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nowadays, health care has been transformed to a patient - centered model, so that the patient plays a more active role in care. in this model, the patients and their relatives attend caregiving process as the main partners and are involved in health providers decision making, treatment, and administration of procedures. in pediatric nursing, family - centered care is also a basic element, which emphasizes on dynamic relationship between the family members and treatment and care providing team, as well as involves the family in care. parents should be clearly active in decision - making process in relation to taking care of children and participation in care. some terms used to describe the concept of participation of the parents are parents participation, involvement of parents in care, participation in care, caring partners, being involved in mutual participation of care, family - centered care, and family participation. application of synonyms for this term has confused the nurses, revealing the lack of a consensus on the concept of parents participation, so that nurses have no clear definition about parents participation. coyne, quoting from darbyshie, states that the concept of parents participation is one of the most controversial issues which should be clarified, as its clarification leads to more efficient application and evaluation of its weak and strong points. finally, it should be mentioned that understanding of this important concept increases its application in action and ultimately improves the quality of pediatric care in hospitals. family unit has a great value in iranian traditions, and strong ties of family members and parents responsibility are highly valuable. in other words, parents participation in taking care of a hospitalized child is the symbol of families power, but despite the importance of this issue, limited research has been conducted on the concept of parents and health providers taking care of hospitalized children in developing countries. clarification of this concept can absolutely help its definition and promotion of nursing care in pediatric wards. therefore, the researcher decided to analyze the concept of parents participation in taking care of hospitalized children. linguistic concepts are the presentation or mental images of phenomena, and are essential for occupational relationship. it is one of the conceptualization, concept involution, and theory development methods, which is used to modify the abstract concept and ambiguity in nursing. this model is generally applicable in all disciplines and practical sciences, especially in nursing. in fact, as hybrid model reveals the general insights and attitudes in a clinical setting, it is specifically used in studying the important nursing phenomena. one of the concepts that has a high application in clinical setting but lacks a clear definition in nursing in iran is the concept of parents participation in taking care of hospitalized children. this issue reveals the necessity of clarification of meaning and dimensions of the above - mentioned concept through hybrid model as the best content analysis method. this content analysis is conducted in three phases : theoretical, fieldwork, and analytical phases. in the theoretical phase, the theory maker vastly and structurally seeks for the study - related issues and application of the concept. the data related to fieldwork are collected through interviews with participants and then analyzed to extract and confirm the concept. comparison and giving a semantic meaning to the results and reviewing the practical and theoretical phases result in the ultimate phase, which is the final analysis. in this phase, for literature review of the articles published until august 2011 in relation with parents participation in taking care of hospitalized children, databases such as pubmed and proquest were searched using the key words, parental partnership, parental participation, family - centered care, parental involvement, hospital, child, and nurse. for literature review, manual article search and the references of the articles the articles with no access to full text were excluded and the rest indicating a definition or concept were selected and similar or opposite cases were reviewed. this phase was conducted through semi - structured interviews with 11 nurses working in two educational pediatric centers affiliated to tehran university of medical sciences. inclusion criteria were being interested in attending the study and sharing the experience and working in pediatric ward with at least 1 year of work experience. it has been used in numerous nursing researches and is applied as a systematic objective research method to explain qualitative phenomena. its qualitative approach is a flexible method for text data analysis, and is used when there is no adequate knowledge about a phenomenon. the transcriptions were reviewed several times line by line and the coded categories and subcategories were revealed by reduction process. after ending the content analysis, the themes related to various dimensions of the concept (parents participation in taking care of a hospitalized child) emerged. for rigor of the data, the researchers returned the transcriptions and extracted codes to the participants and colleagues, and based on their indications, the needed modifications were done. prolonged engagement in interviews transcripts was noticed in data analysis. it was tried to increase rigor of the data through sampling with the upmost variance by conducting interviews with nurses in different wards, with different work experiences, and working in various shits. all participants gave oral informed consent to attend the study and for their voices to be recorded. they were explained about the confidentiality of their information and their option to leave the study whenever they liked, as well as their right to receive the results in case of their request. in this phase, for literature review of the articles published until august 2011 in relation with parents participation in taking care of hospitalized children, databases such as pubmed and proquest were searched using the key words, parental partnership, parental participation, family - centered care, parental involvement, hospital, child, and nurse. for literature review, manual article search and the references of the articles the articles with no access to full text were excluded and the rest indicating a definition or concept were selected and similar or opposite cases were reviewed. this phase was conducted through semi - structured interviews with 11 nurses working in two educational pediatric centers affiliated to tehran university of medical sciences. inclusion criteria were being interested in attending the study and sharing the experience and working in pediatric ward with at least 1 year of work experience. it has been used in numerous nursing researches and is applied as a systematic objective research method to explain qualitative phenomena. its qualitative approach is a flexible method for text data analysis, and is used when there is no adequate knowledge about a phenomenon. the transcriptions were reviewed several times line by line and the coded categories and subcategories were revealed by reduction process. after ending the content analysis, the themes related to various dimensions of the concept (parents participation in taking care of a hospitalized child) emerged. for rigor of the data, the researchers returned the transcriptions and extracted codes to the participants and colleagues, and based on their indications, the needed modifications were done. it was tried to increase rigor of the data through sampling with the upmost variance by conducting interviews with nurses in different wards, with different work experiences, and working in various shits. related permissions were obtained from the heads of related hospitals. all participants gave oral informed consent to attend the study and for their voices to be recorded. they were explained about the confidentiality of their information and their option to leave the study whenever they liked, as well as their right to receive the results in case of their request. the concept of parents participation is not clearly defined in literature and texts, and has been mentioned by different terms such as involvement of the parents, participation in care, mutual participation, parental caregiving, and involving the parents as caregivers, but the simplest definition is parental participation, which means parents participation in taking care of a hospitalized child. this refers to the activities that are done by the parents for their child in hospital, so that they either conduct or share a part of caregiving. parents participation can be in the form of giving physical, psychological, or social care. literature review of the articles in relation with participation can help clarifying the dimensions and characteristics of this concept. table 1 briefly represents the various dimensions of parents participation in taking care of a hospitalized child. characteristics of the concept parents participation in taking care of a hospitalized child and the samples selected by literature review the characteristics, defined in table 1 for the concept parents participation in taking care of a hospitalized child can lead to a theoretical definition, i.e. parents active involvement in specific levels of care based on negotiation, agreement, and mutual open interactive relationship with nurses through exchange of information and increase in parents level of awareness and skills. after analysis of the main content, the dimensions of parents participation were obtained in four themes of parents nurses cooperation (with two subthemes of open communication and trust), education and increase of parents awareness and skill in care, assigning home cares to the parents (with three subthemes of physical, emotional, and safety), and finally, the range of participation and decision making (with two subthemes of domain and time of participation). the first important characteristic of the concept of parents participation in taking care of a hospitalized child is cooperation (with two subthemes of open communication and trust). in this 6 stated : they rely on us through a mutual communication which is made, then, they believe that we are suffering from their pain too. if someone is going to participate in care, he / she should be explained, to be explained means to spend time on him / her, so that the other side can trust on and cooperate with us. to achieve trust and finally patients cooperation, there should be a mutual communication between parents and nurses. having a hospitalized child is a great source of anxiety and stress for the parents as they face a role change. this is when they need help to overcome their role in taking care of their hospitalized child. participation is possible when there is a purposive system with an appropriate and complete planning and education. 5) access to current information about the child 's disease, diagnosis, and treatment helps the parents feel having control and security and adapt with their child 's hospitalization and have a greater share in care, as one of the patients explained : if we can educate the mother well, we are relaxed as we have told her what to do. 3) this is so that the parents, based on their ability, can have a part of care while the child is under supervision of treatment and nursing team. in this concern, i think giving care, personal hygiene and nutritional issues can be assigned to the mothers. the given care should also help us somehow, and the mother should be permitted to take care of her child when there are signs and symptoms of the disease. for example, when the child has a fever, the mother should not lose her heart. 8) assigning a part of care to the mothers decreases their anxiety and frequent and disturbing questions that negatively affect the quality of given care and increases the feeling of independency. the results obtained in the present study, of which some have been presented above, showed that nurses like to assign some of the cares to the parents. this issue requires spending time and supporting the mother. for education, based on mothers and children 's personal needs, care systems should be switched to patient - centered mode instead of duty - centered mode. table 2 presents the list of cares, which the parents can undertake, and are classified into three groups of physical, emotional, and safety cares. list of cares undertaken by mothers in physical, psychological care, and safety of their children in hospitals although the nurses pointed to assigning cares to mothers, they also emphasized on the fact that the level of parents participation must be determined and they should not handle all professional tasks, as nurse no. 5 indicated : they should do primary care and just in general wards as this does not decrease the load of work for the system (health system) the jobs they are aware of should be assigned to them which means primary care like cleaning the baby. on the whole, the ambiguous level and margin of parents participation and their involvement in all cares not only can not solve any problems but also may lead to malpractice and increased load of nurses work and physicians distrust toward nurses. assignment of the care to mothers should be limited to primary and basic care and what they may need in future. mothers should not be allowed to do the jobs which are not related to them, for example, suctioning (procedure), but what you know they can do, and the things not related to child 's recovery. in this phase, the findings of both theoretical and fieldwork phases were compared with each other and their results have been presented in two sections of outcomes and results and concept characteristics subsequently. outcomes and results were obtained in theoretical and fieldwork phases, of which one of the most basic prerequisites of parents participation in taking care of a hospitalized child is attendance of the parents by the child. in numerous studies, it has been emphasized that the parents should attend to their child to participate in care. the other prerequisite for parents participation is parents interest and positivity, as well as the attitude of the staffs to work with them. if parents are not aware of the importance and effects of their participation in care, nurses struggle to involve them in care is useless. on the other hand, the results and effects of parents participation obtained in both phases showed that parents participation has numerous positive effects including their satisfaction, anxiety and pain reduction, children 's sleep recovery, parents peace and encouragement, and appropriate communication with the staffs, feeling of capability in parents, more adaptation of parents with the child 's conditions, promotion of quality of care, and finally reduction of nurses work load. but in some cases, inappropriate results including parents feeling of incapability and stress in doing some of the cares, distance of mothers from family, and their job. this study tried to analyze the concept of parents participation in taking care of a hospitalized child from the viewpoint of the nurses, but as the parents also have a share in this issue, lack of parents experiences can be mentioned as a limitation of the present study. after analysis of the main content, the dimensions of parents participation were obtained in four themes of parents nurses cooperation (with two subthemes of open communication and trust), education and increase of parents awareness and skill in care, assigning home cares to the parents (with three subthemes of physical, emotional, and safety), and finally, the range of participation and decision making (with two subthemes of domain and time of participation). the first important characteristic of the concept of parents participation in taking care of a hospitalized child is cooperation (with two subthemes of open communication and trust). in this 6 stated : they rely on us through a mutual communication which is made, then, they believe that we are suffering from their pain too. if someone is going to participate in care, he / she should be explained, to be explained means to spend time on him / her, so that the other side can trust on and cooperate with us. to achieve trust and finally patients cooperation, there should be a mutual communication between parents and nurses. having a hospitalized child is a great source of anxiety and stress for the parents as they face a role change. this is when they need help to overcome their role in taking care of their hospitalized child. participation is possible when there is a purposive system with an appropriate and complete planning and education. 5) access to current information about the child 's disease, diagnosis, and treatment helps the parents feel having control and security and adapt with their child 's hospitalization and have a greater share in care, as one of the patients explained : if we can educate the mother well, we are relaxed as we have told her what to do. 3) this is so that the parents, based on their ability, can have a part of care while the child is under supervision of treatment and nursing team. in this concern, i think giving care, personal hygiene and nutritional issues can be assigned to the mothers. the given care should also help us somehow, and the mother should be permitted to take care of her child when there are signs and symptoms of the disease. for example, when the child has a fever, the mother should not lose her heart. 8) assigning a part of care to the mothers decreases their anxiety and frequent and disturbing questions that negatively affect the quality of given care and increases the feeling of independency. the results obtained in the present study, of which some have been presented above, showed that nurses like to assign some of the cares to the parents. this issue requires spending time and supporting the mother. for education, based on mothers and children 's personal needs, care systems should be switched to patient - centered mode instead of duty - centered mode. table 2 presents the list of cares, which the parents can undertake, and are classified into three groups of physical, emotional, and safety cares. list of cares undertaken by mothers in physical, psychological care, and safety of their children in hospitals although the nurses pointed to assigning cares to mothers, they also emphasized on the fact that the level of parents participation must be determined and they should not handle all professional tasks, as nurse no. 5 indicated : they should do primary care and just in general wards as this does not decrease the load of work for the system (health system) the jobs they are aware of should be assigned to them which means primary care like cleaning the baby. on the whole, the ambiguous level and margin of parents participation and their involvement in all cares not only can not solve any problems but also may lead to malpractice and increased load of nurses work and physicians distrust toward nurses. assignment of the care to mothers should be limited to primary and basic care and what they may need in future. 4 stated : mothers should not be allowed to do the jobs which are not related to them, for example, suctioning (procedure), but what you know they can do, and the things not related to child 's recovery. in this phase, the findings of both theoretical and fieldwork phases were compared with each other and their results have been presented in two sections of outcomes and results and concept characteristics subsequently. outcomes and results were obtained in theoretical and fieldwork phases, of which one of the most basic prerequisites of parents participation in taking care of a hospitalized child is attendance of the parents by the child. in numerous studies, it has been emphasized that the parents should attend to their child to participate in care. the other prerequisite for parents participation is parents interest and positivity, as well as the attitude of the staffs to work with them. if parents are not aware of the importance and effects of their participation in care, nurses struggle to involve them in care is useless. on the other hand, the results and effects of parents participation obtained in both phases showed that parents participation has numerous positive effects including their satisfaction, anxiety and pain reduction, children 's sleep recovery, parents peace and encouragement, and appropriate communication with the staffs, feeling of capability in parents, more adaptation of parents with the child 's conditions, promotion of quality of care, and finally reduction of nurses work load. but in some cases, inappropriate results including parents feeling of incapability and stress in doing some of the cares, distance of mothers from family, and their job. this study tried to analyze the concept of parents participation in taking care of a hospitalized child from the viewpoint of the nurses, but as the parents also have a share in this issue, lack of parents experiences can be mentioned as a limitation of the present study. when there is no unique perception for a concept, the application of that concept is under question, communication is disturbed, and individuals responses will be based on their personal temporary judgments. clarification of the concept of participation is a difficult task as it has various meanings among people. although the concept of parents participation is clear for teachers and professionals in clinical setting, quality care necessitates its clarification as a basic issue. the results of the present study on investigating and comparing various dimensions of parents participation in taking care of a hospitalized child in two phases of theoretical and fieldwork have been presented in table 3. comparison of the findings in the two above - mentioned phases shows that almost all components of theoretical phase and fieldwork phase coincide with each other. finally, it can be noted that the concept of parents participation in taking care of a hospitalized child means an open and interactive communication between nurses and parents, educating the parents about the process of treatment and care, assigning child 's home needed cares to parents and involving them in caring process, and eventually, determination of the range of parents participation in order to promote the quality of cares. characteristics of the concept of parents participation in taking care of a hospitalized child in theoretical and fieldwork phases in the investigation of the obtained components, the first important and notable element of this concept was open and interactive communication between two sides. this open communication absolutely can help concurrent usage of parents and nurses knowledge and their mutual cooperation. the more active this communication is at the beginning of staffs exposure to the family, the more the possibility of parents trust to negotiate and come on an agreement with nurses is. negotiation is especially valuable when the parents face problems and are disappointed. in such conditions, nurses can help the family resist against problems through an appropriate communication and timely intervention. the second component of this concept was increase of parents awareness and skill in caregiving. nurses determine the level and manner of parents participation through the information they pass on to them, their support toward them, and the method in which they communicate with the parents. another component in the concept of parents participation in taking care of children is their involvement in care. in this regard, coyne states that parental care refers to assignment of a part of cares to the parents based on their capability and with the permission of treatment and nursing team while the child is under their supervision. the clear role of parents in taking care of a hospitalized child is one of the important components of parents participation in care. the difference between patients and nurses perceptions about the behaviors, which are considered as cares, causes conflicts between nurses and parents, lowers the quality of care, and increases dissatisfaction in both sides. parents should be clearly active in decision - making process concerning their childcare, allocation of the person who takes care of their child, or the level of their own participation. meanwhile, nurses should determine their clear expectations for the activities they like the parents to participate in. one of the points discussed in the investigation of the two above - mentioned phases was the difference in parents participation, which means despite identical conditions, the level of parents participation in taking care of children can be different. it can be possibly due to some factors such as parents age, level of education, and cultural backgrounds. the findings of other studies revealed that the parents with higher education as well as younger parents are more eager for participation, while elder parents prefer to leave the care for the staffs. it is concluded that preparation of executive backgrounds and encouraging parents to participate in taking care of a hospitalized child, as well as adequate nursing human resources, spending more time on patients by nurses, and existence of a legal background to support nurses are essential. therefore, conducting studies on the obstacles and facilitating elements for this concept, as well as a similar study on parents viewpoints can help its application. | background : although today parents participation in taking care of hospitalized children is considered as an indispensable principle, it is still among the concepts with no consensus about. the main objective of this study is to define parents participation in taking care of hospitalized children.materials and methods : the concept of parents participation in taking care of hospitalized children was analyzed using a hybrid model in three phases : literature review (theoretical phase), fieldwork, and combination of literature review and fieldwork (analytical phase).results : based on the results of theoretical (literature review), fieldwork, and analytical phases, the best definitions for the concept of parents participation in taking care of hospitalized children are mutual relationship and gaining parents trust toward nurses, giving the required information and education to the parents about care and treatment process, assigning the needed home care to the parents, involving the parents in caregiving process, and finally, defining their participation in decision making (clarifying the parents role) in order to improve the quality of care given to the children.conclusions:the findings of this study showed that the dimensions of parents participation can be applied in pediatric wards, and nurses can improve the quality of care through application of the obtained findings. |
pttm (pulmonary tumor thrombotic microangiopathy), synonymously termed microscopic pulmonary tumor embolism, is a rare condition, and cancer - related pulmonary complications characterized by rapidly progression of dyspnea and pulmonary hypertension occasionally cause sudden death 1. pttm is observed in 3.3% of autopsies of patients with malignant tumors and is usually associated with gastric cancer, although it was reported to affect other sites in a few cases 1,2. rare cases of urothelial carcinoma of the urinary bladder associated with pttm have also been reported 1,3,4. most of the patients in these case reports were diagnosed as having pttm following postmortem examination at autopsy. we report a valuable case of pttm associated with urothelial carcinoma of the urinary bladder in which pulmonary microvascular cytology (pmc) was a useful strategy that aided in the early antemortem diagnosis, permitting the patient to undergo chemotherapy, although the patient ultimately died. the patient was a 77-year - old japanese man with a history of transurethral resection of a bladder tumor at the age of 75 years for urothelial carcinoma of the urinary bladder. pathological findings of the surgical specimens obtained indicated a urothelial carcinoma (g2) that had invaded the muscular layer (fig.1). afterward, he underwent intravesical bacillus calmette - guerin therapy and radiation therapy that resulted in complete remission. in april 2014, he developed a relapse with abdominal metastatic lymph nodes and bone metastasis found, so he underwent chemotherapy with carboplatin and gemcitabine. after 2 cycles this chemotherapy, he developed dyspnea and was referred to our hospital in july 2014. on admission, he had tachycardia (112 beats per minute), tachypnea (31 respirations per minute), and normotensive blood pressure (118/62 mmhg). initial peripheral arterial oxygen saturation (spo2) was 81%, and blood gas analysis showed a pao2 (partial pressure of oxygen in arterial blood) of 46.6 torr and paco2 (partial pressure of carbon dioxide in arterial blood) of 34.2 torr on room air, indicating hypoxemia. his white blood cell count was 5000/l, hemoglobin 7.3 g / dl, and thrombocyte count 10.6 10/l, indicating anemia and thrombocytopenia. c - reactive protein level was elevated at 6.1 mg / dl, and brain natriuretic peptide was 425 pg / ml. a transthoracic echocardiogram showed a normal left ventricular ejection fraction with an elevated estimated systolic pulmonary artery pressure of 89 mmhg. a whole - body contrast - enhanced ct (computed tomography) scan revealed diffuse small nodules, ground - glass opacities, and dilation of the pulmonary arteries in the lungs. other findings included abdominal lymph node swelling and bone metastasis, without obvious pulmonary embolism or deep vein thrombosis (fig.2). microscopic findings of the tumor show urothelial carcinoma of the urinary bladder (hematoxylin and eosin stain). chest ct (computed tomography) showed ground - glass opacities in both lung fields (a). high - resolution ct showed small nodules and ground - glass opacities in the upper lung field (b) and small nodules, ground - glass opacities, and dilation of the pulmonary arteries in the lower lung (c). a tc - maa lung perfusion image showed multiple peripheral defects. during the patient 's hospital course, pmc was performed, for which a swan - ganz catheter was inserted and located in the right pulmonary artery wedge position, and blood was gently withdrawn from the wedged catheter. hemodynamic parameters monitored during the procedure included a mean pulmonary capillary wedge pressure of 14 mmhg ; mean pulmonary artery pressure of 41 mmhg ; and cardiac index of 2.3 l / min / m, which indicated pulmonary hypertension without left cardiac failure. because malignant cells tend to accumulate in the buffy coat of centrifuged blood, slides were made from the buffy coat and were immediately fixed in 95% alcohol and stained using the papanicolaou and giemsa methods. the cytological specimens showed small loose clusters of large atypical cells resembling urothelial carcinoma cells (fig.4). upper and lower gastrointestinal tract endoscopy, small intestine endoscopy, and bone marrow biopsy provided no meaningful results : none of these tests indicated malignant disease or urothelial carcinoma of the urinary bladder. tblb (transbronchial lung biopsy) was performed three times in the right lower lung, but the specimens could not be reveal atypical cells in the vessels. although specimens from tblb also did not provide meaningful results, on the basis of the clinical, radiological, and cytological findings, we diagnosed the patient as having pttm with urothelial carcinoma of the urinary bladder because of the existence of atypical cells resembling urothelial carcinoma cells in the pulmonary microvasculature and the complete absence of malignancy in other organs. chemotherapy with gemcitabine and paclitaxel was started on hospital day 8. however, the patient 's pulmonary hypertension progressed, his breathing rapidly deteriorated, and he died on hospital day 12. pulmonary microvascular cytology showed large atypical cells resembling urothelial carcinoma cells (a : papanicolaou stain, b : giemsa stain). we describe a case of pttm in which pmc detected tumor cells strongly suspicious of urothelial carcinoma of the urinary bladder. pttm should be considered in the differential diagnosis of a patient with cancer who presents with subacute dyspnea. pttm is characterized by the following findings : (1) the presence of tumor emboli in the small pulmonary arteries ; (2) fibrocellular and fibromuscular intimal proliferation in the small pulmonary arteries ; and (3) organization and recanalization of thrombi. the second finding is particularly serious and is very difficult to differentiate from a simple pulmonary arterial tumor emboli. the presence of minute tumor emboli in the peripheral pulmonary arteries can damage the vascular endothelium, leading to thrombus formation and accelerated coagulation 5. pttm is usually associated with adenocarcinoma, particularly gastric adenocarcinoma and adenocarcinomas of the pancreas, breast, lung, and liver 1,2. however, there are a few cases reports of pttm or microscopic pulmonary tumor embolism associated with urothelial carcinoma of the urinary bladder 1,3,4,69. to the best of our knowledge, including the present case, only two cases have been reported in which pmc detected tumor cells that led to a diagnosis of pttm associated with urothelial carcinoma of the urinary bladder 9. the development of clinical signs of pulmonary hypertension result in acute or subacute cor pulmonale and subacute respiratory failure. moreover, stenosis and/or occlusion of the pulmonary arteries occur along with an increase in pulmonary vascular resistance that results in pulmonary hypertension, hemolytic anemia, and disseminated intravascular coagulation 1. the ct findings of pttm include consolidation, ground - glass opacities, small nodules, and tree - in - bud appearance 2. in the present patient, in addition, multiple small and wedge - shaped perfusion detects in a lung perfusion scan are characteristic of pttm 11. on the basis of the clinical and radiological findings, we considered a diagnosis of pttm induced by urothelial carcinoma of the urinary bladder, and pmc, bone marrow biopsy, and tblb were performed. pulmonary microvascular cytology, the cytological study of blood drawn from a wedged pulmonary artery catheter to detect material in the pulmonary microvasculature, was first reported in 1979 in patients with amniotic fluid embolism 12. 13 showed the oxygen saturation of blood withdrawn from pulmonary artery catheters in the wedge position has characteristics of pulmonary capillary blood. in reality, since the catheter lodges in an artery of substantial size, a wedge sample most likely represents a mixture of blood from the terminal pulmonary arteries, arterioles, and capillaries in the subsegmental distribution of the occluded vessel. we also thought that pmc was useful diagnosis method of pttm, which was characterized by the occlusion of arterioles, capillaries, and venules throughout the body by malignant tumor cells. pmc has been extended to the diagnosis of fat embolism, pulmonary microvascular metastasis, and intravascular lymphoma 1417. similar to these disorders, a few cases of pttm were reported in which tumor cells were detected by pmc 14,18. other antemortem diagnostic methods have been suggested, including video - assisted thoracic surgery and tblb 19,20. our patient could not be diagnosed as having pttm from the tblb findings, and we could not perform further examinations because of the patient 's poor condition. however, pmc was successful in detecting tumor cells in the pulmonary microvasculature of our patient. the point where we should be careful about the cytological study is that the presence of megakaryocytes is rarely seen in the peripheral blood. therefore, we should avoid of making mistake such that the megakaryocyte was recognized as atypical cell. although an invasive procedure, pulmonary artery catheterization is generally well tolerated in comparison with video - assisted thoracic surgery and tblb. due to the extremely rapid progression of pttm, few cases have been reported in which systemic chemotherapy has improved the prognosis 19, and no previous cases of chemotherapy administered after the diagnosis of urothelial carcinoma of the urinary bladder associated with pttm have been reported. although the condition of our patient did not improve after chemotherapy and progressed to death, we believe that early diagnosis by pmc may be valuable in improving the prognosis of patients with pttm. on the other hand, anticoagulation therapies and new drugs for the treatment of pulmonary arterial hypertension, such as endothelin antagonists, prostacyclin analogs, and phosphodiesterase type 5 inhibitors, are controversial for the treatment of pttm 21. 22 reported that imatinib may be effective for ameliorating pulmonary hypertension that is caused by pttm. we report a rare case of urothelial carcinoma of the urinary bladder associated with pttm in which an antemortem diagnosis was made with the aid of pmc, permitting the patient to undergo chemotherapy. if the possibility of pttm is considered in cancer patients with a rapidly worsening respiratory condition, we believe that pmc may represent the only chance for diagnosis and achievement of remission in this aggressive disease, particularly for critically ill patients. | key clinical messagepttm (pulmonary tumor thrombotic microangiopathy) is very difficult to diagnose before death. we report a case of urothelial carcinoma of the urinary bladder associated with pttm in which an antemortem diagnosis by pmc (pulmonary microvascular cytology). pmc may represent the only chance for diagnosis and achievement of remission in pttm. |
laparoscopic pyeloplasty has emerged as the most common and widely accepted minimally invasive surgery for ureteropelvic junction obstruction (upjo). this technique has advantages of a high success rate similar to open pyeloplasty and low morbidity similar to endopyelotomy. schuessler were the first authors to report on transperitoneal laparoscopic dismembered pyeloplasty in 5 patients and concluded that the procedure, though time - consuming, was a feasible treatment option for upjo. janetschek were the first authors to report on retroperitoneoscopic dismembered pyeloplasty and concluded that the procedure is too complicated to be a standard procedure. since then, there have been many publications on both types of laparoscopic pyeloplasties, but the choice of whether to perform the procedure by the transperitoneal or retroperitoneoscopic route depends on the surgeon. in transperitoneal laparoscopic pyeloplasty (tplp), the large peritoneal cavity allows for free movement of instruments and intracorporeal suturing, but the rate of bowel - related complications and pain are higher in comparison with retroperitoneoscopic laparoscopic pyeloplasty (rplp). in rplp limited retroperitoneal space restricts free movement of instruments and leads to difficulty in intracorporeal suturing, leading to a longer operating time, but postoperative pain and the rate of bowel - related complications are comparatively lower. most of the published studies are either on tplp or rplp, and some have assessed a combination of both types with nonrandomized comparison and were retrospective in nature. however, the success rate for both procedures is comparable. review of the literature shows only 1 prospective randomized study on the comparison of tplp and rplp for primary upjo. in view of the excellent outcome results of both techniques in isolation with advantages and disadvantages of both procedures, we executed a prospective randomized trial of tplp versus rplp for primary upjo, aiming to determine which technique is better with respect to success rate and postoperative pain and complications. in this prospective randomized study, a total of 112 patients who underwent primary upjo were included, from january 2008 to december 2012. ethical approval was received in accordance with the declaration of helsinki. clinical history and findings of general physical and abdominal examinations patients were examined by routine hemogram, renal function tests, liver function tests, coagulation profile, urine culture, and sensitivity. we performed renal ultrasonography ; intravenous urogram (ivu) ; contrast computed tomography (ct) scans of the kidney, ureter, and bladder ; and diuretic renogram using tc - diethylenetriamine penta - acetic acid (dtpa) scans. for grading of hydronephrosis, we used society for fetal urology universal criteria (table 1). from dtpa scans, split renal function and subrenal obstruction were documented. spiral ct scans showed anterior crossing vessels at the ureteropelvic junction (upj) in 44 patients. patients with a deranged coagulation profile, previous renal surgery, malrotated kidneys, huge hydronephrotic kidneys, or vertebral - spinal deformities were excluded from the study. after providing written informed consent, patients were randomized into 2 groups at a 1:1 ratio based on systematic random sampling. group i consisted of tplp patients, and group ii comprised rplp patients. with the patients under general anesthesia, cystoscopy and retrograde ureteropyelography were performed before surgery. comparison of baseline characteristics in tplp and rplp groups asa = american society of anesthesiologists ; bmi = body mass index. the patient was placed in the kidney position, carbon dioxide pneumoperitoneum created, and the procedure performed with 3 ports. anterior crossing vessels were identified in 21 cases and were dissected to free them from the upj. the stenotic / aperistaltic segment of the ureter was excised, and the ureteric end was spatulated laterally for about 2.5 cm. 40 polyglactin as a continuous suture over a 6f/26-cm double - j stent. in patients with anterior crossing vessels, after this, the rest of the pelvis was closed with continuous no. 40 polyglactin suture. the patient was placed in the kidney position, and the retroperitoneal space was created by a combination of blunt finger and indigenous balloon dissection methods. the stenotic / aperistaltic ureteric segment was excised, the ureteric end was spatulated for about 2.5 cm, and a 6f/26-cm double - j stent was inserted. there were multiple stones (20 stones), which were removed with a flexible cystoscope. the tube drain was removed after 24 to 48 hours depending on the drain output. the visual analog scale (vas) score for pain was determined on postoperative day 1 and day 2. a liquid diet was allowed once bowel sounds had returned, and the diet was increased gradually. the foley catheter was removed on the second to third postoperative day, and the patient was discharged with advice of taking medications including suppressive antibiotic. patients who were symptomatic at follow - up were examined with renal ultrasonography, ivu, and dtpa scans. these patients were then followed up every 3 months, and dtpa scans were performed every 12 months. immediate and long - term postoperative complications were recorded using the modified clavien grading system. statistical analysis was performed with spss software (version 16.0 ; spss, chicago, illinois) using the fisher exact test, test, and mann - whitney u test, and p 0.5 or 0.5 or < 0.5. a total of 112 patients were included, with 56 patients in group i (tplp) and 56 patients in group ii (rplp). open conversion was performed in 1 patient in the tplp group and 2 patients in the rplp group. the laparoscopic procedures were successfully completed in 109 patients : 55 in the tplp group (98.2%) and 54 in the rplp group (96.4%). the demographic characteristics of the patients were comparable in both groups (table 1). anterior crossing vessels were present in 44 cases (39.2%) (table 1). caliceal stones were present in 4 patients in group i and 6 patients in group ii (table 1). the differences in total operative time and intracorporeal suturing time were statistically significant between the 2 groups (table 2). the vas pain score was significantly higher and the requirement for tramadol on the first postoperative day and hospital stay were significantly greater in the tplp group (table 2). postoperative complications (according to the modified clavien grading system) were seen in 14.8% of patients (table 3). clavien grade i complications were seen in 3.6% of patients, grade ii in 18%, and grade iiib in 7.2%. temporary ileus was seen in 10.8% of patients in the tplp group (p <.027) (table 3). secondary upjo was observed in 2 patients in the tplp group at a mean follow - up time of 7 months and in 2 patients in the rplp group at 6 months ; it was managed by open pyeloplasty, and postoperative dtpa scans at 3 and 12 months showed nonobstructive drainage. the mean follow - up period of patients was comparable in both groups (table 2). the overall success rate was 96.4% for tplp versus 96.6% for rplp with a mean follow - up period of 30.75 4.85 months versus 30.99 5.59 months (p <.88). the pearson correlation coefficient for total operative time in rplp patients with different demographic and operative parameters did not show any significant association. comparison of operative and postoperative results between tplp and rplp groups postoperative modified clavien complications in tplp and rplp groups uti = urinary tract infection. schuessler were the first authors to describe tplp for primary upjo, in 1993. in 1996 both of these techniques are now well established, with excellent success rates, and each has its own advantage and disadvantages. in tplp the peritoneal cavity there are risks of bowel injury, ileus, and peritoneal contamination by leaked urine, infection, and carbonic acid. however, tplp provides a larger peritoneal cavity for instrument handling and intracorporeal suturing. in rplp there is minimal risk of bowel - related complications and contamination of the peritoneal cavity. retroperitoneoscopy allows a limited working space for instrument handling and intracorporeal suturing but provides direct access to the ureteropelvic regions. in our study the total operative time and intracorporeal suturing time were significantly higher in the rplp group. the difference in the vas pain score on the first postoperative day, requirement for tramadol, and hospital stay were significantly higher in the tplp group (table 2). relatively greater pain in tplp patients is due to traversing of the peritoneal cavity, manipulation, and reflection of the colon, which requires more tramadol for pain management. anterior crossing vessels were present in 44 patients (39.2%), similar to findings previously reported in the literature. in the tplp group, the ureteropelvic reconstruction was performed anterior to the anterior crossing vessels, whereas in the rplp group, it was performed behind the anterior crossing vessels, in a manner similar to the tplp. our technique was consistent with the basic principle of andersen - hynes open pyeloplasty in which transposition of the ureteropelvic anastomosis must be performed anterior to the anterior crossing vessels. the transposition is easy to perform in tplp, but in rplp this remains a concern. in our study we did not encounter difficulty in achieving this in rplp, and routine transposition was performed. in retroperitoneoscopy the crossing vessels are easily and better seen with simple elevation of the lower pole of the kidney, and transposition and reconstruction are not difficult to perform. in our study, in no cases of retroperitoneoscopy did we encounter any difficulty in performing the dismembered pyeloplasty in the presence of anterior crossing vessels. after dissection from the upj and disconnection of the ureter from the pelvis undergo a cephalad shifting and do not impede the reconstructive procedure. the reconstructed ureteropelvic region, though lying anterior to the anterior crossing vessels, practically lies inferior to the vessels and hence does not create difficulty in performing anastomosis. the crossing vessels have been postulated to be involved in the etiopathogenesis of upj and to be an indicator of poor outcome after endopyelotomy. in some studies on rplp, the transposition was not routinely performed ; rather, in selected cases, the vessels were dissected and placed cephalad to the upj, and ureteropelvic anastomosis was performed. the comparison of results with or without transposition did not show any difference in outcome. however, the selection of cases for not to transpose was not very clearly defined. a 70-fold increased risk of stones developing in primary upjo has been reported by husmann. the increased risk is not because of obstruction leading to delayed washout of urinary solutes and infection ; rather, metabolic abnormalities are the most accepted theory. in our study caliceal stones were present in 4 patients in the tplp group and 6 patients in the rplp group. concomitant stone extraction was performed successfully in all cases. in 1 patient with multiple stones (20 stones), concomitant stone extractions with a flexible nephroscope and cystoscope have similarly been reported by other authors. the important early complications are leakage of urine from the anastomotic site, infection, and bowel - related complications. regarding delayed complications, secondary upjo due to stenosis / stricture of the reconstructed ureteropelvic region is very important and influences the success rate of pyeloplasty. in tplp temporary ileus ileus is a comparatively under - reported complication of transperitoneal laparoscopic surgery, and it must be recognized because it has postoperative implications in patient management. in our study the rate of temporary ileus was significantly higher in the tplp group ; temporary ileus was also observed in some previous studies on tplp. our study ct urogram showed a mean stricture length of 2.2 cm with grade iv hydronephrosis in all patients. open pyeloplasty was performed, and follow - up with dtpa scan showed nonobstructive drainage. the decision to perform open or laparoscopic pyeloplasty depends on the surgeon 's judgment and radiologic findings. in our study the decision to perform open pyeloplasty was made because of the presence of a long stricture length and consent for open surgery. the subjective assessment is based on clinical improvement, and the objective assessment is based on ivu (patency of upj with decrease / absence of hydronephrosis) and nonobstructed drainage on nuclear scans. devenport, in a retrospective analysis, reported a 92% success rate for tplp versus 67% for rplp. the drawbacks of their study were the small number of patients in the rplp group, in whom transposition of the anterior crossing vessels was not performed, and the effect of the learning curve for retroperitoneoscopy in the initial study period. inagaki, in a retrospective study, reported a 95% overall success rate for tplp, including both dismembered and non - dismembered pyeloplasty. soulie reported an 88.9% success rate for rplp, but the main drawback of the study was assessment of success based on excretory urography. abuanz, in a retrospective nonrandomized study, reported an 87% success rate for rplp versus 82% for tplp (85% overall success rate). the drawbacks of their study were the involvement of multiple surgeons, comparatively small sample size, and difficulty in controlling follow - up in some patients. the explanation for the comparatively lower success rate (85%) in this study was the comparatively longer follow - up period (48.93 38.94 months), which might have altered the long - term success of the procedures. shoma, in a prospective randomized comparison between tplp and rplp, showed 95% and 90% success rates, respectively, with mean follow - up periods of 23 months and 20 months, respectively. eden, in a prospective study, reported a 97.5% success rate for rplp with a mean follow - up period of 19.7 months. janetschek, in an interesting study, reported a 98.5% success rate for fenger - plasties at long - term follow - up. in our study the success rate was 96.4% for tplp versus 96.6% for rplp with a mean follow - up period of 30.75 4.85 months versus 30.99 5.59 months (p <.88). follow - up in this study was performed with ivu and dtpa scans at 3 months and then by dtpa scans every 12 months. our study is the first prospective randomized study that included a good sample size with long - term follow - up. all procedures were performed by a single urologist using 3 ports to allow a fair comparison of all postoperative parameters including pain. the vas scoring was performed on the first and second postoperative days by a person who was blinded to the procedure type, and the complications were recorded using the modified clavien grading system. although the operative time is significantly higher for rplp, tplp is significantly associated with postoperative pain, requirement for tramadol, hospital stay, and ileus in comparison with rplp. | background and objectives : to compare laparoscopic transperitoneal versus retroperitoneoscopic pyeloplasty for primary ureteropelvic junction obstruction in a prospective randomized manner and assess overall results with long - term follow-up.methods:in this prospective study, from 2008 to 2012, 112 cases of primary ureteropelvic junction obstruction were randomized in a 1:1 ratio into 2 groups. group i included patients who underwent transperitoneal laparoscopic pyeloplasty, and group ii consisted of patients who underwent retroperitoneoscopic laparoscopic pyeloplasty. demographic and clinical characteristics and postoperative and operative data were collected and analyzed. the statistical analysis was performed with the fisher exact test, 2 test, and mann - whitney u test for independent groups, and p <.05 was considered statistically significant.results:the total operative time and intracorporeal suturing time were significantly higher in group ii than in group i (p <.001). the visual analog scale score for pain on postoperative day 1 and the requirement for tramadol were significantly higher in group i than in group ii (p =.004). the hospital stay and the rate of temporary ileus were significantly greater (p <.036 and p <.02, respectively) in group i than in group ii. the success rate of transperitoneal laparoscopic pyeloplasty versus retroperitoneoscopic laparoscopic pyeloplasty was 96.4% versus 96.6% with a mean follow - up period of 30.75 4.85 months versus 30.99 5.59 months (p <.88).conclusion : transperitoneal laparoscopic pyeloplasty is associated with significantly greater postoperative pain, a higher tramadol dose, a higher rate of ileus, and a longer hospital stay in comparison with retroperitoneoscopic laparoscopic pyeloplasty. although the operative time for retroperitoneoscopic laparoscopic pyeloplasty is significantly longer, the success rate remains the same for both procedures. |
joint hypermobility is often described as an extreme variation of joint laxity causing a complex of musculoskeletal symptoms. the most common criteria often used to identify individuals with joint hypermobility is a modification of the original score reported by beighton., which developed based on the primary work of carter and wilkinson. joint hypermobility with associated musculoskeletal symptoms such as osteoarthritis, joint derangements and instabilities, joint effusions and muscular pain is characterized as benign joint hypermobility syndrome (bjhs) but based on recent studies naming this syndrome as a benign previous studies on generalized joint hypermobility, defined by the criteria of beighton, show an overall wide range of prevalence between 11.2% and 64.6% in children and adolescents. the variability of these results can be due to various methods of evaluation and stringency of criteria among previous studies. excessive joint laxity, or hypermobility, should be considered as a common finding of clinical importance in the management of musculoskeletal disorders. hypermobility is common in young patients and in general is associated with an increased incidence of musculoskeletal injuries. ankle sprains, knee ligament injuries, shoulder instabilities, and osteoarthritis of the hand have been implicated in joint hypermobility. patients with hypermobility often complain of diffuse musculoskeletal pain and injuries with no specific inciting event. orthopedic surgeons and other healthcare providers should be aware of the underlying relationship between hypermobility and musculoskeletal disorders to avoid unnecessary diagnostic tests and inappropriate management. prolonged therapy and general conditioning are typically required, with special emphasis on improving muscle strength and proprioception to address symptoms and prevent future injury. an effective management program may include medications including nonsteroidal anti - inflammatory drugs, acetaminophens, tri - cyclic antidepressants, serotonin / norepinephrine receptor inhibitors, physical therapy, cognitive - behavioral therapy and lifestyle modifications for relieving pain. generalized joint hypermobility has been proposed as a risk factor for injuries to the ankle, knee, and shoulder joints. although the relationship between generalized joint hypermobility and injury to the ligamentous structures in the ankle and knee joints has been examined, less is known about the effect on joint instability. several authors have suggested that generalized joint hypermobility may be related to joint instability, but this relationship has not been adequately investigated. generalized joint hypermobility is implicated in joint instability, but its relationship has not been yet established. individuals with generalized joint hypermobility may be at increased risk of physical activities - related injuries, and one of the high - risk groups for these injuries is military population. hence, this study evaluated the prevalence of bjhs among the active - duty soldiers and the effect of training courses on the related joint instabilities such as the ankle sprain, temporomandibular joint (tmj) and shoulder dislocations. the study was a prospective cohort study conducted on iranian army soldiers of training periods in one of the military bases of isfahan in 2013. inclusion criteria were : soldiers of the training period deployed to the military service during 2013 and not affected by any rheumatologic and connective tissue disorders leading to joint laxity with participant consent. exclusion criteria were : patients with any mental or physical disorders or any history of drug dependency causing joint weakness or severe disability that would have disrupted the examination and may leading to exemption from combat. this study was reviewed and approved in the ethics committee of the aja university of medical sciences and after being justified on the study objectives and consent, soldiers were consciously enrolled in the study. the active - duty soldiers were first examined by a physician and their beighton scores (bs) were obtained. soldiers divided in two groups of healthy and suffered based on their scores. the prevalence of ankle sprain, shoulder and tmj dislocations identified before beginning service by history - taking and reviewing paraclinical documents (such as radiographs, magnetic resonance imaging and surgical report sheets, previous history of ankle sprain, shoulder dislocation and tmj clicks or dislocation). after passing 3 months of military training (including endurance exercises, physical strength practices and specific military educations), soldiers in two groups of healthy and suffered of bjhs were reevaluated in terms of recent occurrence of mentioned diseases. patients suspected to any dislocations were reexamined by the cooperative orthopedist to make a definite diagnosis and control radiographies were taken for ruling out of possible fractures in shoulder or ankle joints. the five criteria of bs are : passive dorsiflexion of the fifth finger of the hands over 90;passive flexion of the thumbs to the flexor surface of the forearms;passive hyperextension of the elbows over 10;passive knee hyperextension over 10 ; andforward trunk flexion as the knees fully extended and palmar surface of the hands resting on the floor. passive dorsiflexion of the fifth finger of the hands over 90 ; passive flexion of the thumbs to the flexor surface of the forearms ; passive hyperextension of the elbows over 10 ; passive knee hyperextension over 10 ; and forward trunk flexion as the knees fully extended and palmar surface of the hands resting on the floor. for each hypermobile joint, one point is given with an additional point for positive trunk hyperflexion for a total score of 9. although there is no universal agreement on the value needed to make a diagnosis for bjhs using the bs, most researchers use a score of 4/9. based on the bs (achieving the minimum score of 4 from beighton criteria), the studied soldiers were divided into two groups with bjhs and group of healthy soldiers. furthermore, based on achieving minimum score of 5 and 6 from beighton criteria, classification for suffering from the syndrome was done, and the studied variables were compared between groups. the results obtained from analyzing variables have been reported as mean standard deviation and frequency (percentage) according to the type of variables. to compare the mean age and bs in individuals with bjhs with the healthy subjects and the frequency of ankle sprain, shoulder dislocation and tmj dislocation, independent - t and chi - square tests were used, respectively. in all cases, out of 721 studied soldiers, two cases were excluded because of exemption and discharge from service before ending the training period and one case was excluded due to desertion from the service. among from 718 soldiers, bjhs (achieving the minimum score of 4 from beighton criteria) was diagnosed in 211 subjects (29.4%) before military training. then, the prevalence of mentioned musculoskeletal complaints before training and their incidence during the training period were studied and analyzed in soldiers with bjhs and healthy soldiers. the study flowchart is given in figure 1. in two groups, the mean age was similar, and there was no significant difference between them. the comparison of bs in soldiers with bjhs (achieving the minimum score of 4 from beighton criteria) with the healthy soldiers shows that in the suffered soldiers, the mean bs was significantly higher than is in the healthy soldiers (p > 0.001) [table 1 ]. comparison the age and bs between healthy soldiers and those with bjhs the prevalence of ankle sprain, shoulder dislocation and tmj dislocation compared in two groups of soldiers before military training, and there was no significant difference between the suffered and healthy groups [table 2 ]. comparison the prevalence of ankle sprain, shoulder dislocation and tmj dislocation in suffered and healthy soldiers before military training at the end of the training period, the incidence of ankle sprain, shoulder dislocation and tmj dislocation in suffered soldiers (achieving the minimum score of 4 from beighton criteria) is given compared with that of healthy soldiers. as it can be observed in figure 2, there was no statistically significant difference between the studied groups in terms of the incidence of shoulder dislocation (p = 0.34) and tmj dislocation (p = 0.08) during the training period. however, the incidence of ankle sprain at the end of the period, in the suffered group, was significantly higher than that in the healthy group (p = 0.03). comparison the incidence of dislocations after 3 months training period between healthy soldier (bs < 4, n = 507) and those with bjhs (bs 4, n = 211). 0.05, p values calculated by chi - square test based on achieving the minimum score of 5 from beighton criteria, the incidence of mentioned dislocations in the suffered soldiers (beigton score 5) compared to other soldiers (beigton score < 5). during the training period, the incidence of ankle sprain (p = 0.005) and tmj dislocation (p = 0.03) in the suffered soldiers was significantly greater than that in the healthy individuals. whereas the incidence of shoulder dislocation between the two groups during the course was not significant again (p = 0.18) [figure 3 ]. comparison the incidence of dislocations after 3 months training period between all soldier with bs 5 (n = 145) and those with bs < 5 (n = 573). bs = beighton score ; p < 0.05, p values calculated by chi - square test moreover, when we considered beigton score 6, the incidence of ankle sprain, shoulder and tmj dislocations in the suffered soldiers (score 6) compared to other soldiers (score < 6) were not statistically significant. difference between the incidence of shoulder dislocation at the end of the training period among studied groups was not significant (p = 0.36). while the incidence of ankle sprain (p = 0.01) and tmj dislocation (p = 0.03) was significantly higher in the suffered soldiers in comparison to healthy ones [figure 4 ]. comparison the incidence of dislocations after 3 months training period between all soldier with bs 6 (n = 77) and those with bs < 6 (n = 641). bs = beighton score ; p < 0.05, p values calculated by chi - square test in this study, the prevalence of bjhs among the active - duty soldiers and the effect of the training period on the three common dislocations including ankle sprain, tmj and shoulder dislocation has been investigated. the frequency of the bjhs found as 29.4% which is higher than what scher. achieved in their study on the incidence of joint hypermobility syndrome (jhs) in the us military population as a raw incidence rate of 0.06/1000 person - years. current literature suggests jhs, defined as generalized hypermobility associated with musculoskeletal symptoms, occurs in approximately 5 - 6% of adults. the lower incidence of jhs in the us military population may be secondary to a protective effect of the muscle strengthening and endurance training programs required by the us military personnel or selection of healthy persons in initial screening for employment. in our small subpopulation in the iranian military population, we found no differences in the prevalence of jhs by age group, this is perhaps because most of our soldiers are a subset of primarily young population without army or athletic training before that does not include either children or the elderly population. previous studies of jhs suggest a trend toward decreasing prevalence of the disorder with increasing age. several authors have speculated on the relationship between generalized joint hypermobility and joint instability ; however, this relationship has not been examined empirically. warner. observed generalized joint hypermobility in 25% of normal control participants and 22% of those with glenohumeral joint instability but did not examine the relationship between glenohumeral joint instability and measures of generalized joint hypermobility. cooper and brems reported that 29 of 38 surgical patients (76%) with multidirectional glenohumeral joint instability demonstrated generalized joint hypermobility, but as in the previous study the relationship between these variables was not examined. in an epidemiologic study of traumatic shoulder dislocations, the estimated incidence rate of shoulder dislocations in the united states was reported as 23.9/100,000 person - years, which was approximately twice the previously reported value. a young age and male sex are risk factors for shoulder dislocation in the united states population. according to our knowledge there are a few studies on the incidence of hypermobility related dislocations among the military force in the world, but regarding the owens. study, the overall incidence rate was 1.69 dislocations per 1000 person - years, and the significant demographic risk factors were male sex, the white race, service in the army, junior enlisted rank, and age of < 30 years. in another studies by owens. the incidence and characteristics of shoulder instability at the united states military academy was reported as a common injury in this population, with subluxations comprising 85% of instability events. in the present study, we have shown a relationship between increased joint hypermobility based on beighton criteria and joint instability among active - duty soldiers during the training period. participants with increased joint hypermobility, defined as a total bs of 4 or greater, reported a higher incidence of two common joint instabilities (ankle and tmj) during the training. moreover, by higher scores in the suffered patients, we observed higher incidence of dislocations in soldiers with joint hypermobility. in the case of shoulder dislocation although no statistically significant difference between two groups was obtained, the incidence of this joint instability in suffered group was higher than that in the control group. thus, our results provide preliminary evidence to support the relationship between bjhs and joint instability when the influences of sex and race were controlled (female are exempt from military service in iran and because the sample population included the duty soldiers there were no significant difference in the age, and all of them were iranian). clinically, this investigation provides preliminary support for increased bjhs as a potential predisposing risk factor for joint instability or dislocation, especially ankle joint, in military force. military training can increase the incidence of ankle sprains and tmj dislocations in hypermobility persons with higher bs during training course in comparison with healthy people, but it does nt seem this course have a significant effect on the incidence of shoulder dislocations. therefore, screening of generalized joint hypermobility may be useful in identifying individuals at increased risk for ankle, tmj or even other joint instabilities, although more prospective researches with larger populations is needed to confirm this. ka and pm carried out the design and coordinated the study, participated in most of the experiments and prepared the manuscript. arh provides assistance in the design of the study, coordinated and carried out all the experiments and participated in manuscript preparation. | background : hypermobile joints are joints with beyond normal range of motion and may be associated with joint derangements. this study aimed to evaluate the prevalence of benign joint hypermobility syndrome (bjhs) among soldiers and effect of training courses on related joint instabilities.materials and methods : in a prospective cohort study on 721 soldiers of iran army in isfahan in 2013 the prevalence of joint hypermobility was obtained by using beighton criteria. soldiers divided in two groups of healthy and suffered based on their scores. the prevalence of ankle sprain, shoulder and temporomandibular joint (tmj) dislocations identified before beginning service by history - taking and reviewing paraclinical documents. after 3 months of military training, a recent occurrence of mentioned diseases was revaluated in two groups. the collected data were analyzed using spss-20 software using independent - t and chi - square tests.results:the frequency of bjhs before military training was 29.4%. after passing military training period, the incidence of ankle sprain was significantly higher in suffered group achieving the minimum beighton score (bs) of 4 (4.3%, p = 0.03), 5 (5.5%, p = 0.005) and also 6 out of 9 (6.5%, p = 0.01). the incidence of tmj dislocation was not significantly different based on a minimum score of 4, while it was higher in suffered group when considering the score of 5 (2.1%) and 6 (2.6%) for discrimination of two groups (p = 0.03). there was no significant difference between two groups in case of shoulder dislocation anyway.conclusion:military training can increase the incidence of ankle sprains and tmj dislocations in hypermobility persons with higher bs in comparison with healthy people. therefore, screening of joint hypermobility may be useful in identifying individuals at increased risk for joint instabilities. |
