigate the factors affecting the recipients ' satisfaction. based on a literature search of previously conducted satisfaction surveys, a satisfaction questionnaire was developed. this instrument was adopted and modified in order to investigate the recipients ' satisfaction. the initial questionnaire contained 24 items, using a 5-point likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). the final questionnaire had 21 items and used the 5-point likert scale. in order to examine factors affecting the recipients ' satisfaction, the recipients were asked about their experiences with meals and services they had received during this study. the survey paper was yellow or blue instead of white because it would be easier for recipients and volunteers to distinguish them among other papers. the questionnaire language was simply written because federal regulations advise a 7th grade reading level for written consent for ordinary adults. questionnaires were prepared in two versions, english and spanish, because almost half of the recipients spoke and read only spanish. another native spanish speaker then translated the questionnaire back into english in order to confirm the accuracy of the spanish questionnaire. original and back translated documents were then compared, and it was confirmed that the spanish questionnaire was suitable for use in the study. originally, the questionnaire used for the pilot test had 20 items and two variables. volunteers delivering lunches for mow were asked to distribute the questionnaires when they delivered meals, and they were also asked to collect the questionnaires either the next day or the next time they completed their routes. the 1-page (two sided) questionnaire was printed on 8.5"11 " paper and folded in half. two different colors were used to differentiate the languages : yellow for english and blue for spanish. no code numbers were used on individual questionnaires to ensure the anonymity of responses. to encourage participation, the researcher made a $ 1 donation to mow for every completed survey. the data was compiled and statistically analyzed using the statistical analysis program spss (statistical package for social sciences) release 15.0 for windows (spss inc., the data were analyzed following anderson and gerbing 's two - step approach : a measurement model and a subsequent structural model. the multiple - item scales of four constructs were subjected to a confirmatory factor analysis (cfa) to determine whether the manifest variables reflected the hypothesized latent variables. the reliability levels (coefficient alpha values) for each construct were above the cutoff of 0.70 proposed by nunnally. after reliability coefficients of the four dimensions reached the required level, the coefficient alpha for total items was calculated within the construct. the adequacy of the items was assessed by composite reliability, convergent validity, and discriminant validity. once the measures were validated, structural equation modeling (sem) this study used several fit indexes : chi - square to df ratio (/df), which is the chi - square fit index divided by degrees of freedom. the comparative fit index, cfi, compares the existing model fit with a null model that assumes the indicator variables (and hence also the latent variables) in the model are uncorrelated (the " independence model "). the normed fit index (nfi), nfi was developed as an alternative to cfi, but one which did not require making chi - square assumptions. the incremental fit index, ifi, is relatively independent of sample size and is favored by some researchers for that reason. root mean square error of approximation, rmsea, is also called rms or rmse or discrepancy per degree of freedom. rmsea is a popular measure of fit, partly because it does not require comparison with a null model and thus does not require the author to posit as plausible a model in which there is complete independence of the latent variables, as does for instance, cfi. the tucker - lewis index, tli, is similar to nfi, but penalizes for model complexity. sem also offers some important, additional benefits over these techniques, including an effective way to deal with multicollinearity, and methods for taking into account the unreliability of consumer response data. there were five latent variables in this model : food quality, volunteer issues, responsiveness, satisfaction, and behavioral intention. based on a literature search of previously conducted satisfaction surveys, a satisfaction questionnaire was developed. this instrument was adopted and modified in order to investigate the recipients ' satisfaction. the initial questionnaire contained 24 items, using a 5-point likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). the final questionnaire had 21 items and used the 5-point likert scale. in order to examine factors affecting the recipients ' satisfaction, the recipients were asked about their experiences with meals and services they had received during this study. the survey paper was yellow or blue instead of white because it would be easier for recipients and volunteers to distinguish them among other papers. the questionnaire language was simply written because federal regulations advise a 7th grade reading level for written consent for ordinary adults. questionnaires were prepared in two versions, english and spanish, because almost half of the recipients spoke and read only spanish. another native spanish speaker then translated the questionnaire back into english in order to confirm the accuracy of the spanish questionnaire. original and back translated documents were then compared, and it was confirmed that the spanish questionnaire was suitable for use in the study. forty questionnaires were distributed, and 20 questionnaires were collected in march 2008. originally, the questionnaire used for the pilot test had 20 items and two variables. unnecessary items were removed and some necessary items were added. for efficiency, extra distributors and collectors were not employed. volunteers delivering lunches for mow were asked to distribute the questionnaires when they delivered meals, and they were also asked to collect the questionnaires either the next day or the next time they completed their routes. the 1-page (two sided) questionnaire was printed on 8.5"11 " paper and folded in half. two different colors were used to differentiate the languages : yellow for english and blue for spanish. no code numbers were used on individual questionnaires to ensure the anonymity of responses. to encourage participation, the researcher made a $ 1 donation to mow for every completed survey. the data was compiled and statistically analyzed using the statistical analysis program spss (statistical package for social sciences) release 15.0 for windows (spss inc., the data were analyzed following anderson and gerbing 's two - step approach : a measurement model and a subsequent structural model. the multiple - item scales of four constructs were subjected to a confirmatory factor analysis (cfa) to determine whether the manifest variables reflected the hypothesized latent variables. the reliability levels (coefficient alpha values) for each construct were above the cutoff of 0.70 proposed by nunnally. after reliability coefficients of the four dimensions reached the required level, the coefficient alpha for total items was calculated within the construct. the adequacy of the items was assessed by composite reliability, convergent validity, and discriminant validity. once the measures were validated, structural equation modeling (sem) this study used several fit indexes : chi - square to df ratio (/df), which is the chi - square fit index divided by degrees of freedom. the comparative fit index, cfi, compares the existing model fit with a null model that assumes the indicator variables (and hence also the latent variables) in the model are uncorrelated (the " independence model "). the normed fit index (nfi), nfi was developed as an alternative to cfi, but one which did not require making chi - square assumptions. the incremental fit index, ifi, is relatively independent of sample size and is favored by some researchers for that reason. root mean square error of approximation, rmsea, is also called rms or rmse or discrepancy per degree of freedom. rmsea is a popular measure of fit, partly because it does not require comparison with a null model and thus does not require the author to posit as plausible a model in which there is complete independence of the latent variables, as does for instance, cfi. the tucker - lewis index, tli, is similar to nfi, but penalizes for model complexity. sem also offers some important, additional benefits over these techniques, including an effective way to deal with multicollinearity, and methods for taking into account the unreliability of consumer response data. there were five latent variables in this model : food quality, volunteer issues, responsiveness, satisfaction, and behavioral intention. the socio - demographic characteristics of respondents in this study are exhibited in table 1. out of 398 questionnaires, 265 (66.6%) were collected, and 209 questionnaires (52.5%) were used for the statistical analysis. the average age of respondents was 75.7 years old, and the largest group (22.0%) out of five age groups was less than 70 years old. more than half (52.1%) of the respondents were older than the average age, above 76 years old. the income level was very low ; the largest portion of the group (44.0%) made less than $ 10,000 per year. except for the respondents who did not answer this question, more than 90% were making less than $ 20,000 per year. most of them had retired long ago, so they were largely relying on a government subsidy or pension. about one fifth (21.5%) of the respondents did not answer this question, possibly because they did not remember how long they had been served meals from this organization. the average length of time respondents had been receiving meals was 2.84 years ; the largest group (23.9%) had been receiving meals between 1 and 2 years. the following average scores of perceptions toward meals and services were calculated : flavor (3.62), taste (3.69), vegetables (3.81), meat / fish (3.71), temperature (3.55), dessert (4.00), drinks (4.04), portion size (3.63), variety (3.82), appearance (4.68), kindness (4.75), attitude (4.72), meal schedule (4.13), and response (4.23). the results show that items for meals were rated slightly lower than those for service. specifically, temperature, flavor, and portion size were quite low compared to others. therefore, if mow organizations address the quality of these items, recipients will be more satisfied with this program. the level of internal consistency in each construct was acceptable, with cronbach 's alpha estimates ranging from 0.698 to 0.939. all of the composite reliabilities of the constructs were over the cutoff value of 0.50, ensuring adequate internal consistency of multiple items for each construct (table 2). in addition, the average variance extracted (ave) of all constructs exceeded the minimum criterion of 0.454, indicating that a large portion of the variance was explained by the constructs. the aves were greater than the squared correlations between any pair of constructs, suggesting discriminant validity. the ratio of to degrees of freedom was 2.433 (p < 0.001). given the known sensitivity of the statistics test to sample size, several widely used goodness - of - fit indices demonstrated that the confirmatory factor model fit the data well (nfi = 0.873, cfi = 0.986, ifi = 0.921, rmsea = 0.083). as a second step, the mow recipients ' perception model 1). the estimation produced the following statistics : (119) = 167.508 (p < 0.005), /df = 1.408, nfi = 0.927, cfi = 0.977, ifi = 0.978, rmsea = 0.044. the model 's fit as indicated by these indexes was satisfactory and path coefficients were analyzed. two paths between (1) volunteer issues and behavioral intention and (2) responsiveness and behavioral intention were not significant. the path for predicting a positive relationship between food quality and satisfaction was supported (11 = 0.733, t = 2.68, p < 0.01). the results show that having high food quality may create recipient satisfaction. also, food quality significantly influenced positive behavioral intention (21 = 0.762, t = 12.38, p < 0.001). while volunteer issues such as appearance, kindness, and attitude had less influence on satisfaction (12 = 0.238, t = 5.68, p < 0.001) than food quality and responsiveness (31 = 0.331, t = 6.38, p < 0.001). moreover, satisfied recipients have positive behavioral intention for mow programs (12 = 0.907, t = 18.38, p < 0.001). one reason for there being no significant association between volunteer issues / responsiveness and behavioral intentions could be related to recipients ' experiences. the average length of time respondents had been receiving meals was from 2.84 years and they were already satisfied and not concerned about retention of the service or word - of - mouth aspects. the structural equation model was re - estimated by constraining the direct effect of satisfaction on behavioral intentions (12 = 0). baron and kenny 's (1986) first three conditions were met in the original structural model (11, 12, and 12 were significant). the fourth condition was also satisfied ; the parameter estimate between satisfaction and behavioral intentions program (12 = 0.907, t = 18.38) in the mediating model became less significant (partial mediation) than the parameter estimate (to bi = 0.31, t = 2.093) in the constrained model. the difference in value between the constrained model ((120) = 171.6) and the mediating model ((119) = 167.508) was statistically significant (d(1) = 4.092, p < 0.05), indicating that the mediating model was a significant improvement over the constrained model. thus, the mediating effects of satisfaction clearly demonstrate that excellent food attributes produce favorable future behavior outcomes through satisfaction. this study aimed to evaluate recipients ' satisfaction with meals and services from the mow program in the usa. this allowed for better understanding of recipient 's satisfaction and behavioral intentions in the mow program. second, this study examined the way in which satisfaction mediates between perceived food quality and consumer behavioral intentions. the mediating effects demonstrate how consumer perceptions of food quality can affect behavioral intentions via the creation of satisfaction. the results also empirically support that satisfaction has a stronger positive indirect effect than the direct effect from other attributes on behavioral intentions. third, the results show that not all the proposed relationships were supported along with the effects of volunteer and responsiveness stimuli in association with satisfaction and behavioral intentions. this empirical evidence could establish the important links among food quality, volunteer issues and responsiveness, satisfaction, and behavioral intentions however, this study has limitations in terms of survey procedures, reliability, and generalization issues.. therefore, the reliability of the response can not be 100% certain because mow recipients may fear exclusion from the service if they respond with dissatisfaction. when the questionnaire was developed, the director of mow restricted some items such as ethnicity and illness records of recipients. moreover, this study did not deal in depth with nutritional issues. according to previous research, therefore, for future studies, greater development of the items and concern about nutritional issues is needed. if this study had been conducted in different segments of the mow program, the results may have shown different relationships among the studied constructs. | this study was conducted to evaluate recipients ' perception of service quality, satisfaction, and behavioral intention in home delivered meals program in the us. out of 398 questionnaires, 265 (66.6%) were collected, and 209 questionnaires (52.5%) were used for the statistical analysis. a confirmatory factor analysis (cfa) with a maximum likelihood was first conducted to estimate the measurement model by verifying the underlying structure of constructs. the level of internal consistency in each construct was acceptable, with cronbach 's alpha estimates ranging from 0.7 to 0.94. all of the composite reliabilities of the constructs were over the cutoff value of 0.50, ensuring adequate internal consistency of multiple items for each construct. as a second step, a meals - on - wheels (mow) recipient perception model was estimated. the model 's fit as indicated by these indexes was satisfactory and path coefficients were analyzed. two paths between (1) volunteer issues and behavioral intention and (2) responsiveness and behavioral intention were not significant. the path for predicting a positive relationship between food quality and satisfaction was supported. the results show that having high food quality may create recipient satisfaction. the findings suggest that food quality and responsiveness are significant predictors of positive satisfaction. moreover, satisfied recipients have positive behavioral intention toward mow programs. |
globally, the production of canned tuna is continuously increasing and the current european market demand far exceeds the availability of raw material that the fishing areas of the european union (eu) are able to provide, forcing fish processing companies to seek new and cheaper sources of supply, such as zero duty africa, caribbean and pacific (acp) countries. there are therefore considerably increased imports of tuna from countries such as the ivory coast, mauritius and papua new guinea and, with regard to italy, also from the seychelles (+ 48%) (fao, 2014), with italian processing companies increasingly supplied by fish from non - european waters (aniol and rupert, 2011). according to a study by the ministry of agriculture, fisheries and food of the spanish government (interatun, 2007), the eu s decision to lower the 12% tax on the importation of fish products from some non - european countries, has significantly reduced the competitiveness of european fishing. moreover, the checks carried out on imported products are not exhaustive and do not always the parameters set by european legislation, in particular regulations (ce) 853/2004, 854/2004, 882/2004 and 558/2010, with the consequent risk that the raw material is not of good quality and that the sterilisation processes normally used in the canning industry may be insufficient to eliminate the microbiological contamination present at the time of processing (landry., 1998 ; warne, 1988). the level of tuna meat contamination is influenced by the environment and the conditions and methods of fishing, time of year, handling, processing and evisceration, but particularly by the handling on board and the delayed cooling of fish during storage and transfer at markets and processing plants (figueroa., 2006 ; grau., 2003 ; doyle., 2000, 2008 ; figueroa., 2006 ; gonzales., 1997), but there are no specific studies on the difference in stability between the same type of canned products manufactured in different production and commercial environments. on the basis of this assumption, we perfomed a study on the comparision of the microbiological stability of canned tuna produced in italy and that manufactured by outside eu processing companies. the investigation covered microbiological laboratory analytical controls, aimed at verifying the presence of bacterial and fungal contamination in a number of commercial samples of canned tuna produced partly in italy and partly in non - european countries and purchased at food retailers during the commercial phase of the food chain. thirty - eight samples of canned tuna in oil from various brands available on the market in some italian regions, were analysed in packs of 80 and 125 g, of which 14 samples were manufactured in non eu factories and marketed on eu markets, while the other 24 were manufactured in italian factories and sold n markets outside eu. samples from outside europe came from ivory coast, colombia, per, ecuador, thailand, mauritius, indonesia and seychelles. the 24 samples of tuna manufactured in italy, belonging to the most popular brands, were from 5 of the most famous canning companies in the domestic market, which will be indicated by letters of the alphabet a, b, c, d and e. samples were displayed on shop shelves for sale at room temperature, before being identified and selected according to the purpose of the investigation. each sample was composed of at least 6 packs of canned tuna in olive oil, of which three were used for testing pre - incubation and three were incubated at 35c for 21 days. during incubation, the samples were checked daily to identify possible impairment (loss of content, can swelling, etc.), but no samples were removed for these reasons before testing began. the samples to be analysed, both those incubated and those non - incubated, were first subjected to a thorough cleaning and cleansing of the external surface of the containers and subsequently disinfected with a 5% chlorine solution. all packaging was equipped with ring pull opening, practiced in sterile conditions under a laminar flow hood. once collected, the samples were subjected to a series of qualitative and quantitative microbiological tests for verification of the possible presence of bacterial and fungal contamination. we proceeded to draw 10 g of content from each sample, diluted in 90 ml of saline solution and subsequently homogenised for 2 min, with the final preparation of decimal dilutions for quantitative tests, while 25 g of matrix was added to 225 ml of peptone water for salmonella spp. the microbiological parameters determined for each sample and the related test methods used were as follows : survey of salmonella spp. (uni, 2004a), total plate count (tpc) horizontal method for the enumeration of microorganisms - colony count at 30c technique (uni, 2013), enumeration of bacillus cereus (uni, 2005), enumeration of coliforms (iso, 2006), enumeration of e.coli (iso, 2001), enumeration of enterobacteriaceae (iso, 2004), enumeration of coagulase - positive staphylococci (uni, 2004b), enumeration of yeasts and molds (iso, 2008), enumeration of sulfite - reducing bacteria growing under anaerobic conditions (iso, 2003). for the specific bacterial colonies developed on the culture media used for counting bacterial colonies (cbc) at 30c, subcultures were carried out on columbia agar base (biolife) supplemented with defibrinated sheep blood and subsequently subjected to gram staining and identification through biochemical testing with an automated system (vitek 2tm compact ; biomrieux, marcy letoile, france). the identification of mold was obtained using morphology and colouring of the colonies, direct microscopic observation and staining with amman s lactophenol / fuchsin lactophenol. before and after pre - incubation at 30c for 15 days, a ph measurement (iso, 1999) was also carried out for all samples. the inspection tests and pre - analytical checks conducted on the containers and content of all 38 samples did not show any abnormality of organoleptic characteristics or outer casing sealing, both in the incubated samples and in those not incubated. table 1 shows that the samples of canned tuna in olive oil from outside the eu do not conform to the microbiological tests performed. in 8/14 non - eu samples (57%) tpc values between 10 and 10 cfu / g were found, as well as the presence of molds in 5/14 samples (35.7%) with mean values of contamination of 2860 cfu / g. in particular, the sample n.3 presented tpc values of 280 cfu / g already in the non - incubated product, while the post - incubation checks showed levels of tpc to be 10 cfu / g, with the simultaneous presence of 10 cfu / g of mold. no level of contamination was found in 6/14 samples. among the individual bacterial species constituting the contaminating flora, gram - positive bacteria such as enterococcus faecalis (120 cfu / g) in one sample and staphylococcus warneri (20 cfu / g) in one sample, gram - negative bacteria such as rhizobium radiobacter (agrobacterium tumefaciens) in two samples and spore - forming bacteria such as bacillus vallismortis in one sample still, with regard to testing on non - eu products, quantitative microbiological tests were performed for sulphite - reducing anaerobes, total coliforms, total enterobacteriaceae and qualitative microbiological tests for salmonella spp., bacillus cereus, e. coli and staphylococcus aureus, all of which constantly gave a negative outcome. as regards the canned tuna produced in italy (table 2), the samples produced from 3 of the 5 fish processing companies considered (companies c, d and e) were free of bacterial and fungal contamination (negative tpc preincubation and post - incubation, negative yeasts and molds numbers, negative detection of pathogens). in 2 samples produced by company a and in 5 produced by company b, however, high values of tpc were recorded and one case also showed fungal contamination. among the mesophilic bacterial flora, gram - negative bacteria (sphingomonas paucimobilis and acinetobacter iwoffii) and granpositive bacteria (streptococcus oralis) were observed. in the light of these results, another 3 samples of the product of a different batch and production date were performed, which showed a complete absence of any level of bacterial and fungal contamination. for company b, 13 different samples were tested, of which 5, from a different batch and production date, were contaminated with values post - incubation between 10 and 10 cfu / g and a fungal load of 100 cfu / g. among the bacterial species present, staphylococcus lentus and leuconostoc mesenteroides were observed, while among the fungi there were penicillium spp. and alternaria spp. as with the non - eu products, tests were performed on all italian production samples for the detection of salmonella spp., bacillus cereus, e. coli and staphylococcus aureus, as well as the determination of total coliforms, sulphite - reducing anaerobes and enumeration of enterobacteriaceae, all of which gave consistently negative results. lastly, a measurement of ph values was performed on all italian and non - eu samples, which gave values consistently between 5.8 and 6.5. the first important fact to consider is that more than 1/3 (39.4%) of the samples of canned tuna examined were contaminated with bacteria and fungi in the microbiological tests performed after an incubation period of 15 days at 30c, thus not being compatible with the specific criteria laid down by regulation (ce) 882/2004 (european commission, 2004c) and directive 97/78/ce (european commission, 1997). in fact, preincubation tests revealed bacterial and fungal contamination in only one sample (280 cfu / g), while in the other samples contaminated in the subsequent post - incubation tests, contamination was likely present in such low concentrations to be undetectable with the microbiological methods used. another important aspect is the absence of the main pathogenic bacteria responsible for food - borne outbreaks, since contaminations were supported in some cases by bacterial and fungal species with non - pathogenic effects in humans or animals (bacillus vallismortis, rhizobium radiobacter) (logan and de vos, 2009 ; hood., 1986), or in some circumstances, responsible for various human diseases such as urinary tract infections, bacteremia, endocarditis, and meningitis (enterococcus faecalis), or infections in patients with an impaired immune system (staphylococcus warneri), (kamath., 1992), nosocomial infections (sphingomonas paucimobilis, streptococcus mitis / oralis, leuconostoc mesenteroides) (shelburne., 2014 ; ryan and adley 2010 ; bou., 2008), and acute gastroenteritis (acinetobacter iwoffii) (nora and greg, 2009). among the species identified and in addition to human infections, only staphylococcus lentus was also associated with subclinical mastitis in sheep (schwendener and perreten, 2012). by then comparing the test results obtained from samples of canned tuna manufactured outside europe and in italy, the assessments must necessarily take into account that the same type of product has been used, although it is manufactured and sold in different social, productive and commercial contexts. if we exclude abnormalities in the thermal treatment process of products and possible contamination after treatment for both production contexts, the results may indicate problems of contamination along the production line or in the use of raw materials with such a high level of contamination to be completely inactivated by the thermal treatment of 116cx30 min, as normally used in the production of canned tuna. in both cases the quality of the raw material might also explain the varied results found in different production batches manufactured by the same company. the contamination of 7/24 italian samples (29%) and 8/14 non - eu samples (57%) represent negative datas and probably lead us to reconsider the concept of commercial stability of canned products as enunciated so far, especially in reference to the non - eu sample that was found already contaminated in the pre - incubation tests and also according to similar results previously obtained by other authors (ali., 2008). this research has revealed the existence of a bacterial and fungal contamination in canned tuna in olive oil commonly found in the market, both of italian and non - eu origin. if the risk could be hypothesised for the non - eu canned products, because of the rules, of the productive standards and of the less strict food security criteria in the countries from which these samples come from ; this risk does not exist for italian canned products thanks to the very good productive level and the established tradition of the italian canning industry, as well as the strict and effective national and eu regulation in force concerning food safety, such as regulations (ec) of the so called hygiene package and subsequent amendments. if we exclude abnormalities of the production process, the risk of contamination of canned products may derive from the quality of health conditions of the raw materials used. the quality levels of raw materials for fish processing industries do not always correspond sufficiently to the advantageous cost proposed by the international market for the supply thereof. this may also explain the irregularities found in the official checks on contamination prevention and food safety, resulting in the activation of the national alert system and in some cases even on a european level. hence, the need for a more incisive monitoring of the import of fresh or frozen tuna intended for processing by the canning industry. lastly, the authors repent the need to reconsider the concept of commercial stability applied to canned products. | the results of a study on the microbiological stability of canned tuna produced by italian companies and similar canned products manufactured in countries outside europe are reported herein. the study involved 38 samples of canned tuna of various brands, of which 14 were produced by companies outside europe and 24 by italian companies. qualitative and quantitative microbiological tests were conducted for the following parameters : bacterial colony counts at 30c, total coliforms, total enterobacteriaceae, sulphite - reducing anaerobes, salmonella spp., bacillus cereus, escherichia coli, staphylococcus aureus, yeasts and molds. bacterial loads and mold contamination were respectivelyin found in 8/14 (57%) samples from outside eu and 7/24 (29%) italian samples. the bacterial flora was represented by gram - positive bacteria (staphylococcus warneri, staphylococcus lentus, streptococcus mitis, enterococcus faecalis, leuconostoc mesenteroides), gram - negative bacteria (sphingomonas paucimobilis, acinetobacter iwoffii, rhizobium radiobacter), spore - forming bacteria (bacillus vallismortis), while the fungal species was represented by penicillium spp., rhizopus spp., rhodotorula spp. and alternaria spp. excluding anomalies in the thermal treatment process of products and any contamination after treatment, the contaminations encountered in both cases were most likely due to insufficient production quality standards and the quality of the raw material used. these results may require a redefinition of the concept of commercial stability as hitherto stated. |
mycotic aortic aneurysms are a rare condition with a relative incidence of 13% of all aortic aneurysms. the patients often present late with nonspecific symptoms, and these aneurysms are prone to rupture ; therefore, early diagnosis and urgent treatment is vital. computed tomography angiography (cta) is the first - line imaging modality for the diagnosis of both mycotic aneurysms and complications such as aortoenteric fistulas (aefs). aefs constitute a devastating clinical condition, which may cause abundant gastrointestinal hemorrhage with or without sepsis. prompt diagnosis and treatment is mandatory because without surgical intervention, the mortality rate approaches 100%. surgical conversion after endovascular aortic repair (evar) is recommended for mycotic aneurysms when the patient is amenable to open surgery. sugimoto. reported that timely surgical intervention after the control of sepsis provided excellent outcomes, whereas the mortality rate of patients with sepsis or rupture was high. a 72-year - old man was admitted to the emergency room with abdominal pain radiating to the back and high fever after extracorporeal shock - wave lithotripsy procedure. cta revealed an irregularly shaped, saccular mycotic infrarenal abdominal aortic aneurysm with a maximum diameter of 59 mm (figure 1a and b). a left - sided psoas abscess (554522 mm) a drainage catheter could not be placed ; hence, the abscess cavity was not liquefied. empiric intravenous antibiotherapy was administered upon clinical diagnosis since the aspirate and blood cultures were negative. (a) axial computed tomography angiography image of the irregularly shaped, saccular mycotic infrarenal abdominal aortic aneurysm (red arrows) and the left - sided psoas abscess (white arrows) is presented here. the infrarenal mycotic abdominal aortic aneurysm was treated with endovascular repair. because it was saccular with adequate proximal and distal lengths, control cta was obtained at the first month, and it demonstrated total thrombosis of the mycotic aneurysm ; however, the psoas abscess persisted. the patient was admitted to the hospital with the same complaints 3 months after evar. abdomen cta examination showed growth and pressurization of the aneurysm sac by type 1b endoleak (figure 2) and expansion of the tubular stent graft at the distal end due to the growth of the aneurysm. the patient was retreated with a bifurcated stent graft. a main body (2513120 mm, endurant medtronic) and a contralateral limb (161380 mm, endurant medtronic) sagittal reformat contrast - enhanced computed tomography images show the growing aneurysm sac by type 1b endoleak and the expanding tubular stent graft at the distal end due to the growing aneurysm. one month after re - intervention, the patient s left leg developed claudication in consequence of left - limb thrombosis due to compression. an extra - anatomic femorofemoral bypass graft was constituted surgically in order to cure the claudication. the mycotic aneurysm sac had disappeared on cta at 7 months follow - up. nonetheless, perigraft infection, air densities within the perigraft soft tissue and the main body of the tubular graft, and the thrombosed limb graft were observed. the psoas abscess remained stable since the diagnosis, and the symptoms relapsed despite the fact that the patient was on antibiotherapy. this time, total evacuation of the abscess cavity by percutaneous aspiration was performed successfully, but the aspirate and blood cultures were negative for the second time. air densities inside the graft and perigraft soft tissue also became prominent during the follow - up period. the patient was admitted to the emergency room with a septic syndrome after 14 months. laboratory values demonstrated leukocytosis, neutrophilia, and increased c - reactive protein and sedimentation levels. abdominal ct scan showed that the left - sided psoas abscess had grown to 9 cm in diameter and contained air fluid levels. the horizontal segment of the duodenum was adhered to the perigraft infectious tissue (figure 3). therefore, stent graft explantation, extensive surgical debridement, aortic ligation, and extra - anatomic right axillofemoral bypass graft reconstruction were performed. the psoas abscess was drained with a percutaneously placed large - bore catheter, and intravenous antibiotherapy was administered. although the abscess completely resolved and the catheter was withdrawn, it relapsed within 10 days. a drainage catheter needed to be introduced once again. multiplanar reformat images of the aortoenteric fistula (red arrows) with negative oral contrast media. in the early postoperative period, symptoms of acute abdomen emerged and small - bowel content was drained out through the surgical drain. ct scan with positive oral contrast media revealed free fluid and air in the peritoneal cavity and contrast media leakage through the surgical drain due to a duodenal fistula. finally the patient recovered and was discharged. when preoperative ct scans were reviewed retrospectively, although the duodenum was free from the process in the beginning, it became adherent to the perigraft infectious tissue as the disease advanced. a fistula could be differentiated in the ct scans with either positive or negative oral contrast media, which were acquired after the giant psoas abscess developed. imaging findings include eccentric, saccular shaped, rapidly growing aneurysms with periaortic soft tissue stranding, free fluid, and gas. the involvement of the adjacent anatomical structures (vertebral body destruction, discitis, psoas abscess, and aef) can accompany these features. open surgical repair with long - term antibiotherapy is accepted as the gold standard of treatment for a mycotic aortic aneurysm. it includes the resection of the infected aortic segment, extensive debridement, and either an extra - anatomical bypass or in situ bypass with a cryopreserved arterial allograft. recently, evar has been considered an alternative for high - risk patients or patients who are in an unstable condition. evar is a minimally invasive procedure with promising early and midterm outcomes ; its long - term durability, however, is still undetermined. based on data currently available, strahm. concluded that the early mortality rate of evar appeared to be lower than that of surgical repair. nevertheless, late aneurysm - related complications and mortality appear to be higher in evar than in surgery because there is no possibility to debride the infectious tissue and the infected aortic wall. infection control gains major importance when an endograft is implanted in an infected hostile environment. preoperative antibiotherapy duration has a major effect on the outcome of evar since persistent infection after evar is associated with a poor prognosis. however jia. reported that in a series of 12 cases treated with evar for a primary mycotic aneurysm, the late relapse rate was high despite adjunctive drainage and aggressive antibiotherapy. in our case, however, the persistence of the infection caused the regrowth of the aneurysm, which was repaired with a bifurcated graft. although the mycotic aneurysm sac vanished and the patient had asymptomatic periods, the third segment of the duodenum became adherent to the periaortic region because of ongoing infection. eventually, a fistula between aorta and duodenum evolved, producing gas within the first tubular graft and its occluded left limb. we evaluated this condition as an exacerbation of the psoas abscess ; nonetheless, not only should it be regarded as a complication of an aef but also other possible sources of infection should be evaluated. the incidence of secondary aefs related to open surgical repair is reported to range between 0.4 and 1.6%. the incidence of aefs due to evar is uncertain. as the proportion of aneurysms managed with evar progressive aortic wall erosion by the rigid extremities of the stent and infectious and inflammatory processes inside the wall of the aneurysm are among the major reasons for fistula formation. therefore, the risk of an aef is further increased in evar - treated mycotic aneurysms. surgical conversion after evar is recommended for mycotic aneurysms when the patient is amenable to open surgery. evar can provide opportunity to stabilize the patient s condition and infection before the operation. sugimoto. reported that timely surgical intervention after the control of sepsis provided excellent outcomes, whereas the mortality rate of patients with sepsis or rupture was high. in mycotic aortic aneurysm patients with aefs, endovascular repair should be accepted as a temporary method to save time and improve the patient s condition for surgery. although the risk of the rupture of the aneurysm is eliminated, there is a predisposition for the recurrence of the mycotic aneurysm and the development of an aef in this hostile environment if the infection source is not eradicated, as was the case in our patient. surgical conversion after evar is recommended for mycotic aneurysms when the patient is amenable to open surgery. our case highlights the importance of a careful follow - up of any possible complications after evar on a mycotic aneurysm and optimal timing for surgical repair. | mycotic aortic aneurysms account for 13% of all aortic aneurysms. the management of this disease is controversial. since open surgical repair is associated with high morbidity and mortality rates, endovascular aneurysm repair is an alternative treatment method with promising early and midterm outcomes, although its long - term durability is unknown.secondary aortoenteric fistulas may occur iatrogenically after either aortic reconstructive surgery or endovascular repair. as the number of aneurysms managed with endovascular aneurysm repair has substantially increased, cases of aortoenteric fistulas referred for endovascular repair are augmented.we report the case of an aortoduodenal fistula manifested with duodenal perforation after staged endovascular and surgical treatment of a mycotic aortic aneurysm. |
birth of a child with ambiguous genitalia often imparts great deal of apprehension and confusion to the involved families. diagnostic workup and timely designation of the gender to these patients clitoromegaly refers to an abnormal enlargement of a female clitoris, thus masquerading as a male penis. most of the cases of clitoromegaly in pediatric age group are attributed to hormonal etiologies with minority of cases affected by nonhormonal causes such as neoplastic infiltration. although complete diagnostic workup of these patients consists of extensive hormonal and chromosomal assays, in practice, pelvic imaging constitutes a vital preliminary step in evaluation. because of unparalleled tissue characterization and multiplaner capabilities, pelvic magnetic resonance imaging (mri) not only adequately depicts the urogenital anatomy in the hormonal causes of female virilization but also aids in surgical planning. moreover, amongst the nonhormonal etiologies, mri can also comprehensively delineate mass lesions infiltrating the clitoris. in this report, we present such a case of ambiguous genitalia in a female baby with congenital clitoromegaly, in which pelvic mri and mr neurography revealed the presence of extensive neurofibromatosis in the clitoris and lumbosacral regions. a 7-year - old child presented to the outpatient department of endocrinology clinic of our hospital for evaluation of ambiguous genitalia and intersex state. on physical examination [figure 1 ], there was presence of multiple light to dark brown patches of pigmentations in the lower abdomen, perineal region, bilateral groins, and upper thighs. the examination of genital region revealed a small penis that looked like an enlarged clitoris, with small scrotum that was separated and looked like labia. based on the clinical examination, the child was presumed to be genetic male and referred for karyotyping and ultrasound of pelvis to look for undescended testes. however, the patients karyotype came out to be 46xx and pelvic sonography revealed normal appearing uterus and bilateral ovaries. endocrine evaluation was done that ruled out precocious puberty, congenital adrenal hyperplasia, and other hormonal causes of clitoromegaly. lumbosacral plain radiographs were obtained which showed widened neural foraminae at the sacral vertebral level [figure 2 ]. mri was performed on a 1.5 t superconducting system (magnetom avanto, seimens medical system, erlangen, germany). the scan was performed in supine position after cushioning the legs with arms folded on the chest. the sequences used were fse t2 sagittal, axial with fat saturation, and coronal with fat suppression, and se t1 coronal and axial with post contrast images. postcontrast volume extrapolated breath - hold examination (vibe) axial and coronal images were also obtained with fat suppression. pelvic mri (sagittal t2 weighted images (wi) and coronal t1w sequences) confirmed the presence of a uterus measuring approximately 5 1.5 1.5 cm (appropriate for age) with normally visualized vagina and bilateral ovaries. t2w images showed homogenous lobulated mass lesions in bilateral sacral neural foraminae extending along bilateral lumbosacral plexus. distal extent of these masses, however, could not be delineated on these images. mr neurography [figures 3 and 4 ] of lumbosacral and thigh regions was performed using three - dimensional (3d) short tau inversion recovery sampling perfection with application - optimized contrasts using varying flip - angle evolutions (stir - space), which revealed the presence of multiple, lobulated, homogenous mass lesions long the nerve roots of bilateral lumbosacral plexus, bilateral sacral neural foraminae, extending into right thigh along the femoral nerve and bilateral broad ligament. a similar signal intensity and enhancement pattern was also noted within the hypertrophied clitoris and bilateral mass lesion measuring approximately 5 2 4 cm was seen involving clitoris and bilateral labial tissue. a provisional diagnosis of neurofibromatosis-1 was given based on the mri picture of plexiform neurofibroma and presence of extensive cafe au lait spots. the patient was referred to pediatric surgery department where excision of the clitoris mass was done with clitoroplasty. multiple brown patches of pigmentations are seen in the lower abdomen, perineal region, bilateral groins, and upper thighs. there was a lobular swelling in the right thigh along with ambiguous genitalia plain lateral radiograph of lumbosacral region shows widened sacral neural foramina (arrow) mr neurography : enlarged lobulated, serpentine, homogenous mass lesions are seen along the nerve roots of bilateral lumbosacral plexus, bilateral sacral neural foraminae, extending into bilateral thighs along femoral nerves which represent plexiformneurofibromas (arrows). note is also made of similar subcutaneous lesion in right medial thigh (arrow) mr neurography : hyperintense mass lesion is seen involving the clitoris (arrow). imaging forms an important step in the assessment of internal urogenital anatomy in a child with ambiguous external genitalia. ultrasound (us) still forms the first step in imaging of these patients for reasons beyond its widespread availability, primarily because complete screening of inguinal, perineal, renal, and adrenal regions can be performed quickly in the same examination. notwithstanding the initial evaluation, mri has a dominant status as the problem solving tool, contributing to the accurate morphologic evaluation of mllerian duct structures, the gonads, and the development of the phallus. a simple and elegant imaging algorithm has been suggested by chavhan. for evaluation of children with ambiguous genitalia. palpable gonads confirmed as testes on us, along with absence of uterus and ovaries should be diagnosed as male pseudohermaphrodite. however, presence of scrotal or abdominal testis along with demonstration of uterus on imaging, points toward the diagnosis of persistent mullerian duct syndrome, mixed gonadal dysgenesis or true hermaphroditism, depending on the absence of ovary, presence of extra streak gonad or an extra ovotestes, respectively. absence of palpable gonad or intraabdominal testes, along with us demonstration of uterus and ovaries should be assigned as female pseudohermaphrodite. in cases where us fails to demonstrate ovary in setting of well visualized uterus, mri should be performed to search for ovarian tissue before labelling the case as pure gonadal dysgenesis in which streak gonads are present instead. clitoromegaly associated with female pseudohermaphroditism (46, xx) is defined as clitoral index (length width) more than 15 mm in a neonate. poorly delineated supporting structures on mri such as bulbospongiosus, transverse perinei muscles provide a clue to differentiate hypertrophied clitoris from a normal penis. congenital enlargement of clitoris is most commonly caused by congenital adrenal hyperplasia or enzyme defect in pathways of steroid biosynthesis. less frequently, various nonhormonal conditions such as neurofibromatosis, epidermoidcysts, hemangiomas, nevuslipomatous cutaneous superficialis are reported causes implicated in clitoromegaly. isolated case reports of other lesions such as abscess, transitional cell carcinoma, melanoma, and lymphoma are also described in the literature. as was in our case, imaging is vital and often first to point out such diagnoses. plexiform neurofibromas, in the setting of neurofibromatosis type i, are the most commonly reported neoplasm of clitoris, thus resulting into female pseudohermaphroditism. although conventional mr sequences have been traditionally considered as the gold standard for diagnosis of neurofibromatosis, recent advent of mr neurography has imparted improved confidence in diagnosis and in aiding surgical planning through its multiplaner capabilities, precise and complete lesion localization, elegant tissue characterization and absence of partial volume averaging compared to conventional sequences. unidirectional motion probing gradient with higher diffusion moment (b value > 800 s / mm) is applied in anteroposterior direction in the axial plane as diffusion is more restricted across the nerve. the authors have used 3d stir space (sampling perfection with application optimized contrasts using varying flip angle evolutions) in the index case, which is used in the imaging of nerve plexuses. other sequences available for neuronal mr imaging are 3d spair space, 3d t2 space, and 3d ciss which are deployed in the evaluation of extremities, spinal cord (mr myelography), and cranial nerves, respectively. as in the present case, 3d images generated by mr neurography can elegantly demonstrate exact extent of the lesion with recognition of even subtle alterations in contour and thickness of the involved nerves. besides differentiation from other mimicking vascular and lymphatic lesions, mr neurography can also display otherwise inconspicuous internal fascicular structure of the neural lesion, disruption of which indicates towards malignant peripheral nerve sheath tumors in nf1. recent literature also describes the use of diffusion tensor imaging as useful adjunct in mr neurography to interrogate microarchitecture of the nerves involved. involvement of genitourinary system is rare in neurofibromatosis syndromes with reported prevalence of 0.65%, commonly involving urinary bladder and ureters. genital involvement in neurofibromatosis is even more infrequently encountered, although reported more commonly in females with clitoromegaly being the most common. however, sutphen. in his study over 236 families of neurofibromatosis-1 concluded that clitoris involvement in neurofibromatosis-1 might be more common than reported. less than 40 cases of genital neurofibromatosis have been reported till date, with most common involvement of clitoris, labia majora, prepuce, and unilateral labia in that order. genital involvement in males is commonly related to the involvement of penis and is mostly associated with concomitant genitourinary involvement. concomitant involvement of external genital system and lumbosacral region is previously only once reported with the involvement of the left half of penis and left lumbosacral plexus in a male patient. to the best of our knowledge, the present case is the first to have concomitantly involved external genitalia and lumbosacral plexus in a female patient. an important aspect of the index case was presence of neurogenic bladder, possibly owing to tumor infiltration of lumbosacral plexus. in the setting of neurofibromatosis, clinical and radiological features of bladder outlet obstruction have been reported in a few isolated case reports in which urinary retention were either attributed to tumor infiltration of bladder neck and prostatic urethra or due to involvement of nerves supplying the bladder. the differential consideration of neurofibromatosis as a possible cause of clitoromegaly may help avoid exhaustive and unnecessary investigations. considering that neurofibromatosis is one of the most common causes when clitoromegaly is caused by tumor infiltration, imaging should always be performed to look for presence of any abnormal mass lesion in the common locations, especially when accompanied by cutaneous lesions. our case report adds to the scant literature about the association of genital neurofibromatosis causing clitoromegaly, and highlights the crucial role of mri in assessment of both common and unpredictable etiologies leading to ambiguous genitalia. the present case also describes the utility of mr neurography as a problem solving tool and a useful adjunct to the conventional mri in augmenting the diagnostic confidence in cases of neurofibromatosis. | genitourinary involvement of neurofibromatosis is uncommon and genital neurofibromatosis is even rarer. involvement of clitoris by neurofibroma can lead to clitoromegaly masquerading as a male penis. we report such a case of ambiguous genitalia in a 7-year - old female child presenting with clitoromegaly since birth, in which magnetic resonance imaging (mri) revealed the presence of extensive neurofibromatosis in the clitoris and lumbosacral regions. we emphasize the central role of mri in evaluation of hormonal and non - hormonal causes of ambiguous genitalia. we further discuss the merits of including mr neurography in the imaging protocol for comprehensive delineation of neurofibromatosis. |
a significant quantity of food and beverages arrive on the market in a robust form of tinplate packaging. hermetically sealed can allow minimization of headspace oxygen and also keep a long and safe shelf life with minimal use of preservatives. however, the use of tinplate for food and beverage packing will result in some tin dissolving into food content. tinplate corrosion depends on many factors including can material, nature of the can linings and coatings, and nature and acidity of the contacting foodstuff. only limited reports are available on the toxicological effects of inorganic tin as present in canned foods, resultant from dissolution on the tin coating. the main potential hazard from acute ingestion seems to be gastric irritation in some individuals exposed to high levels. the maximum limit of 250 mg kg for tin in canned foods and 150 mg kg for tin in canned beverages was recommended by world health organization. within the european union large discrepancies exist between national regulations and maximum permitted levels range from 50 mg kg to 250 mg kg. determination of tin in canned food became very important because it gives information about the contamination process and provides help to increase canned food quality and safety. in order to evaluate tin concentration in canned food and beverages, several analytical methods are described in the recent literature : fluorimetric with use of surfactant reagents to increase the sensitivity [4, 5 ], flame (faas) and electrothermal atomic absorption spectrometry (etaas) [68 ], and inductively coupled plasma optical emission spectrometry (icp - oes) [911 ]. there are also several studies concerning the quality and resistance of tinplate after interaction with foodstuff which performed the use of x - ray microanalysis (eds) and scanning electron microscopy (sem) [12, 13 ]. atomic spectrometry methods in analysis of tin level in canned foods presume decomposition of food samples which is usually performed by acid digestion or dry ashing methods following by measurements in flame or graphite furnace mode of aas or icp - oes. although the preparation step could influence the complete recovery of sn, the sensitivity of applied measurement technique is also essential in correct evaluation of tin content in foods. for example, flame atomic absorption is recommended by european committee for standardization (cen) as a standard analytical method for the determination of tin in fruit and vegetables preserved in cans. in most of routine applications where sn content fall below 35 g g, the flame aas shows limited detection power. in combination with hydride generation system (hg), flame aas technique becomes more sensitive, but it is greatly complicated by the dependency of the hydride species on acidity of sample solution [6, 8, 15 ]. analytical performances of icp - oes technique allow measurements of numerous elements and/or more emission lines of the particular element. consequently, tin content could be determined in combination with other trace elements present in food samples [11, 16 ]. comparable results of different sample preparation procedures followed by icp - oes measurement of tin content were reported for matrices containing more than 30 mg kg of tin. however, interference effects in direct measurements of prepared solutions were not avoided completely, especially those caused by complex matrix. the technique of hydride generation with inductively coupled plasma optical emission spectrometry (hg - icp - oes) is widely used to enhance the sensitivity of semimetal determination and to eliminate the majority of matrix interferences in plasma. the literature survey still suffers from the lack of data concerning the tin determination in canned food by this technique. in the present work a method of the tin determination in canned food samples by hg - icp - oes method the aim of study was to select the most appropriate measurement conditions for tin determination, especially at low - concentration range where faas shows insufficient sensitivity. commercially available samples of fruits and vegetables analyzed by both methods were preserved in variously manufactured tinplate, that is, interior surfaces of cans were fully or partially lacquered. among the chemical and physical factors which influence tin transfer into canned food, therefore, hg - icp - oes as sensitive method for low - content tin determination in canned fruit and vegetable samples should be also helpful in estimating the efficiency and resistance of tinplate package. a prodigy high dispersion inductively coupled plasma optical emission spectrometer (teledyne leeman labs, hudson, nh, usa) was used for the tin content determination in all samples. emission lines used in this work were chosen as most prominent lines given in instrument line library file. a spectraa 220 fs atomic absorption spectrometer (varian, australia) equipped with deuterium background correction and ultraa sn lamp was used for flame aas determinations. tin analysis was performed in acetylene / nitrous oxide flame ; measurements of absorption were made at 235.5 nm with 0.5 nm slit aperture. for the homogenization of food samples a mixer bchi b400 (bchi labortechnik ag, switzerland) was used ; a hot block cs 54 (environmental express, usa) was used for the digestion of previously homogenized samples. high - purity - deionised water (milli - q element system, millipore, usa) was used for the preparation of standard solutions and dilution of samples. in the sample digestion procedures, a hydrochloric acid of suprapure grade (30% m / v) was used. single - element standard solutions of sn 1000 mg l (plasma pure, leeman labs, hudson, nh, usa) was used for the preparation of calibration standard solutions and control of plasma line positioning. all calibration standards for aas measurement were prepared during instrument run by automated dilution of reference tin solution of 50 mg l in 10% v / v hcl. for the determination of tin by hg - icp - oes, a fresh solution of nabh4 0.8% m / v in naoh 0.5% m / v was prepared. tartaric acid for the reduction of tin was prepared by dissolution of 10 g of solid compound in 1 l of ultrapure water. l by dilution with 1% m / v tartaric acid solution to appropriate volume. method of standard addition (msa) included aliquots of prepared samples in which a standard solution of tin was added. the twenty five samples of canned fruits and vegetables stored in original package at room temperature were opened, and the whole content was transferred into a mixer bchi b400 where homogenization step was performed. an amount of 5 g of homogenized sample was transferred into a hot block vessel, and 10 ml of 50% v / v hcl was added. samples were incubated in a hot block cs 54 at 80c for two hours with occasional shaking of vessel content. solution was then transferred by filtrating through filter paper (84 g / m) into 50 ml volumetric flask and filled up to a volume with ultrapure water. reagent blanks were included in each series of digestions. in order to prevent influence of oxidation and losses of analyte, samples stored in glass vessels should be submitted to measurement within 6 hours from preparation. atomic absorption measurements were performed in accordance with recommended standard procedure for the tin determination described in cen / ts 15506:2007. calibration of aas instrument was performed with five solutions which were automatically diluted from reference standard. control of calibration by external standard was performed after eight measuring steps, and recalibration procedure was repeated after twenty fourth measuring step. characteristic concentration (c c) of tin at 1% of signal absorption was 1.24 mg l. blank sample was measured prior to every set of samples. each of two replicates of samples was measured three times subsequently. for the purpose of hg - icp - oes measurements, the calibration standards and 10-fold diluted samples were mixed with a solution of nabh4 by three - channel peristaltic pump at a rate of 0.9 ml min. solutions were introduced into a reaction coil where stannane gas was generated and introduced into plasma through nebulizer and nebulization chamber. the rf power and nebulizer gas flow rate were optimized to give the maximum intensity of sn lines on l - pad detector, that are, 1300 w and 0.9 l min, respectively. this value was selected after collecting a signal by option of time - resolved analysis (tra) of instrument software. the signals started to rise after 25 seconds and plateau of signals was reached after 50 seconds from aspiration start. the precision of signal measurements expressed as relative standard deviation was established in the range 0.1%4.0%. a good linearity of all observed sn lines was obtained (r = 0.99950.9999). intensity measurement in msa mode was performed with those samples which were initially analyzed by aas and showed less than 5 mg l of sn content. an example of obtained msa curves for a selected sample was shown in figure 4. the intensity values in msa operation mode were one order of magnitude greater comparing to those obtained in standard calibration. linear coefficients comprised the same values (r = 0.9996) at each observed line. the method detection limits (c l), which were calculated using 3 criterion, that is, the concentration equivalent to three times standard deviation (3) of the signal (n = 10) of the reagent blank solution, are given in table 2. the limits of quantification (loq) were calculated from calibration curves and expressed in g kg of sample mass. hg - icp - oes measurements of tin content in samples were performed at all selected emission lines. the precision of measurement based on rsd calculations from three replicates showed the smallest disturbance of signal at 235.484 nm (rsd 0.1%) and 189.991 nm (rsd 0.4%), compared to 283.999 nm (rsd 2.6%) and 224.605 nm (rsd 4.0%). the sensitivity of measurements should be examined from the obtained calibration curves and tra signals. by aspirating the solution of low tin concentration, that is, 1 mg l the better sensitivity of signal was noted at 283.999 nm and 189.991 nm lines (figure 2). comparison of detection capability of tin at those lines showed lower detection limit at ionic line of 189.991 nm than at atomic line of 283.999 nm. lower detection limits, good linearity in the observed range of concentrations, and the better precision of measurements favoured the determination of tin content at 189.991 nm. comparing with literature data, the same conclusions from icp - oes determination of tin content without use of hydride generation were already derived, but an order of magnitude better sensitivity and detection capability is obtained here by use of hg - icp - oes system. hydride generation from samples where tartaric acid as interference - retarding agent is added also provides a selective formation of stannane gas which enters into plasma. tin content measured by flame aas and hg - icp - oes in different samples of canned fruits and vegetables is shown in table 3. all results are expressed as a milligram portion of sn per kg of sample mass, along with standard deviation of results (). description of tinplate protection of particular can, that is, fully or partially lacquered interior is also inserted in table 3. accuracy of results was tested by spiking of selected samples with sn standard solution after homogenization step. the selected samples were foodstuffs from complete - or partially lacquered can (nos. 1 and 13). hg - icp - oes measurement of spiked samples gave recoveries of 99% for sample 1 and 102% for sample 13. statistical comparison of results obtained by reference aas method and tested hg - icp - oes method were performed by two - paired t - test at significance level of p = 0.05. calculated t - value of 1.09 is lower than critical value of 2.06, which means that two methods were not significantly different. generally, the results of flame aas and hg - icp - oes measurements showed that tin content in all samples did not exceed maximum permissive level of 200 mg kg in foodstuffs. 11) of pineapples compote reached this level. comparing the results obtained from two applied techniques, it should be noticed that for the most of samples measured by aas and where tin content was above 5 mg kg, the similar results by hg - icp - oes were obtained. the exceptions were samples of pineapples compote (no. 9, 10, 12, 14, and 20) and one sample of peeled plum tomatoes (no 7) with slightly higher tin concentration obtained by hg - icp - oes. by knowing that samples for hg - icp - oes determination include a tartaric acid which also prevents a hydrolysis of possibly present sn (iv) species, observed difference is quite understandable. a sample of peach halves compote (no. 18) showed slightly higher sn concentration when measured by hg - icp - oes compared to concentration obtained by aas. since that longer delay from sample preparation to start of aas measurement could lead to hydrolysis of analyte and forming of insoluble sn (iv) compounds, a significant loss of sn absorption signal might occur. hg - icp - oes measurements include the use of highly reactive reducing agents which convert the majority of tin species in sn (ii) and favour the stannane gas formation. therefore, the much more reliable value in such case is denoted to hg - icp - oes measurement. 25, 7, 8) measured by aas shows limitation of method capability at low - concentration range (table 3). considering the results where aas measurements are limited at 5 mg kg of tin in canned food, the method of standard addition in hg - icp - oes technique showed exclusive advantage. tin content measured in diluted samples was an order of magnitude higher than the loq of 6.4 g kg. it must be mentioned that tin determination in standard calibration mode gave approximately similar results, but with very poor precision (> 20% rsd). changes of surface tension, relative volatility, and viscosity of such solution affect the droplet distribution in hg introduction system and consequently, the amount of tin that reaches the plasma. the complexity of organic matter impact is accomplished by entering solution into plasma where changes in excitation temperature and electronic density of plasma take place. generally, organic matter present in solution can affect the emission which results with enhancement or suppression of signal [18, 19 ]. therefore, method of standard addition allows compensation of matrix effects which is also evident from the improved precision of measurements. applied technique allows the determination of tin presence at low - concentration range where faas, xrf, or icp - oes without hydride system suffer from poor sensitivity [6, 7, 9, 10, 2022 ]. the results obtained for the food samples from fully protected inner walls of cans are very useful in predicting a quality of protection. it is shown that tenth times less portions of sn are present in protected cans than in non - protected or partially protected tinplate. the exceptions, measured as approximately 70 mg kg of tin, are established in samples 1 and 6. the studies concerning the quality of tinplate by sem and eds suggest that defects of lacquer result in tin exposure [12, 13 ] but in much less extent than that was measured in those two samples (nos. therefore, such concentrations might be attributed to the addition of tin chloride which also acts as preserving agent in manufacturing of canned foods [1, 2 ]. in a summary, analysis of tin content in canned fruits and vegetables was performed by use of flame aas which is recommended as a standard analytical method and also by hg - icp - oes. analytical efficiency of hg - icp - oes method was tested on several sn emission lines. low - detection limit, (1.9 g l) and loq of 6.4 g kg, good linearity in the observed range of concentrations, and the better precision of measurements favoured the determination of tin content at 189.991 nm. comparable results of tin content for samples from partially protected tinplate cans were obtained by use of aas and hg - icp - oes. method of standard addition in hg - icp - oes technique was applied on samples from complete lacquered cans where aas method is limited at 5 mg kg. this analytical advantage could utilize hg - icp - oes technique as a helpful tool in certain examination of tinplate protection efficiency. | tin content in samples of canned fruits and vegetables was determined by hydride generation inductively coupled plasma atomic emission spectrometry (hg - icp - oes), and it was compared with results obtained by standard method of flame atomic absorption spectrometry (aas). selected tin emission lines intensity was measured in prepared samples after addition of tartaric acid and followed by hydride generation with sodium borohydride solution. the most favorable line at 189.991 nm showed the best detection limit (1.9 g l1) and limit of quantification (6.4 g kg1). good linearity and sensitivity were established from time resolved analysis and calibration tests. analytical accuracy of 98102% was obtained by recovery study of spiked samples. method of standard addition was applied for tin determination in samples from fully protected tinplate. tin presence at low - concentration range was successfully determined. it was shown that tenth times less concentrations of sn were present in protected cans than in nonprotected or partially protected tinplate. |
the endothelium, the monolayer of endothelial cells lining the lumen of all blood vessels, plays an important role in the regulation of vasomotor tone control with the release of vasoconstrictor mediators.1 endothelial function can be assessed by flow - mediated dilatation (fmd) which be carried out noninvasively with ultrasound techniques on the brachial artery. fmd are widely used as modalities for evaluating atherosclerosis and can be used to predict cardiovascular events.2,3 the ankle - brachial pressure index (abi), the ratio of the systolic blood pressure of the ankle to the systolic brachial pressure, is an index of lower extremity arterial obstruction and a test for the screening of patients with peripheral arterial disease (pad). in healthy subjects, the blood pressure in the ankle artery is higher than that in the brachial, but, when systemic atherosclerosis in an artery of the lower extremities is present, ankle blood flow is barred, resulting in a drop in ankle pressure. the abi is commonly measured at rest, but abi values post - exercise enhance the sensitivity of the test and can be used to identify subclinical atherosclerotic vascular damage.4,5 however, it has not been established whether enhanced post - exercise abi is also associated with endothelial dysfunction. we hypothesized that a decrease in post - exercise abi is related to impaired endothelial function. to test this hypothesis, we investigated changes in post - exercise abi values and fmd in the elderly. the study population comprised 35 men and women aged 5177 years (5 men, 30 women, mean age : 66.2 6.7 years) who were admitted to the wellness 2008 center in osaka sangyo university for exercise prescription and health counseling. of these participants, 1 had angina pectoris, 4 had diabetes, and 3 had a history of smoking. patients who had pad or heart failure were excluded because of severe peripheral vascular damage. this study conformed to the ethical and human principles of research, and written informed consent was obtained from all study participants. resting abi was measured by a doppler flow meter (vasera, vs-1000, fukuda denshi, japan) in the supine position following a standard protocol. abi was also measured immediately post - exercise.6 the exercise protocol consisted of 2.5 minutes of active pedal flexion exercises at a speed of 60 times / min.6 endothelial function was assessed by measuring fmd in the brachial artery using ultrasound imaging (unexef, unex, japan), as previously described.7 data are expressed as mean standard deviation (sd). multivariate correlation analysis included age, body mass index, systolic blood pressure, and the presence of angina pectoris, as well as diabetes and a history of smoking, irrespective of the significance level in univariate analysis. the mean fmd was 5.24% 3.0%, the mean abi at rest was 1.11 0.06, and the mean abi after exercise was 1.04 0.06. the mean reduction in post - exercise abi from baseline readings was 8.6% 4.8% (table 1). no correlation was found between fmd and abi at rest (r = 0.054, p = 0.76). however, a weak correlation was found between fmd and post - exercise abi (r = 0.46, p = 0.06). a strong correlation was observed between fmd and the percentage decrease in post - exercise abi compared to baseline readings (r = 0.52, p = 0.01, fig. these associations could not be explained on the basis of individual differences in age, systolic blood pressure, or a history of angina pectoris, diabetes, or smoking. furthermore, multiple linear regression analysis was used to generate a prediction equation for fmd using the percentage decrease in post - exercise abi [fmd = 0.32 (% decrease in post - exercise abi) = 8.00, r = 0.25, p = 0.001 ]. significant correlations were observed between the ultrasound imaging - measured fmd and the predicted fmd (r = 0.27, p = 0.001). the present study shows that an increased reduction in post - exercise abi is associated with decreased fmd in elderly patients. additionally, we developed a prediction model for fmd using the post - exercise ankle - brachial blood pressure. these results suggest that post - exercise abi, that is, the exercise - induced ankle - brachial pressure mismatch, may be used as a simple surrogate parameter for the determination of endothelial function. generally speaking, endothelial function and blood pressure elevated systolic brachial pressure has been shown to contribute significantly to endothelial dysfunction, which is associated with increasing vasoconstriction. on the other hand, a functional alteration in the endothelium has been shown to result in high systolic brachial pressure. notably, an exaggerated brachial pressure response to exercise is a sign of early stage endothelial dysfunction. mirat suggested that impairment of endothelial function exists in normotensive subjects with exaggerated systolic brachial pressure during the maximal treadmill test.8 tzemos have shown that treated hypertensive subjects with an exaggerated brachial pressure response had endothelial dysfunction, as shown using a simple 3-minute exercise step - test. further, we have previously demonstrated that a low - level exercise - induced increase in brachial blood pressure, but not resting blood pressure, is representative of endothelial dysfunction in postmenopausal females.9 in the present study, we found that abi, the ratio of ankle blood pressure to brachial pressure, dropped after exercise in patients with endothelial dysfunction.10 to the best of our knowledge, this is the first study showing an association between abi and endothelial function. akopov have shown that the synthesis of nitric oxide, important for endothelial - mediated flow regulation, is also impaired in pad to an extent related to the fall in post - exercise abi.11 these findings partly support our results. importantly, endothelial dysfunction has the potential for reversal with risk management or lipid - lowering therapy. therefore, to improve prevention policies, all practitioners should pay attention to the presence of endothelial dysfunction, especially among participants with atherosclerotic risk factors. the measurement of abi has emerged as a simple surrogate marker for atherosclerosis and is highly predictive of total cardiovascular mortality. several studies have demonstrated that abi measured immediately post - exercise enhances this sensitivity. in a prospective cohort study, feringa reported that a reduction in post - exercise abi could identify patients who have normal resting abi values but are at increased risk of long - term mortality.12 our study provides new insights into the potential benefits of measuring post - exercise abi for identifying subclinical participants with endothelial dysfunction. second, we did not obtain relevant clinical data for atherosclerotic risk factors likely to affect endothelial function, such as low - density lipoprotein cholesterol, high - density lipoprotein cholesterol, and hemoglobin a1c level. finally, the number of patients was too small. only a large - scale clinical trial can provide definitive evidence on this interesting clinical topic. elevated systolic brachial pressure has been shown to contribute significantly to endothelial dysfunction, which is associated with increasing vasoconstriction. on the other hand, a functional alteration in the endothelium has been shown to result in high systolic brachial pressure. notably, an exaggerated brachial pressure response to exercise is a sign of early stage endothelial dysfunction. mirat suggested that impairment of endothelial function exists in normotensive subjects with exaggerated systolic brachial pressure during the maximal treadmill test.8 tzemos have shown that treated hypertensive subjects with an exaggerated brachial pressure response had endothelial dysfunction, as shown using a simple 3-minute exercise step - test. further, we have previously demonstrated that a low - level exercise - induced increase in brachial blood pressure, but not resting blood pressure, is representative of endothelial dysfunction in postmenopausal females.9 in the present study, we found that abi, the ratio of ankle blood pressure to brachial pressure, dropped after exercise in patients with endothelial dysfunction.10 to the best of our knowledge, this is the first study showing an association between abi and endothelial function. akopov have shown that the synthesis of nitric oxide, important for endothelial - mediated flow regulation, is also impaired in pad to an extent related to the fall in post - exercise abi.11 these findings partly support our results. importantly, endothelial dysfunction has the potential for reversal with risk management or lipid - lowering therapy. therefore, to improve prevention policies, all practitioners should pay attention to the presence of endothelial dysfunction, especially among participants with atherosclerotic risk factors. the measurement of abi has emerged as a simple surrogate marker for atherosclerosis and is highly predictive of total cardiovascular mortality. several studies have demonstrated that abi measured immediately post - exercise enhances this sensitivity. in a prospective cohort study, feringa reported that a reduction in post - exercise abi could identify patients who have normal resting abi values but are at increased risk of long - term mortality.12 our study provides new insights into the potential benefits of measuring post - exercise abi for identifying subclinical participants with endothelial dysfunction. second, we did not obtain relevant clinical data for atherosclerotic risk factors likely to affect endothelial function, such as low - density lipoprotein cholesterol, high - density lipoprotein cholesterol, and hemoglobin a1c level. finally, the number of patients was too small. only a large - scale clinical trial can provide definitive evidence on this interesting clinical topic. to summarize, a reduction in post - exercise abi from baseline readings is associated with endothelial dysfunction in elderly. post - exercise abi appears to be a simple and reliable surrogate marker for endothelial dysfunction in the elderly, although larger studies are required for validation. | background : the ankle - brachial pressure index (abi), the ratio of the systolic blood pressure of the ankle to the systolic brachial pressure, is commonly measured at rest, but abi values post - exercise enhance the sensitivity of the test and can be used to identify atherosclerotic vascular damage. however, it has not been established whether or not enhanced post - exercise abi is also associated with endothelial dysfunction. we hypothesized that a decrease in post - exercise abi is related to impaired endothelial function.purpose:to investigate alterations in post - exercise abi values and endothelial dysfunction in the elderly.methods:the study population comprised 35 men and women aged 5177 years (mean age : 66 years). patients with peripheral arterial disease or a history of heart failure were excluded. the abi was estimated at rest and immediately after exercise. the exercise protocol comprised 2.5 min of active pedal flexion exercises at a speed of 60 times / min. endothelial function was assessed by measuring flow - mediated vasodilation (fmd) in the brachial artery using ultrasound imaging.results:no correlation was found between fmd and the abi at rest. however, a weak correlation was found between fmd and post - exercise abi (r = 0.46, p = 0.06). a strong correlation was observed between fmd and a decrease in post - exercise abi compared to baseline readings (r = 0.52, p = 0.01). multiple linear regression analysis was used to generate a prediction equation for fmd using the percentage decrease in post - exercise abi. significant correlations were observed between the ultrasound imaging - measured fmd and the predicted fmd (r2 = 0.27, p = 0.001).conclusions : post - exercise abi appears to be a simple surrogate marker for endothelial function in the elderly, although larger studies are required for validation. |