chronic obstructive pulmonary disease (copd) is a progressive disease defined by an airflow limitation that is not fully reversible.1 patients with the disease exhibit pathologic features of emphysema and, to varying degrees, chronic bronchitis. exacerbations, which can be life - threatening for certain patients, are characterized by an amplified inflammatory response and worsening of airflow. these changes in symptoms are sufficient to warrant a change in management and occur an average of approximately three times per year for patients with moderate to severe copd.2 exacerbations are associated with diminished quality of life for copd patients,3,4 and their frequency and severity, as well as the presence of comorbid conditions and the number of medications needed to manage the disease, all contribute to copd severity.1 copd is highly prevalent in the us5,6 and worldwide.7,8 the us prevalence6 was estimated at 10 million adults in 2000, and in 2007, 5.8% of individuals aged 4564 years had physician - diagnosed copd.5 copd is a leading cause of death in the us, and related mortality has been reported to be greater for men than for women, and mortality rates are highest for white patients, followed by black, hispanic, and asian individuals.5 management of stable copd is intended to prevent and control symptoms and to reduce the frequency and severity of exacerbations.1,9,10 maintenance medications for copd include long- and short - acting bronchodilators, and inhaled corticosteroids for patients with repeated exacerbations. fixed - dose combination therapies include short - acting beta agonist (saba)/anticholinergic bronchodilator combinations, and long - acting beta agonist (laba)/corticosteroid combinations.1 patients with very severe copd might receive oxygen therapy. rescue medication can be started at home, but if the exacerbation is not well managed, hospitalization may be required.1,9 patients with severe exacerbations might be managed in the emergency department (ed) or hospital, but life - threatening exacerbations may require immediate intensive care unit (icu) admission.1 treatment for exacerbations includes increasing the dose or frequency of saba administration, adding an anticholinergic bronchodilator, administering oral or intravenous corticosteroids, or oxygen therapy. because exacerbations can be caused by bacterial infection, antibiotics might also be given. these events (ed visit, hospitalization) and changes in treatment (saba, oral / intravenous corticosteroids, antibiotics) are strongly associated with copd exacerbation and have been used to identify exacerbations in clinical research.1114 the high prevalence and morbidity of copd contributed to a projected $ 29.5 billion in related direct costs in 2010 in the us5 and the annual health care cost was estimated at $ 4119 per copd patient in 2000.15 hospitalization is a major cost driver in copd management in various health care systems,5,14,1618 with hospital care projected to account for 45% of direct copd costs in the us in 2010.5 prescription drugs comprise approximately 20% of copd costs.5 disease16 and exacerbation14,17 severity have both been identified as copd cost drivers. however, published data appear to be lacking regarding costs for us commercially insured patients and us costs according to copd severity. our objective was to estimate direct costs of copd- related care for commercially insured patients in the us, including both health plan- and patient - paid amounts. specifically, we wanted to determine the overall costs generated by patients who received care of different intensities, and the costs of different types of copd - related health care visits ranging from outpatient visits to icu stays. we also extrapolated the mean annual direct health care cost of copd for the us managed care population. data for this retrospective analysis were obtained from a managed care claims database which included geographically diverse commercial health plan members in the us. enrollment information and medical and pharmacy claims from calendar year 2006 were used in the analysis. in order to extrapolate cost information from the database population to all commercially insured patients in the us, data from the 2006 medical expenditure panel survey (meps), the most recent available, were also used.19 meps is a set of nationally representative us surveys that collect information on health service use, including demographic characteristics and health insurance coverage. commercially enrolled patients with evidence of copd from january 01, 2006 through december 31, 2006 were included in the study population. copd patients were identified in one of the following ways : an ed or inpatient facility claim with copd (icd-9-cm code 491, 491.0, 491.1, 491.2x, 491.8, 491.9, 492, 492.0, 492.8, 496) in the primary diagnosis position (the diagnosis code must have been on a hospital claim for an inpatient stay) ; an outpatient (routine or urgent care) claim with a primary diagnosis of copd and a second medical claim with a copd diagnosis in any position on a separate service date ; or a physician claim with a primary or secondary diagnosis of copd and a filled prescription for an anticholinergic, laba, or inhaled corticosteroid / laba combination. eligible patients were aged 40 years or older as of 2006 and were continuously enrolled in the health plan with medical and pharmacy benefits during the year - long study period. all data were deidentified and accessed with protocols compliant with the health insurance portability and accountability act.20 privacy board approval was obtained for use of race / ethnicity data. patient demographic data, including age, gender, and geographic region were collected from enrollment information. charlson comorbidity score21 and a flag for asthma diagnosis (icd-9-cm 493.xx), and on fills of respiratory medications (based on pharmacy and medical claims) was collected to characterize further the patients in the study sample. patients were grouped into five mutually exclusive cohorts based on the most intensive type of copd - related care they had received during the study year. patients in the outpatient cohort had at least one medical claim for office or outpatient care with copd indicated in the primary or secondary position, but no evidence of an urgent visit (defined below), ed visit, or inpatient stay for copd. outpatient care included physician office visits, outpatient hospital services, laboratory visits, and urgent care center visits. patients in the urgent outpatient cohort had at least one medical claim for outpatient care for copd (primary or secondary position) followed by a pharmacy claim for an oral corticosteroid or antibiotic within seven days after the visit, but no evidence of more serious care. patients in the ed cohort had at least one medical claim for an ed visit for copd (primary position) but no evidence of an inpatient stay for copd. standard admission cohort comprised patients with at least one inpatient stay for copd (copd diagnosis in the primary position on a hospital claim), but no evidence of icu treatment during any inpatient stay. patients in the icu cohort had evidence of icu care during a copd inpatient stay during the study period. icu care was defined as a medical claim during an inpatient stay with revenue code of 020x, 021x, 0223 or 0234 or a current procedural terminology procedure code for critical care (9929199292). these cohort definitions reflect copd health care encounters associated with regular follow - up and exacerbations of increasing severity.1,11,13 for an additional patient - level cost analysis, study patients were grouped into cohorts based on race / ethnicity as indicated in enrollment information (african american, caucasian, hispanic, other / unknown). cost calculations were based on total paid amounts in 2006, including patient- and health plan - paid amounts, as well as estimated medicare or other payer contributions based on coordination of benefits information. to enable comparisons with current literature, patient- and episode - level costs, as well as costs used in the extrapolation calculation, were adjusted to 2008 us dollars using the annual medical care component of the consumer price index.22 patient- and episode - level costs associated with copd - related (copd diagnosis code on a claim associated with the visit) outpatient visits, urgent outpatient visits, ed visits, standard admissions (no icu), and icu stays were identified. the criteria used to identify these types of visits were the same as those used to define the patient cohorts. costs associated with copd - related care of any intensity that was received by each patient, including the cohort - determining episode type and less serious visits, were included in the analyses. copd - related pharmacy costs were summed from pharmacy claims for any of the following medications : sabas, labas, anticholinergics, methylxanthines, oral / intravenous corticosteroids, inhaled corticosteroids, inhaled corticosteroid / laba combination, other respiratory medication, or antibiotics. in the patient - level analysis, mean annual total health care costs, as well as costs for each type of copd - related visit, other copd - related medical services (from claims with copd diagnosis in the primary position that did not fall into the specified categories), and medications were calculated for each of the patient cohorts. costs per copd - related care episode in 2006 were tabulated based on all episodes of a given type for each cohort during the year, and adjusted episode - level costs were estimated using multivariate models. generalized linear models with gamma distribution and log link were used to account for the skewed distribution of costs.23 the models of ed and inpatient episode - level costs were adjusted for gender, age category, geographic region, race / ethnicity, quan the models of episode - level outpatient costs were adjusted for the variables listed above, as well as copd diagnosis code specific to the visit, provider type, and season of episode. a weighted average of total direct annual copd - related costs (medical and pharmacy) for the us managed care population was extrapolated, based on cost estimates from the sample represented in the study database and demographic data from 2006 meps. patient - level cost distributions were compared among cohorts using the t - test for continuous variables and the chi - square test for proportions and categoric variables. the multivariate models were fitted by stata 10.0 (statacorp, college station, tx) and all other analyses were conducted using sas 9.1 (sas institute, cary, nc). data for this retrospective analysis were obtained from a managed care claims database which included geographically diverse commercial health plan members in the us. enrollment information and medical and pharmacy claims from calendar year 2006 were used in the analysis. in order to extrapolate cost information from the database population to all commercially insured patients in the us, data from the 2006 medical expenditure panel survey (meps), the most recent available, were also used.19 meps is a set of nationally representative us surveys that collect information on health service use, including demographic characteristics and health insurance coverage. commercially enrolled patients with evidence of copd from january 01, 2006 through december 31, 2006 were included in the study population. copd patients were identified in one of the following ways : an ed or inpatient facility claim with copd (icd-9-cm code 491, 491.0, 491.1, 491.2x, 491.8, 491.9, 492, 492.0, 492.8, 496) in the primary diagnosis position (the diagnosis code must have been on a hospital claim for an inpatient stay) ; an outpatient (routine or urgent care) claim with a primary diagnosis of copd and a second medical claim with a copd diagnosis in any position on a separate service date ; or a physician claim with a primary or secondary diagnosis of copd and a filled prescription for an anticholinergic, laba, or inhaled corticosteroid / laba combination. eligible patients were aged 40 years or older as of 2006 and were continuously enrolled in the health plan with medical and pharmacy benefits during the year - long study period. all data were deidentified and accessed with protocols compliant with the health insurance portability and accountability act.20 privacy board approval was obtained for use of race / ethnicity data. patient demographic data, including age, gender, and geographic region were collected from enrollment information. charlson comorbidity score21 and a flag for asthma diagnosis (icd-9-cm 493.xx), and on fills of respiratory medications (based on pharmacy and medical claims) was collected to characterize further the patients in the study sample. patients were grouped into five mutually exclusive cohorts based on the most intensive type of copd - related care they had received during the study year. patients in the outpatient cohort had at least one medical claim for office or outpatient care with copd indicated in the primary or secondary position, but no evidence of an urgent visit (defined below), ed visit, or inpatient stay for copd. outpatient care included physician office visits, outpatient hospital services, laboratory visits, and urgent care center visits. patients in the urgent outpatient cohort had at least one medical claim for outpatient care for copd (primary or secondary position) followed by a pharmacy claim for an oral corticosteroid or antibiotic within seven days after the visit, but no evidence of more serious care. patients in the ed cohort had at least one medical claim for an ed visit for copd (primary position) but no evidence of an inpatient stay for copd. standard admission cohort comprised patients with at least one inpatient stay for copd (copd diagnosis in the primary position on a hospital claim), but no evidence of icu treatment during any inpatient stay. patients in the icu cohort had evidence of icu care during a copd inpatient stay during the study period. icu care was defined as a medical claim during an inpatient stay with revenue code of 020x, 021x, 0223 or 0234 or a current procedural terminology procedure code for critical care (9929199292). these cohort definitions reflect copd health care encounters associated with regular follow - up and exacerbations of increasing severity.1,11,13 for an additional patient - level cost analysis, study patients were grouped into cohorts based on race / ethnicity as indicated in enrollment information (african american, caucasian, hispanic, other / unknown). cost calculations were based on total paid amounts in 2006, including patient- and health plan - paid amounts, as well as estimated medicare or other payer contributions based on coordination of benefits information. to enable comparisons with current literature, patient- and episode - level costs, as well as costs used in the extrapolation calculation, were adjusted to 2008 us dollars using the annual medical care component of the consumer price index.22 patient- and episode - level costs associated with copd - related (copd diagnosis code on a claim associated with the visit) outpatient visits, urgent outpatient visits, ed visits, standard admissions (no icu), and icu stays were identified. the criteria used to identify these types of visits were the same as those used to define the patient cohorts. costs associated with copd - related care of any intensity that was received by each patient, including the cohort - determining episode type and less serious visits, were included in the analyses. copd - related pharmacy costs were summed from pharmacy claims for any of the following medications : sabas, labas, anticholinergics, methylxanthines, oral / intravenous corticosteroids, inhaled corticosteroids, inhaled corticosteroid / laba combination, other respiratory medication, or antibiotics. in the patient - level analysis, mean annual total health care costs, as well as costs for each type of copd - related visit, other copd - related medical services (from claims with copd diagnosis in the primary position that did not fall into the specified categories), and medications were calculated for each of the patient cohorts. costs per copd - related care episode in 2006 were tabulated based on all episodes of a given type for each cohort during the year, and adjusted episode - level costs were estimated using multivariate models. generalized linear models with gamma distribution and log link were used to account for the skewed distribution of costs.23 the models of ed and inpatient episode - level costs were adjusted for gender, age category, geographic region, race / ethnicity, quan charlson comorbidity score, and asthma diagnosis. the models of episode - level outpatient costs were adjusted for the variables listed above, as well as copd diagnosis code specific to the visit, provider type, and season of episode. a weighted average of total direct annual copd - related costs (medical and pharmacy) for the us managed care population was extrapolated, based on cost estimates from the sample represented in the study database and demographic data from 2006 meps. patient - level cost distributions were compared among cohorts using the t - test for continuous variables and the chi - square test for proportions and categoric variables. the multivariate models were fitted by stata 10.0 (statacorp, college station, tx) and all other analyses were conducted using sas 9.1 (sas institute, cary, nc). descriptive characteristics of the patient cohorts are shown in table 1. based on the distribution of patients in the cohorts, approximately 90% of eligible copd patients did not have an ed or inpatient visit during the study year. the source database population comprises heavily caucasian individuals and patients in the south region, and the racial and geographic distribution of the database is reflected among copd patients included in the study, as shown in table 1. age differed significantly among cohorts, with mean age youngest in the urgent outpatient cohort and oldest in the standard admission cohort. charlson comorbidity score differed significantly among the care - intensity cohorts, with the lowest mean scores in the outpatient cohorts and the highest mean score in the icu cohort (table 1). comorbidity scores also differed significantly by race / ethnicity (p < 0.001), hispanic patients had the lowest mean (standard deviation, sd) score at 2.2 (1.8), caucasian patients had a mean score of 2.3 (1.8), and african american patients had the highest mean score at 2.5 (2.0). as expected, the percentage of each cohort that used each type of medication during the study year generally increased across cohorts (table 1). almost half of the patients in the study sample received a saba at some point in 2006, with the lowest percentages in the outpatient cohorts. oral / intravenous corticosteroid receipt ranged from 30% of patients in the outpatient cohort to 76% of patients in the icu cohort. oxygen therapy use increased across the cohorts, from 14% of the outpatient cohort to 57% of the icu cohort. approximately 35% of patients used more than one medication class during the study year and 17% of all patients used no maintenance treatment (inhaled corticosteroid, laba, inhaled corticosteroid / laba combination, anticholinergic, methylxanthine), but did receive rescue medication (ie, saba, oral / intravenous corticosteroid, antibiotic). without regard to cohort, the copd patients included in the study had a mean (sd) of 2.7 (2.9) outpatient visits, 0.7 (1.2) urgent outpatient visits, 0.08 (0.6) ed visits, and 0.05 (0.3) standard admissions during 2006. the patient cohorts were defined based on the most intensive level of care received, but patients also had visits of less serious types. within each cohort, outpatient visits were the most frequently observed type, with the mean count for the study year ranging from 2.5 (3.2) for the urgent outpatient cohort to 4.6 (4.6) for the icu cohort (p < 0.001 across cohorts). the mean count of standard admissions ranged from 0.2 (0.6) for the icu cohort to 1.1 (0.5) for the standard admission cohort (p < 0.001). among patients who had an inpatient stay (ie, patients in the standard admission or icu cohorts), the mean duration of a standard (non - icu) inpatient stay was 10.2 (21.1) days. the icu cohort averaged 1.1 (0.4) icu visits in 2006, and the mean duration of an icu stay was 1.9 (2.9) days. as shown in figure 1, mean (sd) total annual patient - level copd - related costs increased across the cohorts, ranging from $ 2003 ($ 3238) to $ 43,461 ($ 76,159). medical costs, which include costs associated with health care visits, comprised the largest portion of copd - related costs across all cohorts. mean total medical costs increased substantially across cohorts (from $ 1004 per year for the outpatient cohort to $ 41,727 per year for the icu cohort ; p < 0.001 among cohorts) but mean pharmacy costs per year changed relatively little (from $ 999 for the outpatient cohort to $ 1734 for the icu cohort ; p < 0.001 among cohorts). thus, medical costs accounted for a progressively greater percentage of total copd - related health care costs ; up to 90% for the standard admission cohort and 96% for the icu cohort. all together, study patients generated $ 101,284,860 in copd - related medical costs in 2006 and 34% of that amount was attributable to the icu cohort. thus, although patients in the icu cohort comprised 2.2% of the copd population eligible for the study, they accounted for the greatest percentage of copd - related medical costs. mean annual patient - level costs of each type of copd - related visit are shown in figure 2. costs associated with each type of visit differed significantly among the applicable cohorts (p < 0.001 for all among - cohort comparisons). as expected, mean expenditures associated with hospitalization and icu care were substantially greater than costs for other types of visits. as shown in figure 3, mean total copd costs in managed care patients copd- related health care costs were approximately 21% to 25% lower for hispanic patients than for patients of other races (among - group p = 0.135). the adjusted mean costs of a health care episode of each type are shown in figure 4. episode costs increased for more serious types of visits, with the most notable difference between outpatient / ed visits and inpatient / icu visits. the adjusted mean cost of a copd - related standard admission among all patients who had one was $ 9745, and an icu stay increased the cost to $ 33,440 per episode. based on meps data and data from this study, the 2006 prevalence of copd among commercially insured patients in the us was estimated at 2.9 million. this population was predicted to generate $ 15.7 billion (in 2008 us$) in annual direct health care costs, comprising approximately $ 12.4 billion in medical costs and $ 3.3 billion in copd - related pharmacy costs. descriptive characteristics of the patient cohorts are shown in table 1. based on the distribution of patients in the cohorts, approximately 90% of eligible copd patients did not have an ed or inpatient visit during the study year. the source database population comprises heavily caucasian individuals and patients in the south region, and the racial and geographic distribution of the database is reflected among copd patients included in the study, as shown in table 1. age differed significantly among cohorts, with mean age youngest in the urgent outpatient cohort and oldest in the standard admission cohort. charlson comorbidity score differed significantly among the care - intensity cohorts, with the lowest mean scores in the outpatient cohorts and the highest mean score in the icu cohort (table 1). comorbidity scores also differed significantly by race / ethnicity (p < 0.001), hispanic patients had the lowest mean (standard deviation, sd) score at 2.2 (1.8), caucasian patients had a mean score of 2.3 (1.8), and african american patients had the highest mean score at 2.5 (2.0). as expected, the percentage of each cohort that used each type of medication during the study year generally increased across cohorts (table 1). almost half of the patients in the study sample received a saba at some point in 2006, with the lowest percentages in the outpatient cohorts. oral / intravenous corticosteroid receipt ranged from 30% of patients in the outpatient cohort to 76% of patients in the icu cohort. oxygen therapy use increased across the cohorts, from 14% of the outpatient cohort to 57% of the icu cohort. approximately 35% of patients used more than one medication class during the study year and 17% of all patients used no maintenance treatment (inhaled corticosteroid, laba, inhaled corticosteroid / laba combination, anticholinergic, methylxanthine), but did receive rescue medication (ie, saba, oral / intravenous corticosteroid, antibiotic). without regard to cohort, the copd patients included in the study had a mean (sd) of 2.7 (2.9) outpatient visits, 0.7 (1.2) urgent outpatient visits, 0.08 (0.6) ed visits, and 0.05 (0.3) standard admissions during 2006. the patient cohorts were defined based on the most intensive level of care received, but patients also had visits of less serious types. within each cohort, outpatient visits were the most frequently observed type, with the mean count for the study year ranging from 2.5 (3.2) for the urgent outpatient cohort to 4.6 (4.6) for the icu cohort (p < 0.001 across cohorts). the mean count of standard admissions ranged from 0.2 (0.6) for the icu cohort to 1.1 (0.5) for the standard admission cohort (p < 0.001). among patients who had an inpatient stay (ie, patients in the standard admission or icu cohorts), the mean duration of a standard (non - icu) inpatient stay was 10.2 (21.1) days. the icu cohort averaged 1.1 (0.4) icu visits in 2006, and the mean duration of an icu stay was 1.9 (2.9) days. as shown in figure 1, mean (sd) total annual patient - level copd - related costs increased across the cohorts, ranging from $ 2003 ($ 3238) to $ 43,461 ($ 76,159). medical costs, which include costs associated with health care visits, comprised the largest portion of copd - related costs across all cohorts. mean total medical costs increased substantially across cohorts (from $ 1004 per year for the outpatient cohort to $ 41,727 per year for the icu cohort ; p < 0.001 among cohorts) but mean pharmacy costs per year changed relatively little (from $ 999 for the outpatient cohort to $ 1734 for the icu cohort ; p < 0.001 among cohorts). thus, medical costs accounted for a progressively greater percentage of total copd - related health care costs ; up to 90% for the standard admission cohort and 96% for the icu cohort. all together, study patients generated $ 101,284,860 in copd - related medical costs in 2006 and 34% of that amount was attributable to the icu cohort. thus, although patients in the icu cohort comprised 2.2% of the copd population eligible for the study, they accounted for the greatest percentage of copd - related medical costs. mean annual patient - level costs of each type of copd - related visit are shown in figure 2. costs associated with each type of visit differed significantly among the applicable cohorts (p < 0.001 for all among - cohort comparisons). as expected, mean expenditures associated with hospitalization and icu care were substantially greater than costs for other types of visits. as shown in figure 3, mean total copd costs in managed care patients copd- related health care costs were approximately 21% to 25% lower for hispanic patients than for patients of other races (among - group p = 0.135). the adjusted mean costs of a health care episode of each type are shown in figure 4. episode costs increased for more serious types of visits, with the most notable difference between outpatient / ed visits and inpatient / icu visits. the adjusted mean cost of a copd - related standard admission among all patients who had one was $ 9745, and an icu stay increased the cost to $ 33,440 per episode. based on meps data and data from this study, the 2006 prevalence of copd among commercially insured patients in the us was estimated at 2.9 million. this population was predicted to generate $ 15.7 billion (in 2008 us$) in annual direct health care costs, comprising approximately $ 12.4 billion in medical costs and $ 3.3 billion in copd - related pharmacy costs. our study provides estimated episode - level copd - related encounter costs, including costs of an outpatient visit, ed visit, standard hospitalization and hospital admission resulting in an icu stay, as well as patient - level direct annual copd - related health care costs. the results show that costs increase according to the intensity of care received and expand on previous studies with the inclusion of commercial managed care patients, outpatient encounters, and costs stratified according to resource use suggestive of a range of copd severities. stanford and dalal provided hospital - perspective episode - level costs of encounters for a national sample of copd patients for 2001 and 2005 through 2008. these previous studies reported that icu visits, and intubation specifically, accounted for a small proportion of copd- related encounters, but a high proportion of costs. costs associated with an icu stay were approximately triple the costs of a standard admission for their national samples of patients with various health care payers, as well as for the commercial managed care patients in our study. consistent with their hospital - perspective observations,24,25 we found that the icu cohort generated a disproportionate share of health care costs from the perspective of patients and health plan payers. the cost of an icu episode was much higher than costs for other types of visits, and mean annual health care costs were also substantially greater for patients who had an icu encounter. these studies suggest that minimizing the need for hospitalization, and the icu in particular, could lower copd - related health care costs. as guidelines advocate1 and observational studies bear out,2,14 our findings are consistent with reports indicating that escalated use of medical resources drives copd - associated health care costs.5,1418 our results further suggest that care indicative of more severe exacerbations (ie, hospitalization and icu care) is associated with higher episode- and patient - level costs for patients in the us. a canadian study of copd - related health care encounters also determined that severe exacerbations requiring hospitalization were associated with substantially higher episode - level costs than moderate - severity exacerbations, defined according to treatment in an outpatient or ed setting.14 disease management initiatives targeted to preventing exacerbations may reduce health care costs by minimizing copd - related need for inpatient resources.26 in addition, previous research suggests that reducing exacerbation frequency may improve patient quality of life.3 the results presented here must be interpreted in the context of certain limitations. as for all claims - based studies, it should be noted that claims are collected for payment, not research, and the analysis is constrained by the codes and other information contained in claims. if, for example, a critical care code was present on a claim for care that did not actually occur in the icu, then icu episodes might be overestimated based on our criteria. however, we believe that such cases are rare, and that the codes used to identify episode types provide a reasonable estimate of the range of care received by copd patients. because oxygen is often administered in the inpatient setting, this could account for the unexpectedly low proportion of patients in the icu cohort that had evidence of oxygen use. the data limit our cost analysis to direct costs, but because indirect costs of copd are also substantial,5,15,27 the health care costs presented here underestimate the overall burden of copd. in addition, clinical measures of copd severity, such as spirometry, were not available in the claims data to validate the cohort classification based on the level of care the patients received. with regard to the analyses by race, differences in health status and treatment patterns, as well as differences in care - seeking behavior, could affect the reported observations. claims data enabled us to focus on commercial managed care patients, but our findings might not generalize to patients with other types of health care plans, most notably medicare. finally, the extrapolation method assumes that the distribution of copd diagnoses and costs of the population in the research database are similar to the us managed care population. total direct health care costs are driven by medical costs, and copd - related medical resource use may be considered a proxy for exacerbations. patients with care indicative of more severe exacerbations had both higher episode- and patient - level costs. these findings suggest that improved symptom management and reduction in exacerbations would reduce the need for escalated care and subsequently reduce overall costs. | purposeto estimate patient- and episode - level direct costs of chronic obstructive pulmonary disease (copd) among commercially insured patients in the us.methodsin this retrospective claims - based analysis, commercial enrollees with evidence of copd were grouped into five mutually exclusive cohorts based on the most intensive level of copd - related care they received in 2006, ie, outpatient, urgent outpatient (outpatient care in addition to a claim for an oral corticosteroid or antibiotic within seven days), emergency department (ed), standard inpatient admission, and intensive care unit (icu) cohorts. patient- level copd - related annual health care costs, including patient- and payer - paid costs, were compared among the cohorts. adjusted episode - level costs were calculated.resultsof the 37,089 copd patients included in the study, 53% were in the outpatient cohort, 37% were in the urgent outpatient cohort, 3% were in the ed cohort, and the standard admission and icu cohorts together comprised 6%. mean (standard deviation, sd) annual copd - related health care costs (2008 us$) increased across the cohorts (p < 0.001), ranging from $ 2003 ($ 3238) to $ 43,461 ($ 76,159) per patient. medical costs comprised 96% of health care costs for the icu cohort. adjusted mean (sd) episode - level costs were $ 305 ($ 310) for an outpatient visit, $ 274 ($ 336) for an urgent outpatient visit, $ 327 ($ 65) for an ed visit, $ 9745 ($ 2968) for a standard admission, and $ 33,440 for an icu stay.conclusiondirect costs of copd - related care for commercially insured patients are driven by hospital stays with or without icu care. exacerbation prevention resulting in reduced need for inpatient care could lower costs. |
other side effects of chemotherapy are alopecia, stomatitis, immunosuppression, anorexia, nausea, and vomiting which result in a decreased functional capacity and quality of life for cancer patients. 5-fluorouracil (5-fu) is a chemotherapeutic agent used to treat gastrointestinal, breast, pancreatic, and head and neck cancer, among others. the mechanism of cytotoxicity of 5-fu has been ascribed to the misincorporation of fluoronucleotides into rna and dna and to the inhibition of the nucleotide synthetic enzyme thymidylate synthase. 5-fu distributes readily into bone marrow, small intestine, kidney, liver, and spleen [5, 6 ]. in the bone marrow 5-fu, it is incorporated in the dna and induces oxidative stress, which is partly responsible for myelotoxicity [7, 8 ]. it is well known that patients treated with 5-fu are cursed with neutropenia, mucositis, leukopenia, and hematological toxicity [9, 10 ]. because of these side effects, chemoprotective compounds have been used to reduce these problems [1119 ]. bovine dialyzable leukocyte extract or immunepotent crp (icrp) is a dialysate of a heterogeneous mixture of low - molecular - weight substances released from disintegrated leukocytes of the blood or lymphoid tissue obtained from homogenized bovine spleen. icrp was capable of stimulating the immune system in patients with non - small cell lung cancer and increasing their quality of life. also, in vitro studies demonstrated that icrp was an effective therapeutic agent in process involving oxidative cellular damage and clinical inflammatory diseases, through ib / nf-b pathway. in this study, we examined the protector effect of icrp on myelosuppression caused by 5-fu in a mouse model. nine - week - old male balb / c mice were obtained from the bioterium of the laboratorio de inmunologa y virologa de la facultad de ciencias biolgicas. the mice were maintained on pelleted food and water ad libitum and housed in controlled environmental conditions (25c and a 12 h light / dark cycle). the protocol for the animal study was approved by ethic review committee for animal experimentation of the facultad de ciencias biolgicas, uanl. immunepotent crp (icrp) was produced by the laboratorio de inmunologa y virologa, facultad de ciencias biolgicas, uanl (san nicols de los garza, nuevo len, mexico). icrp is a low - molecular - weight product (1012 kda) from bovine spleen. the extract is dialyzed, lyophilized, and determined as pyrogen - free by limulus of amoebocyte lysate assay (endotoxin detection kit, icn biomedical, aurora, oh, usa). the icrp obtained from 1 10 leukocytes is defined as one unit (1 u). mice were randomly divided into 5 groups as follows : control : injected i.p. on day 0 and i.m. with 5-fu in a single dose of 75 mg / kg. nac + 5-fu : injected i.p. with nac in a single dose of 250 mg / kg and one hour later with 5-fu i.p. in a single dose of 75 mg / (5 u), one hour later with 5-fu i.p. in a single dose of 75 mg / kg, and for the 6 consecutive days with icrp (5 u) per day. 5-fu : injected i.p. with 5-fu in a single dose of 75 mg / kg. nac + 5-fu : injected i.p. with nac in a single dose of 250 mg / kg and one hour later with 5-fu i.p. in a single dose of 75 mg / (5 u), one hour later with 5-fu i.p. in a single dose of 75 mg / kg, and for the 6 consecutive days with icrp (5 u) per day. bone marrow cells were flushed with 5 ml of iscove 's modified dulbecco 's medium (imdm) and supplemented with 2% fetal bovine serum (fbs), antimitotic, and antibiotics. cells suspensions were centrifuged at 1600 rpm for 10 min and washed twice in imdm. final suspension was used for total bone marrow cell count, bone marrow colony forming units - granulocyte / macrophage (cfu - gm) assay, cell cycle, and flow cytometric analysis. after the cells were pooled from both femurs and tibias, a count was done by trypan blue exclusion technique, which helps us exclude dead cells from viable cells. to calculate the number of cells obtained from each mouse, 50 l of the cell suspension was taken and this was transferred to 400 l of medium plus 50 l of trypan blue ; 10 l of this suspension was taken and placed in the neubauer chamber (bright line, reichert, usa). a total of 1 10 bm cells were resuspended in 1 ml imdm supplemented with 2% of fbs, and then 300 l of this suspension was added to 3 ml of mouse methylcellulose complete media (r&d systems). subsequently, the mixture was collected with a 3 ml syringe and 1.1 ml of the final mixture was placed in a 35 mm diameter culture dish ; this was done in duplicate. the formation of colonies was observed by microscopy, and the total number of colonies in each dish was counted. the staining procedures were performed using a bd cycle test plus dna reagent kit according to the instructions of the manufacturer. cell cycle phase distributions were analyzed in accuri c6 flow cytometer (bd biosciences, san jose, ca). in addition, the percentage of cells in each phase of cell cycle was analyzed by flowjo software (treestar, inc. bm cells were stained using fluorescent label - conjugated anti - cd71, anti - ter119, anti - cd45, anti - cd11b, and anti - gr-1 antibodies (bd biosciences, san jose, ca). for intracellular staining, nrf2 (d1z9c) xp rabbit mab (pe conjugate) was used, following the technique provided by the manufacturer. for measure of oxidative stress, total the stained cells were analyzed by accuri c6 flow cytometer and cflow plus software (bd biosciences, san jose, ca). the left femoral bone of each mouse was prepared for general histopathological evaluation, including fixation, decalcification, and sectioning (4 m thickness), as well as hematoxylin and eosin (he) staining. blood collection was done by cardiac puncture in edta containing vials for immediate analysis of hematological parameters. hematological analysis was determined by standard clinical procedures using an automatic hematological analyzer (coulter act diff analyzer, beckman coulter). measurement of weight in grams of the mice was performed at the beginning of treatment and seven days later. data was presented as mean sd and statistically analyzed using one - way anova test followed by tukey multiple comparison posttest at p < 0.05 (n = 3) using spss v17 software. the evaluation of the total number of bm cells and the number of cfu - gm was performed 1 and 7 days after the initiating treatment. the number of total bm cells was significantly (p < 0.05) decreased in all the groups treated with 5-fu at day 1. seven days later, the icrp + 5-fu group showed a recovery compared to 5-fu (p < 0.05) and nac + 5-fu groups. on the other hand, the use of icrp treatment by itself did not change, compared to the control (figure 1). when the evaluation of the number of cfu - gm was done, we observed that icrp and nac treatments reversed the side effects of 5-fu related to a decrease in the number of cfu - gm colonies (p < 0.05) on day 1 ; seven days later, nac + 5-fu and icrp + 5-fu groups increased the number of cfu - gm compared to the control (figure 2). treatment with 5-fu significantly decreased s phase and nac and icrp treatments did not change this effect on bm cells. the 5-fu group increased the percentage of sub - g1 phase, which indicates that cells are under apoptosis, on day 1. the cell cycle was not affected (p < 0.05) by treatments on day 7 (table 1). to elucidate the specific population that is protected by the icrp, the percentages of leukocyte (cd45), granulocytic (cd11bgr-1), and erythroid (cd71, ter119) lineages in bm cells were evaluated by flow cytometry. on day one, leukocyte and granulocytic populations were decreased (p < 0.05) by 5-fu treatment ; nac and icrp did not protect bm cells of 5-fu treated mice. the 5-fu group evaluated on day 7 kept low percentages of cd45 and cd11bgr-1 populations but icrp + 5-fu group increased these populations (p < 0.05) similar to the control group (figure 3). the protective effect of icrp on erythroid lineage was evident on days 1 and 7, because icrp + 5-fu group preserves highest percentages in basophilic erythroblast and orthochromatic erythroblast stages of erythroid maturation compared with 5-fu group (p < 0.05). enucleated red blood cells were the predominant population in 5-fu treated mice ; these findings are different to the control and icrp + 5-fu groups (figure 4 and table 2). the ros / superoxide formation and nrf2 activation were induced on day 1 and day 7 by 5-fu treatment and the icrp + 5-fu treatment decreases these parameters on day 7 ; no statistical difference was found on day 1 (figure 5). the effect of icrp on the histopathology of bone marrow at 1 and 7 days is shown in figure 6 and described at continuation ; 5-fu treatment decreased the cell density of bone marrow, creating a hypocellular environment, with marked decrease of megakaryocytes and granulocytic lineage cells, and no large amount of precursor cells was found. nac and icrp treatments protect bone marrow because they present a higher proportion in differentiated precursor cells and cellularity. we examined the effects of icrp on 5-fu treated mice on red blood cell (rbc), hemoglobin (hb), hematocrit (hct), white blood cell (wbc), and platelets (plt) levels 1 and 7 days after initiating treatments. the 5-fu group resulted in anemia and erythrocytopenia and decreased the hematocrit level on day 1 and day 7. the icrp + 5-fu group did not present any of these toxic effects ; their values were similar to the control group (table 3). the 5-fu treated mice gained less body weight (p < 0.05) compared to the control. the icrp + 5-fu group increased body weight similar to the control group (table 4). the chemotherapy with 5-fu is widely used since its discovery to treat a variety of tumors, including colorectal, breast, and liver carcinomas. the hematologic toxicity induced by chemotherapy is related to the dose - limiting side effect, affecting the therapeutic success and quality of life of patients. the 5-fu as a model of bone marrow depletion has been used by many researchers [7, 13, 16, 17, 2428 ] ; in the present study, we corroborated that a single dose of 5-fu (75 mg / kg) reduces the number of cfu - gm, which indicates that bone marrow lineage commitment and proliferative potential are affected. the icrp treatment demonstrates an efficient chemoprotection to 5-fu treatment, due to an increase in bone marrow progenitor cells function, such as those found with the use of amifostine, which is a clinical radioprotector. this effect on progenitor cells can be correlated with the capacity of icrp to protect more committed lineages in bone marrow cells, such as leukocyte (cd45), granulocyte (cd11bgr-1), and erythroid populations (cd71, ter119) which are affected by 5-fu [27, 31 ], and also with normal hematological values of wbc and rbc in a systemic level. other studies have previously used animal models and peripheral blood reconstitution as measure of bone marrow recovery after chemotherapy [32, 33 ]. these results could be used to reduce infections and anemia experienced by patients receiving chemotherapy. in this study, cell cycle analysis was used to determine whether the observed chemoprotection is related to cell cycle arrest at any phase. it is known that the mechanism of cytotoxicity of 5-fu is on actively proliferating cells (s phase) including healthy and cancer cells [35, 36 ]. agents such as tetrapeptide acetyl - n - ser - asp - lys - pro (acsdkp) and tgf- can protect marrow progenitor cells due to the induction of g0/g1 arrest, being an alternative to chemoprotection therapy [37, 38 ]. in our study, icrp and nac did not affect cell cycle in bone marrow cells, suggesting another mechanism of action in the protection of 5-fu treated bone marrow cells. it is know that 5-fu induces oxidative stress in bone marrow cells and this leads to the activation of nrf2 transcription factor [39, 40 ] ; therefore, several researchers explain the mechanism of chemoprotection of different compounds for their ability to activate the antioxidant response and neutralizing ros. our results indicate that icrp decreased ros / superoxide production and nrf2 activation in 5-fu treated bone marrow cells on 7 day ; this could be explained because icrp has an antioxidant capacity by increasing glutathione peroxidase, catalase, and superoxide dismutase enzymes ; further studies are needed to clarify whether these enzymes are responsible for decreasing ros production in bone marrow. these results would suggest that icrp might act as free radical scavenger, similar to aminothiols and phosphorothioates, two protective agents widely used. all these effects of chemoprotection are reflected in our histopathology analysis ; this kind of technique is used to assess the bone marrow architecture, cellularity, estimation of iron stores, and other features. it is important that patients receiving chemotherapy can maintain their weight in order to improve health - related quality of life ; we found that icrp helps to maintain normal weight after 5-fu treatment, which indicates that icrp could improve the quality of life in cancer patients. it is known that nac protects the cytotoxic and apoptotic effects against cisplatin in human tumor cell lines, because nac blocks the death receptor and mitochondrial apoptotic pathways. this is necessary in in vitro and in vivo studies to determinate if icrp has antagonist action against tumor cells treated with chemotherapy due to its antioxidant activity showed in this study. although in previous studies icrp has been administrated to patients with breast and lung cancer as an adjuvant to avoid secondary effects in combination with chemotherapy, there was no effect on the tumor regression and improving the quality of life [20, 46 ]. it is important to investigate new compounds that could be given during chemotherapy treatment and help us to alleviate some side effects, resulting in a significant increase in chemotherapy doses. our results suggest that the icrp can be proposed as a chemoprotective agent because it is able to protect the damage caused by 5-fu in bone marrow cells, ros production, hematological parameters, and weight gain probably by its antioxidant or immunomodulatory capacity. | chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-fluorouracil (5-fu) is wieldy used as myeloablative model in mice. the bovine dialyzable leukocyte extract (bdle) or immunepotent crp (icrp) is an immunomodulatory compound that has antioxidants and anti - inflammatory effects. in order to investigate the chemoprotection effect of icrp on bone marrow cells in 5-fu treated mice, total bone marrow (bm) cell count, bone marrow colony forming units - granulocyte / macrophage (cfu - gm), cell cycle, immunophenotypification, ros / superoxide and nrf2 by flow cytometry, and histological and hematological analyses were performed. our results demonstrated that icrp increased bm cell count and cfu - gm number, arrested bm cells in g0/g1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ros / superoxide formation and nrf2 activation, and also improved hematological levels and weight gain in 5-fu treated mice. these results suggest that icrp has a chemoprotective effect against 5-fu in bm cells that can be used in cancer patients. |
previously our work on the l. casei preq1-ii riboswitch identified by breaker and co - workers resulted in crystals with x - ray diffraction limited to 5.6 resolution. in an effort to improve crystal quality, we conducted blastn refseq rna searches starting with the l. casei riboswitch sequence. our goal was to identify closely related sequences, especially those with shorter joining regions that might be more amenable to high - resolution structural analysis. this approach led to the identification of a putative preq1-ii riboswitch sequence in the 5 utr of a cog4708 gene from l. rhamnosus, which is characteristic of other preq1-ii riboswitches. the cog4708 gene is hypothesized to produce a protein that transports q precursors into the cell. despite genomic synteny and similar probiotic properties, l. casei and l. rhamnosus, l. rhamnosus has also been associated with endocarditis and is not considered strictly beneficial. significantly, two differences were apparent in the respective riboswitch sequences between positions 21 and 77 a region documented previously as important for preq1-binding function. variations included a deletion of l. casei position 34 (c34) in j2/3 of l. rhamnosus, and a c53u change in j2/4. using itc and in - line probing approaches (described below), we found the l. rhamnosus sequence is responsive to preq1, and crystallization trials led to well diffracting crystals. at present, the basis for differences in crystal diffraction resulting from the use of the respective sequences is unknown. 1a, b) and mutants thereof were prepared by in vitro transcription and purified by denaturing page as described. itc measurements were conducted using a vp - itc calorimeter (microcal, inc) at 20 c or 25 c. lyophilized rna was resuspended in 0.010 m na - hepes ph 7.0 or hepps ph 8.3 and 0.10 m nacl or 0.10 m kcl. for mgcl2-free conditions 0.5 mm edta ph 8.0 the rna was heated to 65 c for 5 min, then mgcl2 was added to a final concentration of 6.0 mm, or 20 mm, or 1.0 mm of co(nh3)6cl3 was added, followed by slow cooling to 24 c ; mgcl2 and co(nh3)6cl3 were omitted completely for folding conditions containing edta. the rna was dialyzed against 4 l of 0.10 m nacl or 0.10 m kcl, 6.0 mm, 20.0 mm mgcl2, 1 mm co(nh3)6cl3 or 0.5 mm edta, and 0.050 m na - hepes ph 7.0 or 0.050 m na - hepps ph 8.3 overnight at 4 c, then diluted with dialysis buffer to : 3.3 m for the wild type riboswitch, 13.8 m for the c30u mutant, 23.3 26.2 m for the u41c mutant, 1.5 5.7 m for wild type with 0.5 mm edta, and 3.6 m for wild type with co(nh3)6cl3. measurements were carried out by titrating preq1 into the riboswitch located in the sample cell (cell volume is ~1.7 ml) using 28 or 29 injections of 10 l each, except for the first injection of 3 l, with 120 or 240 s intervals between injections ; the reference power was 15 cal s. the thermograms were analyzed with origin 7.0 (microcal) using a 1:1 binding model. experiments were performed in triplicate for wild type and mc variant l. rhamnosus sequences with mgcl2, and in duplicate for mutant riboswitches and for the wild type in all other conditions (supplementary table 1) ; representative titrations and curve fits are shown in supplementary fig. a solution of 0.25 mm rna (prepared as described above for itc) in 0.01 m na - cacodylate ph 7.0 was heated to 65 c for 3 min. mgcl2 was added to a final concentration of 6 mm and preq1 synthesized as described was added to a final concentration of 0.5 mm followed by heating to 65 c for 5 min. a volume of 1.7 l of pre - folded rna was mixed 1:1 with well solution comprising : 14.414.8% (w / v) poly(ethylene) glycol 6000 (peg6k), 0.140.16 m mgoac2, 0.05 m na - cacodylate ph 6.0, 1 mm spermine, and 0.15 m cscl. crystals grew as rectangular plates of size 0.20 mm 0.05 mm 0.02 mm. cryoprotection was by a 30 s transfer into synthetic mother liquor comprising : 17.217.8% (w / v) peg6k, 168192 mm mgoac2, 0.18 m cscl, 0.06 m na - cacodylate ph 6.0, and 1.2 mm spermine supplemented with 20% (v / v) 2-methyl-2,4-pentanediol and 20% (v / v) ethanol. x - ray diffraction intensities were recorded at stanford synchrotron radiation lightsource (ssrl, menlo park ca) beamline 71 at 173 c and reduced with the hkl2000 software package. a single crystal was used to obtain an 18-fold redundant, 2.6 resolution x - ray data set that was recorded at a wavelength of 1.7 to optimize the cs anomalous signal (supplementary table 2). phenix autosol located 14 site - bound cs atoms, which were used for initial phase calculations in combination with density modification (supplementary fig. the figure of merit before density modification in resolve (as implemented in phenix) was 0.41. initial model building was performed by autobuilding in phenix, followed by iterative rounds of building in coot and refinement in phenix. the trace and solvent content were consistent with a single molecule in the asymmetric unit. a 2.3 resolution native dataset was collected at ssrl on a second crystal at a wavelength of 1.127, which was used to extend the resolution of the sad model. at a late stage, preq1 was modeled into reduced bias 2fo - fc and fo - fc maps (e.g. fig. cs ions were included in the refined model based on their anomalous diffraction signal (supplementary fig. the final rwork / rfree values were 19.3/24.4% with reasonable geometry (supplementary table 2). nearly the entire 77-nucleotide riboswitch was resolved in electron density maps including the ligand - binding pocket and rbs (fig. the area of preq1 that is inaccessible to solvent was calculated using a water probe with a radius of 1.4 using ucsf chimera. the in - line probing reaction was carried out on an extended preq1-ii sequence (supplementary fig. rna was prepared by in vitro transcription (as described above for itc), dephosphorylated with alkaline phosphatase, and radiolabeled with [p]atp (perkinelmer) and t4 polynucleotide kinase (new england biolabs), which was then page purified. rna was folded by heating in 0.05 m tris - hcl (ph 8.3 at 25 c) to 65 c, snap cooling on ice, then adding kcl to 0.10 m, mgcl2 to 0.020 m, and preq1 at one of the concentrations described (supplementary fig. probing reactions each contained 1.0 10 cpm and were incubated at 25 c for ~40 h. reaction products were separated by denaturing 7.5% page, the gel was dried, and then exposed to a phosphor storage screen for ~40 h. imaging was carried out using a ge storm 860, and gel quantification was conducted by use of safa with data normalized against invariant nucleotides selected by safa ; the nucleotides chosen as invariant bases were : 34, 40, 50, 53 and 54. the fraction of rna cleaved was determined by setting the maximum amount cleaved to 1 and the minimum to 0 for all concentrations tested at a given nucleotide position, as described. binding curves were generated in prism 6 by fitting the corrected intensities measured to a dose response curve, with the apparent kd equal to a level of 0.5 cleaved. protein data bank : coordinates and structure factors have been deposited under accession code 4jf2. previously our work on the l. casei preq1-ii riboswitch identified by breaker and co - workers resulted in crystals with x - ray diffraction limited to 5.6 resolution. in an effort to improve crystal quality, we conducted blastn refseq rna searches starting with the l. casei riboswitch sequence. our goal was to identify closely related sequences, especially those with shorter joining regions that might be more amenable to high - resolution structural analysis. this approach led to the identification of a putative preq1-ii riboswitch sequence in the 5 utr of a cog4708 gene from l. rhamnosus, which is characteristic of other preq1-ii riboswitches. the cog4708 gene is hypothesized to produce a protein that transports q precursors into the cell. despite genomic synteny and similar probiotic properties, l. casei and l. rhamnosus, l. rhamnosus has also been associated with endocarditis and is not considered strictly beneficial. significantly, two differences were apparent in the respective riboswitch sequences between positions 21 and 77 a region documented previously as important for preq1-binding function. variations included a deletion of l. casei position 34 (c34) in j2/3 of l. rhamnosus, and a c53u change in j2/4. using itc and in - line probing approaches (described below), we found the l. rhamnosus sequence is responsive to preq1, and crystallization trials led to well diffracting crystals. at present, the basis for differences in crystal diffraction resulting from the use of the respective sequences is unknown. 1a, b) and mutants thereof were prepared by in vitro transcription and purified by denaturing page as described. itc measurements were conducted using a vp - itc calorimeter (microcal, inc) at 20 c or 25 c. lyophilized rna was resuspended in 0.010 m na - hepes ph 7.0 or hepps ph 8.3 and 0.10 m nacl or 0.10 m kcl. for mgcl2-free conditions 0.5 the rna was heated to 65 c for 5 min, then mgcl2 was added to a final concentration of 6.0 mm, or 20 mm, or 1.0 mm of co(nh3)6cl3 was added, followed by slow cooling to 24 c ; mgcl2 and co(nh3)6cl3 were omitted completely for folding conditions containing edta. the rna was dialyzed against 4 l of 0.10 m nacl or 0.10 m kcl, 6.0 mm, 20.0 mm mgcl2, 1 mm co(nh3)6cl3 or 0.5 mm edta, and 0.050 m na - hepes ph 7.0 or 0.050 m na - hepps ph 8.3 overnight at 4 c, then diluted with dialysis buffer to : 3.3 m for the wild type riboswitch, 13.8 m for the c30u mutant, 23.3 26.2 m for the u41c mutant, 1.5 5.7 m for wild type with 0.5 mm edta, and 3.6 m for wild type with co(nh3)6cl3. measurements were carried out by titrating preq1 into the riboswitch located in the sample cell (cell volume is ~1.7 ml) using 28 or 29 injections of 10 l each, except for the first injection of 3 l, with 120 or 240 s intervals between injections ; the reference power was 15 cal s. the thermograms were analyzed with origin 7.0 (microcal) using a 1:1 binding model. experiments were performed in triplicate for wild type and mc variant l. rhamnosus sequences with mgcl2, and in duplicate for mutant riboswitches and for the wild type in all other conditions (supplementary table 1) ; representative titrations and curve fits are shown in supplementary fig. a solution of 0.25 mm rna (prepared as described above for itc) in 0.01 m na - cacodylate ph 7.0 was heated to 65 c for 3 min. mgcl2 was added to a final concentration of 6 mm and preq1 synthesized as described was added to a final concentration of 0.5 mm followed by heating to 65 c for 5 min. a volume of 1.7 l of pre - folded rna was mixed 1:1 with well solution comprising : 14.414.8% (w / v) poly(ethylene) glycol 6000 (peg6k), 0.140.16 m mgoac2, 0.05 m na - cacodylate ph 6.0, 1 mm spermine, and 0.15 m cscl. crystals grew as rectangular plates of size 0.20 mm 0.05 mm 0.02 mm. cryoprotection was by a 30 s transfer into synthetic mother liquor comprising : 17.217.8% (w / v) peg6k, 168192 mm mgoac2, 0.18 m cscl, 0.06 m na - cacodylate ph 6.0, and 1.2 mm spermine supplemented with 20% (v / v) 2-methyl-2,4-pentanediol and 20% (v / v) ethanol. x - ray diffraction intensities were recorded at stanford synchrotron radiation lightsource (ssrl, menlo park ca) beamline 71 at 173 c and reduced with the hkl2000 software package. a single crystal was used to obtain an 18-fold redundant, 2.6 resolution x - ray data set that was recorded at a wavelength of 1.7 to optimize the cs anomalous signal (supplementary table 2). phenix autosol located 14 site - bound cs atoms, which were used for initial phase calculations in combination with density modification (supplementary fig. 3). the figure of merit before density modification in resolve (as implemented in phenix) was 0.41. initial model building was performed by autobuilding in phenix, followed by iterative rounds of building in coot and refinement in phenix. the trace and solvent content were consistent with a single molecule in the asymmetric unit. a 2.3 resolution native dataset was collected at ssrl on a second crystal at a wavelength of 1.127, which was used to extend the resolution of the sad model. at a late stage, preq1 was modeled into reduced bias 2fo - fc and fo - fc maps (e.g. fig. cs ions were included in the refined model based on their anomalous diffraction signal (supplementary fig. the final rwork / rfree values were 19.3/24.4% with reasonable geometry (supplementary table 2). nearly the entire 77-nucleotide riboswitch was resolved in electron density maps including the ligand - binding pocket and rbs (fig. the area of preq1 that is inaccessible to solvent was calculated using a water probe with a radius of 1.4 using ucsf chimera. the in - line probing reaction was carried out on an extended preq1-ii sequence (supplementary fig. 1b) essentially as described. rna was prepared by in vitro transcription (as described above for itc), dephosphorylated with alkaline phosphatase, and radiolabeled with [p]atp (perkinelmer) and t4 polynucleotide kinase (new england biolabs), which was then page purified. rna was folded by heating in 0.05 m tris - hcl (ph 8.3 at 25 c) to 65 c, snap cooling on ice, then adding kcl to 0.10 m, mgcl2 to 0.020 m, and preq1 at one of the concentrations described (supplementary fig probing reactions each contained 1.0 10 cpm and were incubated at 25 c for ~40 h. reaction products were separated by denaturing 7.5% page, the gel was dried, and then exposed to a phosphor storage screen for ~40 h. imaging was carried out using a ge storm 860, and gel quantification was conducted by use of safa with data normalized against invariant nucleotides selected by safa ; the nucleotides chosen as invariant bases were : 34, 40, 50, 53 and 54. the fraction of rna cleaved was determined by setting the maximum amount cleaved to 1 and the minimum to 0 for all concentrations tested at a given nucleotide position, as described. binding curves were generated in prism 6 by fitting the corrected intensities measured to a dose response curve, with the apparent kd equal to a level of 0.5 cleaved. protein data bank : coordinates and structure factors have been deposited under accession code 4jf2. | preq1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preq1 during biosynthesis of the essential trna base queuosine. we report the first preq1-ii riboswitch structure at 2.3 resolution, which uses a novel fold to achieve effector recognition at the confluence of a three - way - helical junction flanking a pseudoknotted ribosome - binding site (rbs). the results account for preq1-ii - riboswitch - mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory rnas. |
newly developed miniaturized vision tests have allowed, in a clinically relevant way, to evaluate the influence of magnification and age on the near visual acuity of dentists. the use of optical aids to improve manual precision has long been a tradition in many medical professions. dentistry seems to be an ideal environment for the use of loupes and microscopes, because operative treatments are performed in a small and narrow environment. however, the idea that magnification devices should be used as standard devices in dentistry is relatively new [24 ]. the body of evidence supporting the general assumption that optical magnification improves the quality of caries detection and filigree therapy is weak. most publications about visual acuity and the influence of magnification [69 ], or concerning microscopes and endodontics [2, 1012 ], are based on expert opinions and case reports. an obvious gap between anecdotal conjecture and solid evidence - based decision making exists with respect to the use of optical aids in dentistry. in a previous study with the help of miniaturized visual tests and a light box, the present authors found a large variability in the near visual acuity of dentists and a significant correlation with the dentists age. yet, the tests were not incorporated in an oral cavity, and thus, the clinical relevance of these miniaturized visual tests was limited. therefore, the aims of the present study were : to test near visual acuity and the influence of magnification and age in a simulated clinical situationto compare the results to the previous study, where the same group of dentists was tested under standardized conditions ex ore with a light box to test near visual acuity and the influence of magnification and age in a simulated clinical situation to compare the results to the previous study, where the same group of dentists was tested under standardized conditions ex ore with a light box the near visual acuity of 40 dentists from the dental school of the university of bern was evaluated using miniaturized visual tests fixed in a dental phantom head unit. the distribution of the dentists in the different age groups was as follows : 2530 years, n = 16 ; 3135 years, n = 9 ; 3640 years, n = 2 ; 4145 years, n = 5 ; 4650 years, n = 1 ; 5155 years, n = 3 ; 5660 years, n = 3 ; 6165 years, n = 1. visual tests with e - optotypes, ranging from 0.05 to 0.5 mm, allowed for the quantification of visual acuity at a typical dental working distance, as described in a previous study. the tests were mounted in cavities in maxillary second premolars and first molars of a dental phantom unit (kavo, biberach, germany) (fig. 1).fig.1the fixation of the visual tests in distal box cavities of phantom teeth allows the testing of visual acuity under simulated clinical conditions the fixation of the visual tests in distal box cavities of phantom teeth allows the testing of visual acuity under simulated clinical conditions the head was positioned on a dental chair. 4, hahnenkratt gmbh, knigsbach, germany) were used, and the operating lamp (delight, planmeca oy, helsinki, finland) for conditions 1 through 4 was individually arranged by the test persons. visual performance was tested under the following conditions : natural visual acuity (nv) : no magnification devices, distance of 300 mm (typical working distance), operating lampfree natural visual acuity (nvf) : no magnification devices and free choice of the distance (typical controlling distance), operating lampgalilean loupe system (g) (evc250n, surgitel, ann arbor, mi, usa) : magnification of 2.5 ; distance of 380 mm ; operating lampkeplerian loupe system (k) (eyemag pro s, zeiss, oberkochen, germany) : magnification of 4.3 ; distance of 400 mm ; operating lampoperating microscope (m4) (leica m300, leica microsystems, heerbrugg, switzerland) : magnification of 4 ; distance of 250 mm ; integrated lightoperating microscope (m6.4) : same as condition 5, but with magnification of 6.4. natural visual acuity (nv) : no magnification devices, distance of 300 mm (typical working distance), operating lamp free natural visual acuity (nvf) : no magnification devices and free choice of the distance (typical controlling distance), operating lamp galilean loupe system (g) (evc250n, surgitel, ann arbor, mi, usa) : magnification of 2.5 ; distance of 380 mm ; operating lamp keplerian loupe system (k) (eyemag pro s, zeiss, oberkochen, germany) : magnification of 4.3 ; distance of 400 mm ; operating lamp operating microscope (m4) (leica m300, leica microsystems, heerbrugg, switzerland) : magnification of 4 ; distance of 250 mm ; integrated light operating microscope (m6.4) : same as condition 5, but with magnification of 6.4. the loupes were fixed on a headband, which allowed the dentists to wear their respective prescription eyeglasses (when necessary) in all groups. during the visual tests, the position of the loupes, the eye object distance, and the reading of the e - optotypes were all controlled by the same expert. the smallest line of the visual test that could be read without mistakes was registered. the distance between the three bars of the letter e (= 1/5 the size of e) corresponds to the smallest detectable structure and allows to calculate the visual acuity. the metric dimension of the bar spacing in the registered line (e.g., 0.03 mm) was converted into the reciprocal value (e.g., 33.3/mm). this allowed to achieve a positive correlation between the metric value and the quality of the visual performance. the influence of the different magnification systems (conditions 16) was tested for all dentists. to investigate the influence of the dentists age, the present authors calculated the correlation between age and visual acuity for the different optical conditions and compared the results of the two subgroups a < 40 years (n = 27) and b 40 years (n = 13). finally, the results of the present study were compared with the results of the previous study, where the visual performance of the same dentists was tested on the light box and not in a setup simulating the clinical situation. for statistical analysis, descriptive statistical analysis was carried out to determine the means and medians as well as the nonparametric 95 % confidence intervals (ci) for all dentists. the influence of the respective groups of dentists and different loupe systems on the visual acuity was analyzed by using rank tests for linear models based on wilcoxon scores, followed by wilcoxon signed rank tests with bonferroni spearman s rank correlation coefficient was used to detect monotone relationships between age and visual acuity. for the third part, spearman s rank correlation coefficient and pearson s correlation coefficient were used to detect monotone and linear relationships between the results of the previous study and those of the present one concerning nv, g, and k. the relative improvement of visual performance with magnification ranged from 250 % (galilean loupe) to 961 % (operating microscope, 6.4), compared to natural visual acuity. statistical analysis revealed significant differences between all tested conditions (p < 0.01) (table 1).table 1visual performance (mean, median, nonparametric 95 % confidence intervals) and the relative improvement of all tested dentists under all test conditionsconditionmeanmedian95 % ciimprovement of visual performance (nv = 100 %) nv8.4311.5211.5213.98100 % nvf15.2018.0416.7921.01180 % g21.0721.0119.5222.90250 % k31.9434.3429.7634.34379 % m457.2753.4853.4858.19679 % m6.481.0181.5975.1387.41961 % note the difference between nv and nvf due to the natural magnification by moving closer to the objectp < 0.01, significant differences within the columns visual performance (mean, median, nonparametric 95 % confidence intervals) and the relative improvement of all tested dentists under all test conditions note the difference between nv and nvf due to the natural magnification by moving closer to the object p < 0.01, significant differences within the columns the correlation between age and visual performance under the different conditions is presented in table 2. the exceptions were nvf with a strong correlation and m6.4 with a weak correlation.table 2the correlation between age and visual performance was medium for all optical conditions except nvf (strong correlation) and m6.4 (weak correlation)conditionspearman s rank correlationnv0.543nvf0.747g0.686k0.563m40.485m6.40.286 the correlation between age and visual performance was medium for all optical conditions except nvf (strong correlation) and m6.4 (weak correlation) the mean visual performance, medians, and 95 % cis for groups a (< 40 years) and b (40 years) are presented in table 3. for all tested conditions, significant differences between the two groups were detected (p < 0.001 or p < 0.05). both groups benefitted from the magnification of the galilean and keplerian loupes and even more from the sophisticated optics of the operating microscope. the visual acuity of group a in natural vision at a closer distance (nvf) was better than the acuity of group b with galilean loupes (g). the difference between conditions nvf and g was not as pronounced for group a as for group b.table 3visual performance (mean, median, nonparametric 95 % confidence intervals) for the two groups < 40 and 40 years under all test conditionsconditionage < 40 years (n = 27) (1/mm)age 40 years (n = 13) (1/mm)meanmedian95 % cimeanmedian95 % cinv11.1611.5211.5214.633.350n.a.nvf20.2921.0118.2721.015.750n.a.g23.3821.0121.0125.3816.7818.0414.7818.04k34.3834.3432.0537.0127.3927.2824.8029.76m460.0758.1955.4964.6152.0648.0848.0855.49m6.485.1587.4182.5994.2173.3175.7662.8481.59note the differences from nvf to g between the two groupsn.a. not available : due to the skewed distribution, a calculation of the 95 % confidence interval was not possible (in the older group when visual performance with the naked eye was testedp < 0.001 ; p < 0.05, significant differences within the columns visual performance (mean, median, nonparametric 95 % confidence intervals) for the two groups < 40 and 40 years under all test conditions note the differences from nvf to g between the two groups n.a. not available : due to the skewed distribution, a calculation of the 95 % confidence interval was not possible (in the older group when visual performance with the naked eye was tested p < 0.001 ; p < 0.05, significant differences within the columns the comparison of the present study with the previous study on a light box revealed a strong correlation between the two setups when no magnification device was used (nv ; spearman s correlation coefficient, 0.698) and when the galilean system was used (g ; pearson s correlation coefficient, 0.725). the correlation was medium for the keplerian system (k ; spearman s correlation coefficient, 0.622). a large variability in natural visual acuity was found (018.04, 1/mm bar spacing), which corroborated the results of a previous study under optimal conditions ex ore. some of the tested dentists had better visual acuity without optical aids than others with galilean loupes. the visual acuity can, as expected, be significantly improved by the use of magnification devices (250961 %, table 1), independent of age and natural visual acuity. when using keplerian loupes, all dentists achieved a significantly higher visual acuity than with galilean loupes. the different magnification factors of the two systems have a clinical rationale : galilean loupes are small and ergonomic but have a magnification limit of 2.5 (3.2 with optical constraints), while the magnification of keplerian loupes is not constrained. the large difference between the microscope and the head - mounted loupe systems, even with comparable magnification factors, was unexpected. the highly superior performance of the operating microscope could be due to the different optical construction of the microscope with a greater angle between the two optical beams. a further, possibly even more important reason could be the static position of the microscope, which offsets any disturbances caused by head movements. this effect is only detectable in a simulated clinical setting as used in this study. presbyopia (loss of accommodation) starts around the age of 40 years and is combined with other changes in the eye, such as decreasing sensitivity to contrast, increasing sensitivity to glare, and lower visual performance [15, 16 ]. the influence of presbyopia on the visual performance was analyzed in two different mathematical approaches. the strong correlation between age and natural visual acuity in a free, i.e., closer, distance (nvf) could be expected due to the loss of accommodation. however, the only marginal correlation between age and acuity using the microscope (m6.4) was unexpected. an explanation could be the fact that the microscope allows a parallel view without any accommodation and is therefore less able to account for presbyopic deficiencies. the division into two groups (< 40 and 40 years old) allowed the quantification of the age - related deficiencies in a second, more comprehensible approach. 40 years old, the condition nvf exhibited a 72 % lower visual performance. this condition has an evident clinical importance : by reducing the working distance, the dentist profits from a natural magnification which is quantified in the difference between the conditions nv and nvf in the younger group a (< 40 years old) (table 3). this natural magnification is widely used in diagnostic situations and mostly compromised with increasing age. the comparison of the extraoral visual test under optimum visual conditions (direct view, light box, supported head) with the intraoral visual test in a phantom head showed a strong correlation for unaided natural vision and galilean loupes. the correlation for keplerian loupes was less pronounced. this could indicate that head - mounted higher magnifications are more sensitive to tremors arising from the head and dental mirrors in a clinical situation. however, an extraoral near vision test seems to be a valid surrogate to judge the performance that would be achieved in the clinical situation. common near vision tests, such as those used by opticians, are physically too large to assess visual acuity at the scale on which dentists work. this is, with one exception, also true for the few research studies identified in the literature [15, 18, 19 ]. the e - optotypes used in the present study ranged from 0.05 to 0.5 mm and were proven to have adequate discriminatory properties. the placement of the tests in artificial teeth provided a realistic simulation of the dental setting, including variables such as the illumination source and the reduced contrast in a cavity, which may influence the visual performance during dental work. however, not all parameters for an objective optical value can be standardized in a simulated clinical setting. the pragmatic question of whether a small structure can be recognized or not seems to be more adequate for a clinical approach. therefore, the visual acuity angle was substituted for the metric dimension of the smallest recognized bar spacing under the given conditions. the reciprocal calculation of bar spacing allowed for a positive correlation between metric values and visual performance. the results of this study suggest the importance of reliable near vision tests for dental personnel. dental students as well as dental personnel with increasing age should be aware of their natural near vision in order to choose adequate optical aids. whether or not an adjusted visual acuity has an impact on the precision of diagnostic and operative procedures remains the subject of further studies. the near visual acuity under simulated clinical conditions varies highly between individuals and decreases over one s lifetime. there is a need for a free accessible visual test at a dental working distance to objectively measure the visual performance of dental students and dental personnel in order to provide personalized recommendations for the use of magnification devices. individuals with poor natural visual acuity or an age 40 years can reliably compensate their visual deficiencies by using optical aids. | objectivesthis study examined the near visual acuity of dentists in relation to age and magnification under simulated clinical conditions.materials and methodsminiaturized visual tests were performed in posterior teeth of a dental phantom head in a simulated clinical setting (dental chair, operating lamp, dental mirror). the visual acuity of 40 dentists was measured under the following conditions : (1) natural visual acuity, distance of 300 mm ; (2) natural visual acuity, free choice of distance ; (3) galilean loupes, magnification of 2.5 ; (4) keplerian loupes, 4.3 ; (5) operating microscope, 4, integrated light ; (6) operating microscope, 6.4, integrated light.resultsthe visual acuity varied widely between individuals and was significantly lower in the group 40 years of age (p < 0.001). significant differences were found between all tested conditions (p < 0.01). furthermore, a correlation between visual acuity and age was found for all conditions. the performance with the microscope was better than with loupes even with comparable magnification factors. some dentists had a better visual acuity without optical aids than others with galilean loupes.conclusionsnear visual acuity under simulated clinical conditions varies widely between individuals and decreases throughout life. visual deficiencies can be compensated for with optical aids.clinical relevancenewly developed miniaturized vision tests have allowed, in a clinically relevant way, to evaluate the influence of magnification and age on the near visual acuity of dentists. |
the main aim of pharmacotherapeutics is the attainment of effective drug concentration at the intended site of action for a sufficient period of time to elicit the response. amongst the various routes of drug delivery, the field of ocular drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist. this is significantly improved over the past 10 - 20 years. as an isolated organ it is very difficult to obtain eye tissue containing drugs from humans, so one is compelled to use animal tissue. due to these human ocular disposition characteristics of virtually important drugs is unknown or incomplete. despite these severe limitations the main objective of the improvement is to maintain the drug in the biophase for an extended period of time. in the ophthalmic drug delivery systems, the physiological constraints imposed by the protective mechanism of the eye lead to the low absorption of drugs and results in a short duration of therapeutic action. a high frequency of the eye drops instillation is associated with patient 's non compliance. after the instillation of eye drop into the eye cavity, the effective tear drainage and blinking action of eye results in a 10 times reduction in the drug concentration within 4 - 20 min. figure 1 explains the fate of the drugs after instillation into the eye. due to the tear drainage, most of the administered dose passes via nasolacrimal duct into the gastro intestinal tract, leading to the side effects. rapid elimination of the eye drops often results in a short duration of the therapeutic effect. the normal volume of tear in the eye is 7 l, a non - blinking eye can accommodate a maximum of 30 l of the fluid where as a blinking eye can hold only 10 l of fluid. the usual single drop size of an instilled drug solution is up to 50 l and thus most of the drug instilled as eye drop is lost. fate of drugs absorbed by ophthalmic route the following characteristics are generally required to optimize ocular drug delivery systems. a good corneal penetration.a prolonged contact time with corneal tissue.simplicity of installation for the patient.a non - irritative and comfortable form (the viscous solution should not provoke lachrymation and reflex blinking).appropriate rheological properties and concentration of viscolyzertarget delivery within the ocular globes so as to prevent the loss to other ocular sites. a good corneal penetration. a prolonged contact time with corneal tissue. a non - irritative and comfortable form (the viscous solution should not provoke lachrymation and reflex blinking). appropriate rheological properties and concentration of viscolyzer target delivery within the ocular globes so as to prevent the loss to other ocular sites. the preceding summary demonstrates that the formulator faces many constraints and prerequisites when developing a modified - release topical ophthalmic drug. in addition to the traditional requirements of oral drugs for safety, efficacy, and stability, ophthalmic products must exhibit additional properties. the regulatory demands for new ophthalmic chemical entities are, most of the time, outweighed by the development efforts and costs compared to the size of the ophthalmic market. most of the conventional ophthalmic dosage forms, i.e., eye solution, eye ointments, eye gels, and eye suspensions are comprised in their effectiveness by several limitations leading to poor ocular bioavailability and the limitations are- they have poor bioavailability because of - rapid precorneal eliminationconjunctival absorptionsolution drainage by gravityinduced lachrymationnormal tear turnover frequent administration is needed, which would increase the risk of drug toxicity and side effectssystemic absorption of the drug and additives drained through nasolacrimal duct may result in undesirable side effects.the amount of drug delivered during external application may vary the drop size of commercial ocular medication is not uniform and those delivered is generally not correct.presence of viscous vehicle can cause blurred vision they have poor bioavailability because of - rapid precorneal eliminationconjunctival absorptionsolution drainage by gravityinduced lachrymationnormal tear turnover rapid precorneal elimination conjunctival absorption solution drainage by gravity normal tear turnover frequent administration is needed, which would increase the risk of drug toxicity and side effects systemic absorption of the drug and additives drained through nasolacrimal duct may result in undesirable side effects. the amount of drug delivered during external application may vary the drop size of commercial ocular medication is not uniform and those delivered is generally not correct. presence of viscous vehicle can cause blurred vision to overcome the drawbacks of conventional ophthalmic dosage form, novel ophthalmic dosage forms such as niosomes, liposomes, pharmacosomes, nanoparticles, contact lenses, and ocular inserts are developed. these dosage forms also comprises of certain limitations as- they cause initial discomfort due to their movement around the eye, especially in case of elderly people. many patients sometimes lose it without noticing it.occasional inadvertent loss during sleeps or while rubbing the eye.interference with vision and a difficult placement they cause initial discomfort due to their movement around the eye, especially in case of elderly people interference with vision and a difficult placement all these disadvantages can be overcome by developing a specific dosage form for ophthalmic drug delivery known as hydrogels systems. previous studies on rabbits by robinson. established that the rate of drainage from the eye of an instilled solution is markedly reduced as the viscosity of the solution is increased. hydrogels can be defined as polymers endowed with the ability to swell in water or aqueous solvents and induce a sol - gel transition. they resemble natural living tissue more than any other class of synthetic biomaterials due to their high water content ; furthermore, the high water content of the materials contributes to their biocompatibility. in this regard, the phase - change polymers, which may trigger drug release in response to external stimuli, are the most investigated. smart hydrogels, or stimuli - sensitive hydrogels, are very different from inert hydrogels in that they can sense changes in environmental properties such as ph and temperature and respond by increasing or decreasing their degree of swelling. the volume - changing behavior of smart hydrogels is particularly useful in drug delivery applications as drug release can be triggered upon environmental changes. these intelligent or smart polymers play important role in drug delivery since they may dictate not only where a drug is delivered, but also when and with which interval it is released. the stimuli that induce various responses of the hydrogels systems include physical (temperature) or chemical (ph, ions) ones. there are many mechanisms have been employed to cause reversible sol - gel phase transition by different stimuli in physiological environmental conditions of human body : the stimuli that induce various responses of the hydrogel systems include- physical stimuli like : change in temperature, electric fields, light, pressure, sound, and magnetic fields.chemical stimuli like : change in ph and ion activation from biological fluid.biological/biochemical (bimolecular) stimuli like : change in glucose level physical stimuli like : change in temperature, electric fields, light, pressure, sound, and magnetic fields. biological / biochemical (bimolecular) stimuli like : change in glucose level in ophthalmic drug delivery three types of stimuli - sensitive hydrogels - temperature sensitive, ph sensitive, and ion - sensitive hydrogels are mainly used. three types of stimuli - sensitive hydrogels with mechanism temperature - sensitive hydrogels are probably the most commonly studied class of environment - sensitive polymer systems in drug delivery research. these hydrogels are able to swell or de - swell as a result of changing in the temperature of the surrounding fluid. for convenience, temperature - sensitive hydrogels are classified into negatively thermosensitive, positively thermosensitive, and thermally reversible gels. negative temperature - sensitive hydrogels have a lower critical solution temperature (lcst) and contract upon heating above the lcst. copolymers of (nisopropylacrylamide) (pniaam) are usually used for negative temperature release. hydrogels show an on off drug release with on at a low temperature and off at high temperature allowing pulsatile drug release. lcst systems are mainly relevant for controlled release of drugs, and of proteins in particular. thermosensitive polymers may be fixed on liposome membranes ; in that case liposomes exhibit control of their content release. positive temperature - sensitive hydrogel has an upper critical solution temperature (ucst), such hydrogel contracts upon cooling below the ucst. polymer networks of poly (acrylic acid) (paa) and polyacrylamide (paam) or poly (acrylamide - co - butyl methacrylate) have positive temperature dependence of swelling. the most commonly used thermoreversible gels are these prepared from poly (ethylene oxide)-b - poly (provpylene oxide)-b - poly (ethylene oxide) (pluronics, tetronics, poloxamer) polymer solution is a free - flowing liquid at ambient temperature and gels at body temperature, such a system would be easy to administer into desired body cavity. in some cases, if lowering the amount of thermogelling polymer is necessary, it may be blended with a ph - sensitive reversibly gelling polymer. recently, the polymers consisting of poly (ethylene glycol)-poly-(dllactic acid - co - glycolic acid)-poly(ethylen glycol) (pegplga- peg) or plga - peg - plga were investigated for sustained injectable drug delivery systems. some of the earliest work with temperature - sensitive hydrogels was done by the group of tanaka pnipaam is the best example of a negative temperature - sensitive hydrogel.. worked with crosslinked pnipaam and determined that the lcst of the pnipaam gels was 34.38c. they also found that the lcst could be increased by mixing small amounts of ionic copolymers in the gels. hoffman proposed the application of pnipaam and its copolymers for temperature - modulated drug release by bulk squeezing and surface regulation. in the bulk squeezing system, the drug that is distributed evenly inside the matrix is squeezed out of the system due to the de - swelling of the hydrogel as a result of increasing the temperature of the environment above the volume phase transition temperature. in the surface regulation system, the swelling ratio of the skin layer is increased as the temperature of the system is lowered below the volume phase transition temperature, and hence, the drug molecules will be able to diffuse through the skin layer. poloxamers are a broad group of compounds that were introduced in the early 1950s as food additives and for pharmaceutical preparations. these water - soluble surfactants are triblock co - polymers prepared from poly (ethylene oxide)-b - poly (propylene oxide)-b - poly (ethylene oxide) commercially available as pluronic are the most commonly used thermosetting polymers and could be applicable for the development of effective ophthalmic drug delivery. depending on the ratio and the distribution along the chain of the hydrophobic and hydrophilic subunits, several molecular weights are available, leading to different gelation properties pluronic f127, which gives colorless and transparent gels, is the most commonly used in pharmaceutical technology. pluronic f 127 is no more damaging to the mouse or rabbit cornea than a physiological saline. the poloxamers are reported to be well tolerated and non - toxic even though large amounts (25 - 30%) of polymers are required to obtained a suitable gel. at concentrations of 20% w / v and higher aqueous solutions of poloxamer-407 remain as a liquid at low temperatures [< 15c ] and yield a highly viscous semisolid gel upon instillation into the cul - de - sac. at low temperatures, the poloxamer forms micellar subunits in solution, and swelling gives rise to large micellar subunits and the creation of cross - linked networks. miller. examined a temperature - sensitive solution of poloxamer used to deliver the miotic pilocarpine. in order to reduce the concentration of polymer and/or to achieve a phase transition temperature higher than room temperature (25c) and gelling at precorneal temperature (35c), the combining pluronic analogs or the addition of further polymer, e.g. peg, paa, methylcellulose (mc), hpmc, attwood. has reported enhancement of the miotic response following sustained release of pilocarpine from the 1% w / w xyloglucan gel. in order to develop a thermosetting gel with a suitable phase transition temperature, wei. gamma scintigraphy demonstrated that the clearance of an optimized formulation containing 21% f127 and 10% f68 was significantly delayed with respect to a phosphate buffer solution. a three - fold increase of the corneal residence time was achieved in the rabbits. three principal mechanisms have been proposed to explain the liquid - gel phase transition after an increase in temperature, including : gradual desolvation of the polymer, increased micellar aggregation, andthe increased entanglement of the polymeric network. gradual desolvation of the polymer, increased micellar aggregation, and the increased entanglement of the polymeric network. despite all the promising results obtained with thermo reversible gels, there remains an important drawback associated with their use ; the risk of gelation before administration by increase in ambient temperature during packing or storage. these gels have ionic groups (which are readily ionizable side groups) attached to impart peculiar characteristics. some of the ph sensitive polymers used in hydrogels preparations are polymethyl methacrylate (pmma), polyacrylamide (paam), polyacrylic acid (paa), poly dimethylaminoethylmethacrylate (pdeaema) and polyethylene glycol. these polymers though in nature are hydrophobic but swells in water depending upon the ph prevalent in the external environment. any change in ph of the biological environment causes changes in the swelling behavior, for example, the hydrogel of caffeine is prepared with poly- mer pdeaema at ph below 6.6. as the polymer shows high swellability but when ph changes to higher side, the polymer showed shrinkage leading to drug release. the other ph - sensitive hydrogels are copolymer of pmma and polyhydroxyethyl methyl acrylate (phema), which are anionic copolymers, swell high in neutral or high ph but do not swell in acidic medium. it was also observed that ph and ionic strength determines kinetics of swelling of phema and guar gum (peppas and peppas, 1990 ; das., 2006). cellulose acetate phthalate (cap) latex, cross linked acrylic, and derivatives such as carbomer are used. cellulose acetate derivatives are the only polymer known to have a buffer capacity that is low enough to gel effectively in the cul - de - sac of the eye. the ph change of about 2.8 units after instillation of the native formulation (ph 4.4) into the tear film leads to an almost instantaneous transformation of the highly fluid latex into viscous gel. the gamma scintigraphy technique was used to monitor the ocular residence time of an ophthalmic preparation based on cellulose acetate phthalate (cap) dispersion. first preliminary investigations of ph - sensitive latexes for ophthalmic administration began in early 1980s and have been extensively studied by boye. cellulose acetate phthalate latex is a polymer with potentially useful properties for sustained drug delivery to the eye because latex is a free running solution at a ph of 4.4, which undergoes coagulation when the ph is raised by the tear fluid to ph. 7.4. the use of ph - sensitive latex nanoparticles has been described by gurny. the poly acrylic acid and its lightly cross - linked commercial forms (polycarbophil and carbopol) exhibit the strongest muco - adhesion. in the pioneering paper, hui and robinson demonstrated that the use of acrylates for ocular delivery of progesterone was based not only on viscosifying but also on bioadhesion properties. carbomer (carbopol) a cross - linked acrylic acid polymer (paa) also shows ph induced phase transition as the ph is raised above its pka of about 5.5. the manufacturer states that carbopol 934 gel has the lowest cross - linking density, while carbopol 981 intermediate and carbopol 940 have the highest. however, the amount of paa required to form stiff gel upon instillation in the eye is not easily neutralized by the buffering action of tear fluid. combination paa with a suitable viscosity enhancing polymer, e.g. hpmc or mc allows a reduction in the paa concentration without comprising the in situ gelling properties. the formulation containing carbopol 940 and methocel e50lv (hpmc) afforded sustained release of ofloxacin over an 8-h period. ion - sensitive polymers belong to the mainly used in situ gelling materials for ocular drug delivery. it is assumed that the rate at which electrolytes from the tear fluid is absorbed by the polymer will depend on the osmotic gradient across the surface of the gel. it is therefore likely that the osmolality of the solution might have an influence on the rate of the sol - gel transition occurring in the eye. one example is gelrite, an anionic extra cellular polysaccharide, low acetyl gellan gum secreted by pseudomonas elodea. gelrite formulations in aqueous solutions form a clear gel in the presence of the mono or divalent cations typically found in the tear fluids. the electrolyte of the tear fluid and especially na+, ca++, and mg++ cations are particularly suited to initiate gelation of the polymer when instilled as a liquid solution in to the cul - de - sac. gelrite has been the most widely studied and seems to be preferred compared to the ph sensitive or temperature setting systems. slightly viscous gellan gum solutions in low concentrations (< 1%) show markedly increase in apparent viscosity, when introduced into presence of a physiological level of cations, without requiring more ions than 1025% of those in tear fluid. gellan - containing formulations of pilocarpine hcl allowed reduction of drug concentration from 2% to 0.5% obtaining the same bioavailability. rozier., found an improvement in the ocular absorption of timolol in albino rabbits when absorption of timolol in albino rabbits when administered in gelrite when compared with an equiviscous solution of hydroxyl - ethyl cellulose. sanzgiri., compared various systems of methyl prednisolone (mp) ; esters of mp with gelrite eye drops, gellan - mp film, and gellan film with dispersed mp. gellan eye drops provided better performance because they afforded the advantage of faster gelation over a high surface area in eye, whereas the results obtained with the gellan - mp film seemed to indicate that the gelation at the surface of the film occurred very slowly, and the surface of release was not controlled. mourice and srinivas measured a two fold increase in the permeation of the fluorescein in humans when using gellan gum compared to isotonic buffer solution. the ability of gel formation at physiological ca2 + levels was used in case of alginic acid as well. presence of this polymer significantly extended the duration of the pressure reducing effect of pilocarpine to 10-h and carteolol to 8-h, allowing only once a day administration in case of carteolol. demonstrated that an aqueous solution of sodium alginate could gel in the eye, without addition of external calcium ions or other bivalent / polyvalent cations. the extent of alginate gelation and consequently the release of pilocarpine were found to be dependent upon the percentage of glucuronic acid residues in the polymer backbone. alginates with g content more than 65%, such as manugel dmb, instantaneously formed gels upon their addition to stf. in vitro release studies indicated the slow release of pilocarpine over a period of 24 hours. recently some other natural polymers believed to be able to form in situ gels by interacting with the lachrymal fluid have been evaluated as potential adjuvant in ophthalmic formulation. this includes carageenans, xyloglucans, and some alginates that are rich in guluronic acid residues. k - carrageenan forms rigid, brittle gels in reply of small amount of k+, i - carrageenan forms elastic gels mainly in the presence of ca. gelation of the low - methoxy pectins can be caused by divalent cations, especially ca. likewise, alginic acid undergoes gelation in presence of divalent / polyvalent cations, e.g., ca due to the interaction with glucuronic acid blocks in alginate chains. sodium alginate consists chiefly of the sodium salt of alginic acid, a linear glycuronan polymer consisting of a mixture of - (1, 4) dmannosyluronic acid and - (1, 4)-lgulosyluronic acid residues. compared the commercial product timoptic xe 0.5% with a timolol mealeate gelforming solution with xanthan gum as the gelling polymer (timolol gfs 0.5% alcon research). the reported data indicated equivalent efficacy in the reduction intraocular pressure (a maintained reduction during long term use) and consequently therapeutic equivalence. keipert reported that the increase in therapeutic effects (i.e., miosis) in rabbits could be due to a permeation enhancing effect of gellan gum comparable to edta. the commercial product timoptol xe preparation containing gelrite remains for a longer period at the eye surface when compared to conventional timolol maleate eye drops. this resulted in an enhanced drug transfer sufficient enough to obtain an intro ocular pressure reduction after a once - daily topical instillation. g / l) is quite sufficient to induce gelation. because the presence of lachrymal fluid is necessary to induce gel formation, accidental gelation during storage hence, stimuli - sensitive hydrogels are better alternative for ophthalmic drug delivery of pharmaceuticals ; they show the following advantages : prolonged drug releasereduced systemic side effectsreduced number of applicationsbetter patient compliance prolonged drug release reduced systemic side effects reduced number of applications better patient compliance temperature - sensitive hydrogels are probably the most commonly studied class of environment - sensitive polymer systems in drug delivery research. these hydrogels are able to swell or de - swell as a result of changing in the temperature of the surrounding fluid. for convenience, temperature - sensitive hydrogels are classified into negatively thermosensitive, positively thermosensitive, and thermally reversible gels. negative temperature - sensitive hydrogels have a lower critical solution temperature (lcst) and contract upon heating above the lcst. copolymers of (nisopropylacrylamide) (pniaam) are usually used for negative temperature release. hydrogels show an on off drug release with on at a low temperature and off at high temperature allowing pulsatile drug release. lcst systems are mainly relevant for controlled release of drugs, and of proteins in particular. thermosensitive polymers may be fixed on liposome membranes ; in that case liposomes exhibit control of their content release. positive temperature - sensitive hydrogel has an upper critical solution temperature (ucst), such hydrogel contracts upon cooling below the ucst. polymer networks of poly (acrylic acid) (paa) and polyacrylamide (paam) or poly (acrylamide - co - butyl methacrylate) have positive temperature dependence of swelling. the most commonly used thermoreversible gels are these prepared from poly (ethylene oxide)-b - poly (provpylene oxide)-b - poly (ethylene oxide) (pluronics, tetronics, poloxamer) polymer solution is a free - flowing liquid at ambient temperature and gels at body temperature, such a system would be easy to administer into desired body cavity. in some cases, if lowering the amount of thermogelling polymer is necessary, it may be blended with a ph - sensitive reversibly gelling polymer. recently, the polymers consisting of poly (ethylene glycol)-poly-(dllactic acid - co - glycolic acid)-poly(ethylen glycol) (pegplga- peg) or plga - peg - plga were investigated for sustained injectable drug delivery systems. some of the earliest work with temperature - sensitive hydrogels was done by the group of tanaka pnipaam is the best example of a negative temperature - sensitive hydrogel.. worked with crosslinked pnipaam and determined that the lcst of the pnipaam gels was 34.38c. they also found that the lcst could be increased by mixing small amounts of ionic copolymers in the gels. hoffman proposed the application of pnipaam and its copolymers for temperature - modulated drug release by bulk squeezing and surface regulation. in the bulk squeezing system, the drug that is distributed evenly inside the matrix is squeezed out of the system due to the de - swelling of the hydrogel as a result of increasing the temperature of the environment above the volume phase transition temperature. in the surface regulation system, the swelling ratio of the skin layer is increased as the temperature of the system is lowered below the volume phase transition temperature, and hence, the drug molecules will be able to diffuse through the skin layer. poloxamers are a broad group of compounds that were introduced in the early 1950s as food additives and for pharmaceutical preparations. these water - soluble surfactants are triblock co - polymers prepared from poly (ethylene oxide)-b - poly (propylene oxide)-b - poly (ethylene oxide) commercially available as pluronic are the most commonly used thermosetting polymers and could be applicable for the development of effective ophthalmic drug delivery. depending on the ratio and the distribution along the chain of the hydrophobic and hydrophilic subunits, several molecular weights are available, leading to different gelation properties pluronic f127, which gives colorless and transparent gels, is the most commonly used in pharmaceutical technology. pluronic f 127 is no more damaging to the mouse or rabbit cornea than a physiological saline. the poloxamers are reported to be well tolerated and non - toxic even though large amounts (25 - 30%) of polymers are required to obtained a suitable gel. at concentrations of 20% w / v and higher aqueous solutions of poloxamer-407 remain as a liquid at low temperatures [< 15c ] and yield a highly viscous semisolid gel upon instillation into the cul - de - sac. at low temperatures, the poloxamer forms micellar subunits in solution, and swelling gives rise to large micellar subunits and the creation of cross - linked networks. miller. examined a temperature - sensitive solution of poloxamer used to deliver the miotic pilocarpine. in order to reduce the concentration of polymer and/or to achieve a phase transition temperature higher than room temperature (25c) and gelling at precorneal temperature (35c), the combining pluronic analogs or the addition of further polymer, e.g. peg, paa, methylcellulose (mc), hpmc, cmc is often necessary. attwood. has reported enhancement of the miotic response following sustained release of pilocarpine from the 1% w / w xyloglucan gel. in order to develop a thermosetting gel with a suitable phase transition temperature, wei. gamma scintigraphy demonstrated that the clearance of an optimized formulation containing 21% f127 and 10% f68 was significantly delayed with respect to a phosphate buffer solution. a three - fold increase of the corneal residence time was achieved in the rabbits. three principal mechanisms have been proposed to explain the liquid - gel phase transition after an increase in temperature, including : gradual desolvation of the polymer, increased micellar aggregation, andthe increased entanglement of the polymeric network. gradual desolvation of the polymer, increased micellar aggregation, and the increased entanglement of the polymeric network. despite all the promising results obtained with thermo reversible gels, there remains an important drawback associated with their use ; the risk of gelation before administration by increase in ambient temperature during packing or storage. these gels have ionic groups (which are readily ionizable side groups) attached to impart peculiar characteristics. some of the ph sensitive polymers used in hydrogels preparations are polymethyl methacrylate (pmma), polyacrylamide (paam), polyacrylic acid (paa), poly dimethylaminoethylmethacrylate (pdeaema) and polyethylene glycol. these polymers though in nature are hydrophobic but swells in water depending upon the ph prevalent in the external environment. any change in ph of the biological environment causes changes in the swelling behavior, for example, the hydrogel of caffeine is prepared with poly- mer pdeaema at ph below 6.6. as the polymer shows high swellability but when ph changes to higher side, the polymer showed shrinkage leading to drug release. the other ph - sensitive hydrogels are copolymer of pmma and polyhydroxyethyl methyl acrylate (phema), which are anionic copolymers, swell high in neutral or high ph but do not swell in acidic medium. it was also observed that ph and ionic strength determines kinetics of swelling of phema and guar gum (peppas and peppas, 1990 ; das., 2006). cellulose acetate phthalate (cap) latex, cross linked acrylic, and derivatives such as carbomer are used. cellulose acetate derivatives are the only polymer known to have a buffer capacity that is low enough to gel effectively in the cul - de - sac of the eye. the ph change of about 2.8 units after instillation of the native formulation (ph 4.4) into the tear film leads to an almost instantaneous transformation of the highly fluid latex into viscous gel. the gamma scintigraphy technique was used to monitor the ocular residence time of an ophthalmic preparation based on cellulose acetate phthalate (cap) dispersion. first preliminary investigations of ph - sensitive latexes for ophthalmic administration began in early 1980s and have been extensively studied by boye. cellulose acetate phthalate latex is a polymer with potentially useful properties for sustained drug delivery to the eye because latex is a free running solution at a ph of 4.4, which undergoes coagulation when the ph is raised by the tear fluid to ph. the poly acrylic acid and its lightly cross - linked commercial forms (polycarbophil and carbopol) exhibit the strongest muco - adhesion. in the pioneering paper, hui and robinson demonstrated that the use of acrylates for ocular delivery of progesterone was based not only on viscosifying but also on bioadhesion properties. carbomer (carbopol) a cross - linked acrylic acid polymer (paa) also shows ph induced phase transition as the ph is raised above its pka of about 5.5. the manufacturer states that carbopol 934 gel has the lowest cross - linking density, while carbopol 981 intermediate and carbopol 940 have the highest. however, the amount of paa required to form stiff gel upon instillation in the eye is not easily neutralized by the buffering action of tear fluid. combination paa with a suitable viscosity enhancing polymer, e.g. hpmc or mc allows a reduction in the paa concentration without comprising the in situ gelling properties. the formulation containing carbopol 940 and methocel e50lv (hpmc) afforded sustained release of ofloxacin over an 8-h period. ion - sensitive polymers belong to the mainly used in situ gelling materials for ocular drug delivery. it is assumed that the rate at which electrolytes from the tear fluid is absorbed by the polymer will depend on the osmotic gradient across the surface of the gel. it is therefore likely that the osmolality of the solution might have an influence on the rate of the sol - gel transition occurring in the eye. one example is gelrite, an anionic extra cellular polysaccharide, low acetyl gellan gum secreted by pseudomonas elodea. gelrite formulations in aqueous solutions form a clear gel in the presence of the mono or divalent cations typically found in the tear fluids. the electrolyte of the tear fluid and especially na+, ca++, and mg++ cations are particularly suited to initiate gelation of the polymer when instilled as a liquid solution in to the cul - de - sac. gelrite has been the most widely studied and seems to be preferred compared to the ph sensitive or temperature setting systems. slightly viscous gellan gum solutions in low concentrations (< 1%) show markedly increase in apparent viscosity, when introduced into presence of a physiological level of cations, without requiring more ions than 1025% of those in tear fluid. gellan - containing formulations of pilocarpine hcl allowed reduction of drug concentration from 2% to 0.5% obtaining the same bioavailability. rozier., found an improvement in the ocular absorption of timolol in albino rabbits when absorption of timolol in albino rabbits when administered in gelrite when compared with an equiviscous solution of hydroxyl - ethyl cellulose. sanzgiri., compared various systems of methyl prednisolone (mp) ; esters of mp with gelrite eye drops, gellan - mp film, and gellan film with dispersed mp. gellan eye drops provided better performance because they afforded the advantage of faster gelation over a high surface area in eye, whereas the results obtained with the gellan - mp film seemed to indicate that the gelation at the surface of the film occurred very slowly, and the surface of release was not controlled. mourice and srinivas measured a two fold increase in the permeation of the fluorescein in humans when using gellan gum compared to isotonic buffer solution. the ability of gel formation at physiological ca2 + levels was used in case of alginic acid as well. presence of this polymer significantly extended the duration of the pressure reducing effect of pilocarpine to 10-h and carteolol to 8-h, allowing only once a day administration in case of carteolol. demonstrated that an aqueous solution of sodium alginate could gel in the eye, without addition of external calcium ions or other bivalent / polyvalent cations. the extent of alginate gelation and consequently the release of pilocarpine were found to be dependent upon the percentage of glucuronic acid residues in the polymer backbone. alginates with g content more than 65%, such as manugel dmb, instantaneously formed gels upon their addition to stf. in vitro release studies indicated the slow release of pilocarpine over a period of 24 hours. recently some other natural polymers believed to be able to form in situ gels by interacting with the lachrymal fluid have been evaluated as potential adjuvant in ophthalmic formulation. this includes carageenans, xyloglucans, and some alginates that are rich in guluronic acid residues. k - carrageenan forms rigid, brittle gels in reply of small amount of k+, i - carrageenan forms elastic gels mainly in the presence of ca. gelation of the low - methoxy pectins can be caused by divalent cations, especially ca. likewise, alginic acid undergoes gelation in presence of divalent / polyvalent cations, e.g., ca due to the interaction with glucuronic acid blocks in alginate chains. sodium alginate consists chiefly of the sodium salt of alginic acid, a linear glycuronan polymer consisting of a mixture of - (1, 4) dmannosyluronic acid and - (1, 4)-lgulosyluronic acid residues. compared the commercial product timoptic xe 0.5% with a timolol mealeate gelforming solution with xanthan gum as the gelling polymer (timolol gfs 0.5% alcon research). the reported data indicated equivalent efficacy in the reduction intraocular pressure (a maintained reduction during long term use) and consequently therapeutic equivalence. keipert reported that the increase in therapeutic effects (i.e., miosis) in rabbits could be due to a permeation enhancing effect of gellan gum comparable to edta. the commercial product timoptol xe preparation containing gelrite remains for a longer period at the eye surface when compared to conventional timolol maleate eye drops. this resulted in an enhanced drug transfer sufficient enough to obtain an intro ocular pressure reduction after a once - daily topical instillation. g / l) is quite sufficient to induce gelation. because the presence of lachrymal fluid is necessary to induce gel formation, accidental gelation during storage hence, stimuli - sensitive hydrogels are better alternative for ophthalmic drug delivery of pharmaceuticals ; they show the following advantages : prolonged drug releasereduced systemic side effectsreduced number of applicationsbetter patient compliance prolonged drug release reduced systemic side effects reduced number of applications better patient compliance the main efforts in ocular drug delivery during the past two decades has been on the design of systems to prolong the residence time of topically applied drugs in conjunctival sac. hydrogels generally offer a moderate improvement of ocular drug bioavailability despite their favorable bioadhesive properties. one of the disadvantages is that hydrogel may result in blurred vision as well as foreign body sensation to patients. stimuli activated gel - forming systems seem to be preferred as they can be administered in drop form and create significantly less problems with vision. thus, the fascinating properties of the stimuli - sensitive polymers seem promising in many future applications and offer possible use as the next generation of materials in biological, biomedical, and pharmaceutical products, because as with non - viscous eye drops, accurate and precise sustained release properties with little or no eye irritation is possible. however, there is still a basic need for more details in this area. | amongst the various routes of drug delivery, the field of ocular drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist for past 10 - 20 years. as an isolated organ, eye is very difficult to study from a drug delivery point of view. despite this limitation, improvements have been made with the objective of maintaining the drug in the biophase for an extended period. a major problem in ocular therapeutics is the attainment of an optimal drug concentration at the site of action. to achieve effective ophthalmic therapy, an adequate amount of active ingredient must be delivered and maintained within the eye. the most frequently used dosage forms, i.e., eye solution, eye ointments, eye gels, and eye suspensions are compromised in their effectiveness by several limitations leading to poor ocular bioavailability. ophthalmic use of viscosity - enhancing agents, penetration enhancers, cyclodextrins, prodrug approaches, and ocular inserts, and the ready existing drug carrier systems along with their application to ophthalmic drug delivery are common to improve ocular bioavailability. amongst these hydrogel (stimuli sensitive) systems are important, which undergo reversible volume and/or sol - gel phase transitions in response to physiological (temperature, ph and present of ions in organism fluids, enzyme substrate) or other external (electric current, light) stimuli. they help to increase in precorneal residence time of drug to a sufficient extent that an ocularly delivered drug can exhibit its maximum biological action. the concept of this innovative ophthalmic delivery approach is to decrease the systemic side effects and to create a more pronounced effect with lower doses of the drug. the present article describes the advantages and use stimuli sensitive of hydrogel systems in ophthalmic drug delivery. |