increased microglial activity facilitates beneficial responses to central nervous system (cns) injuries, including phagocytosis of debris and clearance of apoptotic cells ; however, unregulated microglial activity can lead to production of neurotoxic factors that worsen cns pathology and cause neuronal degeneration [17 ]. microglia constitute the main immune cells in the cns and provide innate immunity under physiological conditions and adaptive immunity under stress, promoting inflammation in response to various signals from apoptotic cells [14 ]. the phenotype of cns resident macrophages is considered activated and designated m1 or classical activation, which describes the proinflammatory phenotypic response. m2 or alternative activation describes phenotypic responses to cytokines, such as interleukin-(il-) 4 and il-13. in many neurodegenerative diseases, persistent injury (such as intraneuronal protein accumulation) promotes the production of proinflammatory molecules (figure 1), like tumor necrosis factor (tnf)-, interleukin (il)-1, il-6, reactive oxygen species (ros), and nitric oxide (no). proinflammatory factors activate microglia [10, 11 ], which may remove not only apoptotic or damaged neurons, but also healthy neurons, aggravating the pathogenic process. the inflammatory response is generally localized to areas of cns injury via communication between immune cells and stressed neurons. innate inflammation is reported in alzheimer 's disease (ad), parkinson 's disease (pd), and the tauopathies (reviewed in). in the healthy brain, microglia have a resting deactivated phenotype (ramified). activated microglia are present in human postmortem brain tissues of patients with tauopathies, including ad, frontotemporal dementia with parkinsonism linked to chromosome-17 (ftdp), progressive supranuclear palsy (psp), and corticobasal degeneration (cbd) [1315 ]. also associated with neurodegeneration in these diseases are hyperphosphorylated tau (p - tau) deposits [1623 ]. it has been demonstrated in animal models of ad that the endotoxin lipopolysaccharide (lps) promotes both inflammation and the accumulation of p - tau and that suppression of microglial activity prolongs survival in ftdp - associated p301l transgenic mice. our laboratory has previously shown a differential increase in microglial activity in response to accumulation of p - tau in lentiviral wild type tau versus mutant p301l mice at 1 month after - injection. cell culture models also demonstrate that proinflammatory cytokines can induce p - tau [2729 ]. these data suggest that microglial activity aggravates p - tau through a common underlying mechanism moderating communication between microglia and neurons. determining how this mechanism is temporally altered in response to p - tau is critical to understanding the beneficial or detrimental role of microglial activity in different stages of disease pathology [3032 ]. a central question in current research pertains to how communication between microglia and neurons, in which pathogenic proteins accumulate, affects the progression of inflammation. one inducer through which neurons and microglia can communicate to regulate inflammation is fractalkine (cx3cl1) (figure 1). cx3cl1 is a 373-amino acid protein that has a chemokine domain located on top of a mucin - like stalk [33, 34 ]. the membrane - bound cx3cl1 can serve as an adhesion molecule for leukocytes expressing the fractalkine receptor (cx3cr1) and soluble cx3cl1 can function as both a proinflammatory chemoattractant that activates receptive inflammatory cells [33, 37 ] and an anti - inflammatory, neuroprotective agent that reduces neuronal apoptosis. the relationship between soluble cx3cl1 in peripheral blood and inflammatory diseases of the cns is unclear. several findings suggest that deletion of cx3cr1 increases microglial activity in various models of acute and chronic neuronal injury [4043 ].. increased levels of serum cx3cl1 are reported in patients with multiple sclerosis [39, 44 ], traumatic brain injury, and human immunodeficiency virus (hiv) with cns complications, but increased levels of serum cx3cl1 are not observed in patients with guillain - barr syndrome and viral and bacterial meningitis. genetic variants with reduced levels of cx3cr1 are linked to age - related macular degeneration in humans. cx3cl1 and its cognate receptor cx3cr1 may play an important role in immunoregulation in animal models of neurodegeneration. cx3cl1 expression is decreased in the cerebral cortex and hippocampus in the aged brains of amyloid precursor protein (app) transgenic mice. decreased cx3cl1 levels are also observed in aged ad transgenic mouse models (tg2576) in association with increased a levels. microglial activity was increased while the levels of a load and cx3cr1 were decreased in myd88 mice, suggesting cx3cl1 involvement in a clearance. cx3cr1 deficiency leads to decreased levels of a deposition and protects against a toxicity in transgenic mouse models of ad [50, 51 ]. lps induces p - tau of both endogenous and transgene - derived tau in nontransgenic mice and in a humanized mouse model of tauopathy, depending on lps dose and cx3cr1 deficiency. additionally, impairment of cx3cl1 signaling pathway leads to deterioration in cognitive function and synaptic plasticity via alteration of il-1 function. although cx3cr1 deficiency exacerbates ad - related neuronal and behavioral pathologies in mice overexpressing human a, these effects are likely to be associated with the level of cytokine production and not a plaque load, suggesting that alteration of proinflammatory factors, including tnf- and il-6 may modulate cx3cl1 signaling. conversely, production of no, il-6, and tnf- may be inhibited by cx3cl1 [53, 54 ]. exogenous cx3cl1 is neuroprotective in some other models of neuroinflammation [55, 56 ], and disruption of cx3cl1-cx3cr1 communication by deletion of the cx3cr1 gene causes neurotoxicity in mouse models of systemic inflammation, pd, and amyotrophic lateral sclerosis but protects against neuronal loss in a mouse model of focal cerebral ischemia. cx3cr1 knockout mice show more toxicity and substantia nigra (sn) degeneration in response to lps treatment following administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp), a neurotoxic precursor of 1-methyl-4-phenylpyridinium (mpp+). together, these studies suggest altered microglial activity through cx3cl1 signaling, which may play a direct role in immunoregulation depending upon the cns insult. cx3cl1-cx3cr1 signaling is therefore a possible mediator of communication between injured neurons and microglia and may play a significant role in the regulation of microglial activity in response to pathogenic protein accumulation in early, or protein secretion, in later stages of disease. a primary feature of ad is the presence of extracellular aggregates of a peptide (plaques) and intracellular inclusions (tangles) containing p - tau [5961 ]. variants of a peptide, including a42 and a40, are produced by the cleavage of app and subsequent cleavage of an intermediate fragment, app c - terminal fragments (ctfs). cleavage of app at an alternative site within the a region by the cleaving enzyme -secretase precludes a formation [63, 64 ]. the causal association between mutations in app and the onset of familial ad supports the role of a in ad pathogenesis. it is likely that in the early stages of disease, a accumulates intraneuronally prior to the formation of extracellular plaques. it is also likely that the intracellular pool of a is externalized as neurons die, contributing to the formation of senile plaques [6770 ]. the presence of intraneuronal a is significant in that such a presence constitutes a preplaque stage of ad pathology. our laboratory has previously shown that intraneuronal a induces microglial and astrocyte activation and increases inflammatory markers in gene transfer models [71, 72 ]. furthermore, it has been shown that intraneuronal a can cause apoptosis and cell death, which stimulate microglial and astrocyte activation independently of extracellular plaques. these results implicate communication between microglia and a expressing neurons in the onset of inflammation in ad. inflammation has been associated with neurodegenerative disease etiology in ad, in which a and tau can act as inflammatory stimuli to promote microglial activity [1, 7476 ]. therefore, inflammation in ad may arise not only from extracellular plaque formation, but also as a consequence of communication between microglia and a-expressing neurons. accumulation of intraneuronal a can induce damage to lysosomes and multivesicular bodies, leading to leakage of a from vesicles into the cytosol and activation of inflammatory mechanisms without extracellular accumulation of amyloid plaques. several studies have suggested that manipulation of chemokines and/or their receptors may be a therapeutic target in neurodegenerative diseases, including ad [7779 ]. microglia treated with recombinant cx3cl1 or il-34 partially protect against a toxicity via enhancement of a clearance and antioxidant production. significant differences in cx3cl1 levels were detected in a cohort of 51 patients with mild cognitive impairment (mci), 51 ad patients and 57 controls. however, the increase in plasma cx3cl1 levels is not congruent with tissue levels, which are decreased in the hippocampus and frontal cortex of advanced ad cases, suggesting variable roles of cx3cl1 in different stages of ad pathogenesis. the level of plasma soluble fractalkine was significantly higher in mci and moderate ad patients compared to severe ad, suggesting that higher levels of soluble plasma fractalkine is associated with greater cognitive impairment. therefore, the fractalkine signaling pathway that mediates communication between microglia and neurons is deficient in ad brains and downregulated by a. the characteristics of pd include death of dopaminergic neurons in the sn [50, 58, 82 ] and formation of lewy bodies (lbs) [8392 ], or inclusions comprised mainly of -synuclein [8399 ]. the simultaneous occurrence of -synuclein and tau pathology is observed in multiple system atrophy (msa), though the mechanisms underlying a possible connection between the two proteins are unknown [100, 101 ]. early onset familial pd arises from mutations in the autosomal recessive genes parkin, pten - induced kinase-1 (pink1), and dj-1 while late onset pd is associated with dominantly - inherited mutations in leucine - rich repeat kinase 2 (lrrk2) and -synuclein. aggregation of -synuclein is implicated in the activation of microglia and subsequent inflammation associated with pd. it was previously thought that -synuclein - related pathology was confined to within neurons, but recent research suggests that microglia are activated following the release of -synuclein aggregates into the extracellular space by apoptotic cells. several microglia - derived inflammatory factors (ros, no, tnf-, and il-1), as well as lps, promote death of dopaminergic neurons [104106 ]. the phagocytosis of -synuclein by microglia induces nadph oxidase activity and the production of ros. inflammation has also been detected in pd brains lacking lbs, such as parkin - linked autosomal recessive early onset pd. these cases, as well as the role of tau as a risk factor for pd, suggest that additional mechanisms regulate inflammation. for example, cx3cl1 suppresses microglial activation and protects against neuronal loss and striatal lesion in 6-hydroxydopamine (6-ohda) rat model of pd. mpp+ increases neuronal cx3cl1 levels in rat sn, but administration of cx3cr1 antagonists blocks pd - like pathology, including loss of dopaminergic neurons and motor behavior, suggesting that fractalkine can modulate microglial activation in pd models. deletion of cx3cr1 aggravates microglial neurotoxicity in response to lps in the mptp model of pd and in the superoxide dismutase 1 (sod1) g93a model of als, suggesting that cx3cl1 signaling may limit microglial toxicity. the level of plasma soluble cx3cl1 also correlates positively with disease severity and progression in human pd patients, suggesting that cx3cl1 can be used as a biomarker to differentiate between neurodegenerative diseases. whether inflammation rescues or exacerbates cell death in neurodegenerative disease likely depends on the stage of disease progression. microglial activation facilitates the removal of apoptotic cells and toxins from the cns, releasing neurotrophic factors that aid in repair following injury. the apparent ambivalence of increased microglial activity is associated with unsuccessful attempts to provide anti - inflammatory treatment in human clinical trials. preliminary clinical trials in which nonsteroidal anti - inflammatory drugs (nsaids) were administered before the development of neurodegeneration suggested that disease risk was reduced by inhibition of the immune response [110, 111 ]. however, later trials found that anti - inflammatory drugs were harmful in ad patients. these conflicting data reflect the current lack of understanding of the role of the immune response in cns diseases and point to the importance of the temporal relationship between the disease stage and the anti - inflammatory intervention. the timing of the immune response in relation to disease progression complicates the use of anti - inflammatory treatment in various cns diseases. for example, the permanence of brain damage following stroke or ischemia depends on the activity of proinflammatory cytokines, the activation of microglia, and the recruitment of leukocytes [112, 113 ]. it has been found that inhibiting tnf- and il-1, which mediate postischemic activity by attracting leukocytes to the injury or by damaging cells directly, confers neuroprotection in animal models of stroke [112, 113 ]. in ad models, the involvement of innate immunity via microglial activation and phagocytosis of a renders anti - inflammatory therapy particularly relevant to the study of ad [114116 ]. in ad patients, however, deficits in toll - like receptors (tlrs) expression inhibit the removal of a from the brain and result in lack of a clearance by macrophages, and tlr2 deficiency in ad mouse models is associated with severe cognitive impairment. in addition to anti - inflammatory treatment, intervention in the hematopoietic system has been suggested as a possible model of treatment for ad. the administration of macrophage colony - stimulating factor, a hematopoietic cytokine, to mouse microglia promotes degradation of internalized a in vitro and protects against cognitive decline in vivo when administered prior to the development of learning and memory deficits, supporting the importance of timing of anti - inflammatory treatment relative to disease progression. taken together, these findings support the targeting of innate immune cells as a therapeutic approach for ad and other neurodegenerative diseases. however, conflicting data from clinical trials necessitate further investigation of the role of the immune response in disease development and progression. research on the suppression of microglial activity has been actively pursued with limited success and strategies to manipulate the protective role of microglia the detection and removal of apoptotic cells have not been fully investigated [122124 ]. these strategies warrant further research, as apoptotic cells that enter secondary necrosis and trigger inflammation [126, 127 ] increase tissue damage. in this context, the role of cx3cl1 in mediating communication between preapoptotic neurons and microglia becomes greatly important. such intervention would be relevant in early stages of disease progression, during which intracellular accumulation of pathogenic proteins anticipates apoptosis and the formation of extracellular protein aggregates. in later stages of disease pathology, decreased cx3cl1 signaling may activate microglia and induce p - tau (figure 2), which exacerbates disease progression by promoting apoptosis. additionally, the use of nsaids to restrain microglial activity may exacerbate pathology due to lack of phagocytic clearance of secreted extracellular amyloids, including -synuclein, a and p - tau. in this context, targeting microglial activity in later disease stages may be detrimental and contributory to disease progression. however, targeting the cx3cl1 pathway in early disease stages could be beneficial, at least in delaying disease progression via restraint of microglial activity. along this line of thought, nsaids administration could regulate key proinflammatory cytokines (figure 2) that would modulate cx3cl1 signaling and microglial activity. it remains to be fully elucidated when and how alteration of proinflammatory markers may increase or decrease cx3cl1 signaling, which may either activate or suppress microglia. one possibility is increased cx3cl1 levels to restrain microglial activity and prevent the exacerbation of p - tau damage. however, this intervention should be timed to avoid interference with microglial activity when patients progress into more advanced stages of disease, during which removal of extracellular deposits becomes necessary. therefore, understanding the critical interplay between proinflammatory (tnf-, il-6, il-1, and il-1), anti - inflammatory cytokines (il-10, tgf-, il-34, etc.), and fractalkine levels to modulate microglial activity is highly significant. furthermore, whether the activation of microglia in the context of neurodegenerative disease is beneficial or detrimental may also depend upon the type of disease. successful anti - inflammatory treatments of cns diseases will likely be specific not only to the stage of disease pathology, but also to the type of disease. it has been found that many of the same cytokines are implicated in the pathology of ad, pd, and als despite distinct patterns of neuronal loss in each disease. previous literature presents contradictory evidence regarding the effects of targeting microglia in various cns diseases. glass., for example, suggest that targeting microglia in pd and als is detrimental while other studies suggest that targeting microglia aids in a clearance in ad. further investigation of the role of the inflammatory response in each disease will determine the potential for anti - inflammatory treatments. here we suggest a temporally - defined strategy of intervention in which early targeting of cx3cl1 signaling slows disease progression and prevents p - tau formation. | the role of inflammation in neurodegenerative diseases has been widely demonstrated. intraneuronal protein accumulation may regulate microglial activity via the fractalkine (cx3cl1) signaling pathway that provides a mechanism through which neurons communicate with microglia. cx3cl1 levels fluctuate in different stages of neurodegenerative diseases and in various animal models, warranting further investigation of the mechanisms underlying microglial response to pathogenic proteins, including tau, -amyloid (a), and -synuclein. the temporal relationship between microglial activity and localization of pathogenic proteins (intra- versus extracellular) likely determines whether neuroinflammation mitigates or exacerbates disease progression. evidence in transgenic models suggests a beneficial effect of microglial activity on clearance of proteins like a and a detrimental effect on tau modification, but the role of cx3cl1 signaling in -synucleinopathies is less clear. here we review the nature of fractalkine - mediated neuronmicroglia interaction, which has significant implications for the efficacy of anti - inflammatory treatments during different stages of neurodegenerative pathology. specifically, it is likely that anti - inflammatory treatment in early stages of disease during intraneuronal accumulation of proteins could be beneficial, while anti - inflammatory treatment in later stages when proteins are secreted to the extracellular space could exacerbate disease progression. |
despite their usually low stability, organic radical cations1, 2 have raised substantial interest in a wide range of research areas owing to their critical importance in a multitude of organic and biological reactions, for which they act as reactive intermediates. furthermore, radical cations that absorb light in the nearinfrared (nir) region have attracted much attention in the materials science community, as this feature may potentially enhance the efficiency of solar cells.3 the core structure of radical cations is usually based on fused rings and/or metal complexes. radical cations with monocyclic rings have received substantially less attention in comparison.4 the core units of these molecules usually contain heteroatoms such as nitrogen, oxygen, and sulfur.1b, 2i, 2j, 5, 6 the properties of triarylaminederived radical cations have especially been studied in great detail in this context.7 in general, their absorption bands at approximately =700 nm are subject to a bathochromic shift. n, ndimethylaminophenylsubstituted triphenylamines show absorption bands at approximately =1300 nm. however, most of these compounds are unstable and/or are present only at low temperature. for example, the radical cation of 1,3,6tris(dipanisylamino)pyrene, which contains fused rings, exhibits a halflife of 3 h.8 in addition, several radical cations generated from sulfurcontaining monocyclic aromatics, such as thiophenes9 and thiazoles,10 have been reported. some thiazole radical cations, such as 1butyl5methylthiazolium,9d have been reported, but their photophysical properties have not been disclosed. furthermore, even the radical cations derived from large molecules with heterocyclic rings, such as azathiahelicene, exhibit halflives of only approximately 8 h.2h in this context, we recently developed 5aminothiazoles with various substituents (scheme 1) that could be readily prepared from thioformamides and secondary thioamides. these 5aminothiazoles represent the first examples of thiazoles that contain diarylamino groups at the 5position,11 and they show a wide range of absorption and fluorescence properties that change upon exposure to external stimuli, such as the addition of acids. thiazoles contain electrondonating (d) and electronaccepting (a) moieties that deviate from coplanar alignment. these da structures usually lead to fluorescence from intramolecular charge transfer (ict) states.12 therefore, the electrons of compounds having the da structure are delocalized over the molecule in stages of absorption and emission. thiazole derivatives moreover exhibit a reversible oneelectron oxidation wave in their cyclic voltammograms (figure s1, supporting information). for example, the halfwave potential of 1 in ch3cn appears at + 0.623 v, whereas that of 3, which contains a 4methoxyphenyl electron donor at the 5position, appears at + 0.407 v. this result indicates that 3 oxidizes more easily than 1. in contrast, the cyclic voltammogram of 4,13 which bears a 4dimethylaminophenyl group at the 5position, exhibits two reversible oxidation waves. the oxidation potential and waveform of 5aminothiazoles can thus be easily controlled by judicious choice of the substituents at the 5position. on the basis of these results, we expected that chemical oxidation of 5aminothiazoles would generate stable radicals, for which we anticipated absorption in the uv / vis nir region. herein, we report the chemical and electrochemical oxidation of 5aminothiazoles to generate radical cations that show uv / vis nir absorption bands. in one case, a halflife time of more than 300 h was observed. to identify appropriate reagents for the chemical oxidation of 5aminothiazole 1, bf2cf3 oet,14 no[sbf6],15 and [(4brc6h4)3n][sbcl6 ] (magic blue, mb)16 were added to solutions of 1 in ch2cl2 (scheme 2). upon the addition of bf2cf3 oet, the color of the solution changed from yellow to orange. however, the thusobtained compounds were very unstable. the use of no[sbf6 ] did not afford any new species, as evident from quantitative recovery of the starting materials. notably, a single chemical species was only obtained with mb, and therefore, we subsequently used mb to generate single chemical species of 14. reactions between thiazole 1 and bf2cf3 oet, no[sbf6 ], and [(4brc6h4)3n][sbcl6 ] (magic blue). we then measured the absorption spectra of the radical cations derived from 14 (figures 1 and s2). whereas 14 showed absorption maxima at approximately =358410 nm (figure 1 a), we expected bathochromic shifts in their absorption bands upon the addition of mb. the addition of 1 equivalent of mb to 1 afforded new absorption bands at =900 and 650 nm (table 1). after the addition of 1 equivalent of mb, the absorption maxima of 2 and 3, both of which contain methoxy groups, were observed at =918 and 932 nm. furthermore, the addition of 1 equivalent of mb to thiazole 4, which contains the strongest electron donor, resulted in a strong absorption in the nir region (=1180 nm). these results suggest that the wavelengths and strength of the absorptions can be adjusted by the substituents introduced on the thiazole rings. absorption spectra of a) 14 and b) 1+mb to 4+mb in ch2cl2, [solute]=110 m uv / vis absorption spectra of thiazoles 14 with mb. [a ] in ch2cl2, [solute]=110 m. the addition of > 3 equivalents of mb to 4 also induced a shift in the absorption band (figure s3). the absorption of 4 gradually changed from =760, 1006, and 1180 nm to 796, 1474, and 2726 nm, whereas the color of the solution changed from red to green. these results corroborate that thiazole 4 generates different radical species, depending on the amount of mb added. to compare the photophysical properties of the chemically oxidized species with those of the electrochemically oxidized species,17 we measured the cyclic voltammograms and absorption spectra of 1. in ch2cl2, 1 showed a oneelectron oxidation wave with peaks at e=1.04 and 0.73 v (figure s4). thus, a potential of 1.04 v was applied to a ch2cl2 solution of 1 for the electrochemical oxidation while the timedependent absorption spectra of this solution were measured (figure 2). the oxidation potentials and the waveforms of the oxidation waves could be controlled by judicious choice of the substituents of the 5aminothiazoles. the observed spectra of electrochemically oxidized 1 were found to be similar to those of chemically oxidized 1 (figure s5). hence, the spectral changes should be attributed to the generation of radical species, and the radical species derived from 1 showed absorption bands at 450 and 650 nm. timedependent absorption spectra of 1 after application of 1.04 v in ch2cl2 ; = 110 m, 0.1 m [nbu4n][clo4 ] ; scan rate=100 mv s ; reference electrode : ag / agcl, counter electrode : pt, and working electrode : pt gauze. to elucidate the properties of the chemically oxidized species, the electron paramagnetic resonance (epr) spectra were measured at room temperature for ch2cl2 solutions of 14 that contained 1 equivalent of mb (1+mb to 4+mb, figure 3). in all cases, the formation of radical species was observed, and the epr parameters are summarized in table 2. the g tensor and hyperfine coupling constant (hfcc, a) values were determined from spectral simulations, whereby a n and a h refer to interactions with nitrogen and hydrogen atoms, respectively. the g values of the spectra for 1+mb to 4+mb (g=2.00302.0034) differ substantially from the g value of a pure mb solution (g=2.0102). thiazoles 3 and 4, with electrondonating groups at the 5positions, showed higher g values than 1 and 2. moreover, upon adding > 3 equivalents of mb to 4, the g value of the 4+mb solution changed (g=2.0044) relative to that observed for the addition of 12 equivalents of mb. this result indicates that the properties of the radical species of 4 drastically change upon adding an excess amount of mb. observed epr spectra of a) 1, c) 2, e) 3, and g) 4 with 1 equivalent of mb, as well as that of i) 4 with 3 equivalents of mb ; the spectra in panels b, d, f, h, and j are the corresponding simulated spectra. signals marked with arrows in panels a, c, e, g, and i represent the mn marker. [a ] in ch2cl2 (distilled from p2o5), [solute]=110 m. the g and a n values were determined from spectral simulations. [b ] calculated by integrating the area of the epr signals, normalized to the peak height of the signals from the mn marker as g standard. to elucidate further details from the epr spectra, we simulated the spectra for 1+mb to 4+mb with software for isotropic epr spectra provided by jeol (figure 3 b, d, f, h, j). in the spectra of 1+mb to 3+mb, only the nitrogen atom at the 5postition affected the spectral line shape. interestingly, the epr spectrum of 4+mb, which exhibited two reversible oxidation waves in its cyclic voltammogram, included components of both the nitrogen atoms and hydrogen atoms, which implies that the unpaired electron is more delocalized in 4+mb than in 1+mb to 3+mb, despite the relatively short halflife of 4+mb (3 h). 5aminothiazoles with electrondonating groups at the 5position and electronaccepting groups at the the smallest a n value among 13 was observed for 2 (0.63 mt), which contains an electrondonating group at the 2position. a slightly higher a n value was observed for 3 (0.78 mt), which contains an electrondonating group at the 5position, and this value is also marginally higher than that of 1 (0.72 mt).18 nevertheless, these a n values are much smaller than that of 2,2,6,6tetramethylpiperidin1oxyl (tempo, 1.59 mt),19 in which an unpaired electron is localized on the nitrogen atom. this result implies that thiazolebased radicals contain more delocalized electrons than, for example, tempo, and hence exhibit higher levels of stability, which is reflected in the relatively long halflives of 1+mb to 4+mb (e.g. 3+mb : 1/2=385 h, 16 days ; table 2). interestingly, 4+mb exhibited, depending on the amount of mb added, two types of epr spectra. upon the addition of 12 equivalents mb, 4+mb showed multiplet signals, whereas upon the addition of > 3 equivalents of mb, the signals associated with 4+mb disappeared. the simulated spectrum of 4+mb (figure 3 h) was very similar to the experimentally observed spectrum (figure 3 g) and reflected interactions with one nitrogen atom (a n=0.64), two other nitrogen atoms (a n=0.34), and eight hydrogen atoms (a h=0.29). in contrast, the addition of > 3 equivalents of mb to 4 furnished another radical species (figure 3 i). the simulated spectrum (figure 3 j) reflects interactions with two nitrogen atoms (a n=0.43) and is in good agreement with the experimentally observed spectrum. timedependent density functional theory (tddft) calculations for neutral 1 showed that the maximum absorption band (=416 nm) should be assigned predominantly to the homolumo transition (97 % contribution). the homo is mainly localized on the substituent at the 5position and the thiazole ring, whereas the lumo is located predominantly on the phenyl group at the 2position and the thiazole ring., we used the longrangecorrected camb3lyp20 functional instead of the b3lyp functional to recalculate the uv / vis absorption spectrum. the recalculated spectrum revealed an absorption maximum at =349 nm, which is in good agreement with the experimentally observed value (=364 nm). we also calculated the uv / vis absorption spectrum for the radical cation of 1. at the b3lyp level, an absorption band at =890 nm was obtained, which is in excellent agreement with the experimental value (=900 nm). this excitation was assigned predominantly to the cationhomosomo transition (97 % contribution) shown in figure s6. the cationhomo is very similar to the homo1 of neutral 1, whereas the somo is essentially the singly occupied homo of neutral 1. a detailed comparison between the homo of neutral 1 and the somo showed a slightly higher contribution from the phenyl group at the 2position for the latter, which was probably caused by increased charge transfer from the substituent at the 5position. as a result, the calculated nc(5position of thiazole) and c(2position of thiazole)c(2position phenyl group) bond lengths contract upon removal of one electron from 1.399 (nc neutral) to 1.367 (nc radical cation) and from 1.472 (cc neutral) to 1.455 (cc radical cation). the cationhomo and somo essentially exhibit contributions from the same region, which suggests that the consideration of longrange corrections for the hybrid b3lyp method is not necessary. the calculated cationhomo somo energy gap (2.10 ev) is much smaller than the homo lumo gap in neutral 1 (b3lyp : 3.56 ev ; camb3lyp : 5.96 ev). this difference should be responsible for the dramatically redshifted uv / vis absorption after the oneelectron oxidation. for organic radicals, the b3lyp method can be used to predict the epr parameters.21 the g tensor shifted from the freeelectron value, and the a n values were predicted by dft calculations for our radicalcation species. the calculated g tensor and the isotropic fermi a n values for the n atom at the 5position (2.0029, 0.74 mt) agree very well with the experimental data (2.0030, 0.72 mt) shown in table s1, which verifies the validity of the results obtained from calculations at the b3lyp/631+g(d) level of theory in this particular case.22 the spin density map for the radical cation of 1 (figure 4) shows positive (blue) and negative (green) spin densities at an isodensity level of 0.004 e bohr. the spin density is mostly delocalized over the n atom at the 5position and the neighboring conjugated c = cn = c fragment in the thiazole ring. the radical character of the cation is also reflected in the occupancy (0.88 e) of the orbital of the lone pair of electrons for the n atom at the 5position analyzed by nbo 6.0.23 mulliken spindensity map for the radical cation of 1 calculated at the b3lyp/631+g(d)/pcm(ch2cl2) level of theory. in conclusion, radical cations 1+mb to 4+mb were generated by adding 1 equivalent of magic blue (mb) to thiazoles 14. the electron paramagnetic resonance (epr) spectra of 1+mb to 4+mb indicated that it was the nitrogen atom at the 5postition that mainly affected the spectral line shape of these thiazolebased radical cations. radical cations with electrondonating groups at the 5position (i.e. 3+mb and 4+mb) showed larger g values than the other radical cations. moreover, upon the addition of 3 equivalents of mb to 4 a different radical species was obtained. radicals 1+mb to 4+mb showed smaller a n values than 2,2,6,6tetramethylpiperidin1oxyl (tempo), which suggested a correspondingly higher level of delocalization and, consequently, more stable radical cations. among these radical cations thus, compounds with strong intramolecular chargetransfer character that contain one moiety that is able to react with oxidants may represent a new concept for the design of stable organic radicals. the absorption wavelengths of 14 showed a bathochromic shift upon the addition of 1 equivalent of mb, and the absorption bands of 1+mb to 4+mb were observed in the nearinfrared region. the absorption spectra of chemically oxidized 1 showed changes similar to those observed in the spectra of electrochemically oxidized 1. therefore, chemical and electrochemical oxidation methods can be chosen for the generation of similar chemical species. further studies on the applications of 5narylaminothiazoles as stable dyes with a wide absorption and emission range are currently in progress in our laboratory. electrochemical measurements were performed by using a platinum working electrode, a platinum gauze working electrode, a platinum wire counter electrode, and a ag / ag [0.01 m agcl ] reference electrode in ch2cl2 with 0.1 m [nbu4n][clo4 ]. measurements were recorded with a bas cs3a cell stand and sec2000 spectra system. prior to use, ch2cl2 was distilled from p2o5. thiazoles 14 were prepared according to literature procedures.11a a ch2cl2 solution of the corresponding thiazole (110 m, 0.1 ml) was diluted in ch2cl2 (0.8 ml), and the mixture was stirred for 1 min. a ch2cl2 solution of magic blue (110 m, 0.1 ml) was then added, which afforded a ch2cl2 solution of the radical cation (110 m, 1 ml). then, an aliquot (0.3 ml) of this solution was transferred to an epr sample tube (diameter=1 mm), and the measurements were conducted. to measure the photophysical properties, a) epr spectrum of a solution of tempo and b) epr simulated spectrum. electrochemical measurements were performed by using a platinum working electrode, a platinum gauze working electrode, a platinum wire counter electrode, and a ag / ag [0.01 m agcl ] reference electrode in ch2cl2 with 0.1 m [nbu4n][clo4 ]. measurements were recorded with a bas cs3a cell stand and sec2000 spectra system. prior to use, ch2cl2 was distilled from p2o5. a ch2cl2 solution of the corresponding thiazole (110 m, 0.1 ml) was diluted in ch2cl2 (0.8 ml), and the mixture was stirred for 1 min. a ch2cl2 solution of magic blue (110 m, 0.1 ml) was then added, which afforded a ch2cl2 solution of the radical cation (110 m, 1 ml). then, an aliquot (0.3 ml) of this solution was transferred to an epr sample tube (diameter=1 mm), and the measurements were conducted. to measure the photophysical properties, a) epr spectrum of a solution of tempo and b) epr simulated spectrum. as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | abstractchemical or electrochemical oneelectron oxidation of 5narylaminothiazoles was found to afford stable radical cations. for chemical oxidation, 1 equivalent of [(4brc6h4)3n][sbcl6 ] (magic blue, mb) was added to ch2cl2 solutions of the thiazoles, and the thusobtained radicals showed light absorption in the nearinfrared region. electrochemical oxidation also led to bathochromic shifts in the absorption bands, and the obtained spectra were similar to those derived from the chemically oxidized species. these radicals afforded electron paramagnetic resonance (epr) spectra that are consistent with the notion of stable nitrogen radicals (halflife385 h). the epr spectrum of a thiazole containing 4dimethylaminophenyl groups on the nitrogen atom at the 5position changed significantly upon adding > 3 equivalents of mb. details of the electronic structures of the experimentally obtained radical cations were generated from theoretical calculations. |
emergency hematopoiesis defines the production of functional hematopoietic cells under nonhomeostatic, proinflammatory, or biologically stressed conditions [14 ]. blood cell production is a tightly regulated process that, after birth and throughout life, starts in a conspicuous hematopoietic stem cell (hsc) subset residing within the bone marrow (bm). our current understanding of how hsc early differentiation is governed by the microenvironment indicates that, besides the stromal cell components of the various hematopoietic niches, not only essential growth and differentiation factors but also microbes and their products can influence differentiation fate decisions [3, 5, 6 ]. of note, emergency hematopoiesis is regulated at the stem and progenitor cell (hspc) level, where conditions such as infection demand the expedited production and activation of innate immune cells to combat noxious extrinsic agents, and the resulting proinflammatory conditions can at the time regulate the earliest steps of the hematopoietic development in favor of the clearance of insulting cues and to further maintain homeostasis. we have previously shown that pathogens and damaged tissue products and proinflammatory cytokines promote emergency hematopoiesis and alter patterns of early lymphoid differentiation in mouse and human [35, 710 ]. in mice, pathogen recognition through toll like receptors (tlr) and the resulting cytokine release induce the expansion of hsc and instruct lineage differentiation fates so immediate innate cell development is guaranteed [6, 7 ]. in general, ligation of tlr2 and tlr4 on these seminal cells promotes redirection toward myeloid cell production, while the sole tlr9 stimulation of primitive common lymphoid progenitors (clp) strikingly induces b cell differentiation blockage while development of dendritic cells (dc), plasmacytoid dendritic cells (pdc), and nk - related interferon killer dendritic cells (ikdc) is substantially enforced [5, 8 ]. in humans, most findings relate to strengthening of myeloid lineage cell production under emergent scenarios, whereas adjustments within the lymphoid branch of the hematopoiesis have been poorly addressed [2, 6 ]. according to what mouse research has shown, human multilymphoid progenitors (mlp) are capable of responding to tlr stimulation by producing dendritic cells, and our recent work suggests that primitive early lymphoid progenitor populations are also capable of microbial components discrimination through tlr, a mechanism that mostly facilitates their differentiation to innate lymphoid lineage cells. of special interest, tlr9 ligation on adult bm progenitors promotes the quick development of nk lineage cells by using mechanisms that involve il-15r upregulation [4, 9 ]. thus, innate immune quick responses against viral threatening infections start in earlier developmental stages than we previously thought. whether the actual tlr - emergent hematopoiesis contributes to innate immunity under pathological conditions and other biological stress settings, including malignant diseases, interestingly, not only conventional pathogen associated molecular patterns (pamps) but also the damage associated molecular patterns- (damps-) like molecules can trigger innate immune sensors and prr signals, including micrornas, histones, fibronectin, and bacterial second messengers like di - gmp (reviewed in [3, 12 ]). even though efficient therapeutic agents have been developed that improve infectious and malignant disease outcomes and increase the overall survival rates, the adjuvant effect of molecules capable of remodeling hematopoietic pathways thus, the possibility of having extensive means of replenishing innate cells opens additional venues for receptor - ligand axes of clinical significance. disruption of normal peripheral blood leukocytes results in the release of heterogeneous mixtures of peptides, among other complex molecules. upon dialysis, the enriched mixture of low - molecular - weight polar and hydrophilic peptides (< 10 kda), named dialyzable leukocyte extracts (dle), has shown a number of therapeutic and adjuvant properties through modulation of immune responses [13, 14 ]. although the precise molecular mechanisms underlying its positive experimental and clinical effects are currently unknown, critical signaling pathways for survival and cellular activation states, including toll like receptor (tlr) and nfb, are apparently involved and often trigger the production of proinflammatory cytokines [1418 ]. of note, a recent investigation using in vitro controlled models of tlr - mediated proinflammation suggested the content of tlr-2 agonist ligands within dle. the exposure of human peripheral mononuclear cells to dle induced the copious secretion of tnfalpha by the monocyte fraction. whether this phenomenon is due to damps or damp - like related peptides within dle is still a matter in question. using an elegant mouse model of experimental tuberculosis, fabre and colleagues demonstrated that the administration of dle (formerly denominated transfer factor) evokes an efficient reconstitution of the cell - mediated immunity, concomitant with a substantial production of ifnalpha, il-2, and inos, an immune protective profile provoking inhibition of bacterial proliferation [19, 20 ]. in contrast, inflammatory injuries of human ocular tissues where limbal epithelial stem cells giving rise to corneal epithelium are compromised have been shown to respond to dle treatment by downregulating the secretion of il-8 and il-6. the same is true for atopic dermatitis, where dle contribute the decrease of inflammatory cells and the severity of the disease. then, despite the proinflammatory potential induction of dle, the net balance induction or suppression may depend on additional biological settings of the damaged tissues, where the dose of dle would be absolutely crucial to get a beneficial result. resolution of herpes virus infections is remarkably benefited from the adjuvant effect of dle treatment [13, 16 ]. in fact, a herpes murine model has become a powerful biological assay to test the functional activity of dle. of special interest, a direct effect on viral replication or infected target cell viability could not been recorded. instead, its protective effects correlated with serum cytokine levels and, most likely, with changes in the cellular immune system. accordingly, as cell - mediated immunity plays a central role in controlling viral - infected and tumor cells, and the capability of dle of strengthening the cellular immunity has been suggested from several studies, dle is considered as a presumptive instrument with adjuvant potential for treating virally induced cancer. moreover, a growing list of viral, parasitic, or fungal infections, as well as acute and chronic diseases, including immunodeficiencies, malignancies, allergies, and autoimmune disorders, seems to favorably respond to dle. however, a more comprehensive understanding of its biological mechanisms is yet needed and will be benefited from less heterogeneous and highly controlled extracts. dle transferon, a blood product licensed for clinical use, has been shown to exhibit batch - to - batch- reproducibility and relatively high homogeneity when ultra - performance liquid chromatography (se - uplc) is used to characterize its content. due to the putative tlr agonist elements within dle and their biological capability of inducing proinflammatory microenvironments, here we sought to examine the dle 's contribution to innate immune replenishment through emergent hematopoiesis. by using in vitro functional assays and controlled early differentiation culture systems, our findings define a powerful route to promote development of a unique cd11c nk cell subset with immune - surveillance capacity. umbilical cord blood (ucb) samples were obtained from normal full - term neonates upon mothers ' written informed consent, while adult bone marrow (abm) was collected from healthy adult donors who entered orthopedic surgery and according to institutional guidelines. mononuclear cells (mncs) were prepared by ficoll - paque plus (ge healthcare bioscience) gradient centrifugation and preserved at 80c until use. all procedures were approved by the ethics and scientific committee of health research at imss (r-2006 - 3602 - 16). cd34 cells from abm and ucb were enriched using the human cd34 progenitor cell isolation kit (miltenyi biotec) according to manufacturer instructions and our previous reports. after staining with pe - conjugated antilineage markers (cd3, cd8, tcr, cd56, cd14, cd11b, cd20, cd19, and cd235a), anti - cd34-apc, anti - cd38-fitc, and anti - cd45ra - pe - txr conjugated antibodies, primitive cell populations were highly purified by multicolor flow cytometry using a facsaria sorter (bd biosciences). hematopoietic stem cells (hsc) were separated as lincd34cd38cd45ra, while multipotent progenitors (mpp) were sorted as lincd34cd38cd45ra, and early lymphoid progenitors (elp) as lincd34cd38cd45ra, as described. upon harvesting from culture, anti - cd56-pe, anti - cd11c - fitc, and anti - cd16-apc (bd biosciences) nk cells were identified by flow cytometry in a facscanto ii equipment (bd biosciences) as cd56cd11c or cd56cd16cd11c cells, while dendritic cells (dc) were detected as cd56cd11c. analysis of flow cytometry data was performed using the flowjo 10 software (treestar inc. dle transferon is manufactured by udimeb at gmp facilities in the national school of biological sciences, national polytechnic institute (ipn), as described [14, 15 ]. cells were disrupted by 5 cycles of freezing and thawing followed by dialysis against water using spectra / pore membranes with a cut - off of 12 kda (spectrum labs, usa). the quality control of transferon comprised (a) endotoxin content, quantified using the endosafe - portable test (charles river laboratories, charleston, sc, usa) according to the manufacturer 's instructions (the specification for endotoxin was established in mexican pharmacopeia, section mga-0316 (4.0 eu / ml)) ; (b) microbiological tests, according to mexican pharmacopeia, section mga-0571 ; and (c) physicochemical characterization by a validated ultra - performance liquid chromatography (uplc) method that analyzes molecular weights and the time of retention of the main peaks compared with those of an internal batch pattern. peptide content per final dose was measured by bicinchoninic acid (bca) method using the pierce bca kit (thermo fisher scientific, waltham ma, usa) according to the manufacturer 's instructions. batch - to - batch reproducibility is consistently analyzed by se - uplc chromatographic profiles, while ifn production from dle - stimulated jurkat cells is quantified as a biological activity test. enriched cd34 cells from ucb and abm were cultured for 24 hours in -mem medium supplemented with 10% fetal bovine serum, 100 u / ml penicillin, and 100 mg / ml streptomycin. dle transferon was used for cell culture stimulation at 5 g / ml. ms-5 stromal cells were grown in presence or not of dle transferon 24 hours before coculturing with cd34 hspc. on the other hand, and upon 24 hours of dle prestimulation, hspc (including the whole enriched cd34 fraction or lincd34cd38cd45ra hsc, lincd34cd38cd45ra mpp, or lincd34cd38cd45ra elp) were centrifuged to remove the medium and cocultured in the presence of ms-5 stromal cells for 30 more days. cocultures were performed using -modified essential medium (-mem) (gibco) supplemented with 10% fetal bovine serum and 100 u / ml penicillin and 100 mg / ml streptomycin. lymphoid lineage cytokines and growth factors were contained throughout coculture : 1 ng / ml flt3-l (fl), 2 ng / ml scf, 5 ng / ml il-7, and 10 ng / ml il-15 (preprotech). coculture systems were incubated at 37c in a humidified atmosphere of 5% co2. enriched cd34 cells from ucb and abm were cultured for 72 hours with 5 g / ml of dle transferon and bromo-2-deoxyuridine (brdu) to a final concentration of 10 m, in -mem serum - free medium supplemented with lymphoid cytokines. after stimulation, cells were stained for the identification of cd34 cell progenitors by multicolor flow cytometry followed by intracellular staining with a monoclonal antibody to brdu according to an established protocol (brdu flow kit, bd biosciences). ucb cd34 cells were pretreated with 20 m of myd88 control or inhibitory peptides (imgenex img-200501) for two hours, followed by 2x washing and staining with 5 m cell trace violet (ctv) dye. dle transferon was included or not at 5 g / ml in a 72 h culture. the dilution of fluorescence intensity was estimated using the application for cell proliferation from the flowjo 7.6.2 software. to investigate the influence of tlr signals in the dle - mediated nk - like cell differentiation, ucb cd34 cells were pretreated with 20 m of myd88 control or inhibitory peptides (imgenex img-200501) for 24 hours, followed by 2x washing and coculturing on ms-5 stromal cells for 15 more days. cocultures were performed using -modified essential medium (-mem) (gibco) supplemented with 10% fetal bovine serum and 100 u / ml penicillin and 100 mg / ml streptomycin (as described above in section 2.4). natural killer cytolytic activity was evaluated using a fluorescence - based assay, as described [9, 23 ]. this assay uses the dye carboxyfluorescein succinimidyl ester (cfse) to distinguish target from effector cells, and the dna intercalating dye 7-aminoactinomycin d (7-aad) (bd pharmingen) for dead / live cells distinction. briefly, the myeloid leukemia k562 cell line (atcc) was kept in log phase growth in rpmi 1640 + glutamax supplemented with 10% of fetal calf serum (fcs). k562 cells were stained with 5 m carboxyfluorescein succinimidyl ester (cfse). in vitro differentiated nk cells from dle - cultures were enumerated by flow cytometry and cocultured with cfse - labeled k562 cells, according to an effector : target ratio curve. il-2 (40 ng / ml) (preprotech) was added to induce nk cell activation, followed by 4 hours of incubation at 37c. cells were washed and 7-aad (bd pharmingen) was incorporated into the cell suspension. multiparametric flow cytometry (facscanto ii, bd biosciences) was conducted to determine functionality. to investigate the capability of dle - newly derived nk - like cells of producing ifn - gamma (ifn) upon stimulation, an ifn assay was performed. after 30-day coculture, the produced cells were stimulated with il-12 (10 ng / ml) and il-18 (50 ng / ml) for 12 hours. nk - like cells were harvested and brefeldin a (bfa, 10 g / ml) was added for 4 hours to inhibit protein intracellular transport. after bfa blockage, cells were carefully washed, followed by incubation with anti - cd56-apc, anti - cd11c - fitc (bd biosciences), and anti - ifn-pe (biolegend) conjugated antibodies to perform extra- and intracellular staining, respectively. gammadelta t peripheral blood lymphocytes (tcr-1) from healthy donors were enriched by the t lymphocyte isolation kit (miltenyibiotec, igg1 clone 11f2) according to manufacturer instructions, followed by staining with 5 m cell trace violet (ctv). simultaneously, newly differentiated cd11ccd56 nk cells from dle - stimulated 30-day cocultures were harvested and purified by flow cytometry sorting in bd facsaria equipment. postsort cell purity was investigated by using the anti - human tcr / mouse igg1 antibody clone b1. the purified t cells were then cocultured with the nk cell population of interest, at a ratio of 2 : 1 cd56cd11c nk : t lymphocyte. supplemented -mem medium with 10% fetal bovine serum, 100 u / ml penicillin, and 100 mg / ml streptomycin was used. t cells proliferation was assessed at 72 hours by dilution of ctv (within the pacific blue channel). the prism software, version 5.01 (graphpad, usa), was used for statistical analysis. comparisons between groups were performed with either the unpaired t - test or the one - way anova. our previous research on mouse and human early hematopoietic fate redirection in response to extrinsic prr agonists prompted us to investigate whether this phenomenon may occur upon exposure to dle transferon. to first determine sensitivity of primitive populations to dle components, we performed brdu incorporation analyses on cd34 cell fractions from cord blood and adult bone marrow. notably, we recorded significant increases of proliferative cell frequencies from both sources after 72 h of dle stimulation (figure 1). furthermore, bone marrow cell stimulation under lymphoid or myeloid culture conditions revealed that adult lymphoid progenitors preferentially expand in response to dle (supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4097642). natural killer (nk) cells constitute a granular innate immune cell type comprising 320% of the human peripheral blood mononuclear cell (pbmc) lymphocyte fraction, which function as major players in innate responses by producing cytokines and chemokines and exerting cytolytic activity against virus - infected or tumor cells [2426 ]. here, dle stimulation of cb cd34 cells rapidly influenced early cell fate decisions and accelerated the differentiation toward a special population of cd56cd11c nk cell (figure 2). although final yields per input progenitor were not indicative of higher net production when the full cd34 compartment is considered, substantially discrepant cd56cd11c cell frequencies suggest the speeding up of such specialized cells (figures 2(a) and 2(b)). in line with the observations in the context of tlr signaling, cells harboring early lymphoid progenitor phenotype were the major producers of dle - newly differentiated nk cells (figure 2(c)). the identity of cd11c nk cells has been a matter in question and a debate point for years. the various studies from dematteo have indicated that cd11c expression defines a distinct subset of murine liver nk cells that respond to adenoviral infection by producing ifn - gamma in a tlr9-il-12-il-18-dependent way. moreover, the same nk dendritic cells (nkdc) phenotype was found to be multifunctional cells with pleiotropic functions, including the ifn-mediated response against bacterial infections and malignant processes, and the migration to central nervous system for viral clearance [2832 ]. belonging to the hardy fraction a of the mouse b cell developmental pathway, the discrete b220cd11cnk1.1 nk subset seemed to meet the biological properties of nkdc, that is, dependency on il-15 and ability of producing highest amounts of ifn. of relevance, two simultaneous reports recently revealed the hybrid phenotypic and functional characteristics of dc and nk within a novel subset of interferon - producing killer dendritic cell (ikdc) population, endowed with strong cytotoxic and antitumor activities, and antigen presenting competence [3437 ]. ikdcs share some properties with other innate lymphoid cells, but their development is unique and is most efficiently generated from l - selectin bm progenitors, while common lymphoid progenitors (clp) are the most effective source of conventional nk cells. even though a ikdc subset has not been formally described in humans, expression of cd11c in nk lineage cells is induced by combination of il-15 and inflammatory cytokines and distinguishes a conspicuous cell subtype participating in immune regulation of chronic diseases. starting from the hsc fraction, dle stimulation could efficiently trigger their production (supplemental figure 2), emphasizing its potential to aim at innate responses but suggesting the divergent origin of dle - derived nk and dc. accordingly, animals treated with dle have showed increased nk cell frequencies and numbers, among other hematopoietic cells [19, 20 ]. due to their fundamental position in viral and tumor immune - surveillance, extensive efforts our data suggest a preliminary strategy to produce high numbers of these cells and, most of all, to robust the in vivo production by remodeling central hematopoiesis. of note, inhibition of myd88 adaptor molecule reduced proliferation and nk - differentiation potentials of dle - stimulated cd34 cells, suggesting the partial contribution of tlr signaling pathway in this emergent phenomenon (supplemental figure 3). despite the wealth of information relevant to transcription factors- and phenotype - associated biological functions of nk lineage cells, we still need to understand the role of environmental niches in their early differentiation under steady - state and pathological conditions. it is well known that they are generated and perform their critical functions in the context of hematopoietic niches within the bone marrow [24, 39 ] or in extramedullar tissues, respectively. indeed, besides the identified developmental niches, an immune response niche, an inflammatory or stress niche, a tumor niche, and a pregnancy niche have been proposed to regulate the biology of nk cells. such niches are presumably controlled by nonhematopoietic cells providing signals triggered from adhesion molecules, chemokines, and cytokines / growth factors [3941 ]. generation of nk cells requires il-15 and fms - like tyrosine kinase 3 ligand, but not il-7. strikingly, the cxcr4/cxcl12 axis conformed by cxcl12-abundant reticular (car) mesenchymal cells and nk precursors might function as the major niche element and is crucial for nk development within bm [39, 41 ]. moreover, production of il-15 is the highest in car cells. our data confirm that microenvironment cues affect the development and biological roles of distinct nk subsets and suggest a substantial positive effect of dle on stromal cell activation that, in turn, may promote cd11cnk and dc expeditious differentiation (figure 3). the additive differentiation effect by stromal cells may result from the action of cytokines and nk growth factors. certainly, a multiplex type assay has shown the differential production of lymphoid factors and vegf (our preliminary unpublished observations). two main subtypes of human nk cells are recognized : cd56cd16 and cd56cd16. while the abundant peripheral cd56cd16 nk cells are phenotypically classified as the most cytotoxic ones, the less copious cd56cd16 subset has been shown to be endowed with noticeable cytokine production capability. their activation is mediated by the net balance between activating and inhibitory receptor engagement signals and in response to cytokines such as il-2, il-12, il-15, and il-18. among activating molecules displayed by the cd56cd16 nk cells, cd16 plays a critical role in targeting antibody fc - bound cells and executing antibody dependent cell - mediated cytotoxicity (adcc). predominantly in tissues and secondary lymphoid organs, the cd56cd16 subset is responsible for production of ifn, tnf, gm - csf, and rantes after activation. the recent reevaluation of functional properties of both cell categories has changed the binary paradigm of nk biology, providing clear evidence of the rapid and simultaneous effector activities exerted by the same cd56cd16 nk cell subset. thus, a prompt and abundant ifn production overlaps with their cytolytic ability and may lead to amplification of macrophage and dc - mediated responses. interestingly, we have now found that most dle - newly produced cells expressed cd16 and that microenvironmental prestimulation induces the density of this molecule (figure 4). the functional competence of nk cells produced from dle - stimulated lymphoid progenitors was tested, demonstrating a very efficient cytolytic potential when a nonradioactive method was used (figure 5(a)). we also observed a copious production of ifn only from the specialized cd11c nk population (figure 5(b)), which is highly relevant to antitumoral and antimicrobial immune protection as this proinflammatory cytokine is pivotal in initiating th1 immune responses against pathogens and tumors [30, 31 ]. thus, these data suggest that early progenitors are promoted to differentiate toward both cytotoxic and regulatory cells in response to dle. recently, cd56cd11c human nk - like cells were shown to induce the proliferation of t cells in an il-18 dependent manner [42, 43 ]. because v2v2 t lymphocytes are key components of the innate immune system that function against infections and human tumor cells, we explore the faculty of dle - nk cells of activating them. of note, dilution of the dye ctv was significantly apparent when t cells were cultured with cd11cnk that are produced upon stimulation with dle. surprisingly, neither unstimulated t cells nor dle - directly stimulated t cells got activated (figures 6 and 7). activation of t cells can be achieved by a number of microorganisms and phosphoantigens and does not require antigen processing, but some atypical costimulatory molecules such as nkg2d play crucial roles in activating them to kill neoplastic or pathogen - infected cells. these innate cells function through a variety of mechanisms, including secretion of cytokines and chemokines, dendritic cell activation, macrophage recruitment, cytolytic activity, and antigen presentation. their contribution to immune - surveillance in a major histocompatibility complex- (mhc-) unrestricted way, by their capability of producing ifn and tnf and via an nk - like pathway, has positioned them as attractive targets for immunotherapy strategies [4548 ]. taken together, our study provides a rational explanation for the adjuvant contribution of dle to the observed effective immune responses against viral and malignant diseases. dle could play an inductor role in the nk lineage emergent hematopoiesis, suggesting the need of elucidating central mechanisms that govern dle - associated tissue regeneration and give an insight into novel cooperative drug activities. while remarkable progress has long been recorded in identifying targets for cellular - based immunotherapeutic strategies to improve acute and chronic disease outcomes, it is becoming clear that definition of extrinsic factors promoting innate cell differentiation processes from the earliest developmental stages may change our vision of immunomodulation. our findings suggest, for the first time, that, in response to dialyzable leukocyte extracts (dle) transferon, human stem and progenitor cells contribute to the emergent production of a special subset of innate cd11c nk cells. of note, this dle - derived nk cell population is endowed with properties such as ifn production, tumor cell cytotoxicity, and the capability of inducing t lymphocyte proliferation that may, in turn, function as coadjuvant component of innate immune responses against virus - infected or tumor cells. | reconstitution of the hematopoietic system during immune responses and immunological and neoplastic diseases or upon transplantation depends on the emergent differentiation of hematopoietic stem / progenitor cells within the bone marrow. although in the last decade the use of dialyzable leukocyte extracts (dle) as supportive therapy in both infectious and malignant settings has increased, its activity on the earliest stages of human hematopoietic development remains poorly understood. here, we have examined the ability of dle to promote replenishment of functional lymphoid lineages from cd34 + cells. our findings suggest that dle increases their differentiation toward a conspicuous cd56+cd16+cd11c+ nk - like cell population endowed with properties such as ifny production, tumor cell cytotoxicity, and the capability of inducing t lymphocyte proliferation. of note, long - term coculture controlled systems showed the bystander effect of dle - stromal cells by providing nk progenitors with signals to overproduce this cell subset. thus, by direct effect on progenitor cells and through activation and remodeling of the supporting hematopoietic microenvironment, dle may contribute a robust innate immune response by promoting the emerging lymphopoiesis of functional cd11c+ nk cells in a partially tlr - related manner. unraveling the identity and mechanisms of the involved dle components may be fundamental to advance the nk cell - based therapy field. |
the accord trial design, inclusion criteria, subject characteristics, and main results have been previously published (2). the accord trial was designed to determine whether cardiovascular event rates in t2 dm could be reduced through intensive glycemic control, intensive blood pressure control, and lipid management targeting triglycerides and hdl cholesterol in addition to ldl cholesterol. each participant was randomized to either intensive (a1c goal 90% of participants in the trial. at the time of discontinuation of the glycemic arm of the trial (and stopping intensive treatment), a large proportion of participants had not completed 3 years in the trial. weight measurements were obtained at every scheduled clinic visit (every 2 months in the intensive arm and every 4 months in the standard arm). weight measurement of participants in the accord trial was standardized by the use of high - quality scales in the clinics with a firm, flat surface and a zeroing system, weighing participants with minimal clothing and without shoes, and with weight distributed over both feet as evenly as possible. information on baseline demographics, clinical characteristics, treatment, and treatment changes was obtained from the accord trial database. we used general linear models to examine the association between each baseline characteristic and weight change at the 2-year visit. for continuous variables, we then added the glycemia randomization arm and included an interaction term between the baseline characteristic and glycemia arm. to examine the relationship between the 1- and 2-year change in weight and change in a1c, we first fit a linear regression of these changes from baseline to year 1 by each arm. we chose to do this analysis at the end of the first year because most of the changes in a1c occurred in the first year. we examined this relationship by tertiles of baseline a1c. to examine the effect of medications on weight change, we considered both baseline and postrandomization medication use. for all analyses, each medication except insulin was represented as an indicator regardless of dosing. we used general linear models to examine the association between each medication at baseline and weight change at the 1- and 2-year visits. due to significant interactions between the effects of medication by arm, we limited our analyses of medications to those that had a significant effect (p 90% of participants in the trial. at the time of discontinuation of the glycemic arm of the trial (and stopping intensive treatment), a large proportion of participants had not completed 3 years in the trial. weight measurements were obtained at every scheduled clinic visit (every 2 months in the intensive arm and every 4 months in the standard arm). weight measurement of participants in the accord trial was standardized by the use of high - quality scales in the clinics with a firm, flat surface and a zeroing system, weighing participants with minimal clothing and without shoes, and with weight distributed over both feet as evenly as possible. information on baseline demographics, clinical characteristics, treatment, and treatment changes was obtained from the accord trial database. we used general linear models to examine the association between each baseline characteristic and weight change at the 2-year visit. for continuous variables, we then added the glycemia randomization arm and included an interaction term between the baseline characteristic and glycemia arm. to examine the relationship between the 1- and 2-year change in weight and change in a1c, we first fit a linear regression of these changes from baseline to year 1 by each arm. we chose to do this analysis at the end of the first year because most of the changes in a1c occurred in the first year. we examined this relationship by tertiles of baseline a1c. to examine the effect of medications on weight change, we considered both baseline and postrandomization medication use. for all analyses, each medication except insulin was represented as an indicator regardless of dosing. we used general linear models to examine the association between each medication at baseline and weight change at the 1- and 2-year visits. due to significant interactions between the effects of medication by arm, all further analyses were performed separately by glycemia arm. we limited our analyses of medications to those that had a significant effect (p 8.5, high baseline bmi, race, high blood pressure, impaired renal function, low hdl cholesterol in males, high triglycerides, baseline insulin use, baseline metformin use, and not using diuretics. on multivariate analysis, younger age, male sex, asian race, no smoking history, high a1c, bmi of 2535, high waist circumference, insulin use, and metformin use were independently associated with weight gain over 2 years. baseline characteristics associated with weight gain at 2 years in both glycemia arms while most of the weight change occurred during the first 2 years, univariate analysis (supplementary table 1) demonstrates that the baseline characteristics of participants associated with increase in weight from years 26 were younger age, diastolic blood pressure, hdl in females, metformin, and sulfonylurea use. the relationship between the baseline and postrandomization a1c and change in weight differed by glycemia arm (supplementary table 2). overall, the relationship between change in a1c and change in weight for those allocated to intensive glucose control was statistically significant (p 8.5, high baseline bmi, race, high blood pressure, impaired renal function, low hdl cholesterol in males, high triglycerides, baseline insulin use, baseline metformin use, and not using diuretics. on multivariate analysis, younger age, male sex, asian race, no smoking history, high a1c, bmi of 2535, high waist circumference, insulin use, and metformin use were independently associated with weight gain over 2 years. baseline characteristics associated with weight gain at 2 years in both glycemia arms while most of the weight change occurred during the first 2 years, univariate analysis (supplementary table 1) demonstrates that the baseline characteristics of participants associated with increase in weight from years 26 were younger age, diastolic blood pressure, hdl in females, metformin, and sulfonylurea use. the relationship between the baseline and postrandomization a1c and change in weight differed by glycemia arm (supplementary table 2). overall, the relationship between change in a1c and change in weight for those allocated to intensive glucose control was statistically significant (p 90% of participants in the trial. at the time of discontinuation of the glycemic arm of the trial (and stopping intensive treatment), a large proportion of participants had not completed 3 years in the trial. thus, although the data suggest some continued weight gain beyond 2 years, the number of participants decreases rapidly over the years. due to the lack of power, none of the statistical tests used are appropriate for use beyond the 2-year time point. some of the results are not surprising, particularly the associations of weight gain with high baseline a1c and insulin use subsequent to randomization. similarly, the greatest gain in weight occurred in participants using combination of insulin and tzd during the trial. this combination was used more extensively in the intensive treatment arm of the trial. those participants who used insulin at entry into the trial tended to gain more weight, perhaps due to substantial increases in their insulin dose and/or the addition of a tzd. change in a1c during the trial did not predict weight gain uniformly, suggesting that it is possible to improve glycemic control without weight gain in some individuals, perhaps using appropriate strategies to change lifestyle (4,5). without adjustment for baseline a1c, a weight gain of 0.65 kg was associated with each 1% (absolute) reduction of a1c. however, assessment within treatment groups and by stratification according to the a1c at baseline showed in some circumstances either little change or a decrease in weight accompanying reductions of a1c. this may reflect the success of strategies to change lifestyle in some participants in the intensive arm, some of whom successfully lost weight while improving glycemic control. the medications taken at baseline and follow - up explain 6 and 14% of the variability in the weight change from baseline during the first year in the standard and intensive arms, respectively, and 10 and 12% of the variability in weight change during the second year, respectively. of interest, during the first year of treatment, variability in the subjects ' baseline medications and baseline a1c account for almost all (88%) of the change in weight in the intensive arm but less than half of the weight change in the standard arm. overall, tzd use accounted for a much higher percent of variability in weight in the intensive than the standard arm. this difference may relate to a greater use of tzds in the intensive compared with the standard arm. it should also be noted that the overall amount of variability explained by medication use after randomization was relatively modest, never accounting for > 15% of the variability in any of the models. however, in clinical practice, what matters most is the net effect on weight. our data suggest that intensive therapy will probably cause weight gain (unless appropriate lifestyle interventions are instituted), and weight gain is likely to be maximal when the intensive therapy consists of a tzd - plus - insulin combination. metformin use at baseline was associated with weight gain during the trial compared with those who did not enter the trial on metformin. this may relate to the known effect of metformin to decrease body weight, which may have already occurred with maximal effect prior to entry into the study. in contrast, we were unable to observe an effect of metformin on attenuation of weight gain when used in conjunction with insulin, as has been seen in other studies (6,7). lack of attenuation of insulin - induced weight gain by metformin has been observed in other studies (8), although these analyses may have been confounded by differences in doses of insulin used between those using or not using metformin. furthermore, the a1c goals in these other trials may have been higher than in accord, leading to less uptitration of insulin. it is important to recognize that while we examined the relationship between the insulin dose (units per kilogram) and body - weight change, we did not do such an analysis for other medications. this is due to the wide range of doses used in insulin titration, whereas dose titration with oral medications were usually two to three steps only and included both increases and decreases in dose. the physiologic mechanisms underlying weight gain in this study are not clarified by our analyses. other studies have suggested that several factors are involved, including a reduction in glycosuria and thus retention of calories otherwise lost, changes in food intake, or changes in energy expenditure (913). in addition, insulin is known to be an anabolic hormone that also has some effects on weight regulation via the central nervous system, and tzds have effects on adipogenesis. drugs that have a lower propensity to cause weight gain or even weight loss, such as those affecting the incretin system or insulin detemir, were used only in a small proportion of participants in the accord trial and for a relatively short duration, as they were not available at the start of the trial. second, we could only include data on participants who had weight data available at 2 years and excluded those whose weight was not recorded or who had died or withdrawn from the study. all patients were advised regarding lifestyle changes according to american diabetes association guidelines, with varying degrees of compliance. we also did not assess factors associated with weight gain beyond the first 2 years in the trial, due to the fact that a large proportion of patients had not completed 3 years in the trial at the time of discontinuation of intensive treatment. we have included the baseline factors associated with weight gain during years 26 (supplementary table 1), although because of the diminishing number of patients, these data should be interpreted cautiously. in summary, we have identified and characterized many features associated with weight gain with intensification of glycemic control. on multivariate analysis, younger age, male sex, asian race, no smoking history, high a1c, bmi of 2535, high waist circumference, insulin use, and metformin use at baseline were independently associated with weight gain over 2 years. following randomization, the intensive group participants with the greatest reduction in a1c gained the most weight. however, all of these factors explain only a small proportion of the weight gain. nevertheless, appreciation of these characteristics may help develop strategies to prevent weight gain when initiating intensive glycemic control in the future. | objectiveidentify determinants of weight gain in people with type 2 diabetes mellitus (t2 dm) allocated to intensive versus standard glycemic control in the action to control cardiovascular risk in diabetes (accord) trial.research design and methodswe studied determinants of weight gain over 2 years in 8,929 participants (4,425 intensive arm and 4,504 standard arm) with t2 dm in the accord trial. we used general linear models to examine the association between each baseline characteristic and weight change at the 2-year visit. we fit a linear regression of change in weight and a1c and used general linear models to examine the association between each medication at baseline and weight change at the 2-year visit, stratified by glycemia allocation.resultsthere was significantly more weight gain in the intensive glycemia arm of the trial compared with the standard arm (3.0 7.0 vs. 0.3 6.3 kg). on multivariate analysis, younger age, male sex, asian race, no smoking history, high a1c, baseline bmi of 2535, high waist circumference, baseline insulin use, and baseline metformin use were independently associated with weight gain over 2 years. reduction of a1c from baseline was consistently associated with weight gain only when baseline a1c was elevated. medication usage accounted for < 15% of the variability of weight change, with initiation of thiazolidinedione (tzd) use the most prominent factor. intensive participants who never took insulin or a tzd had an average weight loss of 2.9 kg during the first 2 years of the trial. in contrast, intensive participants who had never previously used insulin or tzd but began this combination after enrolling in the accord trial had a weight gain of 4.65.3 kg at 2 years.conclusionsweight gain in accord was greater with intensive than with standard treatment and generally associated with reduction of a1c from elevated baseline values. initiation of tzd and/or insulin therapy was the most important medication - related factor associated with weight gain. |
abdominal compartment syndrome (acs) is a life - threatening syndrome with an increase in incidence among critically ill patients. primary acs results from a direct injury within the abdomen and pelvic region (e.g., a blunt or penetrating trauma, ruptured abdominal aortic aneurysm or laparotomy). a direct injury to the abdomen creates an opportunity for hemorrhage and tissue injury. the initial bleeding causes hypoperfusion to the tissues and the accumulating of blood clots within the abdomen initiates intra - abdominal hypertension (iah). acs is most commonly diagnosed in patients with sustaining abdominal or pelvic traumas or in those suffering from some other intra - abdominal hemorrhagic catastrophes. less common etiologic factors are space - occupying lesions (e.g., tumors, obstructed bowel) and bowel / retroperitoneal edema or ascites as well as accompanying mesenteric ischemia or severe pancreatitis. the incidence of acs is reported to be up to 35% in the intensive care population, with reduced survival when compared with other intensive care patients. mortality and morbidity rates are extremely high when patients develop acs, and recognition and treatment are time critical. although the cause of iah can vary, and the degree of organ dysfunction can differ from patient to patient, the reason for this is the effect of decreasing blood flow and increasing pressure on abdominal organs and surrounding organs that creates the end organ dysfunction that defines acs. acs is characterized by a silent physiological derangement and can easily be overlooked or misinterpreted. normal values of intra - abdominal pressure (iap) are considered to be close to atmospheric pressure, represented as 0 - 5 mmhg. pooling of blood in the peripheral circulation occurs as congestion is made in the large veins due to the impeded pathway through the abdomen. as the diaphragm is pushed up by the expanding volume of the abdomen, the heart and the pulmonary veins are compressed, causing increased pulmonary vascular resistance and impaired right ventricular systolic function. the congested heart often produces an elevated central venous pressure and pulmonary capillary wedge pressure. this is not a reflection of the patient 's fluid status but rather an indicator of the rising intra - thoracic pressure and systemic vascular resistance. the increase of thoracic pressures is also manifested by increasing peak airway and plateau pressures that can be measured in a mechanically ventilated patient. these pressures are seen to rise early in the process of acs (iap 15 mmhg), and continue to rise as the iap increases. a decline in cardiac output is an early characteristic of acs as well as an escalating heart rate, which occur in an attempt to compensate for the reduced stroke volume. eventually, cardiac contractility decreases and hypotension becomes apparent after the compensatory mechanisms begin to fail. alveoli continue to collapse as the pressure increases, resulting in decreased oxygen uptake within the blood. indications for iap monitoring include abdominal surgery, traumatic injuries, distended abdomen with acs signs and symptoms such as oliguria, hypoxia, hypotension, unexplained acidosis, mesenteric ischemia and/or elevated intra - cranial pressure. monitoring of iap should be considered for the patients having temporary closure with abdominal packs and receiving large volumes of resuscitation fluids for septic or hypovolemic shock. there needs to be vigilance in assessment of the patient 's iap and organ function to optimize early recognition. every clinician, especially nurses, should remember the risk factors for intra - abdomen hypertension and be vigilant to avoid the progression to acs. by measuring the iap through the bladder, a quick and accurate assessment of abdominal pressure is achieved, and this can be performed by emergency or critical care nurses without a specific medical order or sophisticated invasive monitoring equipments. with regard to the fact that acs is a very lethal condition, the researcher intended to recognize patients affected by acs via iap measurement in patients with severe abdominal and pelvic trauma and then investigate the relationship between pelvic fracture and this hazardous syndrome [figure 1 ]. the clinical presentation of compartment syndrome is also related to heart system, respiratory system and kidney. death rate is high for compartment syndrome. and, the diagnosis of patients in danger, and monitoring them for the symptoms of the syndrome and precocious treatment could help decrease death rate for this syndrome this research was a descriptive analytical study that was performed to characterize patients who develop primary acs and to investigate the relationship between pelvic fracture and acs in abdominal trauma patients (n = 100) referring to the nemazee hospital in 2010. iap was firstly monitored at the time of patient 's entrance to the emergency room and then every 4 h in patients suspected to be at high risk for acs, e.g., those undergoing severe abdominal trauma and pelvic fracture. the bladder pressure method is described as the gold standard and is the most reliable measurement of iap via indirect means. the bladder is considered an excellent vehicle for reflecting the iap as it acts as a passive reservoir when its volume is less than 100 ml. with the patient in supine position, the drainage tube, connected to the foley catheter, is clamped ; 60 ml sterile saline is instilled into the bladder via the aspiration port using an 18-gauge needle and the needle is then attached to a three - way stopcock and water manometer. the manometer is filled with sterile saline and then opened to the patient. the zero mark of the manometer is placed at the level of the pubic symphysis and the pressure is read at the meniscus. the iap is measured in cm h2o and converted to mmhg by the formula : mmhg = cm h2o divided by 1.36. a supine and horizontal position during measurement is most likely to provide reproducible and comparable readings. acs is diagnosed when there is a sustained iah of > 20 mmhg with single or multiple organ dysfunctions such as hypoxia, oliguria or anuria and hypotension, which were not previously present. if the critical care nurse is aware of the high - risk patient groups, suspicion and vigilance may be pivotal in the recognition of the clinical manifestations and early intervention. this highlights the importance of establishing and tracking the iap at an early stage in patients considered to be at risk of acs. during a 12-month period (since november 2010), we measured the iap in 100 patients afflicted with severe abdominal and pelvic trauma, and diagnosed primary acs in 28 of them. about 82.14% (n = 23) of the patients with acs were male and 17.86% (n = 5) were female, and their mean age was 27 years (5 - 50 years). demographics of patient groups by age, sex, mechanism of trauma, associated injury and refer or not refer to operation room about 75% (n = 21) of the patients with acs were referred to the operating room, and underwent laparatomy. among the patients being referred to the operating room, 76.2% (n = 16) expired and 23.8% (n = 5) survived the operation. among the patients not being referred to the operating room, 100% (n = 7) expired. about 46.42% (n = 13) of the patients with acs had pelvic fracture, and the chi - square test showed a significant relationship between pelvic fracture and the percent of acs. acs has been indicated as a complication in serious abdominal trauma for more than 50 years. it develops as a consequence of increased iap not only in abdominal trauma but also in intestinal obstructions, with serous edema of the bowels or a chronically growing ascites, in acute hemorrhagic necrotic pancreatitis and septic peritonitis. in recent years one clinical picture of acs is characterized by abdominal distension, hypoxia and hypercapnia with oliguria up to anuria, when organ dysfunction improves after abdominal decompression. in this research, of these 100 patients, 28 patients with primary acs were identified. among the patients with acs, 82.15% (n = 23) expired and 17.85% (n = 5) survived. malbrain (2005) showed that the incidences of iah and detected patients with acs were 32.1% (n = 85) and 12.9% (n = 11), respectively. patients with iah (iap > 12 mmhg) on admission had a 30-day decrease in survival time compared with those with no iah (62% vs. 79%), but the difference was not statistically significant. regarding age and gender, 82.14% (n = 23) of the patients with acs were male, and their mean age was 27 years (5 - 50 years) in the present study, which is in accordance with walter. they reported that 13 males, aged 45 5 years, developed acs 11.6 2.2 h following resuscitation from a shock. regarding the type of trauma in the present study, 57.14% (n = 16) of the patients with acs had car accidents, 35.71% (n = 10) had motorcycle accidents and 7.14% (n = 2) had suffered from a fall. about 46.42% (n = 13) of the patients with acs had pelvic fracture ; chi - square showed a significant relationship between pelvic fracture and the percent of acs. about 75% (n = 21) of the patients with acs were referred to the operating room and underwent laparatomy, of whom 76.1% expired, but 100% of the patients who were not referred to the operating room expired. among all the patients with acs, 82.15% (n = 23) expired. in a research by pleva., traffic accident trauma together with sporting and leisure time injuries prevailed in 73% of the subjects, and there were 87 abdominal cavity traumas : 31 injuries of the liver, 35 injuries of the spleen, four injuries of the pancreas, 12 injuries of the bowels and five injuries of the retroperitoneum. in the present study, acs occurred in eight patients, of whom six had liver injury. from those six patients, four cases were treated by suture and drainage and two cases and by tamponade in the area of traumatized liver segments seven and eight. in another patient, acs developed as a result of a blunt abdominal trauma associated with pelvic fracture, where decompression laparotomy found no surgical source of bleeding and there was only 500 ml of blood in the douglas space, but extensive distension of thin loops was found together with edema, which was consistent with the findings of our study. immediately after decompression of the abdominal cavity, diuresis was restored and ventilation parameters of the injured improved significantly. in the last patient, acs developed due to extensive retroperitoneal hematoma with injury of soft retroperitoneal tissues and pelvic fracture, where progressive bleeding into the abdominal cavity occurred with developed coagulopathy. every clinician, especially the nurses, should remember the risk factors for iah and be vigilant to prevent its progression to acs. technology and diagnostic capabilities have been developed and refined to optimize the care and treatment of critically ill patients. this has bought a new set of challenges as critically ill patients survive longer and new complications unfold., acs can develop due to extensive retroperitoneal hematoma with injury of soft retroperitoneal tissues. early intervention and diagnosis is essential among these patients as delayed intervention enhances the risk of death extremely. a critical care nurse is well situated to detect subtle signs or changes in the patients clinical condition that may prompt early investigation into the iap. acs is a clinical syndrome that can easily be misinterpreted and a high index of suspicion to iah may be attributed to improved survival from acs. | introduction : an increase in abdominal pressure can lead to so - called intra abdominal compartment syndrome (acs). multiple factors such as an increase in retroperitoneal volume due to pancreatitis, bleeding and edema as a result of pelvic fracture can lead to compartment syndrome. prevention is better than cure in compartment syndrome. by measuring the intra - abdominal pressure (iap) through the bladder, a quick and accurate assessment of abdominal pressure is achieved. therefore, this study aimed to evaluate the relationship between pelvic fracture and acs in traumatic patients.materials and methods : this research was a descriptive analytical study conducted on 100 patients referring to the shiraz nemazee hospital in 2010. iap was monitored every 4 h in patients suspected to be at high risk for acs, e.g., those undergoing severe abdominal trauma and pelvic fracture. the iap was measured via the urinary bladder using the procedure described by kron. data collected were analyzed using spss software.results:the findings showed that acs occurred in 28 of 100 patients. with regard to the associated injuries with abdominal trauma, 19% of all patients and 46/42% of the patients with acs had pelvic fracture. chi - square test revealed a significant relationship between pelvic fracture and incidence rate of acs (p < 0.001).conclusions : according to the collected data, pelvic fracture due to a trauma can be one of the important causes of an increase in iap and acs. in this lethal condition, prevention is better than cure. therefore, serial measurement of iap through the bladder in high - risk patients (those with pelvic fracture by trauma) is recommended to the nurses to diagnose this condition and to decrease the incidence of mortality. |
the prevalence of electronic nicotine delivery systems (ends), such as ecigarettes and vapor pens, is on the rise around the world 1. while ecigarettes may have potential roles in assisting with smoking cessation or tobacco harm reduction 2, they are not currently approved or regulated by the food and drug administration. although several studies show that use of ecigarettes is associated with initial reduction in tobacco use 2, 3, 4, their safety and longterm efficacy as cessation devices are unknown 4. the majority of people who use ecigarettes formerly smoked tobacco cigarettes 5, and many continue to smoke cigarettes while also using ends 5, a phenomenon referred to as dual use. the prevalence of dual use suggests that regular ecigarette use may promote continued tobacco addiction, especially since most of the psychological aspects of cigarette addiction are also operative with ecigarettes and other ends 6. the potentially addictive nature of ecigarettes has received little attention in the research literature, and providers who want to help ends users discontinue use of these devices need guidance on evidencebased methods. the current report presents the case of an individual who had used an ecigarette to assist in quitting smoking but found that he was unable to stop ecigarette use ; he entered our program to quit using ecigarettes. with a combination of behavioral counseling and nicotine replacement therapy (nrt), he became completely tobacco free. a 24yearold caucasian male enrolled in a tobacco treatment program with the stated desire to cease using ecigarettes. he described concerns about his health and fitting in with his social circle, in which smoking and vaping (inhaling vapor from ecigarettes) were not acceptable. he began experimenting with cigarettes around age 17 and smoked 20 cigarettes per day until he was 19, when he quit for 1 year. he resumed smoking at age 20 and then quit for another year at age 22, before resuming smoking at age 23. he had not used tobacco cessation medications during either of these periods of abstinence from smoking. after starting to smoke tobacco cigarettes again, he began using the ecigarette at age 24, thinking that this would be a good step toward permanent cessation. he liked the handtomouth activity of both smoking and vaping and felt that vaping helped him relieve workrelated stress in the same way that cigarettes did. he started vaping within 30 min of waking and vaped up to 10 times a day, averaging one 16 mg bottle of liquid nicotine per week. by the time he entered the tobacco treatment program, the tobacco treatment specialist (tts) used three indicators, based on the fagerstrm scale for nicotine dependence 7, to estimate his ecigarette dependence level as moderate : number of cigarettes smoked per day (approximately 20) when he smoked tobacco cigarettes, time to first ecigarette (30 min), and smoking while ill. the tts initially recommended the daily use of a 14 mg patch, supplemented with 4 mg nicotine lozenges, which he used approximately eight times per day, combined with strategies for behavioral change. after the first week of using both, he reported that he cut his ecigarette use in half. he discontinued the patch after 1 week because he felt he had no control over its dosing. he continued to use approximately eight nicotine lozenges (4 mg) per day, and he reported 2 days without vaping in weeks 3 and 4. six weeks into the program, he asked to try 4 mg cinnamon nicotine gum, as cinnamon was his preferred ecigarette flavor, and he thought the flavor similarity might help him reach his goal. his intuition proved to be correct ; before completion of the 12week program, he had quit using the ecigarette entirely. he used the nrt intermittently for the next 6 months, at which time he discontinued use. more than a year after quitting ecigarettes, he remains ecigarette, tobacco, and nicotine free. we believe this is the first case reported in the literature of successful abstinence from ecigarette dependence achieved using recommended evidencebased treatment for tobacco use and dependence 8. first, no reliable measure similar to the fagerstrm scale for tobacco cigarette dependence exists to assess dependence levels for ecigarette or dual users. as dual use becomes more prevalent 9, clinicians must have objective guidelines to assess dependence levels and ascertain appropriate treatment options for such users. in addition, because no literature exists about using nrt to discontinue ends use, one of the biggest challenges involves estimating the equivalent amount of nrt to recommend 10. nrt works because the patient receives nicotine in an effective, safer, and less addictive form than in cigarettes 11. doityourself refills 12, and patients ' reports on quantity of nicotine consumption may be inaccurate. thus, determining appropriate treatment protocols for patients who wish to quit using ends can be challenging. at the least, clinicians will need to monitor and quantify, as best as possible, both nrt and any continued ends use in order to refine treatment recommendations. further research and improved protocols are needed to help determine the translation of ends use to nicotine absorption. the effects of flavorings in nrt, and their relationship with flavors the individual might have used in ends, also merit further study. in this case, the cinnamon nicotine replacement flavoring and the ability of the patient to adjust the dose himself assisted his attempts to quit and led to optimal dosing. the similarity of flavor of the nicotine gum to an ecigarette flavor that the patient enjoyed helped him be more open to using the gum and may aid others in cessation from ecigarettes, the majority of which are flavored. as ends and dual use increases, clinicians need clearer guidelines to assist those who wish to quit. these guidelines should include protocols for assessing dependence levels and for recommending treatment options, which could include nrt, other pharmaceutical aids, and behavioral therapy. such guidelines are necessary to improve outcomes for ends and dual users who wish to become nicotine free. | key clinical messagealthough use of electronic nicotine delivery system devices, such as ecigarettes and vapor pens, is on the rise, no treatment protocols exist to help such users quit. we report the case of a 24yearold patient in a tobacco treatment program who successfully quit ecigarette use by using nicotine replacement therapy. |
biomonitoring is an important way of evaluating human exposures to selected environmental contaminants and is becoming more widely used in public health work. creatinine is often used for normalizing or adjusting urinary analyte concentrations for dilution in clinical samples and is considered an integral part of monitoring for exposures. creatinine is also commonly used to normalize concentrations of absorbed chemicals in spot urine collection studies like the survey conducted by webs [24 ]. the guidelines of the world health organization (who) set the occupational range at 30 to 300 mg / dl and the us department of transportation measures urinary creatinine down to 5 mg / dl to correct for selective drugs of abuse. there are studies that point out that the limit may need to be lowered to include current environmental toxicants which are now being measured at very trace levels [3, 4 ]. we set our lower calibration range at 20 mg / l (2 mg / dl). there are several published analytical methods for measuring creatinine in urine such as the jaff reaction [57 ], high performance liquid chromatography (hplc) [810 ], enzymatic methods [11, 12 ], and liquid chromatography tandem mass spectrometry (lc / ms / ms) techniques [1315 ]. some of the advantages using the lc / ms / ms method developed at the phl compared to other lc / ms / ms methods include improved linearity, within - day and between - day precision, and lower injection volumes (less system stress). measuring creatinine by lc / ms / ms technology is much more sensitive and selective. enzymatic methods and colorimetric methods such as the jaff reaction are less specific and can be affected by several interfering substances giving results that are too high. for example, erroneously high values can result in these methods from the conditions of the hydrolysis step where urea and sugar combine to form products which also react with picric acid in the jaff reaction. the lc / ms / ms method is not adversely affected by urea, glucose concentrations, or other endogenous substances as discussed elsewhere [57 ]. many of the methods utilized by the center for disease control and prevention (cdc) for measuring environmental analytes for biomonitoring are being developed and analyzed with lc / ms / ms technology. being able to also use lc / ms / ms method for measuring creatinine would eliminate the need to purchase specialized equipment for creatinine analysis. this study was designed to develop a robust, specific, and sensitive method for analyzing creatinine by lc / ms / ms. it was applied to spot urine samples collected by webs and used to normalize the results for pesticide, metal, and other environmental toxicant exposures. a method comparison study performed with the university of washington (uw) medical center 's medicine reference laboratory services (which uses a beckman coulter unicel dxc 800 system that runs an automated jaff reaction) shows a strong positive correlation between the colorimetric jaff reaction and the lc / ms / ms method. all chemicals were of analytical or hplc grade from fisher scientific (fairlawn, nj). creatinine (srm 914a) was from standard reference materials program national institute of standards and technology (nist, gaithersburg, md) and creatinine - d3 was from fisher scientific (fairlawn, nj). water (deionized 18 m) was purified by a nanopure infinity ultrapure water system (barnstead, dubuque, ia) and the carrier grade nitrogen gas was supplied by a peak scientific lab gas generator (billerica, ma). a total of 1576 webs spot urine samples were collected during two different exposure studies and stored at 70c until analysis. 626 samples were collected to assess for the exposure of pyrethroid metabolites in pesticide applicators. 950 samples were collected to assess for the exposure to pyrethroid metabolites, bisphenol a (bpa), and phthalate metabolites in low income households. 50 l of urine was spiked with 450 l of the creatinine - d3 internal standard solution (istd), mixed again and analyzed via lc / ms / ms. the istd was prepared by weighing out 0.0155 g 0.005 g of the creatinine - d3 powder, transferring it to a 500 ml volumetric flask and filling it to volume with a solution of 1 : 1 acetonitrile/18 m di water. the lc / ms / ms analysis was performed on an agilent 1200 hplc stack coupled to an agilent 6410a triple quadrupole mass spectrometer equipped with an electrospray ionization source (esi). the hplc included an in - line degasser, binary pump, temperature controlled column compartment, and two 54-vial tray autosampler racks. the analysis was performed in positive ion mode with a + 4000 v charge on the capillary. the gas temperature was set to 100c with a flow of 12 l / min. the nebulizer was set to 40 psi and the electron multiplier voltage (emv) was set at 0. the acquisition method utilized multiple reaction monitoring scanning (mrm) with a dwell time of 200 ms, a fragmentor voltage of 110 v, and collision energies at 20 v for the creatinine quantifier and internal standard transitions and 8 v for the qualifier transition. 3 c-18 column, 3.0 100 mm, 3.00 m (column : part number ace-111 - 1003), was used with a flow rate of 0.5 ml / min at ambient temperature. isocratic separation was achieved using 75% acetonitrile containing 0.1% formic acid and 25% 18 m water containing 0.1% formic acid. 1 l of the sample (or standard) containing the internal standard the retention time was 0.85 minutes and was set to run at 4 minutes per sample. results from samples were calculated off of the calibration curve which was constructed from the peak area ratios of the analyte to the internal standard for each level. the linearity of this method was determined by preparing eight aqueous calibration standards and analyzing nine of our archived college of american pathology (cap) proficiency testing (pt) urine samples in ten separate runs and then calculating percent recoveries based on the cap assigned mean values for each sample. the calibrators were prepared by serially diluting them with 18 m di water at the following concentrations : 20, 50, 200, 500, 1000, 2000, 3000, and 4000 mg / l. the calibration curve was constructed using the ratio of the peak area of the creatinine to the peak area of the istd plotted against the calibration concentrations with a 1/x weighting applied. the between - day precision was measured and the percent relative standard deviation (rsd) was established at 3 quality control (qc) levels over several days during validation. accuracy was established at the same time using the mean of each qc level and applying 3. a standard addition experiment was designed to establish the within - day precision for the method as well as monitor percent recoveries looking for interferences or recovery issues. 50 l of the 3000 mg / l solution was added to 50 l of the qcl urine and mixed well to create a spiked qc solution. 50 l of the spiked qc solution was transferred into an autosampler vial and 450 l of istd solution was added. the sample was then mixed well and 10 replicate injections of the spiked qc were analyzed. the limit of detection (lod) and limit of quantitation (loq) were calculated from the concentrations of the lowest calibrator (20 mg / l) for each of the 20 validation runs. the lod was calculated statistically as 3 the standard deviation and the loq at 10 the standard deviation. the reproducibility was established by calculating the average results for each qc level established by 2 different analysts during validation. the average of the first analyst was subtracted from the average of the second analyst, divided by the target concentration, and multiplied by 100 to establish the percent error. a comparison study with the uw medical center 's medicine reference laboratory services 50 participant urine samples were homogenized and 2 aliquots per sample were separated for this study. a set of samples was analyzed in - house using the lc / ms / ms method and the duplicates were analyzed at the uw medical center using an automated jaff reaction. the percent recoveries were calculated based on the uw values as the target values and a bland - altman plot was constructed using the results and consisted of the averages of the differences and the 95% limits of agreement for the 2 methods. correlations between the lc / ms / ms and the jaff reaction methods were established using least squares regression analysis in excel 2007. analyses were also conducted in r (r development core team, 2013) to verify the correlation results. the transition ions for creatinine and creatinine - d3 were determined to be 114.0 to 44.1 (quantifier), 114.0 to 86.1 (qualifier), and 117.0 to 47.1 (creatinine - d3) under the esi ionization conditions as shown in figure 1. the retention time for creatinine was 0.85 minutes and the total run time per sample was set to 4 minutes. three webs participant urine samples (table 1), including the one shown in figure 1, were chosen to demonstrate the difference between creatinine corrected and noncorrected results for selected pyrethroid metabolite pesticides, 3,5,6-trichloro-2-pyridinol (an organophosphate insecticide), selected phthalate pesticide metabolites, and bisphenol a. the bland - altman analysis showed that there was no systematic difference between the lc / ms / ms and the colorimetric jaff reaction and only 4% of the points (2 out of 50) were outside of the 95% limit of agreements calculated as the mean of the difference 1.96 times the standard deviation as shown in figure 2. the signal to noise (s / n) ratio was at least 10 : 1 and the qualifier ratios were within 20% of the target ratio. the averages of the percent recovery values were all between 80 and 120% which showed excellent linearity. the results for linearity for the phl method compared to other lc / ms / ms methods researched for this paper was compiled in table 2. the precision was established by calculating the percent rsd for three quality control (qc) levels in urine over the 20 validation runs. the qcl was 1.34%, the qcm was 1.64%, and the qch was 2.59%. the accuracy ranges for qcl, qcm, and qch were 580.00628.71, 775.59855.67, and 1039.481214.74 mg / l, respectively. a comparison of between - day precision values for the phl method compared to other lc / ms / ms methods referenced in this paper was compiled in table 3. l with a standard deviation of 6.17, a percent rsd of 0.33%, and a percent recovery of 103.30%. 49 of the 50 samples were within 20% of the theoretical value showing excellent comparability. the correlation between the 2 methods showed the lc / ms / ms and the colorimetric jaff reaction were associated significantly (pearson r = 0.9898 and r = 0.9797, 0.0001) as shown in figure 3. the purpose of the study was to develop an in - house method for measuring urinary creatinine in random spot urine samples using preexisting lc / ms / ms instrumentation to correct environmental contaminant concentrations affected by the hydration levels of the participating individuals. this study has shown that the phl method is selective, robust, and accurate. it has shown improved linearity, within - day precision, and between - day precision values compared to other published lc / ms / ms methods. one limitation, however, is that, with the simplicity of the sample preparation (no cleanup steps), the instrument tends to get dirtier quicker and may require more cleaning. the 1 l injection volume helps relieve some of that stress to the system. the phl method not only eliminates the need to use hazardous chemicals like picric acid ; it also diminishes affects from endogenous interferences that plague these other techniques. in summary, the lc / ms / ms method developed for the analysis of urinary creatinine in this study has been shown to be quick, simple, and reliable for use in normalizing spot urine samples from exposure biomonitoring surveys. | a viable, quick, and reliable method for determining urinary creatinine by liquid chromatography / tandem mass spectrometry (lc / ms / ms) was developed and used to evaluate spot urine samples collected for the washington environmental biomonitoring survey (webs) : part of the washington state department of health, public health laboratories (phl). 50 l of urine was mixed with a 1 : 1 acetonitrile / water solution containing deuterated creatinine as the internal standard and then analyzed by lc / ms / ms. utilizing electrospray ionization (esi) in positive mode, the transition ions for creatinine and creatinine - d3 were determined to be 114.0 to 44.1 (quantifier), 114.0 to 86.1 (qualifier), and 117.0 to 47.1 (creatinine - d3). the retention time for creatinine was 0.85 minutes. the linear calibration range was 204000 mg / l, with a limit of detection at 1.77 mg / l and a limit of quantitation at 5.91 mg / l. lc / ms / ms and the colorimetric jaff reaction were associated significantly (pearson r = 0.9898 and r 2 = 0.9797, 0.0001). the lc / ms / ms method developed at the phl to determine creatinine in the spot urine samples had shorter retention times, and was more sensitive, reliable, reproducible, and safer than other lc / ms / ms or commercial methods such as the jaff reaction or modified versions thereof. |
we report a case of a 32 week gestation newborn diagnosed with a right ventricular outflow tract mass and subsequently diagnosed with tuberose sclerosis that needed early surgical intervention. while this case needed early intervention, the need for surgical intervention in otherwise asymptomatic cases is debatable as neonatal cardiac rhabdomyomas can spontaneously regress. the need for surgical intervention in otherwise asymptomatic cases is debatable as neonatal cardiac rhabdomyomas can spontaneously regress over time. we report a case of a 32 week gestation newborn diagnosed with a right ventricular outflow tract mass and subsequently diagnosed with tuberose sclerosis that needed early surgical intervention. a 32 week gestation baby born to a primigravida lady with no significant antenatal, maternal medical or family history with a birth weight 1.66 kg (9 - 25 centile) and head circumference of 26.5 cm (0.4 2 centile) was found on routine day 3 examination with a grade 2/6 ejection systolic murmur but otherwise well. subsequent echocardiogram confirmed the presence of multiple small echogenic masses on the inter - ventricular septum and right ventricle free wall, with the largest lesion measuring 8.2 mm 7.1 mm in the right ventricular outflow tract (rvot) in an otherwise structurally normal heart (figure 1, 2 and 3). top - parasternal short axis echocardiography view showing 8.2mm7.1 mm mass in the right ventricular outflow tract. colour doppler of the right ventricular outflow tract the lesion on the rvot caused moderate outflow tract obstruction with a doppler pressure gradient of 70mmhg (figure 4). cardiac rhabdomyomas are associated with tuberose sclerosis complex (tsc), and further investigations were performed to evaluate our suspicion in this case. magnetic resonance imaging (mri) of the brain showed multiple nodules in the subependymal region and cerebral hemispheres bilaterally (figure). mri of the brain showing multiple lesions as seen in tuberose sclerosis ultrasound imaging of her abdominal organs and wood 's light examination of her skin revealed no lesions. she remained otherwise well and was discharged home from our unit at 36 weeks corrected gestational age. further cardiac assessment and serial echocardiograms showed a non - regressing mass of 9 - 11 mm in diameter in the rvot and persistently high doppler derived peak systolic gradient of 60 - 98 mmhg. this necessitated surgical intervention at a corrected gestational age of a month to excise the mass in the rvot and four days later was discharged home after an uncomplicated postoperative period. she continues to thrive with normal developmental milestones at subsequent follow up assessments and serial echocardiograms have shown no reoccurrence on the mass. cardiac tumours are extremely rare in children (0.027 to 0.17%), and cardiac rhabdomyomas the most common type of cardiac tumours. they are usually diagnosed in the antenatal period but can present in the neonatal period with haemodynamic compromise although most neonatal cases remain asymptomatic. cardiac rhabdomyomas are seen in 30 - 80% of patients with tuberose sclerosis and the natural history is one of spontaneous regression over time.1 a meta - analysis showed that most rhabdomyomas associated with tsc are non - obstructive.2 however they can be obstructive and the majority of obstructive lesions affect the left ventricular outflow tract (lvot),2 while our patient had a less common rvot lesion. neonatal cardiac rhabdomyomas causing refractory dysrhythmias or severe haemodynamic compromise are incontrovertible indications for surgery3 but electing to operate in otherwise asymptomatic neonates is debatable. there are few reported cases of right ventricular outflow tract tumours and fewer, possibly under reported cases of asymptomatic rhabdomyomas requiring early surgical intervention. however some cardiac rhabdomyomas will cause outflow tract obstruction in the absence of life threatening arrhythmias or haemodynamic instability and this may be an indication for early surgery to obviate further haemodynamic compromise. regression after the neonatal period reduces the risk of reoperation in the long term.5 a literature search reveals some reports of early surgical intervention in neonatal cardiac rhabdomyomas. erdem referred their 12 day old baby with a right ventricular outflow tract tumour for early surgery as they could not wait for spontaneous regression because of the significant outflow tract obstruction and the mobility of the mass6. ibrahim were of the opinion that conservative management would be inappropriate in their case and opted for early surgery at 36 hours old as they felt that the morphological appearance of the mass increases the risk of it prolapsing through the aortic valve and potentially causing damage. freidberg reported the case of a 3month old referred for surgery with a large prenatally diagnosed cardiac rhabdomyoma in the rvot that failed to regress after a period of observation. however peak gradient across the rvot increased steadily from 40 mmhg to 100 mmhg prompting surgery8. manikoth reported the need for immediate resection on the 5day of life because of the risk of complete obstruction, embolic event and sudden death. none of the neonates in these reports were haemodynamically compromised or unwell prior to surgery. the common factor appears to be the potential for complication necessitating early surgery and in all cases the surgery was uneventful and patients were discharged home not long after surgery. our patient had serial follow - up echocardiograms, which showed that the mass in the rvot and close to the pulmonary valves did not regress and continued to cause significant increase in the rvot pressure gradient. although she was thriving and showing no signs of haemodynamic compromise after detailed multidisciplinary team discussions and counselling of the parents, early surgical intervention was opted for. she had a good result from her operation and made a smooth and excellent recovery from surgery. she is attaining normal developmental milestones and has shown no other complication from the underlying diagnosis of tuberose sclerosis complex. in summary, neonatal cardiac rhabdomyomas can spontaneously regress but early surgical intervention in asymptomatic infants may be necessary. surgery for cardiac tumours carries a mortality of about 5% in children but all the reported cases were uneventful. the choice of management, decision and timing of surgery are largely dependent on the individual case. we recommend that serial echocardiography may help in selecting patients at risk of developing ventricular outflow obstruction and need early surgery and all cases of cardiac rhabdomyomas must be followed up closely for suspected tuberose sclerosis. | backgroundneonatal tuberose sclerosis complex may be associated with symptomatic cardiac rhabdomyomas. cardiac rhabdomyomas are the most common cardiac tumours. the symptoms include haemodynamic instability and life threatening arrhythmias usually requiring early surgical intervention.resultswe report a case of a 32 week gestation newborn diagnosed with a right ventricular outflow tract mass and subsequently diagnosed with tuberose sclerosis that needed early surgical intervention.conclusionwhile this case needed early intervention, the need for surgical intervention in otherwise asymptomatic cases is debatable as neonatal cardiac rhabdomyomas can spontaneously regress. |
the hormones dominating the phases are gonadotropin releasing hormone (gnrh), follicle - stimulating hormone (fsh), luteinizing hormone (lh), estrogen and progesterone. these hormones can be changed by exercise estrogen and progesterone increase after acute exercise. in the menstrual period, progesterone and estrogen secretion is suppressed by chronic exercise1, 8, 13, 26. the performance of female athletes during their menstrual periods has attracted the attention of researchers for many years., studies have not detected any decrease in the performance of female athletes during their menstrual periods4, 6, 10, 24. in this study, the effect of maximal aerobic exercise the luteal phase on some gonadal hormones and alpha - fetoprotein (afp) in female athletes was researched. it has a molecular weight of 70 kd is and it synthesized by the yolk sac, fetal gastrointestinal tract, and liver20. it is an important parameter in pregnancy follow - up as the afp level increases in intrauterine fetal deaths, hydrocephalus, hemolytic diseases, placental separations, and neural - tube defects in pregnant women21. the measurement of afp level is important for tracing prognosis and diagnosis hepatoma and germ cell tumors18, 21. serum afp is also elevated in 524% of malignancies of the pancreas, stomach, and colon20. in a 12-week study performed in 2014, ko. found no change in the afp level of women aged around seventy after 1, 3 and 5 days of an exercise program. our study will contribute to the literature in terms of the relationship between afp and exercise. estrogen is an 18-carbon corticosteroid molecule released from the ovaries of women and from the testis of men. furthermore, it is also affects the cardiovascular system, muscle skeletal system, immune system, and central nervous system. it plays a role in the repair of the skin, nervous system, and hepatic tissues. while the estrogen level in women is not changed by acute exercise, it is suppressed by chronic exercise16. kraemer. found an increase in estradiol with mild exercise. during the luteal phase, the progesterone level increases and exercise nakamura. detected an increase in both estrogen and progesterone after exercise in the mid - luteal phase. the menstrual disorders observed in female athletes result from differences in the release of oligomenore and amonore gonadotropin releasing hormones, and the release of fsh and lh is decreased by exercise11. twelve healthy female soccer players with normal menstrual cycles (regular for one year, 282 days) were included in the study as subjects. all of the subjects were conforming to the luteal phase period (22nd and 25th day) according to body temperatures (table 1table 1.the subjects characteristics (n=12)age (years)19.61.3 (1822)sports age (years)5.40.7 (47)height (cm)1664.7weight (kg)55.45.7). the experimental protocol of this study was approved by the local ethics committee. all the subjects were informed about the purpose and risks of the study before signing a written consent. the test started with slow running (8 km / h), and the subjects ran 2 meters apart from each other between two lines. then, the same distance was run again and the second run was started. the running speed was adjusted by the signals and it was increased by 0.5 km / h per minute. the test was stopped when a subject failed to follow the set pace of the beeps for two successive shuttles or stopped voluntarily14. all the subjects who completed the test were successful. the subjects were instructed not to eat anything three hours before the exercise and not to do any sports from two days before the exercise to control the test and these conditions. afp, estradiol, progesterone fsh, and lh values were measured using an enzyme - linked immunosorbent assay (elisa) and original kits in a laboratory. repeated measure anova was used to compare afp, estradiol, progesterone fsh, and lh serum levels at baseline and immediately and 1hour after the test. afp levels had significantly decreased after 1 h of recovery from exercise in the female soccer players (p<0.05). in the female soccer players, estradiol and lh levels had significantly increased immediately after the exercise but had returned to resting levels after 1 h of recovery (p<0.05). progesterone levels had significantly decreased immediately after the exercise but had returned to resting levels after 1 h of recovery (p<0.05). the value of fsh1 h after exercise was lower than its value before the exercise (p<0.05) (table 2table 2.repeated measure of anova comparison of pre and post (immediately and 1-hour after) shuttle run test effect on hormone level (n=12)pepsep1hseafp (ng / ml)1.50.51.50.381.40.5fsh (mu / ml)5.52.46.22.75.12.4lh (mu / ml)4.83.55.634.94estradiol (pg / ml)96.75.5104.259.19558.9progesterone (ng / ml)6.45.95.456.16.2pe : before exercise, pse : after exercise, p1hse : one hour after exercise). our findings of heart rate before and after maximal aerobic exercise are shown in table 3table 3.before exercise (pe) and after exercise (pse) heart rate values (n=12)pepse heart rate (pulse / min)63.15159.110.3. pe : before exercise, pse : after exercise, p1hse : one hour after exercise in the present study, the effects of acute maximal exercise on afp, estradiol, progesterone, fsh, and lh in female soccer players during the luteal phase of the menstrual cycle were researched. afp is an oncofetal glycoprotein with a molecular mass of approximately 70 kd17, 21. measurements of maternal serum and amniotic fluid afp during pregnancy are important and widely applied in screening methods for the detection of congenital anomalies of the fetus17, 21. measurement of serum afp plays an important role as a diagnostic or in screening tests for in certain malignancies. hepatocellular carcinoma is the most frequently occurring malignant disorder for which afp is used as a biomarker17, 21. other cancer types that increase serum afp levels are pancreatic and gastrointestinal carcinomas, and germ cell tumors of the testis or children s brains17, 21. it is our supposition that decreases in the afp values, a cancer parameter, taking place with exercise, could be used to determine the importance of exercise in cancer treatments. we found only one publication in the literature concerning the change in afp with exercise. in a study of women aged over seventy, it was found that the afp level did not change with exercise5. in our study, estradiol and progesterone are gonadal hormones which decrease with aging, and this may lead to problems such as metabolic syndrome and sarcopenia16. furthermore, estrogen also affects other endocrine responses during exercise9. while the effect of exercise on anabolic hormones is maximal in the young women with normal menstruation, estrogen and progesterone levels decrease more in the women with the menarche disorder11, 25. consequently, female athletes may show menstrual disorders such as amenore (no menstruation), delay in the age of menarche, oligomenore (frequency of menstruation of more than 35 days) and insufficiency of the luteal phase3, 12, 15, 19, 22, 23, 25. in our study, it has been mentioned in the literature that chronic exercise suppresses estrogen and progesterone25. in the present study, the progesterone level had decreased 1 hour after the exercise a result that is consistent with the literature. in female athletes, the pulsatile release of gonadotropin - releasing hormones decreases. this suppresses the release of lh and fsh from the ovaries ; therefore, the release of estrogen decreases. low luteinizing hormone levels explain menarche disorders and secondary amenore in female athletes3, 15. in our study, lh increased immediately after the exercise and returned to its former level after 1 hour. this may show there is a positive effect of acute exercise on lh. on the other hand, the decrease of the fsh level to under its original value 1 hour after the exercise shows that intense exercise suppresses fsh. this result is also consistent with the literature3, 15, 19, 22, 23, 25. in the present study the effect of maximal aerobic exercise in the luteal phase during which athletes show the most menstrual problems on fsh, lh, estradiol and progesterone hormones, and afp was studied. the observed of suppression progesterone and fsh values is the literature consistent with other reports while the increase of estradiol and lh levels immediately after exercise shows that acute exercise can be used as a method of training for female athletes. on the other hand, the significant decrease in the value of afp, which is a cancer parameter, 1 hour after the exercise, may be of valuable in the future studies of the relationship between exercise and cancer. | [purpose ] the performance of female athletes during their menstrual period has attracted the attention of researchers for many years. it is known that the menstrual period changes with exercise. alpha - fetoprotein (afp) is an oncofetal protein. in this study, the effect of maximal aerobic exercise in the luteal phase on some hormones and afp in female athletes was researched. [subjects and methods ] twelve volunteers and healthy female footballers with normal menstrual cycles volunteered for this study as subjects. all the participants performed a shuttle run test. blood samples were taken before, after, and one hour after exercise. serum afp, estrogen, progesterone, follicle - stimulating hormone (fsh), and luteinizing hormone (lh) values were measured using an auto analyzer and original kits. heart rate measurements were performed before and after the exercise. [results ] afp activity had significantly decreased after 1 h of recovery from the exercise in the female soccer players, and estrogen and lh activity had significantly increased immediately after the exercise. progesterone activity had significantly decreased immediately after the exercise. fsh values had significantly increased immediately after the exercise. [conclusion ] the results of the present study show there were significant decreases in the values of afp, which is a cancer parameter, 1 hour after the exercise. this result may be valuable in future physiotherapy studies on the relationship between exercise and cancer. |
chronic heart failure (hf) is a significant health and social problem. in the united states alone, nearly 6 million people suffered from hf in 2008, and this disease is becoming increasingly prevalent with more than 550,000 new cases diagnosed each year. half of hf patients die within 5 years of the first onset of symptoms, and half (50%60%) of the patients diagnosed with severe hf do not live longer than a year. hf is the most common cause of hospitalization in those over the age of 65 years, and 54% of the patients are readmitted to hospital within 6 months of discharge. hf also results in significant morbidity and disability, thereby generating permanent and high health care costs. heart disease is the leading cause of mortality in iran ; unfortunately, further accurate statistics describing the burden of hf are not available. optimal medical management following a cardiovascular event remains underprescribed, and even more so in developing countries. similarly, nonpharmacological management and interventions are infrequently recommended, and patient adherence to lifestyle modifications remains poor. recent european research suggests that the control of cardiovascular risk factors, including tobacco use, obesity, hypertension, hypercholesterolemia, and diabetes, is far below an acceptable level [9, 10 ]. the nature and severity of hf symptoms greatly depend on the patient 's knowledge, cooperation, and active participation in their health management. however, improving health and preventing hf from progressing by adopting self - care skills, adhering to complex treatment regimens, and changing lifestyle behaviours is particularly challenging. according to orem 's self - care theory, effective self - care activities can reduce the need for hospitalization. this theory postulates that nurses interact with patients in three ways according to the patient 's ability to participate in their care : total compensation, partial compensation, and supportive - educational systems. patients in the supportive - educational system are capable of engaging in self - care but require education about the different aspects of therapeutic self - care behaviours. nurses are optimally positioned to identify existing and potential health issues and to provide supportive - educational interventions where appropriate. postdischarge support in the form of patient education is one of the most effective interventions to improve self - care abilities and behaviours among hf patients, which ultimately improves prognosis and reduces hospital readmission rates. the main objective of education for hf patients is to improve the patient 's management of their disease, thereby reducing the onset of complications and morbidity. patient education is generally delivered within the framework of a comprehensive discharge program and covers information about fluid and sodium intake restriction, diet, exercise, adherence to pharmaceutical treatment, monitoring symptoms, and seeking health care when symptoms worsen. according to a recent systematic review, large studies have demonstrated the efficacy of therapeutic education programs in changing cardiac patients ' lifestyles and ultimately improving morbidity and cost - effectiveness. yet, there are no recommendations or standardized guidelines about methods to deliver information and education. strmberg found that most hf patients do not have a clear understanding of recommended self - care behaviours despite receiving related education. rather, hf patients require further assistance to learn self - care behaviours and adapt to living with a chronic illness. providing education and training based on individual patient needs and desires therefore, a thorough assessment of hf patients ' educational needs and preferences, as well as their beliefs and abilities related to medical and lifestyle recommendations, can provide the foundation for personalizing educational efforts. interventions to promote self - care behaviours among hf patients, and corresponding research, must take into account culture, which greatly influences diet, exercise, lifestyle, and attitudes toward medical therapy. the few intervention studies conducted in iran did not evaluate supportive - educational interventions nor did they measure self - care behaviours [1719 ]. the purpose of this research was to evaluate the effect of a supportive - educational intervention on self - care behaviours of iranian hf patients. the research council of the tabriz university of medical sciences gave ethical approval for this randomized control trial. consecutive patients admitted to shahid madani hospital with a diagnosis of hf and who met the inclusion criteria were recruited into the study. a sample size of 80 (40 individuals in the intervention group and 40 in the control group) was deemed sufficient based on a preliminary analysis of self - care scores of 5 hf patients. the following parameters guided the present study ; the optimal self - care behaviour score in the study was 70, the mean and standard deviation of self - care behaviours scores were estimated (mean = 25, sd = 6.15), = 0.05 and power = 0.9 were chosen, and no attrition during followup was anticipated. the participants were randomized into the control and experimental groups using random number software (figure 1). participants who were included were of 18 years age and older, diagnosed with new york heart association class iii or iv hf, had an ejection fraction less than 40%, agreed to predischarge education and follow - up care, and would be available by phone after discharge. participants who were excluded were those who experienced significant worsening of their disease and transfer to the intensive care unit, were hospitalized for greater than 1 month, had a chronic disease other than hf, or were diagnosed with a mental illness. according to orem 's self - care theory, the supportive - educational system is the only system in which patients require assistance in relation to decision - making, behaviour control, and acquiring knowledge and skills. some patients are capable of carrying out self - care behaviours but are in need of guidance and support, while others only require education. the supportive - educational intervention in the present study was tailored to address the appropriate level and type of assistance / care as per the participant 's need. those who needed guidance and support were encouraged to continue to carry out current self - care behaviours, and additional information and support were provided related to reducing salt in the diet, restricting fluids, and increasing physical activity for example. participants requiring education were given information about how to create new self - care behaviours. hf participants randomized to the intervention group received a two - part intervention aimed at improving self - care behaviours. the first phase consisted of a one - hour, nurse - led, in - person hf education session that was customized by the nurse according to the participant 's level of education. an individualized education booklet was reviewed with literate patients, while for illiterate patients this booklet was reviewed with the participant as well as a family member. the intervention was also customized according to the participant 's prior knowledge and learning needs, which was assessed with a learning need inventory. the following information was reviewed : the definition and symptoms of hf, strategies to prevent the worsening of hf symptoms, explanations about medications, and recommendations about dietary changes (i.e., reducing salt intake), exercise, and smoking cessation. these participants were given a booklet at the time of discharge that was based on heart failure society of america (hfsa) 2010 guideline executive summary. the objective of this phase was to reiterate and review information covered during the initial education session and improve the participant 's ability to cope with the disease, as well as enhance self - care behaviours. the first followup telephone call was made by a nurse two days after hospital discharge to verify participant information and determine the next date of contact. the nurse then contacted the participant by phone every two weeks for 3 months. during these phone calls the nurse asked the participant whether they were experiencing any signs or symptoms that would suggest worsening hf. the nurse also reviewed the recommended self - care behaviours and provided support in the form of advice and encouragement when deemed necessary. the participants were also advised to contact the nurse if any question or an acute medical issue arose related to preventing or managing their hf. participants who were randomized to the control group received usual care provided by the hospital and attending physician (nonsystematic and informal teaching). sociodemographic characteristics were gathered through individual interviews and medical data were extracted from medical records. the educational needs of participants in the intervention group were assessed with a self - report questionnaire, which was filled out independently with pen and paper or with the assistance of a nurse during a face - to - face interview. the education needs assessment instrument is specific to hf patients and consists of 42 items, with 7 subscales to assess learning needs in the areas of anatomy, physiology, diet, activity, cognitive factors, risk factors, and pharmaceutical information. this instrument uses a five - point likert scale (ranging from least important to know = 1 to most important to know = 5). baseline self - care behaviour data were collected with the pen and paper method or during face - to - face interviews using the self - care of heart failure index (schfi). subsequent self - care behaviour data were collected with this instrument by telephone at 1, 2, and 3 months following hospital discharge. the maintenance self - care behaviours scores includes 10 questions, the self - care management the scores includes six items, and the self - care confidence the scores includes seven questions. all items are scored on a four - point likert scale from 1 (poor self - care behaviour) to 4 (optimal self - care behaviour), and summative scores are standardized on a scale of 0 to 100. a cutoff point of 70 on each schfi scale is used to judge self - care adequacy. to ensure the accuracy of the farsi translation of these instruments, they were reviewed by three professors (two with english language m.a. the instruments were reviewed for content validity by 12 faculty members of tabriz university of medical sciences (9 with an m.s. and 2 with a ph.d. in nursing, the reliability was determined through test - retest methods, wherein the instruments were given to 10 hf patients twice, 2 days apart. measures of central tendency mean, standard deviation, and percentage were used to describe participant characteristics at baseline. the control and intervention groups ' self - care behaviours were compared at baseline, 1, 2, and 3 months using t - tests. a repeated - measures anova was used to determine time effects and the impact of group on different aspects of self - care behaviours. a total of 200 hf patients were screened for inclusion from july to september 2011. 80 hf patients with an ejection fraction above 40% were excluded, 20 patients with severe hf were transferred to another hospital unit, and 20 patients declined to participate. the 80 remaining patients were enrolled in the study and randomized into the control (n = 40) or intervention (n = 40) groups. of the 40 individuals assigned to the intervention group, two did not complete the study ; one participant required pacemaker implantation, and another became a heart transplant candidate. at study completion, 38 individuals remained in the intervention group and 40 in the control group. 53.8% were men, and 72.5% were married and lived with their spouse and children. ischaemic heart disease (36%) and hypertension (36%) were the main causes of participant 's hf. there was no statistically significant difference between sociodemographic characteristics of the participants in the control and intervention groups. the control and intervention groups did not differ in self - care scores at baseline, prior to delivery of the intervention. each of the self - care scores was significantly higher in the intervention group than the control group at 1, 2, and 3 months (table 2). the anova results showed significant differences in self - care between the control and intervention groups (tables 3 and 5). the results also showed significant difference in self - care behaviours over the three months, such that as time progressed self - care scores among participants in the intervention group continued to increase (table 4). this study provides evidence that a supportive - educational intervention can strengthen and establish new self - care behaviours among hf patients in iran. this intervention, informed by orem 's self - care theory, provided patient education about self - care behaviours to assist in the management of hf, as well as support in the form of telephone followups. this improvement in self - care behaviours is consistent with previous research, wherein self - care skills among hf patients were improved following one educational session and an eight - month followup with a nurse educator. strmberg and colleagues found that the readmission rate and healthcare costs also decreased with educational interventions that included follow - up support for hf patients. this provides support for including education about self - care activities, including nonpharmaceutical interventions, as a part of standard management of hospitalized hf patients. this educational training ought to begin when patients are initially hospitalized and continue following discharge. in the present study, improvements in self - care among participants in the intervention group were not only maintained, but continued to improve over the three months. this finding lends support to comments made by evangelista that education alone does not lead to positive outcomes, and that using behavioural strategies, such as reinforcing behaviours through a follow - up program, can help optimize self - care. however, it is unknown how long beyond the 3-month followup the improvements made in self - care in the present study lasted among participants in the intervention group. there is evidence that the effect of educational interventions on self - care behaviours is not maintained over time [2224 ]. it was likely that the participants gave incorrect answers to the self - report questions. however, by gaining their trust and explaining the confidential nature of the study, we attempted to control false reporting. patient education is currently an essential part of treating chronic diseases such as hf, but, in the busy day - to - day practice of caring for hf patients, education often is not a priority. we recommend that time and resources are to be allocated to enable health care professionals to adequately promote patients ' self - care behaviours through supportive - educational interventions. the present research also suggests that further knowledge about factors that limit or promote effective patient education is needed to improve their quality and effectiveness. findings of this study indicated that patients with hf not only need pharmaceutical management by physicians and nurses, but they also require support to enhance their self - care behaviours and non - pharmaceutical management (e.g., reducing salt in the diet, restricting fluid intake, daily weighing, and monitoring the symptoms). according to the results of the present study, implementing personalized, theoretically driven, supportive - educational programs based on nonpharmacological management strategies might be a useful tool to develop, maintain, and change self - care behaviours of hf patients. this study confirmed that postdischarge supports are very effective in improving self - care activities and reaching optimal behaviours. | background. chronic heart failure is a major health and social problem. the promotion of self - care behaviours can potentially assist patients to effectively manage this chronic condition and prevent worsening of the disease. formal personalized educational interventions that provide support and take into consideration the cultural context are needed. objective. the objective of this research was to evaluate the effect of a supportive - educational intervention on self - care behaviours of heart failure patients in iran. methods. this research was a prospective, randomized trial of a supportive - educational intervention. eighty heart failure patients were randomly assigned to receive the supportive - educational intervention or usual care. the intervention consisted of a one - hour, nurse - led, in - person education session and postdischarge followup by telephone over three months. data were collected at baseline, one, two, and three months. results. the control and intervention groups did not differ in self - care scores at baseline (p > 0.05). each of the self - care scores was significantly higher in the intervention group than the control group at 1, 2, and 3 months (p < 0.001). there were significant differences in self - care behaviours over the three months, among participants in the intervention group. conclusion. this study provides support for the effectiveness of a supportive - educational intervention to increase self - care behaviours among iranian patients suffering from chronic heart failure. |
diabetic muscle infarction (dmi), which was described for the first time by angervall and stener in 1965, is a rare complication of dm. it is usually seen in long - standing dm along with complications, such as, nephropathy, retinopathy and neuropathy. until now it commonly occurs in the thigh muscles and we have been able to find only five patients with upper extremity involvement.[46 ] in this article, we describe a patient with poorly controlled dm and muscle infarction of her forearm. a 58 year - old - woman with a three - week history of left upper forearm pain was admitted to our hospital. she noted a history of type ii dm, which was diagnosed five months ago. her current condition began with a sudden onset of swelling and pain in the left forearm, extending to her fingers. before this recent admission, several antibiotics and non - steroidal anti - inflammatory drugs were prescribed for her, with no improvement. on physical examination the white blood cell count was 17500 (normal, 480010800) with 80% neutrophils (normal, 3375%). fasting blood glucose was 235 mg / dl (normal, 70105) and hemoglobin a1c was 6.5% (normal, < 6%). other laboratory results were as follows : erythrocyte sedimentation rate (esr) 17 mm / hour (normal, 025), creatine kinase (ck) 5930 u / l (normal, 25175), lactate dehydrogenase (ldh) 780 u / l (normal, 0480), aspartate transaminase 89 u / l (normal, < 31), and alanine transaminase 53 u / l (normal, < 31). magnetic resonance imaging (mri) showed diffuse edema and abnormal low and high signal intensities in the soft tissues of the left forearm [figure 1 ]. her muscle enzymes were gradually decreased to 143 and 504 for creatine kinase (ck) and lactate dehydrogenase (ldh), respectively. she was discharged without any swelling or pain eight days later. on a follow - up visit after two months, she did not have any complaint in her forearm. after three months, post - treatment mri was performed that showed no abnormal signal intensity in the soft tissues, or bony structures [figure 2 ]. it shows diffuse edema and low signal intensities in the left forearm muscles, with subcutaneous soft tissue swelling (arrowheads). (b) a sagital t2-weighted magnetic resonance image shows diffuse edema and high signal intensities in the forearm muscles, with subcutaneous soft tissue swelling (arrow) (a) axial t1-weighted and (b) coronal t2-weighted magnetic resonance images after three months show no abnormalities its exact cause is unknown, but several hypotheses have been described, including, arteriosclerosis and microangiopathy, hypoperfusion - induced anoxia, abnormalities of the coagulation system, association with antiphospholipid antibodies, and hypoxia - reperfusion injury. most patients are young and females, with a mean age of 42 years. unlike our patient, they usually have long - standing type i dm. the presentation of dmi ispain and swelling that mostly involves the thigh muscles, and rarely, like in our patient, involves the upper extremity. the best diagnostic tool is the mri, which shows high and low (or normal) signal intensity on t2- and t1-weighted images, respectively. although the diagnosis of dmi can be confirmed by muscle biopsy, and it was done in a majority of the cases in the past, due to nonspecific findings and increased risk of recurrence, it is better for it to be reserved for atypical cases. the differential diagnosis of dmi is broad and the most common ones are thrombophlebitis, soft tissue infection, and diabetic amyotrophy. our patient was afebrile, her blood cultures were negative, and we did not find any evidence of muscle or soft tissue infection. the best method for dmi management is the conservative method, with blood glucose control. she responded well to the rest, with blood glucose control, and without any recurrence in the follow - up visits. diabetic muscle infarction should be noted in the differential diagnosis of any diabetic patient presenting with a swollen and painful limb. | diabetic muscle infarction (dmi) is an unusual complication of diabetes mellitus. it is usually seen in long - standing diabetes mellitus. this article presents a case of dmi in the left forearm of a 58-year - old woman. she had a swollen forearm. the level of creatine kinase was 5930 u / l. her condition was initially suspected for either cellulitis or venous thrombosis. a magnetic resonance imaging (mri) study of the forearm showed diffuse edema and abnormal signals of the left forearm. the diagnosis of dmi was made. she was treated conservatively and her symptoms resolved within a short period of time. dmi should be considered as a differential diagnosis of any painful and swollen limb in diabetic patients. |