lung cancer is the most commonly diagnosed cancer. it is the leading cause of cancer - related deaths in males and the second cause of cancer - related deaths in females worldwide.1 non - small - cell histology comprises ~85% of lung cancers, and 75% of patients are diagnosed at stages iii and iv.2,3 at the advanced stages, taxane chemotherapy regimens are commonly used for the treatment of non - small - cell lung cancers (nsclcs) as first - line options.4 however, long - term survival in patients with advanced nsclc is < 5% and toxic. side effects such as febrile neutropenia, neuropathy, and hypersensitivity reactions are particularly high in taxane formulations. furthermore, the therapeutic results are not satisfactory.4,5 paclitaxel and its semisynthetic form, docetaxel, are isolated from the yew tree and primarily stabilize cytoplasmic microtubules via binding to the -tubulin site, thereby causing cell cycle arrest at the g2/m phase and driving apoptosis.6 several compounds have been tested to determine whether they increase taxane - induced anticancer efficacy. for example, the therapeutic efficacy of paclitaxel is restricted by the increasing frequency of chemotherapeutic resistance in nsclc. pulsing with both taxanes results in approximately fivefold higher resistant clones in nsclc cells, such as a549 and h1299 cells.7 several compounds, such as l - type calcium channel blockers, reverse docetaxel - induced multidrug resistance independent of abcb1 expression in both cell lines.8 additionally, the histone deacetylase inhibitor trichostatin a increases both cell cycle delay at the g2/m phase and apoptosis in docetaxel - treated a549 cells.9 in hormone - refractory prostate cancer (hrpc) models, taxol synergizes with several antioxidants in hrpc cells by inducing cell cycle arrest at the sub g1 phase, apoptosis, and caspase activity and decreasing bcl-2 expression simultaneously.10 by contrast, tp53 mutations are the most frequent gene abnormalities leading to inactivation of p53, which effects paclitaxel sensitivity in nsclc.11 a trace element, zinc, is essential for the wide range of physiological processes, including growth, development, and immune functions as well as the intracellular activities of ~300 enzymes and 2,000 transcription factors.12 it is also effective in decreasing oxidative stress and the generation of inflammatory cytokines such as tnf- and il-1.13 furthermore, the role of zinc in the development and progression of prostate cancer and its widespread antitumor efficacy have been shown in several malignancies.1417 intracellular zinc status is associated with prostate carcinogenesis. for example, zinc deficiency contributes to tumor progression and development in cultured hrpc cells,18 whereas increased levels of intracellular zinc decrease cancer cell proliferation and induce apoptosis.14,15 in a large case - control study, a direct association was found between zinc intake and prostate cancer risk.19 the vast majority of epithelial tumors are associated with reduced intratumoral or plasma zinc levels.20 importantly, zinc deficiency reduces paclitaxel efficacy in cultured prostate cancer cells, whereas increased intracellular zinc concentrations sensitize prostate cancer cells to cytotoxic agents, including paclitaxel, via inhibition of nf-b activation.21,22 therefore, zinc supplementation may have growth inhibitory effects against nsclc cells and may increase docetaxel efficacies. in this study, cellular viability, apoptosis, and cell cycle alterations as well as oxidative stress activities were tested in the nsclc cell lines, a549, and h1299, when zinc was used alone and in combination with docetaxel. nsclc cells, a549 (p53 wild - type), and h1299 (lymph node metastasis, p53 null - type) were obtained from the american type culture collection. the a549 cells were cultured in dmem / ham s f12k medium that containing 10% fbs, 1% penicillin / streptomycin, 1% l - glutamine, and 1% amphotericin b. the h1299 cells were cultured in rpmi-1640 medium containing 10% fbs, 1% penicillin / streptomycin, 1% l - glutamine, and 1% amphotericin b at 37c in a 5% co2 incubator. cell viability was determined using the cck-8 (cell counting kit-8 ; sigma - aldrich, st louis, mo, usa) assay in accordance with the manufacturer s instructions. briefly, when the cells reached 80% confluency, the medium was replaced with fresh medium without zinc or docetaxel. then, the cells were transferred to 96-well plates at a density of 10,000 cells / well. docetaxel (0.001100 m) and zinc sulfate (101,000 m) were added to the cell lines at different concentrations in each of the eight wells. the cck-8 reagents were added to each well at the indicated time after the treatments. at the end of 24 hours and 48 hours, the absorbance was measured at a test wavelength of 450 nm and a reference wavelength of 650 nm using a microplate reader (thermo electron corporation, multiskan ascent). the average inhibitory concentration 50 values (ic50 values) were determined at 24-hour and 48-hour treatment periods. the 24-hour doses were selected due to the lack of significant differences between the 24-hour and 48-hour doses. the cells were plated in six - well plates at a density of 5010 cells / well, and the total volume was brought to 3.2 ml with fresh media. after the initial 72-hour incubation, at which point the cells were ~80% confluent, the cells were treated with the previously established ic50 concentrations of docetaxel, zinc and combinations thereof in fresh medium for an additional 24 hours. the cells were harvested by trypsinization and collected by centrifugation at 1,500 rpm for 5 minutes. following removal of the supernatant, the cells were resuspended in 400 l binding buffer and stained with 5 l annexin - v and 5 l propidium iodide (pi, 50 g / ml) at room temperature for 20 minutes in the dark. after adding 400 l binding buffer to each tube the percentage apoptosis, necrosis, and viable cells were evaluated by using a flow cytometer. a beckman coulterepicsxl - mcl was used for analysis of the h1299 cells, and a becton dickinson (bd) facscanto ii cytometer was used for analysis of the a549 cells. the cycletest plus dna reagent kit (bd pharmingen) the cells were seeded in six - well plates at a density of 5010 cells / well, and their volume was adjusted to 3.2 ml with media. after an initial incubation for 72 hours, at which point the cells were ~80% confluent, the cells were treated with previously defined ic50 doses of zinc and docetaxel and then incubated for 24 hours. then, the medium was removed, and 500 l trypsin was added to the cells, which were transferred into a tube. a volume of 1 ml fresh medium was added to each tube to inactivate the trypsin. the suspension was transferred to 17100 mm tubes and centrifuged at 1,500 rpm for 5 minutes. the supernatant was removed, and the cells were resuspended by adding 1 ml buffer solution (sucrose - citrate buffer containing dimethyl sulfoxide) to the pellet (wash procedure). after removal of the buffer, 250 l solution a (citrate buffer containing trypsin) was added to the pellet in each tube and incubated at room temperature for 10 minutes. then, 200 l solution b (citrate buffer containing trypsin inhibitor and rnase) was added to each tube and incubated at room temperature for 10 minutes. finally, 200 l solution c (pi - staining solution) at 4c was added to each tube and reincubated on ice for 10 minutes. three hours later, the cells were analyzed using double laser - flow cytometry devices. a beckman coulterepicsxl - mcl was used for analysis of the h1299 cells, and a bd facscanto ii cytometer was used for analysis of the a549 cells. the cells in g0/g1, s (synthesis), and g2/m (mitosis) phases were evaluated using the modfitlt3.2 software program. the cell lysates were prepared and kept at 20c before the antioxidant enzyme and malondialdehyde (mda) analyses. for standard preparation, the mda standard (t-9889) a 10 mm stock standard solution was prepared by dissolving the mda in distilled water, and this was then diluted to working concentrations ranging from 1 m to 50 m. for sample preparation, the protein concentrations were measured using a bca protein assay kit (pierce bca protein assay kit 23225 ; thermo fisher scientific, waltham, ma, usa). the samples were lyophilized with a flexi - dry up freeze dryer (fts systems, stone ridge, ny) because of the low protein concentrations. for mda measurement, 100 l distilled water was added to a 40 l volume of all cell samples. then, 20 l of 2.8 mmol / l bht, 40 l of 8.1% sds, 600 l of 8 g / l tba:200 ml / l acetic acid (1:1) were added, and the ph was adjusted with 2 m naoh (ph 3.5). a volume of 200 l distilled water and 1,000 l butanol : pyridine (15:1) were added, and all the samples were vortexed for 1 minute. after 3 minutes, the organic phases were transferred to new eppendorf tubes and centrifuged at 10,000 rpm for 10 minutes. the samples were then placed in an autosampler vial for high - performance liquid chromatography (hplc) analysis. as previously described,23 the analysis was performed using a hplc set prominence lc 20 (shimadzu, kyoto, japan) equipped with sil-20ac autosampler, lc-20ad pump, and rf-10axl fluorescence detector. phosphate buffer (0.01 m in 30% methanol, ph 7.0) was used as the mobile phase. the superoxide dismutase (sod) activity of the cells was determined using a ransod kit (randox labs, crumlin, uk). the absorbance was measured at 505 nm on a microplate reader (bio - tek instruments, inc. the glutathione peroxidase (gpx) activity of the cells was determined using a ransel kit (randox labs, crumlin, uk) and a previously described method.24 briefly, the lyophilized cells and standard solutions were used to assay for gpx. the absorbance was measured at 340 nm on a microplate reader (bio - tek instruments, inc.). in this method, gpx oxidizes glutathione via cumene hydroperoxidase. in the presence of gr and nadph, oxidized glutathione is immediately converted to the reduced form, with concomitant oxidation of nadph to nadp. a 40-l sample was added to the primary reagent (200 l) and cumene (80 l) into a 96-well plate. the plate was read after 1 minute, then again after 2 minutes for the final absorbance (after reaction). the gpx activity of these samples was calculated according to gpx manual included with the ransel kit. the triplicate data were analyzed using the sigmastat 3.5 (systat inc., santa cruz, ca, usa) software program. nsclc cells, a549 (p53 wild - type), and h1299 (lymph node metastasis, p53 null - type) were obtained from the american type culture collection. the a549 cells were cultured in dmem / ham s f12k medium that containing 10% fbs, 1% penicillin / streptomycin, 1% l - glutamine, and 1% amphotericin b. the h1299 cells were cultured in rpmi-1640 medium containing 10% fbs, 1% penicillin / streptomycin, 1% l - glutamine, and 1% amphotericin b at 37c in a 5% co2 incubator. cell viability was determined using the cck-8 (cell counting kit-8 ; sigma - aldrich, st louis, mo, usa) assay in accordance with the manufacturer s instructions. briefly, when the cells reached 80% confluency, the medium was replaced with fresh medium without zinc or docetaxel. then, the cells were transferred to 96-well plates at a density of 10,000 cells / well. docetaxel (0.001100 m) and zinc sulfate (101,000 m) were added to the cell lines at different concentrations in each of the eight wells. the cck-8 reagents were added to each well at the indicated time after the treatments. at the end of 24 hours and 48 hours, the absorbance was measured at a test wavelength of 450 nm and a reference wavelength of 650 nm using a microplate reader (thermo electron corporation, multiskan ascent). the average inhibitory concentration 50 values (ic50 values) were determined at 24-hour and 48-hour treatment periods. the 24-hour doses were selected due to the lack of significant differences between the 24-hour and 48-hour doses. the cells were plated in six - well plates at a density of 5010 cells / well, and the total volume was brought to 3.2 ml with fresh media. after the initial 72-hour incubation, at which point the cells were ~80% confluent, the cells were treated with the previously established ic50 concentrations of docetaxel, zinc and combinations thereof in fresh medium for an additional 24 hours. the cells were harvested by trypsinization and collected by centrifugation at 1,500 rpm for 5 minutes. following removal of the supernatant, the cells were resuspended in 400 l binding buffer and stained with 5 l annexin - v and 5 l propidium iodide (pi, 50 g / ml) at room temperature for 20 minutes in the dark. after adding 400 l binding buffer to each tube the percentage apoptosis, necrosis, and viable cells were evaluated by using a flow cytometer. a beckman coulterepicsxl - mcl was used for analysis of the h1299 cells, and a becton dickinson (bd) facscanto ii cytometer was used for analysis of the a549 cells. the cycletest plus dna reagent kit (bd pharmingen) the cells were seeded in six - well plates at a density of 5010 cells / well, and their volume was adjusted to 3.2 ml with media. after an initial incubation for 72 hours, at which point the cells were ~80% confluent, the cells were treated with previously defined ic50 doses of zinc and docetaxel and then incubated for 24 hours. then, the medium was removed, and 500 l trypsin was added to the cells, which were transferred into a tube. a volume of 1 ml fresh medium was added to each tube to inactivate the trypsin. the suspension the supernatant was removed, and the cells were resuspended by adding 1 ml buffer solution (sucrose - citrate buffer containing dimethyl sulfoxide) to the pellet (wash procedure). after removal of the buffer, 250 l solution a (citrate buffer containing trypsin) was added to the pellet in each tube and incubated at room temperature for 10 minutes. then, 200 l solution b (citrate buffer containing trypsin inhibitor and rnase) was added to each tube and incubated at room temperature for 10 minutes. finally, 200 l solution c (pi - staining solution) at 4c was added to each tube and reincubated on ice for 10 minutes. three hours later, the cells were analyzed using double laser - flow cytometry devices. a beckman coulterepicsxl - mcl was used for analysis of the h1299 cells, and a bd facscanto ii cytometer was used for analysis of the a549 cells. the cells in g0/g1, s (synthesis), and g2/m (mitosis) phases were evaluated using the modfitlt3.2 software program. the cells were plated in six - well plates at a density of 5010 cells / well, and the total volume was brought to 3.2 ml with fresh media. after the initial 72-hour incubation, at which point the cells were ~80% confluent, the cells were treated with the previously established ic50 concentrations of docetaxel, zinc and combinations thereof in fresh medium for an additional 24 hours. the cells were harvested by trypsinization and collected by centrifugation at 1,500 rpm for 5 minutes. following removal of the supernatant, the cells were resuspended in 400 l binding buffer and stained with 5 l annexin - v and 5 l propidium iodide (pi, 50 g / ml) at room temperature for 20 minutes in the dark. after adding 400 l binding buffer to each tube the percentage apoptosis, necrosis, and viable cells were evaluated by using a flow cytometer. a beckman coulterepicsxl - mcl was used for analysis of the h1299 cells, and a becton dickinson (bd) facscanto ii cytometer was used for analysis of the a549 cells. the cycletest plus dna reagent kit (bd pharmingen) was used to determine the distribution of cells between cell - cycle phases. the cells were seeded in six - well plates at a density of 5010 cells / well, and their volume was adjusted to 3.2 ml with media. after an initial incubation for 72 hours, at which point the cells were ~80% confluent, the cells were treated with previously defined ic50 doses of zinc and docetaxel and then incubated for 24 hours. then, the medium was removed, and 500 l trypsin was added to the cells, which were transferred into a tube. a volume of 1 ml fresh medium was added to each tube to inactivate the trypsin. the suspension the supernatant was removed, and the cells were resuspended by adding 1 ml buffer solution (sucrose - citrate buffer containing dimethyl sulfoxide) to the pellet (wash procedure). after removal of the buffer, 250 l solution a (citrate buffer containing trypsin) was added to the pellet in each tube and incubated at room temperature for 10 minutes. then, 200 l solution b (citrate buffer containing trypsin inhibitor and rnase) was added to each tube and incubated at room temperature for 10 minutes. finally, 200 l solution c (pi - staining solution) at 4c was added to each tube and reincubated on ice for 10 minutes. three hours later, the cells were analyzed using double laser - flow cytometry devices. a beckman coulterepicsxl - mcl was used for analysis of the h1299 cells, and a bd facscanto ii cytometer was used for analysis of the a549 cells. the cells in g0/g1, s (synthesis), and g2/m (mitosis) phases were evaluated using the modfitlt3.2 software program. the cell lysates were prepared and kept at 20c before the antioxidant enzyme and malondialdehyde (mda) analyses. for standard preparation, the mda standard (t-9889) a 10 mm stock standard solution was prepared by dissolving the mda in distilled water, and this was then diluted to working concentrations ranging from 1 m to 50 m. for sample preparation, the protein concentrations were measured using a bca protein assay kit (pierce bca protein assay kit 23225 ; thermo fisher scientific, waltham, ma, usa). the samples were lyophilized with a flexi - dry up freeze dryer (fts systems, stone ridge, ny) because of the low protein concentrations. for mda measurement, 100 l then, 20 l of 2.8 mmol / l bht, 40 l of 8.1% sds, 600 l of 8 g / l tba:200 ml / l acetic acid (1:1) were added, and the ph was adjusted with 2 m naoh (ph 3.5). a volume of 200 l distilled water and 1,000 l butanol : pyridine (15:1) were added, and all the samples were vortexed for 1 minute. after 3 minutes, the organic phases were transferred to new eppendorf tubes and centrifuged at 10,000 rpm for 10 minutes. the samples were then placed in an autosampler vial for high - performance liquid chromatography (hplc) analysis. as previously described,23 the analysis was performed using a hplc set prominence lc 20 (shimadzu, kyoto, japan) equipped with sil-20ac autosampler, lc-20ad pump, and rf-10axl fluorescence detector. phosphate buffer (0.01 m in 30% methanol, ph 7.0) was used as the mobile phase. the superoxide dismutase (sod) activity of the cells was determined using a ransod kit (randox labs, crumlin, uk). lyophilized cells and standard solutions the absorbance was measured at 505 nm on a microplate reader (bio - tek instruments, inc. the glutathione peroxidase (gpx) activity of the cells was determined using a ransel kit (randox labs, crumlin, uk) and a previously described method.24 briefly, the lyophilized cells and standard solutions were used to assay for gpx. the absorbance was measured at 340 nm on a microplate reader (bio - tek instruments, inc.). in this method, gpx oxidizes glutathione via cumene hydroperoxidase. in the presence of gr and nadph, oxidized glutathione is immediately converted to the reduced form, with concomitant oxidation of nadph to nadp. a 40-l sample was added to the primary reagent (200 l) and cumene (80 l) into a 96-well plate. the plate was read after 1 minute, then again after 2 minutes for the final absorbance (after reaction). the gpx activity of these samples was calculated according to gpx manual included with the ransel kit. the triplicate data were analyzed using the sigmastat 3.5 (systat inc., santa cruz, ca, usa) software program. for comparison of the groups, first, we determined the ic50 concentrations for zinc and docetaxel on a549 and h1299 cells using a cc8 kit. to investigate the effects of exogenous zinc on cellular viability, apoptosis, and cell cycle alterations, both cell types were treated with various zinc concentrations (50 m, 100 m, and ic50 level). for the combinations, the groups were treated with three different zinc concentrations and the same docetaxel dose (ic50). however, to determine the efficacy of zinc pretreatment on ic50 changes caused by docetaxel, nsclc cells were treated with 50 m and 100 m of zinc. the ic50 values of zinc on a549 and h1299 cells were 287.1 m and 458.2 m, respectively, whereas the docetaxel concentrations were 20.44 m and 33.15 m, respectively (figure 1). the docetaxel ic50 values for a549 cells were reduced to 8.16 m and 6.6 m levels after exposure to 50 m and 100 m of zinc, respectively, whereas these values were reduced to 12.16 m and 14.69 m in h1299 cells. zinc significantly reduced the cellular viability of a549 cells in a dose - dependent fashion when combined with docetaxel and used alone (p<0.001). however, zinc supplementation did not modify h1299 cell viability at all concentrations, although significant suppression was observed for the combination groups. for apoptotic induction, zinc significantly increased the effects of docetaxel on both cell lines (figure 2). as shown in figure 3, the increase of the g0/g1 phase cell population was accompanied by a concomitant s - phase decrease in a549 cells treated with zinc and combinations in a dose - dependent manner. however, zinc and combinations reduced the g0/g1 population and increased s - phase accumulation in h1299 cells. as expected, g2/m phase accumulation significantly increased after docetaxel treatment in both cell lines. h1299 cells showed significantly increased apoptotic / necrotic cell population in the combination groups. to assess the importance of oxidative stress, the sod activity, gpx activity, and mda levels were evaluated in nsclc cells. in both cell lines, the sod values significantly increased all treatment groups compared to the control, but the highest values were observed in the cells treated with 100 m zinc. gpx was not elevated when both the a549 and h1299 cells were treated with 50 m and 100 m doses, but were slightly increased in the docetaxel and combination groups (table 1). the maximum mda response was observed when the a549 cells were exposed to 50 m of zinc and combinations, except the ic50 doses, whereas the maximum mda response occurred when the h1299 cells were exposed to 100 m of zinc and docetaxel (p<0.001, anova followed by holm first, we determined the ic50 concentrations for zinc and docetaxel on a549 and h1299 cells using a cc8 kit. to investigate the effects of exogenous zinc on cellular viability, apoptosis, and cell cycle alterations, both cell types were treated with various zinc concentrations (50 m, 100 m, and ic50 level). for the combinations, the groups were treated with three different zinc concentrations and the same docetaxel dose (ic50). however, to determine the efficacy of zinc pretreatment on ic50 changes caused by docetaxel, nsclc cells were treated with 50 m and 100 m of zinc. the ic50 values of zinc on a549 and h1299 cells were 287.1 m and 458.2 m, respectively, whereas the docetaxel concentrations were 20.44 m and 33.15 m, respectively (figure 1). the docetaxel ic50 values for a549 cells were reduced to 8.16 m and 6.6 m levels after exposure to 50 m and 100 m of zinc, respectively, whereas these values were reduced to 12.16 m and 14.69 m in h1299 cells. zinc significantly reduced the cellular viability of a549 cells in a dose - dependent fashion when combined with docetaxel and used alone (p<0.001). however, zinc supplementation did not modify h1299 cell viability at all concentrations, although significant suppression was observed for the combination groups. for apoptotic induction, zinc significantly increased the effects of docetaxel on both cell lines (figure 2). as shown in figure 3, the increase of the g0/g1 phase cell population was accompanied by a concomitant s - phase decrease in a549 cells treated with zinc and combinations in a dose - dependent manner. however, zinc and combinations reduced the g0/g1 population and increased s - phase accumulation in h1299 cells. as expected, g2/m phase accumulation significantly increased after docetaxel treatment in both cell lines. to assess the importance of oxidative stress, the sod activity, gpx activity, and mda levels were evaluated in nsclc cells. in both cell lines, the sod values significantly increased all treatment groups compared to the control, but the highest values were observed in the cells treated with 100 m zinc. gpx was not elevated when both the a549 and h1299 cells were treated with 50 m and 100 m doses, but were slightly increased in the docetaxel and combination groups (table 1). the maximum mda response was observed when the a549 cells were exposed to 50 m of zinc and combinations, except the ic50 doses, whereas the maximum mda response occurred when the h1299 cells were exposed to 100 m of zinc and docetaxel (p<0.001, anova followed by holm zinc is reported to induce apoptosis and cytotoxicity in different tumor types including prostate, ovarian, hepatoma, pancreatic, and breast cancers,1417,25,26 but it is less studied in lung cancers. here, we found that zinc exhibited growth inhibitory and apoptotic effects in a dose - dependent manner, up to the ic50 concentrations for cultured lung cancer cells. the exact mechanisms underlying this synergism and zinc - induced apoptosis are unclear, but zinc homeostasis and p53 functions appear to be key factors for subsequent physiological processes including dna repair, cell cycle regulation, and response to oxidative stress.27 both exposures to excessive zinc and zinc depletion activate the apoptosis, and also, the zinc - induced apoptosis are commonly associated with the oxidative stress. for example, intracellular zinc deficiency can enhance dna damage due to oxidative stress and also blocks critical cellular signals that drive dna repair and apoptosis,27 such that, zinc deficiency results in increased sensitivity to oxidative stress, increased dna damage, and accelerated carcinogenesis.28 the dna repair functions of p53 are commonly lost, and redox sensitive transcription factors, such as ap-1 and nf-b show reduced nuclear binding as a result of low cellular zinc status.27 the zinc exposure causes intracellular acidosis, increased the reactive oxygen species (ros) generation in several tumor cells,29,30 and stimulates the caspase - dependent and -independent apoptosis.30,31 moreover, the zinc - induced ros generation has been shown in the presence of functionally active p53 in breast cancer cells.29,30 by contrast, reactivation of mutant p53 increases the sensitivity of chemoresistant cancer cells to antineoplastic agents with zinc supplementation, which was demonstrated for glioblastoma and colorectal cancer cells.32 zinc - related apoptosis is associated with decreased bcl-2 and survivin expression in prostate cancer cells, and other studies found that survivin inhibition with deguelin results in enhanced efficacy of docetaxel in breast cancer cells.15,33 therefore, based on these data, we hypothesize that the addition of zinc into docetaxel regimens is a reasonable option to increase docetaxel - induced apoptosis and cytotoxicity in nsclc cells. in the present study, nsclc - derived h1299 (p53-null) and a549 (p53-wild - type) cells were used. the ic50 doses of docetaxel and zinc were higher for the p53-null h1299 cells than a549 cells (figure 1). it is well known that the complete loss of p53 or the expressions of mutant p53 variants are commonly seen in nsclc with 4070% frequencies and are associated with increased resistance to chemotherapy and radiotherapy.11,34,35 functional p53 status may influences docetaxel and zinc - induced cytotoxicity.11 for example, liu demonstrated that docetaxel induces apoptotic cell death in c4 - 2 and lncap prostate cancer cell express wild - type p53 more than du145 (mutant p53) and pc-3 (p53-null) cells. they also shown knockdown of p53 protein in c4 - 2 cells increases docetaxel resistance. the role of p53 on zinc - induced apoptosis has been shown in some similar studies.29,37 in our study, zinc showed cytotoxicity in p53-wild lung cancer cells but not in null cells at different supraphysiological concentrations. these results suggest that zinc - induced cytotoxicity and/or apoptotic induction are associated with the availability of functionally active p53 in nsclc cells. in our study, we observed increased mda levels, which are an indirect indicator of oxidative stress and ros production, using higher zinc concentrations (50 m and 100 m but not the ic50) in p53-wild - type a549 cells, whereas h1299 cells had higher mda levels at 100 m zinc concentrations. based on our data, it is suggested that supraphysiological zinc supplementation induces ros generation and apoptosis in nsclc cells through p53 activation. in the presence of altered intracellular zinc concentrations (low or high), ros stimulates p53 activation and/or p53 itself to stimulate ros production, and finally, it may cause cell death. previous studies demonstrated that when cells are exposed to several zinc chelators, mutant p53 forms start to accumulate, and these forms lose their dna binding capacity. finally, redox - active compounds cause dna - strand breaks, whereas removal of the zinc chelators from the medium returns to cell wild - type p53 form.38 in addition, exposure of mcf7 (p53-wild - type) cells to 1025 m zinc causes an increase in the expression of bax, proapoptotic bcl-2 and, pig3 (p53-induced gene product) and also generation of ros. the synergistic effects of zinc on docetaxel cytotoxicity may occur by inducing ros generation and increasing oxidative stress. the combination of zinc and docetaxel induced apoptosis and cytotoxicity as well as mda levels. interestingly, zinc also reduced the ic50 values of docetaxel and this may related to intracellular action of ros. similar to other chemotherapeutic agents, taxanes induce ros production, and high levels of ros participate in paclitaxel cytotoxicity.39 for example, exposure of a549 cells to paclitaxel resulted in an intracellular increase of o2 and h2o2 levels. then, the addition of n - acetylcysteine (nac) and glutathione, which are h2o2 scavengers, induced a fourfold increase in the paclitaxel ic50 level.40 we also examined the antioxidant enzymes sod and gpx in cell lysates and found significant increases in all treatment modalities compared to the control, with the highest sod values in the combination group. these results may be related to the higher zinc and docetaxel concentrations. zinc is an essential component of cuznsod, which is the first - line defense enzyme to avoid the harmful effects of ros.27,41 in our study, we selected three different zinc concentrations, and zinc concentration was slightly supraphysiological (50 m) along with higher concentrations (100 m and ic50). because, the zinc treatment at ~15 m concentration is accepted as physiological level.42 previous studies showed that zinc - induced apoptosis occurs from 50 m and higher, but zinc - induced ros generation is observed at 100 m doses.14,17,25,43,44 although, we have not counted intracellular free zinc concentrations after the drug and zinc exposures, in a published study, maximum intracellular zinc concentrations have been obtained at 50 m znso4 treatment, and finally decreased to initial level when the concentration of znso4 exceeded 100 m.17 in addition, the cell growth inhibition and apoptosis were evaluated 24 hours after the treatment. apoptotic changes due to zinc treatments (100 ng / ml and 1,000 ng / ml) were remarkably seen in prostate cancer cells in an in vitro study.45 it has also been shown that, the zinc ions translocated into mitochondria 24 hours after the 150 m zinc sulfate treatment and at the beginning of the 18 hours of treatment, cytochrome c started to be seen in the cytoplasm and reached to the peak level in approximately 36 hours of treatment.44 the cell cycle analysis revealed g0/g1 accumulation in a549 cell after zinc treatment in a dose - dependent manner. this results confirmed p53 activation in a549 cells. it is well known that activated p53 causes g1 arrest by inducing p21, followed by cyclin inhibition to induce dna repair before cellular division,46 and the cell cycle analysis showed increased s phase and significantly higher apoptotic / necrotic cell death (~50%) in p53-deficient nsclc cells. the zinc induced - apoptosis and the synergism with docetaxel were speculated through ros generation and p53 activation here. however, one of the major limitations of the present study was the lack of tests that showed the p53 functions or the p53-related protein alterations, as well as the apoptotic protein expressions after the docetaxel and the zinc applications. another important limitation of this study was the absence of the counting of intracellular zinc and ros content after the compound exposures. in conclusion, the exposure to exogenous zinc results in remarkable growth inhibition, increased apoptosis in the nsclc cells at 50 m and 100 m concentrations and zinc supplementation potentially sensitizes the nsclc cells to the docetaxel treatment. | backgroundexposure to exogenous zinc results in increased apoptosis, growth inhibition, and altered oxidative stress in cancer cells. previous studies also suggested that zinc sensitizes some cancer cells to cytotoxic agents depending on the p53 status. therefore, zinc supplementation may show anticancer efficacy solely and may increase docetaxel - induced cytotoxicity in non - small - cell lung cancer cells.methodshere, we report the effects of several concentrations of zinc combined with docetaxel on p53-wild - type (a549) and p53-null (h1299) cells. we evaluated cellular viability, apoptosis, and cell cycle progression as well as oxidative stress parameters, including superoxide dismutase, glutathione peroxidase, and malondialdehyde levels.resultszinc reduced the viability of a549 cells and increased the apoptotic response in both cell lines in a dose - dependent manner. zinc also amplified the docetaxel effects and reduced its inhibitory concentration 50 (ic50) values. the superoxide dismutase levels increased in all treatment groups ; however, glutathione peroxidase was slightly increased in the combination treatments. zinc also caused malondialdehyde elevations at 50 m and 100 m.conclusionzinc has anticancer efficacy against non - small - cell lung cancer cells in the presence of functionally active p53 and enhances docetaxel efficacy in both p53-wild - type and p53-deficient cancer cells. |
over one million deaths occur worldwide each year as a result of lung cancer, and approximately 85% of these are from non - small - cell lung cancer (nsclc).1 cytotoxic chemotherapy remains the mainstay of treatment for the majority of patients with advanced disease ; however, recent pivotal discoveries have led to molecularly defined subtypes of nsclc, particularly in adenocarcinoma. additionally, safe and effective targeted therapies for these specific subgroups have emerged, resulting in a paradigm shift in how clinicians diagnose and treat patients with this disease. the identification of epidermal growth factor receptor (egfr) activating mutations and egfr - tyrosine kinase inhibitors (tkis) led to the 2004 united states food and drug administration (fda) approval of erlotinib as first - line therapy for egfr mutation - positive patients.2,3 more recently, a fusion gene has been discovered between echinoderm microtubule - associated protein - like 4 (eml4) and anaplastic lymphoma kinase (alk) in a small subset of nsclc patients.4 crizotinib, a multitarget tki, has demonstrated overall response rates in approximately 60% of patients with tumors containing the alk gene rearrangement.5 alk is a member of the insulin receptor superfamily and is a transmembrane receptor tyrosine kinase found on the small intestines, testes, and selected neural tissue of adult humans.6 expression of alk during development is tightly regulated and has been implicated to have an important role in differentiation and cell survival. aberrant alk has been described in several neoplasms, including nsclc, rhabdomyosarcoma, neuroblastomas, glioblastoma, melanoma, breast cancer, and neuroectodermal tumors.3 constitutive alk activation in nsclc was first identified by screening a cdna library for genes with transforming capacity from the tumor of a japanese male smoker with adenocarcinoma.4 this screening identified a fusion gene resulting from an inversion of the short arm of chromosome 2 producing the joining of exons 2029 of the alk gene to exons 113 of the eml4 gene. the product of this unique fusion gene is a chimeric protein containing the c - terminus of the alk gene and the n - terminus of the eml4 gene, which ultimately leads to the constitutive activation of alk tyrosine kinase resulting in the activation of multiple downstream signaling pathways, including akt, stat3, and erk (figure 1).7 multiple distinct variants have been identified since the initial discovery of alk - emla resulting from different breakpoints in the eml4 gene.8 the most frequent fusion gene variants of eml4-alk include ; e13;a20 (34%), e6ab;a20 (30%), and e20;a20 (10%).9 alk has also been shown to partner with additional genes such as kif5b and tfg to form other aberrant fusion proteins.10,11 this review article will discuss current diagnostic techniques, development of resistance to alk inhibitors, impact of therapy on quality of life, and future therapeutic directions. detection of the alk gene rearrangement is critical for managing the treatment of nsclc patients with alk - rearranged tumors. a variety of methods are currently available to test for the alk rearrangement, including fluorescence in situ hybridization (fish), immunohistochemistry (ihc), and reverse transcriptase polymerase chain reaction (rt - pcr). rt - pcr has thus far been limited to clinical trials evaluating the sensitivities of ihc and fish. although rt - pcr can identify alk gene rearrangements at the mrna level, its limits include mrna degradation in formalin - fixed, paraffin - embedded tissues ; inability to detect unknown genetic alterations ; and the need for highly specific primers.12 multiplexed rt - pcr may overcome some of these limitations, but its current availability is quite limited. the vysis alk break apart fish probe kit (abbott molecular inc., des plaines, il, usa) was utilized to detect the alk gene rearrangement in studies leading to the approval of the alk inhibitor, crizotinib.13 this test consists of a red - labeled fluorescent probe at the 3 end of alk and a green - labeled fluorescent probe at the 5 end of alk. in tumors with rearranged alk, the red and green probes are split apart (break - apart test). in tumors with a normal alk gene, it is recommended that at least four fields (about 60 cells) are evaluated and if 15% of tumor cells have split alk 5 and 3 probe signals or have isolated 3 signals, the tumor is considered positive for the alk gene rearrangement. detection of the break - apart signal can be challenging, as eml4 and alk normally reside in close proximity to each other on the short arm of chromosome 2. additionally, although fish detects the disruption of the alk locus, it does not identify the resulting partner fusion gene. further, the instrumentation and expertise required for fish are not readily available in all pathology laboratories, thus limiting the applicability of this test in routine care. the use of ihc for detecting alk protein expression has emerged as a conceivable alternative to fish.14 early ihc reports found a large number of false negatives, likely resulting from low - level mrna expression in nsclc versus (vs) anaplastic large cell lymphoma and thus requiring a greater dependence on the primary antibody used in the test. because alk is not normally expressed in lung tissue, any expression of alk in the lung cancer tissue represents an altered status of the alk gene. multiple researchers have demonstrated a strong correlation between ihc and fish.1520 conklin examined 377 tumors by ihc, 273 of which were also evaluated by fish.16 they concluded that the sensitivity and specificity of ihc is antibody dependent, with 5a4 (novocastra with advance detection) and d5f3 (cell signaling with advance detection) clones proving to be the most accurate and consistent among the antibodies tested. additionally, the presence or absence of staining by ihc better correlated with fish than the degree of staining (0 to 3 +) as has been observed with her2 testing. keep in mind, however, that false positives and negatives have been reported.15,18 further optimization of the antibodies and methodology applied is necessary before ihc can be used in routine patient care ; however, ihc may become the preferred test, as it is routinely used in pathology laboratories and is generally simpler and less expensive to perform. the united states fda approved crizotinib in conjunction with the break - apart fish - based assay in august 2011. a year later, the european commission approved crizotinib in conjunction with ihc using the d5f3 rabbit monoclonal primary antibody. trials are ongoing to clarify the alk diagnostic issues, but to date fish remains the standard of care in the united states. another critical question in the management of advanced nsclc involves determining which patient population should be tested for the alk gene rearrangement and in what sequence testing should occur. the frequency of alk rearrangements has been reported to range from 1% to up to 33% of the population.21 this wide range in the prevalence of alk rearrangement can be attributed to the clinical and molecular characteristics of the populations being tested. clinicopathologic and molecular features linked with a higher occurrence of alk rearrangement include adenocarcinoma, light or never smokers, younger age, egfr wild type, and kras wild type.2123 in a report by shaw, the frequency of alk in never / light smokers was 22%, in never / light smokers not harboring an egfr mutation, the frequency was 33%.21 for the most part, the alk gene rearrangement is thought to be mutually exclusive of egfr and kras mutations ; however, tumors containing more than one mutation have been reported.22,24 in one series, 8% of alk - rearranged tumors also contained either an egfr or a kras mutation.24 some investigators have suggested an algorithmic approach to testing, but to date a consensus on an algorithm has not been reached.22,25 currently, the international association for the study of lung cancer recommends egfr and alk testing on adenocarcinomas or tumors with an adenocarcinoma component.26 squamous cell tumors may be considered for testing if the abovementioned clinical features are present. in comparison, the european society for medical oncology guidelines recommends testing in patients with a diagnosis of non - squamous advanced lung cancer or on any type of histology in never smokers who have been screened first for the egfr mutation.27 given the dramatic and rapid impact of alk - targeted therapy, the authors favor erring on the side of testing rather than potentially missing a patient who may derive meaningful benefit from alk inhibition., new york, ny, usa) is an oral small molecule inhibitor of alk, c - met, ros1, and ron tyrosine kinases.28,29 crizotinib was shown to be a potent atp- competitive inhibitor of recombinant human c - met kinase activity resulting in regression of tumors expressing constitutive activity of c - met in preclinical studies. in fact, the drug was initially developed as a c - met inhibitor, but was later found to inhibit tyrosine phosphorylation of alk with a mean half maximal inhibitory concentration (ic50) of 24 nmol / l. the initial phase i dose escalation trial of crizotinib (pf-02341066) included 37 patients with various solid tumors ; dose - limiting toxicities included grade 3 alanine aminotransferase elevation and grade 3 fatigue, and the recommended phase ii dose was established as 250 mg twice daily.5 of the ten patients in the phase i trial with alk - positive nsclc, one had a confirmed partial response (pr), two had unconfirmed prs, and four had stable disease. these results led to a phase i expansion cohort that included 149 alk - rearranged nsclc patients.30 investigators reported a 61% overall response rate (orr), a median progression - free survival of 9.7 months, and a median duration of response of 49.1 weeks. this promising activity gave rise to a phase ii trial (profile 1005) conducted in 901 pretreated patients, the majority of whom had at least two prior therapies.31 mature data is available on 261 patients, 259 of whom are considered response evaluable. the orr was approximately 60%, the median duration of response was 11 months, and the median time to response was 6 weeks. eighteen of the patients had untreated brain metastasis, two patients achieved a cr, two had a pr, and 12 had stable disease. adverse events that were reported in more than 25% of patients included visual disorder, nausea, vomiting, diarrhea, constipation, and peripheral edema. unfortunately, investigators also reported fatalities due to presumed treatment related pneumonitis and hepatotoxicity, occurring in less than 3% of patients. crizotinib was approved by the fda based on data from the expansion cohort of the phase i study and the phase ii study. this approval led to a series of phase iii trials beginning with profile 1007, in which patients were randomly assigned to receive either crizotinib or chemotherapy with pemetrexed or docetaxel.14 three hundred and forty - seven patients with locally advanced or metastatic alk - positive lung cancer who had received one prior platinum - based regimen were enrolled ; crossover was permitted at disease progression. the median progression - free survival more than doubled (7.7 months vs 3 months, p 460 mg twice daily.45 the population included 21 patients with cns metastases with an orr within the brain of 53%.46 a dual alk / egfr inhibitor, ap26113 (ariad pharmaceuticals inc., cambridge, ma, usa), has been developed with tki activity on l1196 m (alk variant) and t790 m (egfr variant) mutations, but not native egfr.47 the updated results of the dose - finding study included 44 patients with advanced malignancies excluding leukemia. the orr in the evaluable alk - positive population was 56%, and 67% in the group with documented exposure to crizotinib. fatigue, nausea, and diarrhea of any grade were reported in over a quarter of patients. grade 3 or 4 toxicities in 4%5% of patients included fatigue, diarrhea, pneumonia, and hypoxia. other emerging strategies against the alk driver mutation include heat shock protein 90 (hsp90) inhibitors. hsp90 is a molecular chaperone that can be present in an abundant amount in tumor cells.48 alk and alk fusion proteins are chaperone proteins of hsp90, and binding of alk to hsp90 inhibits its degradation. thus inhibition of hsp90 will limit the binding of alk to hsp90 and enhance the degradation of alk. in preclinical studies, hsp90 inhibition has led to antitumor activity in alk - positive nsclcs, including tumors with crizotinib resistance.49 ipi-504 and sta9090, both hsp90 inhibitors in development, have shown activity in a limited number of alk - positive nsclc patients. because mechanisms of resistance are varied, it is unclear if these drugs will be effective in all patients or merely in a subset of patients with crizotinib resistance.50 table 1 represents a sample of trials in the alk (+) population available through http://www.clinicaltrials.gov. this process of discovery of alk gene rearrangement, rapid movement of targeted treatment through clinical trials, and early investigation of resistance mechanisms has established a new model for personalized medicine. investigators not only simultaneously gained fda approval and explored resistance mechanisms, they also elucidated the effect crizotinib would have on quality of life. both the phase ii and phase iii trials used the european organization for research and treatment of cancer (eortc) quality of life questionnaire - core 30 (qlq - c30) and the lung cancer module (qlq - lc13).14,51 the qlq - c30 includes five functional domains, eight symptoms, global health, and financial impact.52,53 patient reported outcomes are based on their responses in accordance with a four - point scale from not at all to very much. higher scores reflect a greater symptom burden, but also reflect a higher level of function. the qlq - lc13 includes 13 symptom scales common to lung cancer patients undergoing treatment with chemotherapy or radiation. the phase ii study reported a 10 point improvement in pain, cough, dyspnea, and fatigue ; the majority of these improvements were noted by the second cycle of therapy.51 likewise, the phase iii trial demonstrated a significant reduction in alopecia, cough, dyspnea, fatigue, and pain from baseline in the group treated with crizotinib.14 progression of symptoms was also significantly delayed in the crizotinib group (median 5.6 months crizotinib vs median 1.4 months chemotherapy). finally, there was a meaningful improvement in global quality of life by cycle 4 in the crizotinib group that was not observed in the chemotherapy treated patients. although the frequency of the alk gene rearrangement is relatively low (approximately 4% of nsclc patients), this translates into 70,000 cases annually worldwide. accurately identifying these patients both ihc and fish have been identified as potential diagnostic resources ; however, differences in accuracy, availability, timeliness, and cost need to be completely resolved. additionally, the question of whether to test all advanced non - squamous cell carcinoma patients or to stratify testing based on clinicopathologic and molecular features needs to be answered. one potential pitfall of using an algorithmic approach to answer these important genetic testing questions is that alk has been identified in patients who do not fit the defined standard phenotype and coexisting mutations have been described. given the potential therapeutic impact of alk inhibitor therapy, the authors favor erring on the side of testing while remaining within the bounds of guidelines and good clinical practice. crizotinib is an oral drug that is the first alk inhibitor to show promising clinical activity in tumors with alk activation. crizotinib is generally well tolerated, with nausea, emesis, and visual changes reported as the main adverse events. the molecular alterations that result in resistance appear to be highly diverse, including but not limited to secondary mutations within the alk - tki domain, gene amplification, alternate signaling pathway activation, and coexisting driver mutation. other alk inhibitors currently in development may have activity in some of these crizotinib - resistant tumors. certain second - generation alk inhibitors have shown promising activity in tumors resistant to crizotinib, and several clinical trials are ongoing in this arena. since alk is a chaperone protein of hsp90, inhibitors of hsp90 may prove to be beneficial in patients with alk gene - rearranged nsclc. the discovery of the alk gene rearrangement and effective targeted therapy represents a new model for translational cancer research (figure 2).54,55 only 2 years elapsed from the time of the discovery of alk to the objective tumor responses seen in patients on alk inhibitors. further, it only took 3 years to advance from phase i to phase iii clinical trials, roughly half the time required to move egfr inhibitors forward. finally, mechanisms of resistance are already being elucidated and clinical trials are currently underway in this drug - resistant patient population. thus, in the alk - positive nsclc patient population, the gap between the bench and the bedside has been bridged in record time. | non - small - cell lung cancer (nsclc) is a heterogeneous disease and a challenging malignancy to treat, as many patients have advanced disease at the time of diagnosis. recent advances have led to the identification of molecularly defined subtypes of nsclc, namely for patients with adenocarcinoma histology. the most recently identified molecular target is the anaplastic lymphoma kinase (alk) gene rearrangement, and patient responses to the alk inhibitor crizotinib have led to its approval in this selected patient population. like other tyrosine kinase inhibitors, resistance to crizotinib ultimately develops by various mechanisms requiring alternative therapeutic options. this review article discusses the management of patients with the alk gene rearrangement, mechanisms of crizotinib resistance, and future potential therapeutic options. |
mature microrna (mirna) molecules are approximately 22-nucleotide - long single - stranded rnas that generally repress the expression of protein coding genes. specifically, they preferentially bind to 3 untranslated regions (utrs) of messenger rnas (mrnas) and interfere with their stability and translational efficiency (1,2). the first mirnas and their target genes were identified via classical forward genetic techniques in 1993, but it was not until 2001 that many more mirnas were discovered experimentally and found to be abundant and widespread (35). since then there has been a dramatic growth in the number of annotated mirnas (figure 1). figure 1.the growth of the human mirna genes in mirbase database and the growth of the human experimentally determined mirna target interactions in tarbase. the growth of the human mirna genes in mirbase database and the growth of the human experimentally determined mirna target interactions in tarbase. a crucial aspect of the functional analysis of mirnas is the annotation of their protein - coding targets. a number of computational algorithms have been developed for the prediction of such targets (6). although these programs are very important to guide wet lab experiments, they still lack in sensitivity and specificity (7,8). in parallel, and as support for these programs, a number of experimental procedures have been developed to provide indirect or direct support for predicted mirna target interactions and results from a growing number of such experiments have been published (figure 1). the need for a systematic documentation of such experimentally supported targets was covered by the first version of tarbase (9). in 2006, the database recorded over 550 entries with mirna target interactions in human, mouse, fruit fly, worm and zebrafish. here we present a substantially updated and extended version of this database, tarbase5.0, documenting over 1300 entries. the new database now contains all of the information included in the previous version, plus : specific cells lines (if any) used in the experiments, cell - type - specific expression of the gene product and its potential involvement in carcinogenesis, differential expression of mirnas in specific tissues, developmental or pathological events that a specific mirna is involved in and any annotated types of mirna - related mis - regulation in those events, hgnc symbols as defined by hugo (in order to provide consistent gene naming). additionally, both the underlying sql database and the user interface have been extensively redesigned with several added external links such as a direct link to the paper 's abstract on the pubmed site. specific cells lines (if any) used in the experiments, cell - type - specific expression of the gene product and its potential involvement in carcinogenesis, differential expression of mirnas in specific tissues, developmental or pathological events that a specific mirna is involved in and any annotated types of mirna - related mis - regulation in those events, hgnc symbols as defined by hugo (in order to provide consistent gene naming). the tarbase5.0 database can be directly accessed from the http://microrna.gr/tarbase web page. tarbase5.0 contains data extracted from a total of 203 scientific papers resulting in 1333 entries describing a regulatory interaction between a mirna and a target 3 utr (summarized in table 1). table 1.a list of all tarbase5.0 entriesorganismnumber of papersnumber of entriesmicroarray datapsilac datahomo sapiens110285328474mus musculus2810513d. elegans1814plants2130danio rerio11rat22total203514341474 a list of all tarbase5.0 entries the tarbase5.0 data set contains mirna targets that tested either positive (induces target gene repression) or negative (no influence on target gene expression). for each experiment with a positive outcome the target site is described by the mirna that binds it, the gene in which it occurs, the nature of the experiments that were conducted to test it, the sufficiency of the site to induce translational repression and/or cleavage, and the paper from which all these data is extracted. additionally, for each mirna and protein - coding gene, the database contains links to several other relevant and useful databases such as ensembl (10), hugo (11), ucsc genome browser (12) and swissprot (13). there are a number of direct and indirect experimental procedures that have been developed to test a possible mirna the entries in tarbase5.0 are classified into four categories : true or false in the cases where an assay provides direct experimental evidence, or microarray and/or psilac in the cases that present only indirect evidence from high - throughput techniques to measure mirna - mediated global transcriptomic or proteomic changes. to overexpress a mirna, expression constructs can be engineered using the mature mirna, the precursor (hairpin) mirna, or the pri - mirna sequence for transfection into in vitro or in vivo transformed cells. also, silencing of a specific mirna can be accomplished by introducing chemically modified oligonucleotides that are perfectly complimentary to the mature mirna (antagomirs) (14). these methods for modifying mirna expression allow for several types of follow - up techniques to quantify and interpret differences in target gene expression. below we provide a more detailed description of each of the four categories : true or false : the most commonly used method for providing direct experimental evidence is the reporter gene assay. in its simplest form, an expression vector containing a reporter gene [i.e. luciferase or green fluorescent protein (gfp) ] is first modified by cloning the predicted target 3utr downstream of the reporter gene, and then transfected into a cell line of interest in the absence and presence of the cognate mirna. despite the general utility of this approach to assay for 3utr - mediated effects on reporter protein expression, it is not informative for the precise location of the mirna response element (mre) or number of mirna target sites in the 3utr. integration of the reporter gene assay with site directed mutagenesis of the predicted mre (and, further, restoring the complementarity of the mirna mre interaction by mutating the mature mirna sequence) yields a much more specific and direct result. to measure effects on reporter mrna levels, the most commonly applied technique is quantitative rt - pcr (qrt - pcr). measuring effects on both protein and mrna levels can help provide information about the mode of mirna - mediated silencing : mrna translational repression or immediate risc - mediated mrna cleavage and degradation. a mirna mre interaction is reported as true or false based on the results of the reporter gene assay. microarray and/or psilac : these high - throughput approaches measure global changes in the transcriptome (15) or proteome (8,16) given the presence or absence of a mirna. despite their power for large - scale analysis, these techniques only provide indirect evidence about a mirna 's targets since it is not possible to distinguish between primary direct targets and secondary indirect targets. other high - throughput methods like degradome sequencing (17,18) are also immensely useful but only in the scenarios where a mirna induces risc - mediated mrna cleavage. in order to facilitate user interaction, the query function is divided into several functionally related subgroups. the initial screen of the tarbase5.0 user interface allows users to query based on mirna, gene and organism. for more advanced queries, the user can utilize the extended query options. in this case, the search menus are arranged into four functionally related groups. target interaction : the validity of the interaction (field support type, either true or false), the function of the interaction which can be either translational repression or mrna cleavage (field datatype), the sufficiency of a single target site to exert the specific function (field s_s_s) and the number of mirna response elements present in the specific utr (field mre). the second group contains the fields that refer to the experimental methods that led to the reported result. target interaction (i.e. reporter gene assays) while indirect support refers to experimental procedures that provide more global, system - wide mirna - mediated effects (i.e. microarrays). the third group corresponds to biological properties of the mirna or target gene : biological functions (field protein type), specific expression profiles (field mirna expression) or the physiological processes in which this interaction is involved (field event or pathology). the fourth and final group contains some general query features such as the scientific paper (searchable by author or pmid)., the results screen (figure 2) shows only the repression type, the mirna identifier, the target gene identified by the hgnc symbol (if it is a human gene), the common gene name, the refseq isoform i d (particularly relevant in cases of gene variants or snp haplotypes), the affected biological processes and the paper containing the information presented. users can opt to view more detailed information by clicking on the + box so that the expanded results view is opened (figure 2). the additional information is divided into three categories : mirna information, gene information and experimental conditions. the mirna information category contains the properties of the specific mirna such as the mirna 's sequence [extracted from mirbase (19) ], the number and sequences of the mres, their locations within the gene 's 3utr, and the affected tissues (extracted from the paper). the gene information category gathers mostly biological properties of the target gene like the protein type, ensembl and swissprot ids and chromosome location, providing direct links to ensembl, swissprot and the ucsc browser respectively. moreover, expression profiles and tumor involvement are also provided for human genes (information extracted from the ensembl egenetics database). finally, the experimental conditions category provides the nature of the direct or indirect evidence for the mirna target gene interaction. the cell lines used to carry out the specific experiment are also presented in order to render the experimental conditions more complete and reproducible. even though several computational programs exist to predict mirna targets, the necessity for a systematic collection and description of mirna targets with experimental support led to the development of tarbase. the continuously expanding number of known and newly identified mirnas and their targets, combined with their central role in biological systems, renders this field particularly dependent on centralized information that is accurate, up - to - date, comprehensive and easy to browse or download. in order to satisfy these requirements, we have made extensive updates and modifications and present the new version of the database, tarbase5.0. the tarbase data files can be freely downloaded and used according to the gnu public license. funding for open access charge : aristeia award from general secretary research and technology, greece. | tarbase5.0 is a database which houses a manually curated collection of experimentally supported microrna (mirna) targets in several animal species of central scientific interest, plants and viruses. mirnas are small non - coding rna molecules that exhibit an inhibitory effect on gene expression, interfering with the stability and translational efficiency of the targeted mature messenger rnas. even though several computational programs exist to predict mirna targets, there is a need for a comprehensive collection and description of mirna targets with experimental support. here we introduce a substantially extended version of this resource. the current version includes more than 1300 experimentally supported targets. each target site is described by the mirna that binds it, the gene in which it occurs, the nature of the experiments that were conducted to test it, the sufficiency of the site to induce translational repression and/or cleavage, and the paper from which all these data were extracted. additionally, the database is functionally linked to several other relevant and useful databases such as ensembl, hugo, ucsc and swissprot. the tarbase5.0 database can be queried or downloaded from http://microrna.gr/tarbase. |
geographically and time - limited, they aim to build learning and capacity to inform country - wide implementation. between 2007 and 2011, demonstration projects were the main approach to delivering new vaccines against human papillomavirus (hpv) to prevent cervical cancer in low - income and lower - middle - income countries (llmics). initial pilots, with vaccines donated by merck & co., inc. or glaxosmithkline biologicals to governments and external partners, allowed countries to gain experience vaccinating adolescent girls, who were not routinely targeted for immunisation. with the exception of rwanda and bhutan, vaccine donations for national delivery were not generally available, so demonstration projects enabled resource - poor countries to gain hpv vaccination experience. from late 2012, gavi, the vaccine alliance, began supporting hpv vaccination demonstration projects or national programmes if countries had prior experience vaccinating girls aged 913. learn by doing. gavi provided 2-year funding for vaccines and operational costs to allow time to assess delivery strategies and potential integration with other adolescent services, develop tools, and prepare applications to gavi for national program funding. by mid-2015, gavi had approved demonstration project funding for 25 countries, of which 6 (24%) had already conducted at least one pilot, while another 3 were approved for national support. despite 55 pilots or demonstration projects being completed before 2015, only 7 countries and 3 more in 2015 - 2016 - transitioned from demonstration to national provision. this article aims to examine the value of hpv vaccination demonstration projects to date, including potential drawbacks and how value for informing national scale - up might be increased, drawing from a review of published and unpublished documents from 37 countries and key informant interviews from 23 countries conducted in 2015. countries with data in the public domain are specifically named, while others were anonymised with an identification number. lessons are relevant for countries intending to introduce hpv vaccination and more broadly for funders supporting introduction of new health interventions in llmics. demonstration projects allowed both national and external partners to gain valuable experience in planning and budgeting for a new intervention, enumerating target populations, developing community acceptability and consent procedures for adolescent services, designing and piloting new reporting forms and systems, coordinating with the ministry of education for school - based vaccination, and using standardised evaluation tools. while countries had nuanced experiences and gained from the learning by doing process, lessons from later demonstration projects generally repeated those reported earlier, indicating that while individual countries might require first - hand experience, sufficient collective learning has been generated. first, the limited scale of projects did not allow assessment of potential health system integration, particularly as demonstration projects were often implemented outside routine services. second, communication and social mobilisation activities had to be carefully restricted to avoid perceived inequity among those not receiving vaccination. third, demonstration project was an unclear term for many stakeholders with some communities concluding that the vaccine rather than delivery method was being piloted. fourth, many were conducted in areas primarily selected for convenience (e.g. with higher routine vaccination coverage, more extensive infrastructure, and better education levels than national averages), potentially providing few lessons applicable to national scale - up. fifth, the resource - intensive delivery strategies used in demonstration projects were potentially unsustainable once vaccine donations and external support for operational costs ended. a focus on project evaluation and demonstrating at least 50% vaccination coverage to secure gavi support for national introduction, led some countries to choose safe options (e.g., school - based delivery) known for achieving good coverage but potentially too costly to be sustainably expanded nationally. projects in only 3 countries simultaneously or sequentially tested different vaccination venues, only 2 simultaneously tested different eligibility criteria, and only one tested different vaccination timings. as approximately 13 projects tested hpv vaccination integration (e.g. with tetanus toxoid vaccination, deworming, vitamin a supplementation, health education), learning around combination interventions was largely missed. value would increase substantially if projects were designed to learn specific lessons and inform realistic national scale - up. for example, the prevailing learning by doing objective appeared disconnected from learning for national scale - up, while attaining good coverage, as a performance indicator for funding allocation, could create distortions similarly unsupportive of learning for scale - up. instead, countries could test and cost different delivery strategies in different districts simultaneously, e.g., school - based and health facility - based, to inform sustainable national expansion. alternatively, donors could fund further economic modeling of different strategies to inform both demonstration projects and national scale - up. evaluation is critical for any testing approach and cost and coverage evaluations were included in a few demonstration projects. however, evaluations often lacked sufficient evidence to inform decision - making, with data missing or derived from pre - set grants that generally magnified operational costs. twenty completed demonstration projects before 2015 without announcing plans to scale - up, 5 of which ceased implementing hpv vaccination without attempting to co - finance vaccine and operational costs, 2 conducted further demonstration projects, and the remainder have not indicated a decision. implementation often stalled due to vaccine donations ending, funding constraints, or lack of ownership by national immunisation programmes. demonstration projects may thus delay decision - making or even discourage national scale - up due to the high - cost strategies tested during the demonstration phase. lessons from demonstration projects were not always relevant to scale - up. of seven llmics that scaled - up from demonstration to national implementation, only one reported that project lessons were useful for expansion, while another reported national expansion as more valuable for testing possible implementation strategies. several indicated that demonstration projects were too small to inform national expansion or selected a different delivery strategy than had been tested. of these, 3 used lessons from gardasil access program (gap) projects to test different delivery strategies as part of gavi - funded demonstration projects while 6 conducted multiple, sequential non - gavi projects for reasons unclear from the data. while some scale - up delays are probably due to insufficient national immunisation program engagement with early demonstration projects, an overall discontinuity exists between implementing demonstration projects and national expansion. longer - term comprehensive planning of projects and expansion, or changing to phased national introduction approaches, could accelerate scale - up. the pathway from demonstration to scale - up is not straightforward and major challenges to sustainability persist. interviewees indicated that the expensive campaign - style delivery and reduced external support during scale - up challenged national expansion efforts. however, the question remains whether demonstration projects may have become a way to delay or discourage commitment to scale - up. with many lessons already from 55 demonstration projects, learning may be saturated and further learning likely to be most effective during national implementation, or in the context of phased national expansion. this would maintain political commitment to scale - up and avoid loss of integration with national health systems. well - designed demonstration projects can test multiple delivery strategies, implementation for challenging areas and populations, and integration with national systems. countries implementing new interventions benefit through learning by doing and may need initial experiences to hone social mobilisation, delivery strategies, and reporting. however, demonstrations can distract momentum from national introduction and designs that test alternative scalable options are thus crucial. demonstration projects were valuable when hpv vaccine was first offered to llmics before national funding was available. however, with many lessons already documented, few new lessons observed, and additional funding available, their value has decreased. projects were designed to demonstrate whether llmics could implement hpv vaccination rather than whether they could implement it sustainably and at scale. while the latter is crucial, countries were reportedly reluctant to risk experimenting and potentially lose funding. in the example of hpv vaccination, initial demonstration projects quickly provided consistent lessons. however, scale - up is critical to maximise health impact, and further demonstrations could distract momentum and decision - making. any new demonstration projects should have guidelines that maximise value for national implementation, and flexibility for phased transition to national scale - up, without repeated funding applications. thus, introduction of vaccines or other interventions, particularly those involving new target groups or delivery strategies, needs flexible funding approaches so that pilots can address specific questions of scalability and sustainability. lessons from hpv vaccine introduction are relevant for other potential interventions that may be introduced in the coming years (e.g., menacwy vaccine, rts, s vaccine). nh, smj, kg and sk contributed to data collection, analysis, and interpretation. dwj, dsl, ug, hb and smj designed the study and contributed, along with mf, to data interpretation and critical review. the findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the bill & melinda gates foundation or path. | abstractdemonstration projects or pilots of new public health interventions aim to build learning and capacity to inform country - wide implementation. authors examined the value of hpv vaccination demonstration projects and initial national programmes in low - income and lower - middle - income countries, including potential drawbacks and how value for national scale - up might be increased. data from a systematic review and key informant interviews, analyzed thematically, included 55 demonstration projects and 8 national programmes implemented between 2007 - 2015 (89 years ' experience). initial demonstration projects quickly provided consistent lessons. value would increase if projects were designed to inform sustainable national scale - up. well - designed projects can test multiple delivery strategies, implementation for challenging areas and populations, and integration with national systems. introduction of vaccines or other health interventions, particularly those involving new target groups or delivery strategies, needs flexible funding approaches to address specific questions of scalability and sustainability, including learning lessons through phased national expansion. |