a 43-year - old man had sudden blurring of vision, metamorphopsia, and paracentral scotoma in his left eye on the second postoperative day of dacryocystorhinostomy operation on the same side. ocular examination showed best - corrected visual acuity of 20/20 in the right eye and 20/80 in the left eye. fundus fluorescein angiogram detected multiple leakage sites with smokestack pattern because of pooling of the dye under the neurosensory retinal detachment in the macula [fig. 1 ]. after one week of omission of the nasal drops, there was a gradual resolution of symptoms. fundus photograph and fluorescein angiogram of the left eye showing few hyperfluorescent lesions within the foveal avascular zone and along the superior arcade, from the early venous phase, with evidence of profuse leakage along with pooling of dye during the late phase, indicative of cscr central serous chorioretinopathy is a multifactorial disease, which took many years to reach the precise clinical definition because of controversial etiopathology. it is usually associated with corticosteroid use, type a personality, organ transplantation, pregnancy, hypertension, and psychopharmacologic medication. recently, we have also reported a case of bilateral cscr following one intraarticular injection of corticosteroid. in this patient or case, only a topical instillation of xylometazoline in nasal mucosa produced cscr. it is documented that post - traumatic stress is associated with excessive release of catecholamines. topical application of xylometazoline, an alpha adrenergic receptor agonist, through the nasal route can cause severe systemic side effects. even a single intranasal dose of xylometazoline has been shown to be associated with coma in a neonate and sudden death in a 13-year - old boy due to the rupture of an intracranial aneurysm. in an observational case series of cscr associated with the use of sympathomimetic agents, occurrence of one case of cscr followed the use of oxymetazoline nasal spray three to four times daily for one month. in our case, excessive systemic absorption of xylometazoline from the inflamed nasal mucosa may have enhanced the post - traumatic stress - induced adrenergic stimulation leading to an increase in the choroidal circulation and alteration of the pump action of the retinal pigment epithelium, events which are related to the development of cscr. on the contrary, the intranasal instillation of a few drops of xylometazoline may have been a chance association with the occurrence of cscr, but one interesting point to refute this concept is the spontaneous quick resolution of visual symptoms after discontinuation of this topical medication. this case report widens the spectrum of drugs whose use should be enquired into while examining a patient with cscr. | a 43-year - old man developed central serous choroidoretinopathy in his left eye following dacryocystorhinostomy operation on the same side. he was using xylometazoline nasal drops in his left nostril. action of xylometazoline or the stress related to the operation or the effect of both factors played the role in the causation of this ocular condition. omission of nasal drops or relief from stress resulted in full recovery of vision and complete resolution of symptoms within one month. |
the use of sound information is a growing area of application of signal processing techniques in healthcare and biomedicine. in particular, multiple groups have reported on the development of systems that analyze audio recordings of patients and identify bouts of coughing [15 ] or other sounds indicative of health concerns [69 ]. in addition to their value as a monitoring or screening tool, the automated nature of these systems potentially enables new modes of disease management, especially in conjunction with advances in mobile technology. moreover, the type of approach presented here may be especially suited to improving outcomes and addressing chronic disease in populations that have been previously underserved. this paper studies cough as a symptom of asthma in adolescents. for a variety of reasons, including social and developmental pressures, adolescents with an asthma diagnosis typically have an inaccurate understanding of their disease condition primarily due to poor symptom perception or downplaying symptoms [11, 12 ]. these issues can undermine effective symptom monitoring and medication adherence which can ultimately lead to poor condition management and high healthcare utilization. to address this problem a device is proposed that continuously monitors asthma symptoms, particularly coughing which is the most common symptom in pediatric asthma patients [13, 14 ]. the identified symptoms can be stored and later retrieved by a patient or healthcare provider as an objective reference indicating the levels of asthma control. mobile technology has advanced sufficiently such that the hardware requirements of a personal asthma monitoring device are already met in existing consumer electronics platforms. the problem may, therefore, be approached within the context of mobile application development. owing to its broad install base and mature software development kit (sdk) apple 's ios devices were the target platform (the 4 g ipod touch specifically). like other audio systems for cough monitoring reported in the literature the present approach is based upon proven technology and methods in the fields of speech recognition, keyword and key audio effect detection [1517 ], machine learning, and content - based audio analysis [1823 ]. the symptom recognition algorithm consists of a frame - based feature extractor followed by temporal analysis with a hidden markov model (hmm) based viterbi decoder. mel - frequency cepstral coefficients (mfcc) plus a log - energy measure were employed as the audio features while both left - to - right and ergodic / connected hmm topologies were investigated. the mfcc / hmm recognition framework has also been reported for cough monitoring by, for example, matos. [3, 25 ]. use a similar method however ; in their case, they use an original variant of cepstral analysis dubbed energy cepstral coefficients (ecc) and have inserted an artificial neural network discriminator prior to the hmm decoder. the cough detection algorithm presented here processed the audio data in two steps. in the initial preprocessing step the incoming stream of digital audio samples was rearranged into fixed - length frames from which a set of audio features (feature vector) were computed. the sequence of feature vectors was then passed to a hmm viterbi decoder (using a token passing implementation as described in) using hmms trained on the collected database of symptom and background sounds. as discussed in detail in the following sections the hmm was a composite consisting of separate models for coughing, background, and silence connected together with a simple parallel grammar. prior to development of the automated device for asthma monitoring (adam) application, a large amount of audio data was collected in trial recordings intended to closely approximate the circumstances in which the device would be expected to function. participants with asthma were 1317 years old, had active asthma symptoms within the previous 24 hours, and used english as a primary language. those with other health conditions producing respiratory symptoms (e.g., cold, cardiac disease, or cystic fibrosis) were excluded. twenty - nine adolescents participated in the audio data collection trials ; details of the sample characteristics are provided elsewhere. participants with asthma were 1317 years old, had active asthma symptoms within the previous 24 hours, and used english as a primary language. those with other health conditions producing respiratory symptoms (e.g., cold, cardiac disease, or cystic fibrosis) were excluded. the participants were instructed to continuously record the sounds of their respiration for 24 hours in vivo wearing a personal digital recorder (olympus ws-331 m) and lapel microphone while they went about their daily routines. the external microphone was attached to the shirt collar to optimally capture the respiratory sounds of the subject. the recorder was carried in a pocket or in a carrying case provided by the study. were instructed to place the recorder and microphone near the head of the bed to capture any symptoms that occurred during the night. for the purposes of developing the signal processing component of the adam device the large dataset was split into a series of files of more manageable size. representative selections of the audio containing cough sounds, silence, and background noise across all of the participants were aggregated into individual files. the classification and validation of symptomatic sounds were carried out by the principal investigator and three other clinicians trained in pediatric medicine and pulmonology. coughing sounds that were used to train the recognition algorithm were cropped to only include the sound itself. as a consequence the corresponding audio files had variable length (i.e., the duration of the cough). all other audio files (including the training data for the silence and background hmms) had a fixed duration of 6 seconds. the files were standard mono 16-bit pulse code modulation (pcm) audio format with a sample rate of 11025 hz. to keep training times manageable (especially for the hmms with a large number of mixture components and states) representative portions were selected from the audio database totaling approximately 2 hours for the training of background and silence hmms. the performance metrics that were of most practical interest, given the task, were the false positive rates rfp and true positive rates rtp (sensitivity). to estimate rfp, approximately 6 hours of representative, cough - free, background noise was compiled. to estimate rtp the recognition algorithm was tested on 30 examples of cough that were withheld during training. the preprocessing step consisted of dividing the time - domain digital audio data into frames and then computing a sequence of per - frame descriptive feature vectors. these are implemented natively by the open source hidden markov toolkit (htk) which was used to train the symptom recognition algorithm. frame size was chosen to be 256 samples overlapping by one - half of a frame. the overall frame rate was thus 0.0116 seconds. to compute the mfccs features a 256-point hanning window multiplied the given frame of audio samples and the results were stored in an array a[k ]. a 32-point mel - filterbank (figure 2), setting array sizes to an appropriate power of 2 greatly simplifies programming when discrete fourier transforms are needed. importantly, the indices of a[k ] were reordered to introduce the symmetries needed to compute a discrete cosine transform (dct) with a dft. the result of the second dft was stored in an array c[k ] and the dct coefficients were found via the following formula : (1)mfcc[k]=r{q[k]}r{c[k]}i{q[k]}i{c[k]}2. here, q[k ] is a fixed array arising from the definition of the discrete cosine transform. for k (1,31) q[k ] is equal to a complex exponential with an additional factor of 1/2 when k = 0 : (2)q[k]={22neik/(2n),k(1,31),22neik/(2n),k=0. this value was not retained because a different log - energy measure was used instead to track sound intensity. the base of the logarithm is e, n is the frame size equal to 256, and x[n ] are the values of the time - domain audio samples. although the computation of the mel - cepstrum is not an invertible operation it is reasonable to assume, based upon the performance of the classifier, that these mfccs contained enough discriminative acoustic information for the task. as stated above, the zeroth mfcc is an energy measure. in speech recognition, the higher mfccs, however, are not known to admit any simple perceptual interpretations [27, 28 ]. the first twelve mfccs and the log - energy defined one - half of the feature vector for each frame. an example of these features for a given frame is shown (figure 3). the other half of the feature vector was defined to be the first differences of the mfccs and log - energy. if i is the index of the frames in time then the first difference of any quantity f at time i is defined as (fi+1 fi1)/2. at frame boundaries for simplicity in eventually transferring the recognition algorithm to a mobile application a preemphasis filter was not applied. hidden markov models are often used to describe a sequence of observations as the probabilistic evolution of a finite state machine. associated with each state is a probability distribution that describes the elements of the observation vector. the sequential nature of the relevant data or process is captured in the state - to - state transition probabilities. hmms are partially specified as directed graphs and two example topologies are shown (figure 5). the nodes in the graph represent states and the arrows represent allowed transitions between states. two topologies were examined : an ergodic or connected topology and a left - to - right topology (5). following, connected models were used for background and silence hmms while both the connected and left - to - right topologies were tested for identifying coughs (the key effect). clearly, for a fixed number of states, left - to - right models are much more constrained than connected models as quantified by the total number of possible paths through the model. comparing the two, the role of context partially determines the grammar for the recognition task (equivalently, context determines how the background, silence, and cough models should fit together in a composite hmm). generally, for coarse segmentation of the audio stream, contextual information was not expected to greatly improve the performance of the classifier. this contrasts with speech where context can provide a significant assistance for recognition and even help to overcome poor phoneme level transcription (i.e., possible sequences of phonemes are constrained by the vocabulary of the language and, moreover, the sequence of words is constrained by grammatical rules). the gray boxes at the left and right indicate dummy states that route all of the output states to all of the input states so that the system can be run in a continuous fashion. ultimately, the structure of the composite hmm reflects in the transition matrix pij of the model. in addition to the topology, an hmm is also specified by an output probability distribution in the feature space for each state of the model. usually, for a given hmm, these probability distributions all have the same functional form. for the recognizer, the feature vector is denoted by f = [f1,, fm ]. the mixture model is a weighted sum of such terms : (5)bf(f)=i=1jibf,i,i. the total number of mixture components is denoted by l in this case. the silence model used in this paper was a 3-state connected model with 3 mixtures per state. for background, a variety of connected hmms were tested with the number of states ranging over the values (3,6) and the number of mixture components per state ranging over the values (10,30,60). likewise, for cough, connected and left - to - right topologies were tested with 5 states and the number of mixtures ranging over the values (10,60). after selecting the orders of the hmms the models were trained. as stated above, the hmms reported on in this paper were trained with tools provided in the hidden markov toolkit (htk). specifically, each hmm was initialized using a single pass of viterbi training until convergence followed by a single pass of baum - welch training until convergence. in the nomenclature of htk this is accomplished with the command line programs hinit and hrest, respectively (using the whole - word strategy). to recognize a new audio stream viterbi decoding the viterbi decoding is implemented with token passing [24, 29 ]. in the recognition context the behavior of hidden markov models is best understood using a lattice where the horizontal axis is the discrete time index and the vertical axis an index of the states. a token can be visualized as an entity which moves through the state lattice and keeps a record of the path and the likelihood of the path it has taken. the likelihood is simply the sum of the appropriate values of the emission probabilities b(fn) (where n is a timing variable at the frame rate) and the state - to - state transition probabilities pij. viterbi decoding finds the most likely path through the lattice given the data. using the token idea the decoding algorithm (rule1) to update a new frame of data, or equivalently to move to the next time step in the lattice, compute all possible transitions for the current set of tokens ; (rule2) for each state keep the incoming token that has the highest likelihood. in general, if there are n frames of data and l states then arrays of size n l must be kept in memory. in particular, the likelihoods and the states where the tokens originated are retained in memory. to recover the actual state alignment the algorithm begins at the end of these arrays and works backwards, following the path of the most likely token. the update rule can also be stated mathematically as a recursion : (6)sj[n]=maxi(si[n1]+logpij)+logbj(fn). an example of a decoding pass for some audio data containing coughs is presented (figure 7) (note that for continuity this is the same audio data shown above (figure 3)). a time - domain waveform of the audio being decoded and the resultant state sequence are shown (figure 7). the horizontal axis represents time (at the frame rate) while the vertical axis is an integer of the state index. the black marks indicate states associated with the cough model (in this case, a left - to - right topology). after the viterbi decoder, the rule for deciding whether a frame of audio data contains coughing is simply whether the state sequence ever passed through the cough model. this is equivalent to observing when the maximum likelihood path passed through the middle branch of the composite hmm (figure 6). note that when the cough model uses a left - to - right topology it forces any detected cough event to have visited each state of the cough hmm in a linear manner which is observed in the state sequence (figure 7). as discussed briefly above, this constraint partially explains why the rates of false positive are generally better for the left - to - right versus connected cough models. as stated in the introduction, the ultimate goal of this project was to create a symptom recognition application for asthmatics that could run, continuously, on a mobile device. consequently, apple developer credentials were obtained and the recognition algorithm described above was implemented in the xcode software development environment and ios software development kit. standard core audio apis were used for interfacing to the audio hardware of the mobile platform (4 g ipod touches). all of the required signal processing was facilitated by the fast fourier transform routines provided in the accelerate framework. moreover, custom cabling assemblies were constructed that allowed olympus me-15 tie - clip microphones to interface with the ipod. numerical underflow (or overflow) is a potential pitfall of working with hmms and the viterbi algorithm. equations (4) and (5) show that the probabilities involved can quickly accrue large exponents especially for a greater number of features and especially in conjunction with the viterbi algorithm. therefore, computations are carried out, where possible, with log - likelihoods. by taking care of computing the probability for each mixture component it was found that 64-bit floating point arithmetic could be used without underflowing. the exact algorithm for this computation again, the feature vector was 26-dimensional consisting of twelve mfccs, the log - energy measures, and the first differences of these quantities. here, f,, v, and are, respectively, the feature vector, the mean vector for the given component, a vector of the values along the main diagonal of, and the mixture weight. the likelihood for the entire state is found by running algorithm 1 for each associated mixture component and then taking the natural log. two example instances of the mobile adam application running in a demonstration mode that was used for debugging are shown (figure 8). the data presented in the screenshots is similar to the viterbi decoding examples shown earlier (figure 7) with the exception that the audio is being obtained and processed live on a mobile device. the top trace is the time - domain audio waveform and the bottom trace is a consolidation of the corresponding maximum likelihood state sequence found by token passing. let aseq[m ] for 0 < m < 514 be maximum likelihood state sequence (for the chosen frame rate, 6 second blocks of audio result in 515 feature vectors). from aseq another sequence is created that is defined as (7)acough[m]={1,aseq[m]coughstate,0,aseq[m]otherwise. thus acough[m ] indicates where coughs occur in the audio according to the recognition algorithm. the left screenshot shows the application identifying a sequence of coughs from the database while the right screenshot shows the device discounting some nonsymptom speech (figure 8). these tests were conducted in an anechoic environment with the test sounds played through high - quality active speakers. this is not to claim that such tests recreate the conditions of a patient wearing the device in a nonlaboratory setting. rather, the tests were meant to verify that the recognition framework and application could return positive results in circumstances where any concerns over the reproducibility of test signals were minimized. it should be stated here that the performance measures given in the next section were determined by cross - validation trials with the test database. the logical next step in evaluating the device is to trial it with a population of patients. such a trial will not only measure the accuracy of the recognition algorithm but will also measure the scope of the test database (i.e., how well the algorithm generalizes). the columns in table 1 are from left to right as follows : the number of states in the background model, the number of mixtures per state for the background model, the number of states in the cough model, the number of mixtures per state for the cough model, the topology of the cough model, the rate of false positive per 1 hour, and the sensitivity. it is also noted that the number of states listed in table 1 follows an htk convention of including the dummy states. therefore, the number of states that are actually active during decoding is the given number minus two. firstly, 6 seconds of pcm audio was collected and split into frames with 256 points and half a frame overlap. feature vectors for each frame were computed and the audio was segmented in time by a hidden markov model based viterbi decoder. the entire 6 seconds of audio was classified as either containing a cough or not containing a cough based upon the criterion explained above (figure 7). in the classification task the data is skewed since it was expected that cough would occur relatively infrequently when compared to the frequency of other background noises. here, a pair of metrics was used : the rate of false positive (or false alarms) per 1 hour fp/1 h and sensitivity or true positive rate. the rate of false positive is found by counting the number of false positives and dividing by the total length of time that was analyzed (in units of hours). this can be estimated robustly since it is not a particular challenge to collect large volumes of audio data that are free of coughs (i.e., any hit is guaranteed to be a false alarm, and these are easy to count). let tp, tn, fp, and fn be, respectively, the total number of true positives, true negatives, false positives, and false negatives returned by a classifier. the sensitivity is defined as (8)sensitivity = tptp+fn. sensitivity measures how often the classifier misses symptoms. the composite hmm was tested with 30 examples of cough that were withheld during training. the results presented in the previous section show that certain model topologies and complexities provide a reliable detection and segmentation of cough and background sounds. clearly, the prototype used for the cough hmm has a powerful effect upon both performance measures. as expected, the connected cough models result in a higher false alarm rate (keeping the other variables fixed). also, it is noted that the cough model requires a larger number of mixture components per state to achieve a decent rate of false positive. in trying to select among these alternative algorithms the goal is to provide data to the asthma patients that can give them a clearer picture of their disease state. a significant rate of false alarm will negatively impact the user 's trust in the feedback provided by the device. thus, aiming for low fp/1 h a large background hmm and a left - to - right cough hmm were used. describe the leicester cough monitor (lcm) [3, 25 ]. like the system presented here, the lcm uses a recognition system consisting of preprocessing to calculate the cepstral features followed by hmm analysis. a very high sensitivity (85.7%) and low false positive (0.8 per hour) rate were achieved using this approach. however, in the lcm the audio analysis is performed offline and the ambulatory component of the study consisted strictly of an audio recorder. this differs critically from the present study where all of the processing is performed on the mobile device and the results are provided as feedback in real time. the inherent time and computational restrictions in the present study prevented the use of hmms of the same complexity (in terms of the number of hidden states) as the lcm. like adam and the lcm, shin. report on a cough detection system that uses hmm analysis of audio features. instead of using cepstral features directly shin. processed cepstral features with an artificial neural network before passing them to the hmm viterbi decoder. while their system successfully discriminated cough from environmental noise it was only validated using synthetic examples at most 10 minutes in length so it is not directly comparable to the in vivo situation that is of concern here. also, the present study is the first to examine the feasibility of ambulatory cough detection on a consumer mobile device in an adolescent asthma population. effective asthma management relies upon the accurate monitoring of symptoms by patients. however, current monitoring techniques have had limited success with adolescent groups. around their peers, adolescents with asthma may feel embarrassed about their condition and may also be reluctant to take medication when needed. adam provides reliable and objective symptom monitoring throughout the day that can be accessed privately through an attractive mobile interface. also, since adam is deployed on an already popular consumer platform, the device can be used inconspicuously and the self - management benefits it provides will not be hampered by social pressures. overall, it is expected that adolescents who have access to a technology such as adam will demonstrate greater adherence to treatment regimens, better awareness of symptom severity and, in the long term, require fewer emergency room visits. although cough is the most common symptom found in pediatric patients with uncontrolled asthma [13, 14 ] there are other respiratory symptoms such as wheeze and the initial intention was to design a device to monitor both coughing and wheezing. however, wheeze was encountered infrequently during the data collection phase and the examples of wheeze that were recorded showed significant audio variations between each other. this frustrated attempts to design a reliable algorithm for identifying wheeze and distinguishing it from background noise. in principal, the methods described that were used for detecting cough could be applied to wheeze but it would require a more representative audio database than was available in the present study. firstly, because adam continuously records and processes sounds it is more resource intensive than typical current generation mobile applications. this impacts battery life negatively and may interfere with other applications that may be running on the device concurrently. future iterations of the application with better optimized code and power management will be needed to address these concerns. although mobile technology is becoming increasingly ubiquitous adam is still not appropriate for all situations. the device generally needs to be carried on the person and the lapel microphone must be positioned appropriately. certain activities, such as sports, may require the patient to set the device aside or the patient may be dressed in a manner that makes the adam device awkward to carry (i.e., clothing without a convenient pocket). moreover, in certain social settings, an adolescent may not want to be seen wearing a microphone. these practical limitations are expected however ; as long as patients make reasonable effort to use adam whenever possible the device will provide benefit in terms of asthma self - management. using a large audio database collected in a controlled study an algorithmic approach to the automated assessment of coughing in adolescent asthmatics was explored. this work represents the technical development of a device (adam) capable of providing objective round - the - clock feedback to adolescents with an asthma diagnosis. the eventual goal is to leverage the semantic information that can be gleaned from an audio stream to benefit the patient. in conjunction with the user interface capabilities and also the ubiquity of modern mobile phones this work enables new standards in 24-hour asthma symptom monitoring. | the development of an automated system for asthma monitoring (adam) is described. this consists of a consumer electronics mobile platform running a custom application. the application acquires an audio signal from an external user - worn microphone connected to the device analog - to - digital converter (microphone input). this signal is processed to determine the presence or absence of cough sounds. symptom tallies and raw audio waveforms are recorded and made easily accessible for later review by a healthcare provider. the symptom detection algorithm is based upon standard speech recognition and machine learning paradigms and consists of an audio feature extraction step followed by a hidden markov model based viterbi decoder that has been trained on a large database of audio examples from a variety of subjects. multiple hidden markov model topologies and orders are studied. performance of the recognizer is presented in terms of the sensitivity and the rate of false alarm as determined in a cross - validation test. |
camel milk has been used for centuries as a medicinal drink in middle eastern, asian, and african cultures (13). camel milk products in the world are popular due to increasing demand and are typically available in pharmacies (4). this systematic review has been conducted to evaluate the nutritional value and medicinal properties of camel milk in children, based on historical and clinical evidences. camel milk has been used for centuries in middle eastern societies, and its many medicinal benefits need to be established by conventional medicine. the camel is an animal that has been integrated into daily life of many societies in the pre - modern world and is still greatly respected in the cultures of these societies. thus, traditional medicine books and documents provide many quotes about the camel s mild benefits and nutritional values. avicenna stated therapeutic effects for camel milk in spleen, kidney, liver, cancer, and children diseases have been reported (8). thus, in this comparative review, we aim to investigate the issues discussed with referring to the conventional medicine evidence. according to traditional iranian medicine (tim), the composition of camel milk is closer to human milk and of high nutritional value and therapeutic effects (57). subsequently, the main aim of this research is to understand the various diseases that might be alleviated or prevented with camel milk and relating them to the biochemical content of camel milk and discussing the causality of these benefits based on traditional resources and conventional medicine research. besides, we aim to evaluate camel milk in the cases or conditions for treatment of disease. in this review, the databases, including pubmed, scopus, sid, iranmedex, and google scholar, have been searched using the search strategy of : (camel and milk) and (autism or food allergy or milk allergy or children or diarrhea) for randomized controlled clinical trials, systematic reviews and/or meta - analysis, case series, case reports, and animal studies, which have been published from 1980 to 2014. the abstract of national (iranian) and international congresses / meetings proceedings (including congress of camel) and university dissertations were also explored. materials were sorted according to indications and novelties. out of the 472 searched articles, 35 related studies were chosen for final analysis based on the noted criteria (figure 1). the third reviewer was asked if the agreement (kappa coefficient) was less than 65%. as inclusion criteria, english, persian, and arabic language studies randomized controlled clinical trials, systematic reviews and/or meta - analysis, case series, case reports, and animal studies. irrelevant studies and the studies on other milks, other traditional resources (e.g., chinese medicine), studies on animals, studies rejecting alternative medicine in general or studies with combined methods are excluded. for quality control, all parts of this article, including the abstract, introduction, search strategies and materials, finding, discussion, and results, were evaluated with strobe (strengthening the reporting of observational studies in epidemiology) statement checklist (35). based on the strobe checklist, including 22 items, the strength quality of all articles was assessed. in addition, the sampling method, existence of valid instruments, the appropriateness of the statistical analysis, ethical considerations, identification of the rcts with codes, and presence of inclusion and exclusion criteria were also controlled. the data were based on the search results, which identified 472 articles ; although 434 studies were excluded from the review due to quality difficulties, and 35 articles remained for further consideration. in this review, the databases, including pubmed, scopus, sid, iranmedex, and google scholar, have been searched using the search strategy of : (camel and milk) and (autism or food allergy or milk allergy or children or diarrhea) for randomized controlled clinical trials, systematic reviews and/or meta - analysis, case series, case reports, and animal studies, which have been published from 1980 to 2014. the abstract of national (iranian) and international congresses / meetings proceedings (including congress of camel) and university dissertations were also explored. materials were sorted according to indications and novelties. out of the 472 searched articles, 35 related studies were chosen for final analysis based on the noted criteria (figure 1). the third reviewer was asked if the agreement (kappa coefficient) was less than 65%. as inclusion criteria, english, persian, and arabic language studies randomized controlled clinical trials, systematic reviews and/or meta - analysis, case series, case reports, and animal studies. the titles and abstracts of the relevant studies were evaluated. irrelevant studies and the studies on other milks, other traditional resources (e.g., chinese medicine), studies on animals, studies rejecting alternative medicine in general or studies with combined methods are excluded. for quality control, all parts of this article, including the abstract, introduction, search strategies and materials, finding, discussion, and results, were evaluated with strobe (strengthening the reporting of observational studies in epidemiology) statement checklist (35). based on the strobe checklist, including 22 items, the strength quality of all articles was assessed. in addition, the sampling method, existence of valid instruments, the appropriateness of the statistical analysis, ethical considerations, identification of the rcts with codes, and presence of inclusion and exclusion criteria were also controlled. the data were based on the search results, which identified 472 articles ; although 434 studies were excluded from the review due to quality difficulties, and 35 articles remained for further consideration. camel s milk is generally an opaque white color and has a faint sweetish odor but sharp taste ; sometimes it can be salty (10). in a meta - analysis study (19052006), camel milk was examined and compared with other milks in five manuscripts. the average amount of components of camel milk is protein 3.1% ; fat 3.5% ; lactose 4.4% ; ash 0.79%, and total solids 11.9% (1116). the total solid content is similar to that human milk (17). in previous studies, the total amount of camel milk protein has been reported as being from 2.9 to 4.9. the amount and type of amino acids in camel milk are high except for lysine, glycine, threonine, and valine. the most important proteins in camel milk are whey proteins, which contain albumin, lactoferrin, immunoglobulins, etc. the fat content of dromedary camel milk is between 1.2% and 6.4% (11). short chain fatty acids in camel milk are low, but the long chain fatty acids are high in content. the amount of linoleic acid and unsaturated fatty acids in camel milk is also high, which is important for nutrition (1116). camel milk has a lot of vitamins from different groups : fat and water - soluble, such as vitamin a, e, d, and b, and especially vitamin c (1015). camel milk is a good source of minerals, especially calcium and kalium (1116). therapeutic effects of camel milk were assessed in some clinical trials, case reports, and in vivo/ in vitro studies. six review articles reported on camel milk, citing that it can be used in metabolic and autoimmune diseases, hepatitis, rota viral diarrhea, tuberculosis, cancer, diabetes, liver cirrhosis, rickets, autism, and crohn s disease (1821). related papers to children diseases including autism, food and milk allergies, intolerance to lactose and diarrhea were reviewed in this study as follows : camel milk has lower lactose in comparison with cow s milk (13). only one study indicated whether camel s milk can be consumed by patients intolerant to lactose without undesirable reactions ; in 25 patients, it was seen that camel s milk can be considered an option for an individual s intolerance to lactose, who presents symptoms when ingesting cow s milk (22). camel milk is a remedy for viruses causing diarrhea (such as rota virus). an animal study in 2010 indicated that fermented camel milk had a higher content of sodium and potassium and stopped diarrhea in model rats. it can be concluded that fermented camel milk can be considered as a good food for high nutritive and therapeutic applications (23). a review study reported improvement in crohn s and autism diseases such as diarrhea ; bowel movements were considered normal ; thus, the authors offered a new perspective on the etiology of some diarrheal diseases (24). thus, finding suitable milk for alternative mothers or bovine milks in children was needed. results of a prospective cohort study, performed in pediatric departments between april 2007 and february 2010 on 35 children aged 612 months with a cow s milk allergy (cma), indicated that 80% of children with cma were safely able to consume camel milk without developing any adverse allergic reaction (25). in the second study, entitled to determine whether camel milk is safer than goat milk in cma, 38 children with cma were evaluated by the following tests : cbc, total ige, cow milk - specific ige, and spt. the tests in the children showed less allergic reaction to camel milk and that it can be a safe alternative to goat milk. (26). in another study, researchers tested two patients, aged 3 years 6 months and 2 years 3 months, respectively, with cma, using a prick test with food antigens and prick - by - prick test with powder cm and full cream cow s milk. the patients were positive for the cow s milk and negative for the cm in the prick test. these observations agreed with the results reported by ehlayel on a cohort of 35 patients tested with fresh cm (27). in a similar study six, cma patients aged from 14 months to 13 years using prick test with food antigens and prick - by - prick test with powder cm and full cream cow s milk were tested. all children resulted positive for full cream cow s milk and negative for cm at prick - by - prick test (28). results of the above studies show that cm could represent a possible alternative to infant formulas in cma. the researchers hypothesize that the children were able to tolerate the camel milk because it contains a different protein than cow s milk, which does not elicit an immune response. autism spectrum disorder (asd) is a severe neurodevelopment disorder characterized by impairments in social orientation, communication, and repetitive behaviors (29). a study published in the 2005 observed the effects of camel milk consumption, instead of cow milk, on several cases of children and adults with autism. researchers discovered that, when a 4-year - old female participant consumed camel milk for 40 days, a 15-year - old boy consumed camel milk for 30 days, and several 21-year - old autistics consumed camel milk for two weeks, their autism symptoms disappeared. the authors reported in another study that camel milk consumption in children under 15 has been effective in controlling some of the symptoms, especially in the group under 10 years (30). some parents report that their children were suffering from autism ; then the children used camel milk, some of their symptoms improved such as better sleep ; increased motor planning and spatial awareness ; increased eye contact ; better language and improved gastrointestinal function. in a study conducted on 60 patients with autism (2 to 12 years old) in saudi arabia, the effects of camel and cow s milk were evaluated when 500 ml of milk twice daily for 2 weeks were given to children. in the analysis, results after two weeks showed significant improvement in cognitive and behavioral tests due to camel milk and lowered effects of oxidative stress. the researchers showed that camel milk plays an important role and reduces the effects of oxidative stress by adjusting the antioxidant enzymes and nonenzymatic antioxidant materials levels and improves psychological symptoms (31). in a case report published in 2013, a boy had been introduced in the third year of his life to recognize autism. the mother of this boy started, from the age of nine years, to give him a glass of camel milk at night. he was observed for six years (20072013) to see if camel milk would control the symptoms of autism (32). this report agreed with the results reported by y. shabo (30) that camel milk is especially useful for autistic children (asd). forty - five children diagnosed with autism were randomly assigned to receive boiled cm for group i (n = 15), raw cm for group ii (n = 15), and a placebo for group iii (n = 15) for 2 weeks. cm administered for 2 weeks significantly improved clinical symptoms of autism and decreased serum level of thymus and activation - regulated chemokine (tarc) in autistic children (33). a study has investigated the effect of camel milk on children who were allergic to cow s milk. it appeared that camel milk has a positive effect in children with severe food allergies. a study by el - agamy revealed that, when applying camel s milk protein specific antisera in immunoblotting analysis, there was no immunologic cross reactivity between camel and cow s milk proteins. this study is laboratory work to obtain a better understanding of the competence of the greater camel of cow s milk for food allergies in children. for this purpose, milk proteins of camels, cows, and humans, extracted by two different methods of electrophoresis, and were investigated. the results show that camel milk proteins can be used as a new protein source for food allergies (35). camel s milk is generally an opaque white color and has a faint sweetish odor but sharp taste ; sometimes it can be salty (10). in a meta - analysis study (19052006), camel milk was examined and compared with other milks in five manuscripts. the average amount of components of camel milk is protein 3.1% ; fat 3.5% ; lactose 4.4% ; ash 0.79%, and total solids 11.9% (1116). the total solid content is similar to that human milk (17). in previous studies, the total amount of camel milk protein has been reported as being from 2.9 to 4.9. the amount and type of amino acids in camel milk are high except for lysine, glycine, threonine, and valine. the most important proteins in camel milk are whey proteins, which contain albumin, lactoferrin, immunoglobulins, etc. the fat content of dromedary camel milk is between 1.2% and 6.4% (11). short chain fatty acids in camel milk are low, but the long chain fatty acids are high in content. the amount of linoleic acid and unsaturated fatty acids in camel milk is also high, which is important for nutrition (1116). camel milk has a lot of vitamins from different groups : fat and water - soluble, such as vitamin a, e, d, and b, and especially vitamin c (1015). camel milk is a good source of minerals, especially calcium and kalium (1116). in previous studies, the total amount of camel milk protein has been reported as being from 2.9 to 4.9. the amount and type of amino acids in camel milk are high except for lysine, glycine, threonine, and valine. the most important proteins in camel milk are whey proteins, which contain albumin, lactoferrin, immunoglobulins, etc. the fat content of dromedary camel milk is between 1.2% and 6.4% (11). short chain fatty acids in camel milk are low, but the long chain fatty acids are high in content. the amount of linoleic acid and unsaturated fatty acids in camel milk is also high, which is important for nutrition (1116). camel milk has a lot of vitamins from different groups : fat and water - soluble, such as vitamin a, e, d, and b, and especially vitamin c (1015). camel milk is a good source of minerals, especially calcium and kalium (1116). therapeutic effects of camel milk were assessed in some clinical trials, case reports, and in vivo/ in vitro studies. six review articles reported on camel milk, citing that it can be used in metabolic and autoimmune diseases, hepatitis, rota viral diarrhea, tuberculosis, cancer, diabetes, liver cirrhosis, rickets, autism, and crohn s disease (1821). related papers to children diseases including autism, food and milk allergies, intolerance to lactose and diarrhea were reviewed in this study as follows : camel milk has lower lactose in comparison with cow s milk (13). only one study indicated whether camel s milk can be consumed by patients intolerant to lactose without undesirable reactions ; in 25 patients, it was seen that camel s milk can be considered an option for an individual s intolerance to lactose, who presents symptoms when ingesting cow s milk (22). camel milk is a remedy for viruses causing diarrhea (such as rota virus). an animal study in 2010 indicated that fermented camel milk had a higher content of sodium and potassium and stopped diarrhea in model rats. it can be concluded that fermented camel milk can be considered as a good food for high nutritive and therapeutic applications (23). a review study reported improvement in crohn s and autism diseases such as diarrhea ; bowel movements were considered normal ; thus, the authors offered a new perspective on the etiology of some diarrheal diseases (24). thus, finding suitable milk for alternative mothers or bovine milks in children was needed. results of a prospective cohort study, performed in pediatric departments between april 2007 and february 2010 on 35 children aged 612 months with a cow s milk allergy (cma), indicated that 80% of children with cma were safely able to consume camel milk without developing any adverse allergic reaction (25). in the second study, entitled to determine whether camel milk is safer than goat milk in cma, 38 children with cma were evaluated by the following tests : cbc, total ige, cow milk - specific ige, and spt. the tests in the children showed less allergic reaction to camel milk and that it can be a safe alternative to goat milk. (26). in another study, researchers tested two patients, aged 3 years 6 months and 2 years 3 months, respectively, with cma, using a prick test with food antigens and prick - by - prick test with powder cm and full cream cow s milk. the patients were positive for the cow s milk and negative for the cm in the prick test. these observations agreed with the results reported by ehlayel on a cohort of 35 patients tested with fresh cm (27). in a similar study six, cma patients aged from 14 months to 13 years using prick test with food antigens and prick - by - prick test with powder cm and full cream cow s milk were tested. all children resulted positive for full cream cow s milk and negative for cm at prick - by - prick test (28). results of the above studies show that cm could represent a possible alternative to infant formulas in cma. the researchers hypothesize that the children were able to tolerate the camel milk because it contains a different protein than cow s milk, which does not elicit an immune response. autism spectrum disorder (asd) is a severe neurodevelopment disorder characterized by impairments in social orientation, communication, and repetitive behaviors (29). a study published in the 2005 observed the effects of camel milk consumption, instead of cow milk, on several cases of children and adults with autism. researchers discovered that, when a 4-year - old female participant consumed camel milk for 40 days, a 15-year - old boy consumed camel milk for 30 days, and several 21-year - old autistics consumed camel milk for two weeks, their autism symptoms disappeared. the authors reported in another study that camel milk consumption in children under 15 has been effective in controlling some of the symptoms, especially in the group under 10 years (30). some parents report that their children were suffering from autism ; then the children used camel milk, some of their symptoms improved such as better sleep ; increased motor planning and spatial awareness ; increased eye contact ; better language and improved gastrointestinal function. in a study conducted on 60 patients with autism (2 to 12 years old) in saudi arabia, the effects of camel and cow s milk were evaluated when 500 ml of milk twice daily for 2 weeks were given to children. in the analysis, results after two weeks showed significant improvement in cognitive and behavioral tests due to camel milk and lowered effects of oxidative stress. the researchers showed that camel milk plays an important role and reduces the effects of oxidative stress by adjusting the antioxidant enzymes and nonenzymatic antioxidant materials levels and improves psychological symptoms (31). in a case report published in 2013, a boy had been introduced in the third year of his life to recognize autism. the mother of this boy started, from the age of nine years, to give him a glass of camel milk at night. he was observed for six years (20072013) to see if camel milk would control the symptoms of autism (32). this report agreed with the results reported by y. shabo (30) that camel milk is especially useful for autistic children (asd). forty - five children diagnosed with autism were randomly assigned to receive boiled cm for group i (n = 15), raw cm for group ii (n = 15), and a placebo for group iii (n = 15) for 2 weeks. cm administered for 2 weeks significantly improved clinical symptoms of autism and decreased serum level of thymus and activation - regulated chemokine a study has investigated the effect of camel milk on children who were allergic to cow s milk. it appeared that camel milk has a positive effect in children with severe food allergies. a study by el - agamy revealed that, when applying camel s milk protein specific antisera in immunoblotting analysis, there was no immunologic cross reactivity between camel and cow s milk proteins. this study is laboratory work to obtain a better understanding of the competence of the greater camel of cow s milk for food allergies in children. for this purpose, milk proteins of camels, cows, and humans, extracted by two different methods of electrophoresis, and were investigated. the results show that camel milk proteins can be used as a new protein source for food allergies (35). only one study indicated whether camel s milk can be consumed by patients intolerant to lactose without undesirable reactions ; in 25 patients, it was seen that camel s milk can be considered an option for an individual s intolerance to lactose, who presents symptoms when ingesting cow s milk (22). camel milk is a remedy for viruses causing diarrhea (such as rota virus). an animal study in 2010 indicated that fermented camel milk had a higher content of sodium and potassium and stopped diarrhea in model rats. it can be concluded that fermented camel milk can be considered as a good food for high nutritive and therapeutic applications (23). a review study reported improvement in crohn s and autism diseases such as diarrhea ; bowel movements were considered normal ; thus, the authors offered a new perspective on the etiology of some diarrheal diseases (24). thus, finding suitable milk for alternative mothers or bovine milks in children was needed. results of a prospective cohort study, performed in pediatric departments between april 2007 and february 2010 on 35 children aged 612 months with a cow s milk allergy (cma), indicated that 80% of children with cma were safely able to consume camel milk without developing any adverse allergic reaction (25). in the second study, entitled to determine whether camel milk is safer than goat milk in cma, 38 children with cma were evaluated by the following tests : cbc, total ige, cow milk - specific ige, and spt. the tests in the children showed less allergic reaction to camel milk and that it can be a safe alternative to goat milk. (26). in another study, researchers tested two patients, aged 3 years 6 months and 2 years 3 months, respectively, with cma, using a prick test with food antigens and prick - by - prick test with powder cm and full cream cow s milk. the patients were positive for the cow s milk and negative for the cm in the prick test. these observations agreed with the results reported by ehlayel on a cohort of 35 patients tested with fresh cm (27). in a similar study six, cma patients aged from 14 months to 13 years using prick test with food antigens and prick - by - prick test with powder cm and full cream cow s milk were tested. all children resulted positive for full cream cow s milk and negative for cm at prick - by - prick test (28). results of the above studies show that cm could represent a possible alternative to infant formulas in cma. the researchers hypothesize that the children were able to tolerate the camel milk because it contains a different protein than cow s milk, which does not elicit an immune response. autism spectrum disorder (asd) is a severe neurodevelopment disorder characterized by impairments in social orientation, communication, and repetitive behaviors (29). a study published in the 2005 observed the effects of camel milk consumption, instead of cow milk, on several cases of children and adults with autism. researchers discovered that, when a 4-year - old female participant consumed camel milk for 40 days, a 15-year - old boy consumed camel milk for 30 days, and several 21-year - old autistics consumed camel milk for two weeks, their autism symptoms disappeared. the authors reported in another study that camel milk consumption in children under 15 has been effective in controlling some of the symptoms, especially in the group under 10 years (30). some parents report that their children were suffering from autism ; then the children used camel milk, some of their symptoms improved such as better sleep ; increased motor planning and spatial awareness ; increased eye contact ; better language and improved gastrointestinal function. in a study conducted on 60 patients with autism (2 to 12 years old) in saudi arabia, the effects of camel and cow s milk were evaluated when 500 ml of milk twice daily for 2 weeks were given to children. in the analysis, results after two weeks showed significant improvement in cognitive and behavioral tests due to camel milk and lowered effects of oxidative stress. the researchers showed that camel milk plays an important role and reduces the effects of oxidative stress by adjusting the antioxidant enzymes and nonenzymatic antioxidant materials levels and improves psychological symptoms (31). in a case report published in 2013, a boy had been introduced in the third year of his life to recognize autism. the mother of this boy started, from the age of nine years, to give him a glass of camel milk at night. he was observed for six years (20072013) to see if camel milk would control the symptoms of autism (32). this report agreed with the results reported by y. shabo (30) that camel milk is especially useful for autistic children (asd). forty - five children diagnosed with autism were randomly assigned to receive boiled cm for group i (n = 15), raw cm for group ii (n = 15), and a placebo for group iii (n = 15) for 2 weeks. cm administered for 2 weeks significantly improved clinical symptoms of autism and decreased serum level of thymus and activation - regulated chemokine (tarc) in autistic children (33). a study has investigated the effect of camel milk on children who were allergic to cow s milk. it appeared that camel milk has a positive effect in children with severe food allergies. a study by el - agamy revealed that, when applying camel s milk protein specific antisera in immunoblotting analysis, there was no immunologic cross reactivity between camel and cow s milk proteins. this study is laboratory work to obtain a better understanding of the competence of the greater camel of cow s milk for food allergies in children. for this purpose, milk proteins of camels, cows, and humans, extracted by two different methods of electrophoresis, and were investigated. the results show that camel milk proteins can be used as a new protein source for food allergies (35). these findings revealed that camel milk is safer for children, is effective in the treatment of autism, and improves general well - being. also, the numerous nutrients in camel milk help to promote the body s natural defenses and thus can be considered as a good source of protein, calcium, phosphorus, vitamin c, and niacin and can meet part of the daily nutritional needs of humans. the researchers hope that these reports for pediatricians will lead to increased research on camel milk and its uses for children. | introductioncamel milk is the closest to a human mother s milk. camel milk is different from other milks, however, having low sugar and cholesterol, high minerals (sodium, potassium, iron, copper, zinc and magnesium, and vitamin c). the milk is considered have medicinal characteristics as well. this systematic review is aimed at determining and reporting nutritional values and medicinal characteristics of camel milk in children.methodsthe search strategy of the current review is (camel and milk) and (autism or food allergy or milk allergy or children or diarrhea. the search was conducted via pubmed, scopus, and google scholar. also two persian scientific databases (sid and iranmedex) and international congresses were investigated. full - text papers and abstracts on the topic of camel milk, evaluating nutritional value and medicinal properties, were included in this systematic review.resultsout of the 472 records found in the resources, 35 related studies were included in the final analysis. the result showed that camel milk is highly nutritious and is safe for consumption by children.conclusionit seems that many researchers did not follow a specific guideline for reporting and confirming the therapeutic properties of camel milk in children, but there is evidence denoting the importance, trials, and investigations of its usability and benefits. camel milk as a supplemental treatment seems less invasive and costly than specialist care, medications, alternative treatments, and behavioral interventions. based on our findings, camel milk is safer for children, effective in the treatment of autism, improves general well - being, promotes body natural defenses, is a good nutritional source, and can helps the daily nutritional needs of humans. |
tear film studies have progressed a lot in the past few years, from the traditional quantitative tests like schirmer test, and tear meniscus height measurement, and the qualitative test of tear break - up time (tbut), to anterior segment optical coherence tomography (as oct) measured tear film height and the dynamic tear film studies frequently used nowadays using computerized videokeratoscopy. a lot of studies pointed to the fact that diabetes mellitus greatly affects the tear film function and stability. decreased schirmer 1 test values and shorter but were positively correlated with the subjective severity of dry eye symptoms in type 2 diabetic patients. moreover, the decreased tear film function was found to be more severe in patients with pdr than in those with npdr. other studies tested the corneal sensitivity, corneal epithelial integrity, and conjunctival epithelium (using impression cytology) and showed that the degree of keratoepitheliopathy was marked, and the corneal sensitivity, tbut, and tear secretion were all significantly reduced in the diabetic patients. conjunctival impression cytology showed conjunctival squamous metaplasia and lower goblet cell count in diabetic patients. all these parameters were related to the poor metabolic control, the presence of diabetic neuropathy, and the stage of diabetic retinopathy. in fact, a recent study stated that tear film instability could be a marker of, or rather a predictor for, the occurrence of diabetic neuropathy in type 1 diabetes patients. misra and coresearchers found a positive correlation between tear film stability and corneal subbasal nerve density (measured by corneal in vivo confocal microscopy) which was statistically significant (p = 0.04) ; they also found that decreased tear film stability was associated with increasing age and duration of diabetes. recent tests for assessment of the tear film quantity include as oct measurement of the tear wedge area, or the tear meniscus height of the inferior tear meniscus. other tests for the assessment of tear film stability include the noninvasive but and its subtype, the tear thinning time (ttt), which measures the time taken for the tear film to get thinned out even before the actual break - up of the tear film occurs. the ttt has the advantage of not having to instill any eye drops or fluorescein dye prior to the test, which in itself can cause reflex lacrimation in some people and thus affect the accuracy of the test. another indicator for tear film function is the patient blinking rate per minute which was found to correlate positively to the severity of subjective dry eye symptoms. reduced and incomplete blinking along with increased tear film break - up during normal visual tasks may explain the increased level of ocular discomfort symptoms reported at the end of the day, particularly in dry eye patients. the normal average resting blinking rate per minute is 17, increasing to around 26/min during conversation and decreasing to around 4.5/min while reading [6, 7 ]. tear film function and stability in diabetic patients are an area of ongoing active research. to our knowledge, a few studies have investigated the correlation between the quantity and quality of tears [8, 9 ] but a few or almost no studies examined this correlation in diabetic patients and compared them to age matched controls. the purpose of this study is to study the tear film quantity (represented by as oct measured inferior tear meniscus height) and correlate it with the quality (represented by the ttt for stability, and patient blinking rate per minute) in diabetic patients and age matched controls. the study was done in accordance with the ethical standards in the declaration of helsinki 1964. an informed consent was taken from all patients before participating in the study. in this case control prospective series, 22 diabetic patients (9 males and 13 females), that is, group a, and 16 age matched controls (7 males and 9 females), that is, group b, were recruited from kasr al aini (cairo university hospital) ophthalmology and diabetology out - patient clinics. the age of the patients ranged from 40 to 70 years (mean 57.22 8.5 sd) for the diabetic patients (group a) and from 30 to 77 years (mean 57.12 12.22 a full history was taken, including detailed ocular and medical history, as well as a thorough full ophthalmological examination. the inclusion criteria for patients were as follows : a history of diabetes mellitus for a duration 5 years for cases in group a and no history of any systemic disease for group b.no history of chronic eye diseases and/or previous ocular surgery.no symptoms of dry eye.no history of associated systemic conditions.no use of systemic or topical medications that may affect the tear film secretion like parasympathomimetics or parasympatholytics.the patient blinking rate per minute was measured in all patients, by the same observer who attended during all patient examinations and was asked to count the rate of blinking while the patient was having a routine conversation with his doctor. a history of diabetes mellitus for a duration 5 years for cases in group a and no history of any systemic disease for group b. no history of chronic eye diseases and/or previous ocular surgery. no history of associated systemic conditions. no use of systemic or topical medications that may affect the tear film secretion like parasympathomimetics or parasympatholytics. for assessment of tear film quantity the tear meniscus height (tmh) was measured by the spectral domain optical coherence tomography (rtvue100 - 2, optovue, ca, usa) with the cornea - anterior segment lens long attached. the tear meniscus height (tmh) was measured through applying a vertical line scan passing the cornea, the inferior tear meniscus, and the lower eyelid at the 6 o'clock position. in the resulting triangular image of the inferior tear meniscus, the height of the limb opposite the acute angle formed between the inferior cornea and the lower lid margin was measured manually using the machine calipers and expressed in microns. for the assessment of tear film stability, a subtype of the noninvasive but each subject was instructed to place their chin on the chin rest of the keratometer (ciom srl, milano, italy). the patient was then asked to blink several times and then open his eyes and stop blinking. the observer then counted the time elapsed in seconds between the last blink and the earliest change in shape of the keratometer 's mires reflected upon the patient 's corneal surface. the time was then recorded in seconds. the tear thinning time is usually normally shorter than the tbut, as tear film thinning happens seconds before actual tear break - up. for each measured parameter, three successive readings were taken for each patient, and the average was taken for each case. data were statistically described in terms of mean standard deviation (sd), median and range, or frequencies (number of cases) and percentages when appropriate. comparison of numerical variables between the study groups was done using mann - whitney u test for independent samples. for comparing gender, correlation between various variables was done using pearson moment correlation equation for linear relation in normally distributed variables and spearman rank correlation equation for nonnormal variables / nonlinear monotonic relation. all statistical calculations were done using the computer program spss (statistical package for the social science ; spss inc., chicago, il, usa). this is a nonrandomized controlled case study, where twenty - two diabetic patients (group a), attending the out - patient clinic for routine checkup, who met the inclusion criteria for the current study were recruited. the obtained data under study were compared to those obtained from a control group of nondiabetic patients (group b), who still met the inclusion criteria for the current work. the demographic data of patients enrolled in the study is summarized in table 1. the duration of diabetes in group a patients ranged from 5 to 30 years duration with a mean duration of 13.5 7.5 (sd). ttt was found to range from 9 to 22 seconds with a mean value of 14.36 sec 3.38 in the diabetic patients (group a), as compared to a mean value of 14.75 sec 3.43 in the control group (group b) ; the difference between the two groups was found to be statistically insignificant (p = 0.6). moreover, the recorded mean value was 13.85 sec 2.99 in the diabetic females in group a, as compared with a mean value of 15.11 sec 3.95 in the diabetic males within the same group ; the difference was noted to be of no statistical significance (p = 0.5). the rate of blinking per minute was recorded to vary from 18 to 40 blinks / min with a mean value of 25.32/min (6.15 sd) in group a patients as compared to a range from 15 to 24 blinks / min with a mean of 20.44/min (2.25 sd) in group b ; the difference between the two groups was found to be statistically highly significant (p = 0.002). moreover, the rate of blinking was found to be lower among diabetic females when compared to diabetic males in group a, with mean rates of 22.85/min (3.75) and 28.89/min (7.35), respectively. the tmh ranged from 163 to 740 m with a mean of (365.64 148.6 sd) in group a patients as compared to a range from 171 to 727 m (356.38 155.75 sd) in group b patients, with no significant statistical difference (p = 0.96). regarding the sex differences, the tmh was found to be less in diabetic males as compared to diabetic females in group a with a mean value of 349.56 m 140.07 and 376.77 m 158.89, respectively, although the difference was statistically insignificant (p = 0.81). generally, a negative correlation of varying strength was found between the age of the patient and the three studied variables (the tmh, ttt, and blinking rate / minute) as shown in (table 2). a strong negative correlation (r = 0.625) was found between the age of patient and the tmh in microns, a correlation that was found to be statistically highly significant (p = 0.002). the weak negative correlation between the age and the rate of blinking (r = 0.39) was of no statistical significance (p = 0.06) ; however it was close to being significant. the weak negative correlation between age and ttt was of no statistical significance (p = 0.67). upon correlating the three tear film parameters with each other, a weak positive correlation (r = 0.23) was recorded between the ttt and blinking rate / min (representing stability) and the tmh in microns (representing quantity), although it was statistically not significant (p = 0.28). the rate of blinking was found to be positively correlated to the ttt, again with no statistical significance (p = 0.83). a negative correlation was elicited between the studied tear film parameters and the duration of diabetes. this correlation was of statistical significance regarding the blinking rate and tmh (p = 0.03), although it failed to achieve a statistical significance regarding ttt (p = 0.35). a summary of the correlation between the different studied parameters in group a is elaborated in table 2. a weak negative correlation between the age of the patient and ttt as well as tmh was elicited (table 3) that was noted to be statistically nonsignificant. however, a weak positive correlation between the rate of blinking and age was noted as well, again with no statistical value. moreover, a weak negative correlation was recorded between ttt and blinking rate / min (r = 0.21) that was considered to be of no statistical significance (p = 0.4) as compared to the moderate positive correlation between ttt and tmh (r = 0.46) that was noted to be statistically close to being significant (p = 0.069). the rate of blinking was found to be negatively correlated to the tmh, again with no statistical significance (p = 0.14). a summary of the correlation between the different studied parameters in group b is elaborated in table 3. diabetes mellitus is a systemic disease that affects mainly the microcirculation and can affect the ocular surface integrity through different mechanisms. the prevalence of dry eye in diabetic patients has been reported to be about 50% in type 2 diabetes. 7% of children with type 1 diabetes were reported to have dry eye manifestations compared to 0% of age matched control children. in the present study, we tried to study the impact of diabetes on the tear film quality and quantity in diabetic patients with no subjective symptoms of dry eye. we also tried to find a correlation between the different studied tear film parameters in diabetics versus normal controls. in the present study, the mean ttt was found to be less in the diabetic group as compared to the control group, although not reaching a statistically significant value ; this finding agrees with multiple previous studies, which concluded that diabetes can affect the tear film stability [9, 12, 13 ]. the reason why we preferred to perform the ttt over the regular tear break - up test (tbut) with fluorescein is the postulated limitations, including the need to instill fluorescein dye, lack of standardization of fluorescein concentration or amount, and the possible induction of reflex tearing that might alter the results of the test. tmh is typically used to detect the amount of the aqueous layer of the tear film. in our study it was noted to have a mean value higher in the diabetic than in the control group, yet with no statistical significance. again, this agreed with previous published results that diabetic patients might show a decrease in but, a decrease in basal secretion yet with a normal overall tear secretion. diabetes is one of the well - established causes of excessive blinking (blinking eye syndrome). however, some authors reported a decrease in the blinking rate in diabetics but with an increase in the interblinking interval. in our study, the rate of blinking per minute was found to be significantly higher in the diabetic as compared to the control group. we owe this to the changes in ocular surface integrity (namely, recurrent epithelial defects) associated with diabetes with subsequent irritation. furthermore, blinking was found to be significantly higher in females as compared to males within the diabetic group. this gender difference in blinking rate needs to be further investigated in more detail, as other hormonal factors may be involved. several studies demonstrated that dry eye could be correlated with the duration of diabetes as well as the severity of retinopathy [15, 16 ] ; however, others postulated that the metabolic control might be of great impact rather than the duration [2, 8 ]. in our study, we did not study the correlation with the severity of the disease ; however, a statistically significant impact could be elicited regarding the effect of duration of diabetes on tear production (i.e., tmh) as well as on the rate of blinking and the ttt. the absence of subjective symptoms of dryness (as all our patients did not complain of dry eye symptoms) despite the low values of ttt in the diabetic group can be explained by the fact that the associated decrease in corneal sensitivity caused by diabetic peripheral neuropathy in those patients might mask the symptoms of dryness. previous studies demonstrated that the impact of diabetes on ocular surface integrity and subsequently on dry eye associated with the disease might be affected with the decreased circulating sex hormones in the postmenopausal diabetic females, a thing that might add to the severity of the condition. however, we noted that the ttt values were found to be less in diabetic females as compared to males, despite higher tmh values. whether this is or is not related to menopause or is merely a gender difference needs to be further investigated in detail in subsequent studies. the mechanisms by which diabetes affects the integrity of the ocular surface, and the tear film stability and tear production have been previously investigated, whereas the impact of diabetes might also be related to the microcirculation of the lacrimal gland, a point which needs to be further correlated with tear film parameters [17, 18 ]. some studies suggested that the decreased corneal sensitivity and corneal neuropathy to be a cause of decreased basal tear secretions in diabetics. moreover, an inflammatory theory, with the postulation that dry eyes might be secondary to an inflammatory process mediated through t - call lymphocytes was also suggested by some authors. in conclusion, our study showed that the tear film integrity was found to be affected in diabetic patients, as compared to age matched controls, especially when it comes to the blinking rate. a significant negative correlation was found between the duration of diabetes and all studied tear film parameters. the correlation between the volume and stability of the tear film in diabetics was found to be a positive one. a significant difference in blinking rate was found between males and females in the diabetic group, the exact cause of which needs further investigation. the associated neuropathy with the secondary decrease in corneal sensitivity might add to the severity of the condition, yet with minimal symptoms. our study thus recommends that a routine examination for dry eye should be considered in all diabetic patients even in the absence of subjective symptoms. in diabetics the tear film stability might be affected, despite an increase in tear production that is, tmh, due to the fact that diabetes can affect the basal tear production mainly, rather than the total tear production. this again should not trick us into excluding dry eye in a diabetic patient depending only on the normal tmh measurements. the mechanism of dry eye in diabetes is a multifactorial one, where the diagnosis should be based on the measurement of multiple tear parameters rather than a single one, as not all the parameters are affected in a similar pattern. the severity of the condition might be related to the severity of diabetes and the metabolic control of the disease ; however, the duration of the disease plays a significant role as well. | purpose. to assess the tear film quantity and correlate it with the quality and stability of the tear film in diabetics and compare them to age matched controls. introduction. diabetes affects tear film parameters in multiple ways. poor metabolic control and neuropathy are postulated factors. to further understand how diabetes affects tear film parameters this study was conducted. subjects and methods. tear meniscus height was measured by anterior segment oct, along with tear thinning time, a subtype of noninvasive tear break - up time, and blinking rate per minute which were all recorded for 22 diabetic patients. correlations between these tear film parameters were studied and then compared to 16 age matched controls. results. a statistically significant difference was found in blinking rate between the diabetic and the control group (p = 0.002), with higher blinking rate among diabetics. all tear film parameters were negatively correlated with duration of diabetes. a positive correlation was found between tear film volume and stability. conclusion. diabetes affects the tear film in various ways. diabetics should be examined for dry eye signs even in absence of symptoms which may be masked by associated neuropathy. duration of diabetes has an impact on tear film status. |
freeze - fracture electron microscopy was established as a major technique in ultrastructure research over 30 years ago. when biological specimens are in the frozen state, cellular bilayer lipid membranes have a plane of weakness in their hydrophobic interior ; so if the sample is fractured, the fracture plane will split the membrane into half - membrane leaflets, each corresponding to a phospholipid monolayer with associated proteins. the result is a three - dimensional perspective of the membranous organization of the cell, with en face views of the membrane interior. these details are made visible in the electron microscope by making a very fine platinum - carbon replica of the fracture plane. the platinum is evaporated onto the specimen at an angle, so that it is deposited in varying thickness according to the topography at the fractured surface. in this way, high resolution details of membrane structure are revealed that can not be seen by other techniques. by freeze - fracture, the distribution and organization of integral membrane proteins (seen as intramembrane particles), and other specialized features, the freeze - fracture technique revolutionized the way we look at membranes, and its contribution to our knowledge of membrane structure was unrivalled, but one limitation remained the need to identify the chemical nature of the structural components visualized. without this knowledge, the functions of newly discovered structural features remained speculative. thus, the combination of cytochemistry with freeze - fracture was a widely recognized goal. the technical challenges involved in developing effective techniques in freeze - fracture cytochemistry were considerable and took several decades to be overcome. of all the techniques in freeze - fracture cytochemistry attempted and tested over the last three decades [47 ], the freeze - fracture replica immunolabeling (fril) technique has proven to be the most successful, and is currently having a substantial impact in solving questions in cell biology that have hitherto been impossible to address with other ultrastructural, cell biological, or molecular approaches [810 ]. to illustrate the potential of this approach, we present here image data on the localization of caveolin-1 and pat family proteins (the collective term for perilipin, adipophilin and tip 47), which are associated with lipid droplets. by revisiting the lipid droplet and its associated proteins with fril, new perspectives have emerged which challenge prevailing concepts on a number of fronts and open up new questions for future research. an understanding of the principles and methodology of freeze - fracture cytochemistry requires a basic knowledge of how standard freeze - fracture works. the utility of freeze - fracture depends critically on the tendency of the fracture plane to follow a plane of weakness in the hydrophobic interior of frozen membranes, splitting them into half - membrane leaflets (figure 1(a)). there are four essential steps in making a standard freeze - fracture replica : (1) rapid freezing of the specimen ; in routine application, the freezing step is often preceded by pretreatment with glutaraldehyde fixation and glycerol cryoprotection (2) fracturing it at low temperature (100c or lower) (3) making a replica of the newly exposed frozen surface by vacuum - deposition of platinum and carbon (4) cleaning the replica to remove the biological material. for a detailed protocol on how to carry out freeze - fracture, details of membrane structure are seen at macromolecular resolution. in particular, the distribution and organization of integral membrane proteins (seen as intramembrane particles) an important landmark in the progress to develop an effective technique in freeze - fracture replica immunolabeling was the introduction of colloidal gold labeling to electron microscopy in the early 1980s. gold particles, because of their high electron density and small size, were quickly recognized to be ideal markers to use in conjunction with replicas. for the purposes of the present paper, a key foundation to the development of fril was the recognition that half - membrane leaflets could be retained attached to the platinum - carbon replica without significantly interfering with visibility of structure, and that these could provide a source of epitopes for immunogold labeling [8, 9, 11 ]. the specimen is rapidly frozen, freeze - fractured and platinum - carbon replicas made following the standard protocol used for freeze - fracture electron microscopy. however, instead of removing the biological material from the replica with bleach or acids as in the conventional technique, the replica is treated with sodium dodecyl sulphate (sds) [8, 9 ]. the sds removes the bulk of the biological material from the replica so that structure is visible by electron microscopy, leaving a single lipid monolayer and associated integral and surface proteins adherent to the replica. the spatial distribution of the targeted epitopes can be viewed superimposed upon the membrane interior. both membrane leaflets of the plasma membrane and those of intracellular membranes are accessible to labeling. understanding the topological relationships between the different membrane systems of the cell and the conventions used in describing freeze - fractured membranes is essential for interpreting freeze - fractured specimens. to apply the fril technique and interpret results, the practitioner has to be acquainted with the nomenclature used for describing standard freeze - fracture images. this nomenclature is best explained by first thinking of the membrane as consisting of two halves a p half which lies adjacent to the protoplasm (i.e., cytoplasm or nucleoplasm), and an e half which lies adjacent to the extracellular, exoplasmic or endoplasmic space. the term fracture face is applied to the interior views exposed as a result of splitting the membrane by freeze - fracture. the fracture face of the p half is thus termed the p face, while that of the e half is termed e face. the true surfaces of the p half and the e half of the membranes are termed the p surface and the e surface, respectively (figure 1). to apply this nomenclature to the lipid droplet requires some adaptation because of the unusual structure of this organelle. standard freeze - fracture nomenclature can not be applied to lipid droplets as these organelles do not have a limiting phospholipid bilayer membrane as normally found enveloping organelles. the lipid droplet consists of a hydrophobic neutral lipid core surrounded by a single monolayer of phospholipids. concavely fractured droplets thus show an aspect of this monolayer that is equivalent to the p face of a normal membrane. the complementary aspect, revealed in convex fractures, would be termed an e face in the case of a normal lipid bilayer, but this designation is not appropriate since there is no e half to the enveloping structure, hence convex fractures show the outer - facing aspect of the lipid core. some fractures through lipid droplets expose multilayers of lipid, giving an onion - like morphology. lipid droplets that are cross - fractured reveal a simple homogeneous content or stacked internal fracture faces (figure 2). they consist of a core of hydrophobic neutral lipids (triglycerides and sterol esters) surrounded by an envelope of polar lipids (phospholipids, cholesterol, and fatty acids). the lipid droplet envelope is thought to be essentially a membrane monolayer derived from the cytoplasmic leaflet of the er membrane, but hard - and - fast evidence for this origin is sorely lacking. associated with lipid droplets are characteristic proteins including the pat - family proteins (perilipin, adipophilin, and tip 47, and others) and the caveolins, and recent proteomic analyses have revealed a myriad of as - yet - to - be confirmed or identified protein species including histones in lipid droplets. specific lipid droplet proteins regulate various lipid droplet functions, and abnormalilties in the complement of lipid droplet proteins are hence directly reflected in diseases like obesity, diabetes, atherosclerosis, and disorders with lipid storage dysfunction. where are all the lipid droplet proteins then ? it is the expected and accepted location, because lipid droplet proteins and envelope lipids are both amphipathic and can conveniently mingle in the envelope. and after all, we are used to proteins integrated into cell membranes although, as explained above, the droplet envelope is not a membrane at all, only half of one. until recently, pat family proteins and caveolins were considered to be associated exclusively with the droplet envelope, not with the core or other organelles. consequently, regulation of intracellular lipid metabolism was hypothesized to occur at the interface between the lipid droplet and the cytosol. as we will see below, adipophilin is an ubiquitously expressed lipid droplet - associated protein of all mammalian cell types [2022 ]. tip 47 was originally described as a cytosolic protein which binds to the cytoplasmic domains of the cation - dependent and cation - independent mannose 6-phosphate receptor. subsequent studies reported tip 47 at the lipid droplet surface of hela cells and other cell types. our recent studies revealed that tip 47 is not restricted to cytosolic compartments but is also localized in the lipid droplet core and in the plasma membrane of macrophages in a similar manner to that found with adipophilin and perilipin. the function of adipophilin and tip 47 in foam cell formation is only partly understood. a number of other proteins have been reported in association with lipid droplets, in particular caveolin-1, a protein best known as a structural component of caveolae. electron microscopy of immunogold - labeled replicas localize adipophilin (figures 3(a) and (4) and perilipin (figures 3(b) and 3(c))) to the periphery of the lipid droplet. while cross - fractured lipid droplets reveal a peripheral ring of label (figure 3(a)), in concavely fractured droplets en face views of this surface label are seen, allowing its distribution and extent to be appreciated (figures 3(b) and 3(c)). the convexly fractured lipid droplets are devoid of label (figure 3(c)). figure 4 illustrates a survey freeze - fracture view of lipid droplets in a lipid - laden macrophage after immunogold labeling for adipophilin. results such as these, in which the pat family proteins are localized to the outer surface of the lipid droplet, suitably position these proteins to function in the mobilization or accretion of lipids, at the interface between the droplet and the cytoplasm. however, apart from positive labeling in the periphery of lipid droplets prominent label for adipophilin in lipid - laden macrophages is also apparent in er membranes and plasma membranes (figure 5). the labeling in these membranes is confined to the p face ; no label is detected in the e face. adipophilin label is also seen in the p - face of the outer nuclear membrane (figure 6) with which the er membrane p face is continuous. no label is apparent in the e face of the outer nuclear membrane or in either fracture face of the inner nuclear membrane. the membranes of the golgi apparatus, mitochondria, and vesicles were consistently devoid of label (figure 7). apart from disclosing differential labeling in the leaflets of the various membrane systems of the cell, freeze - fracture reveals three - dimensional aspects of intracellular membranes, permitting unique views of the spatial relationship of these membranes. such perspectives enable the nature of the intimate association between lipid droplets and er segments to be appreciated (figure 8). figure 8(a) presents freeze - fracture images of the sites of er - lipid - droplet association. freeze - fracture immunocytochemistry reveals distinctive labeling patterns for adipophilin at the sites of lipid droplet association. abundant labeling for adipophilin is seen in the p face of the er membrane that lies closest to the lipid droplet ; no label is detected in the e face of the partner er membrane distant from the droplet. the mechanism of lipid droplet formation that has gained general acceptance holds that neutral lipids accumulate within the lipid bilayer of the er membrane from where they are budded - off, enclosed by a protein - bearing phospholipid monolayer originating from the cytoplasmic monolayer of the er membrane, to give a cytoplasmic lipid droplet. how the lipids are channeled to budding domains, how budding occurs mechanistically, and how the directionality of the budding is achieved are not understood. indeed, little direct experimental evidence supports the budding model, and alternative models have been proposed [20, 25 ]. an alternative model was recently put forth in which lipid droplets are excised from both leaflets of the er membrane bilayer as a bicelle. droplets are initially formed within small bilayer vesicles, utilizing the machinery of vesicle formation of the secretory pathway. this might occur in a specialized domain of er that is distinct from vesicular transport and dedicated to lipid synthesis. newly formed lipids might fill the bilayer of existing membrane vesicles, effectively filling the vesicles with neutral lipids. in a variation of this model the er bilayer is filled with neutral lipids and the resulting bulge does not detach from the er. freeze - fracture, by permitting unique three - dimensional views of the spatial relationship of membranes and organelles, demonstrates unequivocally that at sites of close association, the lipid droplet is not situated within the er membrane, but adjacent to it. both er membranes clearly lie external to and follow the contour of the lipid droplet, enclosing it in a manner akin to an egg - cup (the er) holding an egg (the lipid droplet) (figure 8(c)). fril further demonstrates that adipophilin is concentrated in prominent clusters in the p half of the er membrane at the site of the closely opposed lipid droplet (figures 8(b) and 8(d)), as well as in the lipid droplet surface opposed to the er. adipophilin is thus strategically placed to play a role in lipid droplet growth by facilitating lipid transfer from the er to the droplet. the evidence from these studies indicates that lipid droplets originate and develop adjacent and external to specialized domains of the er membrane enriched in adipophilin, not within the bilayer of the er as previously supposed. the caveolins, originally described as ubiquitous integral proteins of cell surface pits called caveolae, are of immense physiologic interest, because they are involved in signal transduction and cellular cholesterol homeostasis. just how caveolins assist in these capacities is currently an area of particularly active research. the best studied caveolin, caveolin-1, cycles constitutively between the plasma membrane and several intracellular compartments. cavelion-1 binds directly to cholesterol and is believed to participate facultatively in the bidirectional shuttling of free cholesterol between the cell surface and the various intracellular compartments, including er, golgi, and particularly lipid droplets. at its main location at the cell surface, caveolin-1 is an unusual protein, because both of its hydrophilic ends project into the cytosol, while the intervening region is anchored into the lipid phase of the membrane bilayer. caveolin-1 does not entirely span the lipid bilayer, but is confined essentially to one leaflet of the membrane. after immunolabeling replicas with anticaveolin-1, caveolin-1 label is found in the p face of the plasma membrane of adipocytes (figure 9(a)). deep caveolae are generally strongly marked on their rims, whereas labeling of shallow caveolae and morphologically undifferentiated regions of the plasma membrane is usually weaker. immunolabeling shows that caveolin-1 is also present in intracellular organelles of adipocytes including lipid droplets. the prevailing wisdom holds that caveolin-1 and also the pat family proteins are confined exclusively to the droplet surface [2931 ]. fril, however, demonstrates that these proteins are distributed not only in the surface but also throughout the lipid droplet core. double labeling of perilipin and caveolin-1 reveals extreme heterogeneity with respect to the distribution of these two lipid droplet - associated proteins in lipid droplets of adipocytes. figure 9(b) illustrates an example of one lipid droplet in which the fracture has exposed the outermost monolayer (p face) and another that has been cross - fractured in the same cell. the p face exhibits colocalization of perilipin and caveolin-1, but the core of the other droplet contains almost exclusively caveolin-1. the key finding of these studies is that in mature lipid droplets, lipid droplet - associated proteins clearly gain access to the successive interior lamellae and the amorphous interior of the lipid droplets, as indicated by the presence of specific label in these zones. labeling of the droplet - associated proteins in lipid droplets is far from being confined to the p face. proteins now acknowledged to be inside the lipid droplet core are pat family proteins [32, 33 ], caveolins, flotillins and cyclooxygenase, among others [37, 38 ]. reports of lipid droplet proteins in the lipid droplet core appear to be studiously neglected. why ? because the presence of lipid droplet proteins in the core raises several disconcerting questions. lipid droplet proteins could gain access to the core if both neutral lipids and lipid droplet proteins in the er are transferred across the phospholipid layer of the droplet envelope during lipid droplet biogenesis. indeed, one compelling hypothesis suggests that lipid transfer between the er and the droplet does take place across the droplet envelope and depends on the recruitment of rab 18 to the droplet in response to stimulation of lipolysis by -adrenergic agonists. furthermore, at least one pat protein, adipophilin, which is present in er, seems to be conferred to the nascent droplet from adjacent clusters of adipophilin in the cytoplasmic leaflet of the er membrane. second, how can those amphipathic proteins ever be accommodated among the hydrophobic lipid molecules of the core ? freeze - fracture studies reveal that the lipids of the cores are preferentially arranged in thin layers like the leaves of an onion. therefore, one explanation is that lipid droplet proteins may be forced into place onto the interfaces between the internal lamellar structures of the core during droplet biogenesis, even though they do not normally mix with neutral lipids. taken together, these two explanations would mean that regulation of intracellular lipid metabolism occurs between the lipid droplet and the er rather than between droplet and cytosol as currently preached. how much longer are we going to ignore the presence of lipid droplet proteins in lipid droplet cores ? after all, studying how they get there might help us to appreciate better how lipids and proteins are exchanged across phospholipid barriers, how the er takes part in lipid droplet biogenesis, and how lipid droplet - associated proteins regulate intracellular lipid metabolism in health and disease. we have seen that pat family proteins are not, as previously supposed, specific to lipid droplets but also occur in specialized er membranes, adjacent to the droplet at the proposed sites of biogenesis and growth. through application of fril, however, it is clear that sites similar to those identified in the er are also present in other cellular locations. another area to which fril has shed new light is the finding of pat family proteins as integral components of the plasma membrane of macrophages and adipocytes. under normal culture conditions, these proteins are dispersed in the p half of the plasma membrane (figure 10(a)). stimulation of lipid droplet formation by incubation of the cells with acetylated low - density lipoprotein leads to clustering of the pat family proteins in raised plasma membrane domains (figure 10(b)). fractures penetrating beneath the plasma membrane demonstrate that lipid droplets are closely opposed to these domains (figure 10(c)). the discovery by fril of pat family proteins in the plasma membrane came as a surprise, given that no evidence for such a localization was apparent from immunofluoresence microscopy or subcellular fractionation. however, in normal cells, because fril shows perilipin and adipophilin to be widely dispersed in the plasma membrane, the proteins are unlikely to be above the concentration threshold required for clear detection by immunofluorescence. once clustered, the potential for detectability is increased but as the clusters are extremely closely opposed to lipid droplets, immunofluorescence microscopy has insufficient resolution to determine whether any labeling observed derives from the lipid droplet itself or the immediately adjacent plasma membrane domain. as perilipin and adipophilin are envisaged as characteristic proteins of the lipid droplet, it is understandable that any such signal observed would be assumed to derive from the lipid droplet. similarly, lack of plasma membrane localization of adipophilin and perilipin in subcellular fractionation may arise in part from adherence of the closely opposed plasma membrane domains to lipid droplets during their isolation. the relationship between lipid droplets and pat family protein - enriched plasma membrane domains is strikingly similar to that of the lipid droplets and the er membranes discussed above. at times of extreme lipid loading, the number of sites in the er may be insufficient to cope with demand for lipid droplet synthesis and growth, and thus equivalent machinery in the plasma membrane may be brought into play. it may be speculated that aggregation of the pat family proteins into plasma membrane assemblies may facilitate carrier - mediated lipid or fatty acid influx directly from the extracellular environment into the growing lipid droplet. whatever the functional role of these aggregates is, the findings suggest a common cellular mechanism of intracellular lipid loading in the macrophage as part of the pathogenesis of atherosclerosis and in the adipocyte during development of obesity. by revisiting the lipid droplet with freeze - fracture electron microscopy and the fril technique, new findings have emerged which challenge previous assumptions on where its associated proteins are localized within the cell and how these proteins target to lipid droplets, and on how lipid droplets form and grow. our aim here has been to present an integrated survey of a range of these new findings with a view to stimulating debate on their functional significance. we are, of course, mindful that morphology alone does not explain functional processes. equally, however, a knowledge of structure underpins and provides the framework for understanding function, and without that knowledge, functional assumptions may be led seriously astray. the unique advantages of fril have yielded new information on the structure, composition and protein organization of the lipid droplet. we know now that pat family proteins and caveolins are not confined to the surface of the lipid droplet as previously believed, but pervade the droplet core. there is no irrefutable evidence for the widely held view that the lipid droplet is formed within the er membrane bilayer ; our finding that lipid droplets appear to develop enclosed by but external to specialized sites of the er membrane bilayer that are enriched in adipophilin challenges the long held concept that they are formed within the er membrane bilayer. pat family proteins are not specific to the lipid droplet, but are widely present in the plasma membrane where, under conditions of lipid loading, they adopt a similar configuration to the specialized sites in the er. the examples discussed illustrate the recent impact of the fril technique in advancing our understanding of selected aspects of lipid droplet biology. the information that this approach provides is unique and further exploitation of the fril technique may be expected as its scope and power become more widely appreciated. | lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet - associated proteins. this realization has made the study of proteins, such as pat family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. freeze - fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet - associated proteins. in this paper illustrative examples of how freeze - fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of pat family proteins and caveolin-1 are presented. by revisiting the lipid droplet with freeze - fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies. |
thyroid eye disease (ted) is an autoimmune disease involving the retroocular tissues associated with graves ' disease [1, 2 ]. typical signs and symptoms include proptosis, retroorbital pain, tearing, conjunctival redness and edema, corneal lesions, impaired extraocular motility with or without diplopia, periorbital edema, visual impairment, and, rarely, blindness. treatment options for ted include immunosuppressive agents, radiotherapy, and various surgical procedures such as orbital decompression, squint surgery, and correction of eyelid retraction [35 ]. glucocorticoids are still the most widely used immunosuppressive agents for the treatment of ted and appear to be the most effective for associated soft tissue inflammation, optic neuropathy, and extraocular muscle impairment [6, 7 ]. the main disadvantages of glucocorticoid therapy are the potential recurrence of the disease after discontinuation and the side effects in long - term treatment. several alternative therapies have been proposed to manage resistant ted such as orbital radiation therapy, several other immunosuppressive agents, and biological drugs. however, the effectiveness of these treatments is still widely debated in the literature [815 ]. the aim of this study is to evaluate the efficacy of methotrexate used as single therapy in a group of patients with recurrence of the active ted after withdrawal of corticosteroids due to the onset of side - effects. this is a retrospective comparative case series of 36 patients, from 2001 to 2010, who experienced reactivation of ted after withdrawal of corticosteroid therapy because of side - effects. patients with sight threatening for compressive optic neuropathy were excluded from this study as they needed urgent treatment such as high - dose steroids or surgical decompression to prevent optic atrophy. before methotrexate therapy, all patients received as initial treatment intravenous pulse - methylprednisolone therapy at a dose of 10 mg / kg twice a week for 6 weeks (on tuesdays and thursdays), followed by oral prednisone at a dose of 30 mg / day, tapered to discontinuation over approximately 2 months. patients with recurrence of inflammatory signs (7-point clinical activity score (7-cas) 3 and in patients with asymmetric presentation the eye with the worse score was considered) received as maintenance oral prednisone at a dose of 10 mg / day. from this group, 36 patients who stopped corticosteroids due to the occurrence of side - effects or intolerance to maintenance steroid therapy were commenced on methotrexate therapy. during a time period of 9 years reasons for cessation of steroid therapy included mood swings (25% of patients), hyperglycemia (21% of patients), hypertension (19% of patients), fecal blood (7% of patients), and complaining of puffiness, sleeplessness, facial flushing, slight tremor, and agitation (28% of patients). length of previous corticosteroid therapy ranged from 2 months to 1 year (median 5.12 months, table 1). time between pulse therapy and second - line treatment ranged from 2 months to 1 year (median 8 months, table 1). patients were aged from 35 to 70 years with a mean age of 52 years (table 1). at the onset of ted, hyperthyroidism was treated in 20 patients with thiamazole, 8 with a combination of thiamazole and thyroidectomy, 3 with radioiodine therapy, and 5 with thiamazole and radioiodine (table 1). thyroid function at time of treatment with methotrexate was as follows : 22 patients (61%) were euthyroid (12 still using thiamazole and 10 without treatment), 3 patients (8%) were still hyperthyroid (using thiamazole), and 11 patients (30%) were hypothyroid (using levothyroxine). pretreatment laboratory investigations included complete blood count ; assessment of liver, kidney, and lung functions ; serum immunoglobulin levels ; and hepatitis b and c serology. methotrexate was administered at two different weekly doses depending on the weight of the patient : 7.5 mg for patients weighing less than 60 kg and 10 mg for those weighing more than 60 kg. the weekly dose of 7.5 mg was orally administered and fractionated as follows : two tablets of 2.5 mg on thursday and one on friday, while the dose of 10 mg was administered and fractionated as three tablets of 2.5 mg on thursday and one on friday. to minimize the potential toxicity, a dose of 15 mg of folic acid was prescribed for each patient on sundays. laboratory evaluation including a complete blood count and liver enzyme testing was performed every 2 weeks. the following parameters were measured at baseline (premethotrexate treatment) and at 3, 6, and 12 months after commencement of methotrexate treatment : 7-cas [17, 18 ], ocular motility, exophthalmos, eyelid aperture, and visual acuity (va). ocular motility was measured using the light reflex method, recently validated for ted motility evaluation : a score of 45 was given when the light 's reflex was at the limbus, 30 at half way between the limbus and pupil edge and 15 at the pupil edge. improvement in ocular motility was considered if variation was at least of 15, as previously reported. exophthalmos was measured with hertel exophthalmometry : a decrease of 2 mm was considered significant. where different scores or measures of severity existed for the two separate eyes of one individual, scores of the worse eye were considered for analysis. mean 7-cas, exophthalmometry, and mrd measurements were compared before and after methotrexate treatment using student 's t - test. the chi squared test () was used to compare the results of va and ocular motility data. there was a significant improvement in 7-cas score at 3, 6, and 12 months (p < 0.0001) (table 2). at 3 months, 24 patients (67%) had a reduction of 7-cas in both eyes ; at 6 months, 29 patients (80%) had a reduction of 7-cas in both eyes ; at 12 months, 34 patients (94%) had a reduction of 7-cas in both eyes. before methotrexate therapy, 18 patients (50%) had a va of 20/20, 8 patients (22%) had 20/25, 8 patients (22%) had 20/30, and 2 patients (6%) had 20/50. after 6 months, 2 patients had an improvement of one line on the snellen chart. after 12 months of followup only 1 patient had an improvement of three lines on the snellen chart. 15 patients (42%) had some degree of gaze restriction at baseline. at 3 months, 4 patients improved (27% of patients with ocular motility disturbances), and at 6 and 12 months a total of 10 patients improved (67% of patients with ocular motility disturbances). the reduction of ocular motility restriction after 6 and 12 months of treatment was significant (p < 0.001) (table 3). mean hertel exophthalmometry measurement was 24 mm (range from 17 to 27, sd 3). there was no significant change at 3, 6, and 12 months after treatment (table 3). mean mrd was 6 mm (range from 4 to 7, sd 1.5). there was no significant change at 3, 6, and 12 months after treatment (table 3). the orbital disease usually starts with a dynamic period of active inflammation characterized by pain, redness, and swelling of the eyelids. during the active phase, disease severity increases and then plateaus. after this, disease severity slowly decreases until stable end stage, called inactive phase. although the shape of the curve is representative of disease course for majority of patients, the time axis of rundle 's curve shows high individual variability : it can take from a few months to more than 5 years to reach the static phase. prompt restoration of stable euthyroidism and immunosuppression may decrease the duration of the dynamic phase and reverse the tendency to progression. management of active inflammation in ted in the present study was based on the 7-cas. when the inflammatory grade was 3 or greater, or if there was evidence of progression in the inflammation, immunosuppressive treatment was necessary. systemic steroids are generally considered first - line treatment for ted as they can be given with systemic (oral or intravenous) or local (subconjunctival or retrobulbar) administration. the efficacy of high - dose intravenous corticosteroid pulse therapy has been widely reported. in our study all patients initially received corticosteroid pulse therapy, which is routinely used in our unit for patients with 7-cas higher than 3. for those patients with persistent ted or reactivation after pulse therapy, long - term corticosteroid therapy is commonly used. however long - term use of corticosteroid can lead to complications such as high blood pressure, cushing syndrome, diabetes, osteoporosis, fragility fractures, and in rare cases acute hepatitis. patients included in this study developed such side - effects over the treatment course, and when therapy was withdrawn, they experienced an increase in the inflammation score. many alternative therapies have been reported for such patients, although their efficacy is still under debate [12, 14, 15 ]. the efficacy of methotrexate has been reported for many autoimmune diseases requiring a long - term maintenance therapy such as rheumatoid arthritis, inflammatory bowel disease, and wegener 's granulomatosis. methotrexate is an immunosuppressive drug that inhibits dihydrofolate reductase enzyme, leading to the inhibition of the dna, rna, and protein synthesis [15, 22 ]. cytotoxic and antiproliferative effects can be seen at high doses, while anti - inflammatory and immunomodulatory effects can be observed with chronic low - dose treatment [23, 24 ]. the use of methotrexate is proposed on the basis of the autoimmune nature of ted [11, 15, 25, 26 ]. patients who received methotrexate showed an early significant improvement of 7-cas at 3, 6, and 12 months (p < 0.0001). although none of patients included in this study had an optic neuropathy, va improvement was reported but it was not significant. the improvement in ocular motility restriction was statistically significant after 6 and 12 months of methotrexate treatment (p < 0.001). however, side effects such as gastrointestinal disturbances, increased risk of opportunistic infections, bone marrow depression, interstitial pneumonitis, liver toxicity, asthenia, and fecal occult blood should always be considered since they have been reported in the literature [27, 28 ]. the addition of folate may reduce methotrexate toxicity, without affecting its effectiveness [14, 23 ]. the glucocorticoid - sparing effect of methotrexate is confirmed in our study, but this drug should not be used for more than 2 years in order to avoid the risk of megaloblastic anemia and lymphomas. all the participants into this study had experienced recurrence of inflammatory signs of ted after withdrawal of corticosteroid treatment such as ocular pain and discomfort due to chemosis, hyperemia, and eyelid edema. these patients, as reported in previous studies, seem to have a phase of activity longer than usual. early surgical decompression could be considered for these patients even though its effectiveness needs to be confirmed. in addition, patients with this condition may refuse surgery and methotrexate may be offered to them as a second - line therapy in order to contain the inflammatory process and prevent further complications due to fibrosis process. hence, the indication for treatment with methotrexate should be a recurrent ted with 7-cas 3 after withdrawal of steroids because of onset of steroid side - effects or intolerance. our study suggests that methotrexate therapy is useful for reducing the signs and symptoms of inflammation in ted, shortening the active phase, and reducing eye discomfort since all patients had a statistically significant reduction in inflammatory score. methotrexate appears to be a suitable second - line treatment for patients with recalcitrant ted to control the clinical profile and delay surgery until the disease stability. | background / aim. to evaluate the efficacy of methotrexate for the treatment of thyroid eye disease (ted). methods. 36 consecutive patients with active ted, previously treated with corticosteroids but stopped due to the occurrence of side effects, were commenced on methotrexate therapy. two different weekly doses were administered depending on the weight of the patient (7.5 mg or 10 mg). clinical activity score (7-cas), visual acuity (va), ocular motility, exophthalmos, and eyelid position were retrospectively evaluated at 3, 6, and 12 months and compared with baseline data. results. there was a statistically significant improvement in 7-cas at 3, 6, and 12 months after treatment (p < 0.0001). there was no significant change in visual acuity. ocular motility disturbances improved at 6 and 12 months (p < 0.001). there was no significant change in exophthalmos (mean 24 mm, sd 3 mm) or eyelid position (marginal reflex distance mean 6 mm, sd 1.5 mm) during the follow - up period. no side effects were registered. conclusions. methotrexate therapy is effective in reducing cas and ocular motility disturbances. no significant improvement in proptosis or eyelid retraction should be expected from this treatment. eventually, it might be considered a suitable alternative treatment in ted for patients who can not tolerate steroids. |
genital herpes is most commonly caused by infection with herpes simplex virus type 2 (hsv-2), and is one of the most prevalent sexually transmitted diseases (std) worldwide [13 ]. the incidence of new infections is about 1 million cases per year in the united states. at least 50 million persons in the united states have hsv-2 infection. however, 86% of these individuals are unaware of their diagnosis, either because they do not have symptoms or because they have symptoms that go unrecognized [3, 6 ]. previous studies demonstrated both viral shedding and recurrences are more frequent in the first year after initial hsv-2 infection [711 ]. in one natural history study, women who acquired genital herpes within a year before enrollment had more frequent shedding compared to women who had been infected for one year or more. another study found that individuals diagnosed for 6 months were twice as likely to shed asymptomatically compared to those diagnosed for more than 6 months. collectively, higher viral shedding and recurrence rates in subjects with newly acquired infection may translate to an increased risk of transmitting virus to uninfected sex partners. in studies of immunocompetent adults with a known recurrence pattern, hsv-2 dna as detected by polymerase chain reaction (pcr) testing was present on approximately 10% of days [12, 13 ]. several investigations have supported the effect of suppressive antiviral therapy on the reduction of viral shedding in patients with an established recurrence pattern [9, 11, 1417 ]. more recent studies have shown that suppressive antiviral therapy is as effective at controlling symptomatic disease in recently infected persons as it is in those with prevalent infection [18, 19 ]. because clinical and subclinical reactivation of hsv may result in the sexual transmission of hsv to a partner, suppressing viral shedding is important at both the individual and population level. the current study was conducted to provide additional natural history data in individuals who have been given a new diagnosis of genital herpes, and to provide the first evaluation of the efficacy of valacyclovir 1 g once daily in reducing hsv-2 shedding in this population. this was a double - blind, randomized, multicenter, two - way crossover study conducted at 14 centers in the united states (us) between february and november 2006. subjects were randomized to receive valacyclovir 1 g or matching placebo once daily for 60 days in each of two treatment periods in a two - way crossover design. in our study, the term val - pbo is used to describe the treatment order for subjects randomized to receive valacyclovir in the first treatment period followed by placebo in the second treatment period ; the term pbo - val is used to describe the treatment order for subjects randomized to receive placebo in the first treatment period followed by valacyclovir in the second treatment period. the 1 g dose of valacyclovir was selected to optimize treatment in this population, 20% of whom would be expected to have 10 or more outbreaks per year based on previous studies. the primary endpoint was the percent of all days with any hsv-2 viral shedding over 60 days defined, for each subject as the percent of all days with pcr data for which hsv-2 shedding was detected by a positive pcr result. secondary endpoints included percent of all days with subclinical shedding (without a genital lesion), percent of all days with clinical shedding (with a genital lesion), proportion of subjects with no shedding, proportion of subjects with at least one symptomatic recurrence and time to first symptomatic herpes recurrence. eligible subjects were diagnosed with a first recognized episode of herpes at the screening visit or within 4 months of randomization. subjects were required to have documented signs and symptoms of genital hsv-2 infection, as well as laboratory confirmation (positive culture, pcr, or type - specific serology) of hsv-2 infection to be eligible for the study. subjects that were enrolled on the basis of a positive culture / pcr, but in the absence of hsv-2 antibodies, represented true incident hsv-2 infection. it was accepted that a subject 's first recognized episode of genital herpes might not represent new infection as prior episodes may have escaped detection. all subjects signed an informed consent document approved by an appropriate review committee prior to any study procedures. a subject 's participation in the study included a screening evaluation, a randomization visit, and eight study visits (four visits per treatment period, with a 15-day interval between each visit). hsv-1 and once randomized, subjects attended the clinic for additional visits if they suspected they had a genital herpes recurrence. a genital examination was conducted at the screening visit for subjects who presented with genital herpes signs or symptoms. genital examinations also were conducted at the randomization and genital herpes recurrence visits for all subjects. during each 60-day treatment period and during washout, subjects collected a single daily swab from their genital / anal - rectal area for hsv-2 detection by pcr. on day 1 of a genital herpes recurrence, swabs of the lesion also were collected in the clinic for hsv-2 detection by culture and pcr. during a documented recurrence, a subject temporarily discontinued blinded study medication and received open - label episodic treatment with valacyclovir 500 mg twice daily for 3 days, after which double - blind therapy resumed. subjects used a telephonic, interactive voice response system daily to capture information on genital lesions, genital symptoms, and oral outbreaks. adverse events, concomitant medications, and treatment compliance information were collected at each clinic visit, based on recall and discussion with the subject as well as pill counts. blood and urine samples were collected for hematology, clinical chemistry, and urinalysis at the screening visit and at the end of each treatment period. the herpeselect elisa glycoprotein g - based assay (focus diagnostics, cypress, calif, usa) was used to detect the presence of hsv-1 and hsv-2 antibodies at screen (quest diagnostics, van nuys, calif, usa). dna was assessed by qualitative florescent - based real time pcr assay at the university of washington virology research laboratory, seattle, wash, usa. in this study, it was estimated that 47 completed subjects provided at least 90% power to detect a reduction in the mean percent days of hsv-2 shedding from 9.6% in the placebo treatment phase to 2.8% in the valacyclovir treatment phase, assuming standard deviations of 9.8% and 5.6%, respectively, and a correlation of 0.4. forty - seven completed subjects provided 90% power to detect a reduction in the mean percent days of subclinical hsv-2 shedding from 7.0% in the placebo treatment phase to 2.8% in the valacyclovir treatment phase, assuming standard deviations of 9.3% and 5.4%, respectively, and a correlation of 0.4. to allow for dropouts the populations considered in the overall analysis included the intent - to - treat (itt) exposed, consisting of all subjects who received at least one dose of investigational product. also considered was the intent - to - treat crossover (ittc) population consisting of all itt subjects who had at least one pcr result in each treatment period. the first period efficacy population consisted of all patients with efficacy data in the itt - exposed, with the exception of 2 subjects whose data was compromised (see section 3). the treatment comparison for continuous endpoints, including the primary endpoint, was tested using nonparametric crossover analysis methods in the ittc population. the test of treatment difference was preceded by a test of carryover effect using a wilcoxon rank - sum test at the 5% level of significance. if this test was significant, then the treatment comparison would be tested using the first period efficacy population only. this analysis was carried out as a supporting analysis in any case because the test of carryover may have relatively low power. time to first genital recurrence was evaluated with kaplan - meier estimates and log - rank test using the first period efficacy population and data in treatment period 1. seventy subjects from 14 centers in the united states were randomized into the study, 35 subjects to the val - pbo treatment sequence, and 35 subjects to the pbo - val treatment sequence. during the course of routine monitoring, errors in sample identification the error was felt to compromise the integrity of the data ; therefore, swab samples from two subjects in the val - pbo sequence group were excluded from the efficacy analysis. an additional three subjects were lost to follow - up early in treatment period 1. these three subjects had some swabbing samples but all pcr assay results were missing, leaving 65 subjects in the first period efficacy population. twenty of the 70 (29%) subjects in the itt population prematurely withdrew from the study (11 while receiving valacyclovir, 9 while receiving placebo). the primary reason for withdrawal was lost to followup (n = 8, 11%), followed by other reason (n = 4, 6%), and adverse event (n = 2, 3%). at least one major protocol deviation was recorded for 35 (50%) subjects randomized. deviations included failure to take at least 80% of double - blind study medication (n = 13, 37%) and genital / anal - rectal swab pcr results on fewer than 85% of study days (n = 20, 57%). the study population was predominantly female (70%) ; 51% were white (table 1). of the 70 subjects randomized, 9 (13%) were hsv-2 seronegative at entry indicating newly acquired hsv-2 infection. note that one of these 9 subjects did not have efficacy data in treatment period 1. of the 8 in the first period efficacy population, 6 were also hsv-1 negative, indicating primary infection. all nine subjects presented with laboratory confirmation of hsv-2 infection by culture or pcr at screen or upon initial diagnosis made within 4 months of randomization. these subjects represent a subset of the population who had a clearly documented recent hsv-2 infection. the primary efficacy endpoint was the difference in mean percent of all days with hsv-2 shedding as determined by a type - specific hsv pcr assay. for the ittc population, shedding was reported on a mean 13.5% of all days in subjects while receiving placebo compared to a mean 2.9% of days in subjects while receiving valacyclovir (p 6 months prior to study entry), participants received oral valacyclovir 500 mg bid, acyclovir 400 mg bid, and placebo in random order for 7 weeks each, in a three - period crossover design. there was a one - week washout between treatment periods. however, 27 subjects (39%) had a diagnosis of a first episode of genital herpes 6 months before randomization. overall, for the 69 subjects randomized, hsv dna was detected by pcr from at least one anatomical site on 40.2% of the days while receiving placebo, compared to 8.0% of days while receiving acyclovir (80% reduction) and 7.2% of days while receiving valacyclovir (82% reduction). in the current study, the mean percent of days with total hsv-2 shedding was 2.9% while receiving valacyclovir, compared to 13.5% while receiving placebo. these results are similar to those of the first two studies [12, 13 ] noted above except that the proportion of days with shedding while on placebo was slightly higher in the current study. it is not clear if that difference is meaningful, but it could be because our subjects were recently infected and these persons shed virus more frequently than those with established disease [8, 9 ]. the study by gupta. reported rates of shedding in both the placebo and treatment phases that were higher than those of the current study. however, the proportional reduction of shedding in subjects on suppression in our study was similar to that reported by gupta. the reason for the discrepancy between the results reported by gupta. and other studies, including the one reported here, is uncertain, but could be related to the multiple swab technique used in the earlier study or in the selection of study subjects. of the 70 subjects in this study, 9 (13%) these subjects clearly had new hsv-2 infections while the remaining study subjects had been infected long enough to mount an antibody response by the time they enrolled in the study. this provided us with the opportunity to explore the differences in viral shedding between those with and without antibodies to hsv-2 in the group randomized to placebo treatment in the first study period. as was previously reported in a study that evaluated viral shedding by culture in initially hsv-2 seronegative subjects, our post - hoc analysis showed shedding rates 4-to5-fold higher among those initially hsv-2 seronegative subjects compared to the broader group of subjects who were newly diagnosed but already hsv-2 seropositive. given the limited number of patients in the hsv-2 seronegative subset, the viral shedding results in this cohort must be interpreted with caution, yet the observation suggests the potential importance of early treatment in persons with new hsv-2 infection. the cdc advocates the use of suppressive therapy for reducing genital herpes recurrences in patients with frequent recurrences (6 recurrences / year), and states treatment also is effective in patients with less frequent recurrences (5). in addition, sexual transmission of hsv can occur during asymptomatic periods, and asymptomatic viral shedding is most frequent during the first 12 months after acquiring hsv-2 (5). two recent studies have shown that suppressive therapy with valacyclovir in subjects with newly diagnosed hsv-2 genital herpes has clinical efficacy in reducing outbreaks, similar to that seen in persons with established recurrent disease [18, 19 ]. the current study extends those findings by showing that the impact of suppressive valacyclovir on viral shedding is also similar to that found in persons with established recurrent genital herpes. taken together, these studies suggest that there is no reason to wait until a person with newly diagnosed genital herpes establishes a recurrence pattern to consider suppressive therapy. in fact, because of the high rate of viral shedding and high probability of clinical recurrences, persons newly diagnosed with hsv-2 infection may be among the best candidates for suppressive therapy. | objective. genital herpes (gh) recurrences and viral shedding are more frequent in the first year after initial hsv-2 infection. the objective of this study was to provide the first evaluation of valacyclovir 1 g once daily compared to placebo in reducing viral shedding in subjects newly diagnosed with gh. methods. 70 subjects were randomized to receive valacyclovir 1 g daily or placebo in a crossover design for 60 days with a 7-day washout period. a daily swab of the genital / anal - rectal area was self - collected for hsv-2 detection by pcr. subjects attended the clinic for routine study visits and gh recurrence visits. treatment differences were assessed using a nonparametric crossover analysis. results. 52 subjects had at least one pcr measurement in both treatment periods and comprised the primary efficacy population. valacyclovir significantly reduced hsv-2 shedding during all days compared to placebo (mean 2.9% versus 13.5% of all days (p <.01), a 78% reduction). valacyclovir significantly reduced subclinical hsv-2 shedding during all days compared to placebo (mean 2.4% versus 11.0% of all days (p <.01), a 78% reduction). however, 79% of subjects had no gh recurrences while receiving valacyclovir compared to 52% of subjects receiving placebo (p <.01). conclusion. in this study, the frequency of total and subclinical hsv-2 shedding was greater than reported in earlier studies involving subjects with a history of symptomatic genital recurrences. our study is the first to demonstrate a significant reduction in viral shedding with valacyclovir 1 g daily compared to placebo in a population of subjects newly diagnosed with hsv-2 infection. |
dengue is an arboviral disease endemic in most of the tropical countries with recurrent epidemics. although dengue is known to cause hepatic involvement commonly, it only occasionally results in fulminant liver failure (flf), which has a high mortality. n - acetyl cysteine (nac), a precursor of glutathione, is an effective antioxidant scavenging for oh radicals have shown to be effective mainly in acetaminophen and even in non - acetaminophen - induced flf. activated recombinant factor vii (rfviia) was initially developed to overcome the limitations of existing treatments for patients with congenital hemophilia and inhibitors. clinical success in this arena led to experimental use in other coagulopathies as well. to the best of our knowledge, this is the first reported case of dengue hemorrhagic fever complicated by fulminant liver failure and massive hemorrhage successfully managed with nac and rfviia. a 37-year - old previously healthy man who was a teetotaler admitted with fever, repeated vomiting, body aches, and fatigue for four days. examination revealed a temperature of 100f, tachycardia (106/min), normotension, and tachypnea (28/minute). investigations showed leukocyte count ; 5.1 10/l (neutrophils 53%, lymphocytes 41%), and platelets ; 60 10/l, hemoglobin ; 14.1 g / dl with hematocrit of 43.4%, aspartate aminotransferase (ast) ; 220 u / l, alanine aminotransferase (alt) ; 157 u / l on admission. presumptive diagnosis of dhf was made considering the clinical picture and prevailing outbreak in the country. close monitoring with replacement of maintenance volume of fluids was started in both oral and intravenous means. during next 36 hours, he had clinical and hematological evidence of ongoing plasma leakage but remained hemodynamically stable. next day, he was shifted to intensive care unit due to agitation and drowsiness. platelet count dropped to less than 10 10/l during day 5 and 6 of the illness and subsequently started rising gradually. u / l) in seventh day of illness with prolongation of prothrombin time of 19.8s (inr ; 1.7) activated partial thromboplastin time (89s), and elevated serum bilirubin. chest x - ray and ultrasonography of abdomen showed bilateral pleural effusions and moderate ascites. liver failure treatments started with intravenous vitamin k, thiamine, lactulose, and oral metronidazole. in the same day, worsening agitation and sensorium (grade iii hepatic encephalopathy) necessitated elective intubation and mechanical ventilation. blood sugar was monitored, and his serum sodium levels maintained at 145 - 150 mmol / l range. an internal jugular central venous access was secured following transfusion of platelets and fresh frozen plasma (ffp). twenty percent manitol infusion was administered 12 hourly for 48 hours, and nac intravenous infusion was continued for 72 hours. bleeding was difficult to control with transfusion of platelets, fresh frozen plasma, and pack red cells alone. this prompted us to administer him with 3 doses of rfviia following transfusion of cryoprecipitate to replenish fibrinogen levels in serum. patient 's central venous pressure started rising (24 - 29 cmh2o) by ninth day with of illness necessitating a considerable dose of frusemide to achieve a safe level of cvp (8 - 12 cmh2o). dengue igm and igg (elisa) were positive, while malarial smear, hepatitis panel, leptospira serology, and hiv elisa became negative. however, the course of his illness was complicated initially by ventilator - associated pneumonia with acenetobacter and later by pseudomonas, necessitating antibiotic cover with netilmycine and ceftazidime. he was extubated following 7 days of ventilatory support and managed to be discharged after 18 days of hospital stay. dengue hemorrhagic fever is defined as the presence of fever, thrombocytopenia, and evidence of capillary leak with serological evidence of acute infection by dengue virus. current management of dhf is mainly focused on meticulous fluid management during leakage phase of the illness. in dhf, an antibody - dependent immune mechanism is in play, by which non - neutralizing antibodies of previous dengue infection complexes with the new infecting serotype enhancing its uptake. liver damage is due to viral replication in hepatocytes, cytokine - related damage, and partly due to ischemic hepatitis. fulminant liver failure is defined as rapid onset of acute encephalopathy and coagulopathy (inr 1.5) in the setting of liver failure less than 6 weeks duration. since the liver is capable of regenerating to a large extent as underlying cause of hepatocyte injury is controlled with supportive medical therapy, fhf in principle sustains potential for a complete recovery. fulminant liver failure due to dengue infection with subsequent complete recovery has been reported rarely. flf may be associated with changes in systemic hemodynamic, i.e., tissue hypoxia, which contributes to multiple - organ failure. recent studies have shown that nac administered to patients with flf (non - paracetamol - induced) increases oxygen delivery and improves survival. morbidity and mortality in dhf is mainly associated with either prolonged shock or fluid overload. activated recombinant factor vii is a coagulation protein that induces hemostasis by directly activating factor x. there were some evidences on safety and efficacy of rfviia for controlling hemorrhage in patients without hemophilia. since inadvertent administration of blood products would worsen the fluid overload in dhf, the place for rfviia seems to be invaluable to arrest life - threatening bleeding as in our patient. so far, there was no clear information about the prophylactic or therapeutic use of rfviia in dhf. the use of nac and rfviia despite limited evidence presumably had a greater impact on recovery of our patient. therefore, this case highlights the potential use of rfviia and nac along with aggressive supportive measures in cases of near - fatal dhf for a successful outcome. prevention of ischemic injury of liver by meticulous fluid resuscitation during the leakage phase of dengue fever is of paramount importance. dengue may occasionally lead to flf, has to be treated promptly with aggressive supportive measures. indication of rfviia to arrest uncontrolled internal bleeding and the place of nac in non - acetaminophen - induced flf in complicated dhf is a platform for discussion. | consensus on management of complicated cases of dengue infection is evolving. dengue hemorrhagic fever (dhf) occasionally progress to fulminant liver failure with high fatality rate. inadvertent use of blood products to control massive bleeding in dengue shock syndrome may worsen fluid overload and subsequently the multi - organ dysfunction. we report a case of 37-years - old sri lankan man who developed fulminant liver failure and massive bleeding associated with dhf, subsequently recovered completely with supportive measures including administration of n - acetyl cysteine and activated recombinant factor vii. in conclusion, prevention of ischemic injury to liver and adoption of early aggressive supportive measures in complicated cases of dengue hemorrhagic fever is crucial for a favorable outcome. indications for rfviia to arrest uncontrolled internal bleeding and use of nac in non - acetaminophen - induced acute liver failure in complicated dhf are a platform for discussion. |
the choroid, a well - vascularized connective tissue, resides between the retina pigment epithelium and the sclera and extends from the ora serrata anteriorly to the optic nerve posteriorly. the choroid receives most of the ocular blood flow (~85%) and has one of the highest metabolic activities in the body. the physiological functions of the choroid include providing vascular supply to the anterior optic nerve head and retina, emmetropization, thermoregulation, and waste - product removal.1,2 thus, a structurally and functionally healthy choroid plays an essential role in the function of the macula and peripapillary area. the abnormal choroid has been associated with many macular diseases, such as age - related macular degeneration, central serous chorioretinopathy, diabetic retinopathy, behet s disease, and retinitis pigmentosa.37 some previous studies have also reported that abnormal peripapillary choroidal blood supply may have a role in the development of glaucomatous optic neuropathy.810 in this respect, determination of peripapillary choroidal thickness (ct) in a healthy population may be helpful for displaying the pathological alterations in peripapillary areas, such as glaucoma, and might be a useful tool in clinical practice. until recently, the choroid could only be evaluated by indocyanine green angiography and ultrasonography. the development of spectral - domain optical coherence tomography has recently presented enhanced depth imaging optical coherence tomography (edi - oct), which allows in vivo examination and quantification of the choroid.11 the aim of the study reported here was to determine the normal peripapillary ct, measured by edi - oct, in healthy turkish volunteers. participants were recruited from the gaziantep university school of medicine department of ophthalmology between january and july 2014. the ethics committee of gaziantep university school of medicine approval and informed consent from all patients in accordance with the declaration of helsinki were obtained. each participant was subjected to comprehensive ophthalmic assessment, including visual acuity, refraction (using a kr 8900 refractometer [topcon corporation, tokyo, japan ]), axial length (al ; with a echoscan us-500 [nidek co, ltd, aichi, japan ]), slit - lamp biomicroscopy, intraocular pressure (with a goldmann applanation tonometer), and dilated fundus examination. exclusion criteria were diabetes mellitus ; hypertension ; and/or history of intraocular surgery, photodynamic therapy, transpupillary thermotherapy, focal laser treatment, ionizing radiation, uveitis, retinal disorders, glaucoma, amblyopia, high myopia, and/or poor image quality due to severe cataract. the same clinician (bo) performed edi - oct imaging of all subjects in the morning (between 9 am and 12 pm) with heidelberg spectralis equipment (heidelberg engineering inc, heidelberg, germany) as described previously : images, each comprising 100 scans, using the automatic averaging and eye tracking features, were obtained.11 a 3.4 mm diameter peripapillary circle scan centered on the optic disc was used for retinal nerve - fiber layer measurement. ct was assessed manually from the retinal pigment epithelium to the inner face of the sclera, in the inferior, superior, nasal, and temporal quadrants. two clinicians (bo and sk), who were masked to clinical patient data, measured the ct in all subjects and the mean value of the measurements was calculated. the eyes having more than 15% difference in measurement between the two clinicians were excluded from the study. statistical analysis was performed using spss software (v 22.0 ; ibm corporation, armonk, ny, usa). participants were recruited from the gaziantep university school of medicine department of ophthalmology between january and july 2014. the ethics committee of gaziantep university school of medicine approval and informed consent from all patients in accordance with the declaration of helsinki were obtained. each participant was subjected to comprehensive ophthalmic assessment, including visual acuity, refraction (using a kr 8900 refractometer [topcon corporation, tokyo, japan ]), axial length (al ; with a echoscan us-500 [nidek co, ltd, aichi, japan ]), slit - lamp biomicroscopy, intraocular pressure (with a goldmann applanation tonometer), and dilated fundus examination. exclusion criteria were diabetes mellitus ; hypertension ; and/or history of intraocular surgery, photodynamic therapy, transpupillary thermotherapy, focal laser treatment, ionizing radiation, uveitis, retinal disorders, glaucoma, amblyopia, high myopia, and/or poor image quality due to severe cataract. the same clinician (bo) performed edi - oct imaging of all subjects in the morning (between 9 am and 12 pm) with heidelberg spectralis equipment (heidelberg engineering inc, heidelberg, germany) as described previously : images, each comprising 100 scans, using the automatic averaging and eye tracking features, were obtained.11 a 3.4 mm diameter peripapillary circle scan centered on the optic disc was used for retinal nerve - fiber layer measurement. ct was assessed manually from the retinal pigment epithelium to the inner face of the sclera, in the inferior, superior, nasal, and temporal quadrants. two clinicians (bo and sk), who were masked to clinical patient data, measured the ct in all subjects and the mean value of the measurements was calculated. the eyes having more than 15% difference in measurement between the two clinicians were excluded from the study. statistical analysis was performed using spss software (v 22.0 ; ibm corporation, armonk, ny, usa). a total of 57 eyes of 25 female and 32 male subjects were included in the study. the mean (sd) age of the subjects was 30.910.6 years (range : 1856) (figure 1). the mean spherical equivalent refraction error was 0.421.06 (range : 3.75 to 3.00). the mean peripapillary ct in the superior, inferior, nasal, and temporal quadrants was 22557, 18347, 22057, and 23359 m, respectively (figure 2). the inferior peripapillary ct value was significantly lower than superior, nasal, and temporal peripapillary ct values (p0.05). the correlation analyses between the superior, inferior, nasal, and temporal peripapillary cts are shown in table 2. all peripapillary ct values were significantly correlated with each other. there were no significant correlations between age, al, or spherical equivalent, and each peripapillary ct measurement (table 3). when the data from the male and female groups were assessed as two different groups, the mean age was 3312 years in female subjects and 298 years in male subjects (p=0.198). the mean al was 23.40.8 mm in male subjects and 23.20.7 mm in female subjects (p=0.534). although each peripapillary ct value was thicker in males than in females, only the nasal peripapillary ct reached statistical significance (p=0.016). the blood supply of the prelaminar area of the optic nerve head originates from the branches within the peripapillary choroid. the peripapillary choroid may have a role in several ocular pathologies such as pathological myopia and glaucoma.1214 evaluation of peripapillary ct can be used in elucidating physiopathological changes and monitoring of disease progression. currently, edi - oct technology allows sensitive assessments of peripapillary and subfoveal cts. to the best of our knowledge, we have been the first to report comprehensive data about the peripapillary ct of a healthy turkish population by edi - oct. in our study, the peripapillary ct was thickest at the temporal quadrant, followed by the superior, nasal, and inferior regions. our results are very similar to those in a study by huang who evaluated the peripapillary ct in a healthy chinese population.9 they determined same order for peripapillary ct thickness distributions. tanabe found that the peripapillary ct was the thickest at the superior quadrant, followed by the temporal, nasal, and inferior regions in healthy subjects.15 moreover, we found that only the inferior peripapillary ct showed statistically significant differences when compared with the temporal, superior, and nasal areas. similarly, huang and tanabe indicated that the inferior peripapillary choroid was significantly thinner than all other sectors.9,15 the most prominent finding of our and previous studies is the thinner ct in the inferior peripapillary area. although the reason for this finding is not exactly known, embryological processes may have a role in inferior choroidal thinning. it has been found that the optic fissure, which is finally closed during ocular development, is placed in the inferior side of the optic cup.16 future studies are needed to determine why the inferior peripapillary ct is significantly lower than the ct in other quadrants. due to the prelaminar portion of the optic disk head being nourished by peripapillary choroidal circulation and the choroid being thinner in the inferior sector, the inferior optic disk may be more vulnerable to changes in the choroidal circulation flow. this claim is supported by previous studies that have shown that glaucoma influences the superior hemifield of the optic disc more frequently and more severely than the inferior hemifield of optic disc.8,15 age and al are well - known factors associated with ct in various studies.11,17,18 studies have demonstrated that a decrease of nearly 1113 m per decade occurs in peripapillary ct.9,19 on the other hand, ho reported that the age of their subjects did not correlate with the peripapillary ct.20 huang found no correlation between the peripapillary ct and al.9 in this study, we did not find a correlation between the peripapillary ct and al or age. this may be because of the small sample size used in this study. also, when considering the sex factor, each of the peripapillary ct values was thicker in males than in females, but only the difference in nasal peripapillary ct between the two sexes was statistically significant. moreover, the heidelberg spectralis optical coherence tomography equipment does not provide automatic segmentation of the choroid ; hence, all identifications of bruch s membrane and the inner scleral border were conducted manually. the peripapillary ct in the turkish population has some similarity to that in populations in previous studies. the findings of the study show that turkish people have significantly lower peripapillary ct values in the inferior quadrant than in the superior, nasal, and temporal quadrants. further studies are needed to elucidate the reason for this thinning in the inferior area. | aimthe objective of the study reported here was to investigate the normal peripapillary choroidal thickness (ct), measured by enhanced depth imaging optical coherence tomography (edi - oct), in healthy turkish volunteers.materials and methodsin this prospective cross - sectional study, 57 eyes of 57 healthy turkish subjects were enrolled. each participant underwent a comprehensive ophthalmic examination and peripapillary ct measurement using edi-oct.resultsthe mean age of the 25 female and 32 male patients in the study was 30.910.6 years (range, 1856 years). the mean peripapillary ct at the superior, inferior, nasal, and temporal sites was 22557, 18347, 22057, and 23359 m, respectively. the inferior peripapillary ct value was significantly lower than the peripapillary ct values (p<0.001 for all), whereas no significant differences were found between the superior, nasal, and temporal peripapillary ct values.conclusionthe findings of the study revealed that turkish people had significantly lower peripapillary ct values in the inferior quadrant than in the superior, nasal, and temporal quadrants. |