the most common causes of severe mitral regurgitation (mr) in developing countries is rheumatic heart disease. chronic left ventricular (lv) volume overload as a result of mr leads to compensatory dilatation of the left ventricle. although this response initially maintains cardiac output, myocardial decompensation eventually results in symptoms of heart failure and an increased risk of sudden death. in addition, backflow into the left atrium (la) results in enlargement of the la, atrial fibrillation, and elevated pulmonary pressures. echocardiography allows accurate evaluation of the presence or absence, severity, and cause of mr. although doppler echocardiography provides several methods of quantifying the severity of regurgitation, none have been shown to predict the clinical outcome. the most important aspect of the echocardiographic examination is the quantitation of lv systolic performance. although calculation of the ejection fraction (ef) is an imperfect means of assessing contractility, from a practical point of view, the ef in conjunction with the end - systolic dimension provides a clinically useful measure of ventricular performance. in recent years, the measurement of natriuretic peptides in patients with the valvular disease and heart failure has become important. the plasma level of b - type natriuretic peptide (bnp) is known to increase with lv dysfunction from many causes, and elevated plasma levels of bnp have been shown to indicate early states of myocardial deterioration in various diseases. such findings raised hopes that natriuretic peptides may also be useful in valvular heart disease. hence, the use of multiple echocardiographic and hormonal parameters in combination can provide an accurate assessment of mr severity and lv dysfunction in most cases. to study bnp level as an index of symptoms and severity of chronic rheumatic mr. to study bnp level as an index of symptoms and severity of chronic rheumatic mr. our study included 140 patients with rheumatic mr, as diagnosed by clinical examination, and conventional echocardiography. patients were classified into three groups ; group i : forty patients with mild rheumatic mr, group ii : fifty patients with moderate rheumatic mr, and group iii : fifty patients with severe rheumatic mr. patients with moderate to severe mitral stenosispatients with moderate to severe aortic valve stenosis and regurgitationpatients with ischemic heart diseasepatients with hypertensionpatients with serum creatinine > 2.5 mg / dl. patients with moderate to severe mitral stenosis patients with moderate to severe aortic valve stenosis and regurgitation patients with ischemic heart disease patients with hypertension patients with serum creatinine > 2.5 mg / dl. all patients included in this study were subjected to : detailed history including age, sex, smoking, new york heart association (nyha) functional classcomplete general and cardiological examinationsbnp was measured using the raybio bnp enzyme immunoassay (eia) kit. it is an in vitro quantitative assay for detecting bnp peptide based on the principle of competitive eia. the microplate in the kit was precoated with anti - rabbit secondary antibody. after a blocking step and incubation of the plate with anti - bnp antibody, both biotinylated bnp peptide and peptide standard or targeted peptide in samples interacted competitively with the bnp antibody. uncompleted (bound) biotinylated bnp peptide then interacted with streptavidin - horseradish peroxidase (sa - hrp), which catalyzed a color development reaction. the intensity of colorimetric signal was directly proportional to the amount of biotinylated peptide - sa - hrp complex and inversely proportional to the amount of bnp peptide in the standard or samples. this is due to the competitive binding to bnp antibody between biotinylated bnp peptide and peptides in standard or samples. a standard curve of known concentration of bnp peptide was established and the concentration of bnp peptide in the samples was calculated accordinglycomplete transthoracic echo doppler study : echocardiograms were recorded at rest, using acuson cv70 echo doppler machines equipped with a 2.5/3.25-mhz annular array transducer. patients were studied in left lateral position and electrocardiogram leads were connected to define timing of the cardiac cycle. parasternal long axis and short axis view and apical 4, 5, and 2 chambers views were obtained the following parameters were studied in each patient : detailed history including age, sex, smoking, new york heart association (nyha) functional class complete general and cardiological examinations bnp was measured using the raybio bnp enzyme immunoassay (eia) kit. it is an in vitro quantitative assay for detecting bnp peptide based on the principle of competitive eia. the microplate in the kit was precoated with anti - rabbit secondary antibody. after a blocking step and incubation of the plate with anti - bnp antibody, both biotinylated bnp peptide and peptide standard or targeted peptide in samples interacted competitively with the bnp antibody. uncompleted (bound) biotinylated bnp peptide then interacted with streptavidin - horseradish peroxidase (sa - hrp), which catalyzed a color development reaction. the intensity of colorimetric signal was directly proportional to the amount of biotinylated peptide - sa - hrp complex and inversely proportional to the amount of bnp peptide in the standard or samples. this is due to the competitive binding to bnp antibody between biotinylated bnp peptide and peptides in standard or samples. a standard curve of known concentration of bnp peptide was established and the concentration of bnp peptide in the samples was calculated accordingly complete transthoracic echo doppler study : echocardiograms were recorded at rest, using acuson cv70 echo doppler machines equipped with a 2.5/3.25-mhz annular array transducer. patients were studied in left lateral position and electrocardiogram leads were connected to define timing of the cardiac cycle. parasternal long axis and short axis view and apical 4, 5, and 2 chambers views were obtained the following parameters were studied in each patient : assessment of the lv : left ventricular end diastolic diameter (lvedd) and left ventricular end - systolic diameter (lvesd) were measured and the lv ef was calculated using, m - mode and biplane modified simpson 's rule. conventional pulsed wave doppler echocardiography was used to evaluate trans - mitral lv filling velocities. peak early diastolic flow velocity (e), peak late diastolic velocities (a), and the ratio of e / a were determined. lv myocardial tissue doppler velocities were assessed, the sample volume was placed at the lateral and septal margin of the mitral annulus on the apical four chamber view, systolic myocardial velocity (s), early diastolic myocardial velocity (e '), and late diastolic myocardial velocity (a ') were calculatedassessment of the la : la size was assessed by m - mode or two - dimensional anteroposterior la linear dimension which was obtained from the parasternal long axis view. la volume may also be measured using simpson 's ruleassessment of the severity of mr according to the recommendation of the american society of echocardiography : assessment of the lv : left ventricular end diastolic diameter (lvedd) and left ventricular end - systolic diameter (lvesd) were measured and the lv ef was calculated using, m - mode and biplane modified simpson 's rule. conventional pulsed wave doppler echocardiography was used to evaluate trans - mitral lv filling velocities. peak early diastolic flow velocity (e), peak late diastolic velocities (a), and the ratio of e / a were determined. lv myocardial tissue doppler velocities were assessed, the sample volume was placed at the lateral and septal margin of the mitral annulus on the apical four chamber view, systolic myocardial velocity (s), early diastolic myocardial velocity (e '), and late diastolic myocardial velocity (a ') were calculated assessment of the la : la size was assessed by m - mode or two - dimensional anteroposterior la linear dimension which was obtained from the parasternal long axis view. la volume may also be measured using simpson 's rule assessment of the severity of mr according to the recommendation of the american society of echocardiography : vena contracta : the degree of mr was classified according to width of vena contracta into : mild mr if vena contracta 0.7 cmregurgitation jet area : the degree of mr was classified according to jet area into : mild mr if the jet area 8 cmeffective regurgitation orifice area (ero) : mild mr if the ero 0.4 cmregurgitation volume (rv) : mild mr if the rv 60 ml / beat vena contracta : the degree of mr was classified according to width of vena contracta into : mild mr if vena contracta 0.7 cm regurgitation jet area : the degree of mr was classified according to jet area into : mild mr if the jet area 8 cm effective regurgitation orifice area (ero) : mild mr if the ero 0.4 cm regurgitation volume (rv) : mild mr if the rv 60 ml / beat statistical package for social sciences (version 15.0, spss inc, chicago, ill) was used for data analysis. data were statistically described in terms of range, mean standard deviation (sd), frequencies (number of cases), and relative frequencies (percentages) when appropriate mean and sd were estimates of quantitative data. patients with moderate to severe mitral stenosispatients with moderate to severe aortic valve stenosis and regurgitationpatients with ischemic heart diseasepatients with hypertensionpatients with serum creatinine > 2.5 mg / dl. patients with moderate to severe mitral stenosis patients with moderate to severe aortic valve stenosis and regurgitation patients with ischemic heart disease patients with hypertension patients with serum creatinine > 2.5 mg / dl. all patients included in this study were subjected to : detailed history including age, sex, smoking, new york heart association (nyha) functional classcomplete general and cardiological examinationsbnp was measured using the raybio bnp enzyme immunoassay (eia) kit. it is an in vitro quantitative assay for detecting bnp peptide based on the principle of competitive eia. the microplate in the kit was precoated with anti - rabbit secondary antibody. after a blocking step and incubation of the plate with anti - bnp antibody, both biotinylated bnp peptide and peptide standard or targeted peptide in samples interacted competitively with the bnp antibody. uncompleted (bound) biotinylated bnp peptide then interacted with streptavidin - horseradish peroxidase (sa - hrp), which catalyzed a color development reaction. the intensity of colorimetric signal was directly proportional to the amount of biotinylated peptide - sa - hrp complex and inversely proportional to the amount of bnp peptide in the standard or samples. this is due to the competitive binding to bnp antibody between biotinylated bnp peptide and peptides in standard or samples. a standard curve of known concentration of bnp peptide was established and the concentration of bnp peptide in the samples was calculated accordinglycomplete transthoracic echo doppler study : echocardiograms were recorded at rest, using acuson cv70 echo doppler machines equipped with a 2.5/3.25-mhz annular array transducer. patients were studied in left lateral position and electrocardiogram leads were connected to define timing of the cardiac cycle. parasternal long axis and short axis view and apical 4, 5, and 2 chambers views were obtained the following parameters were studied in each patient : detailed history including age, sex, smoking, new york heart association (nyha) functional class complete general and cardiological examinations bnp was measured using the raybio bnp enzyme immunoassay (eia) kit. it is an in vitro quantitative assay for detecting bnp peptide based on the principle of competitive eia. the microplate in the kit was precoated with anti - rabbit secondary antibody. after a blocking step and incubation of the plate with anti - bnp antibody, both biotinylated bnp peptide and peptide standard or targeted peptide in samples interacted competitively with the bnp antibody. uncompleted (bound) biotinylated bnp peptide then interacted with streptavidin - horseradish peroxidase (sa - hrp), which catalyzed a color development reaction. the intensity of colorimetric signal was directly proportional to the amount of biotinylated peptide - sa - hrp complex and inversely proportional to the amount of bnp peptide in the standard or samples. this is due to the competitive binding to bnp antibody between biotinylated bnp peptide and peptides in standard or samples. a standard curve of known concentration of bnp peptide was established and the concentration of bnp peptide in the samples was calculated accordingly complete transthoracic echo doppler study : echocardiograms were recorded at rest, using acuson cv70 echo doppler machines equipped with a 2.5/3.25-mhz annular array transducer. patients were studied in left lateral position and electrocardiogram leads were connected to define timing of the cardiac cycle. parasternal long axis and short axis view and apical 4, 5, and 2 chambers views were obtained the following parameters were studied in each patient : assessment of the lv : left ventricular end diastolic diameter (lvedd) and left ventricular end - systolic diameter (lvesd) were measured and the lv ef was calculated using, m - mode and biplane modified simpson 's rule. conventional pulsed wave doppler echocardiography was used to evaluate trans - mitral lv filling velocities. peak early diastolic flow velocity (e), peak late diastolic velocities (a), and the ratio of e / a were determined. lv myocardial tissue doppler velocities were assessed, the sample volume was placed at the lateral and septal margin of the mitral annulus on the apical four chamber view, systolic myocardial velocity (s), early diastolic myocardial velocity (e '), and late diastolic myocardial velocity (a ') were calculatedassessment of the la : la size was assessed by m - mode or two - dimensional anteroposterior la linear dimension which was obtained from the parasternal long axis view. la volume may also be measured using simpson 's ruleassessment of the severity of mr according to the recommendation of the american society of echocardiography : assessment of the lv : left ventricular end diastolic diameter (lvedd) and left ventricular end - systolic diameter (lvesd) were measured and the lv ef was calculated using, m - mode and biplane modified simpson 's rule. conventional pulsed wave doppler echocardiography was used to evaluate trans - mitral lv filling velocities. peak early diastolic flow velocity (e), peak late diastolic velocities (a), and the ratio of e / a were determined. lv myocardial tissue doppler velocities were assessed, the sample volume was placed at the lateral and septal margin of the mitral annulus on the apical four chamber view, systolic myocardial velocity (s), early diastolic myocardial velocity (e '), and late diastolic myocardial velocity (a ') were calculated assessment of the la : la size was assessed by m - mode or two - dimensional anteroposterior la linear dimension which was obtained from the parasternal long axis view. la volume may also be measured using simpson 's rule assessment of the severity of mr according to the recommendation of the american society of echocardiography : vena contracta : the degree of mr was classified according to width of vena contracta into : mild mr if vena contracta 0.7 cmregurgitation jet area : the degree of mr was classified according to jet area into : mild mr if the jet area 8 cmeffective regurgitation orifice area (ero) : mild mr if the ero 0.4 cmregurgitation volume (rv) : mild mr if the rv 60 ml / beat vena contracta : the degree of mr was classified according to width of vena contracta into : mild mr if vena contracta 0.7 cm regurgitation jet area : the degree of mr was classified according to jet area into : mild mr if the jet area 8 cm effective regurgitation orifice area (ero) : mild mr if the ero 0.4 cm regurgitation volume (rv) : mild mr if the rv 60 ml / beat statistical package for social sciences (version 15.0, spss inc, chicago, ill) was used for data analysis. data were statistically described in terms of range, mean standard deviation (sd), frequencies (number of cases), and relative frequencies (percentages) when appropriate mean and sd were estimates of quantitative data. our study included 140 patients with chronic rheumatic mr, 84 female (60%), and 56 male (40%), with mean age 31.8 12.0 years (range, 1557 years). weight 62.59 12.4 (range, 3591 kg), height was 155.13 6.87 (range, 140167 cm), bsa 1.61 0.16 m (range, 1.31.92 m), 131 patients has normal sinus rhythm (94%), nine cases has atrial fibrillation (6%), 21 patients were asymptomatic (15%), 35 patients (25%) had nyha class i symptoms, 45 cases (32%) had nyha class ii symptoms, and 39 cases (28%) had nyha class iii symptoms, 40 patients (28%) had mild rheumatic mr, 50 patients (36%) had moderate rheumatic mr, and 50 patients (36%) had severe rheumatic mr. there was significant correlation between groups regarding lv dimensions and volumes and contractility, la dimensions and volumes, degree of mr measured by (vena contracta, regurgitant jet area regurgitant volumes, and effective orifice area), degree of tricuspid regurgitation, and pulmonary artery systolic pressure. no significant correlation between groups regarding septal wall thickness, posterior wall thickness, rv dimensions, rv functions measured by (fractional area change, tricuspid annular plane systolic excursion method, and s wave velocity of the tricuspid annulus) as shown in table 1. echocardiographic characteristics of the study groups the plasma levels of bnp rose with increasing severity of mr and with increasing la dimensions and volumes, lv dimensions and volumes, echocardiographic parameters of mr severity (width of the vena contracta, regurgitation jet area, ero, and regurgitant volume), and e waves. no significant correlation was found between bnp level and septal wall thickness, and posterior wall thickness [tables 2 and 3 ]. correlation between bnp level and echocardiographic variables correlation between mean values of bnp and echocardiographic parameters bnp was significantly higher in patients with severe mr compared with moderate and mild mr (p 12 (pmol / l) and reported 75% sensitivity and 85% specificity for identifying symptoms in patient with organic mr. furthermore, our study found a good significant correlation between bnp and la size (p < 0.0001) and la volume index (p < 0.0001), week significant correlation between bnp and ef measured by m mode (p = 0.03), and strong significant correlation with lvedd, lvedvi, lvesd, and lvsdvi (p < 0.001). these results suggest that bnp testing may serve as a marker for lv remodeling in chronic rheumatic mr and may be useful in identifying the earliest stages of lv decompensation. when the echocardiographic assessment is technically difficult, low bnp level would suggest that mr is not severe. in patients with rheumatic mr, plasma levels of bnp rise with increasing severity of mr and are higher in markedly symptomatic than in mildly symptomatic patients, even after adjustment for echocardiographic measures of the severity of regurgitation. separate analysis of bnp level based on the presence of associated comorbidities or other valvular lesions are needed. larger studies are required for assessment of bnp as a prognostic marker in patients with severe rheumatic mr. the cost of bnp affects the number of patients enrolled in the study. despite these difficulties, separate analysis of bnp level based on the presence of associated comorbidities or other valvular lesions are needed. larger studies are required for assessment of bnp as a prognostic marker in patients with severe rheumatic mr. the cost of bnp affects the number of patients enrolled in the study. despite these difficulties, | background : the most common causes of severe mitral regurgitation (mr) in developing countries are rheumatic heart disease. the plasma level of b - type natriuretic peptide (bnp) is known to increase with left ventricular (lv) dysfunction.aim of the work : to study bnp level as an index of symptoms and severity of chronic rheumatic mr.patients and methods : one hundred and forty patients with rheumatic mr and lv ejection fractions (efs) of > 55% underwent assessment of symptoms, transthoracic echocardiography, and measurement of bnp. results : the level of bnp rose with increasing left atrium (la) dimensions and volumes, lv dimensions and volumes, echocardiographic parameters of mr severity (width of the vena contracta, regurgitation jet area, effective regurgitation orifice area, and regurgitant volume), and e waves.results:bnp was significantly higher in patients with severe mr compared with moderate and mild mr (p < 0.001), and using cutoff point of 61 pg / ml mm had 97% sensitivity and 89% specificity for predicting patients with severe mr (0.99, 95% confidence interval [ci ] 0.91). bnp was significantly higher in patients with new york heart association (nyha iii) compared with nyha ii, i and asymptomatic patients (p < 0.001) and using cutoff point of 53 pg / ml had 97% sensitivity and 87% specificity for predicting symptomatic patients with symptomatic mr (0.81, 95% ci 0.700.92).conclusions : bnp level increase with increasing severity of rheumatic mr and are higher in symptomatic compared to asymptomatic patients, even in the presence of normal ef%. |
the integrated exposure uptake biokinetic (ieubk) model, recommended for use by the u.s. environmental protection agency at residential superfund sites to predict potential risks to children from lead exposure and to establish lead remediation levels, requires an interindividual geometric standard deviation (gsdi) as an essential input parameter. the gsdi quantifies the variability of blood lead concentrations for children exposed to similar environmental concentrations of lead. estimates of potential risks are directly related to the gsdi, and therefore the gsdi directly impacts the scope of remediation at superfund sites. site - specific gsdi can be calculated for sites where blood lead and environmental lead have been measured. this paper uses data from blood and environmental lead studies conducted at the bingham creek and sandy, utah, superfund sites to calculate gsdi using regression modeling, box modeling, and structural equation modeling. gsdis were calculated using various methods for treating values below the analytical method detection and quantitation limits. treatment of nonquantifiable blood lead concentrations affected the gsdi more than the statistical method used to calculate the gsdi. for any given treatment, the different statistical methods produced similar gsdis. because of the uncertainties associated with data in the blood lead studies, we recommend that a range of gsdis be used when analyzing site - specific risks associated with exposure to environmental lead instead of a single estimate. because the different statistical methods produce similar gsdis, we recommend a simple procedure to calculate site - specific gsdi from a scientifically sound blood and environmental lead study.imagesfigure 1 |
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warts are mucocutaneous growths (exo- or endophytic) caused by human papilloma virus (hpv). common therapeutic modalities ablate the growth without the generation of specific immunity against the wart. this often leads to irritation, pain, recurrence, scars, and prolonged morbidity (especially in palmoplantar warts). the stimulation of the immune system by exposing the virus to immune mediators forms an ideal modality of treatment due to spontaneous regression of the wart with long - lasting immunity. however, serotypes other than those in vaccine (hpv 6, 11, 16 and 18) also cause warts and hence this modality may not be effective always. autoimplantation is a novel, one - time procedure which treats the warts by stimulating an immune response against hpv. as it is an autograft technique, it is more likely that immunity against the same serotype is elicited (in contrast to the hpv vaccine where immunity is developed only against hpv 6, 11, 16 and 18). this is done by extracting a subcutis deep wart tissue and implanting it into the uninvolved skin. the autowart injection involves multiple sessions of injection of wart suspension. in this study, we extracted the donor tissue by paring the wart instead of removing a part of the wart under local anaesthesia. this was adopted to reduce the discomfort, risk of infection and hasten the time taken for performing the procedure. this study was conducted in the dermatology outpatient department of a tertiary care rural hospital. thirty - five patients with multiple (more than five warts), recurrent verruca vulgaris (warts which have recurred after any modality of treatment) and palmoplantar warts were enrolled in the study after an informed consent and ethics committee approval. pregnant, lactating mothers, immunocompromised individuals and those with verruca plana were excluded from study. donor tissue for autograft was harvested by paring a verrucous lesion or a palmoplantar wart. the lesion to be pared was cleansed with spirit - povidone iodine spirit to achieve asepsis. 11 autografting was done either on the non - dominant flexural forearm or the upper anteromedial thigh. the site for engraftment was cleansed with spirit - povidone iodine - spirit and infiltrated with about 0.5 ml of lignocaine with adrenaline (1:200,000). a subcutis deep stab incision of about 35 mm was made using the same surgical blade of no. the pared tissue was introduced deep into the subcutis using adson 's forceps or an insulin syringe used for infiltration [figure 3 ]. the margins of the wound were approximated by pressure and a micropore plaster was applied on it. amoxycillin 500 mg + clavulanic acid 125 mg, bid) and if required, analgesics orally. 11 used to pare the wart introducing the pared tissue deep into the subcutis using adson 's forceps patients were assessed monthly, and resolution of all warts within 3 months was taken as complete clearance. out of 35 patients, 8 were lost to follow - up and 27 patients were available for evaluation. the commonest age group affected was that of 2040 years (13 patients, 48.1%). a total of 20 (74.1%) patients showed the resolution of the warts within 3 months [figures 4 and 5 ], 5 (18.5%) patients had no improvement at all [table 1 ] and in 1 patient (3.7% ; male, 17 years of age) with multiple warts, except one lesion, others had resolved completely. one (3.7%) male patient aged 16 years had relapse, with a new lesion occurring at a different site. he underwent autoimplantation of the new wart, which healed over a period of 1 month. recurrent wart on the lateral aspect of the right second toe complete resolution of the wart after autoimplantation summary of characteristics of non - responding patients three (11.1%) patients developed erythematous tender nodules at the site of engraftment, with a purulent discharge from them in one patient. warts are one of the common viral infections of the mucocutaneous surfaces and often induce papillomatous growth. the commonest modality of treatment for warts is ablation using electrocautery, radiofrequency, laser or cryogens. these treatments destroy the wart - containing tissue but may not stimulate the immune system against the pathogen. hence, for the effective treatment of warts, there should be immune stimulation which can lead to long - term immunity against hpv. specific immune stimulation against hpv has been tried by autoimplantation of the wart tissue into the uninvolved skin, injecting the suspension of the crushed wart into the muscle or skin [table 2 ] and by quadrivalent hpv vaccines. patients with regression of warts either spontaneously or following successful therapy or injection of antigens showed the development of cell - mediated immunity and appearance of virus - specific igm and igg antibodies. comparison of the present study with previous studies in their study on the autoimplantation technique, shivakumar. extracted a subcutis deep part of the wart using a 18 g needle and implanted it into the skin elsewhere. this resulted in two wounds. also, hpv is an epidermal infection and there is no added advantage with the extracting donor tissue containing dermis and subcutis. hence, in this study, we harvested the donor by paring the wart to get the stratum corneum tissue containing virus. by this method, we avoided wound at the donor site and also circumvented the issue of taking the unwanted deeper tissues for engraftment. in contrast to the autowart injection therapy, this is a single sitting procedure and avoids the risk of injection abscess. most of our patients were males and belonged to the 20- to 40-year age group in parlance with other studies. a complete clearance of warts was observed in 74.1% of patients [table 2 ], greater than that observed by usman. we had one patient with multiple warts in whom one of the warts persisted at the end of 3 months. non - responders were minimal in the study, using autowart injection, by srivastava. one of our patients had clearance of warts after autoimplantation but developed a new lesion at a new site. this probably could be due to infection by a different serotype of hpv. as we were unable to isolate the species of hpv, this further supports the hypothesis. in one patient, warts healed with transient hypopigmentation. of the 27 patients, only 2 had anogenital warts in addition to verruca vulgaris and these also cleared completely with the autoimplantation of warts. hence, the final outcome was much better in our study (74.1%) when compared with other autoimplantation as well as autowart injection techniques (4473.3%). reaction at the site of engraftment in the form of erythematous tender nodules was observed in three patients, with a purulent discharge in one from the nodules. shivakumar. also noticed reactions at the injection site in some patients but have not disclosed the exact number. other researchers of the autoimplantation or autowart injection technique have not commented on any such reactions. this could be due to intense inflammation caused by implanted squames which act as a foreign body in the dermis and subcutis analogous to an inflammatory reaction which occurs in the case of a ruptured follicle. a quadrivalent hpv vaccine has shown promising results in the treatment of warts. however, the presence of only anecdotal reports, partial clearance in one of the two studies and a limited access in resource - restricted countries are the drawbacks of this vaccine. limitations of our study include a small sample size, lack of controls to assess spontaneous resolution of warts and lack of serotyping of persistent warts or checking for serospecific hpv antibodies. we also need to study the cause for the partial clearance or occurrence of new warts in spite of the clearance of all previous warts. the modified technique of autoimplantation using the pared stratum corneum tissue of the wart instead of the subcutis deep wart tissue for autografting is a safe, efficacious, less traumatic and rapid procedure for the treatment of multiple, recurrent and palmoplantar warts. | background : ideal treatment for warts should be effective, safe, have less morbidity and provide long - lasting immunity against human papilloma virus. this can optimally achieved by the stimulation of the immune system against the virus. the autoimplantation of warts, autowart injection and quadrivalent vaccines have been used for this purpose. autoimplanatation is a simple technique where the subcutis deep wart tissue is harvested as a donor and implanted into the uninvolved skin. however, this led to two wounds, at donor and recipient sites.aim:the aim was to evaluate the safety and efficacy of a novel modification of autoimplantation therapy in the treatment of multiple, recurrent and palmoplantar warts.subjects and methods : thirty - three patients with multiple, recurrent and palmoplantar warts were enrolled. instead of taking a bit of the wart tissue, the donor tissue was harvested by paring the wart. the pared tissue was implanted deep into the subcutis by stab incision done using the same surgical blade no. 11. the resolution of all warts within 3 months after the procedure was considered successful. patients with complete clearance were followed up for 1 month for any recurrence.results:out of 35 patients, 27 patients were available for follow - up. a total of 20 (74.1%) patients showed a complete clearance of warts within 3 months. partial clearance was seen in 1 patient. erythematous nodules developed at the site of implantation in 3 (11.1%) patients. there was relapse in one patient.conclusion:a modified technique of autoimplantation of warts employing the pared stratum corneum tissue from the wart is a simple, effective, less traumatic and rapid procedure in the treatment of multiple, recurrent and palmoplantar warts. |
a classic dichotomy can illustrate this point : a given duration, say a minute, is not experientially invariant. specifically, when awaiting for something, or feeling bored or blue, time drags ; when entertained or absorbed in skillful performance, time flies (i.e., we are hardly aware of the minutes passing by). the allocation of attention to time constitutes one major factor influencing whether the subjective flow of time will speed up or slow down : when attention is focused on time, perceived time slows down and experienced duration expands ; when distracted away from it, it speeds up and duration contracts. traditionally, cognitive models of time perception account for the speeding up or slowing down of time passing by positing the existence of an internal clock composed of a pacemaker (internal pacing of time units) and an accumulator (collection of time units) whose combined function is to represent subjective duration (treisman, 1963 ; gibbon., 1984). in the attentional gate clock models, the produced time units or pulses are only registered when attention is directed to time (zakay and block, 1997) leading to the opening of the gate and feeding of pulses into a counter. yet, two mechanisms can lead to alterations of subjective duration : (1) an increased (decreased) attention to time leads to an accumulation of more (less) pulses over a given time span or (2) an arousal - related increase (decrease) of the rate of pulses emitted by the pacemaker (faster or slower clock rate, respectively), leads to a faster (slower) accumulation of temporal units over time (burle and casini, 2001 ; droit - volet and meck, 2007 ; wittmann and paulus, 2008). for instance, heightened emotional states are physiologically accompanied by an increase in arousal, which would lead to a higher pacemaker rate (droit - volet and gil, 2009) : indeed, subjects tend to overestimate the duration of highly arousing pictures with emotional valence (e.g., angry faces or accidents) (angrilli., 1997 ; droit - volet. an increase in body temperature is also associated with an overestimation of duration (wearden and penton - voak, 1995) and in dangerous or life - threatening situations, people often report that events are prolonged in time and that everything occurs in slow motion (for such a personal report see popper and eccles, 1986, p. 529). during those moments, one experiences strong emotional distress and needs to react as quickly as possible. for these reasons, if mental processing speeds up considerably, external events seem to slow down accordingly as is popularly portrayed in the matrix combat scenes. all together, the rate of the pacemaker in clock models determines the perceived duration of an event whether due to arousal or attention : as a rule of thumb, the faster the rate, the more units accumulated and the longer the perceived duration. however, there is presently no real consensus on the how, when and where of such clock components in the brain (wittmann and van wassenhove, 2009) albeit specific functional implementations have been proposed, notably as a centralized and dedicated system for time (buhusi and meck, 2005). in controlled laboratory settings, subjective alterations of time (temporal illusions) can be systematically elicited. we now highlight a few cases which remain challenging for a general clock model framework and which could be parsimoniously accounted for by recent alternative proposals. one class of subjective duration effects are observed right before saccadic eye movement : in chronostasis, a backdating mechanism for temporal labeling of the saccadic onset depends on the duration of the preceding eye movement and leads to subjective dilation of duration (yarrow., 2001) ; analogous effects have been reported in the tactile domain during action (yarrow and rothwell, 2003). during saccadic movement such phenomena have been suggested to be tied to a neural remapping of temporal events compensating for the delay in producing a movement. however, subjective dilation of time can be seen without voluntary action for salient auditory events for instance (alexander., 2005) ; more generally, the subjective duration of unexpected or infrequent stimuli is overestimated as compared to expected and frequent stimuli (rose and summers, 1995 ; tse., 2004 conversely, stimulus repetition can lead to a subjective temporal compression of events thereby eliciting an apparent subjective dilation in the non - repeated stimulus (pariyadath and eagleman, 2007 ; van wassenhove. these effects have been accounted for by an energy efficiency model relying on well described neural suppression effects (eagleman and pariyadath, 2009) additionally, visual adaptation paradigms can be used to locally distort the apparent duration of events (johnston. 2010) further suggesting that these effects do not necessitate attention and can be specific to stimulus feature or low - level properties of a given stimulus (see for instance johnston (2010) for a local content - dependent clock). in line with this, a recent computational model has also been put forward in which no dedicated system is necessary and which relies on the natural statistics of events (ahrens and sahani, 2011). consistent with local adaptation mechanisms and content - related timing, whereas a looming (expanding) disc embedded within a series of standard discs leads to subjective time dilation (tse. 2004 ; van wassenhove., 2008), a standard disc embedded within a series of looming discs leads to subjective time compression (van wassenhove., 2008). an attention allocation mechanism would predict temporal dilation not only in the looming condition but also in the receding condition ; an adaptation mechanism would rather predict such perceptual inversion (van wassenhove. hence, several explanations are currently debated : (1) the attentional gate model (in line with clock models) : the subjective dilation of duration results from an increase of attention directed to the deviant stimulus (tse., 2004 ; new and scholl, 2009) ; (2) the energy efficiency coding : a greater amount of energy expenditure for the encoding of a deviant stimulus leads to subjective duration dilation in comparison to higher coding efficiency and less energy expenditure for standard stimuli (eagleman and pariyadath, 2009) ; (3) the local neural computations attuned to inherent stimuli properties : temporal effects can be driven by the intrinsic dynamics of the stimulus, namely, faster moving stimuli or stimuli with higher flicker frequency last subjectively longer (kanai., 2006 ; new and scholl, 2009). new computational schemes for time estimation are thus emerging albeit with different neural implementations (karmakar and buonomano, 2007 ; johnston, 2010 ; ahrens and sahani, 2011). surprisingly little to no neuroimaging data are currently available that would provide insights on the neural mechanisms mediating such temporal illusions. here, we report an event - related functional magnetic resonance imaging (fmri) study (wittmann.,, subjects viewed a stream of five visual events, all of which were static and of identical duration except for the fourth one, which was a deviant target consisting of a looming or a receding disc (van wassenhove., 2008). the use of an experimental paradigm using a looming signal as a (deviant) target in a stream of steady (standard) events allowed us to test (i) the specificity of temporal dilation and (ii) the neural underpinnings of time perception with respect to self. looming signals are not only salient and attention - drawing events (yantis and egeth, 1999 ; franconeri., 2005 ; for a refined hypothesis see skarratt., 2009) for instance, rhesus monkeys show a persistent avoidance response to looming stimuli (schiff., 1962) and brain responses specific to the dynamics of this stimulus develop in the first year of life in humans (van der weel and van der meer, 2009). looming signals are natural self - referential events : the time - to-(self) contact of such stimuli needs to be computed rapidly for planning an adequate escape behavior. our hypothesis was that looming signals engage brain structures involved in the processing of time in self - referential coordinates. this bears particular relevance in the context of a recent proposal pertaining to the experience of time as a self - referential process (craig, 2008, 2009a, b). one predicted implicated neural structure was the insular cortex which is functionally involved in interoception and is a key area for the integration of information originating within the body and for a meta - representation of homeostatic feelings. the insular cortex would naturally be at the core of the experienced self at one moment in time, providing a continuity of subjective awareness across time through a series of elementary emotional moments (craig, 2009a). it is noteworthy that although activation of the insular cortex has been repeatedly shown in neuroimaging studies on time perception (e.g., pouthas., 2005 ; livesey., 2007 ; 2010), it is only recently that the insular cortex has been discussed as causally relating to the processing of duration and the experience of time (craig, 2008, 2009a ; wittmann, 2009b ; kosillo and smith, 2010 ; wittmann., 2010a). psychophysical and fmri data of 15 right - handed participants (seven female ; mean age : 26 years) were analyzed in this study. first, participants underwent a psychophysical test outside the scanner (pre - fmri) followed by a similar psychophysical test during fmri. in the pre - fmri session, participants were presented with a stream of five visual events consisting of three consecutive standard discs (standard), one looming, receding or steady target (loom, recede, steady) and one last standard disc (figure 1). in the fmri session, the target stimulus consisted of either loom or recede (i.e., the target was never steady). the target was systematically varied in duration and always the fourth event in the stream of 500 ms standard discs (fmri condition : 494.2 ms). the stimuli consisted of gray disks centered on the monitor screen displayed on a black background. participants judged in a two - alternative forced choice whether the target was shorter or longer than all other standard in the trial (i.e., the first, second, third, and fifth stimuli). due to hardware constraints (refresh rate) slight differences in standard and target durations were chosen in the pre - fmri and the fmri sessions : in the pre - fmri session, standard was 500 ms and targets were 23.3, 10, or 3.3% of this duration ; in the fmri session, standard was 494.2 ms and targets were 23.8, 7.1, or 2.4% of the standard duration. experimental design : a trial consisted of a stream of five visual events, four standards (in first, second, third and fifth position) and one target (fourth position). all standards were static discs of 500 ms duration (standard) ; all targets were presented in fourth position in the sequence and varied in duration. in the pre - fmri session, a target could be static (steady), looming (loom) or receding (recede). in the fmri session, the inter - stimulus intervals (isi) and the inter - trial intervals (iti) were pseudo - randomly chosen from 500 to 1000 ms (isi), 2 to 4 s (iti pre - fmri), and 12 to 14 s (iti fmri). the pre - fmri session was conducted to (1) train the subjects in the task, (2) evaluate the perceived duration of a steady target to control for the compression of duration due to adaptation to repetition (eagleman and pariyadath, 2009), and (3) replicate prior findings with respect to time dilation observed in deviant looming but not receding stimulus presentation (van wassenhove. 2008). to preserve a dynamic control (size, motion, integrated luminance, etc.), the receding signal was the control for the looming condition in the fmri task, pending replication of the perceptual effects. brain activations corresponding to each stimulus were contrasted (loom vs. standard, recede vs. standard, and loom vs. recede) using the estimated voxel - wise response amplitude and function for each regressor of interest covering the specific stimulus duration. t - tests were used to probe for activation differences between the selected stimulus contrasts. a threshold adjustment method using monte - carlo simulations provided significant regions of interest which were corrected both on the voxel and cluster level (p standard disc. regions that are also active in the contrast receding > standard disc are highlighted in bold. regions of significant activation (p standard disc. regions that are also active in the contrast looming > standard disc are highlighted in bold. regions of significant activation (p receding disc and receding > looming disc. the psychophysical results replicated prior findings (van wassenhove., 2008) : a temporal dilation effect was observed for loom but not for recede targets as can be seen in figure 2a (percent of longer the points of subjective equality (pse ; figure 2c) significantly differed in the loom vs. the steady conditions [t(1,10) = 3.846, p standard disc. regions that are also active in the contrast receding > standard disc are highlighted in bold. regions of significant activation (p standard disc. regions that are also active in the contrast looming > standard disc are highlighted in bold. regions of significant activation (p receding disc and receding > looming disc. this is the first fmri attempt at delineating brain regions involved in a visual temporal illusion, namely subjective time dilation with looming signals. the behavioral results replicated earlier findings (tse., 2004 ; van wassenhove., 2008) and showed a profound temporal dilation effect. one goal was to use a predictable temporal locus for the targets (in each trial the target was consistently the fourth in the sequence of five events) in order to avoid any temporal expectation effect due to a sudden allocation of attention to the target : specifically, the overestimation of duration observed here depends on the nature of the stimulus as no effect was found with a receding signal (for a thorough discussion, see van wassenhove., 2008). in this context, one possible interpretation of the dilation illusion fits the cognitive pacemaker - accumulator model of time perception : an emotional response accompanied by increased arousal (looming) leads to an increased pacemaker rate and a larger accumulation of pacemaker ticks (droit - volet and meck, 2007 ; wittmann and paulus, 2008). however, the fmri analysis shows that similar brain regions were activated in loom and recede conditions when contrasted with standard. the outcome of these contrasts can be interpreted as indicating regions of interest involved in the estimation of duration for dynamic or salient targets versus static standards. the insular cortex, the anterior cingulate cortex, the basal ganglia as well as the dorsolateral prefrontal cortex were activated in both contrasts. this is consistent with prior neuroimaging findings on time perception (lewis and miall, 2003 ; rubia and smith, 2004 ; wittmann, 2009b, c). has been ascribed a role as coincidence detector of oscillatory phases (mattell and meck, 2004 ; buhusi and meck, 2005) ; dorsolateral frontal regions have been associated with working memory related integration, and anterior cingulate activity has been related to attention processes in time perception (lewis and miall, 2006). this network may thus highlight the comparison process between the duration stored in memory (the standard duration or reference duration ; gallistel and gibbon, 2000) with the target duration to be compared irrespective of its content. it is noteworthy that activation of the insular cortex for both targets in the stream of standards is consistent with the insular cortex mediating alertness in the presence of salient stimulation (sterzer and kleinschmidt, 2010). the regions found in this contrast is thus consistent with the implications of neural structures classically implicated in the clock model but such contrast does not offer an explanatory account with regards to the specificity of the illusory percept found with looming versus receding signals. one hypothesis considered in this study was that the experience of time is fundamentally self - referential (craig, 2008, 2009a ; wittmann, 2009b), i.e., uses the internal representation of self as a frame of reference (van de grind, 2002 ; northoff., 2011). in this context, the use of looming stimuli provides an explicit means to test the effect of a self - referential stimulus. in the decisive loom vs. recede contrast, differential activation was found in the mid- and posterior cingulate regions of the left hemisphere. a growing number of empirical studies indicate that mid- and posterior midline cortical structures are associated with the default brain network which is active when an individual is in a resting state and presumably engaged in self - relevant thoughts and beliefs (raichle., 2001 ; wicker., 2003) a recent study addressing the issue of mentalizing about self vs. others (lombardo., 2010) reported the activation of medial prefrontal and posterior cingulate cortices as key structures. in another fmri study, direct (one 's own self - beliefs) and reflected appraisals (one 's perception of how others view him or her) recruited the mid and posterior cingulate cortex (ochsner.,, the involvement of midline structures in the temporal perception of looming (but not receding) signals might be related to the potential threat carried by these signals to the perceiver (schiff., 1962). future studies will have to delineate more carefully subregions within the cingulate cortex and their respective functions (northoff. as can be seen in figure 3, a retrospenial region of the posterior cingulate cortex is also active during the presentation of the standards as compared to the dynamic targets, whereas more anterior and dorsal regions are also active in the looming versus receding contrast. the left lateralized activation of the anterior insula found in recede vs. loom is in line with an asymmetric representation of emotional feelings in the anterior insular cortex (craig, 2008). in primates, the insular cortex functions as the primary receptive area for homeostasis. re - representations of homeostatic afferent activity have been suggested to form the basis for the subjective awareness of emotional states (craig, 2009b). the anterior insular and cingulate cortex are conjointly engaged during task performance as complementary limbic sensory and motor regions. recent evidence suggests that the anterior insula and the anterior cingulate are part of the core control network. a system for task - dependent control of sensory information and goal - directed behavior (cole and schneider, 2007 ; craig, 2009b). this control network is also engaged in the perception of time : the anterior insula has been shown to be related to dysfunctions in timing behavior (rubia., 2009) and specifically involved in multisensory time synchronization (bushara., 2001). the homeostatic awareness model (craig, 2008, 2009b) provides a neuroanatomical framework for forebrain emotional asymmetry in which the left forebrain is predominantly related to parasympathetic activity (with approach, safety, positive affect), and the right forebrain is predominantly related to sympathetic activity (with arousal, danger, negative affect). thus, activation in the left anterior insula for the recede condition could be related to the feelings evoked with the receding (and thus non - threatening) stimulus, which is virtually moving away from the perceiver. however, this interpretation is weakened by the fact that no right - sided anterior insula activation (related to negative affect) was found in the loom condition, which would have been predicted by this model. research in the field of time perception is far from reaching a consensus on the two main questions of how and where in the brain time is processed (wittmann and van wassenhove, 2009). this is likely due to the complexity of time research as time encompasses a large number of aspects including duration on different time scales, synchrony, order etc., this study is a first attempt at addressing the difficult issue of content and stimulus - specificity in temporal illusions. our results provide some hints as to why loom signals last subjectively longer than recede signals and highlight how two simplistic, albeit surprisingly powerful stimuli, can engage differential mechanisms in representing time. one limitation concerning the choice of our receding stimulus may have weakened our findings : an initial jump to a large size for the receding target, which then shrinks to a smaller size, could have led to a smaller effect size. future experimental designs will likely use 3-d objects for more realistic stimulus rendering, which would be emotionally more salient. a better understanding of such temporal illusions will also largely benefit from temporally resolved neuroimaging techniques (magnetoencephalography, in particular). nevertheless, the temporal illusion was robust across participants and we were thus able to explore one fundamental aspect of time perception. the findings presented here are noteworthy in offering a new view for the role of self - related processes in the perception of time. 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. | for a given physical duration, certain events can be experienced as subjectively longer in duration than others. try this for yourself : take a quick glance at the second hand of a clock. immediately, the tick will pause momentarily and appear to be longer than the subsequent ticks. yet, they all last exactly 1 s. by and large, a deviant or an unexpected stimulus in a series of similar events (same duration, same features) can elicit a relative overestimation of subjective time (or time dilation) but, as is shown here, this is not always the case. we conducted an event - related functional magnetic neuroimaging study on the time dilation effect. participants were presented with a series of five visual discs, all static and of equal duration (standards) except for the fourth one, a looming or a receding target. the duration of the target was systematically varied and participants judged whether it was shorter or longer than all other standards in the sequence. subjective time dilation was observed for the looming stimulus but not for the receding one, which was estimated to be of equal duration to the standards. the neural activation for targets (looming and receding) contrasted with the standards revealed an increased activation of the anterior insula and of the anterior cingulate cortex. contrasting the looming with the receding targets (i.e., capturing the time dilation effect proper) revealed a specific activation of cortical midline structures. the implication of midline structures in the time dilation illusion is here interpreted in the context of self - referential processes. |