applying optimum amount of force during orthodontic treatment is a challenging task for the orthodontist. amount of force to be applied during orthodontic treatment is one of the problems encountered in orthodontics.1 the quantification of stress in the periodontal ligament (pdl) is an important concept, as stress in this tissue is transmitted to alveolus with subsequent bone remodeling and tooth movement. infinite element method (fem) studies, the linear models are widely used. as compared to the linear static analysis, non - linear analysis gives more realistic results as they closely reflect the dynamic nature of the oral emvironment.2 for the present study maxillary central incisor was modeled into a three - dimensional (3d) fem model because it undergoes the most detailed tooth movement and is at the second - highest for root resorption after the maxillary lateral incisor.3 therefore, the purpose of this study is to analyze the stresses in pdl by applying intrusive and torque movements by a 3d fem and to compare these stresses with linear and non - linear analyses in a maxillary central incisor model. to evaluate the stress distribution in pdl on application of orthodontic load (vertical intrusive force and lingual root torque) on maxillary central incisors with a 3d fem analysis using linear properties and then application of equivalent force magnitude using non - linear properties.to compare the differences of stress distribution between linear and non - linear properties. to evaluate the stress distribution in pdl on application of orthodontic load (vertical intrusive force and lingual root torque) on maxillary central incisors with a 3d fem analysis using linear properties and then application of equivalent force magnitude using non - linear properties. to compare the differences of stress distribution between linear and non - linear properties to evaluate the stress distribution in pdl on application of orthodontic load (vertical intrusive force and lingual root torque) on maxillary central incisors with a 3d fem analysis using linear properties and then application of equivalent force magnitude using non - linear properties.to compare the differences of stress distribution between linear and non - linear properties. to evaluate the stress distribution in pdl on application of orthodontic load (vertical intrusive force and lingual root torque) on maxillary central incisors with a 3d fem analysis using linear properties and then application of equivalent force magnitude using non - linear properties. to compare the differences of stress distribution between linear and non - linear properties in this study, the 3d fem model of maxillary central incisor used in the previous study was used to calculate the stress in the pdl by application of intrusive and torque movements and compare these stresses in linear and non - linear analyses. construction of a geometric modelconversion of the geometric model to a finite element modelmaterial property data representationdefining the boundary conditionloading configurationsolving the system of non - linear algebraic equationinterpretation of the results. construction of a geometric model conversion of the geometric model to a finite element model material property data representation defining the boundary condition loading configuration solving the system of non - linear algebraic equation interpretation of the results. the same geometric model which was used in part i of the study was used, meshing was done using hypermesh and then a fem model was created.4 each structure was then assigned a specific material property. the different structures in the finite element model are tooth, pdl, cortical bone and cancellous bone. the material properties used in this study have been taken from the finite element studies previously conducted.5,2 these material properties were linear and isotropic for linear analysis and non - linear mechanical properties were assigned for non - linear analysis (tables 1 and 2).5,6 linear material properties used in fem. piecewise linear mechanical property values describing non - linear elastic stress - strain behavior of pdl. the boundary condition in the finite element model was defined at all the peripheral nodes of the bone with three degree of movement in all directions. application of the forces (intrusion and lingual root torque) was same as the previous study. construction of a geometric modelconversion of the geometric model to a finite element modelmaterial property data representationdefining the boundary conditionloading configurationsolving the system of non - linear algebraic equationinterpretation of the results. construction of a geometric model conversion of the geometric model to a finite element model material property data representation defining the boundary condition loading configuration solving the system of non - linear algebraic equation interpretation of the results. the same geometric model which was used in part i of the study was used, meshing was done using hypermesh and then a fem model was created.4 the different structures in the finite element model are tooth, pdl, cortical bone and cancellous bone. the material properties used in this study have been taken from the finite element studies previously conducted.5,2 these material properties were linear and isotropic for linear analysis and non - linear mechanical properties were assigned for non - linear analysis (tables 1 and 2).5,6 linear material properties used in fem. piecewise linear mechanical property values describing non - linear elastic stress - strain behavior of pdl. the boundary condition in the finite element model was defined at all the peripheral nodes of the bone with three degree of movement in all directions. application of the forces (intrusion and lingual root torque) was same as the previous study. the forces were applied to the maxillary central incisor for intrusive and lingual root torque movements and the equations were solved with linear and non - linear properties by ansys workbench software and sequentially the stress patterns produced in pdl were analyzed. the results obtained were divided into the following : application of intrusive force of 0.30 n using linear properties.application of intrusive force starting with 0.30 n and doing iterations using non - linear properties to get the equivalent stress as obtained using linear properties.application of lingual root torque of 1.0 n using linear properties.application of lingual root torque of 1.0 n and doing iterations using non - linear properties to get the equivalent stress as obtained using linear properties. application of intrusive force starting with 0.30 n and doing iterations using non - linear properties to get the equivalent stress as obtained using linear properties. application of lingual root torque of 1.0 n and doing iterations using non - linear properties to get the equivalent stress as obtained using linear properties. when 0.30 n of intrusive forces were applied along the long axis of the tooth it produced maximum compressive stress (denoted as minus) of 0.02006 n / mm at the apex of the pdl and the maximum tensile stress was 0.001528 n / mm at the cervical margin. when 0.30 n of intrusive forces were applied parallel to the long axis of the tooth using non - linear properties it produced minimum compressive stresses of 0.0293 n / mm at the apex of the pdl and maximum tensile stresses was 0.00264 n / mm at the cervical margin (figure 1). force levels were iterated till 0.2 n, where an equivalent stress similar to the analysis done using linear properties was obtained (figure 2). distribution of principle stress in periodontal ligament on the application of the intrusive force of 0.30 n using non - linear properties. distribution of principle stress in periodontal ligament on the application of the intrusive force of 0.20 n using non - linear properties. when 1.0 n with a 15 n / mm moment load was applied using linear properties, it produced a minimum compressive stress of 0.01646 n / mm and maximum tensile stress of 0.01832 n / mm. when 1.0 n with a 15 n / mm of moment load was applied using non - linear properties, the minimum compressive stresses was 0.0260n / mm and maximum tensile stresses was 0.0297 n / mm (figure 3). the moment value was iterated till 0.8 n, where an equivalent stress similar to the linear properties was obtained (figure 4). distribution of principle stress in periodontal ligament on the application of lingual root torque of 1.00 n / mm using non - linear properties. distribution of principle stress in periodontal ligament on the application of lingual root torque of 0.80 n / mm using non - linear properties. when 0.30 n of intrusive forces were applied along the long axis of the tooth it produced maximum compressive stress (denoted as minus) of 0.02006 n / mm at the apex of the pdl and the maximum tensile stress was 0.001528 n / mm at the cervical margin. when 0.30 n of intrusive forces were applied parallel to the long axis of the tooth using non - linear properties it produced minimum compressive stresses of 0.0293 n / mm at the apex of the pdl and maximum tensile stresses was 0.00264 n / mm at the cervical margin (figure 1). force levels were iterated till 0.2 n, where an equivalent stress similar to the analysis done using linear properties was obtained (figure 2). distribution of principle stress in periodontal ligament on the application of the intrusive force of 0.30 n using non - linear properties. distribution of principle stress in periodontal ligament on the application of the intrusive force of 0.20 n using non - linear properties. when 1.0 n with a 15 n / mm moment load was applied using linear properties, it produced a minimum compressive stress of 0.01646 n / mm and maximum tensile stress of 0.01832 n / mm. when 1.0 n with a 15 n / mm of moment load was applied using non - linear properties, the minimum compressive stresses was 0.0260n / mm and maximum tensile stresses was 0.0297 n / mm (figure 3). the moment value was iterated till 0.8 n, where an equivalent stress similar to the linear properties was obtained (figure 4). distribution of principle stress in periodontal ligament on the application of lingual root torque of 1.00 n / mm using non - linear properties. distribution of principle stress in periodontal ligament on the application of lingual root torque of 0.80 n / mm using non - linear properties. for the non - linear analysis of pdl, elastic property in the form of a stress the load was subdivided into a series of multiple increments and then applied to the constructed geometric model. in each step, the program performed a linear solution and was checked for convergence. if the convergence criteria were not satisfied, the load vector was re - evaluated, and a new solution was obtained. this iterative procedure continued until the problem converged.7 this study shows that intrusive forces and lingual root torque produces stress at the root apex. similar results were found in previous investigations for vertical tooth movement that used fem.8 - 10 the results obtained in the part i of this study using linear properties were taken into consideration. the current study offers unique information about the non - linear elasticity of the pdl in the range of orthodontic forces for intrusion. as compared to the linear analysis the non - linear analysis is known to provide more accurate and reliable results. the use of non - linear mechanical properties for the pdl resulted in a dramatic departure from the stresses predicted by the linear models which is in agreement with the study by toms and eberhardt.11 on application of intrusive force of 0.30 n with non - linear properties, the compressive stress was mainly concentrated at the apex whereas tensile stress was distributed throughout the pdl which is in agreement with rudolph and wilson.8,10 once the stresses were obtained with linear properties, the same force load was applied using non - linear properties. the minimum compressive stress was 0.0293 n / mm and the maximum tensile stress was 0.00264 n / mm. the compressive and the tensile stress levels were increased with the same amount of load in non - linear analysis. therefore to get the equivalent stress as linear properties, iterations were done with non - linear properties and the force level was reduced to 0.2 n. this shows that less force is required in non - linear analysis compared to the linear analysis. the studies of toms, eberhardt and durkee found the similar results.11,12 toms and eberhardt found out that stresses at the apex with non - linear analysis was 2.4 times more than that of linear analyses for extrusion.11 durkee observed that non - linear nature of the pdl results in higher compressive stresses being generated, for both lateral and axial loading, than a comparable linear model.13 when 1.0 n / mm moment load was applied for lingual root torque maximum compressive stress was concentrated at the apex and near the buccal part of alveolar crest whereas the tensile stress was distributed at the mid region of pdl which is in agreement with dorrow and sander ; hohmann.13,14 when 1.0 n / mm of moment load was applied using non - linear properties, a minimum compressive stresses of 0.0260n / mm and maximum tensile stresses of 0.0297 n / mm was obtained. therefore to get the same stress as linear, iterations were done with non - linear properties. these results were in agreement with the study by toms, eberhardt, and durkee which tells that the stresses produced in the pdl are more with non - linear analysis compared to the linear analysis with application of same amount of load.11,12 so, this shows that less force is required in non - linear analysis compared to the linear analysis (graph 1). stresses in the pdl were evaluated with intrusive and lingual root torque movements by a 3d fem and these stresses were compared in linear and non - linear analyses in a maxillary central incisor model. 0.30 n was the load applied for intrusion and 1.0 n / mm was the moment load for torque. for intrusive and lingual root torque movement distribution of stress over the pdl was within the range of optimal stress value as proposed by lee, for orthodontic tooth movement with linear properties.15 when same force load was applied in non - linear analysis, stresses were more compared to linear analysis for both intrusive and lingual root torque. to get the same stress as linear, iterations were done using non - linear properties and the force level was reduced to 0.2 n and 0.8 n for intrusion and torque respectively, which is within the range of optimal orthodontic force as suggested by proffit. this shows that the force level required for non - linear analysis is lesser than that of linear analysis. therefore in case of non - linear analysis, 1.5 times less force was applied for intrusion and 1.25 times less force was applied for torque as compared to the linear analysis. the mobility of tooth and its recovery increases in a quasi - logarithmic manner which is suggestive of the viscoelastic nature of pdl. this property of pdl needs to be addressed in the future studies. in fem analysis, the simulation of materials with complex geometry and dimensions using requires the determination of elastic, plastic and viscoelastic material properties. the exact idea of stress and strain in the pdl for different types of tooth movements can be determined with the non - linear simulation. however, the non - linear fem analysis has only been recently used in the field of orthodontics, its reliability and credibility in studying biologic structures has yet to be fully established. | background : simulation of periodontal ligament (pdl) using non - linear finite element method (fem) analysis gives better insight into understanding of the biology of tooth movement. the stresses in the pdl were evaluated for intrusion and lingual root torque using non - linear properties.materials and methods : a three - dimensional (3d) fem model of the maxillary incisors was generated using solidworks modeling software. stresses in the pdl were evaluated for intrusive and lingual root torque movements by 3d fem using ansys software. these stresses were compared with linear and non - linear analyses.results:for intrusive and lingual root torque movements, distribution of stress over the pdl was within the range of optimal stress value as proposed by lee, but was exceeding the force system given by proffit as optimum forces for orthodontic tooth movement with linear properties. when same force load was applied in non - linear analysis, stresses were more compared to linear analysis and were beyond the optimal stress range as proposed by lee for both intrusive and lingual root torque. to get the same stress as linear analysis, iterations were done using non - linear properties and the force level was reduced.conclusion:this shows that the force level required for non - linear analysis is lesser than that of linear analysis. |
cryptophane - a was the first cryptophane synthesized by collet. in 1981. it is composed of two equivalent cyclotribenzylene (ctb) caps bound together by three ethylene - dioxy linkers. on each phenyl ring a has been the subject of many interesting studies in the field of host it has a hydrophobic three - dimensional cavity capable of binding small organic molecules such as chloromethanes as well as xenon atoms. the previously investigated chloromethane complexes of cryptophane - c revealed a surprisingly high affinity constant for dichloromethane and relatively low affinity constant for chloroform. this has brought up the question of what structural properties have the largest effect on the complexation. cryptophane - a and -c are both anti - isomers, but the latter has nonequivalent caps with only one of the caps carrying the methoxy substituents on the phenyl groups (see molecules 1 and 2 in scheme 1). apart from this, the two molecules 1 and 2 are identical. they also have the same cavity volumes. as will be discussed in the present work, cryptophane - a in solution is in exchange between its conformers similarly to cryptophane - c. the encapsulation of a chloromethane guest changes the probability distribution of the conformers as it has been seen before. nuclear magnetic resonance (nmr) is a perfect tool for characterizing the encapsulation process but also for following the guest - induced changes in the host. these chemical events can be explained, for instance, by means of nmr spin echo experiments. in this work, translational diffusion was investigated in order to reveal the eventual presence of another guest, water, inside the host cavity. meiboom gill (cpmg) relaxation dispersion methodology was dedicated to shed light on the host conformational exchange. obviously, upon encapsulation, both h and c spectra displayed changes of the peak positions. it is not only the peak position that is very informative but also the intensity and broadening of the guest h signal in the free and bound position, in particular if the host and the guest are present in close to the 1:1 molar ratio. this concentration ratio also allows a better estimation of the affinity constant from the h spectrum. the comparison of the data to cryptophane - c is not sufficient to draw the final conclusion concerning the relation between the structure of the caps and the affinity. to complete the analysis, another cryptophane - a derivative was chosen in which the methoxy groups attached to the ctb rings were replaced by butoxy groups, resulting in a third cryptophane with the same cavity volume (see molecule 3 in scheme 1). the trans - conformers of cryptophane - a are shown in figure 1, and its analogue with butoxy groups replacing the methoxy substituents, denoted cryptophane - but, is shown in figure 2. (a) the t1t1t1 conformer of cryptophane - a with the atom numbering. cryptophane - a and cryptophane - but were synthesized by brotin.c labeled chloroform and the deuterated solvent, 1,1,2,2-tetrachloroethane - d2, were obtained from cambridge isotope laboratory. the raw solid cryptophanes contained chcl3, ch2cl2, and ethanol coming from the recrystallization. a 30 mm solution of cryptophane - a was prepared without any purification (sample 1). another solution, sample 2, with 10 mm cryptophane - a containing 60 mm added nonlabeled chloroform was prepared with no purification. for quantitative analysis, the material was dipped into nonlabeled chloroform or dichloromethane and the solvent was then let to evaporate. the following solutions in 1,1,2,2-tetrachloroethane - d2 were prepared:(3)12 mm cryptophane - a and 13 mm ch2cl2(4)11 mm cryptophane - but and 13 mm ch2cl2(5)10 mm cryptophane - a and 11 mm chcl3(6)11 mm cryptophane - but and 12 mm chcl3(7)20 mm cryptophane - a and 15 mm ch2cl2(8)10 mm cryptophane - a and 62 mm chcl3(9)10 mm cryptophane - but and 90 mm chcl3(10)10 mm cryptophane - but and 25 mm chcl3(11)10 mm cryptophane - but, 9 mm chcl3, and 6.5 mm ch2cl2(12)30 mm cryptophane - a and 150 mm chcl3 12 mm cryptophane - a and 13 mm ch2cl2 11 mm cryptophane - but and 13 mm ch2cl2 10 mm cryptophane - a and 11 mm chcl3 11 mm cryptophane - but and 12 mm chcl3 20 mm cryptophane - a and 15 mm ch2cl2 10 mm cryptophane - a and 62 mm chcl3 10 mm cryptophane - but and 90 mm chcl3 10 mm cryptophane - but and 25 mm chcl3 10 mm cryptophane - but, 9 mm chcl3, and 6.5 mm ch2cl2 30 mm cryptophane - a and 150 mm chcl3 the solubility of cryptophane - a in tetrachloroethane is much higher than that of cryptophane - c and cryptophane - but. h and c experiments were performed with bruker avance spectrometers operating at 9.4, 14.1, and 16.5 t using 5 mm (bbi and bbo at 9.4 t, txi and bbo at 14.1 t, and cryo - txi at 16.5 t) probe heads. at 9.4 t, the temperature calibration was done using a standard methanol calibration sample, while a resistance detector made of copper wire dipped into silicon oil contained in a 5 mm nmr tube was used at the two higher fields. the accuracy of the temperature determination is estimated at 1 k. all the experiments measuring build - up or decay were repeated at least twice. the peak assignment was based on dqf - cosy, (2d) noesy, (2d) roesy, as well as h c edited hsqc experiments. the exsy measurements were performed at 250 k and 16.5 t using the implementation as the dpfgsenoe sequence with two selectively refocusing shaped pulses and one hard pulse in the middle of the mixing time interval. the semi - selective inversion pulses were implemented as gaussian g3 cascades with a duration of 1820 ms. experiments were performed with 64 accumulated signal transients, using a relaxation delay of 3540 s. only the doublets of the c - labeled chloroform were evaluated. the purpose of the label is to enhance the proton spin lattice relaxation. the evaluation of the exchange rate of the forward (complexation) reaction was based on the approach proposed originally by macura. and described by hu and krishnamurthy. the exchange rate of the backward (decomplexation) reaction was based on the principle of detailed balance, in order to avoid the error coming from small intensities at very short mixing times in the initial rate regime. c spectra were recorded with waltz16 proton decoupling at 9.4 t, while waltz65 was used at 14.1 and 16.5 t. the decoupling power corresponded, on average, to the nutation frequency of 2.8 khz. lattice relaxation times of the guest (chcl3) were measured by the inversion recovery method using 1619 recovery delays ranging from 0.5 ms to 30 s with a relaxation delay of 35 s. the heteronuclear overhauser enhancement was measured with the dynamic noe sequence. the noe build - up period was set to 5t1 and the relaxation delay to 10t1. in the spectrum with no noe enhancement, the 2d noesy and roesy at 9.4 and 14.1 t were recorded at 235, 240, 255, and 258 k. the detection method used was states -tppi. the mixing times were 0.020.24 s for the noesy and 0.040.2 s for the roesy. the spin lock power was set to 3 khz. in the direct dimension, 8 scans were used with 16 dummy scans, with a relaxation delay of 57 s. the size of the direct fid was 4096 data points, giving an approximately 0.6 s acquisition time in the t2 domain. 512 and 768 data points were used in the indirect (t1) dimension, which were zero filed to 4096 points. the noesy pulse sequence contained a hard -pulse in the middle of the mixing time and two z gradients with a power of 40% and 40% of the maximum value. the h diffusion experiments were performed at 258 and 268 k using a pulse - gradient stimulated echo (pgste) sequence with a longitudinal eddy - current delay (led) and bipolar gradient pulses. the calibration of the gradients was accomplished by measuring the diffusion coefficient of a well - known sample at 298 k. in our case, the bruker standard sample which is composed of 1% h2o in d2o with 0.1 g / l gdcl3 was used. during the diffusion experiments, the gradient strength was linearly incremented in 32 steps from 2 to 95% of its maximum value ; the diffusion time and the gradient pulse duration were kept constant. the c cpmg measurements were performed at two static magnetic fields (b0 = 14.1 and 16.5 t) and two temperatures (t = 298 and 310 k). the carbons 1 and 2 corresponding to the cryptophane - a linkers and the aromatic carbon 6 (see figure 1) were considered. gill (cpmg) sequence with proton decoupling during acquisition and a proton 180 pulse at every second echo in the c cpmg pulse train to avoid the csa - dd cross - correlation effects. for each r2 measurement, the delay between the 180 pulses in the cpmg pulse train, cpmg, was kept constant while the number of 180 pulses was increased. every experiment was recorded with 1024 scans and 16 dummy scans with a relaxation delay of 2 s. the c cpmg relaxation dispersions were established for cpmg (= 1/(2cpmg)) values ranging from 166.7 to 2500 hz. all calculations were performed in a similar way as reported for cryptophane d and c. the gaussian 09 package was used. geometries were optimized at the level of the dft - b3lyp functional with the basis set 6 - 31g(d). each optimized structure was then taken and a single point energy calculation was done, together with the calculation of the c chemical shift using the giao method, with the larger basis set 6 - 311+g(2d, p). the calculated chemical shifts reported are referenced to the calculated chemical shift of tms at the same level of theory. all the calculations were performed using the conductor polarizable continuum model (cpcm) with the parameters appropriate for dichloroethane solvent. two sets of calculations were made : one for the empty host and one with a chloroform molecule inside the cavity. the van der waals interaction was taken into consideration in all structures optimized with the b3lyp functional by adding, after the geometry optimization, the empirical term to the dft energies. following our earlier work, the complex formation in the host guest (h g) systems is described in terms of the reaction1 nmr methods allow investigation of the reaction kinetics at equilibrium, when the concentrations of various species are constant in time. the forward and backward processes are individually described by the rate expressions:23 the (pseudo) first - order rate constants, kfb (free - to - bound) and kbf (bound - to - free), are defined according to4 the equilibrium constant for the complex formation (the affinity constant) is given by5 equation 5 can be reformulated in terms of the principle of detailed balance:6 equations 16 form the basis of the discussion of the kinetics and thermodynamics of our systems. the solutions with approximately 1:1 host : guest ratios (samples 36) are very informative compared to the solutions with high guest excess (samples 810 and 12). in the case of samples 36, the intensity and the integral of the bound guest peak are higher than those for the free guest. this implies a relatively high affinity to the host. in order to extract more information on kinetics, namely, the exchange rates, it is possible to perform line - shape fitting on the spectra in which the exchange caused line - broadening is higher than 1/t2, i.e., the combined effect of the natural line width in the absence of exchange and magnetic field inhomogeneity. this is not a problem in the case of the dichloromethane guest, since, even at the lowest temperature (235 k), the exchange - caused line - broadening is large (see figure 3). the proton spectrum of ch2cl2@cryptophane - a in 1,1,2,2-c2d2cl4 (sample 3) at 14.1 t and 235 k. the exchange rates in the case of chcl3 at 235, 245, and 255 k (bold font) were determined by exsy experiments, in other cases by line - shape fitting (samples 3 and 5). the equilibrium constant (k) was calculated from the concentrations of the species. in the case of chcl3@cryptophane - a at low temperatures, the exchange of the guest is not fast enough for line - shape fitting. the rate constants kfb reported in table 1 at 235, 245, and 255 k were obtained from the exsy experiments, while kbf were calculated from the principle of detailed balance. the rates and populations for chcl3@cryptophane - a at higher temperatures are based on the line - shape fitting. this can be explained by the size difference between the two guests (indeed, the van der waals volumes of dichloromethane and chloroform are, respectively, equal to 55 and 72). for both guests, the only difference between the two hosts is that cryptophane - c has nonequivalent caps, since the methoxy groups are missing from one of them. a hypothesis formulated in our recent cryptophane - c study is that the methoxy groups block the entrance and exit of the cavity. in the case of cryptophane - a, the methoxy groups from the caps (see figure 1) present an even more severe hindrance for the guest. thus, the corresponding exchange processes are expected to be slower for cryptophane - a, in agreement with the present observations. in the case of both guests, this means that the affinity of cryptophane - a is very high both to chloroform and to dichloromethane. knowing the total concentrations of the compounds, one can calculate the concentration of the species ([h ], [g ], [hg ], see the supporting information) and calculate the equilibrium constant at every temperature. from table 1, the affinity constant is higher for chloroform than for dichloromethane at 235 k. this is interesting, since chloroform is the bigger guest, which might be expected to fit worse inside the host cavity. it is also noteworthy that the equilibrium constant determined here for the dichloromethane guest is much higher than it was reported in earlier work from our laboratories, where a large excess of dichloromethane compared to the cryptophanes led to a large uncertainty in the free host concentration. the data analysis of the solutions containing cryptophane - but and dichloromethane is difficult because the exchange at low temperatures is rather slow and only the integrals can be used for estimating the approximate concentration of the species present. an example of the h spectrum of sample 4 at 245 k is shown in figure 4. the proton spectrum of ch2cl2@cryptophane - but in 1,1,2,2-c2d2cl4 (sample 4) at 14.1 t and 245 k. nevertheless, it is possible to calculate the populations of the free site pf and of the bound site pb (where pb = 1 pf) at this temperature range (see table 2). at higher temperatures, overlap occurs between the bound guest and the signals from the butoxy groups. for chcl3@cryptophane - but, it was possible to measure signal integrals at the low temperatures (235 and 245 k) and to perform the line shape analysis between 255 and 305 k. comparing the values of the bound site population pb in tables 1 and 2, one can notice that the bound site population is the highest out of the four complexes in the case of ch2cl2@cryptophane - but. the equilibrium constant values for both complexes are very high, falling actually in the range where nmr is not the most suitable technique. the values reported here for the dichloromethane complex carry a big error, since the concentration of the host is very low (see the supporting information) and the baseline and integration error sum up. nevertheless, it is still possible to draw the conclusion that the equilibrium constant is very high. surprisingly, the exchange is faster in the case of chcl3@cryptophane - but than chcl3@cryptophane - a, while the affinity constants are similar. a possible explanation of this observation (as suggested by one of the reviewers) is that the bulky butoxy groups can not organize in the closed conformation (see below). summarizing the exchange rates between free and complexed sites, one can state that the chcl3 guest is typically in slow exchange, while ch2cl2 exchanges faster on the chemical shift time scale. using the arrhenius and vant hoff equations, it is possible to estimate the activation energy and the reaction enthalpy of the complexation reactions (see the supporting information). the activation energy obtained for the ch2cl2@cryptophane - a system agrees with previous studies. the activation energies for the chcl3@cryptophane - a are the highest, which agrees well with structure and size considerations such as the previously mentioned blocking effect of the methoxy groups connected to the ctb rings. the lower activation energy of chcl3@cryptophane - but indicates also that the butoxy groups play a role in the complexation dynamics, but it is not as obvious as in the case of cryptophane - a. looking at these results, it is worthwhile to not forget to take account of experimental errors coming from the sample preparation, the integration, and the line - shape fitting. the estimated error of total concentrations is 20% (based on repeated sample preparations) and is obviously propagated to the association constant k and the exchange rate constant k1 (the other parameters, determined by integration or by line - shape fitting, carry a lower error which can be estimated around 5%). the samples containing close to 1:1 molar ratio of the host and the guest also suffer from the low intensity of the peak of the free site. this is, on the other hand, the consequence of the high affinity constant which was hidden in earlier work by the high excess of the guest. in order to probe the mobility of the guest within the cryptophane cavity, relaxation of chloroform, both in the free and the bound state, the longitudinal relaxation rate r1 (inverse of the longitudinal relaxation time t1) and the heteronuclear noe were measured for the c of the guest, in analogy with earlier work from our laboratories. one can see that, when chloroform is free in the solution, extreme narrowing conditions are achieved ; indeed, the c spin - relaxation rate r1 is independent of the magnetic field and a full noe is retrieved. concerning the bound chloroform, the reported values show a field dependence of the longitudinal relaxation rate r1 as well as a noe enhancement less than full. in order to explain these results, the motion of chcl3 encapsulated in the cryptophane - for the global correlation time, the value of 2.7 ns, estimated from the plot of r versus the temperature established earlier, was used. the resulting square of the generalized order parameter, s, is also shown in the table. it can be seen that the lipari szabo order parameter for the guest within the cryptophane cavities agrees quite well with the corresponding quantities from the solid state. the s value found is high, indicating that the motion of the chcl3 molecule is restricted inside the host cavity. as there is no doubt concerning the encapsulation of dichloromethane or chloroform within cryptophane, one can wonder if other species present in our solutions can enter the host cavity. the question can especially arise for water (all the samples contain a small amount of water coming from the walls of the glass materials), as its molecular size is smaller than the one of dichloromethane or chloroform. to bring out the possible encapsulation of water, h diffusion experiments were performed on sample 7 and were compared to the results obtained on a sample containing dichloromethane in the same solvent but without cryptophane. self - diffusion coefficients for cryptophane - a, dichloromethane, and water are given in table 5 for the two samples investigated and for the two temperatures considered (258 and 268 k). first, one can observe that the diffusion coefficient of each species varies as expected with the temperature. the self - diffusion coefficient of ch2cl2 in the sample containing the host (sample 7) is much lower than the one in the sample without cryptophane - a (roughly 4 times smaller at 258 k and 3 times smaller at 268 k), indicating changes in the translational motions of ch2cl2. moreover, its diffusion coefficient at 258 k is close to the value found for cryptophane, which definitely proves that dichloromethane and cryptophane diffuse as one entity at this temperature. at 268 k, d is roughly 2 times larger than d. this larger diffusion coefficient can be explained by the fact that d can be seen as an apparent diffusion coefficient to which the diffusion of both bound and free molecules contributes. indeed, as the exchange is getting faster and the association constant decreases with the temperature, the contribution coming from the free dichloromethane is more important, leading d to increase. looking now at the self - diffusion coefficients of water, d, in the samples with and without cryptophane, it is interesting to notice that they do not evolve in the same way as for dichloromethane. even if they slightly decrease, they are still 10 times larger than the diffusion coefficients of cryptophane. consequently, water may be interacting with the cryptophane but does not enter the cavity in solution, thus proving the hydrophobicity of the host cavity. this observation differs from the conclusions obtained in the solid state by taratula., which were however obtained in the absence of other suitable guests. in our earlier studies, it was shown that the complexation of chloromethane changes the probability distribution of the host conformers. this has an effect on the chemical shifts in both the h and c spectra because they are the weighted averages of the chemical shifts of all the conformers in fast exchange in solution. the exchange between the host conformers is always fast in the investigated temperature range, as opposed to the guest exchange (discussed above) which can be both fast and slow in this range. in the case of cryptophane - a and cryptophane - but, the equilibrium constant for both guests, chloroform and dichloromethane, is higher than in the case of previously investigated compounds. this means that, at low temperatures, when the equilibrium is shifted toward complex formation, even at low concentration of the guest (1:1 host : guest ratio) the complexed forms dominate the spectra. in order to investigate the influence of complexation on the conformational distribution, it is an advantage to look at spectra with low guest concentrations, at least lower than the total concentration of the host. one way to create these conditions is to work on samples containing more than one guest. for cryptophane - a, the h and c spectra of sample 1 (cryptophane - a containing solvents of purification) at 240 k are shown in figures 5 and 6. the h spectrum of the solution containing 30 mm cryptophane - a, 8 mm chcl3, 7 mm ch2cl2, and 10 mm ch3ch2oh in 1,1,2,2-c2d2cl4 (sample 1). the spectrum was recorded at 14.1 t and 240 k. the capital letter a denotes the chcl3@cryptophane - a, b the ch2cl2 complex, and c the ch3ch2oh complex. the c spectrum of the solution containing 30 mm cryptophane - a, 8 mm chcl3, 7 mm ch2cl2, and 10 mm ch3ch2oh in 1,1,2,2-c2d2cl4 (sample 1). the spectrum was recorded at 14.1 t and 240 k. the capital letter a denotes the chcl3@cryptophane - a, b the ch2cl2 complex, and c the ch3ch2oh complex. nevertheless, one can see three sets of signals for the three complexed forms of the host. the chloroform complex is labeled with the capital latter a, the dichloromethane complex with b, and ethanol with c. both spectra are very informative. at this temperature, the free peaks of the guest are too small to allow reliable integration or not even observable. this is not a surprise for chloromethanes, knowing the high affinity of the host to these guests.. it can be further seen in figure 5 that the chloroform complex has only one peak for the linker protons (figure 5, peak (a)1,2), in contrast to the other guests which have two peaks for the linker protons (figure 5, peaks (b, c)1,2). the two proton peaks in the case of other guests are dipolarly coupled to each other (cross - relaxed by each other), according to noesy and roesy spectra (see below). it can be seen that the difference between protons 3 and 6 is the biggest in the case of the chloroform complex and the smallest in the case of the ethanol complex. in figure 6, the c spectrum of the same sample is shown. it is also possible to see here three separate sets of peaks for the three complexes (figure 6, peak sets a, b, c). the spectrum shown here is a proton - decoupled c spectrum which is easy to interpret ; every peak represents a chemically and magnetically nonequivalent site in the molecule in question. the peak of the methoxy group is shifted upfield going from ethanol to chloroform guest (figure 6, peaks (a)m, (b)m, and (c)m). the opposite behavior is true for the linker carbons. the signals belonging to the linkers are shifted downfield, and the chloroform complex has the highest chemical shift value. as in the case of the proton spectrum, the difference between the signals of the aromatic carbons is the highest in the case of the chloroform complex and the lowest in the case of the ethanol complex. if a high excess of chloroform (host : chloroform = 1:6, sample 2) is added to the same solution, it will push the other guests out of the cavity and the free guest peaks are observable again (see the supporting information). figures 7 and 8 show the h and c spectrum of solution number 11 for the cryptophane - but complexes. the h spectrum of the solution containing 10 mm cryptophane - a, 9 mm chcl3, and 6.5 mm ch2cl2 in 1,1,2,2-c2d2cl4 (sample 11). the spectrum was recorded at 16.5 t and 250 k. the capital letter a denotes the chcl3 and b the ch2cl2 complex. the c spectrum of the solution containing 10 mm cryptophane - a, 9 mm chcl3, and 6.5 mm ch2cl2 in 1,1,2,2-c2d2cl4 (sample 11). the spectrum was recorded at 16.5 t and 250 k. the capital letter a denotes the chcl3 and b the ch2cl2 complex. in this case, the sample does not contain any ethanol but only dichloromethane and chloroform in close to equimolar amount. both spectra show exactly the same behavior concerning the chemical shift changes as in the previous case. by calculating the chemical shift differences of the aromatic carbon signals 3 and 6 (= 3 6), together with the full width at half - height (fwhh) of carbons 1, 3, and 6 for the two hosts (see the supporting information), it can be seen that the signals at low temperatures are broadened by both conformational exchange of the hosts and the exchange of the guest. by increasing the temperature this is due to the increasing amount of noncomplexed host, characterized by smaller -values. at temperatures higher than 275 k, the peaks start to broaden again and, in the case of chloroform complexes, the peaks broaden into the baseline and further analysis is very difficult. the spectra displayed in figures 7 and 8 (sample 11) were recorded at 250 k. in the h spectrum (figure 7), it can be seen that the position of the bound ch2cl2 peak is very close to signal number 4 and other impurities, making, unfortunately, the integration difficult. nevertheless, one can try to fit a lorentzian to the peaks and extract information on the concentration ratio between bound and free ch2cl2, because in this case not only the quantitative information is useful. the quantitative information can be used to get a better picture of the affinity of the host. one can see that the ratio [hg]/[g ] = kfb / kbf is much lower than that in table 2. this is the effect of having two guests present at the same time. the data in table 6 indicate that the affinities of cryptophane - but to both chloromethanes are rather similar. the experimental information provided by the simple spectra is not sufficient to draw a definite conclusion on the type of conformers preferred by the different guests. in order to give a more detailed answer, one needs to look at the structure and symmetry properties of the conformers. in the case of cryptophane - a, the quantum chemical optimization was done previously by brotin. the present study complements that work by reporting also the chemical shifts of the c sites in the conformers. moreover, an attempt to correct the calculated energies with the van der waals contribution is performed here. as it was described in detail earlier the all - trans conformers for cryptophane - a and cryptophane - but are shown in figures 1 and 2, respectively. there are two types of trans conformers (both characterized by the -o-1-2-o- dihedral angle close to 180) which differ in the relative orientation of the bond connecting carbons 1 and 2 with respect to the methoxy or butoxy groups on the ctb rings. this means that there is no chemical shift difference between carbon number 1 and 2these nuclei become symmetry - equivalent. a complication occurs when chcl3 enters the cavity : since it belongs to the c3v symmetry group, it reduces the symmetry of the complex to c3. there are two basic types of gauche conformers, denoted g+ and g, with the only difference in the sign of the -o-1-2-o- dihedral angle (+ 60, 60). this means that the equivalence of the linker carbons no longer holds : they have different chemical shifts. additionally, there are also two types of g+ and g conformers, which are denoted g+,g+b, g, and g b. the difference between the conformers with and without subscript b is the orientation of the linker 12 bond with respect to the methoxy groups and, more importantly, to the other linkers. the three linkers can take different conformations independently of each other, which results in a large number of possible species. g conformer, and the green framework depicts the g bg g. the g g conformer is the molecule with black colored carbons, while the green molecule is the g bg g. because of the equivalence of the caps, if one exchanges all the linkers from g,+ to g,+b, one gets back the original conformer ; this means that the all - gauche conformers are pairwise equivalent (e.g., g bg bg b = g g g). the gauche conformers have one very important and useful property in common : the linker carbons are nonequivalent and their chemical shifts are different. b denotes a conformer optimized with a methoxy dihedral of around 110. the calculated shifts for the individual carbons differ by a few tenths of a ppm. here, the average values are given. as it can be seen in the table, when the gauche conformer changes to gauche b, the chemical shifts of the corresponding carbons 1 and 2 exchange places. this would not cause any complication, if the exchange of the linkers was synchronized, meaning that they only change simultaneously. this is not the case and the dynamics of the linkers can average the chemical shift to only one single peak, even if the symmetry of the conformers is only c3. the chemical shifts and homonuclear j - couplings between the linker protons (there are four of them in each linker, with in principle different shifts and coupling, but they show up as two multiplets ; see, e.g., figure 5) are not averaged out for the dichloromethane (and ethanol) complex. the observed chemical shifts for the proton - carrying aromatic carbons also display conformation dependences. the situation of the chemical shifts of carbons 3 and 6 is more complicated, because of the close vicinity of the methoxy groups to number 6. the one with lower energy corresponds to the methoxy groups in the plane of the aromatic rings, with the dihedral angle (6 - 1-o - m) around zero. when the same dihedral is around 110, the methoxy group points out of the plane of the aromatic ring. the energy difference between these two methoxy group conformers is only about 6 kj / mol, more or less independent of the linker conformation. the dynamic behavior of the methoxy groups plays an important role both in the kinetics of the complexation (as discussed in the case of cryptophane - c) and in the chemical shift of the carbon number 6. the calculated chemical shifts and the difference between them are shown in table 7. there are two important conclusions that can be drawn looking at table 7. the first is that the chemical shift difference between carbons 3 and 6 decreases going from the all - trans conformers to the all - gauche conformers. the second conclusion is that the orientation of the methoxy groups affects this difference dramatically. namely, in the case of trans and gauche conformers, the order of the peaks (the sign of the -value) is reversed. in the case of gauche+,, it is known that the order of the peaks 3 and 6 in the carbon spectrum is never exchanged. the explanation of this observation is that it almost never happens that all six of the methoxy groups are oriented with a dihedral angle of 110. this, in turn, has an effect on the kinetics of the complex formation, since the reaction can only occur when two methoxy groups on the opposite sides of a cavity window point out of the aromatic plane. this is shown in figure 10, where the conformer g g g is displayed in the van der waals representation when two methoxy groups on the opposite sides have a dihedral angle of zero (figure 10a) and 110 (figure 10b). (a) the molecule with the 6 - 1-o - m dihedral angle close to zero, blocking the exit. (b) the molecule with the 6 - 1-o - m dihedral angle close to 110, out of the way. it can be seen that it is not possible for chcl3 to exit when the methoxy groups block the way. as anticipated above, this increased steric hindrance can be seen as the origin of the slower dynamics of both guests@cryptophane - a compared to cryptophane - c. cryptophane - but is a larger molecule, and the dft calculations are time - consuming. due to this, the chemical shifts of only three noncomplexed (g+g+g+, t1t1t1, t2t2t2) and two chloroform - complexed (g+g+g+, t1t1t1) conformers were calculated (see the supporting information). the shift difference between the proton - carrying aromatic carbons is highest in the case of the trans conformer and lowest in the case of the gauche conformer. the chemical shifts of the linker carbons are moved downfield (to higher shift values), if the trans character of the complex increases, and the shift difference,, between the aromatic carbons also increases. however, with this information at hand, it is still not possible to decide which trans conformer is preferred by the complex. the only conclusion possible to draw is that the trans character increases in the case of binding of both chloromethane guests. one possible help to decide which trans conformer is preferred by the complex can be provided by the dft - calculated energies of the different complexes. the problem with the energies is that they are basis - set - dependent and that the contribution coming from the dispersion correction is an empirical estimate. first, there are two energy values coming from the small and large basis set and containing only the zero point correction and the thermal contribution ; in addition, there are the energies containing the dispersion contribution. from the relative energies of the noncomplexed cryptophane - a conformers with respect to the lowest values within the respective approximation level (see the supporting information), it can be seen that there are big differences between different calculations. in the case of no dispersion correction, the minimum occurs at the t1t1t1 conformation with both basis sets and the dispersion - corrected energies have their minimum at the g, one can calculate the probability distribution of the conformers and weight the calculated chemical shifts with it. the calculated weighted average -values for the small and large basis set are 11.6 and 10.6 ppm, respectively, which does not match with the measured values. calculating the weighted chemical shifts in the same way, but using the dispersion - corrected energy data, one obtains 6.7 and 6.5 ppm. one has to keep in mind that in reality there was never any noncomplexed cryptophane - a in the solutions studied. the situation is more complicated in the case of chcl3@cryptophane - a and chcl3@cryptophane - but complexes. first, as it was mentioned before, all the gauche conformers have at most c3 symmetry. this also means that there is a difference in energy, dependent on the orientation of the chloroform molecule within the cavity. the two possible orientations of chcl3 inside the cavity of the g g g conformer of cryptophane - a. it is also possible to change the linkers to gauche+b conformation and let the orientation of chloroform to remain unchanged. this complication arises only because the chloroform molecules can not turn around in the cavity (see relaxation data above). from the energies of the chloroform - complexed cryptophane - a (see the supporting information), it can be seen that, without the dispersion correction, a minimum occurs at the t2t2t2 with the small basis set and at the g bt1t1 conformer with the large basis set. for the dispersion - corrected data, looking at the probability - weighted sum of the chemical shift differences between the proton - carrying aromatic carbons 3 and 6, one obtains similar values as before : 11.3 and 10.5 ppm without the dispersion correction and 10.5 and 8.6 ppm with the dispersion correction. it is thus confirmed again that the complexation of chloroform will increase this difference, but still it is not possible to select the preferred conformers. so far, the only conclusion that can be drawn is that some guests prefer different conformations of the host. thus, guests can either fit into major or minor states of the host. the main concern is that, due to a fast conformational exchange of the host (see above), minor states are not observable on the recorded spectra. only a set of peaks corresponding to an average of these major and minor states is detectable. in order to get more information on this conformational or chemical exchange, the c cpmg relaxation methodology, which allows one to characterize physicochemical parameters of systems under conformational exchange, was applied on sample 12. as observed in figures 12 and 13, the transverse relaxation rate r2 of the linkers (singlet peak for carbons 1 and 2, as seen in figure 6) and of the aromatic carbon 6 varies as expected with the repetition rate cpmg (= 1/(2cpmg)), indicating the presence of dynamical processes occurring in the micro- to millisecond range. c relaxation dispersion curves obtained for the cryptophane - a linkers (triangles) and the aromatic carbon 6 (lozenges) at 298 k (sample 12). filled symbols, transverse relaxation rates r2 obtained at 14.1 t ; empty symbols, transverse relaxation rates r2 obtained at 16.5 t. c relaxation dispersion curves obtained for the cryptophane - a linkers (triangles) and the aromatic carbon 6 (lozenges) at 310 k (sample 12). filled symbols, transverse relaxation rates r2 obtained at 14.1 t ; empty symbols, transverse relaxation rates r2 obtained at 16.5 t. the two - field dispersion data obtained at 14.1 and 16.5 t for both linker and aromatic carbons were fitted according to the luz meiboom equation for a two - site fast exchange:7where r20 is the transverse relaxation rate in the absence of exchange, pmajor is the relative population of the major state in exchange, corresponds to the resonance frequency difference between the spins in the major and minor states, and texch is the exchange time between the two states. of course, the assumption of a two - site exchange is probably an oversimplification, but this model, which is usually the first employed for analyzing experimental data, is adequate for having an estimation of the parameters of interest. in order to get accurate parameters, the two - field dispersion curves were fitted simultaneously for each case (linker and aromatic carbons) by using a nonlinear least - squares fitting procedure. the thermodynamic parameter pminor (= 1 pmajor), the structural parameter, the kinetic parameter texch, and the relaxation rate in the absence of exchange r20 deduced from the fitting of the c transverse dispersion curves are listed in table 8 for the two fields and the two temperatures investigated. at 16.5 t, corresponds to (16.5/14.1) times the value obtained at 14.1 t. errors are deduced from monte carlo iterations. considering the calculated errors, the results obtained for the linker and the aromatic carbons are quite similar as expected, showing the reliability of the method. in both cases, the population of the minor state is not affected by the temperature. the value found for pminor is around 0.05, indicating the presence of a low - populated state. the exchange time texch is in the millisecond range, and the exchange rate, defined as (1/texch), is increasing with temperature, agreeing with the arrhenius equation. concerning the exchange - free relaxation rate r20, it varies only little with the changes of temperature and field, with the transverse relaxation of the aromatic carbon being slightly faster. according to the values found for the linker and the aromatic carbons, the cryptophane - a minor state (if it would be visible on the spectrum) should appear a few thousand hertz away (roughly 67 ppm at 298 k and 810 ppm at 310 k) from the peaks corresponding to the major state. by comparing these results to those obtained by quantum chemical calculations (see table 7), one can see that the values are consistent with the calculated chemical shifts when going from the trans conformers (which should correspond to the major state) to the gauche conformers (which could be attributed to the minor state). the analysis of the c relaxation dispersion curves revealed that a fast conformational exchange is occurring in the complexed cryptophane - a system. to complete this study and to give a possible solution of the above - mentioned conformational problem, it is relevant to measure the cross - relaxation rates between the linker protons and the aromatic proton number 3. after this, one can make comparisons with the distances from the dft - optimized conformer structures : the cross - relaxation rates are proportional to the inverse sixth power of the corresponding proton one can see that a high cross - relaxation rate is expected in the case of the gauche conformers, since the distances are the shortest there. it is interesting to notice that, in the case of dichloromethane complexes, there are no big differences between the two peaks in the spectrum. however, there is a big difference between the ch2cl2 and chcl3 complexes in cryptophane - a. the cross - relaxation rate decreases by half in the case of the chloroform guest. this suggests that the conformational changes are much smaller in the case of the latter host. this is also indicated in the spectra : there are always two peaks in the proton spectra of cryptophane - but, and the chemical shift difference between carbons 3 and 6 is also smaller. looking at the cross - relaxation data at 258 k (the same temperature as used in cryptophane - c measurements), it can be noted that the cross - relaxation rates in ch2cl2@cryptophane - c are by a factor of 4 smaller. the small cross - relaxation rate of chcl3 means that probably the t1t1t1 conformer is preferred over the t2t2t2. the high cross - relaxation rates of the chcl3 guest with both hosts (cryptophane - a, cryptophane - but) are very surprising and shed light on the possible problems with dft calculations, namely, that the optimized structures are static and the systems are not. consistently with the carbon-13 relaxation data, chloroform can be thought of as moving in a cone inside the cavity. it is impossible that one measures such a high cross - relaxation rate at this distance. in reality this also means that the position of chloroform predicted by dft is not fully correct. this can, in turn, perhaps be traced back to the lack of van der waals contribution during the geometry optimization procedures. the investigation of chloromethanes@cryptophane with the help of different nmr methodologies sheds light on the complexation process. besides the fact that the chloromethane and the cryptophane diffuse as a single entity, it was also proven that a conformational variability / selection of encapsulation of chloromethane guests into the host cavity is also occurring this phenomenon, already observed on variable temperature spectra, was studied further by dft calculations and cpmg relaxation dispersion. these techniques gave structural information about the host but also allowed the determination of both kinetic and thermodynamic properties related to the conformational changes of the host. nevertheless, the results obtained in this study for the solution state differ from the previously proposed induced fit model by taratula. in the solid state. both the conformations of the methoxy groups and of the linkers play an important role in the case of cryptophane - a guest encapsulation, as in the case of cryptophane - c. this model was supported by the dynamical measurements of guest exchange and the activation energies. moreover, the conformational changes of the host are different from those experienced in cryptophane - c. here, the trans character plays an important role in the case of cryptophane - a, which can be caused by the methoxy groups being too close to each other when lying in the plane of the aromatic rings, which is their most stable position. in the case of cryptophane - but, the gauche character of the linkers is more pronounced and, as shown in the proton and carbon spectra, there is less change experienced by the host upon complex formation. both hosts have a surprisingly high affinity constant to chcl3, compared to other cryptophane hosts. this can be explained by the volume differences of the conformers. in the case of cryptophane - c, the gauche conformers were more populated, while the trans conformers are more important in the case of cryptophane - a. however, it should be noted that the explanation of the thermodynamic stability by the cavity size does not work in the case of cryptophane - but. it does explain the difference between dichloromethane complexes but not that between the chloroform encapsulation in various hosts. the stabilities of the chloroform complexes are very similar which is, at the present stage of the research, surprising and requires more investigation. | host guest complexes between cryptophane - a as host and dichloromethane and chloroform as guests are investigated using 1h and 13c nmr spectroscopy. moreover, a related cryptophane, with the methoxy groups replaced by butoxy units (cryptophane - but), and its complexes with the same guests were also studied. variable temperature spectra showed effects of chemical exchange between the free and bound guests, as well as of conformational exchange of the host. the guest exchange was studied quantitatively by exchange spectroscopy or line shape analysis. extraction of kinetic and thermodynamic parameters led to the characterization of the affinity between guests and hosts. on the other hand, the host exchange was investigated by means of 13c carr purcell meiboom gill (cpmg) relaxation dispersion which aims at the determination of the transverse relaxation rate r2, the inverse of the transverse relaxation time t2, as a function of the repetition of the pulses in a cpmg train. the variation of the measured transverse relaxation rate with the repetition rate cpmg indicated conformational exchange occurring on the microsecond millisecond time scale. structural information was obtained through measurements of cross - relaxation rates, both within the host and between the host and the guest protons. the nmr results were supported by dft calculations. |
acute myocardial ischemic injury is associated with various pathophysiological responses, including inflammatory activity with migration of neutrophils to the ischemic site. although early blood reperfusion to the ischemic myocardium is undoubtedly beneficial in the setting of acute myocardial infarction, there is much evidence that re - establishment of coronary blood flow helps propagate inflammatory processes, enhancing tissue destruction and contributing to myocardial dysfunction.1,2 attempts to limit myocardial necrosis and improve outcomes in acute myocardial infarction by modulating the inflammatory process have long been proposed. around thirty years ago, investigators demonstrated that some, but not all, anti - inflammatory agents could reduce the size of myocardial infarction in laboratory animals.3 on the other hand, no clear benefits and even evidence of deleterious effects were seen in human studies of anti - inflammatory drugs in acute coronary syndromes,47 although meloxicam was associated with a better outcome in a small trial.8 more recently, with the advent of selective cyclooxygenase (cox)-2 inhibitors, the issue of anti - inflammatory drugs in acute myocardial infarction has again been brought to the forefront. cox is an enzyme that converts free arachidonic acid into prostaglandins, which mediate inflammation. differing from the cox-1 isoform, which is constitutively expressed and has a housekeeping role, cox-2 is usually absent or minimally present in most tissues but is induced under certain conditions such as inflammation. indeed, cox-2 is typically considered a pro - inflammatory enzyme, and selective cox-2 inhibitors have been widely prescribed for acute and chronic inflammatory disorders. importantly, this class of drugs has been associated with adverse cardiac events in humans, raising serious concerns about their safety.913 the mechanisms by which cox-2 inhibitors may harm the heart have not been fully elucidated. it has been shown that under certain circumstances, cox-2 possesses other properties, including inhibition of fibrosis, thrombosis and even inflammation1416 which may play a cardioprotective role. investigations focusing on cox-2 and cox-2 inhibitors in different models of myocardial ischemia / infarction in diverse animals have elicited contradictory findings, bringing more controversy to this issue. while several studies have shown that cox-2 inhibition protects the heart and preserves cardiac function in murine models of myocardial ischemia / infarction,1721 celecoxib was shown to deteriorate myocardial function and increase mortality after myocardial infarction in pigs.22 importantly, these studies evaluated the consequences of complete ligation of a coronary artery, without reperfusion, on cardiac function over several days or weeks. in a clinical setting, early coronary reperfusion is not only desirable but also increasingly frequent, now that fibrinolytic therapy and percutaneous intervention have become more accessible. moreover, it is known that it is during the first moments after an acute myocardial infarction that the subjects are the most susceptible to complications. the effects of cox-2 inhibition in the very early phase after an acute myocardial infarction with coronary reperfusion have not been well addressed. in rabbits, it appears that cox-2 has an essential role in cardioprotection conferred by ischemic preconditioning23 ; accordingly, cox-2 inhibition was shown to exacerbate ischemia - induced acute myocardial dysfunction in an in vitro perfused rabbit heart model.24 this study was designed to help elucidate the role of cox-2 and the mechanisms involved in the effect of selective cox-2 inhibitors in the very early phase of acute myocardial infarction followed by coronary reperfusion. specifically, this study aimed to evaluate the effect of pre - treatment with rofecoxib, a selective cox-2 inhibitor, on the extent of infarction, biomarkers of cardiac necrosis and hemodynamic behavior in a well - established canine model of myocardial ischemia - reperfusion. this study was approved by the ethics in research committee of our institution (protocol 2165/02/172), according to the american heart association regulations for research involving animals, based on the guide for the care and use of laboratory animals (national health council publication, national academy press, washington, d.c 1996.)64 the dogs used in this experiment were obtained from the public health system, urban zoonosis department. after being properly cleaned, the animals were housed in the biotery in isolated cages with veterinary care for several days before the surgery was carried out. they were fed commercial dog feed and water ad libitum, and they were vaccinated against rabies (hydrophobia) and given a dose of vermicide. forty - eight adult male dogs (canis familiaris) were studied : 28 dogs were part of the study group treated with commercial rofecoxib (vioxx, mk-966) at a single 50-mg / day dose per os with their chow during the 48 hours preceding the surgical procedure, while 20 dogs were part of the control group. the weight of the animals ranged from 15 to 30 kg. rofecoxib is rapidly absorbed after oral administration. in dogs, a peak concentration of 207 ng / ml (0.6585 m) was achieved 2 to 3 hours after oral administration of a single 25-mg dose.6163 in both groups, the animals were anesthetized, intubated and artificially ventilated. the following hemodynamic parameters were evaluated : coronary blood flow, average blood pressure and heart rate. immediately before the occlusion, blood was collected from the venous sinus to evaluate creatine phosphokinase - mb (cpk - mb), cpk - mb mass and troponin - i levels. after completing the 30-minute reperfusion, a new blood sample from the venous sinus was collected to evaluate the same biochemical markers. at the end of each experiment, all dogs received a dose of 30 mg / kg of sodium pentobarbital before euthanasia with potassium chloride. following this procedure, their hearts were excised and submitted to evans blue and triphenyltetrazolium chloride staining to define the risk areas and areas of infarction. a 7f polyethylene catheter was positioned in the left carotid artery for continuous monitoring of the average blood pressure (abp) and heart rate (hr). the catheter was connected to a biopac systems transducer, tsd-104 model (biopac systems, santa barbara, ca, usa), linked to a computer with specific software (acknowledge) to read the hemodynamic values. central venous access was obtained through cannulation of the left internal jugular vein by means of another 7f polyethylene catheter to infuse liquids and anesthetic. coronary flow was measured with placement of an electromagnetic flow - transducer (transonic systems inc., probe 2s) in the ada in its proximal third, immediately after the emergence of the first diagonal branch. quantitative determination of the mb isoform of creatine kinase was performed by means of in vitro immune assays, using an automatic analyzer roche / hitachi model 912 (roche diagnostics, indianapolis, in, usa) this method presupposes that ck - mb is made up of two subunits : ck - m and ck - b, both with an active site. the test consists of adding a polyclonal antibody directed against the ck - m site to the serum, which almost completely (99.6%) inhibits the catalytic ability of the ck - m subunit without affecting the ck - b subunit. the remaining activity of the ck - b subunit is equal to half of the activity of ck - mb. the catalytic activity of ck - mb can be calculated as the measured ck - b activity multiplied by 2, since the ck - bb isoform rarely appears in the serum.53,54 for the measurement of creatine kinase - mb mass, immunochemiluminescence with laboratory automation was used by means of immulite dpc equipment (diagnostic products corporation los angeles, ca, usa). in this method, the serum is put into contact with small spheres covered by anti - ck - mb murine monoclonal antibody (turbo ck - mb test unit - scp1). a second test unit is then added (turbo ck - mb scp2), containing alkaline phosphatase conjugated to an anti - ck - mb murine polyclonal antibody. upon reacting, both units cause the release of luminescence, quantified by the immulite equipment.55,56 for measurement of troponin i, immuno - chemiluminescence with laboratory automation was used by means of immulite dpc equipment (diagnostic products corporation the serum is put into contact with small spheres covered by anti - troponin i rat monoclonal antibody (turbo troponin i sti1 test unit). sti2) is added, containing alkaline phosphatase conjugated with anti - troponin i goat polyclonal antibody. upon reacting, the regions of the left ventricular myocardium with normoperfusion, ischemia and infarction were evaluated at the end of each experiment by the techniques described by jolly and chagas.58 at the end of the protocol, the hearts were excised. the ada was then occluded at the same place in which the occlusor was positioned and cannulated distal to the occlusion. evans blue stain (1 mg / kg) was infused through a catheter positioned in the right coronary artery and in the left coronary artery trunk, and simultaneously, a solution at 2% ctt was injected into the ada. thus, the areas that were not perfused by the ada were stained by evans blue, delimiting the region at risk supplied by the ada. the heart was then sectioned longitudinally and incubated in a ctt solution at 37c for 30 minutes. this technique stains the viable myocardium in bright red and the necrotic tissue in a pale yellow shade. the regions that did not show a bright red color and remained a pale yellow shade were defined as necrotic myocardium.59,60 analysis was carried out by observation of the minimum and maximum values and the calculation of averages and standard deviations. for the qualitative variables, absolute and relative frequencies were calculated. to analyze the hypothesis of average equality between the two groups, a student s t - test25 was used, which allows the evaluation of variables at a single period between the control and treated groups. this method was used to analyze the weight of the fragments of the left ventricle. in the hemodynamic analysis of the groups, the analysis of variance (anova) technique with repeated53 measurements initially, the study included 61 dogs, but 13 dogs were excluded because they exhibited ventricular fibrillation during the coronary artery occlusion. electric cardioversion could not be used because it can alter serum levels of necrosis markers. the arterial blood pressure drop in the basal period as compared to the arterial blood pressure in the 1 minute of occlusion showed a sharp drop (p<0.001) in both groups. there was a significant arterial blood pressure increase (p<0.001) between the 1 and 30 minute in the control group ; however, this was not observed in the treated group (p=0.2257). it was also observed that the arterial blood pressure at the 30 minute did not significantly differ from that at the 180 minute, in either the control group (p=0.2576) or the treated group (p=0.0546). it was observed, however, that the arterial pressure at the 180 minute differed from that at the 1 minute of reperfusion in the control group (p=0.0056), while the same drop in pressure was not observed in the treated group (p=0.3973). using anova with repeated measurements, we observed that both groups showed the same heart rate throughout the study period (p=0.1015). the groups did not show significant differences between their averages (p=0.2013) and showed the same significant changes throughout the study (p < 0.001), indicating that there was homogeneity in heart rate among the groups. the two groups also showed the same cardiac flow behavior throughout the experiment (p=0.5315). they did not show significant differences between their averages (p=0.4118) and showed the same significant changes throughout the experiment (p < 0.001). the basal period differed from the 1 minute of reperfusion (p < 0.001), and this differed from the 30 minute of reperfusion (p < 0.001) in both groups, showing that both groups had reactive hyperemia, characteristic of reperfusion. no significant changes were observed in the normoperfused areas between the studied groups (p=0.1648). the same results were obtained for the ischemic areas (p=0.3651) and the infarction area (p=0.2793). using anova with repeated measurements, we observed that the two groups showed a significant increase in cpk - mb between pre- and post - infarction (p<0.001), indicating that both groups suffered acute myocardial infarction. the control group showed significantly higher averages of cpk - mb than the treated group during the post - infarction period (p=0.0445) (figure 1). the control and treated groups showed a significant increase in the level of the cpk - mb mass between the pre- and post - infarction periods (p<0.001), with no difference between their averages during pre - infarction (p=0.2408). during the post - infarction period, however, we observed a significant cpk - mb mass increase in the control group (p=0.007), as compared to the treated group (figure 2). the two groups showed a significant difference in troponin i levels during the pre- and post - infarction periods (p<0.001). the groups showed no difference between levels during pre - infarction (p=0.6275), but troponin i was increased in the control group (p<0.001) during the post - infarction period, as compared to the group treated with icox-2 (figure 3). the current study shows that the use of a specific cyclooxygenase-2 inhibitor in experimental acute myocardial ischemia models results in a protective effect on cardiac muscle. in the analysis of blood pressure, two periods of pressure instability were observed : one occurred during the period in which the coronary occlusion was performed (detected at 1 min), and the other occurred during the period in which the coronary was reperfused (detected in the 1 minute of reperfusion). this statistically significant pressure drop was only observed in the control group, as the treated group did not show arterial pressure instability during these two periods. this effect of a selective cyclooxygenase-2 inhibitor on the systolic, diastolic and average blood pressure has also been observed in other studies.2628 the most commonly accepted hypothesis is related to the effect of sodium reabsorption by the kidneys when the cyclooxygenase-2 inhibitor is administered, without affecting glomerular filtration. in a recent study by hcherl.,29 cardiac dysfunction and blood pressure were evaluated in rats undergoing intravenous infusion of lipopolysaccharides. these animals were divided into two groups : the treated group received a specific cyclooxygenase-2 inhibitor (rofecoxib), and the control group was untreated. a significant decrease in blood pressure and myocardial dysfunction was observed in the control group. the blood pressure observations of hcherl.29 were similar to those obtained in our experiment, in which the group treated with icox-2 did not show a sharp drop in blood pressure during the periods when the coronary occlusion and reperfusion were performed. despite these results, the exact mechanism by which selective cyclooxygenase-2 inhibitors interfere with blood pressure remains to be explained. our results were similar to those of a study by verma.,30 who used plethysmography in the forearm of volunteers and healthy patients to evaluate the use of rofecoxib, as compared to infusion of acetylcholine and sodium nitroprussiate. it was observed that the patients did not show blood flow changes, suggesting that the selective cox-2 inhibitor did not stop the vasodilatation mechanisms mediated by the endothelium. in our biochemical analysis such methods are also valid for laboratory animals.31,32 each animal served as its own control between the pre - infarction and the post - infarction period, and the obtained values were in accordance with those found in other studies.31,32 considering the kinetics of the analyzed biochemical markers in the circulation (e.g., cell compartmentalization, linkage to plasma molecules, distribution, degradation and elimination), the subjects only have detectable plasma levels in the peripheral circulation from four to five hours after the coronary ischemic event.31 due to the metabolic and distributive kinetics of cpk - mb, cpk - mb mass and troponin i, we collected blood samples by central venous access (from the coronary sinus) after 30 minutes of coronary reperfusion, i.e., three and a half hours after the start of the ischemic event. previous studies36,37 making use of non - steroid anti - inflammatories showed that these medications did not have a cardioprotective effect during ischemic injury, but there is still no exact definition of their role in cardiomyocytes.38,39 ischemic inflammatory myocardial injury began during the period in which the blood supply was interrupted and increased during the period of coronary reperfusion40,41, along with a greater expression of cox-234 mrna. a study by metais showed that increased cox-2 levels in the microvasculature of pig ventricles were related to an increase in local vasoconstriction. this vasoconstriction, however, was inhibited by the use of a selective cyclooxygenase-2 inhibitor. these results could explain, in part, the results of our current study, in which we observed biochemical signs of myocardial cytoprotection in acute myocardial infarction with treatment with a selective cyclooxygenase-2 inhibitor. we found significantly lower values for the biochemical markers in the treated group, as compared to the control group, due to the use of rofecoxib. the values of cpk mb were obtained through the least sensitive detection method (p=0.0445). immunochemiluminescence with electronic reading, however, which was used to detect cpk mb mass (p=0.007) and troponin i (p<0.001), is a more sensitive assay. although significant differences were observed between the groups for the biochemical markers, indicating possible myocardial cytoprotection in the setting of an acute ischemic stimulus, these differences were not observed in the quantification of the normoperfused, ischemic or infarcted tissue.2123,42 some questions are still open. is there really a relationship between coxibs and the increase in the number of ischemic cardiac events ? if so, how can we explain this pathophysiology ? are these pathophysiological mechanisms dependent on a coxib class effect, or are they only caused by a specific kind of selective cyclooygenase-2 inhibitor ? the vigor study (vioxx gastrointestinal outcomes research trial) was the first great study to discuss the presence of cardiovascular events with the use of icox-2. meanwhile, results from a metanalysis43 involving rofecoxib showed that naproxen, used in the vigor study, has a protective effect, whereas the rofecoxib group went without this protection. another important piece of information pertaining to this study was that the analyzed population consisted of patients with rheumatoid arthritis. these patients have a higher risk of cardiovascular events, and this compromises the results of the vigor study. on the other hand, a study44 of 150 patients using rofecoxib, who underwent isolated coronary artery bypass graft surgery, showed that there was no clinical evidence for a deleterious effect of rofecoxib on myocardium in these patients. differences in the chemical structures of the various coxibs result in different pharmacological and pharmacokinetic proprieties that could theoretically explain why one coxib is more toxic than another of the same class. there are no studies, however, that evaluate cardiovascular events and compare one coxib with another of the same class, which might support this theory.45 on the other hand, a study developed by andersohn.46 in which patients with acute myocardial infarction were evaluated, suggested that the toxicity could be related to the entire coxib class. however, these authors compared icox-2 drugs to a non - selective cox inhibitor (diclofenac), similar to the vigor study. meanwhile, an experimental study conducted by delgado. demonstrated that cyclooxygenase-2 inhibition could attenuate the development of cardiac heart failure after myocardial lesions, as evidenced by improved left ventricular ejection fraction and a reduction in animal mortality.47 further ongoing trials are investigating the newer coxibs as well as the older drugs, but unfortunately, they are mostly being performed in patients with rheumatoid arthritis, as in the class and the target studies. it is already known that rheumatoid arthritis itself is a risk factor for cardiac disease, most likely due to a chronic increase in circulating cytokine concentrations as well as in c reactive protein and other proatherogenic inflammatory markers, with activation of leucocytes in both processes.48 another study showed that the use of icox-2 was able to reduce endothelial oxidative stress and the generation of ionic forms (nitric oxide induced synthase), peroxynitrite and superoxide and the formation of interleukins 6 and 8. these reductions in endothelial oxidative stress provoked improvement in endothelium - dependent vasodilatation, resulting in the improvement of heart failure disease.49 moreover, an increase in serum vegf (vascular endothelial growth factor) was not suppressed by treatment with icox-2, indicating that vegf was induced in a cox-2-independent manner.50, 51 recently, another experimental study52 showed, for the first time, a functional improvement with the administration of parecoxib, a selective cox-2 inhibitor, in an animal model of ischemic congestive heart failure. confirming these findings, a functional improvement with cox-2 inhibitors in a model of non - ischemic heart failure was already shown and was paired with a significant reduction in mortality.46 cumulative data currently suggest that the mechanisms of acute myocardial infarction and inflammation have different phases in which the same component (e.g., cyclooxygenase-2) can be beneficial in a given moment but have a different effect at another moment. thus, further studies are needed to clarify the pathophysiological mechanisms linking inflammatory activity to acute myocardial infarction. some questions are still open. is there really a relationship between coxibs and the increase in the number of ischemic cardiac events ? if so, how can we explain this pathophysiology ? are these pathophysiological mechanisms dependent on a coxib class effect, or are they only caused by a specific kind of selective cyclooygenase-2 inhibitor ? the vigor study (vioxx gastrointestinal outcomes research trial) was the first great study to discuss the presence of cardiovascular events with the use of icox-2. meanwhile, results from a metanalysis43 involving rofecoxib showed that naproxen, used in the vigor study, has a protective effect, whereas the rofecoxib group went without this protection. another important piece of information pertaining to this study was that the analyzed population consisted of patients with rheumatoid arthritis. these patients have a higher risk of cardiovascular events, and this compromises the results of the vigor study. on the other hand, a study44 of 150 patients using rofecoxib, who underwent isolated coronary artery bypass graft surgery, showed that there was no clinical evidence for a deleterious effect of rofecoxib on myocardium in these patients. differences in the chemical structures of the various coxibs result in different pharmacological and pharmacokinetic proprieties that could theoretically explain why one coxib is more toxic than another of the same class. there are no studies, however, that evaluate cardiovascular events and compare one coxib with another of the same class, which might support this theory.45 on the other hand, a study developed by andersohn.46 in which patients with acute myocardial infarction were evaluated, suggested that the toxicity could be related to the entire coxib class. however, these authors compared icox-2 drugs to a non - selective cox inhibitor (diclofenac), similar to the vigor study. meanwhile, an experimental study conducted by delgado. demonstrated that cyclooxygenase-2 inhibition could attenuate the development of cardiac heart failure after myocardial lesions, as evidenced by improved left ventricular ejection fraction and a reduction in animal mortality.47 further ongoing trials are investigating the newer coxibs as well as the older drugs, but unfortunately, they are mostly being performed in patients with rheumatoid arthritis, as in the class and the target studies. it is already known that rheumatoid arthritis itself is a risk factor for cardiac disease, most likely due to a chronic increase in circulating cytokine concentrations as well as in c reactive protein and other proatherogenic inflammatory markers, with activation of leucocytes in both processes.48 another study showed that the use of icox-2 was able to reduce endothelial oxidative stress and the generation of ionic forms (nitric oxide induced synthase), peroxynitrite and superoxide and the formation of interleukins 6 and 8. these reductions in endothelial oxidative stress provoked improvement in endothelium - dependent vasodilatation, resulting in the improvement of heart failure disease.49 moreover, an increase in serum vegf (vascular endothelial growth factor) was not suppressed by treatment with icox-2, indicating that vegf was induced in a cox-2-independent manner.50, 51 recently, another experimental study52 showed, for the first time, a functional improvement with the administration of parecoxib, a selective cox-2 inhibitor, in an animal model of ischemic congestive heart failure. confirming these findings, a functional improvement with cox-2 inhibitors in a model of non - ischemic heart failure was already shown and was paired with a significant reduction in mortality.46 cumulative data currently suggest that the mechanisms of acute myocardial infarction and inflammation have different phases in which the same component (e.g., cyclooxygenase-2) can be beneficial in a given moment but have a different effect at another moment. thus, further studies are needed to clarify the pathophysiological mechanisms linking inflammatory activity to acute myocardial infarction. the results of this study have shown that the use of a specific cyclooxygenase-2 inhibitor, rofecoxib, might be beneficial in reducing the intensity of myocardial injury induced by acute coronary occlusion in dogs. new clinical studies related to endothelial dysfunction and inflammation are necessary, however, in order to elucidate the complete role of inflammatory mechanisms in acute myocardial infarction. | backgroundacute myocardial infarction is associated with tissue inflammation. early coronary reperfusion clearly improves the outcome but may help propagate the inflammatory response and enhance tissue damage. cyclooxygenase-2 is an enzyme that catalyzes the initial step in the formation of inflammatory prostaglandins from arachidonic acid. cyclooxygenase-2 levels are increased when ischemic cardiac events occur. the overall function of cox-2 in the inflammatory process generated by myocardial ischemic damage has not yet been elucidated.goalthe objective of this study was to determine whether a selective cyclooxygenase-2 inhibitor (rofecoxib) could alter the evolution of acute myocardial infarction after reperfusion.methods and resultsthis study was performed with 48 mongrel dogs divided into two groups : controls and those treated with the drug. all animals were prepared for left anterior descending coronary artery occlusion. the dogs then underwent 180 minutes of coronary occlusion, followed by 30 minutes of reperfusion. blood samples were collected from the venous sinus immediately before coronary occlusion and after 30 minutes of reperfusion for measurements of cpk - mb, cpk - mbm and troponin i. during the experiment we observed the mean blood pressure, heart rate and coronary flow.the coronary flow and heart rate did not change, but in the control group, there was blood pressure instability, in addition to maximal levels of cpk - mb post - infarction. the same results were observed for cpk - mbm and troponin i.conclusionin a canine model of myocardial ischemia - reperfusion, selective inhibition of cyclooxygenase-2 with rofecoxib was not associated with early detrimental effects on the hemodynamic profile or the gross extent of infarction ; in fact, it may be beneficial by limiting cell necrosis. |