attentional problems are among the most consistently reported cognitive impairments related to prematurity (aarnoudse - moens., 2009 ; anderson, 2014 ; bhutta., 2002 ; hack., 2009 ; jaekel., 2013b ; mulder., 2009 ; wilson - ching., 2013), this is supported by many behavioral studies that assess specific attentional functions in children (anderson., 2011 ; geldof., wilson - ching., 2013) or adults (eryigit - madzwamuse., 2015 ; nosarti., 2007 ; solsnes., 2014) who were born very (vp, 3sd from mean), and six because of excessive scan - to - scan movements (> 2 mm in any direction). a further five cases where excluded due to unavailable iq data at 26 years. a comparison sample of term - born born infants (ga > 36 weeks) was recruited from normal postnatal wards in the same obstetric hospitals. of the initial 916 control children, 350 were randomly selected as term controls within the stratification variables sex and family socioeconomic status (ses) to be comparable with the vp / vlbw cohort at age 6 years 3 months. of these, 308 were eligible for 26 year follow - up assessments, with 229 (74.4%) participating in the psychological assessments, and 110 (35.6%) undergoing the additional mri examination. for two of these participants, ant imaging data were not available, one was excluded because of severe image artifacts, two due to high error / omission rates (> 3 sd above mean), and two because of excessive scan - to - scan movements (> 2 mm in any direction). a further three cases were excluded due to unavailable iq data at 26 years. in total, the final sample included 100 controls. background information for neonatal parameters was drawn from earlier assessments (gutbrod., 2000 ; riegel., 1995) : these included ga, bw, multiple births, maternal age, duration of neonatal intensive treatment (dnti) and intensity of neonatal intensive treatment (inti), duration of ventilation and duration of hospitalization. moreover, standardized optimality scores (prechtl, 1967) summarized the number of prepregnancy complications (08, e.g., prior disabled child, prior preterm birth), pregnancy complications (014, e.g., nicotine addiction, anemia), perinatal complications (015, e.g., no spontaneous labor, anesthesia), and neonatal complications (021, e.g., ventilation / intubation, neonatal seizures, sepsis ; cf. schmid., 2011). for vp / vlbw only, intraventricular hemorrhage (ivh) was assessed with ultrasound examination, graded 14. ses at birth was based on a weighted composite derived from the occupation of the highest educational qualification held by either parent and the occupation of the self - identified head of the family (bauer, 1988). developmental cognitive measurements included the german adaptations of the griffiths scales of baby abilities (brandt, 1983) at 5 and 20 months, and the kaufman - assessment battery for children (melchers and preuss, 1991) at 6 and 8 years. at 26 years, a short version of the german wechsler adult scale of intelligence, third edition (wais - iii : von aster., 2006) was administered to derive estimates for the full scale intelligence quotient (fsiq). the battery included the subtests vocabulary, similarities, digit symbol coding, block design, matrix reasoning, and letter number sequencing (cf. in addition, adult handedness was examined with the edinburgh handedness inventory (ehi : oldfield, 1971). participants were classified as left - handed (ehi : 100 to 61), ambidexter (ehi : 60 to + 60), or right - handed (ehi : + 61 to + 100) (cf. dragovic, 2004). to examine dropout - related selection biases, neonatal and developmental scores for the presented vp / vlbw and term - born subsamples were compared with respective data from those participants in the original cohort who were not included in the following analyses. visual stimuli were projected onto a display positioned inside the scanner room, which was viewed through a mirror system mounted on the mr head coil., 2005 ; fan., 2002), presented in a fast event - related design (supplementary fig. right orientation of a centrally presented arrow which was flanked by four additional arrows of the same size that pointed either into the same (congruent condition) or opposite direction (incongruent condition). in contrast to the original behavioral paradigm (fan., 2002 ; see also : neufang., 2011), but similar to other fmri adaptations of the task (fan. 2009), no neutral target stimulus condition (e.g., dashes instead of arrow flankers) was included. the target stimuli were presented either above or below a fixation cross which was continuously visible in the center of the display. each target stimulus was presented for a duration of 1050 ms, followed by a fixation cross baseline for 1950 ms. participants were instructed to indicate the direction of the central arrow as fast and accurately as possible, by pressing a left or right button (which corresponded to the index or middle finger of the dominant hand). approximately 600 ms before each target stimulus, a cue stimulus could appear for a duration of 150 ms to inform about the impending target stimulus. there were three cueing conditions : in the double cue condition, two asterisks appeared both above and below the fixation cross, indicating the timing, but not the location of the upcoming target. in the spatial cue condition, a single asterisk appeared at the same location as the upcoming target stimulus, indicating both the timing and the location of the upcoming target (i.e., no invalid cues were included ; see also fan.,, only the default fixation cross was visible during the 600 ms phase before target presentation. in total, the task design included six task conditions (3 [no cue / double cue / spatial cue ] 2 [congruent / incongruent ]). each run included 96 active trial events (16 trials for each of the six task conditions), and additional 32 null events (fixation cross, presented for 2000 ms) randomly interspersed between the trials. the presentation order of the different trial types was pseudorandomized, using optseq2 (http://surfer.nmr.mgh.harvard.edu/optseq)., armonk, ny, usa). frequency distributions for categorical variables were analyzed using tests (or fisher exact tests). unless stated otherwise, mean differences for continuous variables were analyzed with student 's t - tests for independent samples (including welch satterthwaite correction for unequal variances), using bootstrapping (efron and tibshirani, 1993) estimates of p values (based on 5000 samples, bias - corrected and accelerated method). in line with earlier reports of ant behavioral data (e.g., fan., 2005 ; pizzo., 2010), an executive network score was computed that represented the difference between median reaction time (rt, in ms) for the incongruent minus congruent target stimuli (averaged across cue conditions). therefore, higher executive network score values indicate a stronger rt increase for the incongruent target stimuli, which is supposed to reflect stronger response interference, and, hence, weaker efficiency of the executive network. high accuracy rates in both groups (i.e., highly skewed data distributions) precluded complementary analyses for accuracy rate data. instead, accuracy rates for incongruent and congruent task conditions, respectively, were collapsed to derive simple summary scores. we used univariate and stepwise multiple regressions to examine whether executive network scores in the vp / vlbw group were predicted by neonatal variables which are known risk factors for impaired neurological outcome : ga and bw, dnti, and duration of ventilation (e.g., aanes., mr data were initially acquired on identical philips achieva 3 t tx systems (philips, best, netherlands), using 8-channel sense head coils. due to a scanner upgrade, bonn had to switch to a complementary philips ingenia 3 t system after n = 15 participants, while munich had to do the same switch after n = 105 participants (supplementary table 1). yet, the identical sequence parameters were used on all scanners. to account for possible confounds introduced by the scanner - specific differences, all second - level functional data analyses included dummy regressors for scanner identity as covariates of no interest. during each run, 215 t2 -weighted epi volumes were acquired (tr = 2000 ms, te = 35 ms, flip angle = 82, parallel imaging with sense = 2 (a p) ; 32 interleaved oblique axial slices with a slice thickness = 4 mm (no gap) ; field of view = 220 220 128 mm ; reconstruction matrix = 96 96 ; reconstructed voxel size = 2.29 2.29 4 mm). five additional dummy scans were acquired (to achieve longitudinal magnetization equilibrium), but were already discarded before image reconstruction. for image registration purposes, high - resolution t1-weighted 3d - mprage volumes were acquired (ti = 1300 ms, tr = 7.7 ms, te = 3.9 ms, flip angle = 15 ; 180 sagittal slices, field of view : 256 256 mm, reconstructed voxel size = 1 mm). data were analyzed using spm8 (wellcome trust centre for neuroimaging, university college london, uk : http://www.fil.ion.ucl.ac.uk/spm), under matlab 8.2 (mathworks, natick, ma, usa). preprocessing of the functional data included slice time correction, realignment and unwarping of the epi series, co - registration of the t1-weighted image with the mean epi volume, segmentation of the t1-weighted image using unified segmentation (ashburner and friston, 2005), application of segmentation - derived normalization parameters to the co - registered functional data (interpolated to an isotropic voxel size of 2 mm), and spatial smoothing of the normalized epi series with a gaussian kernel of 6 mm fwhm. functional time series were modeled using general linear modeling (glm : friston., 1994). the first - level design matrix included separate event regressors for each of the six task conditions, with each event onset corresponding to the onset of the cue (i.e., for the no cue conditions, event onset sampled the fixation cross baseline stimulus presented 600 ms before target presentation : cf. an additional error regressor that captured trial events with false responses (or omissions) was included as a covariate of no interest. to capture residual movement - related artifacts, six regressors for the individual realignment parameters additionally, we included the time course of the average signal from white matter for each participant as a nuisance covariate (linzenbold and himmelbach, 2012 ; martin., 2015) : individual white matter masks from t1 segmentation were thresholded with a probability value of 0.99, and the mean signal time course within the white matter volume was read out from the realigned and normalized epi series, using marsbar (brett., 2002). task - related regressors were convolved with the spm8 canonical hemodynamic response function. to remove slow frequency signal drifts, high - pass filtering with 128 s cut - off was applied. parameter estimates were generated using restricted maximum - likelihood estimation, modeling temporal autocorrelation with an ar(1) model. for each participant, a first - level contrast for the critical incongruent > congruent comparison was computed (which measures brain activity evoked by stimulus conflict for the correct trials, and thus is supposed to reflect the executive attention component of the task). for this purpose, the contrast maps were subsequently entered into second - level random effect analyses (e.g., penny., 2003). to compare the spatial pattern of task - induced activations in both groups, initial one - sample t - test analyses were conducted for each group. then, activation differences between groups were analyzed using two - sample t - tests. each analysis included additional covariates for scanner identity (coded by three dummy variables), sex and age at examination (which varied slightly between groups). moreover, an additional rt regressor (representing the average of the median rts for the six individual task conditions) and a regressor for the total number of missing and errors were added to account for potential confounding influences of global rt (see supplementary fig. 2) and accuracy. since cognitive testing showed significant group differences in global cognitive function (see section 3.1), which may explain some variance in attentional processing (e.g., eryigit - madzwamuse., 2015), group comparisons also included fsiq as an additional covariate (cf. de kieviet., 2014). moreover, regression models for the vp / vlbw group with neonatal variables (ga, bw, dnti and duration of ventilation) as covariates were set up to examine whether these parameters predicted activation changes within the vp / vlbw group. contrast maps were set at a cluster extent threshold of p vp / vlbw, nor for vp / vlbw > controls. post hoc inspections of contrast estimates indicated that vp / vlbw the vp / vlbw group tended to show weaker thalamic activations than the controls (which therefore became visible only at more liberal voxelwise thresholds), but this was not sufficiently robust to produce significant group differences. regression analyses indicated that activation in the right dorsal acc (mnimax [14, 12, 42 ]) showed a negative association with ga, indicating that those vp / vlbw born more prematurely showed stronger activations in this region (table 4, fig. a similar pattern was found for a cluster in the right superior occipital lobe, extending from the superior and middle occipital gyrus to precuneus / cuneus (mnimax [28 68 20 ]). moreover, there was a positive association with dnti in an adjacent region of the dorsal acc (mnimax [2, 22, 28 ]), indicating that those vp / vlbw with longer intensive treatment also showed stronger activations in this region. while the areas showing a negative association with ga substantially overlapped with the group main effect (suggesting functional modulation of common task - relevant brain areas), the cluster showing a positive association with dnti was located in a non - overlapping aspect of the dorsal acc (supplementary fig. 4). additionally, a cluster in the right middle frontal gyrus showed a negative association with dnti (mnimax [34 20 42 ] : supplementary fig. 5), indicating weaker activation for vp / vlbw with longer intensive treatment, but cluster extent only approached the statistical threshold (k = 86, p =.055 fwe, cluster - level corrected). for the other neonatal variables, to the best of our knowledge, this is the first functional neuroimaging study investigating the neural underpinnings of executive attention in adults born very preterm. while we found preliminary evidence for subtle performance deficits during the processing of incongruent arrow flanker stimuli in the context of an ant fmri paradigm, which converges with previous findings in vp children (de kieviet., 2014 ; geldof., 2013 ; pizzo., 2010), there was no systematic compensatory recruitment, or even reorganization of the implicated fronto - cingulo - parietal processing networks. this could relate to the fact that we investigated a relatively high - functioning sample of vp / vlbw adults, which may have limited the need for compensatory functional reorganization : yet, we also found that both behavioral performance and activations in some acc and occipital areas were associated with lower gestational age and longer postnatal intensive treatment in the preterm - born group, suggesting at least subtle long - term influences of premature birth. contrary to our a priori assumptions, we found no imaging evidence for altered brain activation in the vp / vlbw adults on the group level. compared with term - born controls, there were no significantly reduced activations that may indicate functional impairments in task - relevant networks (e.g., griffiths., 2013), nor over - activations in the task - typical or supplementary brain areas that could be interpreted as compensatory activity (e.g., peterson., 2002) indeed, the activation main effects (incongruent > congruent arrow flanker trials) were strikingly similar in the two cohorts (fig. 2), revealing similar activations in expected fronto - cingulo - parietal networks, which are consistent with observations from earlier ant studies (fan., 2005 ; neufang., 2011), and meta - analyses for similar interference processing tasks (cieslik., 2015). while meta - analyses for executive control tasks in general suggest a domain - general cognitive control network that may extend further into rostral dlpfc regions (niendam., 2012), the caudal focus of frontal activations is in line with previous studies. additionally, both groups showed activations in extrastriate occipital regions, which have also been observed in earlier studies (backes., 2011 ; kellermann., 2011 ; korsch., 2014), and may reflect top - down modulation of stimulus processing in these sensory areas. although there were some regions where term control individuals showed more robust activations, e.g., in the thalamus (consistent with fan., 2005), these differences were not stable enough to be confirmed by direct group comparisons. in sum, the observations provide no evidence for a systematic reorganization of the brain networks implicated in executive attention, at least not in this relatively high - functioning vp / vlbw cohort (see section 4.5.1). despite the absence of categorical group differences, we found that brain activations were partially predicted by neonatal risk variables (fig. 3) : we found a significant inverse association between ga and activation in the right dorsal acc and lateral occipital cortex for the preterm born group, indicating a stronger activation of these task - relevant areas with increasing prematurity that would be compatible with a compensatory over - recruitment (see nosarti., 2009 for a similar this is further corroborated by the observation that vp / vlpbw adults with longer neonatal intensive care showed stronger dorsal acc activations, yet in a non - overlapping region (supplementary fig. 4), which may reflect a compensatory recruitment of additional acc regions. this would be consistent with a broader imaging literature implicating the dorsal acc in monitoring aspects of cognitive control (e.g., botvinick., 2001). conversely, there was suggestive evidence that vp / vlbw with longer neonatal intensive care showed weaker activity in a right - sided middle frontal region. yet, this area was located adjacent to task - typical processing networks for the ant and similar interference processing tasks (cieslik., 2015 ; fan., 2005), making clear inferences about its functional relevance difficult. yet, it seems to overlap with earlier meta - analytic findings of a domain - general cognitive control network (niendam. 1), and could therefore indicate an impaired supplementary recruitment of this control - related region. our findings do not clearly support the impaired recruitment of task - relevant brain networks that was observed by griffiths. (2013) in 11-year - old ep / elbw children who performed a combined stroop / n - back task : these ep / elbw children showed reduced activations in supplementary motor areas (sma) and the dorsal acc, the anterior insula and occipital brain regions. 2) suggested reduced thalamic activations in our vp / vlbw group, which would converge with other studies finding thalamic alterations (buml., 2014 ; nosarti., methodological differences between the studies may influence the divergent findings, including the varying developmental stage (11 vs. 26 years, with the possibility of function catch - up in the adult sample) and severity of prematurity (while the griffith study focused on ep / elbw children, we only had few ep / elbw survivors in our cohort). regarding task design, the present study used a simple flanker paradigm, while the other study combined stroop color - word interference stimuli with additional working memory demands : from a cognitive workload perspective (jaekel., 2013a ; just and varma, 2007), the simultaneous multi - item processing may have provoked an earlier breakdown of processing capacities. on the other hand, (2014) who used a flanker task paradigm to examine 8-year old vp and term - born children, and did not find any groupwise activation differences despite significant impairments in task performance : this applied to both a whole brain analysis, and a subsequent roi analysis for dorsal acc, left parietal and right parietal regions, which showed significant activations in both groups. the latter aspect is similar to our observations, although we found more extended activations, including lateral prefrontal and anterior insular regions (which may result from our substantially larger sample size), and only subtle decrements in behavioral task performance. de kieviet and colleagues speculate whether the potential for a compensatory over - recruitment of task - relevant brain networks may be limited in preterm - born individuals, and might therefore not translate into clear - cut group differences. yet, our observation that preterm - born adults with lower gestational age (and longer duration of neonatal intensive treatment) showed stronger activations in dorsal acc regions (table 4, fig. 3) suggests that there may be at least some compensatory potential in older preterm - born individuals. in sum, the available fmri literature provides inconclusive evidence for functional alterations of brain networks implicated in executive attention, and the present data extend these observations into early adulthood. our data suggest that the basic functional organization of the relevant networks is largely preserved, although individuals with lower ga may be more likely to show at least subtle behavioral and functional alterations. yet, our observations compare with fmri studies that investigated other aspects of executive functioning (e.g., fluency, working memory, motor inhibition) in preterm - born individuals, and found variable evidence for activation differences within the dorsal fronto - cingulo - parietal networks linked with cognitive control (niendam., 2012) : similar to the present experiment, some find no clear group differences in these brain regions (daamen., 2014), or mixed patterns of both lower and higher activations (nosarti., 2006 ; nosarti., 2009). whether these inconsistencies can be explained, e.g. by the varying degree of prematurity in the studied populations, and/or variations in task - specific processing demands (e.g., cognitive workload : jaekel., 2013a) needs to be examined more systematically. previous behavioral studies using the ant found significant deficits for the executive network (geldof. the fact that we could only detect subtle impairments of executive network function in the vp / vlbw participants, as indicated by marginally lower accuracy rates, and stronger rt increases for incongruent vs. congruent targets, could relate to specific requirements of the fmri task adaptation, but, more likely, reflects an incidental positive selection of less impaired vp / vlbw individuals (see section 4.5.1). meanwhile, there still was evidence that the efficiency of the executive network in the vp / vlbw group was linked to neonatal risk factors. both lower gestational age and longer duration of ventilation predicted worse performance, which concurs with observations that showed a quadratic effect of gestational age on behavioral ratings of attention problems in childhood (eryigit - madzwamuse and wolke, 2015). stepwise regression suggests that both predictors explain overlapping behavioral variance, with duration of ventilation (as an indicator for neonatal intensive treatment) providing better predictive value than ga (as an indicator for biological maturity). after controlling for fsiq, only ga was left as a significant predictor, which might suggest that the influence of ventilation duration is largely mediated by its effects on global cognitive function, while ga may explain some additional variance independent from fsiq. this does not only reduce the probability of missing differences due to a lack of power, but may also be less sensitive to reporting bias (i.e., reporting of more activation clusters than could be expected due to sample size) than conventional small - scale studies (for further discussion : david., 2013 ; ioannidis., 2014). another distinctive feature is that vp / vlbw participants were not drawn from hospital - based cohorts (e.g., gimenez., 2005), but came from a prospective epidemiological sample (griffiths., 2013), which should promote generalizability (kukull and ganguli, 2012), and offered the opportunity to systematically evaluate the possible impact of selection biases. actually, drop - out analyses showed a methodological drawback of the study : there was a positive selection of vp / vlbw with relatively high levels of cognitive functioning, lower neonatal complications, and lesser neurological impairment, as indicated by a significantly lower rate of individuals with severe cerebral palsy. the fact that we did not observe an increased rate of non - right handers in the vp / vlbw group, which is frequently reported in the preterm literature (domellf., 2011), may also support the assertion of lesser neurological impairment, although the fact that wm and csf proportion was decreased and increased, respectively, in vp / vlbw adults illustrates that deviant white matter development was still present in our sample. while the average iq in the vp / vlbw group was significantly lower than for controls, supporting previous studies (bhutta., 2002), and findings for the whole bls sample (breeman., 2015), drop - out analyses confirmed that the vp / vlbw of the present mri sample showed a higher iq than those who were not scanned, or had to be excluded (e.g., due to excessive motion). positive iq attrition is also observed in other studies that followed up preterm - born populations into adulthood (nosarti., 2007), and in some fmri studies, the examined populations actually show average iq performance (lawrence., 2010 ; narberhaus., 2009 ; while positive selection is not completely invalidating results, the group differences in our cognitively (and medically) fitter subsample can only provide a conservative estimate of possible differences, and are limited with regard to their generalizability (kukull and ganguli, 2012) : possibly, individuals with stronger cognitive deficits and neonatal adversities would have presented quantitatively or qualitatively different activation patterns that deviate more clearly from term - born controls. on the other hand, the inclusion of individuals who are potentially overcharged by the task (resulting in a substantial increase of error rates) would also introduce new analytical problems (price and friston, 1999). the present study found only subtle attentional differences, which is most plausibly explained by a positive selection of meanwhile, some studies observe that attention problems in vp / vlbw children and adolescents may not impair task performance in a continuous manner, but mainly express as intermittent lapses of attention, i.e. higher rates of omissions, or outlier responses (e.g., nosarti., 2007). actually, brain activity coinciding or preceding errors and lapses may be quite informative. yet, the brevity of the presented paradigm and the high accuracy rates in both groups precluded the collection of a sufficient number of trial events for systematic statistical analyses. to achieve this goal, a slow event - related design with irregular target presentation over an extended time course (similar to a continuous performance task) may be more appropriate. one may derive two main conclusions from the reported data : first, task - evoked brain activity mediating interference processing in vp / vlbw adults is not per se deviant at the macroscopic anatomical level, although there was evidence for subtle performance decrements, confirming recent observations in preterm - born children. second, the observation of modest associations between brain activations and task performance during flanker task processing and gestational and neonatal adversities in vp / vlbw adults suggest that preterm birth has at least a subtle long - term effect on executive attention, which may become more salient in more severely affected preterm - born individuals. due to the increasing survival rates of babies with extremely low gestational age and/or birth weight, this is a concern that deserves further examination. | very preterm birth is associated with an increased prevalence of attention problems and may especially impair executive attention, i.e., top - down control of attentional selection in situations where distracting information interferes with the processing of task - relevant stimuli. while there are initial findings linking structural brain alterations in preterm - born individuals with attention problems, the functional basis of these problems are not well understood. the present study used an fmri adaptation of the attentional network test to examine the neural correlates of executive attention in a large sample of n = 86 adults born very preterm and/or with very low birth weight (vp / vlbw), and n = 100 term - born controls. executive attention was measured by comparing task behavior and brain activations associated with the processing of incongruent vs. congruent arrow flanker stimuli. consistent with subtle impairments of executive attention, the vp / vlbw group showed lower accuracy and a tendency for increased response times during the processing of incongruent stimuli. both groups showed similar activation patters, especially within expected fronto - cingulo - parietal areas, but no significant between - group differences. our results argue for a maintained attention - relevant network organization in high - functioning preterm born adults in spite of subtle deficits in executive attention. gestational age and neonatal treatment variables showed associations with task behavior, and brain activation in the dorsal acc and lateral occipital areas, suggesting that the degree of prematurity (and related neonatal complications) has subtle modulatory influences on executive attention processing. |
migraine originated from a greece term meaning hemicraine or half of the head (1), usually associated with episodes of strong unilateral pulsating headache (2). migraine disorder shows a high - related relevancy ; approximately 50% of migraine cases have a first - degree related as a migraine sufferer (3). familial clustering migraine points to importance of genetic factors in this illness, but its inheritance pattern is argumentative and supposed it is likely multifactorial, although autosomal dominant inheritance can not account for it (3). the prevalence of migraine impressed about 10%12% of the white population (4), 24% of us populations and 12% of adults (5) contains both sexes (4), affected women more higher than men (17.1% in women and 5.6% in men) (5) and often involves the middle - aged people (6). migraine is observed as an inherited brain disturbance, specified by neurotransmitter imbalances, especially, serotonin 5-hydroxytryptamine (7) that contribute to neuronal dysfunctions (8). this disorder is generally characterized by strong and recurrent head pains which typically lasting about 472 h and attended by some symptoms like vomiting, neurological disturbance, photophobia and phonophobia. according to the classification of international headache society (ihs), two main classes of migraine consist : migraine without aura (mo), which included 70% of all migraineurs and migraine with aura (ma), which affected the rest of migraine population (about 25%) (9), both sub - groups have an intense and valid genetic background, but according to recent epidemiological information, the genetic factors in increasing development of ma (25%30%) are stronger than mo (11). the differences between ma and mo refer to the clinical symptoms of ma, it can include signals such as food craving, mood changes, neck stiffness, fatigue, reversible visual system symptoms, sensory and aphasic aura signs, each symptom might last from 5 min to 1 h (9). since migraine is a complicated and multifactorial disease, no distinct marker is in available to diagnosis the patient s status, yet. although, studies discovered prostaglandins synthesized by cyclooxygenase (cox) pathways, play a significant role in pathogenesis of this disorder, cox enzymes involved in pain mechanisms and migraine attacks, are the most important mediators of inflammation and pain (2). cox plays a noticeable role in prostanoids synthesis from arachidonic acid and arises this chemical reaction in both constitutive (cox-1) and inducible (cox-2) isoforms (12) which leads to sensibility and chronic pain in neuronal cells (13). prostaglandin e2 (pge2) and prostaglandin i2 (pgi2) are two important prostanoids that effect on pain signal considerably. some pharmaceutics combination like nonsteroidal anti - inflammatory drugs (nsaids) can inhibit cox-1 and cox-2 pathway and degrade pain in migraineur by reducing the production of prostanoids consisting pge2. thereby, one of the important factor in migraine therapy is cox-2 modulation, hence, applying non - selective cox suppressors such as acetylsalicylic acid was been high usage. moreover, a selective cox-2 inhibitor as rofecoxib is so effective in patient suffering from migraine with or without aura to tolerate the pain (2). as for the substantial role of cox-2 in synthesize pge2 and pathogenesis of migraine, we hypothesized that cox-2 gene basic polymorphisms may increase susceptibility to migraine. therefore, we did this research project for the first time in iran (bushehr province) and second time in the world. the aim of study was to determine the association of cox-2 - 765g > c (rs20417) and cox-2 - 1195a > g (rs689466) promoter polymorphisms to migraine susceptibility in iranian migraine patients for the first time in this region using rflp method. in this case - control study a total of 100 migraine patients (79 females, 21 males) and 100 controls (77 females, 23 males) were collected from july to nov 2013 from bushehr province, eastern iran and examined for promoter polymorphisms of cox-2 gene variant. controls were chosen among the people volunteered to donate their blood in the bushehr blood transforming center. the ethics board of hospital for migraine approved this study and all individuals providing samples signed informed consents covering aspects of the experiments conducted. the numbers of 20 patient samples were the individuals suffered from ma and 80 samples from mo. inclusion criteria were as follows : 1) patients with migraine referred to the mentioned clinic ; 2) signing informed consents covering by patients itself ; 3) diagnosis migraine by consultant. exclusion criteria were as follows : 1) discontentment of migraine patients or control group ; 2) unverified of migraine patients or control group by consultant. both control and patients were interviewed and examined by a specialist neurologist, all of them answered to a complete and perfect questionnaire and expressed their specifications such as their sex, age of onset, inbreeding or outbreeding marriage, related migraine history and pain severity. after obtaining awarded consent, the blood samples were transported to the laboratory and stored at 20 c until needed for analysis. pcr products for cox-2 gene on agarose gel with cox-2 - 765g > c (rs20417) and cox21195a > g (rs689466) primers showed fragments about 309 and 273 bp respectively (fig. 1 and 2). the anticipated results after restriction for each gene are also mentioned in table 1. the agarose gel electrophoresis of pcr products after digested with acii and pvuii enzymes are showed in fig. 3 and 4, respectively. agarose gel stained with ethidium bromide, pcr products of cox-2 gene by using the cox-2 - 765g > c (rs20417) primer. line 16 is 309 bp fragments, line m is 100 bp dna ladder agarose gel stained with ethidium bromide, pcr products of cox-2 gene by using the cox21195ag (rs689466) primer. line 16 is 273 bp fragments, line m is 100 bp dna ladder result of enzyme digestion by acii. lane m is 100 bp ladder, lane 1 and 2 : heterozygote polymorphic dnas (gc) contained 309, 209 and 100 bp fragments, lane 3 and 6 : 309 bp normal dnas (gg), lane 4 and 5 : homozygote polymorphic dnas (cc) contained 209 and 100 bp fragments result of enzyme digestion by pvuii. lane m is 100 bp ladder, lane 1, 3 and 4 : 273 bp normal dnas (aa), lane 2 : homozygote polymorphic dnas (gg) contained 220 and 53 bp fragments, lane 5 and 6 : heterozygote polymorphic dnas (ag) contained 273, 220 and 53 bp fragments pcr and rflp procedures and products of cox-2 - 765g > c and cox-2 - 1195a > g genes total dna was extracted from blood samples using dna purification kit (dnp, cinnagen, iran) according to the manufacturer s recommendation. the total extracted dna was measured at 260 nm optical density (14). in order to amplify the individual s respective gene, therefore, the final volume of 25 l pcr reactions in 0.2 ml tubes containing 500 ng/l of dna template, 2 mm of mgcl2 concentration, 2 mm dntps, 10 pmol/l of each primer, 5 l of 10x pcr buffer and 1 u of taq dna polymerase (fermentas, germany) was performed. thermal pcr conditions consisted of denaturation phase for 5 min at 95 c, followed by 30 cycles of 94 c for 1 min, temperature of 59 c for cox-2 - 1195a > g (rs689466) primer and 56 c for cox-2 - 765g > c (rs20417) primer for 1 min, and 72 c for 1 min, with a final extension for 5 min at 72 c. pcr - amplified products were examined by 2% agarose gel and analysis of pcr products for presence of cox-2 - 1195a > g gene revealed 273 bp fragments (fig. after electrophoresis, dna was observed and photographed in ultraviolet imager (uvi) doc gel documentation systems (uk). restriction fragment length polymorphisms (rflps) were used for analysis sequence polymorphisms of proliferated dna. the pcr product from second step digested for 4 h at 37 c by acii and pvuii restriction enzymes for cox-2 - 765g > c (rs20417) and cox-2 - 1195a > g (rs689466) snps, respectively, according to the manufacturer s instructions. the results of enzymatic digestion were identified in a 2% agarose gel by electrophoresis and ethidium bromide staining. molecular weight 100 bp plus marker (fermentas, co.) and undigested pcr products was included in each analysis. the genotypes were deduced from the fragmentation patterns of the amplified dna, observed in uvidoc gel documentation systems. statistical analyses were performed by the chi - square test using the spss 20 (spss inc. chicago, il, usa) software. related risk at 95% confidence intervals (ci) was calculated as the odds ratio (or). linkage disequilibrium between cox-2 - 765g > c and cox-2 - 1195a > g polymorphisms was performed using shesis software (http://analysis.bio-x.cn/myanalysis.php) (15). in this case - control study a total of 100 migraine patients (79 females, 21 males) and 100 controls (77 females, 23 males) were collected from july to nov 2013 from bushehr province, eastern iran and examined for promoter polymorphisms of cox-2 gene variant. controls were chosen among the people volunteered to donate their blood in the bushehr blood transforming center. the ethics board of hospital for migraine approved this study and all individuals providing samples signed informed consents covering aspects of the experiments conducted. the numbers of 20 patient samples were the individuals suffered from ma and 80 samples from mo. inclusion criteria were as follows : 1) patients with migraine referred to the mentioned clinic ; 2) signing informed consents covering by patients itself ; 3) diagnosis migraine by consultant. exclusion criteria were as follows : 1) discontentment of migraine patients or control group ; 2) unverified of migraine patients or control group by consultant. both control and patients were interviewed and examined by a specialist neurologist, all of them answered to a complete and perfect questionnaire and expressed their specifications such as their sex, age of onset, inbreeding or outbreeding marriage, related migraine history and pain severity. after obtaining awarded consent, the blood samples were transported to the laboratory and stored at 20 c until needed for analysis. pcr products for cox-2 gene on agarose gel with cox-2 - 765g > c (rs20417) and cox21195a > g (rs689466) primers showed fragments about 309 and 273 bp respectively (fig. 1 and 2). the anticipated results after restriction for each gene are also mentioned in table 1. the agarose gel electrophoresis of pcr products after digested with acii and pvuii enzymes are showed in fig. 3 and 4, respectively. agarose gel stained with ethidium bromide, pcr products of cox-2 gene by using the cox-2 - 765g > c (rs20417) primer. line 16 is 309 bp fragments, line m is 100 bp dna ladder agarose gel stained with ethidium bromide, pcr products of cox-2 gene by using the cox21195ag (rs689466) primer. line 16 is 273 bp fragments, line m is 100 bp dna ladder result of enzyme digestion by acii. lane m is 100 bp ladder, lane 1 and 2 : heterozygote polymorphic dnas (gc) contained 309, 209 and 100 bp fragments, lane 3 and 6 : 309 bp normal dnas (gg), lane 4 and 5 : homozygote polymorphic dnas (cc) contained 209 and 100 bp fragments result of enzyme digestion by pvuii. lane m is 100 bp ladder, lane 1, 3 and 4 : 273 bp normal dnas (aa), lane 2 : homozygote polymorphic dnas (gg) contained 220 and 53 bp fragments, lane 5 and 6 : heterozygote polymorphic dnas (ag) contained 273, 220 and 53 bp fragments pcr and rflp procedures and products of cox-2 - 765g > c and cox-2 - 1195a > g genes total dna was extracted from blood samples using dna purification kit (dnp, cinnagen, iran) according to the manufacturer s recommendation. in order to amplify the individual s respective gene, pcr test was accomplished using specific primers (table 1). therefore, the final volume of 25 l pcr reactions in 0.2 ml tubes containing 500 ng/l of dna template, 2 mm of mgcl2 concentration, 2 mm dntps, 10 pmol/l of each primer, 5 l of 10x pcr buffer and 1 u of taq dna polymerase (fermentas, germany) was performed. thermal pcr conditions consisted of denaturation phase for 5 min at 95 c, followed by 30 cycles of 94 c for 1 min, temperature of 59 c for cox-2 - 1195a > g (rs689466) primer and 56 c for cox-2 - 765g > c (rs20417) primer for 1 min, and 72 c for 1 min, with a final extension for 5 min at 72 c. pcr - amplified products were examined by 2% agarose gel and analysis of pcr products for presence of cox-2 - 1195a > g gene revealed 273 bp fragments (fig. dna was observed and photographed in ultraviolet imager (uvi) doc gel documentation systems (uk). restriction fragment length polymorphisms (rflps) were used for analysis sequence polymorphisms of proliferated dna. the pcr product from second step digested for 4 h at 37 c by acii and pvuii restriction enzymes for cox-2 - 765g > c (rs20417) and cox-2 - 1195a > g (rs689466) snps, respectively, according to the manufacturer s instructions. the results of enzymatic digestion were identified in a 2% agarose gel by electrophoresis and ethidium bromide staining. molecular weight 100 bp plus marker (fermentas, co.) and undigested pcr products was included in each analysis. the genotypes were deduced from the fragmentation patterns of the amplified dna, observed in uvidoc gel documentation systems. statistical analyses were performed by the chi - square test using the spss 20 (spss inc. chicago, il, usa) software. related risk at 95% confidence intervals (ci) was calculated as the odds ratio (or). linkage disequilibrium between cox-2 - 765g > c and cox-2 - 1195a > g polymorphisms was performed using shesis software (http://analysis.bio-x.cn/myanalysis.php) (15). the mean age was 33.1610.38 for patients (1869 age), and 34.2010.16 yr (1860 age) for controls. there were symmetry between patient and control subjects in terms of sex prevalence and mean age and most of patients experienced intensive pain, which disorganized their functions and activities. table 2 shows the frequencies of cox-2 - 765g > c and cox-2 - 1195a > g genotype distributions in controls, ma and mo patients separately. there were statistically considerable differences in cox-2 - 765g > c and cox-2 - 1195a > g genotypes between the controls and patients. carriers of cox-2 - 765 cc and gc genotypes (polymorph types) in patients were further than in the controls and frequencies of cox-2 - 765 gg genotype (the wild type) in controls were higher than in all the patients (p g genotype distributions in patients and controls p1 : p value of migraine with aura vs control. polymorphism, only aa genotype (the wild type) was statistically higher in the controls than in the all patients (p c polymorphism (p g polymorphism (p c and cox-2 - 1195a > g polymorphisms. in this analysis, no significant difference was observed in frequencies of cox-2 - 765 g : 1195 g haplotype in patients as compared with controls (p c and cox-2 - 1195a > g genotype distributions in patients with related or unrelated parents separately (table 5). frequencies of cox-2 - 765g > c and cox-2 - 1195a > g genotype distributions in patients with related and unrelated marriage parents p1 : p value of patients with related parent vs control. c, quantity of cox-2 - 765 gg genotype (the wild type) in controls were higher as compared with the patients with related parents and unrelated parents, however, quantity of cox-2 - 765 cc and cox-2 - 765 gc genotypes in patients with related and unrelated parents were more than in migraine- free subjects (p c and cox-2 - 1195a > g snps between the ma patients, mo patients, patients with related parents, patients with unrelated parents as compared with control individuals, so these variants and consanguineous type of marriage can increase risk of migraine susceptibility. we have studied the impression of genetic polymorphisms of cox-2 gene and the risk of migraine susceptibility. in this study, there is a positive relation between the -765 gc, -765 cc, -1195 ag and -1195 gg and being adventured to migraine attacks. in other words c allele of cox-2 - 765g > c and g allele of cox-2 - 1195a > g play key role in increasing migraine risk and besides -765 gg and -1195 aa have protective effect against migraine. we showed this association on iranian population as the first study in iran and second in the world. recent studies are available now and have recognized four new genetic variants related to migraine. a new variant known as rs1835740 moderates glutamate homeostasis is associated with concepts of neurotransmitter disorders. this new variant may be more specific for acute sorts of migraine such as ma than mo (8). another variant exists as rs11172113, involves the lipoprotein receptor lrp1 which may interact with neuronal glutamate receptors and pathway however the specify function of third variant rs2651889 (prdm16) in migraine is unknown yet (8). the fourth variant that for the first time connected gene impressions on migraine pain pathways, was rs10166942 is in close proximity to trpm8, which encodes for pain and cold receptors (8). cox-2 derived prostanoids enumerated as crucial clinical mediators of pain and other inflammatory symptoms of knee osteoarthritis (2) suggested that cox-2 polymorphisms could contribute susceptibility to sarcoidosis (16). cox-2 - 765g > c and cox-2 - 1195a > g would alter the mrna levels and transcriptional pattern (17). there is a major relevance between cox-2 expression and the risk of vitiligo (19, 20). correlation of cox-2 functional polymorphisms (cox-2 - 1195a > g, -765g > c, -8473t > c) and the risk of vitiligo has been reported (21). furthermore, expression of the two main isoforms of cyclooxygenase (cox-1 and cox-2) in human cancerous prostatic tissues has been studied and overexpression of both of them is reported (23). moreover, an increased propensity for lung cancer development was observed in individuals who caring the c allele of a polymorphism in the 3-utr of cox-2 (24). in a study on iranian population, there was no significant relation between ptgs2 (cox-2)-765g > c gene polymorphism and the risk of sporadic colorectal cancer (25). moreover, 75% of patient with poorly differentiated tumors and 84.6% of patient with moderately differentiated tumors had high scale of cox-2 in their primary tumor cells (26). induction of cox-2 in dementia of alzheimer type and down s syndrome may contribute to the integration of free radicals and then may be related to neuronal degeneration (27). selective cox-2 inhibitors can increase the risk of vascular events (28) myocardial infarction (29) and cardiovascular occurrences (30). conversely, selective cox-2 inhibitors made a significantly reduction in the risk of breast cancer (31), this reality can be related to the finding that elevated cox-2 expression in breast cancers was seen as a common and ordinary point (32). cox-2 may be a target for the prohibition, treatment and abatement the risk of squamous cell carcinoma of the head and neck (hnscc) (33). the cox-2 pathway can lead to the growth and apoptosis of pancreatic cancer (34) and interesting results are in available, pointed to that overexpression of cox-2 in multiple myeloma (mm) is correlated with reduced survival (3536). as the first study in iran, demonstrates that cox-2 gene polymorphisms would increase the risk of affliction to migraine disorder. further studies on other races in the other regions with different climate are necessary to compare various results. regarding effective role of genetic factors in ma and related clustering migraine, more genetically based studies can be helpful in diagnosing the reasons, symptoms and inheritance prognosis of illness. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors. | background : migraine is a common debilitating primary headache disorder with current head pain attacks, which contributes to physical activity dysfunctions in chronic pain phase. pge2 and pgi2 are two important prostaglandins synthesised by cox-2 enzymes, involved in migraine pain signals. cox-2 modulation is essential in treatment and pathogenesis of migraine. this study aimed to investigating the association between cox-2 gene polymorphisms with the risk of migraine susceptibility in migraine patients with related and unrelated parents.methods:this case- control study was based on 100 migraine patients and 100 non - migraine subjects in bushehr province, iran in 2013. genomic dna of blood samples was extracted and genotyping of cox-2 - 765g > c (rs20417) and cox-2 - 1195a > g (rs689466) gene variants was investigated by pcr - rflp method. statistical analyses were accomplished using the spss software package.results:there was a significant differences in the frequencies of the cox-2 - 765g > c and cox-2 - 1195a > g genotypes between migraine patients and controls (p0.05).conclusion : cox-2 - 765cc, cox-2 - 765cg, cox-2 - 1195gg and cox-2 - 1195ag genotypes can increase the risk of migraine significantly. as the first study in iran, we are hopeful to achieve greater results about the relevancy of cox-2 gene, migraine and pain signals pathway by repeating these experiments on more samples. |
it can be classified into two broad categories : acute - onset and delayed - onset. according to endophthalmitis vitrectomy study, infection occurring within 6 weeks after cataract surgery was defined as acute - onset and after 6 weeks as delayed - onset.1 these two categories differ in their microbiological spectrum.2 the purpose of the current study was to investigate the spectrum of organisms causing culture proven delayed post - cataract surgery endophthalmitis and their antimicrobial susceptibilities at our center between 2006 and 2013 and compare it with available western literature. microbiology records were reviewed of all the culture proven delayed post - cataract surgery endophthalmitis cases treated at l v prasad eye institute, hyderabad, india between january 2006 and march 2013. the study was approved by the institutional review board and adhered to the guidelines of the declaration of helsinki. vitreous samples from all patients had been investigated for bacteria and fungus using institutional protocol. bacterial isolates were identified using analytical profile index system until 2010 and vitek-2 compact system (biomrieux, craponne, france), thereafter. isolates included 12 (36.4%) gram - positive cocci, one (3.0%) gram - positive bacilli, seven (21.2%) gram - negative bacteria, two (6.1%) nocardia sp., and eleven (33.3%) fungi (table 1). most common isolates identified were coagulase negative staphylococci, aspergillus sp. and candida sp. (5/33, 15.2% each) followed by streptococcus pneumoniae (3/33, 9.1%) and burkholderia cepacia (3/33, 9.1%). onset of endophthalmitis occurred 30 days to 15 years (median 150 days) post - operatively in streptococcal cases, 30 to 168 (median 72) days in coagulase - negative staphylococcal cases, 60 to 157 (median 84) days in gram - negative cases, and 60 to 365 (median 76) days in fungal cases. gram - negative isolates were most susceptible to ofloxacin (85.7%) followed by ceftazidime, ciprofloxacin, gatifloxacin, and moxifloxacin (71.4% each). isolates included 12 (36.4%) gram - positive cocci, one (3.0%) gram - positive bacilli, seven (21.2%) gram - negative bacteria, two (6.1%) nocardia sp., and eleven (33.3%) fungi (table 1). most common isolates identified were coagulase negative staphylococci, aspergillus sp. and candida sp. (5/33, 15.2% each) followed by streptococcus pneumoniae (3/33, 9.1%) and burkholderia cepacia (3/33, 9.1%). onset of endophthalmitis occurred 30 days to 15 years (median 150 days) post - operatively in streptococcal cases, 30 to 168 (median 72) days in coagulase - negative staphylococcal cases, 60 to 157 (median 84) days in gram - negative cases, and 60 to 365 (median 76) days in fungal cases. gram - negative isolates were most susceptible to ofloxacin (85.7%) followed by ceftazidime, ciprofloxacin, gatifloxacin, and moxifloxacin (71.4% each). the microorganisms in delayed post - cataract surgery endophthalmitis are believed to be less virulent as indicated by its insidious onset and low grade of inflammation. in most of the studies published in the literature, propionibacterium acnes were most commonly isolated from the vitreous samples of delayed - onset endophthalmitis.25 in the current study, p. acnes were not isolated from any sample. one of the reasons for this could be that anaerobic culture was not done in all cases. from india, there are mostly case reports on delayed - onset endophthalmitis after cataract surgery,6 thus it is not possible to comment on the incidence of p. acnes associated delayed post - cataract surgery endophthalmitis in this region. a higher incidence of fungal isolates noted in the current study is consistent with other studies from the same geographic region.2 fungi, comparison of microorganisms identified in various delayed - onset post - cataract surgery endophthalmitis studies is given in table 2. the antimicrobial susceptibility of the organisms is comparable to that of the same organisms in different clinical settings like acute post - cataract endophthalmitis and post - traumatic endophthalmitis in the same geographic region.7,8 gram - positive organisms are most susceptible to vancomycin and gram - negative organisms to fluoroquinolones and ceftazidime. low susceptibility of gram - negative organisms to amikacin was due to b. cepacia being the most common cause and they have been shown to be less susceptible to amikacin as compared to ceftazidime and ciprofloxacin.9 this is the first study from india on the antimicrobial spectrum and antimicrobial susceptibility of delayed post - cataract surgery endophthalmitis. less virulent and slow growing organisms like coagulase negative staphylococci and fungi are the most common causative agents of delayed - onset endophthalmitis. | the objective of this study was to evaluate the microbiological spectrum and antimicrobial susceptibility of isolates in delayed post - cataract surgery endophthalmitis. a retrospective review of 33 consecutive patients with culture proven delayed post - cataract surgery endophthalmitis was done from january 2006 to march 2013. there were 22 bacterial and eleven fungal cases. common isolates were streptococci (seven cases), coagulase - negative staphylococci (five), gram - negative bacilli (seven), nocardia (two), aspergillus (five), candida (five). gram - positive cocci were most susceptible to vancomycin and gatifloxacin (91.7%). gram - negative isolates were most susceptible to ofloxacin (85.7%). fungi being slow growing organisms are an important cause of delayed post - cataract surgery endophthalmitis. |
endothelial nitric oxide synthase (enos) is a crucial enzyme for vascular physiology ; its reduced activity during ageing is associated with increased susceptibility to cardio- and cerebro - vascular diseases. moreover, in mice lacking enos, caloric restriction does not exert its positive effects in delaying ageing and increasing life span. long - living individuals (llis) have a favorable genetic profile characterized by an enrichment of alleles associated with the protection from ageing and cardiovascular disease. we have recently shown for three different populations that llis are enriched for rs2070325 (i229v), the minor allele of bactericidal / permeability - increasing fold - containing family b member 4 (bpifb4).rs2070325 was one of four single - nucleotide polymorphisms on bpifb4 that variously combined to generate bpifb4 isoforms, such as the wild type (wt) protein and a longevity - associated variant (lav). of note, the lav - bpifb4 was associated with potentiated enos activity in cells, an effect correlated with increased binding of bpifb4 to 14 - 3 - 3through an atypical - binding site for the protein and increased phosphorylation of bpifb4 at serine 75a site recognized by protein kinase r - like endoplasmic reticulum kinase (perk). heat shock protein 90 (hsp90) was also recruited to the lav14 - 3 - 3 complex as part of the enos activation machinery. indeed, hsp90 co - immunoprecipitated with bpifb4, and a specific hsp90 inhibitor blocked the potentiation of endothelial function and enos activation exerted by the lav. despite these findings, further characterization is needed to define how lav - bpifb4 transduces upstream signals to enos. on this point this function is mediated by protein kinase c alpha (pkc), which stimulates nitric oxide (no) production in endothelial cells and plays a role in regulating blood flow in vivo. in the present study, we demonstrate that pkc is part of the signalling pathway activated by lav - bpifb4 to potentiate vascular function. in particular, we show that lav - bpifb4 activates enos - dependent endothelial function through ca - mediated potentiation of pkc. moreover, when pkc and/or enos is inactivated e.g. by exposing cells to ca - free media or knocking out enos lav - bpifb4 can still enhance vasorelaxation through an endothelium - derived hyperpolarizing factor (edhf)-mediated pathway. all experiments involving animals conformed to the guidelines for the care and use of laboratory animals published with directive 2010/63/eu of the european parliament and were approved by the review board of irccs inm neuromed (ref. mice were sacrificed by intraperitoneal injection of ketamine / xylazine (respectively, 150 and 20 mg / kg bw), and second - order branches of the mesenteric arterial tree were surgically removed and mounted on a pressure myograph for experiments. endothelium - dependent relaxation was assessed by measuring the dilatory responses of mesenteric arteries to cumulative concentrations of ach (from 10 to 10 m) in vessels pre - contracted with u46619 at a dose necessary to obtain a similar level of pre - contraction in each ring (80% of initial kcl - evoked contraction). ach - evoked vasorelaxation was also tested in the presence of the pkc inhibitor g6976 (0.5 m) or the akt inhibitor il6-hydroxymethyl - chiro - inositol-2-(r)-2-o - methyl-3-o - octadecyl - sn - glycerocarbonate (10 m) (no. the endothelium was mechanically removed by inserting a tungsten wire into the lumen of the vessel and rotating it back and forth before mounting the vessel on the pressure myograph. another experimental series was performed on vessels transfected in presence of ca and then studied in the absence of external ca, using ca - free krebs, in presence of apamin (apa)a potent inhibitor of atp - type ca - activated k channels and skca, and charybdotoxin (ctx)a potent and selective inhibitor of the voltage - gated ca - activated k channel (kv1.3) and bkca channel (both were purchased from sigma - aldrich). for facs analysis, transfected arteries were digested with type 2 collagenase (0.05% ; worthington cls2) for 45 min at 37 c in a shaking incubator. freed cells were washed with pbs and passed through a 100-m strainer (bd falcon). afterwards, cells were stained with anti - cd31-fitc (1:100, bd biosciences - pharmigen) at 4 c for 20 min and then permeabilized with cytofix / cytoperm (bd biosciences - pharmigen) at 4 c for 20 min. subsequently, cells were incubated with anti - bpifb4 (1:100 ; abcam) at 4 c for 1 h and then an allophycocyanin (apc)-conjugated anti - mouse secondary antibody (1:200 ; biolegend). for non - directly conjugated antibody to bpifb4, a staining mix without anti - bpifb4 antibody but with inclusion of the fluorescent secondary antibody was used as negative control. analysis of cell populations was performed using a facs canto ii equipped with facs diva software (bd biosciences) and the flowlogic (miltenyi biotec) analysis program. bpifb4 cdna (wt and lav isoforms) was cloned from prk5 expression plasmids into the lentiviral vector pcdh - ef1-msc - pa - pgk - cop - green fluorescence protein (gfp)-t2a - puro (system biosciences). lentiviral particles were generated by transfection of pcdh constructs along with the packaging vectors pmd2.vsv.g, prsv - rev, and pmdlg / prre (kindly provided by prof luigi naldini, san raffaele scientific institute, milan, italy) into human embryonic kidney (hek293 t) cells by calcium phosphate transfection. lentiviral particles were concentrated by ultracentrifugation (25 000 rpm for 4 h at 4 c) and stored at 80 c until immediately prior to use. lentivirus titration was performed by transducing hek293 t cells with concentrated particles in the presence of 4 g / ml polybrene and measuring gfp expression after 3 days by flow cytometry. for ca mobilization and confocal microscopy assay, human umbilical vein endothelial cells (huvecs) (lonza) were grown in complete egm2 medium (lonza) and infected with empty lentiviral vectors or particles encoding either wt- or lav - bpifb4 [at 5 multiplicity of infection (moi) ]. after 72 h, cells were selected with 2 g / ml puromycin for 48 h. hek293 t cells were grown in dulbecco s modified eagle s medium supplemented with 10% (v / v) fetal bovine serum and 1% non - essential amino acids at 37 c in a 5% co2 atmosphere. for co - immunoprecipitation, 1.4 10 cells were plated in 10-cm dishes and transfected with prk5 vector encoding lav - bpifb4 or with an empty plasmid, using lipofectamine 2000 (life technologies) according to the manufacturer s protocol. twenty - four hours post - transfection, hek293 t cells were incubated with g6976 (0.3 m) for another 24 h, harvested, and solubilized in lysis buffer (20 mm tris - hcl ph 7.5, 650 mm nacl, 500 mm edta, 250 mm egta, and triton x-100). lysates were cleared at 13 000 rpm for 20 min at 4 c, and 700 g protein incubated overnight with 2 g of mouse anti - gfp (invitrogen), mouse anti-14 - 3 - 3 (abcam), or mouse anti - igg (millipore) for the control. antigen complexes were precipitated with glinked sepharose protein (ge healthcare) for 4 h at 4 c and the beads washed three times with lysis buffer. the denatured co - immunoprecipitation products were resolved with sds - page, electro - blotted onto pvdf membranes, and hybridized with 1:1000 rabbit anti-14 - 3 - 3 (abcam). each sample was a pool of mesenteric arteries (length, 2 mm ; diameter, 250 m) excised from four mice. huvecs infected with lentiviral particles encoding wt- bpifb4, lav - bpifb4, or gfp (empty vector) were starved in serum- and growth factor - free egm-2 for 4 h and then stimulated with 100 m ach for 10 min. protein extracts were separated on 810% sds - page at 100 v for 1 h or on 412% sds - page at 100 v for 2 h and then transferred to a nitrocellulose or pvdf membrane as previously described in. western blots were analysed using imagej software (wayne rasband, national institutes of health, usa) to determine optical density (od) of the bands. the od readings of phosphorylated proteins are expressed as a ratio relative to total protein or to beta - actin. free intracellular ca concentration ([ca]i) recordings were obtained by time - resolved digital fluorescence microscopy on infected huvecs loaded with the ca indicator x - rhod-1 am (excitation, 550 nm ; emission, 610 nm) to avoid overlapping of fluorescence signals due to the presence of gfp. briefly, cells were incubated for 45 min at 37 c with 2 m of x - rhod-1 am. cells were then placed in standard mammalian ringer solution (in mm : nacl, 140 ; kcl, 2.5 ; cacl2, 2 ; mgcl2, 2 ; hepes - naoh, 10 ; and glucose, 10 ; ph 7.3), and continuously superfused with a gravity - driven fast perfusion system (biologique 100). most cells were infected and displayed clear gfp fluorescence that did not interfere with the fluorescent signal of the ca dye. the time courses of ca transients were quantified by measuring at each time point the fluorescence emission in the region of interest surrounding each cell, and then transforming the obtained values as follows : the amplitude of the atp - induced ca transient was evaluated as the difference between maximal and basal f / f values. huvecs infected with lentiviral particles encoding wt - bpifb4, lav - bpifb4, or gfp (empty vector) were fixed in 4% paraformaldehyde in pbs for 20 min, washed twice in 50 mm nh4cl in pbs, and permeabilized for 5 min in 0.2% triton x-100 in pbs. immunofluorescence analysis was performed on an inverted, motorized microscope (axio observer z.1) equipped with a 63x/1.4 plan - apochromat objective (carl zeiss). the attached laser - scanning unit (lsm 700 4x pigtailed laser 405 - 488 - 555 - 639, carl zeiss) enabled confocal imaging. for excitation, fluorescence emission was revealed by a mbs (main dichroic beam splitter) and a vsd (variable secondary dichroic beam splitter). triple staining fluorescence images were acquired separately using zen 2012 software in the blue (hoechst 33258), green (egfp), and red (alexa fluor 594) channels at a resolution of 1024 1024 pixels, with the confocal pinhole set to one airy unit, and then saved in tiff format. densitometry data were analysed with one - way anova followed by bonferroni posthoc analysis, as appropriate, using dedicated software (graphpad prism, v5.0). mice were sacrificed by intraperitoneal injection of ketamine / xylazine (respectively, 150 and 20 mg / kg bw), and second - order branches of the mesenteric arterial tree were surgically removed and mounted on a pressure myograph for experiments. endothelium - dependent relaxation was assessed by measuring the dilatory responses of mesenteric arteries to cumulative concentrations of ach (from 10 to 10 m) in vessels pre - contracted with u46619 at a dose necessary to obtain a similar level of pre - contraction in each ring (80% of initial kcl - evoked contraction). ach - evoked vasorelaxation was also tested in the presence of the pkc inhibitor g6976 (0.5 m) or the akt inhibitor il6-hydroxymethyl - chiro - inositol-2-(r)-2-o - methyl-3-o - octadecyl - sn - glycerocarbonate (10 m) (no. the endothelium was mechanically removed by inserting a tungsten wire into the lumen of the vessel and rotating it back and forth before mounting the vessel on the pressure myograph. another experimental series was performed on vessels transfected in presence of ca and then studied in the absence of external ca, using ca - free krebs, in presence of apamin (apa)a potent inhibitor of atp - type ca - activated k channels and skca, and charybdotoxin (ctx)a potent and selective inhibitor of the voltage - gated ca - activated k channel (kv1.3) and bkca channel (both were purchased from sigma - aldrich). for facs analysis, transfected arteries were digested with type 2 collagenase (0.05% ; worthington cls2) for 45 min at 37 c in a shaking incubator. freed cells were washed with pbs and passed through a 100-m strainer (bd falcon). afterwards, cells were stained with anti - cd31-fitc (1:100, bd biosciences - pharmigen) at 4 c for 20 min and then permeabilized with cytofix / cytoperm (bd biosciences - pharmigen) at 4 c for 20 min. subsequently, cells were incubated with anti - bpifb4 (1:100 ; abcam) at 4 c for 1 h and then an allophycocyanin (apc)-conjugated anti - mouse secondary antibody (1:200 ; biolegend). for non - directly conjugated antibody to bpifb4, a staining mix without anti - bpifb4 antibody but with inclusion of the fluorescent secondary antibody was used as negative control. analysis of cell populations was performed using a facs canto ii equipped with facs diva software (bd biosciences) and the flowlogic (miltenyi biotec) analysis program. bpifb4 cdna (wt and lav isoforms) was cloned from prk5 expression plasmids into the lentiviral vector pcdh - ef1-msc - pa - pgk - cop - green fluorescence protein (gfp)-t2a - puro (system biosciences). lentiviral particles were generated by transfection of pcdh constructs along with the packaging vectors pmd2.vsv.g, prsv - rev, and pmdlg / prre (kindly provided by prof luigi naldini, san raffaele scientific institute, milan, italy) into human embryonic kidney (hek293 t) cells by calcium phosphate transfection. lentiviral particles were concentrated by ultracentrifugation (25 000 rpm for 4 h at 4 c) and stored at 80 c until immediately prior to use. lentivirus titration was performed by transducing hek293 t cells with concentrated particles in the presence of 4 g / ml polybrene and measuring gfp expression after 3 days by flow cytometry. for ca mobilization and confocal microscopy assay, human umbilical vein endothelial cells (huvecs) (lonza) were grown in complete egm2 medium (lonza) and infected with empty lentiviral vectors or particles encoding either wt- or lav - bpifb4 [at 5 multiplicity of infection (moi) ]. after 72 h, cells were selected with 2 g / ml puromycin for 48 h. hek293 t cells were grown in dulbecco s modified eagle s medium supplemented with 10% (v / v) fetal bovine serum and 1% non - essential amino acids at 37 c in a 5% co2 atmosphere. for co - immunoprecipitation, 1.4 10 cells were plated in 10-cm dishes and transfected with prk5 vector encoding lav - bpifb4 or with an empty plasmid, using lipofectamine 2000 (life technologies) according to the manufacturer s protocol. twenty - four hours post - transfection, hek293 t cells were incubated with g6976 (0.3 m) for another 24 h, harvested, and solubilized in lysis buffer (20 mm tris - hcl ph 7.5, 650 mm nacl, 500 mm edta, 250 mm egta, and triton x-100). lysates were cleared at 13 000 rpm for 20 min at 4 c, and 700 g protein incubated overnight with 2 g of mouse anti - gfp (invitrogen), mouse anti-14 - 3 - 3 (abcam), or mouse anti - igg (millipore) for the control. the antibody antigen complexes were precipitated with glinked sepharose protein (ge healthcare) for 4 h at 4 c and the beads washed three times with lysis buffer. the denatured co - immunoprecipitation products were resolved with sds - page, electro - blotted onto pvdf membranes, and hybridized with 1:1000 rabbit anti-14 - 3 - 3 (abcam). each sample was a pool of mesenteric arteries (length, 2 mm ; diameter, 250 m) excised from four mice. huvecs infected with lentiviral particles encoding wt- bpifb4, lav - bpifb4, or gfp (empty vector) were starved in serum- and growth factor - free egm-2 for 4 h and then stimulated with 100 m ach for 10 min. protein extracts were separated on 810% sds - page at 100 v for 1 h or on 412% sds - page at 100 v for 2 h and then transferred to a nitrocellulose or pvdf membrane as previously described in. western blots were analysed using imagej software (wayne rasband, national institutes of health, usa) to determine optical density (od) of the bands. the od readings of phosphorylated proteins are expressed as a ratio relative to total protein or to beta - actin. free intracellular ca concentration ([ca]i) recordings were obtained by time - resolved digital fluorescence microscopy on infected huvecs loaded with the ca indicator x - rhod-1 am (excitation, 550 nm ; emission, 610 nm) to avoid overlapping of fluorescence signals due to the presence of gfp. briefly, cells were incubated for 45 min at 37 c with 2 m of x - rhod-1 am. cells were then placed in standard mammalian ringer solution (in mm : nacl, 140 ; kcl, 2.5 ; cacl2, 2 ; mgcl2, 2 ; hepes - naoh, 10 ; and glucose, 10 ; ph 7.3), and continuously superfused with a gravity - driven fast perfusion system (biologique 100). most cells were infected and displayed clear gfp fluorescence that did not interfere with the fluorescent signal of the ca dye. the time courses of ca transients were quantified by measuring at each time point the fluorescence emission in the region of interest surrounding each cell, and then transforming the obtained values as follows : f / f = [f(t) f(0)]/f(0). for each cell, the amplitude of the atp - induced ca transient was evaluated as the difference between maximal and basal f / f values. huvecs infected with lentiviral particles encoding wt - bpifb4, lav - bpifb4, or gfp (empty vector) were fixed in 4% paraformaldehyde in pbs for 20 min, washed twice in 50 mm nh4cl in pbs, and permeabilized for 5 min in 0.2% triton x-100 in pbs. immunofluorescence analysis was performed on an inverted, motorized microscope (axio observer z.1) equipped with a 63x/1.4 plan - apochromat objective (carl zeiss). the attached laser - scanning unit (lsm 700 4x pigtailed laser 405 - 488 - 555 - 639, carl zeiss) enabled confocal imaging. fluorescence emission was revealed by a mbs (main dichroic beam splitter) and a vsd (variable secondary dichroic beam splitter). triple staining fluorescence images were acquired separately using zen 2012 software in the blue (hoechst 33258), green (egfp), and red (alexa fluor 594) channels at a resolution of 1024 1024 pixels, with the confocal pinhole set to one airy unit, and then saved in tiff format. vessel reactivity is given as mean s.e.m. and analysed by two - way anova. densitometry data were analysed with one - way anova followed by bonferroni posthoc analysis, as appropriate, using dedicated software (graphpad prism, v5.0). we previously reported that lav - bpifb4 enhances no - mediated vasorelaxation evoked by ach. ach - evoked vasodilation of isolated mesenteric vessels has been reported to require intact pkc activity. here, found that pkc was more phosphorylated at threonine 497an activation site of the enzyme in lav - bpifb4-overexpressing vessels than in those expressing the wt protein or only gfp (figure 1a). expression of lav - bpifb4 protein was detected through facs analysis in 79 4% cd31 endothelial cells (data not shown). to confirm that the mechanisms recruited by lav - bpifb4 take place in endothelial cells, we performed western blotting on huvecs infected with lentiviral vectors encoding gfp (empty), wt- bpifb4, or lav - bpifb4. also in this experimental setting, overexpression of lav - bpifb4 was associated with activation of pkc and enos (figure 1b). representative western blots of (a) ex vivo c57bl/6 mouse mesenteric arteries, (b) huvecs, and (c) vessels with (e+) or without (e) endothelium, transfected with an empty vector (e) or vectors for the expression of wt bpifb4 or lav - bpifb4. graphs on the right show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. n = 6 experiments for a ; n = 3 experiments for b and c. statistics was performed using one - way anova, following bonferroni s multiple comparison test. representative western blots of (a) ex vivo c57bl/6 mouse mesenteric arteries, (b) huvecs, and (c) vessels with (e+) or without (e) endothelium, transfected with an empty vector (e) or vectors for the expression of wt bpifb4 or lav - bpifb4. graphs on the right show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. n = 6 experiments for a ; n = 3 experiments for b and c. statistics was performed using one - way anova, following bonferroni s multiple comparison test. p < 0.05. to better characterize the role of the endothelial and smooth muscle layers, we performed experiments on endothelium - denuded vessels : the loss of endothelium was confirmed by the absence of enos upon western blotting (figure 1c) and by the absence of ach - evoked vasorelaxation in functional studies (data not shown). overexpression of lav - bpifb4 upregulated the phosphorylation of enos by about 2.5-fold and evoked the activation of pkc regardless of the presence or not of endothelium (figure 1c). of note, treatment with the pkc inhibitor g6976 significantly blunted ach - evoked vasorelaxation in control vessels (figure 2a) and abolished both endothelial vasorelaxation and enhanced enos phosphorylation in lav - bpifb4-expressing vessels (figure 2b and c). based on these results, we can assert that pkc is recruited by lav - bpifb4 to modulate enos and vascular tone. response curves to ach in ex vivo c57bl/6 mouse mesenteric arteries transfected with (a) an empty vector or (b) a vector for the expression of lav - bpifb4, with and without the pkc inhibitor g6976. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4.. statistics was performed using one - way anova, following bonferroni s multiple comparison test ; p < 0.05. e, empty vector ; lav, vector for the expression of lav - bpifb4 ; g6976, pkc inhibitor. inhibition of pkc abolishes the vascular effects of lav - bpifb4. response curves to ach in ex vivo c57bl/6 mouse mesenteric arteries transfected with (a) an empty vector or (b) a vector for the expression of lav - bpifb4, with and without the pkc inhibitor g6976. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. statistics was performed using one - way anova, following bonferroni s multiple comparison test ; p < 0.05. e, empty vector ; lav, vector for the expression of lav - bpifb4 ; g6976, pkc inhibitor. well - known ca - dependent processes we investigated how agonist - induced ca mobilization was influenced by the expression of the bpifb4 isoforms in huvecs. atp was used to elicit ca transients (figure 3a), so avoiding interaction of ach with the nicotinic receptors present on the huvecs. overexpression of the lav - bpifb4 isoform determined clear increases in the number of responsive cells (figure 3b) and the mean amplitude of ca transient upon stimulation (figure 3c). moreover, overexpression of lav - bpifb4 was associated with increased localization of pkc to the plasma membrane (figure 3d), a hallmark of its activation. the percentages of cells with membrane - localized pkc in each setting were : empty, 6.5% ; wt - bpifb4, 10% ; lav - bpifb4, 60%. figure 3overexpression of lav - bpifb4 sensitizes endothelial cells to agonist - induced ca mobilization, and the vascular effects do not require recruitment of mncs. (a) typical time - courses of [ca]i changes elicited by 100 m atp (horizontal bar, 2 min application) in huvecs overexpressing the wt- or lav - bpifb4 isoforms (average from 40 cells in individual optical fields). for the empty vector, a time - course averaged from 25 individual cells in a single optical field was shown. histograms of (b) the percentage of responding cells (n = 134, 113, and 129 cells, respectively) and (c) their mean ca transient amplitudes after atp application (empty, n = 3 independent experiments ; wt and lav, n = 5 independent experiments). pkc (red) was mainly cytosolic in huvecs infected with an empty vector (empty) and with a lentiviral vector encoding wt - bpifb4 ; in contrast, pkc was located mainly to the plasma membrane in huvecs overexpressing lav - bpifb4, a clear hallmark of pkc activation. arrows indicate regions of plasma membrane - localized pkc ; blue, hoechst - stained nuclei ; green, gfp expression. (e) western blot of ex vivo mouse mesenteric arteries from c57bl/6 mice treated with lps (20 mg / kg for 16 h) and of vessels from untreated c57bl/6 mice after transfection with empty vector (e) or overexpressing lav - bpifb4. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. overexpression of lav - bpifb4 sensitizes endothelial cells to agonist - induced ca mobilization, and the vascular effects do not require recruitment of mncs. (a) typical time - courses of [ca]i changes elicited by 100 m atp (horizontal bar, 2 min application) in huvecs overexpressing the wt- or lav - bpifb4 isoforms (average from 40 cells in individual optical fields). for the empty vector, a time - course averaged from 25 individual cells in a single optical field was shown. histograms of (b) the percentage of responding cells (n = 134, 113, and 129 cells, respectively) and (c) their mean ca transient amplitudes after atp application (empty, n = 3 independent experiments ; wt and lav, n = 5 independent experiments). pkc (red) was mainly cytosolic in huvecs infected with an empty vector (empty) and with a lentiviral vector encoding wt - bpifb4 ; in contrast, pkc was located mainly to the plasma membrane in huvecs overexpressing lav - bpifb4, a clear hallmark of pkc activation. arrows indicate regions of plasma membrane - localized pkc ; blue, hoechst - stained nuclei ; green, gfp expression. (e) western blot of ex vivo mouse mesenteric arteries from c57bl/6 mice treated with lps (20 mg / kg for 16 h) and of vessels from untreated c57bl/6 mice after transfection with empty vector (e) or overexpressing lav - bpifb4.. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. gap junctions allow exchange of ca ions between cells, and connexin-43 (cx43) plays a prominent role in this mechanism. we found increased expression of cx43 in vessels overexpressing lav - bpifb4 (figure 3e). based on this finding, we speculate that cx43 could be involved in the effects of lav - bpifb4 on ca mobilization. in previous work, we reported that mononuclear cells (mncs) from homozygous rs2070325 carriers (which express lav - bpifb4) have significantly upregulated enos activity vs. those from heterozygous and wt carriers. to exclude that this mechanism was responsible for the above findings, we assessed recruitment of mncs to vessels. evaluation of the mnc marker cd45 indicated that mncs were present in vessels treated with lipopolysaccharide (lps) (a well - known stimulus that induces mncs recruitment) but not in those overexpressing lav - bpifb4 (figure 3e). to clarify the role of ca in the vascular action of lav - bpifb4, we conducted vascular reactivity studies on mesenteric arteries in the absence of external ca. in the ca - free condition, lav - bpifb4 was hypo - phosphorylated and not able to enhance pkc and enos phosphorylation (figure 4a). (a) western blot of ex vivo c57bl/6 mouse mesenteric arteries transfected with lav - bpifb4 or empty (e) expression vectors, in the presence or absence (ca) of external ca. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4.. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. dose response curves to ach of mouse mesenteric arteries from ex vivo wt c57bl/6 mice (b, c) or from enos ko mice (d) transfected with empty vector (e) or with a vector for the expression of lav - bpifb4 in the absence of external ca ([ca ]) and in the absence or presence of the edhf inhibitors apa + ctx (100 nm each)., n = 11 experiments for b ; n = 8 experiments for c and d. statistics was performed using two - way anova ; p < 0.05 ; p < 0.01 ; p < 0.05 ; p < 0.01 vs. after lav + apa + ctx ; p < 0.05 ; p < 0.01 vs. before. (a) western blot of ex vivo c57bl/6 mouse mesenteric arteries transfected with lav - bpifb4 or empty (e) expression vectors, in the presence or absence (ca) of external ca. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4.. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. dose response curves to ach of mouse mesenteric arteries from ex vivo wt c57bl/6 mice (b, c) or from enos ko mice (d) transfected with empty vector (e) or with a vector for the expression of lav - bpifb4 in the absence of external ca ([ca ]) and in the absence or presence of the edhf inhibitors apa + ctx (100 nm each)., n = 11 experiments for b ; n = 8 experiments for c and d. statistics was performed using two - way anova ; p < 0.05 ; p < 0.01 ; p < 0.05 ; p < 0.01 vs. after lav + apa + ctx ; p < 0.05 ; p < 0.01 vs. before. in addition to no, endothelium generates other mediators involved in the regulation of vascular tone, among which is edhf. thus, we inhibited edhf release using apa which blocks atp - type ca - activated k channels and skca plus ctx which blocks voltage - gated ca - activated kchannels (kv1.3) and bkca channels. we found that when edhf release was inhibited in the absence of external ca, lav - bpifb4 failed to enhance endothelial vasorelaxation (figure 4c). taken together, these findings demonstrate that in the presence of external ca, lav - bpifb4 enhances endothelial function via a pkcenos - mediated mechanism, whereas in the absence of ca, lav - bpifb4 functions via an edhf - mediated pathway. this was supported by experiments performed on enos vessels : indeed, lav - bpifb4 was still able to enhance endothelial vasorelaxation in the absence of enos, but this effect was blunted in the presence of edhf inhibition (figure 4d). we have previously reported that phosphorylation of serine 75 in bpifb4 by perk which is enhanced in the presence of the lav isoform, induces binding to 14 - 3 - 3 and activation of enos. detailed amino acid sequencing analysis revealed that serine 75 is also within a potential phosphorylation substrate motif for pkc (amino acids 7375 : sxr / sir). thus, we hypothesized that pkc contributes to enos activation also through its phosphorylation of bpifb4. we first evaluated signalling in mesenteric vessels in which lav - bpifb4 could not be phosphorylated by perk (namely by overexpressing a protein mutated in the perk - phosphorylation site lav - bpifb4or by overexpressing the lav isoform in the presence of the perk inhibitor gsk2606414) or bound to 14 - 3 - 3 (by overexpressing a protein mutated in the 14 - 3 - 3-binding site lav - bpifb4). in these setting, bpifb4 was hypo - phosphorylated and enos was inactive, but pkc remained phosphorylated (figure 5a). when the phosphorylation of pkc was inhibited, lav - bpifb4 did not co - immunoprecipitate with 14 - 3 - 3 (figure 5b). indeed, as shown earlier, inhibition of pkc significantly reduced phosphorylation of lav - bpifb4 at serine 75 and phosphorylation of enos (figure 2c), indicating that pkc is needed for the activation of lav - bpifb4 and enos. figure 5activation of pkc is independent of phosphorylation of bpifb4 by perk at serine 75. (a) western blot of ex vivo mouse mesenteric arteries overexpressing lav - bpifb4, lav - bpifb4 (ser75ala variation), lav - bpifb4 (ser82asn variation), or lav - bpifb4 plus gsk2606414 (a perk inhibitor). on the right, graphs of quantification of p - enos (s1177), p - pkc - alpha (t497), p - bpifb4, and bpifb4.. statistics was performed using one - way anova following bonferroni s multiple comparison test ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. or lav - bpifb4 plus gsk2606414. (b) co - immunoprecipitation of bpifb4 and 14 - 3 - 3 in hek293 t cells overexpressing lav - bpifb4 tagged with gfp protein and treated with the pkc inhibitor g6976. immunoprecipitation was performed with anti - gfp (directed toward lav - bpifb4-gfp), anti-14 - 3 - 3, and anti - igg (as negative control) antibodies followed by immunoblotting with anti-14 - 3 - 3 (n = 2 independent experiments). (c) dose response curves for ach of ex vivo c57bl/6 mouse mesenteric arteries transfected with empty vector (e) or with lav - bpifb4 in the presence (+) or absence of an akt inhibitor. (a) western blot of ex vivo mouse mesenteric arteries overexpressing lav - bpifb4, lav - bpifb4 (ser75ala variation), lav - bpifb4 (ser82asn variation), or lav - bpifb4 plus gsk2606414 (a perk inhibitor). on the right, graphs of quantification of p - enos (s1177), p - pkc - alpha (t497), p - bpifb4, and bpifb4.. statistics was performed using one - way anova following bonferroni s multiple comparison test ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. or lav - bpifb4 plus gsk2606414. (b) co - immunoprecipitation of bpifb4 and 14 - 3 - 3 in hek293 t cells overexpressing lav - bpifb4 tagged with gfp protein and treated with the pkc inhibitor g6976. immunoprecipitation was performed with anti - gfp (directed toward lav - bpifb4-gfp), anti-14 - 3 - 3, and anti - igg (as negative control) antibodies followed by immunoblotting with anti-14 - 3 - 3 (n = 2 independent experiments). (c) dose response curves for ach of ex vivo c57bl/6 mouse mesenteric arteries transfected with empty vector (e) or with lav - bpifb4 in the presence (+) or absence of an akt inhibitor. statistics was performed using two - way anova ; p < 0.05. because akt signalling is one on the most important pathways modulating enos function, we performed experiments in the presence of akt inhibition. in this experimental condition, vessels transfected with empty vector had significantly impaired ach - evoked vasorelaxation, whereas those overexpressing lav - bpifb4 were still able to enhance ach vasorelaxation (figure 5c). these results clearly demonstrate that the vascular effects mediated by lav - bpifb4 are independent of akt signalling. we previously reported that lav - bpifb4 enhances no - mediated vasorelaxation evoked by ach. ach - evoked vasodilation of isolated mesenteric vessels has been reported to require intact pkc activity. here, found that pkc was more phosphorylated at threonine 497an activation site of the enzyme in lav - bpifb4-overexpressing vessels than in those expressing the wt protein or only gfp (figure 1a). expression of lav - bpifb4 protein was detected through facs analysis in 79 4% cd31 endothelial cells (data not shown). to confirm that the mechanisms recruited by lav - bpifb4 take place in endothelial cells, we performed western blotting on huvecs infected with lentiviral vectors encoding gfp (empty), wt- bpifb4, or lav - bpifb4. also in this experimental setting, overexpression of lav - bpifb4 was associated with activation of pkc and enos (figure 1b). representative western blots of (a) ex vivo c57bl/6 mouse mesenteric arteries, (b) huvecs, and (c) vessels with (e+) or without (e) endothelium, transfected with an empty vector (e) or vectors for the expression of wt bpifb4 or lav - bpifb4. graphs on the right show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. n = 6 experiments for a ; n = 3 experiments for b and c. statistics was performed using one - way anova, following bonferroni s multiple comparison test. representative western blots of (a) ex vivo c57bl/6 mouse mesenteric arteries, (b) huvecs, and (c) vessels with (e+) or without (e) endothelium, transfected with an empty vector (e) or vectors for the expression of wt bpifb4 or lav - bpifb4. graphs on the right show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. n = 6 experiments for a ; n = 3 experiments for b and c. statistics was performed using one - way anova, following bonferroni s multiple comparison test. p < 0.05. to better characterize the role of the endothelial and smooth muscle layers, we performed experiments on endothelium - denuded vessels : the loss of endothelium was confirmed by the absence of enos upon western blotting (figure 1c) and by the absence of ach - evoked vasorelaxation in functional studies (data not shown). overexpression of lav - bpifb4 upregulated the phosphorylation of enos by about 2.5-fold and evoked the activation of pkc regardless of the presence or not of endothelium (figure 1c). of note, treatment with the pkc inhibitor g6976 significantly blunted ach - evoked vasorelaxation in control vessels (figure 2a) and abolished both endothelial vasorelaxation and enhanced enos phosphorylation in lav - bpifb4-expressing vessels (figure 2b and c). based on these results, we can assert that pkc is recruited by lav - bpifb4 to modulate enos and vascular tone. response curves to ach in ex vivo c57bl/6 mouse mesenteric arteries transfected with (a) an empty vector or (b) a vector for the expression of lav - bpifb4, with and without the pkc inhibitor g6976. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4.. statistics was performed using one - way anova, following bonferroni s multiple comparison test ; p < 0.05. e, empty vector ; lav, vector for the expression of lav - bpifb4 ; g6976, pkc inhibitor. inhibition of pkc abolishes the vascular effects of lav - bpifb4. response curves to ach in ex vivo c57bl/6 mouse mesenteric arteries transfected with (a) an empty vector or (b) a vector for the expression of lav - bpifb4, with and without the pkc inhibitor g6976. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. statistics was performed using one - way anova, following bonferroni s multiple comparison test ; p < 0.05. e, empty vector ; lav, vector for the expression of lav - bpifb4 ; g6976, pkc inhibitor. well - known ca - dependent processes we investigated how agonist - induced ca mobilization was influenced by the expression of the bpifb4 isoforms in huvecs. atp was used to elicit ca transients (figure 3a), so avoiding interaction of ach with the nicotinic receptors present on the huvecs. overexpression of the lav - bpifb4 isoform determined clear increases in the number of responsive cells (figure 3b) and the mean amplitude of ca transient upon stimulation (figure 3c). moreover, overexpression of lav - bpifb4 was associated with increased localization of pkc to the plasma membrane (figure 3d), a hallmark of its activation. the percentages of cells with membrane - localized pkc in each setting were : empty, 6.5% ; wt - bpifb4, 10% ; lav - bpifb4, 60%. figure 3overexpression of lav - bpifb4 sensitizes endothelial cells to agonist - induced ca mobilization, and the vascular effects do not require recruitment of mncs. (a) typical time - courses of [ca]i changes elicited by 100 m atp (horizontal bar, 2 min application) in huvecs overexpressing the wt- or lav - bpifb4 isoforms (average from 40 cells in individual optical fields). for the empty vector, a time - course averaged from 25 individual cells in a single optical field was shown. histograms of (b) the percentage of responding cells (n = 134, 113, and 129 cells, respectively) and (c) their mean ca transient amplitudes after atp application (empty, n = 3 independent experiments ; wt and lav, n = 5 independent experiments). pkc (red) was mainly cytosolic in huvecs infected with an empty vector (empty) and with a lentiviral vector encoding wt - bpifb4 ; in contrast, pkc was located mainly to the plasma membrane in huvecs overexpressing lav - bpifb4, a clear hallmark of pkc activation. arrows indicate regions of plasma membrane - localized pkc ; blue, hoechst - stained nuclei ; green, gfp expression. (e) western blot of ex vivo mouse mesenteric arteries from c57bl/6 mice treated with lps (20 mg / kg for 16 h) and of vessels from untreated c57bl/6 mice after transfection with empty vector (e) or overexpressing lav - bpifb4.. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. overexpression of lav - bpifb4 sensitizes endothelial cells to agonist - induced ca mobilization, and the vascular effects do not require recruitment of mncs. (a) typical time - courses of [ca]i changes elicited by 100 m atp (horizontal bar, 2 min application) in huvecs overexpressing the wt- or lav - bpifb4 isoforms (average from 40 cells in individual optical fields). for the empty vector, a time - course averaged from 25 individual cells in a single optical field was shown. histograms of (b) the percentage of responding cells (n = 134, 113, and 129 cells, respectively) and (c) their mean ca transient amplitudes after atp application (empty, n = 3 independent experiments ; wt and lav, n = 5 independent experiments). pkc (red) was mainly cytosolic in huvecs infected with an empty vector (empty) and with a lentiviral vector encoding wt - bpifb4 ; in contrast, pkc was located mainly to the plasma membrane in huvecs overexpressing lav - bpifb4, a clear hallmark of pkc activation. arrows indicate regions of plasma membrane - localized pkc ; blue, hoechst - stained nuclei ; green, gfp expression. scale bar = 10 m. (e) western blot of ex vivo mouse mesenteric arteries from c57bl/6 mice treated with lps (20 mg / kg for 16 h) and of vessels from untreated c57bl/6 mice after transfection with empty vector (e) or overexpressing lav - bpifb4. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. gap junctions allow exchange of ca ions between cells, and connexin-43 (cx43) plays a prominent role in this mechanism. we found increased expression of cx43 in vessels overexpressing lav - bpifb4 (figure 3e). based on this finding, we speculate that cx43 could be involved in the effects of lav - bpifb4 on ca mobilization. in previous work, we reported that mononuclear cells (mncs) from homozygous rs2070325 carriers (which express lav - bpifb4) have significantly upregulated enos activity vs. those from heterozygous and wt carriers. to exclude that this mechanism was responsible for the above findings, we assessed recruitment of mncs to vessels. evaluation of the mnc marker cd45 indicated that mncs were present in vessels treated with lipopolysaccharide (lps) (a well - known stimulus that induces mncs recruitment) but not in those overexpressing lav - bpifb4 (figure 3e). ach - evoked enos phosphorylation requires influx of ca. to clarify the role of ca in the vascular action of lav - bpifb4, we conducted vascular reactivity studies on mesenteric arteries in the absence of external ca. in the ca - free condition, lav - bpifb4 was hypo - phosphorylated and not able to enhance pkc and enos phosphorylation (figure 4a). however, endothelial vasorelaxation was still enhanced (figure 4b). (a) western blot of ex vivo c57bl/6 mouse mesenteric arteries transfected with lav - bpifb4 or empty (e) expression vectors, in the presence or absence (ca) of external ca. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4.. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. response curves to ach of mouse mesenteric arteries from ex vivo wt c57bl/6 mice (b, c) or from enos ko mice (d) transfected with empty vector (e) or with a vector for the expression of lav - bpifb4 in the absence of external ca ([ca ]) and in the absence or presence of the edhf inhibitors apa + ctx (100 nm each)., n = 11 experiments for b ; n = 8 experiments for c and d. statistics was performed using two - way anova ; p < 0.05 ; p < 0.01 ; p < 0.05 ; p < 0.01 vs. after lav + apa + ctx ; p < 0.05 ; p < 0.01 vs. before (a) western blot of ex vivo c57bl/6 mouse mesenteric arteries transfected with lav - bpifb4 or empty (e) expression vectors, in the presence or absence (ca) of external ca. right graphs show quantification of p - enos (s1177), p - pkc (t497), p - bpifb4 (s75), and bpifb4. statistics was performed using one way anova, following bonferroni s multiple comparison test ; p < 0.05. dose response curves to ach of mouse mesenteric arteries from ex vivo wt c57bl/6 mice (b, c) or from enos ko mice (d) transfected with empty vector (e) or with a vector for the expression of lav - bpifb4 in the absence of external ca ([ca ]) and in the absence or presence of the edhf inhibitors apa + ctx (100 nm each)., n = 11 experiments for b ; n = 8 experiments for c and d. statistics was performed using two - way anova ; p < 0.05 ; p < 0.01 ; p < 0.05 ; p < 0.01 vs. after lav + apa + ctx ; p < 0.05 ; p < 0.01 vs. before. in addition to no, endothelium generates other mediators involved in the regulation of vascular tone, among which is edhf. thus, we inhibited edhf release using apa which blocks atp - type ca - activated k channels and skca plus ctx which blocks voltage - gated ca - activated kchannels (kv1.3) and bkca channels. we found that when edhf release was inhibited in the absence of external ca, lav - bpifb4 failed to enhance endothelial vasorelaxation (figure 4c). taken together, these findings demonstrate that in the presence of external ca, lav - bpifb4 enhances endothelial function via a pkcenos - mediated mechanism, whereas in the absence of ca, lav - bpifb4 functions via an edhf - mediated pathway. this was supported by experiments performed on enos vessels : indeed, lav - bpifb4 was still able to enhance endothelial vasorelaxation in the absence of enos, but this effect was blunted in the presence of edhf inhibition (figure 4d). we have previously reported that phosphorylation of serine 75 in bpifb4 by perk which is enhanced in the presence of the lav isoform, induces binding to 14 - 3 - 3 and activation of enos. detailed amino acid sequencing analysis revealed that serine 75 is also within a potential phosphorylation substrate motif for pkc (amino acids 7375 : sxr / sir). thus, we hypothesized that pkc contributes to enos activation also through its phosphorylation of bpifb4. we first evaluated signalling in mesenteric vessels in which lav - bpifb4 could not be phosphorylated by perk (namely by overexpressing a protein mutated in the perk - phosphorylation site lav - bpifb4or by overexpressing the lav isoform in the presence of the perk inhibitor gsk2606414) or bound to 14 - 3 - 3 (by overexpressing a protein mutated in the 14 - 3 - 3-binding site lav - bpifb4). in these setting, bpifb4 was hypo - phosphorylated and enos was inactive, but pkc remained phosphorylated (figure 5a). when the phosphorylation of pkc was inhibited, lav - bpifb4 did not co - immunoprecipitate with 14 - 3 - 3 (figure 5b). indeed, as shown earlier, inhibition of pkc significantly reduced phosphorylation of lav - bpifb4 at serine 75 and phosphorylation of enos (figure 2c), indicating that pkc is needed for the activation of lav - bpifb4 and enos. figure 5activation of pkc is independent of phosphorylation of bpifb4 by perk at serine 75. (a) western blot of ex vivo mouse mesenteric arteries overexpressing lav - bpifb4, lav - bpifb4 (ser75ala variation), lav - bpifb4 (ser82asn variation), or lav - bpifb4 plus gsk2606414 (a perk inhibitor). on the right, graphs of quantification of p - enos (s1177), p - pkc - alpha (t497), p - bpifb4, and bpifb4. statistics was performed using one - way anova following bonferroni s multiple comparison test ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. or lav - bpifb4 plus gsk2606414. (b) co - immunoprecipitation of bpifb4 and 14 - 3 - 3 in hek293 t cells overexpressing lav - bpifb4 tagged with gfp protein and treated with the pkc inhibitor g6976. immunoprecipitation was performed with anti - gfp (directed toward lav - bpifb4-gfp), anti-14 - 3 - 3, and anti - igg (as negative control) antibodies followed by immunoblotting with anti-14 - 3 - 3 (n = 2 independent experiments). (c) dose response curves for ach of ex vivo c57bl/6 mouse mesenteric arteries transfected with empty vector (e) or with lav - bpifb4 in the presence (+) or absence of an akt inhibitor. (a) western blot of ex vivo mouse mesenteric arteries overexpressing lav - bpifb4, lav - bpifb4 (ser75ala variation), lav - bpifb4 (ser82asn variation), or lav - bpifb4 plus gsk2606414 (a perk inhibitor). on the right, graphs of quantification of p - enos (s1177), p - pkc - alpha (t497), p - bpifb4, and bpifb4. statistics was performed using one - way anova following bonferroni s multiple comparison test ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. lav - bpifb4 ; p < 0.001 vs. or lav - bpifb4 plus gsk2606414. (b) co - immunoprecipitation of bpifb4 and 14 - 3 - 3 in hek293 t cells overexpressing lav - bpifb4 tagged with gfp protein and treated with the pkc inhibitor g6976. immunoprecipitation was performed with anti - gfp (directed toward lav - bpifb4-gfp), anti-14 - 3 - 3, and anti - igg (as negative control) antibodies followed by immunoblotting with anti-14 - 3 - 3 (n = 2 independent experiments). (c) dose response curves for ach of ex vivo c57bl/6 mouse mesenteric arteries transfected with empty vector (e) or with lav - bpifb4 in the presence (+) or absence of an akt inhibitor.. statistics was performed using two - way anova ; p < 0.05. because akt signalling is one on the most important pathways modulating enos function, we performed experiments in the presence of akt inhibition. in this experimental condition, vessels transfected with empty vector had significantly impaired ach - evoked vasorelaxation, whereas those overexpressing lav - bpifb4 were still able to enhance ach vasorelaxation (figure 5c). these results clearly demonstrate that the vascular effects mediated by lav - bpifb4 are independent of akt signalling. the main finding of this study is that the enhanced ability of the lav isoform of bpifb4 to stimulate no production in the endothelium is due to activation of pkc signalling. indeed, the overexpression of lav - bpifb4 in huvecs augmented ca mobilization, increasing translocation of pkc to the plasma membrane, a step necessary for the activation of the kinase. in the absence of external ca, the ability of lav - bpifb4 to enhance both pkc and enos phosphorylation was abolished. we demonstrated previously that phosphorylation of bpifb4 at serine 75 by perk and the binding to 14 - 3 - 3 protein are fundamental for activation of enos and endothelial function. so, to further clarify the mechanisms elicited by the lav isoform, we investigated potential players, focusing our attention on pkc, a major pkc family member expressed in endothelial cells and involved in regulating enos function. we found that inhibition of pkc impeded the enhancing effects of lav - bpifb4 on enos and endothelial function, positioning this kinase between bpifb4 and enos. of note, mutation of lav - bpifb4 at its perk - phosphorylated site or at its 14 - 3 - 3-binding site blunted enos activation but did not interfere with the ability to activate pkc. these findings indicate that the activation of pkc is independent of ser75 phosphorylation and binding to 14 - 3 - 3. we also found that serine 75 is within a pkc phosphorylation motif (amino acids 7375 : sxr / sir), indicating that the kinase works also upstream of bpifb4. indeed, when we inhibited pkc, the site serine 75 became hypo - phosphorylated and bpifb4 did not co - immunoprecipitate with 14 - 3 - 3. all these findings suggest a mechanism whereby the stimulation of ca influx by lav - bpifb4 leads to the activation pkc, which in turn increases phosphorylation of bpifb4 at serine 75 ; this hyper - phosphorylation results in enhanced binding of lav - bpifb4 to 14 - 3 - 3 and hsp90, allowing enos to binding with the complex and become phosphorylated by pkc (figure 6). mechanism recruited by lav - bpifb4 to modulate enos function in endothelial cells (up). lav - bpifb4 enhances ca influx, leading to pkc activation and consequential phosphorylation of lav - bpifb4 at serine 75. this hyper - phosphorylation results in enhanced binding of lav - bpifb4 to 14 - 3 - 3 and hsp90, steps necessary for interaction of the complex with enos and its phosphorylation by pkc. alternatively, in the absence of external ca or functional enos, lav - bpifb4 can still enhance vasorelaxation by stimulating edhf signalling. possible involvement of lav - bpifb4 in the activation of pkc in smooth muscle cells (bottom). bpifb4 val-229, lav - bpifb4 ; pkc, protein kinase c - alpha ; hsp90, heat shock protein 90 ; enos, endothelial nitric oxide synthase ; no, nitric oxide ; edhf, endothelium - derived hyperpolarizing factors ; ca, calcium ion. mechanism recruited by lav - bpifb4 to modulate enos function in endothelial cells (up). lav - bpifb4 enhances ca influx, leading to pkc activation and consequential phosphorylation of lav - bpifb4 at serine 75. this hyper - phosphorylation results in enhanced binding of lav - bpifb4 to 14 - 3 - 3 and hsp90, steps necessary for interaction of the complex with enos and its phosphorylation by pkc. alternatively, in the absence of external ca or functional enos, lav - bpifb4 can still enhance vasorelaxation by stimulating edhf signalling. possible involvement of lav - bpifb4 in the activation of pkc in smooth muscle cells (bottom). bpifb4 val-229, lav - bpifb4 ; pkc, protein kinase c - alpha ; hsp90, heat shock protein 90 ; enos, endothelial nitric oxide synthase ; no, nitric oxide ; edhf, endothelium - derived hyperpolarizing factors ; ca, calcium ion. in addition, careful analysis of our data revealed that in the absence of external ca, lav - bpifb4 still enhanced endothelial vasorelaxation despite blunted phosphorylation of pkc and enos. we found that this effect was dependent upon the recruitment of edhf, which is known can substitute for no in settings where enos is dysfunctional. to definitively clarify the concept that lav - bpifb4 is able to recruit alternative mechanisms protecting endothelial function in the absence of enos, we performed experiments on vessels from enos deficient mice. also in this experimental setting, the expression of lav - bpifb4 was associated with enhanced endothelial vasorelaxation. more studies are needed to clarify the mechanism through which lav - bpifb4 mediates edhf release. our findings highlight that ca mobilization is a key signal necessary for lav - bpifb4 to enhance endothelial no release. the agonist - induced ca entry observed in huvecs is an example of capacitive ca influx triggered by metabotropic receptors. lav - bpifb4 is not able to mobilize ca itself, but its expression strongly increases the amplitude of agonist - evoked ca signals, likely by interacting with one or more elements of the molecular machinery regulating [ca]i. these findings also indicate a possible broader role of lav - bpifb4 in modulating other functions where pkc is involved, such as control of stem cell maintenance, development, and differentiation, functions that are lost during ageing and that could be preserved in llis. finally, our study defines the effects of lav - bpifb4 on the modulation of enos function in endothelial cells ; we can not exclude a contribution by smooth muscle cells in enos activation. the lav of bpifb4 has tremendous potential for exploitation as an important tool for the treatment of ageing - related diseases because of its effect on no metabolism. moreover, the effect of lav - bpifb4 on edhf release opens up new scenarios for enhancing endothelial function via therapeutic targets other than enos. this work was supported by italian ministry of university and research (miur / firb automed. l. milanesi is supported by flagship interomics pb05 and a.ferrario is a fellow of this project. | aimsageing is associated with impairment of endothelial nitric oxide synthase (enos) and progressive reduction in endothelial function. a genetic study on long - living individuals who are characterized by delays in ageing and in the onset of cardiovascular disease previously revealed i229v (rs2070325) in bactericidal / permeability - increasing fold - containing - family - b - member-4 (bpifb4) as a longevity - associated variant (lav) ; the lav protein enhanced endothelial no production and vasorelaxation through a protein kinase r like endoplasmic reticulum kinase/14 - 3 - 3/heat shock protein 90 signal. here, we further characterize the molecular mechanisms underlying lav - bpifb4-dependent enhancement of vascular function.methods and resultslav - bpifb4 upregulated enos function via mobilization of ca2 + and activation of protein kinase c alpha (pkc). indeed, the overexpression of lav - bpifb4 in human endothelial cells enhanced atp - induced ca2 + mobilization and the translocation of pkc to the plasma membrane. coherently, pharmacological inhibition of pkc blunted the positive effect of lav - bpifb4 on enos and endothelial function. in addition, although lav - bpifb4 lost the ability to activate pkc and enos in ex vivo vessels studied in an external ca2 + -free medium and in vessels from enos/ mice, it still potentiated endothelial activity, recruiting an alternative mechanism dependent upon endothelium - derived hyperpolarizing factor (edhf).conclusionswe have identified novel molecular determinants of the beneficial effects of lav - bpifb4 on endothelial function, showing the roles of ca2 + mobilization and pkc in enos activation and of edhf when enos is inhibited. these results highlight the role lav - bpifb4 can have in restoring signals that are lost during ageing. |
intraocular pressure (iop) is a major risk factor for the development of glaucoma. it is generally believed that postural changes have a greater effect on the iop in glaucomatous eyes than in normal eyes [3, 68 ]. in addition to the supine position being an important contributor to the iop elevation observed at night, this position has also been shown to be associated with the progression of open - angle glaucoma [6, 9, 10 ]. thus, when treating glaucoma patients, it is very important to be able to ascertain the iop changes that occur due to postural changes. however, timolol maleate, latanoprost, and brinzolamide do not decrease the magnitude of the iop elevation associated with postural change. trabeculectomy is the recommended surgical therapy for eyes with glaucoma, as this procedure leads to a greater iop reduction than other therapeutic interventions. furthermore, it has been reported that eyes that have undergone trabeculectomy have less iop fluctuation during the diurnal iop changes than medically treated glaucoma eyes [12, 13 ]. some investigators have additionally reported that trabeculectomy not only decreases the iop but also reduces the degree of the posture - induced iop changes [1416 ]. however, when the bleb function fails, it has been shown that the postural iop changes return to baseline levels. the purpose of the current study was to investigate the correlation between the sitting iop and the degree of postural iop changes in relation to the time course after trabeculectomy. between august 2011 and august 2012, this prospective, consecutive series examined 30 eyes of 30 open - angle glaucoma patients who were treated with trabeculectomy and followed up at kagawa university hospital, kagawa, japan. males and females, aged 50 to 87 years, were eligible for this study. the study group consisted of 21 primary open - angle glaucoma (poag) and 3 normal - tension glaucoma (ntg) patients, with each patient treated with a topical hypotensive agent in both eyes. all patients underwent visual acuity, refraction, central and peripheral field, slit - lamp, and gonioscopy examinations. glaucomatous eyes were defined as eyes exhibiting structural glaucomatous changes (vertical cup - disc asymmetry between fellow eyes of 0.2, a cup - to - disc ratio of 0.6, and neuroretinal rim narrowing, notches, localized pallor, or retinal nerve fiber layer defects with glaucomatous visual field (vf) loss in the corresponding hemifield). a glaucomatous vf was defined as a glaucoma hemifield test outside normal limits on at least two consecutive baseline tests and the presence of at least three contiguous test points within the same hemifield on the pattern deviation plot at p < 1%, with at least one at p < 0.5% when excluding points on the edge of the field or those directly above and below the blind spot. patients with an iop 22 mmhg were diagnosed as poag while ntg was diagnosed when the untreated peak iop was 21 mmhg, which included 24-hour fluctuations. all patients signed an informed consent form in accordance with the principles embodied in the declaration of helsinki. this study was approved by the institutional review board of the kagawa university hospital. throughout the experimental period, a single examiner used the icare rebound tonometer (icare ; tiolat oy, helsinki, finland) to measure the iop in the morning for each of the patients before the trabeculectomy and at 1, 2, 3, 6, and 12 months and then every 6 months postoperatively. during the actual measurements, each subject was asked to sit comfortably at the edge of an examining bed in a quiet room. all iops were measured with the icare rebound tonometer while the patients remained in a sitting position. after this reading, patients were asked to lie on a bed and then turn to a lateral decubitus position with their heads placed on a soft pillow. the body was positioned so that the eye scheduled for the surgery was located directly above the other eye. after the examiner asked the patients to gaze straight ahead and look at a fixation point, the iop measurement was made by touching the transducer to the center of the patients ' cornea. the average of each set was automatically calculated, with the averaged values used for the statistical analysis. patient visits tended to be more frequent during the early postoperative period. at each visit, all patients underwent a standard ophthalmologic examination that included slit - lamp, goldmann applanation tonometry, and binocular fundus examinations. to maintain good control of the iop, laser suture lysis (performed within 1 month after trabeculectomy) statistical analyses were performed using spss version 19.0 (ibm, new york, ny). iop differences before and after the trabeculectomy were compared using a paired t - test. iop differences between the operated eye and the nonoperated other eye were evaluated by an unpaired t - test. out of the 30 subjects examined, 29 eyes of 29 patients completed the protocol. the one patient who did not complete the study developed serious hypotonic maculopathy at 3 months after the filtering surgery. figure 1 shows the correlation between the goldmann applanation tonometer and icare on subjects in the sitting position at baseline. both were in close agreement with a correlation coefficient of 0.86 (p < 0.001). at baseline, the mean iop in the sitting position was 16.7 5.5 mmhg (range ; 930 mmhg) (figure 2). after assuming the lateral decubitus position, the iop increased in all patients. prior to surgery, the iop in the lateral decubitus position was significantly higher than that in the sitting position (p < 0.001). the mean change in the iop between the two body positions was 3.0 1.6 mmhg. one month after trabeculectomy, there were significant reductions of the iop in the sitting position to 7.8 3.2 mmhg (range ; 215 mmhg) and in the lateral decubitus position to 8.6 3.9 mmhg (range ; 318 mmhg) (figure 2). the difference in the iop between the sitting and lateral decubitus positions was statistically significant (p < 0.001 ; paired t - test). postoperative changes in the iop for the sitting and lateral decubitus positions were 7.8 3.3 mmhg and 8.8 4.1 mmhg at 2 months (p < 0.001 ; paired t - test), 8.1 3.6 mmhg and 9.4 5.3 mmhg at 3 months (p = 0.002 ; paired t - test), 7.7 2.8 mmhg and 9.0 3.6 mmhg at 6 months (p < 0.001 ; paired t - test), 8.9 3.2 mmhg and 10.3 4.1 mmhg at 12 months (p < 0.001 ; paired t - test), and 8.3 2.8 mmhg and 9.1 3.6 mmhg at 18 months (p = 0.03 ; paired t - test), respectively. the degree of reduction of the posture - induced iops relative to the difference before the surgery was significant at 1, 2, 3, 6, 12, and 18 months postoperatively (p < 0.001, p < 0.001, p = 0.001, p < 0.001, p = 0.002, and p = 0.001, resp. ; bonferroni test). needle revision was required in seven cases (more than 15 mmhg in the sitting position). in 22 patients who did not require needle revisions at every visit, table 2 shows both the sitting position iops and the postural iop changes in the patients who required needle revision. all of these patients initially showed increased posture - induced iop changes (greater than 3 mmhg), after which increased iops were then observed in the sitting position. the mean change in the iop between the two body positions was 0.9 0.6 mmhg (range ; 02 mmhg) in the success group and 5.0 1.2 mmhg (range ; 47 mmhg) in the failure group. the difference in the change in the mean postural - induced iop between the two groups was statistically significant (p < 0.001, unpaired t - test). the results of this study confirm earlier reports [1416 ] that trabeculectomy not only reduces the iop in the sitting position but also reduces the degree of the posture - induced changes in the iop. the main finding of our current study was that the measurement of postural iop changes might be a beneficial method for assessing whether a filtering bleb is functioning. there has been a lot of speculation on the potential mechanism responsible for the postural iop rise. although some investigators have hypothesized that it is due to choroidal vascular congestion and increased episcleral venous pressure [17, 18 ], it has also been suggested that it might be unrelated to aqueous production. anderson and grant and parsley. reported that the iop changes that occurred when moving from a sitting to supine position were greater following glaucoma surgery than in nonoperated, medically treated glaucomatous eyes. however, we have recently reported that after trabeculectomy there was a decrease not only for the iop in the sitting position but also for the magnitude of the iop elevation associated with the postural change.. found that the posture - induced iop change was significantly lower in trabeculectomized eyes than in the nonoperated contralateral eyes. sawada and yamamoto showed that trabeculectomy not only decreased the iop but also reduced the degree of the posture - induced iop changes if patients had a cystic filtering bleb. however, they also found that if the bleb function failed, the postural iop changes returned to baseline levels. some investigators have suggested that the posture - induced iop decrease after trabeculectomy might mainly be due to the ability of the trabeculectomy to lower the iop [15, 21 ]. however, sawada and yamamoto recently reported that eyes that underwent successful trabeculectomy had smaller posture - induced iop changes as compared to nonoperated, medically treated eyes, with the iop in both groups found to be 12 mmhg or less. trabeculectomy creates a new aqueous pathway via the filtering bleb that is independent of the episcleral veins. therefore, it seems reasonable that trabeculectomy could cause a suppression of the posture - induced iop alternations. prior to surgery, the mean change in the iop between the two body positions was 3.0 1.6 mmhg. the most important finding in our current study was that there was an increase in the posture - induced iop prior to the increase in the iop in the sitting position. posture - induced iop changes have been shown to rapidly occur within 5 minutes after a position change [23, 24 ]. when the filtering bleb function is working, posture - induced iop changes are suppressed within 5 minutes after changing the position. however, when the filtering bleb function worsens, it appears that more time is required (at least more than 5 minutes) in order to reduce or fail to reduce the posture - induced iop changes, even when the iop in the sitting position is still low enough. furthermore, when there is continued worsening of the filtering bleb function, not only posture - induced iop but also the iop in the sitting position increases. although the use of 5-fluorouracil (5-fu) and mitomycin c has been shown to improve the outcome of filtration surgeries, additional treatments are commonly required in order to manage failed filtration blebs. several reports have shown needle revision to be an effective and relatively simple method for reestablishing filtration in eyes with failed filtration blebs [2527 ]. however, failure of the initial 5-fu needling revision has been shown to be significantly correlated with higher preneedling iop [26, 28 ]. thus, it has been recommended that the needle revision be performed as soon as there is an increase in the iop or detection of bleb flattening. if it were possible to predict a failing filtering bleb before the iop begins to increase, then simple monitoring of patients followed by a quick reaction to any changes in the iop after the surgical procedure could lead to a greatly improved success rate for needle revision. the limitations of our present work include having only a short follow - up period and a small sample size. a second limitation was that we did not evaluate the inner structure of the filtering bleb using ultrasound biomicroscopy or anterior segment optical coherence tomography. in conclusion, assessment of postural iop changes after trabeculectomy might be potentially useful in predicting iop changes in the sitting position after trabeculectomy. however, further long - term followups and large - scale investigations will need to be undertaken in order to definitively clarify this issue. | background. to investigate the correlation between intraocular pressure (iop) fluctuation with postural change and iop in relation to the time course after trabeculectomy. methods. a total of 29 patients who had previously undergone primary trabeculectomy with mitomycin c were examined. iop was obtained at 1, 2, 3, 6, and 12 months and then every 6 months postoperatively. results. the postural iop difference before surgery was 3.0 1.8 mmhg, which was reduced to 0.9 1.1 mmhg at 1 month, 1.0 1.0 mmhg at 2 months, 1.3 2.0 mmhg at 3 months, 1.3 1.4 mmhg at 6 months, 1.4 1.5 mmhg at 12 months, and 1.1 0.7 mmhg at 18 months after trabeculectomy (p < 0.01 each visit). the filtering surgery failed in 7 out of 29 eyes. postural iop changes were less than 3 mmhg in those patients who did not require needle revision at every visit. however, in patients who did require needle revision, the increase in the posture - induced iop was greater than 3 mmhg prior to the increase in the sitting position iop. conclusions. assessment of postural iop changes after trabeculectomy might be potentially useful for predicting iop changes after trabeculectomy. |
cervical cancer is the second most frequent cancer in women in the world, comprising about 10% of all human cancers. a current summary report from who indicates that there are about 500 thousand women diagnosed with cervical cancer, of whom about 270 thousand women die from the disease every year. in china, a total of 500 million women are at risk of cervical cancer. after more than 30 years of research into the infectious nature of cervical cancer, it is well established that human papillomaviruses (hpvs) are the primary cause for cervical cancer. up till now, over 130 different types of hpv have been found, where hpv-16 and hpv-18 are of high risk, contribute to over 70% of all cervical cancer cases. therefore, prevention of hpv-16 and hpv-18 's infection among women becomes an important issue for scientists and epidemiologists to lower the incidence of cervical cancer. hpvs are double - stranded dna viruses, which target the basal cells of squamous epithelia for infection. the structure of its circular dna genome are composed of 2 major oncogenes e6 and e7, 2 major structural protein genes l1 and l2. based on these elements, the l1 protein of hpv expressed recombinantly in vitro can self - assemble into virus - like particles (vlps). vlps have hpv type - specific conformational neutralizing epitopes and thus induce type - specific neutralizing antibodies to protect against hpv infections. in line with this fact, large pharmaceutical companies like merck and gsk have developed vlp - based vaccine products and successfully put them into the market, using yeast and insect cell expression systems, respectively. both of these two products include vlps for hpv-16 and hpv-18. in our company, an l1 vlp - based vaccine candidate aiming at hpv-16 and hpv-18 was also produced using yeast protein expression system [9, 10 ]. it includes vlp-16 and vlp-18 and thus should prevent against over 70% of cervical cancers. for l1 vlp - based vaccines, the efficacy is largely dependent on the number of neutralizing antibodies induced by the conformational neutralizing epitopes existing on vlp surfaces. the more the neutralizing antibodies are induced, the more the vaccine should be effective to protect against hpv infections. to date, different methods have been developed to quantitate the type - specific neutralizing antibodies for hpvs, such as pseudoneutralization assays and crias. for this reason, several studies have used a luminex - based competitive immunoassay [13, 14 ] which could be used to quantify different type - specific neutralizing antibodies in a single reaction simultaneously. in the assay, different types of vlps were bound with different kinds of microspheres, then the known, hpv type - specific, phycoerythrin - labeled neutralizing monoclonal antibodies (mabs) were mixed with vlp - coupled microspheres and the vaccine challenged serum samples for reaction, where the pe - labeled neutralizing mabs compete with the serum antibodies to bind to the conformational epitopes on vlps. after that, the microspheres were put into luminex systems for detection of the pe signal. therefore, strong signals mean more pe - labeled mabs binding, and thus less neutralizing antibodies existed in the serum sample. in order to quantitatively evaluate the high and low levels of neutralizing antibodies, serum from african green monkeys hyperimmunized with hpv l1 vlps was reported to be used as reference standards successfully. an alternative is to make use of normal human serum spiked with unlabeled type - specific neutralizing monoclonal antibodies as reference standards. in this study, a 2-plex luminex - based competitive immunoassay was developed to evaluate the efficacy of our hpv-16 and hpv-18 l1 vlp - derived vaccine candidates. the reference standard was prepared using normal human serum spiked with various amounts of no - fluorescence labeled type - specific neutralizing mabs rather than hpv - positive serum. the fluorescence - labeled neutralizing mabs were not coupled directly with pe but with biotin first, and an amount of streptavidin - pe was added then for detection, which makes the assay more flexible. in developing the assay, at first, the coupling ratio of vlps to microspheres and the coupling ratio of biotin to type - specific neutralizing mabs were optimized, the mixed vlp - coupled microspheres were blended with the mixed biotinylated neutralizing mabs to confirm the lack of cross - neutralizing activity, and finally a 2-plex standard curve for the competitive immunoassay was constructed for neutralizing antibody detection in serum samples. in this study, the microspheres bio - plex cooh bead 038 and bio - plex cooh bead 053 were purchased from bio - rad corporation, hercules, calif. the hpv-16 l1 vlp and hpv-18 l1 vlp were produced by yeast protein expression system in the laboratory of shanghai zerun biotechnology co., ltd. the neutralizing monoclonal antibodies (mabs) h16.v5 for hpv-16 and h18.j4 for hpv-18 were kindly provided by dr. the luminex 200 system was purchased from luminex corporation, and data was collected and analyzed with luminex is 2.3 and prism 4. microspheres were incubated in 96-well 1.2 m pvdf filter microtiter plates (millipore, billerica, mass) and rotated in ms 3 digital (ika, janke & kunkel - str. the washing steps were carried out in bio - plex pro ii wash station (bio - rad, hercules, calif). vlps were coupled to microspheres according to the previously published methods with a few modifications [13, 14 ]. l1 vlp (vlp-16) and hpv-18 l1 vlp (vlp-18) were covalently conjugated to bio - plex cooh bead 038 (# 38 beads) and bio - plex cooh bead 053 (# 53 beads), respectively, as follows. initially carboxylated beads (1.25 10 beads / ml) were fully suspended by vortexing and sonication, and then about 500 l beads were transferred to a 1.5 ml protein lobind tube (eppendorf) for coupling. after centrifuging the tube at 14000 g for 4 min, the supernatant was removed. then the beads were washed once with 500 l 0.1 m nah2po4 (ph 6.2) by vortexing and sonication, followed by an incubation with 50 l 50 mg / ml sulfo - nhs (pierce, rockford, il), 70 l 0.4 m 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (edc) (ge, shanghai, china), and 380 l pbs (ph 7.4) for activation, where amine - reactive esters of carboxylate groups were formed for labeling. the activation lasted in darkness for 30 min, at 8001000 rpm, rt. after that, the beads were washed twice with 50 mm mes buffer (sigma, ph 6.0) and pelleted in mes buffer before counting using a hemocytometer. the hpv l1 vlps were transferred to 50 mm mes buffer (ph 6.0) by ultrafiltration, and then its concentration was determined by bca (bca protein assay kit, pierce, rockford, ill) before use. in coupling, 1 10 activated beads and buffer with transferred vlps were mixed in mes buffer to a final volume of 100 l200 l. the mixture was rotated in darkness, at 8001000 rpm, rt, overnight. consequently, the beads were washed twice with pbs plus 0.05% tween-20 (ph 7.4), followed by blocking in histidine buffer (20 mm histidine (sigma), 0.5 m nacl, 1% bsa, ph 6.2) for 30 min in darkness, rt, at 8001000 rpm. after washing the beads twice with histidine buffer, they were counted by hemocytometer and diluted to 1 10 beads / ml in histidine buffer for preservation in darkness, at 4c. the hpv-16 and hpv-18 neutralizing mabs h16.v5 and h18.j4 were biotinylated using micro sulfo - nhs - lc - biotinylation kit (pierce). the procedure followed the manufacturer 's instructions. in brief, neutralizing monoclonal mabs were transferred to pbs (ph 7.27.4) and subjected to bca (bca protein assay kit, pierce) to determine concentration before biotinylation. an amount of active sulfo - nhs - lc - biotin for different mole ratios of biotin and mab was then added, and the mixture was rotated at 8001000 rpm in darkness, rt, for 45 min. finally, the biotinylated beads were put at 20c immediately to stop the reaction and stored before use. the biotinylation of h16.v5 and h18.j4 was confirmed to determine the optimal mole ratio of biotin and mabs for reaction. 5000 vlp-16 conjugated # 38 beads (25 l, 2 10 beads / ml, coupling ratio = 40 g protein per million beads) or vlp-18 conjugated # 53 beads (25 l, 2 10 beads / ml, coupling ratio = 40 g protein per million beads) were mixed with 25 l of diluted biotinylated h16.v5 (biotin / h16.v5 mole ratio = 20/1, 40/1, 80/1, 160/1, 320/1, 640/1, 1280/1) or biotinylated h18.j4 (biotin / h16.v5 mole ratio = 20/1, 40/1, 80/1, 160/1, 320/1, 640/1, 1280/1) and 50 l pbs - bsa to a final volume of 100 l, where the biotinylated mabs were 20-fold diluted, followed by a 2-fold serious dilution in a total of 11 different dilutions. then the microtiter plate was rotated at 8001000 rpm, in darkness, for 3 h, followed by 3 times of wash in wash station. after that, 50 l 2.5 g / ml streptavidin - pe was added for incubation at 8001000 rpm, in darkness, for 20 min. the beads were then washed 3 times and resuspended in 125 l pbs - bsa for reading in luminex 200 system (luminex corporation, austin, tex). after the coupling reaction of hpv l1 vlps to microspheres has been completed, the coupling efficiency was confirmed. in brief, 5000 vlp-16 conjugated # 38 beads (25 l, 2 10 beads / ml) or vlp-18 conjugated # 53 beads (25 l, 2 10 beads / ml) were mixed with 25 l biotinylated h16.v5 (biotin / h16.v5 mole ratio = 100/1) or biotinylated h18.j4 (biotin / h18.j4 mole ratio = 100/1) and 50 l pbs - bsa buffer (pbs, 1% bsa, ph 7.4) to a final volume of 100 l, for incubation in a 96-well prewetted filter plate, in darkness, at 8001000 rpm for 3 h, where the biotinylated mab was 1 : 50, 1 : 100, 1 : 1000 diluted in pbs - bsa buffer (pbs, 1% bsa, ph 7.4). then the beads were washed 3 times in bio - plex pro ii wash station (bio - rad) using pbs - bsa buffer. after that, 50 l 2.5 g / ml streptavidin - pe was added, followed by incubation in darkness, at 8001000 rpm for 20 min. after washing the beads again for 3 times with the wash station, 125 l pbs - bsa buffer was added for reading in luminex 200 system (luminex corporation). the normal human serum spiked with different amounts of the no - biotin labeled neutralizing mabs was used as the standard. it was prepared as follows : initially the normal human serum (nhs) was blended with certain amounts of unlabeled h16.v5 and h18.j4 to a final concentration of 20 g / ml. then, the mixture was 2-fold diluted with nhs to make h16.v5 and h18.j4 in a series of 15 different concentrations. for each 50 l diluted sample, 5000 vlp-16 conjugated # 38 beads (coupling ratio = 40 g protein per million beads), 5000 vlp-18 conjugated # 53 beads (coupling ratio = 40 g protein per million beads), 500-fold diluted (final dilution) biotinylated h16.v5 (biotin / h16.v5 mole ratio = 320/1), and 200-fold diluted (final dilution) biotinylated h18.j4 (biotin / h18.j4 mole ratio = 640/1) were added to a final volume of 100 l, followed by an incubation in 96-well prewetted filter plate, in darkness, at 8001000 rpm for 3 h. after washing in triplicate by wash station using pbs - bsa buffer, the beads were added with 50 l 2.5 g / ml streptavidin - pe and rotated at 8001000 rpm, in darkness, for 20 min for staining. finally the beads were washed again in triplicates and resuspended in 125 l pbs - bsa buffer before reading in luminex 200 system (luminex corporation). all assays were conducted in duplicates. in brief, 5000 vlp-16 conjugated # 38 beads (coupling ratio = 40 g protein per million beads) and 5000 vlp-18 conjugated # 53 beads (coupling ratio = 40 g protein per million beads) were added with 40-fold diluted (final dilution) biotinylated h16.v5 (biotin / h16.v5 mole ratio = 320/1) and 200-fold diluted (final dilution) biotinylated h18.j4 (biotin / h18.j4 mole ratio = 320/1) or both. different ratios (g / million beads) of hpv-16 l1 vlp or hpv-18 l1 vlp and # 38 or # 53 microspheres were used for coupling, respectively. the vlp coupled microspheres were then reacted with the same amount of related neutralizing mabs. the coupling ratio at which the vlp - conjugated beads can generate the strongest signal was then selected as the optimal. the coupling reaction for vlp-16 and # 38 beads was saturated when coupling ratio was bigger than 40 g protein per million beads (figure 1(a)), while for vlp-18 the optimal coupling ratio was 40 g protein per million beads (figure 1(b)). another concern for choosing 40 g protein per million beads was to find a robust way for coupling ; more vlps used could ascertain the same quality of coupled beads at each time. it would be important in qc and clinical trial. for biotinylation of neutralizing mabs h16.v5 and h18.j4, different coupling mole ratios of amine reactive biotin and mab were tried, and the biotinylated mabs were then mixed with vlp - conjugated microspheres for reaction. the optimal coupling mole ratio was the one at which the reaction could generate the strongest signal. for both h16.v5 and h18.j4, the optimal coupling ratios of biotin to mabs were 320/1 or higher (figures 2(a) and 2(b)), where the biotinylation reaction was saturated at the current detection system. when changing our system, for example, the added amount and concentration of streptavidin - pe, the optimal coupling ratio for biotinylation may be higher, because there may be not enough streptavidin - pe for staining. before mixing the 2 vlp - conjugated microspheres together for immunoassay, studies were carried out to make sure that no cross - reactivity existed between the 2 analytes. just as predicted, with our produced hpv vaccine candidates, biotinylated h16.v5 could only bound to vlp-16 coupled microspheres for signaling while biotinylated h18.j4 only bound to vlp-18 coupled microspheres (figure 3). the results ascertained the establishment of the 2-plex immunoassay with high sensitivity and specificity. in this section, the saturated vlp coupled beads plus the saturated biotin coupled mab just generate no cross - reactivity. the coupling ratios for vlps to microspheres were both 40 g protein per million beads. not surprisingly, standard curve preparations (figure 4) for the 2-plex luminex - based competitive immunoassay were established successfully with good fit (r > 0.99) for both analytes and wide dynamic range (9.8 ng / ml to 5.0 g / ml for vlp-16 neutralizing antibodies, while 39.0 ng / ml to 5.0 g / ml for vlp-18 neutralizing antibodies), where the no - biotinylated mabs (standards) and biotinylated mabs competitively bind to hpv vlp coupled microspheres. luminex xmap system is a technology combined with flow cytometry, microspheres, and lasers together, which allows the quantitation of up to 100 different analytes simultaneously using quite small sample volumes in a single reaction. it has been applied to hini detection, kidney biomarkers detection, human leukocyte antigen genotyping, cytokine detection and quantitation, and so forth. with high sensitivity and specificity. cervical cancer, as the second prevalent cancer in women in the world, was caused by hpv infections. some studies have revealed that hpv l1 vlp - based vaccine could effectively induce type - specific neutralizing antibodies to hpvs and thus protect against their invasions. in order to make the vaccine more powerful, it was often designed to target more than one type of hpvs, for instance, merck 's gardasil aimed at hpv-6, -11, -16, and -18 simultaneously. as a sensitive, economic and high - throughput method, luminex system was reported [13, 14 ] to evaluate the efficacy of the hpv l1 vlp - based vaccine products. compared to the traditional elisa- or facs - based method, luminex has many advantages. it could be used to quantitate type - specific neutralizing antibodies induced by various hpvs simultaneously and thus was more practical for clinical use, where large number of serum samples should be analysed in a short time. here, in order to establish an assay for efficacy determination of our hpv-16, -18 l1 vlps based vaccine candidate, also for further clinical use, a 2-plex luminex - based competitive immunoassay was also developed. although the reported method represents an important reference for this study, some modifications were tried to make the assay more flexible in the practical use. when developing the immunoassay, first of all, the amount of vlp needed for coupling with microsphere should be determined to generate constant and strong signals. for the 2-plex method, 2 different beads were prepared for hpv-16 l1 vlp and hpv-18 l2 vlp, respectively. interestingly, the optimal coupling ratio for vlp-18 was 40 g / million beads, while for vlp-16 the coupling would be saturated if the ratio was bigger than 40 g / million beads, which may be due to the different neutralizing epitopes existed in vlp-16 and vlp-18. on the other hand, in this step, it was also found that low - binding tube was very essential for coupling of high quality (data not shown), while the conventional tube may absorb microspheres during the reaction and thus lower the recovery. the amount of biotin needed for biotinylation of neutralizing mabs should also be optimized. in the reported method, the signal dye pe was bound directly to mabs, which we thought was inflexible for the assay 's practical use. sometimes, it is difficult to obtain a stable pe labeled mab or other molecules when several new analytes are to be added to this multiplex assay. in contrast, biotin labeling technology is highly developed, and it is simple to obtain stable biotinylated mab. in fact, biotinylated mab plus streptavidin - pe was commonly used for signaling in most elisa, luminex, or other immunoassay - based systems. in this study, just as predicted, with our produced hpv-16 and hpv-18 l1 vlps based vaccine candidate, no cross - activity was found between h16.v5 and h18.j4, which were well - characterized, strong type - specific hpv-16 and hpv-18 neutralizers. after that, the 2-plex standard curve (std) of the competitive immunoassay for quantifying antibodies neutralizing hpv-16 and hpv-18 simultaneously was then tried to be constructed. unlike the published paper from merck, normal human serum spiked with no biotin labeled neutralizing mabs was used for standards, instead of vlp challenged monkey serum. the major reason for that is the difficulty in preparing enough well - characterized and standardized serum for the assay. in this study, the 2-plex std for hpv-16 and hpv-18 was well established with high sensitivity and wide dynamic range. later the assay was tried to measure the induced neutralizing antibodies in vlp challenged mice serum. it could be distinguished with different antibody levels for hpv-16 and hpv-18 simultaneously, of samples derived from mice challenged with our developed vlp vaccine candidate of different doses (data not shown). therefore, the 2-plex luminex - based assay provided a fast, sensitive, and effective approach to evaluate the efficacy of our hpv l1 vlp - based vaccine candidate. at present, the dilution buffer used for assay was normal human serum for clinical sample use. when applying the assay to monkey or mouse serum samples, the dilution buffer should better use normal monkey serum or normal mouse serum accordingly because it is commonly used as the dilution matrix or blank. the published paper suggests the use of antibody - depleted human serum (adhs) as dilution buffer because of its reliable and stable resources, which we thought could also be tried in our developed systems in the future. in addition, the unit used for characterizing the level of neutralizing antibodies in serum samples should be defined. in doing this, large number of serum samples with neutralizing antibodies of low, medium, and high levels should be applied to the same std curve, to further validate the assay. a 2-plex luminex - based competitive immunoassay was developed to evaluate the efficacy of our hpv l1 vlp - based vaccine candidate in inducing neutralizing antibodies against hpv-16 and hpv-18. the assay was convenient, sensitive, effective, and requiring only small amount of samples. recently, additional studies from our laboratory are underway to further validate the assay and also determine the method limitation with plenty of serum samples. the correlations of the assay to other characterization method for hpv l1 vlp - based vaccines like pseudoneutralization assay and ivrp were also investigated. moving forward, as researches on the assay become more comprehensive, the 2-plex luminex assay will be routinely applied for evaluating and quantifying neutralizing antibodies in clinical use. | human papillomavirus (hpv) l1 virus - like particles (vlps) were proven an effective vaccine candidate to prevent against hpv-16 and -18 infections. in order to evaluate the potency of our produced hpv-16 and -18 l1 vlps - based vaccine candidates, also to quantify neutralizing antibodies induced by them, a 2-plex luminex - based competitive immunoassay was developed. unlike the published paper, the no - biotin conjugated neutralizing mabs spiked normal human serum (nhs) was used for standard curve preparation, while phycoerythrin (pe) was not labeled directly to neutralizing mabs for signaling. after the coupling optimization of vlps to microspheres and the neutralizing mabs biotinylation, the 2-plex standard curve was prepared with good fit and high dynamic range. in addition, no cross - reactivity was also confirmed. the 2-plex luminex - based immunoassay represents good potential not only for vaccine candidate 's evaluation but also for its further clinical use. |