migraine is a highly prevalent illness, and women of reproductive ages are more affected by it than men or women in other age groups (1). many researchers have supported the view that behavioral interventions such as biofeedback and relaxation, either alone or in combination, can be effective for the treatment of headache (2 - 7). especially, thermal biofeedback with or without combined autogenic training is known to be effective for the treatment of migraine headache (5, 8). in addition, some studies showed that the effect of the behavioral treatment is durable (9, 10). there have been no controlled studies about the efficacy of biofeedback and relaxation training for patients with migraine in korea, and few studies have been conducted to investigate the direct association between mood states, including anxiety or depression, and headache activity. the mechanism underlying the efficacy of biofeedback treatment and the role of psychological factors in migraine treatment are still unclear, though various mood states, including anxiety and depression, and some cognitive factors are known to be related to pain regulation (11 - 13). psychological conditions including anxiety and depression have been reported to have a strong association with migraine (12 - 14). thus, in this study, we examined the hypothesis that biofeedback - assisted autogenic training is effective for the treatment of korean female patients with migraine and that the improvement of psychological factors, such as anxiety and depression, is associated with the reduction of headache activity in migraine patients who are given biofeedback treatment. thirty - two patients, who had migraine headache with or without aura, as defined by the international headache society criteria (15), participated in this study, from march 2003 until december 2006. the patients were female subjects aged 20 to 40 yr who had a body mass index (bmi) ranging from 18 to 27.5. the patients were randomly allocated to one of two patient groups, consisting of a treatment group receiving biofeedback - assistant relaxation treatment (n=17) and a monitoring group in which the headache activities and biofeedback parameters of the patients were simply measured (n=15). these two groups did not show any significant difference in the duration of headache, headache index, duration of medication use, or other headache - related variables at the baseline (table 1). twenty - one normal female volunteers were recruited by advertisement and were matched with the migraine patients by age (treatment group : 31.125.49 yr, monitoring group : 31.874.70 yr, normal control group : 29.384.64 yr), and mood states and mean skin temperature of the patients before the biofeedback treatment were compared with those of the normal control subjects. each subject underwent a comprehensive medical and psychiatric assessment including careful history taking, physical examination, neurological examination, mental status examination, and electrocardiography. those patients who seemed to suffer from secondary headache due to medical or psychiatric illnesses were not included in the study. they did not take regular medications for migraine headache, and only pro re nata (p.r.n.) this study was approved by the institutional review board of samsung medical center, and all of the subjects gave their written informed consent. the patients in the treatment group received 8 sessions of biofeedback - assisted autogenic training (two sessions per week) during a period of 4 weeks, while those in the monitoring group received no active intervention except for simple biofeedback measurements (1st, 4th, and 8th session) during the treatment - waiting period. biofeedback treatment was administered with the patient sitting on a comfortable arm - chair in a quiet room in the psychiatric outpatient clinic of samsung medical center. a procomp+system and biograph version 2.1 program (thought technology ltd., quebec, canada) were used for the biofeedback treatment and measurements. the autogenic training procedures consisted of 6 standard exercises combining both relaxation and auto - suggestion (limb heaviness exercise, limb warmth exercise, cardiac exercise, respiration exercise, solar plexus warmth exercise, and " forehead cooling " exercise) (16). during the 1st session, after the baseline profiles were obtained, education in relaxation techniques was given to the patients in the treatment group. during the 2nd to 8th sessions, thermal biofeedback combined with autogenic training for the first 15 - 20 min, after collecting the headache diary data, the patients were rated on psychological variables and headache severity. the patients were trained to increase their skin temperature by an experienced biofeedback therapist for about 15 min according to the procedures described in a treatment manual. during the last 15 min, they were instructed to continue the relaxation exercise by themselves, while listening to a prerecorded tape. the headache severity was rated on a 6-point rating scale with 0 representing " no pain " and 5 representing " pain as severe as it can be ". the average frequency, duration, and intensity of pain during the 7 consecutive days were compiled as headache indexes. headache indexes at baseline, after 2 weeks, and after 4 weeks of biofeedback treatment were used to compare the two groups. secondary outcomes were evaluated using the mpq (17) and the clinical global impression severity scale (cgi - s) administered by the therapist. the baseline headache variables were recorded for the previous 1 week before the treatment and the follow - up headache variables as the mean of the symptoms during the previous week. due to the possibility of there being a change in the perception of pain according to the menstrual cycle (18), the menstruation phase was determined by asking the subjects the onset of their last menstruation. before the biofeedback treatment, there were no differences in the headache indices and mood states between the treatment group and monitoring group according to the menstruation phase. the psychological assessments for anxiety and depressive symptoms were performed using the hamilton rating scales for anxiety (ham - a) (19) and depression (ham - d) (20), and the spielberger state anxiety inventory (stai - s) (21). the cgi - s, ham - a, and ham - d were performed by a well - trained psychiatrist who was blind to the patients ' clinical states. to compare the baseline values, chi - square test, fisher 's exact test, and analysis of variance (anova) were used.. repeated measures anova was performed to analysis mean changes of skin temperature, psychological variables, and secondary pain outcomes. student 's t - test with bonferroni 's correction was performed for post hoc analysis. univariate and multivariate logistic regression analyses were used to whether the baseline or changes of depression and anxiety level could predict the treatment outcome. thirty - two patients, who had migraine headache with or without aura, as defined by the international headache society criteria (15), participated in this study, from march 2003 until december 2006. the patients were female subjects aged 20 to 40 yr who had a body mass index (bmi) ranging from 18 to 27.5. the patients were randomly allocated to one of two patient groups, consisting of a treatment group receiving biofeedback - assistant relaxation treatment (n=17) and a monitoring group in which the headache activities and biofeedback parameters of the patients were simply measured (n=15). these two groups did not show any significant difference in the duration of headache, headache index, duration of medication use, or other headache - related variables at the baseline (table 1). twenty - one normal female volunteers were recruited by advertisement and were matched with the migraine patients by age (treatment group : 31.125.49 yr, monitoring group : 31.874.70 yr, normal control group : 29.384.64 yr), and mood states and mean skin temperature of the patients before the biofeedback treatment were compared with those of the normal control subjects. each subject underwent a comprehensive medical and psychiatric assessment including careful history taking, physical examination, neurological examination, mental status examination, and electrocardiography. those patients who seemed to suffer from secondary headache due to medical or psychiatric illnesses were not included in the study. they did not take regular medications for migraine headache, and only pro re nata (p.r.n.) this study was approved by the institutional review board of samsung medical center, and all of the subjects gave their written informed consent. the patients in the treatment group received 8 sessions of biofeedback - assisted autogenic training (two sessions per week) during a period of 4 weeks, while those in the monitoring group received no active intervention except for simple biofeedback measurements (1st, 4th, and 8th session) during the treatment - waiting period. biofeedback treatment was administered with the patient sitting on a comfortable arm - chair in a quiet room in the psychiatric outpatient clinic of samsung medical center. a procomp+system and biograph version 2.1 program (thought technology ltd., quebec, canada) were used for the biofeedback treatment and measurements. the autogenic training procedures consisted of 6 standard exercises combining both relaxation and auto - suggestion (limb heaviness exercise, limb warmth exercise, cardiac exercise, respiration exercise, solar plexus warmth exercise, and " forehead cooling " exercise) (16). during the 1st session, after the baseline profiles were obtained, education in relaxation techniques was given to the patients in the treatment group. during the 2nd to 8th sessions, thermal biofeedback combined with autogenic training for the first 15 - 20 min, after collecting the headache diary data, the patients were rated on psychological variables and headache severity. the patients were trained to increase their skin temperature by an experienced biofeedback therapist for about 15 min according to the procedures described in a treatment manual. during the last 15 min, they were instructed to continue the relaxation exercise by themselves, while listening to a prerecorded tape. the headache severity was rated on a 6-point rating scale with 0 representing " no pain " and 5 representing " pain as severe as it can be ". the average frequency, duration, and intensity of pain during the 7 consecutive days were compiled as headache indexes. headache indexes at baseline, after 2 weeks, and after 4 weeks of biofeedback treatment were used to compare the two groups. secondary outcomes were evaluated using the mpq (17) and the clinical global impression severity scale (cgi - s) administered by the therapist. the baseline headache variables were recorded for the previous 1 week before the treatment and the follow - up headache variables as the mean of the symptoms during the previous week. due to the possibility of there being a change in the perception of pain according to the menstrual cycle (18), the menstruation phase was determined by asking the subjects the onset of their last menstruation. before the biofeedback treatment, there were no differences in the headache indices and mood states between the treatment group and monitoring group according to the menstruation phase. the psychological assessments for anxiety and depressive symptoms were performed using the hamilton rating scales for anxiety (ham - a) (19) and depression (ham - d) (20), and the spielberger state anxiety inventory (stai - s) (21). the cgi - s, ham - a, and ham - d were performed by a well - trained psychiatrist who was blind to the patients ' clinical states. to compare the baseline values, chi - square test, fisher 's exact test, and analysis of variance (anova) were used. treatment outcome analysis in terms of headache index was preformed using chi - square test. repeated measures anova was performed to analysis mean changes of skin temperature, psychological variables, and secondary pain outcomes. student 's t - test with bonferroni 's correction was performed for post hoc analysis. univariate and multivariate logistic regression analyses were used to whether the baseline or changes of depression and anxiety level could predict the treatment outcome. the fisher 's exact test revealed a significant difference in the response rate in terms of headache index between the two groups (=4.979, df=1, p=0.029). ten of the 17 patients (58.9%) in the treatment group showed a significant improvement in their headache index corresponding to a 50% or greater reduction in their headache activity, whereas only 3 of the 15 subjects (20%) in the monitoring group achieved a significant improvement (fig. 1). repeated measures anova indicated that there were significant interactions between time and group in the mpq - s (f=6.994, p=0.014), mpq - a (f=9.978, p=0.006), and cgi - s (f=16.160, p=0.001). mean resting skin temperatures did not show any differences between the treatment and monitoring group, nor pre- and post - treatment (all p values > 0.1). the results of the comparisons of the psychological states of the three groups are shown in table 2. the anxiety and depression levels in the migraine patients were significantly higher than those in the normal control subjects at the baseline (all p values 0.1) in the biofeedback - assisted training group. however, the multivariate logistic regression revealed that the greater reduction of the ham - a score was associated with the treatment response controlling for age, bmi, and the baseline ham - a in the biofeedback - assisted autogenic training group (or=1.52, ci=1.01 - 2.33, p=0.048) whereas the reduction of the ham - d score nor that of the stai - s were not (p=0.088 and p=0.080, respectively). we found that biofeedback - assisted autogenic training is effective in management of female migraine patients in korean population. they also exhibited significant differences in their mood states measured by the ham - a, ham - d, stai - s, as compared with the normal healthy women. this finding is consistent with the results of previous studies which showed that migraine patients had high levels of anxiety and depression (12, 14, 22). it is also consistent with a previous report (23) that negative mood states such as anxiety and depression are related to physical complaints and headache. the anxiety and depression scores in the migraine patients were reduced after 4 weeks of biofeedback treatment. furthermore, the reduction in anxiety level after the biofeedback treatment was related to the treatment response of the patients. to our knowledge, there have been no reports about the association between mood states, including anxiety and depression, and improvements in headache activity in migraine patients receiving biofeedback treatment. our result is also consistent with the finding of previous studies (24 - 27) that mood states might affect the experience of pain through the cognitive processing of nociceptive information, although this hypothesis has not been directly examined in headache patients. the present result is in contrast with the previous report (2) that decreased depression level rather than anxiety was related to the outcome of biofeedback - assisted training in patients with chronic tension - type headache. taken together, reduction of anxiety may be related to the biofeedback treatment response of migraine headache, whereas reduction of depression may be related to the biofeedback treatment response of chronic tension - type headache. further studies will be necessary to confirm this difference between migraine and tension type headache. mean skin temperatures between the pre- and post - treatment changes were not statistically different. our finding also suggests that the reduction of anxiety rather than depression level is more important in the biofeedback treatment of migraine. this result appears to confirm the previous findings that the treatment mechanism of biofeedback is mediated by an indirect process rather than a direct physiological change (28, 29). cognitive factors such as increased self - efficacy belief or coping style can affect the success of biofeedback treatment, and anxiety is known to be closely associated with cognitive factors (11). however, since migraine is known to be inter - related to anxiety (30), our tentative finding has the limitation that it does not confirm whether an improvement in anxiety causes a decrease in headache activity. further studies are needed to examine the relationship among mood states, cognitive factors, and headache. migraine is an illness with a fluctuating course in which the symptoms wax and wane, and psychophysiological treatment may have a delayed effect. secondly, the severity of headache in the recruited patients was relatively low and the patients in this study may not represent the general population with migraine headache. however, to the best of our knowledge, this is the first controlled study to examine the efficacy of biofeedback treatment and the relationship between mood states and headache activity in korean migraine patients. in conclusion, biofeedback treatment was found to cause an improvement of headache and mood states such as anxiety and depression in female migraine patients. in addition, the reduction of anxiety level was related to the biofeedback treatment response. these results suggest that biofeedback treatment can be an effective non - pharmacological treatment for migraine patients and that the improvement of the anxiety states afforded by biofeedback treatment may play a key role in reducing headache activity. | biofeedback with or without combined autogenic training is known to be effective for the treatment of migraine. this study aimed to examine the effect of biofeedback treatment on headache activity, anxiety, and depression in korean female patients with migraine headache. patients were randomized into the treatment group (n=17) and monitoring group (n=15). mood states including anxiety and depression, and psychophysiological variables such as mean skin temperature of the patients were compared with those of the normal controls (n=21). we found greater treatment response rate (defined as 50% reduction in headache index) in patients with biofeedback - assisted autogenic training than in monitoring group. the scores on the anxiety and depression scales in the patients receiving biofeedback - assisted autogenic training decreased after the biofeedback treatment. moreover, the decrease in their anxiety levels was significantly related to the treatment outcome. this result suggests that the biofeedback - assisted autogenic training is effective for the treatment of migraine and its therapeutic effect is closely related to the improvement of the anxiety level. |
the emerging discipline of cancer survivorship research includes the study of the consequences of cancer diagnosis and its treatment among children and adults who have experienced cancer. while the united states (usa) has conducted the majority of research on adult cancer survivorship [26 ], the united kingdom and canada have conducted large - scale cohort studies of childhood cancer survivorship [7, 8 ]. as more countries are beginning to address issues related to adult survivorship [920 ], it will be important to consider how transferrable findings will be across cultures and nations. identifying generalizable and nongeneralizable research among cultures will hasten what we learn about cancer survivorship. this essay presents an overview of cancer survivorship research and its challenges and discusses in detail three examples of pronounced cross - cultural challenges currently facing the field. finally, future courses of action within the field of comparative cancer survivorship research are suggested. according to the us institute of medicine, cancer survivorship research encompasses the physical, psychosocial, and economic consequences of cancer diagnosis and its treatment among both pediatric and adult survivors of cancer. it seeks to identify, examine, prevent, and control adverse cancer- and treatment - related outcomes and to provide a knowledge base regarding optimal follow - up care and surveillance of cancer survivors. lastly, cancer survivorship research aspires to optimize health after cancer treatment, seeking to promote healthy lifestyles and behaviors. a cancer survivor is a person diagnosed with cancer, current or past, who is still living. originally, the medical community limited the term to denote persons who remained alive and disease - free for a minimum of five years, thus reflecting cure. today 's cancer survivors may be disease - free or not and may experience no, minor or major short- or long - term effects such as pain, sexual dysfunction, second cancers, or poor quality of life from cancer illness and/or treatment. cancer survivorship denotes the phase following primary treatment until end - of - life care or death. family members and caregivers may also be impacted by the survivorship experience and are therefore included in the survivorship definition, as are their processes related to coping and/or grief [1, 5 ]. cancer is one of the most common serious illnesses in the developed world today, and between one in three and one in two people can be expected to be diagnosed with cancer during their lifetime. cancer incidence increases with age and is consequently rising in societies where life expectancy is increasing. concurrently, the number of cancer cases is increasing due to cancer screening and improved diagnostic tests, population growth, and changes in underlying risk factors, such as environmental exposures, obesity, and physical inactivity [1, 5, 21 ]. at the same time, prognosis is improving for most cancers, and the overall five - year relative survival after cancer is above 60% in most developed countries due to early detection and improved treatment [5, 21 ]. europe has the highest number of cancer survivors close to 14 million, while there are about 12 million in canada and the usa, about 8 million in south - eastern asia, but only about 1 million in oceania [21, 22 ]. this results in an estimated population of more than 35 million cancer survivors in the developed world alone, corresponding to a population prevalence of about 3.5%. the absolute number of cancer survivors is predicted to increase nearly threefold over the next few decades, while the number of cancer deaths is expected to double. this dramatic increase will have profound implications for the individuals directly affected by cancer and for societies who attend to their needs. cancer incidence and cause - of - death registration is essential for identifying cancer survivors and for understanding the complex interplay. in many parts of the developing world (e.g., most of africa) resources for cancer registration are not available. less than 20% of the world 's population is covered by cancer registries and around 30% by mortality registration, and the total number of cancer survivors in the world is thus unknown. research on cancer survivorship is currently primarily undertaken in countries with adequate cancer surveillance and sufficient resources available to care for persons after a diagnosis has been made. cancer survivorship research is currently conducted in many ways, reflecting the complexities of the subject matter. like in other areas of health research data, data may be collected prospectively or retrospectively, and both objective and subjective [10, 24 ] measures of exposures and outcomes are relevant. the data sources span from administrative records collected for other purposes such as billing, to hospital, state and national cancer registry information [11, 12, 26 ], clinical trials [27, 28 ], as well as information from questionnaires, interviews, or observation [12, 18 ]. in certain instances, more precise answers and further tests may be required from subjects, including biological samples (e.g., blood, urine, saliva) or physical stress tests or tests to measure cognitive performance [29, 30 ], as well as different imaging techniques. the majority of studies to date have included only cancer survivors, but some questions are impossible to answer without noncancer comparison groups [10, 19, 25 ]. essential components of cancer survivorship research are the study of short - term, long - term, and late organic and somatic effects following cancer, treatment, and cancer control interventions. the most commonly reported long- and short - term physical complaints after cancer are pain, digestive problems, neuropathies in the extremities, cardiovascular problems, immune deficiency, anemia, lymphedema, impaired vision due to cataracts, impaired mobility, and fatigue [10, 32 ]. research has focused on adverse effects of cancer treatment on cognitive functioning such as memory, attention, and concentration. a relatively new avenue of research focuses on the economic and legal issues, particularly the economic sequelae of cancer and cancer treatment on individuals, families, and societies [15, 35, 36 ]. cancer survivorship research also includes the promotion of healthy lifestyles and behaviors studying the effects of interventions to reduce smoking, increase physical fitness, and reduce weight. benefits of having survived cancer have been identified through research, that is, posttraumatic growth [24, 38 ]. research on the role of families on survivors ' health - related and psychosocial outcomes as well as cancer survivorship 's impact on health and wellbeing on family units, members, and caregivers is also emerging [12, 39 ]. lastly, issues related to followup care, access, and health care delivery are in the forefront, with concerns about disparities related to poverty - related or other sociodemographic patient characteristics, with a focus on underserved populations [4042 ]. in cancer survivorship research, diverse information must be collected and assessed over extended time periods and from multiple sources, including the patient, family members, caregivers, clinics, health care systems, and policy. this is both practically and methodologically difficult and costly. as is the case for many emerging research fields, the challenges facing the field of cancer survivorship research are both substantive and methodological. one important substantive challenge is to decide which research questions to ask and what hypotheses to test in this relatively new field. economic, legal, social, physical, spiritual, emotional aspects, among others, have been identified as content areas, but the relative importance placed on each of these may vary between cultures because cultures view and value illness, treatments, and caregiving differently [20, 42, 43 ]. are there common beliefs and attitudes among different cultures that can be studied to benefit multiple societies ? what research questions and findings transfer across societies ? which questions must be answered within subcultures due to large contrasts between cultures or societies ? the answers to these questions impact on how limited research funding can be distributed to optimize the knowledge gained and thus augment cancer survivorship care, cross - culturally. we must learn how to best measure the effects of surviving cancer and how to increase overall and subgroup research participation and followup. to be efficient we need to use existing data sources while identifying new sources, and apply existing research methods, while designing and testing new methods. assessing effects from multiple sources and over time may call for new and advanced research methods. researchers with expertise in research design, measurement, and analyses must be included. finally, when considering design and methods we must keep in the forefront the need to review costs and benefits as they relate to the generalizability of the findings to different populations. the application of research findings to individuals and subgroups across different societies requires an understanding of the substantive concerns of each culture and may thus need to rely on study designs and methods that are acceptable across cultures. these issues are also involved in how one disseminates and uses the results to improve cancer care across different societies. figure 1 exemplifies challenges of either substantive or methodological character and also provides an example of an area where there is overlap. the substantive and methodological challenges described above may exist within one culture. as we proceed to describe challenges across cultures, broadly defined to include diverse countries, societies, or groups within these that share distinct features, matters become more complicated. different cultures may face distinctive substantive challenges. also methodological challenges may in part vary across cultures, given different infrastructures, data availability, and legal restrictions, whereas other methodological challenges may have more general relevance. across cultures, one example of a substantive challenge could be which research questions and findings may be transferable from one culture to another ?, as is shown on the left - hand side in figure 2. on the right - hand side in this figure, an example of a methodological challenge could be how to make data comparable across studies to facilitate larger samples and validity of results in universal versus specific contexts ? an example of a challenge that cuts across this categorization could be how to design studies that may generalize to diverse populations in different societies ? where more specifically one 's concern could be how to ensure that the data collected represent similar constructs and features across societies. equally important is to recognize what cultural aspects are so unique that they can not be compared across cultures. each entails components of both substantive and methodological issues, and we believe that all need to be adequately addressed for cancer survivorship research to move forward. the second challenge concerns how one may assure that what is being measured is what one wants to measure across cultures lastly, the challenge of securing nondifferential participation cross - culturally, initially, and during followup will be addressed. certain research questions are important to ask cross - culturally, whereas others may be adequately addressed within (sub)cultures. research hypotheses, questions, and findings that generalize to other societies and thus translate to larger / diverse populations need not be replicated to the same extent as those that are culturally specific. distinguishing between unnecessary and important replications is essential to minimize study costs and ensure optimal outcomes for cancer survivors and requires knowledge and insight into the different cultures, systems, and societies. certain phenomena might need to be studied in different cultures and at various levels to determine whether questions pertain cross - culturally and across levels. examples of study questions that might be valid across cultures include for instance the presence of short, long, and late physical effects following cancer and its treatment, as there is little reason to believe that for instance drug side effects, late effects of radiation, or the prevalence of second cancers differ markedly between cultures unless treatment regimens have been substantially different [10, 26, 32 ]. this was in part the case for pediatric cancer treatment regimens in europe and the usa a few decades ago, and may have contributed to differences in the type and prevalence of somatic late effects across regions. perceptions and implications of these late effects, may, however, differ vastly between cultures, depending on health and welfare systems, expectations and existing norms [16, 17, 46, 47 ]. an example of a question that is less likely to be valid across cultures would be one that addresses economic sequelae of cancer. both the direct costs associated with becoming ill and the indirect costs associated with illness - related work absence are likely to vary as health insurance, followup care, access, and health care delivery differ across cultures [15, 18, 48, 49 ]. research on disparities in this area tends to show that the least well off tend to fare the poorest, and poverty or low socioeconomic resources thus becomes one possible target where differential care or followup may be necessary [4042 ]. at the community level, one might ask whether results from one hospital are likely to pertain to different hospitals, and similarly for regions and nations. for instance, is research conducted in the usa relevant in european countries and vice versa ? cultural differences in employment behavior, perhaps driven by differences in health and welfare systems, between the usa and norway are highlighted in this example where we compare employment after cancer. norway provides universal access to cancer care without costs to individuals, regardless of employment status and a fair percentage stop working after their cancer diagnosis. in the usa, employment rates remain relatively high after cancer, perhaps in part because many who are ineligible for medicaid or medicare have their health insurance linked to their employment or the employment of their spouse, frequently with large deductibles and copayments that can forestall treatment and bankrupt families. further, us breast cancer patients with health insurance tied to their own employment have been found to be more likely to work than those who are covered through their spouse. this would be an irrelevant study question in norway and other welfare states with universal health care. likewise may cross - cultural and international comparisons facilitate discussions of the relative importance of organizational structures across systems and disclose effects that could be modified within systems. the aboriginal people in australia have, for instance, higher cancer mortality than the nonindigenous population, and a study has identified transportation and accommodation problems, travel and health care expenses, removal from family, and lack of appropriate support persons as important factors for treatment decisions and access. the challenge of securing construct validity and thus contributing to internal validity is present within societies, and even more so across societies. how well we are able to capture the exposures and the outcomes that we want to assess is of outmost importance for our results and conclusions. physical consequences of cancer and its treatment can be measures both subjectively and objectively, but perceptions of physical consequences or psychosocial issues may only be subjectively reported. the exact relationship between these different types of measures and outcomes has not yet been established within the field of cancer survivorship research. to what degree these measurements capture what they aim to, within or across cultures, remains largely unknown. because this new research area is in a rush to find results, some studies have performed validation and reported outcomes simultaneously. it is, however, important to distinguish scale validation research from studies that attempt to provide information on outcomes as scale development moves forward [5355 ], and it is also important to clarify the extent to which scales are likely to be valid across cultures. to further exemplify, the study of physical late effects and psychosocial consequences requires monitoring of large and diverse groups of cancer survivors as the individual adverse events are relatively rare (i.e., second cancers or divorce) and occur at various points in time from diagnosis [25, 26 ]. furthermore, some late effects are relevant only for subgroups of survivors, such as females who received radiation to the chest area for non - hodgkin 's lymphoma during their reproductive years and later developed breast cancer. in these instances, cross - cultural studies may enable a pooling of study subjects and thus provide sufficient power to answer more questions, as has been done in for instance the after breast cancer pooling project, established to examine the role of physical activity, adiposity, dietary factors, supplement use, and quality of life in breast cancer prognosis among us and chinese survivors. in some instances, for instance could pooling of female cancer survivors ' employment data from cultures with vastly different female employment patterns yield results that are representative for neither culture. in addition, there is a lack of evidence - based guidelines for which tests to use and when for surveillance, complicating research measurements. as it is difficult to distinguish effects of normal aging, lifestyle behaviors, and comorbidity from that related to cancer, comparable control subjects need to be assessed and followed simultaneously. at present there is a scarcity of prospectively collected data within most cultures, which limits the possibility to draw valid conclusions about causality. on the other hand, prospective studies might lock the data collection to measurement techniques that become obsolete during the observation period. in addition, new statistical methods may be needed to account for sequences of events and the longitudinal nature of late and long - term effects. an equally or perhaps more important aspect pertains to the time span in question. due to the late onset of adverse events, information that becomes available is in many instances the consequences of yesterday 's treatment regimens, as advances take place and protocols are updated regularly. the pace may, however, vary across cultures, which may further hamper comparative research efforts. the information obtained may be outdated, although relevant to the longer - term survivors exposed in the past. new treatments are often in part based on chemical compounds used earlier, and although not identical perhaps similar enough that conclusions may be drawn nonetheless. this has resulted in an increased focus on the potential role of intermediate outcomes. with rapid improvements in diagnostic procedures and treatment regimens sophisticated mathematical modeling can predict outcomes before they actually occur, and thus allow for appropriate interventions in advance. intermediate outcomes may also help in determining the relative importance of different treatment regimens versus earlier diagnosis, helping to determine the value of screening. cancer survivorship research thus benefits from multiple measurement techniques and designs because various research methods have the potential for providing diverse insights, within as well as across cultures. previous studies in the field of cancer survivorship have by and large utilized information provided by affected individuals through interviews or questionnaires. unfortunately, response rates have been relatively low and not representative of the entire population of survivors [1, 58 ]. participation and response rates are in general related to many of the exposures and outcomes of interest in cancer survivorship research, and are likely to differ within and across cultures [27, 28, 58 ]. in studies based on membership in groups, organizations, or catchment areas surprisingly, not all cancer survivors are aware of or accurately report their diagnosis when asked specifically to do so [60, 61 ]. these individuals are unlikely to participate in studies that are not registry - based and tend to have different characteristics than those who report their diagnosis. furthermore, similar baseline characteristics among responders and nonresponders have been shown to be insufficient evidence of nonresponse bias, as the two groups experience different long - term outcome rates. in general, participation rates are on the decline today, across studies and in most societies [27, 63 ]. with high social mobility in many western countries, recent administrative and legal changes across countries have required studies to have complicated informed consent. this may increase the potential for bias, as a more complex consent process may make it more likely for healthier, more resourceful persons to participate [63, 64 ]. as study aims and measures become more sophisticated, more precise answers and more tests may be required from subjects. this can be time and energy consuming, and persons in poorer health or those experiencing adverse outcomes may be less likely to have the ability to participate [27, 64 ]. sensitive topics are, in addition, more likely to invoke differential nonresponse rates, for instance sexual preferences and/or behavior. cultural norms and expectations any or all of the factors listed above limit our ability to capture complete cohorts. this is unfortunate, as although the detailed tests, examinations, questionnaires, and/or interviews may reduce participation, they may at the same time increase construct validity for those participating. achieving a more optimal balance between the two within and across cultures is thus a challenge. cross - cultural comparisons are increasingly important to maximize the cost - benefit of conducting research without duplication of effort in cancer survivorship research. these comparisons will also help to determine the relative importance of cultural and societal structures. although many consequences of cancer may be culturally dependent, it is likely that research questions and results may be transferred to cultures with similar populations, cancer burdens, health systems, and welfare structures. how to interpret findings for populations that differ substantially in any of these respects remains unclear, and culture - specific research in these areas remain warranted. cancer survivor research encompasses the fields of clinical, behavioral, sociologic, anthropologic, economic, and health services research. we need an increased focus on genuine interdisciplinary research efforts, where persons from different backgrounds work together in teams to solve problems, thus coalescing their unique and shared experiences to create new theoretical and conceptual models. the longitudinal nature of cancer survivorship research remains challenging, as cultures ' rate of uptake of new detection methods, treatments, as well as research ideas and methods differ. current findings may reflect earlier diagnostic criteria and treatment options and not necessarily improvements in treatment or methods to simultaneously incorporate aspects of survivorship at individual, group, and societal levels, separately as well as jointly, and changes that occur within and across these levels over time are needed. as populations grow and age so will the number of cancer survivors, requiring an increased emphasis on optimal resource management. reasonable health care costs, adequate personnel and services, and equal access to care will be important for both the individual cancer patient and the greater community. it is thus pertinent that the current focus on individuals, directly or indirectly affected, shifts to also include effects at societal levels. in this effort we recommend that whenever possible, data should be made comparable across studies, cultures, regions, and systems to facilitate larger and more diverse studies and valid results in universal and specific contexts. to facilitate this, cancer registries can expand to include agreed upon factors important for survivorship as a foundation for prospective research, nationally and cross - nationally. further, analytic tools must be identified or developed that will enable us to capture needs of survivors, families, caregivers, clinicians, health and welfare policy makers and enforcers and also taking this a step further make findings at group levels relevant at an individual level for those affected and those who provide care. to address the broad aspects of survivorship we need data, methods, and study designs that can be used to compare across cultures. survivorship registries, currently being developed in norway, and in vermont, usa, and likely other places, will yield important information on the psychosocial needs and resources, use of diagnostic tests, treatments received, and risks of recurrences and morbidities, along with other relevant long - term outcomes and thus provide a knowledge base for further survivorship research. in vermont over 2000 adult cancer survivors representing nine different cancers are available to be invited into research studies. national survivorship registries for prostate and colorectal cancer which contain information on both initial and recurrent cancer and treatment have been established at the cancer registry of norway. these registries may be linked to several other national registries to obtain for instance cause of death, medication use, income, education, employment and marital status. they can also be used to identify patients to conduct more in depth studies, for example, whether persons ' quality of life after rectal cancer depend on whether or not they have a stoma. research hypotheses, questions, and findings that generalize to other larger or more diverse populations can be identified to increase the rate of new findings. to optimize data collection we must strive to increase the comparability across studies, cultures, regions, and systems to enhance validity in universal and specific contexts and thus achieve power to make valid conclusions also on negative results. the needs of survivors, families / caregivers, clinicians, health and welfare policy makers and enforcers can be identified and addressed in cross - cultural comparative research. new study designs and statistical methods will enhance all aspects of cancer survivorship research. prioritizing the expansion of cancer registries to include survivorship data elements will facilitate prospective research, nationally and cross - culturally. the relative importance of research that benefits individuals versus the larger society needs to be assessed, and findings at group levels can be made relevant at an individual level for those affected and those aiming to assist., we hope that a comparative perspective will be considered when new survivorship cohorts are established and in the planning and implementation of new survivorship studies. in cancer survivorship care cross - cultural comparisons may translate to health and welfare policy through health services research on cancer survivorship. | cancer survivorship research includes the study of physical, psychosocial, and economic consequences of cancer diagnosis and treatment among pediatric and adult cancer survivors. historically, the majority of cancer survivorship studies were from the united states, but survivorship issues are increasingly being addressed in other developed countries. cross - cultural studies remain, however, scarce. the degree to which knowledge attained may or may not be transferred across cultures, countries, or regions is not known. some important challenges for comparative research are therefore discussed in a cross - cultural perspective. several substantive and methodological challenges that complicate the execution of cross - cultural cancer survivorship research are presented with examples and discussed to facilitate comparative research efforts in the establishment of new survivorship cohorts and in the planning and implementation of survivorship studies. comparative research is one key to understanding the nature of cancer survivorship, distinguishing modifiable from nonmodifiable factors at individual, hospital, societal, and system levels and may thus guide appropriate interventions. lastly, suggested future courses of action within the field of comparative cancer survivorship research are provided. |
currently, the uniprotkb protein sequence database contains > 17 million protein sequences (1). this wealth of data is helping us to understand biology at an ever increasing rate. a large fraction of these sequences can be grouped into a few thousand common protein families. proteins within these families often share common functions that can allow information experimentally gleaned on one protein to be transferred to uncharacterized ones. this process of transitive annotation is essential to make sense of the rapidly growing amount of sequence data. there are concerns about transitive annotation not being robust and thus leading to numerous annotation errors (2). although this phenomenon does occur it seems clear that high - quality manual curation of the protein sequence databases, the careful use of databases of protein families for annotation and feedback from users of protein databases have largely kept the gross errors in check. for example, incorrect protein function assignments from large - scale genome projects in general have not been transferred to hundreds or thousands of other proteins as feared. on the other hand, subtler misannotations such as assigning an incorrect but related enzymatic activity to a protein (for example phosphorylating the wrong substrate) occur. due to the lack of experimental work on most proteins a recent estimate for six large enzyme superfamilies studied suggested a range of 563% of incorrect annotations (3). a further source of error in the sequence databases is the prediction of spurious genes (4). automatic gene prediction methods in prokaryotes are increasingly accurate, glimmer3, for example, both improves start site prediction relative to glimmer2 and reduces the high false - positive rate for high gc genomes. however, given the large number of proteins being deposited in the sequence databases, it is still likely that many thousands of the included sequences are either wholly spurious or improperly extended, past their true start sites. as the capacity to manually curate gene predictions diminishes, it has been noted that certain alternate reading frames seem more likely to give rise to long spurious open reading frames (orfs) (6). normark. (7) found that frames + 3 and 1 were most likely to give rise to long spurious orfs. although alternative overlapping reading frames are used in viral genomes, there are relatively few confirmed cases found in prokaryotes or eukaryotes. during the construction of the pfam database of protein families (8), we have occasionally been alerted to the presence of families that were entirely composed of spuriously predicted orfs. once one gene has been spuriously predicted and put in the sequence database, there is a danger that future genome projects will annotate new protein - coding genes by similarity to the first spurious orf. this can lead to entire families of spurious orfs. in the worst - case scenario, (9) where a spuriously predicted gene on the opposite strand of ribosomal rna had been given the incorrect function of cell wall hydrolase (pf10695). it may seem surprising that spuriously predicted orfs would appear to have conservation like bona fide proteins. however, at the protein level the alignment of spurious orfs can look like a normal protein alignment. in figure 1, we show the multiple sequence alignment for former pfam family pf10695, showing a protein - like conservation pattern. this conservation is actually due to the selective forces conserving the opposite strand rrna sequence and structure. once these errors are propagated to pfam and other databases, then there is a danger that the error will be widely transferred and hence difficult to correct. the first example (figure 2a) shows a pair of proteins with correctly identified homology domains but with an uncharacteristically long tail - to - tail overlap. the second is an example of a hidden markov model (hmm)-based domain definition identifying a region in a spurious gene call that overlaps a true gene (figure 2b). figure 1.seed alignment for the antifam family derived from pf10695. amino acids are colored by average similarity according to the blosum62 amino acid substitution matrix from most similar (light blue) to less similar (gray). s and e in the first row stand for sequence start and sequence end, respectively. (a) shows two proteins from the corynebacterium efficiens genome that encode components of a restriction system. (b) two highly overlapping predicted proteins from the rhodopirellula baltica genome coded on opposite strands of dna. the q7uy10 protein contains two pfam duf1596 domains. green boxes represent regions matched by pfam families, the red shaded areas represent transmembrane domains predicted by phobius (10) and the blue shaded areas represent regions of low complexity (11). amino acids are colored by average similarity according to the blosum62 amino acid substitution matrix from most similar (light blue) to less similar (gray). s and e in the first row stand for sequence start and sequence end, respectively. (a) shows two proteins from the corynebacterium efficiens genome that encode components of a restriction system. (b) two highly overlapping predicted proteins from the rhodopirellula baltica genome coded on opposite strands of dna. green boxes represent regions matched by pfam families, the red shaded areas represent transmembrane domains predicted by phobius (10) and the blue shaded areas represent regions of low complexity (11). antifam matches to predicted proteins in some cases will suggest that modifications to the extent of the coding region are needed rather than complete deletion of the protein from the sequence database. most prfb genes, encoding the bacterial translation release factor 2, have a + 1 programmed frameshift early in the coding region (12). the region downstream of the frameshift site is easily identified by gene finders, but unreconstructed extension 5 to the frameshift results in translation of the wrong reading frame. some of the multiple sequence alignments used are taken from the pfam database seed alignments for families identified as spurious orfs. these alignments are kept as they appeared in the final release of pfam before they were withdrawn (table 1). several additional custom families have been created to identify other commonly recurring spurious orfs (table 2). the profile hmms have been constructed using the hmmer3 package with default parameters (13). the profile hmm library can be searched against any set of protein sequences using the due to the speed of the hmmer3 package, searching a sequence database such as uniprotkb will take a few minutes and searching a complete proteome will take seconds. table 1.antifam entries derived from pfam familiespfam accession number (identifier)last pfam release presentreason for deleting from pfamno. of matches in uniprotno. of matches in metagenomics data evidence for homology to known protein on opposite strand30pf07616 (duf1578)15.0proteins may not be expressed. evidence for homology to known protein on opposite strand66pf07630 (duf1591)15.0proteins may not be expressed. evidence for homology to known protein on opposite strand60pf07633 (duf1594)15.0proteins may not be expressed. evidence for homology to known protein on opposite strand50pf11370 (duf3170)25.0probable shadow orf of clp protease167pf11194 (duf2825)25.0probable crispr repeat regions15918pf11664 (duf3264)25.0probable crispr repeat regions2113pf10695 (cw - hydrolase)25.0antisense to rrna (9)2251,654pf10919 (duf2699)26.0shadow orf of pf00665 (integrase core domain 1)2511pf07641 (duf1596)26.0dubious genome annotation. family comprises only three sequences from rhodopirellula baltica, two overlapping30the final two columns show the number of matches of each antifam entry to uniprotkb and to a metagenomic data set.the metagenomic set of sequences is the same as that used by pfam (14).crispr, clustered regularly interspaced short palindromic repeats. table 2.antifam entries derived from custom multiple sequence alignmentidentifiertype of spurious familyno. of matches in uniprotno. of matches in metagenomics data setspurious_orf_10translated bacterial trna, trna01196795spurious_orf_11translated bacterial trna, trna0289170spurious_orf_12translated bacterial trna, trna03143408spurious_orf_13translated bacterial trna, trna0477671spurious_orf_14translated bacterial trna, trna05156191spurious_orf_15translated bacterial trna, trna063163spurious_orf_16translated bacterial trna, trna074017spurious_orf_17translated bacterial trna, trna08510spurious_orf_18translated bacterial trna, trna09439spurious_orf_19translated bacterial trna, trna10712spurious_orf_20translated bacterial trna, trna114328spurious_orf_21prfb frameshift245spurious_orf_22from a lncrna, linc00174261the metagenomic set of sequences is the same as that used by pfam (14). antifam entries derived from pfam families the final two columns show the number of matches of each antifam entry to uniprotkb and to a metagenomic data set. the metagenomic set of sequences is the same as that used by pfam (14). antifam entries derived from custom multiple sequence alignment the metagenomic set of sequences is the same as that used by pfam (14). antifam is primarily a tool that is aimed at bioinformaticians to be used as part of genome annotation projects. the antifam alignments and profile hmms can be downloaded from the following url : ftp://ftp.sanger.ac.uk/pub/databases/pfam/antifam/ of the 1310 proteins identified in uniprotkb as probably being spurious the large majority were from trembl, the unreviewed part of uniprotkb. they had been automatically created from the records in the european nucleotide archive, genbank or dna data bank of japan (ddbj). one spurious protein found in the reviewed swiss - prot section of uniprotkb was y114_chlmu (q9pli5) that is an uncharacterized protein from chlamydia muridarum. this belonged to the previously mentioned spurious cw - hydrolase family and was removed in uniprot release 2011_10. an additional 13 spurious proteins in the reviewed portion of uniprotkb are also identified, of which 8 are due to non - coding rna translations : o67358.1 aquifex aeolicus trigger factor contains frameshift extension;p19773.1 mycobacterium tuberculosis protein matching duf2699;p47080.1 yeast protein yjl007c product of a dubious gene prediction;p92540.1 arabidopsis protein;q04100.1 yeast protein ydr445c product of dubious gene prediction and partly overlaps ydr444w;q52m62.3 human product of a dubious coding sequence (cds) prediction. o67358.1 aquifex aeolicus trigger factor contains frameshift extension ; p19773.1 mycobacterium tuberculosis protein matching duf2699 ; p47080.1 yeast protein yjl007c product of a dubious gene prediction ; p92540.1 arabidopsis protein ; q04100.1 yeast protein ydr445c product of dubious gene prediction and partly overlaps ydr444w ; q52m62.3 human product of a dubious coding sequence (cds) prediction. probable non - coding rna ; q9cjr2.1 pasteurella multocida trna - derived match ; q9cmd0.1 p. multocida trna - derived match ; q9cmx0.1 p. multocida trna - derived match ; and q9cmz6.1 p. in addition to the families reported by pfam users, we tried to identify if further spurious families existed. the large majority of proteins in the trembl portion of uniprotkb come from translations found in entries in the european nucleotide archive, genbank or ddbj. thus, we scanned trembl entries to identify uniprotkb entries that overlapped with each other in the nucleotide entry. we confined our scan to the prokaryotic entries because the nature of overlaps is relatively simple compared to the complex patterns of interlacing and nesting found in eukaryotic gene structures. this list of proteins was then used to identify further pfam families that contained numerous overlapping genes. this measure means that large well - known families that are likely to have many overlaps by chance are not at the top of the list. the first release of antifam contains only a modest number of families. however, we see a number of ways to increase this in the future. the first of these is to increase the number of non - coding rna - based families. we currently have only one ribosomal rna - based family and we can add many further families. we can identify proteins related to ribosomal rnas initially using tblastn, which compares a protein to a nucleotide sequence considering all six reading frames. in addition, we could also consider comparing a large database of rna sequences to the protein sequence databases to identify further potentially spurious proteins. to date, we have only been able to investigate the pfam families with the highest fraction of overlapping proteins. but in the coming months, we will investigate this list more thoroughly to identify if any further pfam families should be deleted and added to antifam. the first release of antifam contains 23 families derived from pfam as well as a small number of non - coding rnas that were erroneously translated into protein sequences. we expect that this number will grow in the future and we have several ideas to help us to achieve this. this should increase the power of antifam to reduce the number of spurious orfs finding their way into the sequence databases. we hope that antifam will become an indispensible tool for quality control in metagenomic and genomic studies. we are particularly keen for biocurators and experimental biologists to remain vigilant and alert us to new cases of spurious orfs so that we can add them to this resource. wellcome trust (grant number wt077044/z/05/z) ; national human genome research institute (grant number r01 hg004881). funding for open access charge : wellcome trust (grant number wt077044/z/05/z). | as the deluge of genomic dna sequence grows the fraction of protein sequences that have been manually curated falls. in turn, as the number of laboratories with the ability to sequence genomes in a high - throughput manner grows, the informatics capability of those labs to accurately identify and annotate all genes within a genome may often be lacking. these issues have led to fears about transitive annotation errors making sequence databases less reliable. during the lifetime of the pfam protein families database a number of protein families have been built, which were later identified as composed solely of spurious open reading frames (orfs) either on the opposite strand or in a different, overlapping reading frame with respect to the true protein - coding or non - coding rna gene. these families were deleted and are no longer available in pfam. however, we realized that these may perform a useful function to identify new spurious orfs. we have collected these families together in antifam along with additional custom - made families of spurious orfs. this resource currently contains 23 families that identified 1310 spurious proteins in uniprotkb and a further 4119 spurious proteins in a collection of metagenomic sequences. uniprot has adopted antifam as a part of the uniprotkb quality control process and will investigate these spurious proteins for exclusion. |
luminescent metal complexes are increasingly employed as biomolecular and cellular probes due to their unique photophysical properties that make them particularly well suited to monitor biological processes. examples include not only complexes of transition metals such as pt, ir, and ru but also those that incorporate lanthanides, most commonly eu and tb. in particular, the long luminescence lifetime of the emitting metal mitigates the interference of background fluorescence originating from the biological sample. moreover, since the large energy difference between the absorbing and emissive states of these complexes removes self - absorption issues, their luminescence signal intensity is proportional to their concentration over a wide range of values. with this in mind, lanthanide - based molecular probes have been applied to the detection of reactive oxygen species (ros), redox - active metals, ph, and intracellular analytes, such as atp. the continued applications of such metal - based luminescent probes, however, further rely on the ability to limit their cellular association altogether for certain tissue imaging experiments or, alternatively, to direct them to specific cellular regions. prior studies indicate that structural properties of metal complexes, such as their hydrophobicity and size, influence their cell viability and cellular association, including their membrane permeability. although cell - penetrating peptides have been applied to increase the cell uptake of lanthanide complexes, the goal of this project is to modify the intrinsic properties of the metal complex to control cellular association. presented here is a systematic study to evaluate the effects of structural variations on a core structure common to most luminescent lanthanide probes and an investigation of the practicality of such probes for cellular and tissue applications requiring extracellular imaging agents. the most common platform of luminescent lanthanide complexes features a chelating polyaminocarboxylate ligand substituted with one or more carboxamide pendant arms from which additional moieties are attached, notably the probe s antenna. the laporte - forbidden nature of the f f transition dictates that, for practical applications in biologically relevant settings, the probe must incorporate a sensitizing antenna. in terms of the design of a responsive probe, the luminescence intensity of the lanthanide complex can then be modulated by altering, among other parameters, the excited triplet state energy level of the antenna and the antenna lanthanide distance. for biological applications, it is thus important to select an antenna with suitable energetic properties for lanthanide sensitization that is also resistant to quenching by the medium. work by parker and co - workers identified that the nature of the antenna, specifically its point of attachment to the cyclen backbone of the ligand, affects the subcellular localization of the complexes. given the increasing use of phenanthridine and isophthalamide antennas for the design of luminescent lanthanide probes, a deeper understanding of the structural parameters influencing their effect on cellular viability and cell association is necessary to further optimize their structures for biological application both extra- and intracellularly. herein, two parameters were evaluated for their effect on cell viability and association : the nature of the antenna and that of the pendant amide arms. the macrocyclic polyaminocarboxylate ligand provides kinetic inertness and thus predicted lower toxicity on cell viability. furthermore, the overall positively charged lanthanide complexes of dota tetraamide (dotam) ligands are kinetically more inert than the dota analogues as a result of the decreased basicity of the nitrogen atoms. moreover, the macrocyclic framework is less susceptible to enzymatic degradation. the high kinetic inertness of macrocyclic lanthanide complexes and their ability to resist degradation is critical for their use in vivo. three different antennas were investigated : the hydrophilic 2-hydroxyisophthalamide (iam), a reported sensitizer of both tb and eu, the closely related and synthetically more facile 2-methoxyisophthalamide (iam(ome)), and the extended aromatic 6-methylphenanthridine (phen) (chart 1). for each complex, the quantum yield, cellular viability, and cell association were evaluated. the effect of charge and hydrophobicity on cell viability and association was subsequently evaluated with complexes comprising the iam(ome) antenna functionalized with varying carboxamide arms (chart 2). the isophthalamide complexes tb - dota - iam(ome) ([tb-1 ]) and tb - dota - iam ([tb-2 ]) were synthesized according to scheme 1. briefly, the oxidation of bis(hydroxymethyl)-p - cresol with mno2 and koh generated the diacid 11. subsequent treatment with methyl iodide generated the methoxy - protected diester 12, which was then converted to the diamide 13 with methylamine. the aldehyde 14 was generated via a riley oxidation of the aryl methyl group in naphthalene at 215 c. periodinane oxidation methods (dess martin and 2-iodoxybenzoic acid), permananganates, and chromates (jones reagent and pcc) were unsuccessful mediators of this transformation. reaction of the aldehyde 14 with hydroxylamine hydrochloride generated the oxime 15, which was crystallized by addition of diethyl ether to an acidic solution of the crude reaction mixture in ethyl acetate and methanol and then reduced to the amine form of the iam antenna 16. the tris - boc - protected cyclen 17 was synthesized as reported and further coupled to the amine 16 using hatu as the activating agent. boc deprotection in a hydrochloric acid / methanol mixture afforded the iam(ome) cyclen conjugate 19, which served as a common intermediate in the synthesis of complexes [tb-1 ], [tb-2 ], and [tb-4][tb-10 ]. reagents and conditions : (a) mno2, ch3cl, 60 c, 20 h ; (b) koh (s), 230 c, 1 h ; (c) ch3i, k2co3, acetone, 56 c, 16 h ; (d) nh2ch3, ch3oh, 65 c, 18 h ; (e) seo2, naphthalene, 215 c, 2.5 h ; (f) hydroxylamine hydrochloride, pyridine, 22 c, 24 h ; (g) pd / c (10%), hcl, ethanol / h2o, 5 bar, 16 h ; (h) hatu, dipea, dmf, 22 c, 40 h ; (i) hcl, ch3oh, 22 c, 18 h ; (j) cs2co3, ch3cn, 40 c, 18 h ; (k) hcl, ch3oh, 22 c, 18 h ; (l) bbr3, ch2cl2, 25 c, 18 h ; (m) ch3oh, 65 c, 16 h ; (n) tbcl3, h2o / ch3oh, ph 7, 45 c, 48 h ; (o) 0.2 m koh, h2o, 22 c, 20 h. alkylation of the cyclen derivative 19 with tert - butyl bromoacetate yielded the protected ligand 20 (scheme 1). deprotection in a hydrochloric acid / methanol mixture yielded the free ligand dota - iam(ome) (1). harsher deprotection conditions with boron tribromide removed both the tert - butyl groups and the aryl methyl ether to yield dota - iam (2). the final complexes [tb-1 ] and [tb-2 ] were obtained from their respective ligands, 1 and 2, by heating with tbcl3 in aqueous solutions at neutral to slightly basic ph for 2 days or more. the phenathridine complex [tb-3 ] was synthesized according to the published procedure. in order to evaluate the role of charge and hydrophobicity of the lanthanide complex in its cellular compatibility, seven derivatives of tb - dota - iam(ome) ([tb-1 ]) were synthesized (chart 2 and scheme 1). the synthesis was designed to take advantage of the common intermediate 19, described above. each bromoacetamide arm was synthesized using a biphasic method in which bromoacetyl bromide in dichloromethane and aqueous potassium carbonate were simultaneously added dropwise to a solution of the corresponding amine in dichloromethane at 0 c. this method resulted in moderate to high yields (6585%) of the desired products in high purity. the ligands were obtained upon reaction of the bromoacetamide arms with the intermediate 19. the carboxylic acid derivatives [tb-9 ] and [tb-10 ] were obtained by saponification of their corresponding esters [tb-7 ] and [tb-8 ], respectively. the nature of the sensitizing antenna of a lanthanide complex can affect its function in a biological environment. here, we compare [tb-1 ], [tb-2 ], and [tb-3 ], three complexes that feature either an isophthalamide- or phenanthridine - based antenna for sensitizing the lanthanide emissions (chart 1). all three complexes luminesce in the green region of the visible spectrum, with maximum emission at 545 nm upon excitation at 350 nm (figure s1 (supporting information)). these three antenna platforms have different properties in terms of hydrophobicity and interactions with biomolecules, including nucleic acids, reducing agents, and proteins. the hydrophilic isophthalamide and methoxyisophthalamide are composed of a single aromatic ring and have no known interactions with biomolecules, while the phenanthridine is an extended aromatic system capable of base stacking with nucleotides and dna intercalation. the quantum yield,, of the three tb complexes in pbs was investigated by comparing the luminescence intensity of each complex to that of a quinine sulfate standard in 0.1 m sulfuric acid (r = 0.577) according to the optically dilute method. at ph 7.8 and 22 c, the quantum yields of [tb-1 ] and [tb-3 ] are statistically identical (4.6 1.2% vs 4.7 2.2%, respectively). the quantum yield of the phenathridine derivative [tb-3 ] is comparable to that of similar complexes featuring either triamide- or triphosphinate - substituted dota ligands. however, [tb-2 ] is a more efficient emitter with an average quantum yield of 9.8 1.5%, indicating that substituting the methoxy for the hydroxyl group on the isophthalamide antenna substantially affects the quantum yield of the complex. 50% have been reported by raymond and co - workers for tb complexes with tetra - iam - substituted ligands. in contrast with the case for [tb-2 ], in the tetra - iam complex h(2,2)-iam the isophthalamide directly coordinates the lanthanide, thereby enabling a dexter - type energy transfer from the antenna to the tb that is much more efficient than the frster - type, through - space mechanism likely taking place in [tb-2 ]. on the other hand, [tb-2 ] has substantially higher water solubility (m range) than the cryptand h(2,2)-iam (m range) and enables the design of responsive probes. nonetheless, the quantum yields of [tb-1 ], [tb-2 ], and [tb-3 ] are comparable to those of luminescent d - block compounds used for cellular imaging. for example, the quantum yields of ir complexes featuring 2-phenylpyridine ligands are between 4 and 8% in pbs buffer. not surprisingly, the quantum yields of tb - centered luminescence are lower than those of commonly used fluorescence dyes such as cy3 (4% in pbs), cy5 (30% in pbs), alexa750 (12% in phosphate buffer), and texas red (30% in water). however, in comparison with phosphorescent metal complexes, organic dyes suffer from short fluorescence lifetimes and small stokes shifts. as for any luminescent or fluorescent probe, emission is susceptible to quenching by reducing agents, proteins, and other components of biological media. in the case of luminescent tb and eu complexes, the quenching pathways can involve electron or charge transfer from the excited states to intracellular reducing agents such as urate, ascorbate, and glutathione that are present in mm concentrations in cells. in addition, luminescence quenching by proteins, such as human serum albumin, has been reported. the degree of luminescence quenching was evaluated by measuring the time - delayed luminescence intensity of [tb-1 ] with respect to its concentration in pbs and whole cell lysate at two different protein concentrations. in whole cell lysate containing 0.25 mg of protein / ml, the luminescence intensity of [tb-1 ] is quenched by 22 2.8% ; at 0.5 mg of protein / ml the luminescence intensity is reduced by 30 2.4% (table 1 and figure s2 (supporting information)). advantageously, the isophthalamide antenna is less susceptible to quenching than complexes containing electron - poor sensitizing moieties. for instance, the metal - centered emission of terbium azaxanthone complexes is quenched by 3060% in the presence of 0.2 mm human serum albumin. experimental conditions : [tb-1 ] = 015 m, pbs, ph 7.8, time delay 0.1 ms, excitation wavelength 345 nm, slit widths (excitation and emission) 5 nm, integrated emission intensity from 470 to 635 nm, t = 20 c. whole cell lysate was generated from l6 myoblasts ; the protein concentration was determined by a bca assay. percent quenching was calculated using the percent change in the slope of the integrated luminescence intensity versus the [tb-1 ] (m) plots (figure s2 (supporting information)). the effect of [tb-1 ], [tb-2 ], and [tb-3 ] on the viability of l6 rat muscle myoblast cells was investigated using an mtt assay. it was hypothesized that the 2-methoxyisophthalamide would have a reduced effect on cell viability due to the protecting group on the phenol that could block nonspecific interactions with the cell proteins and coordination of cellular metals, such as iron and copper. additionally, the phenathridine complex [tb-3 ] was expected to have a greater effect on cell viability due to its ability to intercalate dna and rna. following a 24 h incubation with 0300 m tb complex, the phenanthridine derivative [tb-3 ] decreased cell viability to 74% at 100 m and to 63% at 300 m. on the other hand, the similar profiles indicate that the methoxy protecting group on the isophthalamide does not significantly improve cell viability at the doses studied here. this is in contrast to a pair of rh probes chelated by chrysenequinone diimine and n - functionalized dipyridylamine ligands, for which small structural changes were shown to have a substantial impact on cell viability. in that case, altering the ethanol substituent to a propyl reduces the viability of hct116n and hct116o cells from 80% to 60% after a 24 h incubation with 40 m complex. obtained by an mtt assay after a 24 h incubation with 100 m complex. results are mean sd (n = 3). determined by icp - ms after a 4 h incubation with 50 m complex. viability (24 h) of l6 myoblasts treated with 0300 m [tb-1 ] (solid squares), [tb-2 ] (open circles), and [tb-3 ] (solid triangles) as determined with an mtt assay. overall, the complexes in this study influence cell viability slightly less than complexes containing tetraazatriphenylene or azaxanthone antennas with phenyl or ethyl ester substituted carboxamide arms, which have ed50 values in the 100 m to > 240 m range. they affect cell viability similarly to ru(iii) polypyridine complexes, which also have ed50 (72 h) values 300 m in mcf-7 and ht-29 cell lines. it is interesting to note that the europium compound of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid containing no antenna moiety, [eu - dota ], has a negligible effect on cell viability from 0 to 500 m with a 72 h incubation. thus, with respect to cell viability, [tb-1 ] and [tb-2 ] are preferred for cellular imaging applications compared to [tb-3 ]. the nature of the antenna was also expected to affect the cellular association of the complexes. following a 4 h incubation with 50 m of tb complex, the cellular accumulation of the metal was determined by icp - ms and expressed relative to the protein concentration as determined by a bca assay. importantly, it should be noted that the quantitative nature of measuring cellular association of metal complexes by icp - ms is balanced by the inability of the technique to distinguish between internalized or membrane - bound complexes. it was anticipated that the extended aromatic antenna of [tb-3 ] would facilitate membrane permeability, as suggested by the increased cellular uptake of more lipophilic ru complexes and phenanthridine - substituted cisplatin. however, the cell association values of the hydrophilic tb - dota - iam(ome) ([tb-1 ]) and tb - dota - iam ([tb-2 ]) are statistically not different from the values for the hydrophobic tb - dota - phen ([tb-3 ]) (table 2). small structural changes to the ligands of metal complexes have been reported to exert varied effects on the intracellular accumulation and distribution. using a group of lanthanide complexes with tetraazatriphenylene- or azaxanthone - based antennas, parker identified that how the antenna was linked to the cyclen ring affected membrane permeability and localization more than small structural modifications on the antenna itself. for the isophthalamide complexes, however, conversion from the methoxy group in [tb-1 ] to the phenolate of [tb-2 ] does affect cellular association. this observation is more in concert with the findings of barton, who measured substantial differences in cell association between an alkyl - substituted dipyridyl rh complex (705 ng of rh / mg of protein) in comparison to a complex that introduced a terminal alcohol on the alkyl group (165 ng of rh / mg of protein). the cellular association values measured for the hydroxyisophthalamide [tb-2 ] are comparable to the values reported for a tb complex with a tetraazatriphenylene antenna and phenyl - substituted amide arms, which had a maximum accumulation of 10 mol / g of protein into nih 3t3 cells after a 4 h incubation at 50 m. unfortunately, further comparisons with cellular accumulation values reported in the literature are limited due to differences in the method of data collection (icp - ms vs flow cytometry), and data presentation of metal per amount of protein or per cell. notable differences among the three metal complexes include the increased effect of [tb-3 ] on cell viability above 100 m in spite of its only modest cell association. the synthesis of [tb-2 ] requires a synthetically challenging boron tribromide deprotection to produce the 2-hydroxyisophthalamide antenna, and the resulting complex has a cellular association only slightly higher than that of its analogue [tb-1 ]. the more facile synthesis of the 2-methoxyisophthalamide of [tb-1 ] in comparison to [tb-2 ] thus became a significant factor in selecting an antenna for further studies evaluating the effects of variable arms on the cellular compatibility of lanthanide complexes. the role of charge and hydrophobicity of the polyaminocarboxylate arms on the cell viability and association of the lanthanide probes was evaluated with a library of derivatives of [tb-1 ]. complexes [tb-4][tb-10 ] share a common cyclen backbone with a 2-methoxyisophthalamide antenna but differ in the nature of the remaining substituents (chart 2). the alkyl, benzyl, and trifluoro groups impart increasing hydrophobicity to the complexes, whereas alanine and glutamine amino acid based substituents in either an ethyl ester or carboxylic acid form render greater hydrophilicity. the resulting complexes also differ in overall complex charge ; neutral carboxamide arms generate the 3 + charged complexes [tb-4][tb-8 ], the addition of monoanionic arms forms the zwitterionic complex [tb-9 ], and the dianionic glutamate arm creates [tb-10 ] bearing a net 3 charge. as with the previous complexes that differ with respect to the antenna, the cell viability (24 h) was measured for the library of complexes featuring pendant arms with structural and electronic variations. of the hydrophobic complexes, only [tb-5 ] decreases cell viability significantly to 60%, while the values for [tb-4 ] and [tb-6 ] are comparable to that of the parent complex [tb-1 ], maintaining a viability above 80% in the concentration range measured (figure 2a and table 2). each of the amino acid derivatives [tb-7 ], [tb-8 ], [tb-9 ], and [tb-10 ] also sustain viability above 75% even in the presence of 300 m tb complex (figure 2b and table 2). interestingly, the notable reduction in cell viability upon addition of medium - length alkyl chains matches the results of a previous study comparing a family of ir(iii) cyclometalated probes. in that study, the ir complexes featuring the shortest (c2) and longest (c18) alkyl chains had comparably minimal effects on hela cell viability (ed50 15 m), while the median length alkyl chain (c10) was significantly more cytotoxic (ed50 = 2 m). the hexyl alkyl derivative [tb-5 ] excluded, the relatively low effect on cell viability (ed50 values > 300 m) of this entire class of tb complexes suggests they have only limited interactions with cellular components and bodes well for their application. viability (24 h) of l6 myoblasts treated with 0300 m tb complexes as determined with an mtt assay : (a) [tb-1 ] (solid squares), [tb-4 ] (solid triangles), [tb-5 ] (open circles), and [tb-6 ] (open inverted triangles) ; (b) [tb-7 ] (solid squares), [tb-8 ] (solid triangles), [tb-9 ] (open circles), and [tb-10 ] (open inverted triangles). the role of the pendant arms on the cellular association of the tb complexes was also investigated by icp - ms. it was hypothesized, on the basis of the results of barton, that [tb-4 ], [tb-5 ], and [tb-6 ] would penetrate cells more efficiently due to their lipophilic substituents and cationic character that would assist transport across the membrane. indeed, higher tb accumulation is observed with the hydrophobic hexyl - substituted [tb-5 ] and trifluoro - containing [tb-6 ] than with the more hydrophilic amino acid derivatives (table 2). however, this conclusion can not be universally applied to all hydrophobic arms, since the cellular association of the benzyl - substituted [tb-4 ] is less than that of the parent complex [tb-1 ]. the alkyl - substituted ir complexes discussed previously also do not illustrate a direct relationship between hydrophobicity and cellular association. in that study, each complex investigated displayed measurable cellular association by icp - ms, but the c10 version afforded the greatest association, followed by the c2 and then the c18 derivative. thus, it is clear that the lipophilicity of the complex alone can not be used to predict the cellular association of metal complexes. of the amino acid derivatized complexes, the alanine - based [tb-7 ] and [tb-9 ] have higher cell association values than the glutamine - based compounds [tb-8 ] and [tb-10 ]. positively charged complexes containing esters are reported to have enhanced cell association in comparison to carboxylic acid derivatives, and this is observed with the slightly higher cell association values of the ethyl ester complex of alanine [tb-7 ] in comparison to the neutral acid complex [tb-9 ]. this reflects the finding that the cell association of [eu - dotam ] is 5 times that of [eu - dota ], which showed negligible cellular accumulation. interestingly, this trend is not repeated for the glutamine ethyl ester and acid complexes, [tb-8 ] and [tb-10 ], respectively, which both have low cell association values despite their large differences in overall complex charge. this is in contrast with to lanthanide complexes studied by parker in which the intracellular concentration of a 3 charged glutamate - functionalized dota with a tetraazatriphenylene antenna was approximately 3 times that of the neutral ln - dota - tetraazatriphenylene. an important conclusion is that the parameters influencing cellular association of lanthanide complexes are multifaceted and include both structural features (lipophilicity, nature of the antenna, terminal functional groups) and, to a lesser extent, overall complex charge. this study investigated the effect of structural modifications of the ligand on cell association in the absence of targeting moieties ; however, conjugating ln complexes to cell - penetrating peptides or receptor targeting groups has successfully increased the cellular uptake. progress in the field of cell - penetrating, lanthanide - based contrast agents has been reviewed and includes the use of polyarginine and tat - based peptides. this technique has also been applied to luminescent metal complexes and dual imaging agents. additional approaches to increase cellular accumulation include coupling complexes to moieties that facilitate interactions with cellular membrane receptors, transporters, or special groups such as exofacial thiols. the translation of these approaches to the luminescent macrocyclic polyaminocarboxylate complexes described herein is being further investigated by our group. the cellular association of the tb complexes was also investigated with epifluorescence microscopy. l6 myoblasts were treated with tb complex (200 m for 4 h), washed with pbs at room temperature, and fixed with formaldehyde prior to imaging. results of representative cells indicate the presence of weak cellular association for the trifluoro - substituted [tb-6 ], while the fluorescence intensity of phenanthridine - containing [tb-3 ] is comparable to that of the control cells (figure 3). the remaining tb complexes exhibit similar weak cellular fluorescence (figure s3 (supporting information)). due to reports that different staining patterns can be observed in experiments that differ only in whether cells were imaged in a live or fixed state, the cellular association of [tb-6 ] was also examined in live cells via fluorescence microscopy (figure s4 (supporting information)). in these images, an overall decrease in the fluorescence to levels comparable to those for the untreated control cells was observed. fluorescence microscopy images of representative l6 myoblasts treated with 200 m [tb-3 ] and [tb-6 ] for 4 h at 37 c : (a) fluorescence images, 60 objective, 325375 nm excitation filter, 470750 nm emission filter, 0.4 s exposure time ; (b) bright field images, 0.04 s exposure time. scale bars represent 10 m. the reduced punctate staining in live cells in comparison to fixed cells has also been observed by belitsky. however, unlike our 2-hydroxyisophthalamide complexes, lanthanide complexes featuring tetraazatriphenylene antennas containing phenyl, ester, or carboxylate substituents are taken up by cho or nih 3t3 cells and can be visualized with fluorescence microscopy regardless of the nature of their pendent arms. likewise, bnzli observed lanthanide helicates ranging in overall charge from 6 to 6 + localized to endosomes and lysosomes, consistent with endocytotic uptake mechanisms irrespective of the polarity or overall charge of the complex. regardless of the reasons, the low cell associations exhibited by this entire class of complexes render them particularly useful for monitoring extracellular species. a family of luminescent lanthanide complexes featuring different sensitizing antennas varying in charge and hydrophobicity and pendant arms of variable structural and electronic properties were synthesized. the cell viability and association of this family of complexes have been evaluated. with respect to the antenna, the 2-methoxyisophthalamide of [tb-1 ] was selected as the ideal candidate for further studies due to its synthetic ease (in comparison to [tb-2 ]), suitable quantum yield, little effect on cell viability, and modest cell association. surprisingly, the addition of hydrophobic moieties (benzyl, hexyl, and trifluoro) did not increase cell association. all of the complexes investigated minimally affect cell viability and exhibit low cellular association, regardless of the overall complex charge or relative hydrophobicity ; thus, it is presumed that it is the hydrophilic nature of the 2-methoxyisophthalamide antenna that confers the low membrane permeability of this class of compounds. thus, the probes based on the structural framework presented here are well - suited for monitoring extracellular analytes such as group i ions, polysaccharides, hormones, or other signaling molecules. unless otherwise noted, starting materials were obtained from commercial suppliers and used without further purification. water was distilled and further purified by a millipore cartridge system (resistivity 18 m). elemental analyses were performed by inductively coupled plasma mass spectrometry on a thermo scientific xseries 2 icp - ms fitted with an esi pc3 peltier cooled spray chamber, sc - fast injection loop, and sc-4 autosampler at the department of geology at the university of minnesota. samples were diluted appropriately and analyzed in the presence of a 20 ppb of in internal standard using the he / h2 collision - reaction mode. data were collected between 220 and 800 nm using a quartz cell with a path length of 10 mm. luminescence data were recorded on a varian eclipse fluorescence spectrophotometer using a quartz cell with a path length of 10 mm. the absorbance and integrated luminescence intensities of [tb-1][tb-3 ] in pbs (ph 7.8, n = 1.3345) were compared to the reference fluorescence of quinine sulfate (r = 0.577) in 0.1 m sulfuric acid (n = 1.333). absorption spectra were collected on a cary 100 bio uv visible spectrophotometer ; emission spectra were recorded on a cary eclipse fluorescence spectrophotometer. quantum yields () were calculated according to the optically dilute method using the equationwhere a is the absorbance at the excitation wavelength (), n is the refractive index, and i is the integrated intensity. all data were collected using the same spectrophotometer on the same day, resulting in a constant intensity of the excitation light (ex = 350 nm). measurements were obtained at 22 c in a quartz cell of 1 cm path length in degassed solutions using an excitation wavelength of 350 nm and excitation and emission slit widths of 10 nm for both complexes. the fluorescence response of quinine sulfate was reported as the integrated emission intensity from 365 to 665 nm ; the tb luminescence of each complex was collected with a time delay of 0.1 ms and reported as the integrated emission intensity from 470 to 688 nm. rat muscle l6 myoblast cell line was obtained from atcc (manassas, va). cells were cultured in 25 or 75 cm vented culture flasks in dulbecco s modified eagle medium (dmem) supplemented with 10% (v / v) bovine serum (bs) and 10 g of gentamicin ml at 37 c and 5% co2. the cells were maintained by splitting every 34 days before reaching confluence ; cells were rinsed with pbs, lifted using 0.25 g l typsin for 5 min, and diluted in fresh medium. all cells were propagated at low passage numbers (< 20) to maintain their myoblastic character. l6 cells were harvested from two 75 cm culture flasks grown to 85% confluence and centrifuged at 400 g for 10 min at 4 c. the resulting pellet was rinsed with ice - cold pbs (2 ml) and centrifuged. the cells were then suspended in pbs (1 ml) and transferred to a tight - fitting homogenizer (b clearance 0.00080.0022 in.). approximately 90% of the cells were ruptured after 120 strokes, as determined by cell counting with trypan blue and a hemocytometer. the total protein concentration of the whole cell lysate was determined with a bicinchoninic acid (bca) assay (bca protein assay kit, thermoscientific) using bovine serum albumin (bsa) protein standard solutions. the whole cell lysate was then diluted to a protein concentration of 1.0 mg / ml, and aliquots (450 l) were stored at 20 c for later use. an aqueous suspension of [tb-1 ] (100 m) in whole cell lysate diluted in pbs (0.25 or 0.5 mg of protein / ml) was titrated into an aqueous solution of whole cell lysate in pbs (0.25 or 0.5 mg of protein / ml). the time - delayed emission profile was recorded in the presence of 015 m of tb complex. the titration was repeated in pure pbs buffer (ph 7.8) without cell lysate. measurements were recorded with an excitation wavelength of 345 nm, a time delay of 0.1 ms, and excitation and emission slit widths of 5 nm at a temperature of 20 c. the luminescence response was reported as the integrated emission intensity from 470 to 640 nm. the percent quenching of the luminescence was calculated from the slopes of the integrated luminescence intensity versus the concentration of tb complex (m) plots in pbs and whole cell lysate at the two protein concentrations. l6 myoblasts (225 l) were plated at 4.0 10 cells ml in a 48-well cell culture plate. cells were allowed to recover 24 h, after which tb complexes in pbs (25 l) were added to the culture media (final concentrations of 0, 1, 3, 10, 30, 100, and 300 m). for the negative control, pbs (without tb complex) was used. the mtt assay was performed during the last 3 h of the compound exposure time as follows. mtt (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, 25 l of 5 mg / ml stock solution, 0.5 mg / ml final concentration) was added to the media, and the plate was returned to the cell culture incubator for 3 h. when a purple precipitate was clearly visible, 250 l of 0.04 m hcl in isopropyl alcohol was added to each well. after an incubation of 4 h at room temperature in the dark, the samples were centrifuged at 10000 g for 5 min to pellet cellular debris. for each sample, a portion of the supernatant (200 l) was transferred to a corresponding well of a 96-well plate, and the absorbance at 570 and 690 nm was recorded using a microtiter plate reader. the cell viability was calculated according to the following equation using the adjusted absorbance of the cells treated with tb complex in comparison to the control cells. the cellular association of [tb-1][tb-10 ] was investigated by seeding in triplicate l6 myoblasts (225 l) at 4.5 10 cells ml in a 48-well plate (10000 cells ml). cells were allowed to recover for 48 h and had grown to 80% confluence, at which time a concentrated solution of tb complex in pbs (25 l) was added to achieve a final concentration of 50 m in the cellular media. cells grown in tb complex free media (addition of only pbs) were included as a negative control. following 4 h of incubation, the media were removed, and the wells were washed twice at room temperature by adding pbs and rocking the culture plate back and forth several times. the addition of a lysis buffer (75 l of 10 mm tris, ph 7.5, 100 mm nacl, 1 mm na2edta, and 1% triton x-100) followed by a 20 min incubation at 4 c ruptured the cell membranes. aliquots (3 8 l) from each well were removed for a bca assay to determine the protein concentration. samples (50 l) were combined with concentrated nitric acid (50 l) and heated to 95 c for 18 h in flame - sealed glass ampules prior to icp - ms analysis. the tb content (ppb) was determined by icp - ms, and the cellular association of each tb complex (ctb), expressed as mol of tb / g of protein, was calculated according to the equationwhere cm is the measured metal concentration (ppb) of the cell samples, m is the molecular mass of tb, and cp is the protein concentration (mg / ml). l6 myoblasts (passage 9 or 10) were seeded in a polylysine - coated 8-well glass - bottom plate and allowed to grow for 24 h to 50% confluence at 37 c in 5% co2. then tb complexes [tb-2][tb-8 ] were added in pbs for a final concentration of 200 m. negative control wells containing cells grown in tb complex free media (addition of only pbs) were included. following a 4 h incubation, the media were removed, and cells were washed three times with pbs at room temperature by rocking the culture plate back and forth gently. cells were fixed with 4% formaldehyde in pbs at room temperature for 15 min. additionally, live cell imaging of [tb-6 ] was performed in dmem phenol red free media. epifluorescence images of representative cells were acquired on an olympus ix81 inverted microscope equipped with a 60 (n.a. excitation was supplied by an exfo xcite 120 metal halide lamp source coupled to the microscope via a liquid light guide, images were collected with a c910001 ccd camera, and hcimage software was utilized to control the microscope and camera and to process images. an excitation filter (325375 nm), dichoric filter (460 nm), and emission filter (470750 nm) were employed with exposure times of 0.4 s for fixed cells and 0.1 s for live cells. bright field images of cells were also collected to determine the position of cells (exposure time 0.04 s). the contrast of bright field images was adjusted to a contrast range of 0100 (normal range 0255) using hcimage software. | a systematic study of the effect of hydrophobicity and charge on the cell viability and cell association of lanthanide metal complexes is presented. the terbium luminescent probes feature a macrocyclic polyaminocarboxylate ligand (dota) in which the hydrophobicity of the antenna and that of the carboxyamide pendant arms are independently varied. three sensitizing antennas were investigated in terms of their function in vitro : 2-methoxyisophthalamide (iam(ome)), 2-hydroxyisophthalamide (iam), and 6-methylphenanthridine (phen). of these complexes, tb - dota - iam exhibited the highest quantum yield, although the higher cell viability and more facile synthesis of the structurally related tb - dota - iam(ome) platform renders it more attractive. further modification of this latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic amino acid based moieties generated a family of complexes that exhibit high cell viability (ed50 > 300 m) regardless of the lipophilicity or the overall complex charge. only the hexyl - substituted complex reduced cell viability to 60% in the presence of 100 m complex. additionally, cellular association was investigated by icp - ms and fluorescence microscopy. surprisingly, the hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. it is thus postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (iam(ome)) disfavors the cellular association of these complexes. as such, responsive luminescent probes based on this scaffold would be appropriate for the detection of extracellular species. |
mandibular angle fracture is the most common mandibular fracture, accounting for 40% of all mandibular fractures1. mandibular angle fracture is defined as a fracture line between the anterior border of the mandibular ramus and the body of the mandible2. the management of angle fracture is difficult because of its thin cross - sectional area and its attachment to the masticatory muscles applying forces in different vectors, its abrupt change in curvature, and the presence of lower third molars3. hanson.1 have reported that the presence of a lower third molar doubles the risk of angle fracture, and rajandram.4 showed the presence of lower third molars in 60.4% of mandibular angle fractures. partially erupted third molars produce an increased risk of mandibular angle fracture. as a result, previous studies have recommended that individuals such as sports players, who are exposed to high risk of facial trauma, wear protective facial gear and mouth guards4. some researchers support the extraction of the tooth in the fracture line, as they believe it decreases the risk of infection and the need for removal of the third molars and plate in the future5. other researchers have shown that extraction of the tooth can lead to loss of bone, increase the difficulty in reduction and plating, and increase both the surgical time of the procedure and the risk to the inferior alveolar nerve6. the aim of this study was to evaluate the implication of the third molars in the postoperative infection, postoperative nerve injury, bone healing, change in occlusion, and temporomandibular disorder (tmd) of the mandibular angle fracture with open reduction and internal fixation (orif). a retrospective review of patients who presented with mandibular angle fracture at our department of oral and maxillofacial surgery, inje university busan paik hospital (busan, korea) between january 2011 and december 2015 was conducted, and data were collected by reviewing medical records and radiographs. of the 63 total patients who underwent orif and perioperative intermaxillary fixation (imf) with an arch bar, 49 patients were identified as having third molars in the fracture line and were followed up with until plate removal. the inclusion criteria were as follows : patients having third molars in the fracture line, patients who underwent orif under general anesthesia and perioperative imf with an arch bar, and patients who participated in follow - up until plate removal. panoramic radiographs and facial bone computed tomography of the patients were obtained, and demographic data, including age, sex, and medical history, were documented. analgesics and antibiotics were administered in all cases for about 6 days from admission until discharge. examination findings related to postoperative alveolar nerve function, change in occlusion, tmd, infection, and delayed union or nonunion were recorded, and additional images of panoramic radiographs (fig. the data were statistically analyzed using the ibm spss statistics version 22.0 for windows (ibm co., armonk, ny, usa), and comparative analyses of the two groups were performed using the test. differences were considered statistically significant if the confidence interval p - value was 0.05).(table 2) this study included 49 patients, 43 males (87.8%) and 6 females (12.2%), divided into a non - extraction group and an extraction group. of these patients, 17 patients (34.7%) had a mandible angle fracture on the right side, while 32 patients (65.3%) had a mandible angle fracture on the left side. patel.3 showed that the overwhelming majority of patients were men, comprising 85 of 103 patients (82.5%), whereas women accounted for 18 of 103 patients (17.5%) with mandibular angle fracture. most fractures occurred at the left mandibular angle (34.9%), followed by the right side (34.9%) and both sides (5.8%). in this study, all patients underwent imf for the management of mandibular angle fracture. imf is a reliable technique for reduction and stabilization before plate fixation, although the use of interdental wiring may have adverse effects on the teeth or surrounding tissue and require additional time78910. bhagol.11 explained that although superior and inferior plates are typically required for adequate fixation, a superior border plate placed at the point of maximal tension is sufficient for mandibular angle fracture. in this study, preoperative imf using an arch bar was applied and used for approximately 3 to 5 days after surgery, with 1 or 2 miniplates placed at the superior area during orif surgery. only 1 patient with malocclusion because of left mandible deviation after surgery required an additional operation. yadavalli.6 proposed that the presence of third molars in fracture lines may be of great value in the repositioning of fractures because the presence of third molars prevents further injury to the bone tissue. the extraction of third molars makes it difficult to reduce the contact between fracture segments when the fragments are highly mobile6. because extraction of the tooth increases the risk of contamination through the empty alveolus612, yadavalli.6 used the following criteria for surgical removal of the third molars in the line of fracture : pericoronal or periodontal infection, cross caries, extensive periapical lesions, mobility, or exposure of the apical half or more of the root fracture. mcnamara.5 noted that the risks involved with extraction of third molars includes loss of bone, greater difficulty in reduction and plating, increased surgical time, and increased risk to the inferior alveolar nerve. in their study, the incidence of nerve injury was 16% for the retention group compared to 39% for the removal group. therefore, they stated that the removal of third molars creates an additional risk of nerve injury and increases operating time4. in this study, 4 patients in the non - extraction group and 1 patient in the extraction group complained of postoperative nerve injury, and there was no significant difference between the groups. all of the patients exhibited gradual recovery of the nerve during the follow - up period. the issue of postoperative infection has long been debated and represents a common complication of mandible angle fracture3. a 1994 study by ellis and walker12 found that the extraction of third molars in fracture lines seemed to increase postoperative infection3413. in 2002, they proposed that the risk of postoperative infection increased if teeth were left in the fracture line3413. patel.3 defined infection as purulent discharge from the intraoral incision through a sinus tract to the skin, or a closed area of swelling that required incision and drainage of purulent material. they found no statistically meaningful relationship between tooth management in the line of the fracture and rate of postoperative infection3. they stated that the differences in rates of infection might be attributed to inherent differences in the socioeconomic status of patients, tobacco and alcohol use and abuse, nutritional status, and other medical co - morbidities3. however, in this study, the patients were mostly young and free of significant medical history such as uncontrolled diabetes, alcohol abuse, or severe smoking. also, antibiotics were administered upon admission to the hospital until about 6 days from admission until discharge. as a result, there seemed to be no immediate infection or other complications. during the follow - up period, 5 patients in the non - extraction group received medication and underwent intra or extra - oral incision and drainage because of infection. after treatment for 5 days, acute symptoms and sign of infections subsided. all infected third molars underwent extraction during the plate removal surgical procedure. none of the patients in the extraction group exhibited infection in the third molar extraction area or fracture line. this may be attributed to standard protocol at the time of surgery such as gentle curettage, copious irrigation, interrupted sutures offering strength and flexibility of the extraction site, and antibiotics. alterations of the masticatory system caused by dislocation of body fragments and conservative treatment of mandibular fractures showed that inaccurate immobilization of the mandible after trauma resulted in a new bite. consequently, conditions for the development of tmj dysfunction occurred as a result of adapted dynamical masticatory movements and new occlusion1415. instances of deviation upon opening and the presence of joint sounds were higher in cases treated with miniplate fixation and were more susceptible to development of traumatic arthritis16. in this study one of the patients complained of tmj noise, while the other patient complained of intermittent tmj pain after surgery. however, there was no statistically significant difference between the two patients, and both of them improved during the follow - up period. one limitation of this study is the small number of patients in the extraction group. there was no statistically significant difference in postoperative complications between the extraction and non - extraction groups of third molars in the fracture line. if the third molar is partially impacted or completely nonfunctional, likely to be involved in pathologic conditions later in life, or possible to remove it with the plate simultaneously, extraction of the third molar in the fracture line should be considered during orif surgery of the mandible angle fracture. | objectivesthe aim of this study was to evaluate the implication of third molars in postoperative complications of mandibular angle fracture with open reduction and internal fixation (orif).materials and methodsdata were collected on patients who presented with mandibular angle fracture at our department of oral and maxillofacial surgery between january 2011 and december 2015. of the 63 total patients who underwent orif and perioperative intermaxillary fixation (imf) with an arch bar, 49 patients were identified as having third molars in the fracture line and were followed up with until plate removal. the complications of postoperative infection, postoperative nerve injury, bone healing, and changes in occlusion and temporomandibular joint were evaluated and analyzed using statistical methods.resultsin total, 49 patients had third molars in the fracture line and underwent orif surgery and perioperative imf with an arch bar. the third molar in the fracture line was retained during orif in 39 patients. several patients complained of nerve injury, temporomandibular disorder (tmd), change of occlusion, and postoperative infection around the retained third molar. the third molars were removed during orif surgery in 10 patients. some of these patients complained of nerve injury, but no other complications, such as tmd, change in occlusion, or postoperative infection, were observed. there was no delayed union or nonunion in either of the groups. no statistically significant difference was found between the non - extraction group and the retained teeth group regarding complications after orif.conclusionif the third molar is partially impacted or completely nonfunctional, likely to be involved in pathologic conditions later in life, or possible to remove with the plate simultaneously, extraction of the third molar in the fracture line should be considered during orif surgery of the mandible angle fracture. |
the incidence of hepatocellular carcinoma (hcc) is increasing in the united states, europe, and worldwide, and has become a serious problem in many countries. patients with chronic liver disease related to hepatitis b or c virus infection are at high risk for developing hcc, and the risk of hcc development becomes higher as fibrosis of the liver advances. indeed, it is reported that the risk of hcc development in cirrhotic patients with hepatitis c virus (hcv) infection is as high as 7% per year in japan. interferon (ifn) has a potent antiviral effect and has been widely used for the treatment of chronic hcv infection. ifn therapy improves inflammation and fibrosis in the liver of the patients and reduces the risk of hcc development, particularly when it results in sustained virological response. on the other hand, there are several reports that describe hcc development long after the clearance of hcv by successful ifn therapy. therefore, it is still uncertain how long the patients hold the risk of hcc development after the resolution of chronic hcv infection. in the current report, we describe a patient who developed hcc 11 and a half years after the clearance of hcv. a 41-year - old japanese man was found to be seropositive for anti - hcv antibody (dainabot, tokyo, japan) with abnormal liver function tests at a health check - up in 1994. he had no history of blood transfusion or drug abuse, but had been consuming approximately 25 g / day of alcohol for 20 years. serum hepatitis b surface antigen (hbsag) and antibody to hbsag (anti - hbs) were negative. a liver biopsy specimen was consistent with the feature of chronic hepatitis showing moderate fibrosis and mild necroinflammatory reaction, diagnosed later as f2/a1 according to the classification by desmet.. he was diagnosed to have chronic hepatitis c (ch - c) and received ifn monotherapy with natural ifn alpha (human lymphoblastoid ifn ; sumitomo pharmaceutical co., osaka, japan), 6 million units (mu), every day for the first 2 weeks and three times a week for the following 22 weeks. hcv rna became negative during therapy, and the negativity of hcv rna continued during 6 months of follow - up after discontinuation of therapy. he abstained from alcohol during and after the ifn therapy, but his body weight showed a gradual increase with slight elevation of serum aminotransferase levels. he was diagnosed to have fatty liver and diabetes mellitus, but treatment was not introduced. in january 2006, he was found to have a hepatic nodule 4.0 cm in diameter at liver s4/8 region by plain abdominal ct at an annual follow - up examination. he was admitted to our hospital (department of gastroenterology, aichi medical university hospital, aichi, japan) in february 2006. on admission, physical examination was unremarkable ; the liver and spleen were not palpable, and jaundice, ascites, peripheral edema and other signs of chronic liver disease were not observed. complete blood counts (cbc) were normal (white blood cell count 6,900/l, hemoglobin 14.4 g / dl, and platelet count 253,000/l). liver function tests were normal ; serum levels of transaminases and total bilirubin were within normal range (ast 22 iu / l, alt 23 iu / l, and total bilirubin 0.9 mg / dl). other laboratory tests, including liver fibrosis markers such as type iv collagen 7s, type iii procollagen - n - peptide and hyaluronic acid, were also within normal range. however, fasting blood sugar (128 mg / dl) and hba1c (6.8%) were slightly elevated (table 1). serum tumor makers of hcc were within normal range, prothrombin induced by vitamin k absence - ii (pivka - ii) level was 18 mau / ml (normal range < 40 mau / ml), and a - fetoprotein level was 4.4 ng / ml (normal range < 10 ng / ml). he was seropositive for anti - hcv by a second - generation enzyme immunoassay (dainabot, tokyo, japan) and seronegative for hcv rna by amplicore hcv test (roche diagnostic inc., tokyo, japan). on dynamic ct, the nodule on liver s8 area revealed a tumor staining of 4.0 cm in diameter in the early arterial phase and modest washout density in the post - vascular phase (fig. on contrast - enhanced us scan, blood inflow into the same nodule was detected in the early arterial phase, and the nodule became hypoechoic compared to the liver parenchyma in the post - vascular phase. celiac angiography revealed a hypervascular tumor with proliferation of fine tumor vessels (which also showed nodular and mosaic features) at the s4/8 region of the liver, just below the dome of right the diaphragm (fig. percutaneous liver biopsy was performed at the noncancerous lesion, and the specimen showed fibrosis of stage f2 and mild inflammation of grade a1 with mild steatosis (fig. the tumor was curatively resected without any complication, and the histological study of the resected specimen revealed that the tumor was a moderately differentiated hcc (fig. in the present study we describe a patient with hcc who developed hcc 11 and a half years after the eradication of hcv. there are many reports aimed to elucidate the risk factors for liver carcinogenesis, and some of them have tried to examine the incidence of hcc in patients with ch - c after ifn therapy. examined the risk of hcc development in ch - c patients treated by ifn and showed that the cumulative incidence of hcc in relapsers (transient responders) to ifn therapy was almost equal to that in sustained responders, and that it was significantly higher in nonresponders than in sustained and transient responders. the seventh - year cumulative incidence rates of hcc in sustained responders, relapsers, and nonresponders were estimated to be 4.3, 4.7, and 26.1%, respectively. they suggested that patients in the high - risk group of hcc after ifn therapy were those who showed no response, those who were older, or males. sustained responders to ifn therapy usually show normal alt levels continuously. in relapsers, alt values usually become normal when hcv rna becomes seronegative during therapy. thus, the reduction in the necroinflammatory activity in the liver appears to be an important factor to prevent the development of hcc even when the eradication of hcv is not achieved. another report indicated that the group at high risk for developing hcc in sustained responders to ifn therapy were older, more often male, and had more advanced histological disease stages. in addition, kasahara. showed that the risk of death from liver - related diseases in sustained and nonsustained virological responders was significantly lower compared with that in untreated patients. furthermore, sustained and transient biochemical responders were shown to have a reduced risk of liver - related death compared to biochemical nonresponders. thus, it is likely that ifn treatment prevents liver - related death and improves survival of ch - c patients with virological as well as biochemical response. in the present case, the liver biopsy specimen at the time of operation (before operation) revealed liver damage of f2/a1. this finding was almost the same compared with that at the start of ifn therapy 12 years earlier. we considered the possibility that the presence of nonalcoholic fatty liver disease (nafld) might contribute, at least in part, to the development of hcc. nafld is a clinicopathological term that encompasses a disease spectrum ranging from simple triglyceride accumulation in hepatocyte (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis), fibrosis, and cirrhosis. judging from the clinicopathological features in the current case, we could not exclude the influence of nafld for the development of hcc, while the pathological role of nafld might be limited. although it has been reported that hcc rarely develops long after eradication of hcv, all patients with chronic hcv infection should be followed as long as possible for the potential development of hcc, even sustained virological responders to ifn therapy. more careful follow - up is recommended, particularly when patients have complications such as nafld. | a 41-year - old japanese man had received successful interferon (ifn) therapy against chronic hepatitis c in 1994. since then, serum hepatitis c virus (hcv) rna had been negative, and aminotransferase levels had been continuously normal. he had abstained from alcohol. however, his serum aminotransferase levels showed slight elevation as his body weight increased gradually. he was diagnosed as having fatty liver and diabetes mellitus. in january 2006, 11 and a half years after the resolution of hcv infection, he was found to have a hepatic nodule 4.0 cm in diameter at liver s4/8 region by plain abdominal ct at an annual follow - up examination. he was diagnosed as having hepatocellular carcinoma (hcc) by angiography. the tumor was curatively resected and its histological diagnosis was moderately differentiated hcc. noncancerous lesion of the liver revealed fibrosis of stage f2 and mild inflammation of grade a1 with mild steatosis. this case suggests that all patients with chronic hcv infection should be followed as long as possible for the potential development of hcc even after clearance of the virus. |
over 40% of patients being treated with oral anticoagulation are prescribed a direct oral anticoagulants (doacs) according to a recent study assessing prescribing patterns 1. pivotal trials for the doacs have reported noninferiority or superiority of these agents to warfarin, a vitamin k antagonist (vka), for multiple indications including nonvalvular atrial fibrillation (af) and venous thromboembolism (vte) 2, 3, 4, 5. furthermore, several of these studies also demonstrated similar or lower rates of minor and major bleeding and eliminates the need for frequent laboratory monitoring of these doacs in comparison with warfarin. despite these benefits, dabigatran, a direct thrombin inhibitor, is the only doac with an fdaapproved reversal agent idarucizumab currently on the market 6. currently, other doacs such as rivaroxaban, apixaban, and edoxaban, lacks an fdaapproved reversal agent, although there are currently multiple agents in the pipeline for reversing these newer anticoagulants 7. as a result, the current mainstay management of factor xa inhibitorinduced bleeding is supportive care with traditional blood products such as packed red blood cells (prbc), as the anticoagulant effects of rivaroxaban can be expected to last 24 h or more 8. fourfactor prothrombin complex concentrate (4fpcc), the only pcc formulation currently available in the united states, contains factors ii, vii, ix, and x, proteins c and s, and heparin. this agent is currently only fdaapproved for the management of acute major bleeding or need for urgent surgery or invasive procedures in patients on vka, with dosing recommendations based on international normalized ratio (inr) values. the 2012 chest antithrombotic guideline provides a level 2c recommendation for 4fpcc in the management of vkaassociated major bleeds over plasma products 9, although the guideline supplement is published 1 year after rivaroxaban is initially approved in the united states. we report the following case of a 69yearold caucasian male anticoagulated with rivaroxaban 20 mg orally every morning with meals for chronic nonvalvular af (cha2ds2vasc=5), admitted to the cardiothoracic intensive care unit for emergent onpump coronary artery bypass graft due to stelevated myocardial infarction and hemodynamic instability secondary to confirm thrombosis of a previously inserted left anterior descending (lad) artery stent. the patient had a past medical history of chronic nonvalvular af, coronary artery disease with two coronary stents of the lad and first diagonal branch arteries, systolic heart failure, type ii diabetes mellitus, and hypertension. he had been experiencing angina that wax and wane for the past week that intensified prompting admission to an outside facility. he deteriorated overnight and was initiated on intravenous (iv) unfractionated heparin and bivalirudin, and sedated and intubated at the facility. emergent left heart catheterization was performed and found thrombosis of the lad stent with a left ventricular enddiastolic pressure of 15 mmhg necessitating placement of a 1:1 intraaortic balloon pump placement. overnight at the facility, bivalirudin was discontinued, and in the morning, he received rivaroxaban 20 mg once at that facility, and he was immediately transferred to our facility via the air ambulance for emergent onpump coronary artery bypass graft (cabg). on admission at 5 h estimated time prior to the scheduled emergent onpump cabg, iv heparin was continued at 1200 units / h. the patient 's calculated creatinine clearance (crcl) via the cockcroft gault equation is 95 ml / min based on his admission serum creatinine of 0.8 mg / dl and actual weight of 77.1 kg ; actual weight was used as his ideal body weight is 77.6 kg. urine output captured 5 h as admission is 500 ml, which is equivocal to 1.2 ml / kg / h based on his actual body weight. relevant admission hematologic laboratory markers revealed an elevated inr and activated partial thromboplastin time (aptt) (table s1). fourfactor prothrombin complex concentrate 2500 units (30 units / kg) iv at 200 units / min for one dose was administered 3 h prior to the scheduled cabg ; the dose of 30 units / kg was chosen based on the review of available literature demonstrated survival for majority of patients that have received 4fpcc dosing ranging from 25 to 40 units / kg (table s2). twovessel onpump cabg of the lad and first diagonal branch was performed and required ~4 h of intraoperative time. in the operating room, the patient lost a total of 650 ml of blood necessitating 2 units of both prbc and platelets, respectively. following the procedure, he was transferred to the cardiothoracic intensive care unit for hemodynamic optimization with fluids, vasopressors, and inotropes. on postoperative days (pods) 13, he received additional units of prbc for optimization of oxygen delivery by maintaining target hemoglobin of 10 g / dl. by pod 2, the intraaortic balloon pump was weaned off, he was successfully extubated from mechanical ventilation, and was initiated on a regular enteral diet. no significant events occurred between pods 27 and by pod 7, and he was transferred to the cardiothoracic stepdown unit for observation. he was scheduled for discharge on pod 8, but due to case management issues, he was discharged by pod 9 without any complications and was prescribed for rivaroxaban 20 mg orally every morning with meals. according to the patient, he prefers to take it in the morning despite recommendation by the manufacturer to take the medication in the evening for the indication of nonvalvular af. a followup visit 1 month after discharged did not reveal any new significant event, and a chest xray obtained during the visit revealed an almost complete resolution of postoperative effusions and atelectasis. rivaroxaban inhibits coagulation factor xa, hindering the downstream coagulation cascade that involves activation of factor ii, i, and xiii thus preventing thrombogenesis 10, 11, 12. in patients with a crcl greater than 50 ml / min, the recommended dosing of rivaroxaban for nonvalvular af is 20 mg by mouth every evening with meals. the drug 's reported halflives with a crcl greater than 50 ml / min 59 h in adults and 1115 h in the elderly. approximately 66% of rivaroxaban undergoes renal elimination, and the drug has a time to peak plasma concentration of 24 h. in addition, it has a large volume of distribution at approximately 50 l and is highly protein bound to albumin between 92% and 95% making it virtually nondialyzable. several blood products like 4fpcc have been suggested by experts as reversal agents for the doacs. despite being approved for urgent reversal of vka, some experts believe that 4fpcc may still be beneficial when used for severe bleeding or urgent reversal with doacs as it replenishes factor ii, vii, ix, x, and proteins c and s based on published case reports and series across different patient population, although the dosing and outcomes of these publications have been conflicting, and very limited or no information was provided on potential confounders (e.g., inr, concurrent disease states) for several of these cases (table s2) 13, 14, 15, 16. while bivalirudin is known to cause false elevation in inr, it is unlikely contributory to the initial inr elevation upon admission as the medication was discontinued overnight, and the serum concentration of the drug on admission is most likely negligible based on the drug 's reported halflife of 25 min with a crcl greater than 90 ml / min 17. while there is conflicting publications regarding the utility of 4fpcc in the reversal of bleeding from rivaroxaban and other doacs, the case presented demonstrated both the efficacy and safety of 4fpcc for the migration of rivaroxabaninduced bleeding for emergent cabg. our patient received 30 units / kg of 4fpcc, 2 units of prbc, and 2 units of platelets which differ from other cases where patients have received between 25 and 50 units / kg of 4fpcc, and between 1 and 10 units of prbc. as there is no established standard on the dosing of 4fpcc for doacs across hospitals, the risk of administration and excessive dosing must be weighed as 4fpcc carries a black box warning for thromboembolic events. as the cases evaluated demonstrated survival between 25 and 40 units / kg, we have chosen 30 units / kg as an approximate median value based on risk versus benefit. furthermore, cost should also be a consideration for appropriate utilization of the drug as the average wholesale price for each vial of 1000 units is approximately $ 1400, and most patients will require multiple vials. ultimately, more data are needed to determine the risks versus benefits of 4fpcc in the optimal dosing of 4fpcc and the management of severe bleeding from doacs. recently, there is a proposed management algorithm for patients on doacs based on urgency, including the use of alternative reversal agent such as activated charcoal, although the algorithm lacks dosing recommendation for 4fpcc, any information in regard to the pipeline factor xa inhibitor antidote such as, and any concrete evidence supporting its use 18. we present the first case of 4fpcc for the alleviation of bleeding for emergent onpump cabg with the patient discharged by pod 9 with no sequelae. while 4fpcc may play a role in the management of rivaroxabaninduced bleeding in surgical procedures, safe and effective therapies for reversing such bleeds are urgently needed as the outcomes of 4fpcc on other doacs have been conflicting. ml : substantial contributions to the case report : identified case, reviewed literature, drafted and revised manuscript, and final approval of the version to be published ca : reviewed literature, drafted and revised manuscript ud : cardiothoracic surgeon for the case, provided expert feedback. | key clinical messagewe presented the first case of fourfactor prothrombin complex concentrate (4fpcc) for the alleviation of bleeding for emergent onpump coronary artery bypass graft (cabg) with the patient discharged by postoperative day (pod) 9 with no sequelae. until direct antidotes are available, 4fpcc may play a role in the management of mitigating rivaroxabaninduced bleeding in surgical procedure. |
the neural mechanisms that underlie l - dopa - induced dyskinesias in parkinson s disease (pd) remain poorly understood [14 ]. dyskinesias have been associated with pulsatile stimulation of dopamine receptors, downstream changes in genes and proteins, and changes in nondopamine transmitter systems [46 ]. all these changes lead to alterations in the firing patterns within the basal ganglia, causing overactivations of the primary motor cortex and premotor cortical areas [7, 8 ]. activation studies in pd patients (h2 o positron emission tomography (pet) and xe single photon emission computed tomography (spect)) are in line with these results, and indicate that l - dopa - induced dyskinesias are associated with inappropriate overactivity of striatofrontal projections both at rest and during volitional actions [2, 9 ]. comparison of rest with dyskinesias versus rest without dyskinesias showed a relative activation of the motor cortex, the lateral and mesial premotor areas including the supplementary motor area (sma), the dorsal prefrontal cortex, and the basal ganglia. the comparison of dyskinetic versus nondyskinetic states during voluntary movement revealed additional activation of the same brain regions. unfortunately, in the pet study of brooks. mentioned above, regional cerebral blood flow (rcbf) in pd patients with and without l - dopa induced dyskinesias was not compared to that of healthy subjects, and therefore, it is difficult to speculate whether the relative increases in rcbf observed in patients with dyskinesias were in fact above or still below that of controls in all the reported areas including the dlpfc. repetitive transcranial magnetic stimulation (rtms) can produce changes in excitability of cortical circuits that outlast the period of stimulation, opening the possibility of intervening directly with the mechanisms of cortical plasticity in the human cortex. rtms is also a suitable tool to investigate plasticity within a distributed functional network since conditioning effects of rtms are not limited to the stimulated area but give rise to functional changes in interconnected cortical areas. the magnitude and the direction of rtms - induced plasticity depend on extrinsic factors (i.e., the variables of stimulation such as intensity, frequency, and total number of stimuli) and intrinsic factors (i.e., the functional state of the cortex targeted by rtms). high frequencies of rtms applied over the motor cortex lead to facilitatory aftereffects on corticospinal excitability while low - frequency stimulations lead to opposite (i.e., inhibitory) aftereffects (for review, see 10). the main problem remains, that the basic mechanisms mediating the effects of rtms are still poorly understood. recenly, several studies have suggested the therapeutic efficacy of rtms in pd while other studies have found no clinical improvement of motor performance in pd [1115 ]. using rtms over the sma, an italian group showed that 900 pulses of 1 hz rtms with an intensity set at 90% of the resting motor excitability threshold markedly reduced drug - induced dyskinesias in 8 advanced pd patients. in spite of this promising result, a year later the same group discovered that 5 repeated sessions of the 1 hz rtms failed to enhance and/or prolong the beneficial effects of the procedure, and thus could not be considered clinically useful. very similar results were reported by a toronto group who used the same stimulation parameters but targeted the primary motor cortex contralateral to the side with more severe dyskinesias in 6 pd patients. although the benefit was evident at day 1 after a two - week course of rtms, the authors have not managed to obtain more sustained clinical improvement after the treatment. we recently reported the results of a pilot study that investigated whether 5 repeated sessions of high frequency rtms at 10 hz applied over the motor cortex (mc) of the relevant leg or over the left dorsolateral prefrontal cortex (dlpfc) might result in a modification of off-related freezing of gait and motor symptoms of pd in 6 advanced pd patients. the selection of mc and dlpfc targets for rtms was based on dopamine release in the putamen and caudate, respectively [19, 20 ]. the target selection was further supported by the results of lomarev. who had reported positive cumulative benefit of high - frequency rtms applied over the same targets for improving gait, as well as reducing upper limb bradykinesia in pd patients. in our study, rtms was well tolerated. despite a reported trend towards improvement of the stroop test interference after rtms over the left dlpfc, interestingly, patients who had undergone the dlpfc treatment reported a subjective improvement of their dyskinesias. therefore, in this paper, we present a post hoc analysis of the data from the same group of patients that focuses on cortical excitability and on the possible modification of l - dopa - induced dyskinesias by repeated sessions of rtms over the dlpfc. we studied 6 patients (5 men and 1 woman, mean age 67.37.7 years) with advanced pd according to the uk parkinson s disease brain bank criteria [21, 22 ], a hoehn - yahr stage between 2.5 and 4 while in the off state, and without dementia (mini - mental state examination score > 24). a montgomery - asberg depression rating scale cut - off score of 14 was used to exclude patients with depression. patients with a predominant tremor form of pd were also excluded from the study. the daily l - dopa dose equivalent was 1145.8413.2 mg ; all patients were on l - dopa entacapone plus a dopamine receptor agonist. the antiparkinsonian medication was stable for at least 4 weeks prior to the study commencement and during the study. all patients signed the informed consent form, which was approved by the ethics committee of saint anne s hospital in brno. rtms was applied over the left dlpfc (5 cm anterior to the optimum scalp position for activation of the contralateral first dorsal interosseus muscle) or over the optimal position for the tibialis anterior muscle contralateral to the foot which was most frequently used by the patient to make the first step after freezing of gait (i.e., mc), using the magstim super rapid stimulator and a figure - of - eight coil with a mean diameter of 7 cm. the coil was placed tangentially to the scalp with the handle pointing backwards and laterally at 45 angle away from the midline inducing a posterior - anterior current in the brain. nowadays, the frameless stereotaxy has been used in order to target the region of brain corresponding to cytoarchitectonic area 9/46 as defined by petrides and pandya. nevertheless, this part of the brain was found to overlap with the region targeted by the standard procedure (rektorova and paus, unpublished data), used in most studies. rtms was delivered in the on state (i.e., when pd medication was working and providing benefit) without dyskinesias according to the individual patients diaries, at the same time of day. one treatment consisted of 5 sessions over 5 consecutive days. we chose a repeated sessions design, and our patients were stimulated while being in their on state since both conditions have been associated with better rtms outcomes in literature [12, 14 ]. one session consisted of 1350 pulses delivered at 10 hz frequency, at an intensity of 90% of the resting motor excitability threshold (for the right first dorsal interosseus muscle when stimulating the left dlpfc, and the appropriate tibialis anterior muscle when stimulating the mc). although we aimed at a crossover design, only two patients completed both treatments (with a one - month interval between them). in order to ensure the method was safe with regard to cognitive functions, a brief neuropsychological battery of tests was administered prior to, immediately after, and one week after each rtms treatment. experienced neurologists who were blinded to the stimulation site performed a neurological evaluation prior to, immediately after, and one week after appropriate rtms treatment. the evaluation was conducted during the patients relative off state (i.e., when pd medication was not improving pd symptoms resulting in a lack of mobility) according to the individual patients diaries, at the same time of day. it consisted of the updrs, part iii (motor examination) and part iv (treatment complications). a more detailed evaluation and analysis of gait and off-related freezing of gait has been reported elsewhere. single - pulse and paired - pulse tms was performed while the patient was on to study the effects of rtms treatments on cortical excitability. we used the paired - pulse tms paradigm of ridding. at interstimulus intervals (isi) we evaluated motor evoked potentials (meps) elicited by single and paired - pulse tms pulses over the mc contralateral to either the right first dorsal interosseus muscle (prior to, immediately after, and one week after the rtms treatment over the left dlpfc) or the appropriate tibialis anterior muscle (prior to, immediately after, and one week after the rtms treatment over the appropriate mc). the motor cortical sites were chosen to specifically accommodate the primary goal of our freezing of gait protocol design. mep size reflects more globally the corticospinal input - output balance, excitatory inputs from high - threshold glutamatergic pathways to the motor cortex lead to intracortical facilitation, whereas inhibitory inputs from low - threshold gaba - a - mediated pathways lead to intracortical inhibition at short isi. the effects of rtms treatments on single mep amplitudes and updrs before and after the rtms treatments were analysed by a two - tailed paired - sample t test. a two - factor repeated measures anova, with the factors of time (3 levels : baseline, immediately after the rtms treatment, and 1 week after the rtms treatment) and isi (3 levels : averaged data from the inhibiting isi (1 and 3 milliseconds), the facilitating isi (15 milliseconds), and the intermediate isi (7 milliseconds)) was performed to evaluate the effects of the rtms treatment applied over the mc and the dlpfc, respectively. despite our aim to perform a randomised crossover design, the study was stopped prematurely as several patients withdrew the consent form before completing both treatments. a subjective lack of treatment effect on freezing of gait (i.e., the primary outcome) was the reason for the study withdrawal. this study presents the results from four patients that completed the rtms treatment over the mc and four patients that completed the treatment over the dlpfc. despite the lack of effect of rtms on freezing of gait and motor symptoms of pd, the patients reported a decreased frequency and intensity of dyskinesias after 5 consecutive sessions of rtms targeted over the left dlpfc. these changes were reflected by the mean updrs iv decrease immediately after 5 sessions of the stimulation as compared with the baseline scores (see table 1). the result did not reach statistical significance (p=.06) but could be suggestive of a mild improvement of dyskinesias. there was no cumulative effect of the rtms treatment over the mc on corticospinal excitability as measured by mep responses produced by single tms pulses over the left mc contralateral to the appropriate tibialis anterior muscle (see table 2). however, we found that 5 consecutive sessions of high frequency rtms of the left dlpfc produced a significant decrease in the amplitude of mep responses produced by single tms pulses over the left mc contralateral to the right first dorsal interosseus muscle (see table 2). there were no effects of any rtms treatments on the time course of intracortical inhibition or intracortical facilitation in the appropriate motor cortex ; this was verified with a two - factor repeated measures anova with the factors time and isi : f(4,26) = 0.72, p=.6 for rtms treatment over the dlpfc ; f(4,10) = 2.4, p=.1 for rtms treatment over the mc. the results of our study point to a possible impact of repeated sessions of rtms applied over the left dlpfc on l - dopa - induced dyskinesias in advanced pd patients. the mean change in the updrs iv scores did not reach a statistical significance, but we did not evaluate dyskinesias by more appropriate standardized scales since the assessment of dyskinesias was not the primary outcome of our pilot study. on the other hand, updrs iv was used to test dyskinesias in a pet study by brooks.. there are a number of hypothetical mechanisms that might underlie the possible effects of rtms of the dlpfc on l - dopa - induced dyskinesias in advanced pd. it appears that changes of the excitability of the primary motor cortex may be more efficiently performed through stimulation of premotor and/or other more anterior brain regions, for review, see. we found that 5 consecutive sessions of high frequency rtms of the left dlpfc produced a significant decrease in the amplitude of mep responses produced by single tms pulses over the left mc for the right first dorsal interosseus muscle (i.e., decrease of the cortico - spinal excitability). these results are in accord with the results of rollnick. who also demonstrated that stimulation of the dlpfc via subthreshold 5 hz rtms induces motor cortex depression in healthy subjects. it was posited by the authors that this finding might be explained by the fact that prefrontal brain areas contribute to motor cortex inhibition through antagonisms between frontal and parietal lobes [32, 33 ] but the exact mechanisms are not fully understood. was found by means of pet and functional magnetic resonance imaging (fmri) while studying simple or complex motor hand tasks [34, 35 ]. it was also observed in spect and pet studies of pd patients with l - dopa - induced dyskinesias [2, 9 ]. electrophysiological data furhter support this notion : increased mep aplitudes at rest were found in pd patients with l - dopa induced dyskinesias [36, 37 ]. neuroimaging studies have shown that subthalamic nucleus stimulation acts through the reduction of abnormal overactivity in the motor system at rest [34, 38 ]. even though dyskinesias were not systematically monitored in these studies, it has been well known that subthalamic nucleus stimulation surgery is able to reduce l - dopa - induced dyskinesias. taken together, normalizing (i.e., reducing) the excitability of the primary motor cortex, for example, by rtms of the dlpfc might be one possible factor in improving l - dopa - induced dyskinesias in advanced pd patients. rtms applied over the left dlpfc can induce significant increases of rcbf in the stimulated area. functional imaging studies (pet and fmri) report decreases in rcbf and mri signal in the dlpfc and the rostral sma in advanced pd patients compared to controls [354145 ]. the dlpfc is connected with the rostral sma. after unilateral pallidotomy, glucose metabolism increased in the dlpfc, in addition to metabolic changes in other cortical and subcortical areas. rcbf increases were reported in the dlpfc and the rostral sma during apomorphine pumps. again, pallidotomy, subthalamic nucleus stimulation and continuous apomorphine are able to reverse dyskinesias [394951 ]. therefore, the direct involvement of the dlpfc and the degeneration of mesofrontal dopaminergic afferents might also be hypothesized to play some role in the development of dyskinesias. finally, involvement of the dorsolateral the prefrontal circuit encompasses the dorsal prefrontal cortex, the dorsal caudate nucleus, the dorsal medial globus pallidus, and the ventral anterior nucleus of the thalamus. have shown that high frequency rtms of the dlpfc leads to dopamine release in the ipsilateral caudate nucleus [19, 20 ]. we used the same parameters as strafella. with regard to frequency and intensity of stimulation we have shown for the first time that repeated sessions of subthreshold 10 hz rtms of the dlpfc induced significant reductions of the ipsilateral motor cortex excitability in advanced pd population, and might possibly be suggestive of an improvement of l - dopa - induced dyskinesias. it has to be pointed out that this was a small observational study not specifically designed to assess dyskinesias in pd. dlpfc stimulation was not controlled by the placebo rtms and therefore a possible placebo effect can not be excluded either. on the other hand, the dlpfc stimulation was controlled by yet another active stimulation over the mc. improvement of dyskinesia could not have been expected by patients since changes in the motor complications score were not included in the primary outcomes of our pilot study. further research is warranted to explore more precisely whether the unilateral or bilateral stimulation of the dlpfc via repeated sessions of high frequency rtms induces beneficial effects on l - dopa - induced dyskinesias in advanced pd patients, and whether it could be considered clinically useful. | we studied whether five sessions of 10 hz repetitive transcranial magnetic stimulation (rtms treatment) applied over the dorsolateral prefrontal cortex (dlpfc) or the primary motor cortex (mc) in advanced parkinson 's disease (pd) patients would have any effect on l - dopa - induced dyskinesias and cortical excitability. we aimed at a randomised, controlled study. single - pulse transcranial magnetic stimulation (tms), paired - pulse transcranial magnetic stimulation, and the unified parkinson 's disease rating scale (updrs parts iii and iv) were performed prior to, immediately after, and one week after an appropriate rtms treatment. stimulation of the left dlpfc induced a significant motor cortex depression and a trend towards the improvement of l - dopa - induced dyskinesias. |
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