stroke is the most common cerebrovascular disease and the most frequent cause of death as a single disease despite advancements in modern medical technology and increased awareness. activities of daily living are limited by the disability in sensory, motor, and cognitive and emotional control functions seen following a stroke1. due to muscular stiffness and weakness, stroke patients have difficulties in supporting their weight on the lower extremity on the affected side, thereby causing disabilities in balance control while standing, which affects quality of life negatively2. in addition, significant reductions in the function of the upper extremity on the affected side are experienced by stroke, resulting in significant difficulties in independent movements and performing the activities of daily living3. during arm stretching movements by stroke patients, muscle activity is reduced more in the affected side than in the unaffected side, leading to problems with postural adjustment due to the weakness of trunk muscles4. as static and dynamic balance abilities that can maintain the body s center of gravity within the basal plane in the standing position are reduced, asymmetrical posture of the trunk is revealed in order to compensate for the instability. the trunk muscles are involved with trunk movements and spinal stability5 and extend in a wide area in both vertical and horizontal directions. they also adjust trunk motions in various spatial directions6 and are involved with postural control by maintaining balance through maintaining a normal lumbar curvature7. upper extremity training in the standing position improves postural control while maintaining the body s center of gravity8. trainings of the maintenance of a standing position and upper extremity are more beneficial to the performances of activites of daily living if they are conducted simultaneously rather than independently9. task training in the standing position can improve postural control thereby helping stroke patients to pay more attention to their safety in complex environments as well as maintaining safety while improveing their ability to perform various activites of daily living10. functional and dynamic balance training is important to improve the gait and functional balance of stroke patients11. trunk alignment is affected by weakness in trunk muscles and abnormal postures following a stroke. therefore, this study aimed to identify the effect of upper extremity training in a standing position on trunk alignment and provide a scientific foundations for upper extremity training in a standing position, which is frequently used clinically. this study conducted experiments with 12 stroke patients who were admitted to rehabilitation hospital in incheon gyeonggi - do in korea. the sitting group had four males and two females patients, while the standing group had three males and three females patienta. the mean was 62 years for the sitting group and 60 years for the standing group. the mean duration of disease was 13 months for sitting group and 15 months for standing group. the numbers of subjects with right and left hemiplegia were seven and five respectively. signed consent was given voluntarily by the subjects involved in the study. the selection criteria for subjects : were hemiplegic patients, moderate severity according to the fugl - meyer upper extremity test, and being able to maintain a standing position without assistance for five min or longer. this study was conducted from march 31, 2015 to may 30, 2015. upper extremity training in either a sitting or a standing positions was given for 30 min per day, five times a week, for six weeks. upper extremity training involved the following : grabbing a cone on the table and moveing it to a specified place, moveing a cone on a table to a shelf, stretching an upper extremity to grab a ball to put it into a basket, and stretching an upper extremity to grab a ring and put it onto bar. this study evaluated postural alignment before and after the upper extremity training between sitting control group and standing experimental group. postural alignment evaluation was performed using the formetric 4d system (diers, international gmbh, schlangenbad, germany, 2010), which provides an alternative to radiographic examination for the analysis of spinal alignment. the formetric 4d system provides a high reproducibility rate and objective data, as well as accurate analysis on spinal curvature. it can analyze the ear surface of the trunk in three dimensions and examine spinal shapes rapidly using surface contour lines without the need for radiation exposure. the measurement principle is as follows : the surface of the back is reconstructed via raster lines, followed by camera capture thereby locating the estimated spinous process, in order to measure the curvature through anatomical markers and the spinous processes. the normal distribution of data was demonstrated using the kolmogorov - smirnov test. to compare changes in measured variables before and after the upper extremity training in both groups, the independent t - test was used to compare changes in outcome measures between the two groups. no significant changes in lordosis or kyphosis were seen in the results of the sitting group, before and after the upper extremity training (p>0.05). in the standing group, no significant changes in lumbar lordosis was found (p>0.05) but significant change in thoracic kyphosis was found (p0.05) but significantly improved thoracic kyphosis (p0.05). the comparison between the sitting and standing position groups showed that there was no significant difference in the change lumbar lordosis (p>0.05) but had a significant difference in the change in thoracic kyphosis (p<0.05). the asymmetrical posture of stroke patients in the median plane causes unstable posture, however, balance training in the standing position can help normal weight distribution and recover of a symmetrical posture12. the static standing posture can also help the recover of asymmetrically affected trunk muscles and reduce thoracic kyphosis. the trunk muscles necessary for postural control are activated by the reduction in thoracic kyphosis, thereby having an effect on trunk control and balance adjustment13. activities of daily living are negatively affected by hyperkyphosis, significantly degarding quality of life. twently to forty percent of the elderly population is affected by hyperkyphosis, and it is closely related with aging. in addition, it can slow gait, reduc balance, cause trunk shaking, and increase risk of falls14. an increase in thoracic kyphosis is characterized by a bending posture and center of gravity of the trunk moveing in front of body, as well as affecting alignment of the joint of the lower extremity. thus, difficulties in accurate postural control due to reduced reaction to postural sway and the normal gait pattern is reduced due to the disability in postural control can be caused by hyperkyphosis. the bending posture due to hyperkyphosis can increase risk of falls risk and is one of the main risk factors fall due to the disability in postural control, especially while walking15. it is important to improve balance during the rehabilitation of stroke patients because it allows the independent performance of activities of daily living. standing on two legs without postural sway is easy for a healthy persons whereas it can be difficult for stroke patients and may require a long period of rehabilitation to reacquire. the cpo in sitting and standing positions had more regular fluctuations in the standing position than the sitting position and greater balance is required to maintain postural control while standing16. thus, a standing position is more effective in balance training for postural control than a sitting position. results of dual task training for the affected upper extremity side and gait training given to stroke patients in a standing position showed improvements in ability and that long - term rehabilitation was needed17. the present results showed greater activation of trunk muscles during upper extremity training in the standing position thereby reducing thoracic kyphosis. however, upper extremity training in a standing position can reduce the risk of falls by decreasing hyperkyphosis, demonstrating that rehabilitation in the standing rather than sitting position is more beneficial. during forward stretching movements of the upper extremity by stroke patients, trunk muscle activation differed between both sides and disabilities in anticipatory postural adjustments occurred due to the weakening of trunk muscles on the affected side. disabilities in feedforward movement adjustments such as anticipatory postural adjustments, lead to patients not being able to cope with unexpected trunk swaying due to the weakening of trunk muscles. upper extremity training can also provide training for anticipatory postural adjustments and improve the ability to perform activities of daily living18. heads of stroke patients is typically positioned more forwardly and it emphasizes a postures with round shoulders and thoracic kyphosis while sitting affecting trunk alignment. however, a standing position can maintain the body s center of gravity in a vertical direction and increase changes in the nervous system and muscle strength in trunk, thereby improving muscle strength and balance19. the present study result showed that upper extremity training in a standing position had a greater effect on trunk alignment by reducing thoracic kyphosis than training in a sitting position. upper extremity training in a standing position strengthened trunk muscles while maintaining the body s center of gravity in a vertical direction and activated trunk muscles during upper extremity training thereby improving postural adjustment due to the effect on trunk alignment. furthermore, it can help independent daily living through anticipatory postural adjustments as well as reducing the risk of fall. the limitations of this study were the small number of subjects and no comparison of changes in scoliosis because of the focus only on lumbar lordosis and thoracic kyphosis - related variables when examining trunk alignment. in the future, comparison of changes in scoliosis as well as trunk alignment and balance | [purpose ] this study aimed to examine the effect of upper extremity training in the standing position on trunk alignment of patients with stroke. [subjects and methods ] twelve stroke patients were enrolled in the study and divided into two groups : a group of six patients in a sitting position and a group of six patients in a standing position. upper extremity training for 30 min per day, five times a week for six weeks was given to subjects in both groups. in order to assess trunk alignment, lumbar lordosis and thoracic kyphosis were examined before and after upper extremity training using formetric 4d. [results ] after training the standing position group had no significant change in lumbar lordosis but a significant change in thoracic kyphosis. the sitting position group showed no significant changes in either lumbar lordosis or thoracic kyphosis. the comparison between groups showed there was no significant difference in the change in lumbar lordosis but there was a significant difference in the change in thoracic kyphosis. [conclusion ] examination of trunk alignment showed that upper extremity training conducted in a standing position reduced thoracic kyphosis more than in a sitting position. |
disease registries are important tools for tracking disease trends or treatment outcomes.123 the health / disease registry could be hospital / clinic - based or population based. the former is used for a specific disease irrespective of the location of the case. alternately, a population based registry is used to compile information on specified diseases by region, community, and state in which they are diagnosed.4 they exist in different formats ; paper based registries, the most common types of registries in the past were complemented over time by computerized records. in recent years, web - based registries have become popular because they are user friendly and can be managed from different locations. additionally, the web - based approach overcomes data management issues and allows for periodic generation of information for monitoring and policy making. health managers at hospitals, national, and international levels use web - based registries to compile information on different health issues.567 since 1983, paper and computer - based retinoblastoma and tumor registries had been in place at the king khaled eye specialist hospital (kkesh) riyadh, saudi arabia.89 on the basis of the computer - based congenital glaucoma registry, baseline features of primary and secondary congenital glaucoma were described.10 saudi arabia has one of the highest incidences of primary congenital glaucoma worldwide.10 however, there is a relative paucity of information on the outcomes of managed children. a simple, yet effective web - based registry was created to prospectively collect data on congenital glaucoma. to the best of our knowledge, web - based registries have not been used to track outcomes or disease trends in congenital glaucoma (cg). this study was undertaken between january 2012 and march 2013. the approval of research department of (kkesh) a registry committee comprising of ophthalmologists, epidemiologist, statistician, information and a technology specialists, and a registry manager was formed. two glaucoma specialists prepared a case report form (crf) that included a questionnaire on congenital glaucoma [table 1 ]. information technology specialists provided the lime survey form (lime survey 2.05, germany) that is available without cost.11 all the crf questions were incorporated as an electronic form in the lime survey software. to minimize manual entry by the end user, drop down boxes and check boxes the biostatistician checked the validity of the case report form to ensure that the questionnaire could gather sufficient information from end users as per the registry manual that was prepared for web - based data entry. a person with basic knowledge of database and programming should be able to use mysql software and create an electronic crf using lyme survey. the ecrf was then linked to this system in kkesh is subscription based and is used by numerous departments in the hospital. hence, use of this system for cgr was a small fraction of the total cost of this service. cost analysis by full time equivalent for establishing a web - based congenital glaucoma registry to make the registry web based, a uniform resource locator (url) was created for the cgr.13 the website could only be accessed with a unique login identification and password. the cgr data was tested and could be exported to common analytical software such as statistical package for social studies (spss, ibm corp., new york, ny, usa) and r - language.1415 the individuals involved in establishing the cgr were interviewed to determine the approximate time they spent in developing the online registry. from their responses, we calculated the full time equivalent (fte). a cost analysis based on fte spent in establishing a web - based cg registry the time required for completing one cgr form was calculated. the time to analyze and interpret the cgr by a data manager an example of populated drop boxes in the electronic crf is presented in figure 2. flow chart of web - based glaucoma registry populated drop boxes in electronic case report form (crf) for congenital glaucoma registry the data of 80 patients from an existing paper / computer based cgr at kkesh (2000 to 2003) was entered in the electronic crf as a pilot project. descriptive analyses of the 80 cases were performed and plotted using an automatic outcomes function available in the lime survey software. the time for a clinician or data entry personnel to complete the crf during clinical assessment was less than 4 minutes. in addition, automated descriptive analysis in a graph format could be generated [figure 3 ]. web - based congenital glaucoma registry generated automated graphical display of outcomes the cost analysis based on the full time equivalent (fte) spent in establishing web - based cg registry is presented in table 1. web - based registries have been previously established in other fields of medicine.256 most involve conversion of existing manual and computer - based registries to web - based registries. experience in other fields of medicine indicated that these registries are sustainable because they are user friendly, easily accessible from any part of the world, provide epidemiological data with minimum errors, and have important applications. in this study, we outline the process of creating a web - based cgr using off the shelf software, and based on our experience it was relatively easy to establish. some of the key components in making the endeavor successful was to have a knowledgeable ophthalmic and it team combined with an efficient data entry manager and biostatistician. a cost analysis of the registry included factors such as human resources, equipment, and supplies and it was determined that a product was created at a fraction of the cost of commercially available or customized databases. some of the cost savings achieved during this project were related to the existing infrastructure in the hospital 's information technology department and the free downloadable software. an accurate estimate of actual cost of commercially available or customize databases was not available despite attempts to procure information on specific costs. however, rough estimates from personal communication with local institutions in saudi arabia that maintain registries suggested that the costs of customizing and maintaining registries / databases are much higher. from the clinician 's perspective, the feedback received was that the data entry was intuitive, quick, and easy, and that allied health personnel could be trained to enter the data. also of importance was the data could be entered at any workstation connected to the internet or intranet. we believe that this process which includes additional data entry other than what is entered in the medical record will not decrease the efficiency of clinical practice. the software feature of generating automated descriptive analysis plotted on graphs will help track data in real time enabling the data manager to prepare policy briefs in an efficient manner. due to the relatively simple it infrastructure required, we suggest that establishment of similar registries would be feasible in most developing countries individuals from each country that plan to use such a registry would need to calculate costs per fte. since the registry is web - based, it is feasible for such a registry to be used to collect data from multiple centers nationally or internationally and could be created for any ophthalmic condition. the web - based registry has already been expanded to other diseases and pathology such as retinoblastoma, ocular tumors, retinal dystrophies, and keratoconus at our institute. these registries will soon cover the kingdom and then the gulf countries. since the it infrastructure is simple it can be located at only one institution, and the costs associated with creating and maintaining the registry will be further reduced. to the best of our knowledge, such a web - based registry has been created for diabetic patients with data on 10,000 cases.16 it is possible that one of the weaknesses of establishing such a registry might be accommodating large data sets. in addition, a potential weakness is the discontinuation of free downloadable software or the absence of technical support for this product in the future may cause difficulties in continuing additional data entry using the same format. however, since the data stored on a my sql server internally, data that was collected would not be lost. congenital glaucoma registry information was collected from both glaucoma and pediatric ophthalmology units at kkesh. the pilot was based on transferring retrospective data from a paper - based registry to a web - based registry. if many institutions are involved in the registry, the time required to register a case could be longer than that described in the current study. cloud testing by external institutions could further strengthen the web - based cgr. using cloud computing, the development for a larger storage capacity | in this brief communication, we present the steps used to establish a web - based congenital glaucoma registry at our institution. the contents of a case report form (crf) were developed by a group of glaucoma subspecialists. information technology (it) specialists used lime survey softwaretm to create an electronic crf. a my structured query language (mysql) server was used as a database with a virtual machine operating system. two ophthalmologists and 2 it specialists worked for 7 hours, and a biostatistician and a data registrar worked for 24 hours each to establish the electronic crf. using the crf which was transferred to the lime survey tool, and the mysql server application, data could be directly stored in spreadsheet programs that included microsoft excel, spss, and r - language and queried in real - time. in a pilot test, clinical data from 80 patients with congenital glaucoma were entered into the registry and successful descriptive analysis and data entry validation was performed. a web - based disease registry was established in a short period of time in a cost - efficient manner using available resources and a team - based approach. |
the synthesis of high - quality colloidal core shell quantum dots (qds) is usually performed in organic solvents. from these solvents, they can be processed and used in numerous applications in optoelectronics and photovoltaics. alternatively, the qds can be subsequently transferred into aqueous solutions in order to increase their range of applications, particularly in biological labeling and environmental sensing. such a transfer requires either hydrophilic bifunctional ligands to be exchanged for the native hydrophobic monofunctional coordinating ligands, usually octadecylamine (oda) or the addition of amphiphilic ligands or diblock copolymers where the hydrophobic regions intercalate with the native ligands and hydrophilic regions are exposed to the aqueous environment. there are advantages and disadvantages to each approach, but it mainly comes down to a choice of size vs stability. the long - chain native ligands and the amphiphilic nature of ligands used in the intercalation approach lead to qds with relatively large overall diameters. the ligand - exchange approach can, in principle, use extremely small ligands. however, when using these qds for fluorescence applications, it has been found that fluorescence quenching depends on both ligand size and the functional group used, so a balance is usually found between size, stability, and fluorescence quantum yield. as a compromise, small polydentate ligands such as dihydrolipoic acid (dhla) have been used to improve colloidal stability over monodentate ligands, although we have recently found that the lower density of dhla ligands on the surface compared to monodentate mpa ligands leads to higher nonspecific adsorption of target thiol molecules at low concentrations. in any case, the colloidal stability of ligand - exchanged quantum dots is usually lower than the amphiphilic polymer - coated qds and depends strongly on the nature of the ligands and the solvent conditions. this can be a particularly significant problem at low qd concentration and under reductive or oxidative conditions, as is often the case in biological fluorescence labeling applications. combining the advantages of ligand exchange using compact, water - soluble ligands with cross - linking has the potential to allow a better compromise in which the size increase is modest while the improvement in colloidal stability should be significant. only a few examples of cross - linking compact, ligand - exchanged qds have been reported, and they have used a chemical cross - linking approach. however, a major disadvantage with chemical cross - linking is that it is difficult to inhibit interparticle cross - linking, which means that a purification step may be required and overall yield will be compromised. photochemical cross - linking is an attractive alternative to localize the cross - linking to within a particle, particularly if the cross - linking group is not exposed to the solvent environment. self - assembled monolayers (sams) of diacetylene ligands on gold were directly photo - cross - linked using high - energy ultraviolet light (uv, 254 nm) and were shown to result in surfaces that are extremely robust for use in harsh environments. in this study, we demonstrate that qds themselves can initiate cross - linking of bifunctional diacetylene ligands using lower energy visible light to generate radicals (scheme 1). photoexcited cdse has been previously used to generate radicals on adsorbed species for charge separation in photovoltaic applications and in solid - state polymer nanocomposite blends. however, to the best of our knowledge, using a qd - mediated photo - cross - linking approach in solution to limit interparticle cross - linking while, at the same time, promoting intraparticle cross - linking to improve the colloidal stability of those same qds has not yet been demonstrated. we show that taking advantage of the qd as a visible - light initiator leads to much better cross - linking of the ligands than direct cross - linking of the diacetylene groups with uv light, since competing ligand dissociation reactions induced by uv exposure are avoided. our approach results in extensive intraparticle cross - linking but with practically no interparticle cross - linking, and more importantly, it leads to excellent long - term colloidal stability of the qds in aqueous environments while maintaining relatively small hydrodynamic sizes. the synthesis of the bifunctional diacetylene (da) ligand followed a combination of procedures described by xu. and kim. details of the da ligand synthesis and nmr peak assignments are provided in the supporting information. cdse / zns (core / shell) quantum dots (em = 597 nm) coated with hydrophobic octadecylamine (oda) ligands were purchased from ocean nanotech (springdale, ar) as a dried powder and dissolved in toluene (emd chemicals inc., billerica, ma). prior to the ligand exchange procedure, the as - purchased qds were purified from excess ligands by precipitating from toluene with acetone (emd), centrifuging at 14 100 g (14 500 rpm, minispin plus, eppendorf), and discarding the supernatant. then, the precipitated qds were redissolved into chloroform (drisolv, emd), mixed with methanol (emd), and centrifuged again and the supernatant was discarded. a ligand solution was prepared by adding 10 mg of the da ligand to 1 ml of chloroform or by adding 2.66 l of mercaptopropanoic acid (mpa) (alfa aesar, ward hill, ma) in 1 ml of methanol. the ph was adjusted to 11 by adding microliter quantities of a stock solution of tetramethylammonium hydroxide (tmaoh) (alfa aesar) in methanol. the ligand solution was added to the precipitated qds, and stirred for 24 h at room temperature in the dark. it has been shown that 254 nm uv exposure can be used to photopolymerize diacetylenes. the fluorescence of the sample as a function of 254 nm (short wave) uv exposure time was monitored to determine the optimal cross - linking time in terms of the fluorescence properties. emission spectra were obtained by exciting at 530 nm in a 100 l quartz fluorescence cuvette (starna cells). from all the samples, those exposed for 0, 20, 100, and 210 min were then analyzed with an ft - ir spectrometer (bruker, vertex 70) equipped with a dtgs detector. a 40 l aliquot of the sample was taken from the stock solution at the specified time of exposure and placed in the exact center of a caf2 window which was then left to dry under a n2 atmosphere so that the ir beam diameter completely probed the whole sample spot. to transfer the water - soluble qd - da and qd - mpa to water, methanol and acetone were added, respectively, and the mixture was centrifuged to precipitate the qds from the reaction solution. having removed the supernatant, the qds were redissolved into millipore (18.2 mcm) water. each solution was transferred to a four - windowed quartz fluorescence cuvette with a stopper (starna) for further analysis. photographs to show qd fluorescence and colloidal dispersion in water were taken with a 10.1 mp camera (pentax) whereby a hand - held uv lamp operating at 366 nm (long wave) was shone from beneath the four - windowed quartz fluorescence cuvettes containing the samples. single particle burst analysis and fluorescence lifetimes were acquired on a picoquant microtime 200 fluorescence microscope as previously described. a pulsed laser operating at 485 nm, 15 w, and 5 mhz was used for excitation and focused though the objective (planapo 63xw, olympus) to a diffraction - limited spot. the emission was collected by the same objective and passed through a 100 m pinhole and a 585/55 m filter before being detected on a single photon counting avalanche diode (pdm series, microphotonic devices, bolzano, italy), and the data was saved in time - tagged time - resolved format to enable offline calculation of fluorescence bursts, fluorescence lifetime, and fluorescence correlation spectroscopy (fcs) using the symphotime software (version 5.3.2, picoquant gmbh). the absorption and photoluminescence (pl) spectra of the qds before ligand exchange (qd - oda) and after (qd - mpa, or qd - da) are shown in figure 1. in addition to the characteristic qd exciton absorbance peak at 583 nm and the broad absorbance that increases with increasing energy that is observed in each sample, the absorbance of qd - da shows another intense peak between 400 and 430 nm, which is assigned to transitions in the diacetylene moiety. it was found that using the same initial qd concentration for the ligand exchange resulted in better solubilization efficiency compared to qd - mpa, as evidenced by the stronger color of the solutions shown in the inset of figure 1. this could be a consequence of the diacetylene ligands favoring a close - packing arrangement on the qd surface, resulting in a sam - driven ligand exchange process, whereby hydrophobic interactions between chains result in a higher ligand density compared to the shorter and more soluble (i.e., labile) mpa ligands. in both cases, however, the fluorescence quantum yield decreased by approximately half, which is common for thiolated ligands on qds even those with a core the da ligands decreased the quantum yield a little more than mpa, consistent with a more complete ligand exchange (more thiols) because of sam packing. absorption and photoluminescence spectra of qds before (black) and after ligand exchange with mpa (blue) and da (red). inset : photographs of qds under room light after ligand exchange using mpa (left) and da (right) show relative solubilization efficiency. we hypothesized that using high - energy uv light to directly photo - cross - link diacetylene ligands on qds would not be ideal, since the qd thiol coordination bond would be prone to photooxidation but that qds excited with visible light could be used to initiate photo - cross - linking. to test our hypothesis, we measured the absorption spectra of qd - da in water as a function of time left exposed to room light. one qd - da sample was first exposed to 30 min of uv light, while the other was not. this data is presented in parts a and b of figure 2, respectively. at day 0, the characteristic diacetylene peak between 400 and 430 nm was stronger for the unexposed qd - da compared to the uv - exposed qd - da. this could be a consequence of da ligands undergoing partial cross - linking and/or uv - induced dissociation (vide infra). as the qds were left exposed to room light for up to 2 weeks, significant changes in the absorption spectra were observed for both samples, although the details are somewhat different. both samples show the formation of a new peak at 650 nm, which begins to form within the first day and increases steadily. this increase is concomitant with a decrease in the absorbance in the 350450 nm region. we assign this 650 nm peak to transition of the long, conjugated -electron system that results from extensive cross - linking of the da ligands on the qd induced by visible light. a similar peak has also been observed for similar ligands on gold particles that were irradiated with uv light for 30120 min. in the case of the qds studied here, qd - da that had been exposed to uv light first showed an increase in the 650 nm peak within 7 days and then saturated, indicating that cross - linking stopped prematurely. for the qd - da samples that had not been exposed to uv light first, the 650 nm peak continued to increase for the full 14 days and the decrease in the 350450 nm region was much more pronounced, suggesting that visible - light - induced cross - linking was slower but more extensive, without the competing process of uv - induced ligand dissociation that prematurely halts cross - linking. in both cases, the excitonic qd peak at 583 nm showed a moderate blue - shift : to 572 nm in the case of uv - exposed qd - da and to 568 nm for the non - uv - exposed qd - da. it is interesting to note that, for the uv - exposed samples on day 0, the peak at 650 nm was absent, suggesting that the uv exposure was not able to induce much conjugation even though the 400430 nm peak was attenuated. this is consistent with our interpretation of a competition between uv - induced cross - linking and ligand dissociation which limits the extent of cross - linking that can occur. such a competition was probably not observed in the case of gold, since uv light does not excite gold as it does for semiconductor qds and uv - induced ligand dissociation does not occur for the stable au s bond. uv vis absorption spectra of qd - da in water as a function of exposure to room light over a 2-week period that (a) had been and (b) had not been exposed to 30 min of uv light prior to transfer to water. further evidence that uv light leads to only partial cross - linking of qd - da and competing uv - induced dissociation of the da ligands from the qd is found using ft - ir spectroscopy. figure 3 shows the ft - ir transmittance spectra in three regions of interest as a function of exposure time to uv light. in figure 3a, the 31002500 cm region shows the sp c h stretching peaks and the asymmetric stretch at 2922 cm is used to normalize the spectra. the peak at 3012 cm first increased slightly and then decreased upon uv exposure. this peak is usually assigned to alkenyl c h peaks from c = c bonds. however, cross - linking of diacetylenes should not result in such a peak, unless side - reactions occurred that led to incomplete cross - linking. this may be possible if there was no adjacent diacetylene to cross - link to, due to partial ligand removal, leaving a reactive c = c radical that may have led to the formation of a c = c h bond. figure 3b shows the 21002300 cm region as a function of exposure time of the qd - da to uv light, where cc bonds have a weak but isolated absorbance. in fact, two peaks are observed in this region, one at 2153 cm and another at 2254 cm (black arrows), which are characteristic of diacetylenes. after 20 min of uv exposure, the 2254 cm peak effectively disappeared, while the 2153 cm peak diminished only slightly. as expected from the cross - linking reaction (scheme 1), the cc cc moieties become (c = c cc)n, and the change from a double peak to a single peak within 20 min may reflect this. however, the fact that both peaks decreased within 100210 min suggests that neither type of ligand was present on the particles after extended uv exposure. figure 3c highlights the 14501700 cm region, which shows peaks related to c = c and c = o stretching as well as c h bending modes. the peaks at 1573 and 1604 cm are assigned as stretches from the c = o group from deprotonated and protonated carboxylic acids, respectively, forming a close - packed hydrogen - bonded network of ligands. h bending modes, which remained constant within 20 min of uv exposure and reduced in the 20100 min time window. since the c h stretching frequency was normalized, this reduction may indicate a significant change in the ligand arrangement on this time scale that partially reduces the oscillator strength of bending modes, which could be the result of the cross - linking process increasing the rigidity of the sam. a small peak at 1652 cm appearing after 20 min of uv exposure (black arrow), which was not present before cross - linking, may also be indicative of c = c bonds forming during cross - linking, but due to several overlapping peaks in this region, it is difficult to make a definitive assignment at this point. the ft - ir data can be summarized as follows : changes in the diacetylene peaks show that the uv light caused these groups to react within 20 min and that they are completely gone within 100 min. these changes were also accompanied by small increases in peaks associated with alkenyl c h and c = c bonds in 20 min that then decreased within 100 min, suggesting side reactions limited the extent of cross - linking. in the 020 min time period, the c = o peaks remained but also started to decrease in the 20100 min period. finally, changes in the c h bending modes suggest significant ligand rearrangement on this 20100 min time scale. taken together, the ft - ir data supports the fact that diacetylenes can be partially cross - linked on qd - da within 20 min of uv exposure, but more complete cross - linking using uv light was inhibited by the fact that there was a competing process of ligand rearrangement and dissociation that took over on the 20100 min time scale of uv exposure. the photoluminescence intensity as a function of time exposed to uv and visible light (sequentially) is presented in the supporting information, which shows the effect of these partial cross - linking and ligand dissociation processes on the pl properties. this data provides more evidence that the cross - linking mechanism can be initiated using both uv and visible light and that radicals can actually improve the pl of the qds, in agreement with a recent study, but that continued exposure to uv light is extremely detrimental to the particles. in a previous study, radicals were generated by external initiators to improve the pl of the qds, but the supplementary pl data here shows that radicals generated by the photoexcitation of qds themselves can have a similar effect. ft - ir transmittance spectra of qd - da as a function of uv exposure time showing (a) c h stretching, (b) cc stretching, and (c) c = c and c = o stretching, as well as c the significant advantage of using visible - light qd - mediated photocatalysis for cross - linking rather than uv light is highlighted in figure 4. photographs of the fluorescence from qd - da with or without 30 min prior exposure to uv light dispersed in water and then exposed to room light were taken over several days. as a comparison, qds solubilized with one of the most commonly used, non - cross - linking thiolated ligands, qd - mpa, is shown. qd - mpa showed extensive precipitation from solution even after the first day and the effect was worsened by prior uv exposure, which would be expected for a uv - induced ligand dissociation process. both the qd - da samples showed significant improvement in colloidal stability over qd - mpa. however, the non - uv - exposed qd - da showed better long - term stability than the uv - exposed qd - da, highlighted by the fact that the particles remained suspended in solution for the whole time period. the uv - exposed qd - da began to precipitate from solution within 57 days, and by day 11 was considerably worse than the non - uv - exposed qd - da. fluorescence photographs under 366 nm (long wave) uv light of qd - mpa and qd - da samples that either were or were not exposed to 30 min of 254 nm (short wave) uv light first to show colloidal stability and fluorescence intensity as a function of time. a fluorescence layer at the bottom of the vial indicated extensive aggregation of the qds. only the non - uv - exposed qd - da samples show no visible aggregation over time. the degree of aggregation or dispersion in uv - exposed and non - uv - exposed qd - da was then analyzed using single particle fluorescence spectroscopy on a picoquant microtime 200 fluorescence confocal microscope, after being left in aqueous solution for 14 days under room light. samples were stirred and diluted to pm concentrations and analyzed using burst integrated fluorescence and fluorescence correlation spectroscopy (fcs), the results of which are shown in figure 5. example burst integrated fluorescence traces of particles diffusing through the confocal volume of a fluorescence microscope for (a) visible - light - exposed (black) and (b) uv - light - exposed (red) qd - da, obtained after they have been sitting in water for 14 days and (c) fcs autocorrelation analysis of each qd - da sample to show average diffusion times of particles through the confocal volume. for the non - uv - exposed qd - da, fitted curves with (thick black) and without (thin black) taking blinking into account are included, with both fits recovering at the same diffusion times. blinking is not needed to be taken into account for uv - exposed qd - da (red) due to aggregation. figure 5a shows an example of a burst integrated fluorescence trace from visible - light - exposed qd - da, while figure 5b shows an example from uv - exposed qd - da. the data is binned into 1 ms time points, with the intensity of each point corresponding to the average detector count rate during that 1 ms time bin. the peaks in fluorescence intensity arise from fluorescent particles diffusing through the diffraction - limited focused laser beam ; a single peak corresponding to a single particle diffusing through the focus. the non - uv - exposed samples show much lower intensity bursts, with each peak being narrow, compared to the wide, intense peaks from the uv - exposed samples. these intense, wide peaks are due to large, highly fluorescent aggregates of qds slowly diffusing through the laser focus. the average diffusion time of particles diffusing through the focus is analyzed by calculating the autocorrelation function (acf) from the burst integrated fluorescence traces, which is shown in figure 5c. the black curve shows the acf for the non - uv - exposed qd - da, while the red curve shows uv - exposed qd - da. the diffusion coefficients are calculated by fitting these curves to extract diffusion times through the confocal volume. the data were fit to eq 1, to take blinking into account as previously described.1where2with w0 and z0 describing the width (500 nm) and height (2 m) of the confocal beam, f describes the fraction of particles that blinked during the transit time in the beam and a and describe the power - law blinking dynamics, as previously described. fits both with and without taking blinking into account were performed (i.e., either by setting f = 0 or allowing it to be fit). for the non - uv - exposed samples, the data are fit better at shorter times by taking blinking into account, but both recovered the same diffusion times. blinking was not a factor for the uv - exposed samples, which is expected if particles have aggregated. diffusion times (d) through the confocal volume of 3.09 0.11 ms for the non - uv - exposed qd - da (i.e., cross - linked by qd - mediated visible - light photocatalysis) and 265 5 ms for the uv - exposed qd - da were found. these translate into diffusion constants of 20 1 and 0.236 0.004 m / s, respectively, at the lab temperature of 20 c. using the stokes einstein relation in water at 20 c, these represent hydrodynamic radii of 10 nm for the qd - mediated visible - light cross - linked qd - da and 900 nm for the uv - exposed qd - da. on the basis of the sizes of the core shell qd and the ligand, 10 nm is a reasonable hydrodynamic radius to be expected for single particles, while 900 nm highlights the extensive aggregation of the uv - exposed qd - da. the lack of aggregation of visible - light cross - linked qd - da is also evident from the fact that the blinking parameters are needed to fit the data. we also measured the fluorescence lifetime of these samples to determine the extent of fluorescence quenching caused by the aggregation (supporting information). the qd - mediated visible - light photocatalysis resulted in cross - linked qds that show a fluorescence lifetime in water of 9.2 ns, while the uv - exposed qds show a shorter fluorescence lifetime of 6.9 ns. it is important to reiterate here that no purification step from potential aggregates was performed before taking either of these measurements. the results from this study are schematically depicted in figure 6, in which using a qd - mediated visible - light cross - linking approach for the ligands was able to both significantly avoid interparticle cross - linking and avoid ligand dissociation that resulted from uv exposure. ligand dissociation results in qd aggregation over time, while extensive cross - linking of ligands is able to maintain colloidal stability for long periods of time, highlighting the major outcome from this study. schematic of the differences observed using our visible - light qd - mediated approach to cross - linking compared to using uv light to directly cross - link the ligands. under the high energy uv light, ligand dissociation competes with cross - linking that leads to only partial cross - linking of the ligands and eventual aggregation of the qds. we have designed a novel visible - light photo - cross - linking approach to qd long - term stabilization in aqueous solution. this was achieved by synthesizing a photo - cross - linkable diacetylene ligand and using the qd to improve the cross - linking of the ligands on the particle by a visible - light photocatalytic mechanism which, in turn, improved the colloidal properties of the same qd that helped cross - link the ligands. this method combines the advantage of using ligand exchange to render qds water - soluble with the advantage of controlling cross - linking so that it remains intraparticle rather than interparticle, thus avoiding the formation of aggregates and the need for purification. this new approach has the potential to lead to smaller particle sizes than is possible using amphiphilic polymers by further reducing the size of the bifunctional ligand, although it will be necessary to optimize the correct balance between chain length and sam packing density to achieve such a goal. furthermore, other water - soluble bioreactive functionalities can be incorporated, since the cross - linking moiety is completely separated from the bioreactive functional group on the particle, and does not rely on any other reagents added to the solution to achieve the cross - linking. | ligand cross - linking is known to improve the colloidal stability of nanoparticles, particularly in aqueous solutions. however, most cross - linking is performed chemically, in which it is difficult to limit interparticle cross - linking, unless performed at low concentrations. photochemical cross - linking is a promising approach but usually requires ultraviolet (uv) light to initiate. using such high - energy photons can be harmful to systems in which the ligand nanoparticle bond is fairly weak, as is the case for the commonly used semiconductor quantum dots (qds). here, we introduce a novel approach to cross - link thiolated ligands on qds by utilizing the photocatalytic activity of qds upon absorbing visible light. we show that using visible light leads to better ligand cross - linking by avoiding the problem of ligand dissociation that occurs upon uv light exposure. once cross - linked, the ligands significantly enhance the colloidal stability of those same qds that facilitated cross - linking. |
a 51-year - old male patient with diabetes was admitted to the division of pulmonology with the chief complaints of fever and hemoptysis. a chest computed tomography scan was performed for the evaluation of persistent fever and recurrent pleural effusion, and it showed pulmonary embolism at bilateral multiple segmental arteries and segmental infarction of the involved lung segments. based on the results, echocardiography was additionally performed and revealed isolated pulmonary valve infective endocarditis with severe pulmonary regurgitation. after the diagnosis was made, the patient was treated with antibiotics, followed by echocardiography. the postoperative course was uneventful and the patient was followed up at the outpatient clinic regularly with anticoagulation. after 6 months, he was brought to the emergency unit presenting with a high fever (> 39) and general weakness that had lasted for 3 days. methicillin - sensitive staphylococcus aureus (s. aureus) was isolated from his blood culture, and the echocardiography showed a hypoechoic movable mass on the prosthetic pulmonary valve (fig. 1) and the pressure gradient of the prosthetic pulmonary valve was 16.2 mmhg (maximum pressure gradient)/9.4 mmhg (mean pressure gradient). we did a redo - pulmonary valve replacement under the impression of prosthetic valve infective endocarditis. a secure portion for the new prosthetic pulmonary valve replacement was hard to find because of the risk of pulmonary vessel wall injury. moreover, complete debridement of the remaining infected tissue seemed impossible. thus a valved graft was made by placing a 21 mm tissue valve in the middle of a 22 mm vascutek artificial graft (fig. 3). after completely obliterating the proximal portion of the pulmonary arterial lumen with simple suturing for isolation of probable remnant infected debris, the valved graft was used to perform the redo - pulmonary valve replacement similar to the rastelli operation with right ventricular outflow tract reconstruction (fig. immediate postoperative echocardiography showed intact prosthetic pulmonary valvular function with a peak pulmonary valve pressure gradient of 34 mmhg and a mean pulmonary valve pressure gradient of 16 mmhg. follow - up echocardiography was performed on postoperative day # 19, and the peak pulmonary valve pressure gradient was 20.71 mmhg, while the mean pulmonary valve pressure gradient was 11.14 mmhg. the patient is currently being treated with anticoagulant (coumadin), presenting no respiratory or cardiovascular symptoms during follow - up in outpatient clinic for 2 years after surgery. the last echocardiography was performed at 15 months after surgery ; it showed intact prosthetic valve function with a peak pulmonary valve pressure gradient of 19 mmhg and a mean pulmonary valve pressure gradient of 10 mmhg. prosthetic valve infective endocarditis is a serious and potentially fatal complication of valve replacement. according to the data by arvay and lengyel published in 1988, prosthetic valve endocarditis developed in 19 of 329 patients with bioprostheses (5.8%) and in 8 of 583 patients with mechanical valves (1.4%). the incidence of infective endocarditis that solely involves the pulmonary valve is known to be lower than that involving additional valves. furthermore, the incidence of prosthetic pulmonary valve infective endocarditis occurring after pulmonary valve replacement is also known to be lower than that occurring after replacement of other valves. it is reported that approximately 1% of prosthetic valve endocarditis involves only the pulmonary valve and the total cases reported for isolated pulmonary valve prosthetic infective endocarditis are few in number. early prosthetic valve endocarditis and late prosthetic valve endocarditis can be classified according to the period in which prosthetic valve infective endocarditis occurs after initial valvular surgery. early prosthetic valve endocarditis has been classified as prosthetic valve endocarditis occurring within 12 months after surgery and late prosthetic valve endocarditis as occurring later than 12 months after surgery. also, nosocomial infective endocarditis has been defined as an infection occurring > 72 hours after admission to the hospital or infective endocarditis acquired in association with a significant invasive procedure performed during a recent hospitalization < 8 weeks before the current hospitalization. therefore, this case can be classified early prosthetic valve endocarditis without evidence of nosocomial infection. although there are slight differences among published reports, s. aureus is found to be the most common pathogen causing native valve endocarditis, responsible for approximately 1/4 of all cases. along with coagulase negative staphylococcus, s. aureus is also the most common pathogen found in prosthetic valve endocarditis. in early and late prosthetic valve endocarditis, methicillin sensitive s. aureus is found to be a more frequent cause than methicillin - resistant s. aureus. prosthetic valve endocarditis caused by s. aureus is a fatal complication with mortality rates reaching as high as 40%. the reported incidence of prosthetic valve endocarditis is about 3.1% for 12 months after surgery and 5.7% for 60 months after surgery. however, it is reported that central nervous system complications due to embolization is not an independent risk factor. this is a case report of a 51-year - old male patient with early prosthetic valve endocarditis due to methicillin - sensitive s. aureus who underwent a redo - pulmonary valve replacement using the complete vegetation isolation method for the prevention of recurrence of isolated prosthetic pulmonary valve infective endocarditis that had occurred six months after his initial pulmonary valve replacement with tissue valve. the infection extended to the annulus of the pulmonary valve, so we decided to anastomose to the right ventricular free wall and distal portion of the pulmonary artery, which were not infected, rather than the annulus or proximal portion of the pulmonary artery. however, we could not preserve the annulus of the pulmonary valve, so we needed a vascular conduit for prosthetic valve implantation. after the surgery, we managed follow - up taking into consideration that the artificial graft could have become a re - infection source in the future, so the patient was treated with multiple antibiotics for 12 weeks, postoperative echocardiography 3 times, and repeated blood culture even though he did not have a fever. the patient received effective and successful surgical treatment and was discharged without any significant symptoms or complications. this case suggests that when complete removal of vegetation is difficult either structurally or technically, more diverse surgical techniques should be considered for the prevention of recurrence of infective endocarditis by the remnant vegetation. the limitation of this report is that no such surgery of the same method has been reported, so this method has not yet been proven to be generalizable. also, a lack of long - term follow - up data on the surgery could be another limitation. | a 51-year - old male was admitted to the hospital with complaints of fever and hemoptysis. after evaluation of the fever focus, he was diagnosed with pulmonary valve infective endocarditis. thus pulmonary valve replacement and antibiotics therapy were performed and discharged. he was brought to the emergency unit presenting with a high fever (> 39) and general weakness 6 months after the initial operation. the echocardiography revealed prosthetic pulmonary valve endocarditis. therefore, redo - pulmonary valve replacement using valved conduit was performed in the rastelli fashion because of the risk of pulmonary arterial wall injury and recurrent endocarditis from the remnant inflammatory tissue. we report here on the successful surgical treatment of prosthetic pulmonary valve endocarditis with an alternative surgical method. |
child abuse is a serious social health problem all over the world with important adverse effects. this is an issue concerning millions of children and families which influence significantly children of any ethnicity, color, social status and religion and encompasses all ages (before birth, in infancy, childhood, adolescence, etc.) this is in the range of 4.7 to 91.6% according to the type of maltreatment and place of study (2 - 5). relationship between child abuse and mental disorder is well known in different countries (6 - 9). evidence shows that child abuse and neglect are related to various disorders in adolescence, which include mental disorders, psychological and behavioral disorders, substance abuse, and poor physical health (10 - 14). results in one review found emotional, sexual and physical child abuse as the most serious risk factors for depression. and sexual child abuse and family violence as the greatest risk factors for anxiety disorders (15). other studies assessed the association between childhood sexual abuse and major depression and anxiety at higher age, and found that severe forms of sexual abuse were correlated with a greater risk of outcomes (16 - 19). in iran, our knowledge about various effects associated with child abuse types and specific mental disorders is not sufficient. our overall objective was to extend our understanding of the relation between mental conditions including anxiety, stress and depression, and child abuse and neglect. maybe dispelling this gap in knowledge would disclose findings that could be as an alarm for health professionals and decision makers for prevention and intervention programming aimed at reducing both child abuse and poor mental health outcomes in adolescents. in 2013, we conducted a cross - sectional survey in two secondary school levels (6th and 7th level) in yazd province located in central iran. of the 745 students from 25 classes eligible to participate, 700 completed the questionnaire with a response rate of 94%. the first part of the questionnaire included 20 demographic questions. in the second part a standard self - reported, valid and reliable (minimum internal consistency for sexual domain : 0.87) questionnaire was applied for recording child abuse information in neglect, psychological, physical and sexual domains (20). all questions could be answered by a 4-level scale (never, sometimes, often and almost always). to score the questionnaire, children with positive answer to at least one question in each domain of emotional, physical and neglect were considered victims of that domain ; children with positive answers to at least one question in each domain of emotional, physical and neglect were included in the total number of child abuse victims of all domains. another questionnaire used for measuring mental health situation among the adolescents was dass (depression anxiety stress scales)-42. the dass is a 42-item self report instrument designed to measure the three related negative emotional states of depression, anxiety and tension / stress in adolescents. dass is made up of 42 self - report items to be completed over five to ten minutes, each reflecting a negative emotional symptom. each of these is rated on a four - point likert scale of frequency or severity of the participant s experiences over the last week with the intention of emphasizing states over traits. these scores ranged from 0, meaning that the subject believed the item did not apply to him at all, to 3, meaning that the subject considered the item to apply to him very much, or most of the time (21). in iran validity and reliability of dass-42 questionnaire were assessed (22). correlation coefficient with beck anxiety inventory and beck depression inventory was 0.81 and 0.74 and alpha coefficient for depression, anxiety and stress 0.94, 0.85 and 0.87 respectively (23). adolescents with scores less than 9, 7 and 14 for depression, anxiety and stress were considered as normal by coding zero in dependent outcome, and the other scores as faced with mental disorders by coding one in logistic regression model. mental health and child abuse questionnaire score could vary from 0 to 126, and 38 to 152 respectively. in both of them the collected data was analyzed using spss software and chi - square test, independent t - test and logistic regression analysis. in order to identify predicting factors for mental disorders in students, each variable was entered separately, and in the last step for multivariate analyses, those variables were entered which were significant in the univariate analyses to find the set of best predictors of mental disorders in adolescents using forward conditional model. considering the importance of the issue and the necessity of taking students satisfaction and trust to cooperate honestly in responding to the questions, at first, nature and purpose of the questionnaire were clarified for students and anonymity was also emphasized. of the 700 respondents aged 12 to 18 years (mean 13.3 years ; sd 0.9), 57% were females. the majority (92.8%) of students did not have another job and few (3.8%) of them said they used alcohol or drug. among the adolescents 6.5% were single child in the family whereas 40.7%, 21.7% and 31.1% were first, mean mental disorder score was 30.824.1 (range : 0 - 119) and it was 56.1 6.5 (range : 38 - 81) for child abuse. there was a statically significant correlation between mental disorder and child abuse score (spearman rho : correlation coefficient = 0.2 ; p - value < 0.001) (figure 1). the highest correlation between mental disorders and child abuse was found in psychological domain, spearman s rho coefficients were 0.46, 0.41 and 0.36 for depression, anxiety and stress respectively (p - value < 0.001). other coefficients for correlation between mental disorder (i.e. depression, anxiety and stress) and four domains of child abuse are shown in table 2. p value < 0.001 for all correlations. among adolescents with experience of at least one type of child abuse, suffering from some degree of depression was 45.3% vs 6.8 % in adolescents without it. there were statically significant relationships between severity of different types of mental disorders and experience of at least one type of child abuse (chi square : p - value < 0.001) (table 3). chi square : p value < 0.001. based on the results of simple logistic regression for mental disorder, related variables including type of school (governmental / non profit), sex, birth order, student s or parent s substance abuse and parents living situation in model 1 and four domains of disorders (all p < 0.05), were candidates to enter in the multivariate analysis in two models. however, of the above mentioned variables, sex, birth order, parent s substance abuse in model1 and neglect severity, emotional abuse severity and having experience of sexual abuse (all p < 0.05) in model 2, were significant in the multivariate analysis. based on these results, females, last born adolescents and subjects with parental drug or alcohol abuse had mental disorder odds of 3, 0.4 and 1.9 times compared to others. odds of mental disorder for students with child abuse and neglect in various severities are shown in table 4. model.1 : hosmer and lemeshow test : chi square (6) = 1.4, p - value = 0.96 ; a total of 76.8% of subjects were correctly classified. model.2 : hosmer and lemeshow test : chi square (7) = 6.3, p - value = 0.5 ; a total of 78.8% of subjects were correctly classified. 93.5% of the adolescents had experienced at least one type of neglect, psychological, physical and sexual abuse ranging mild to severe. all 4 types of child abuse and neglect were associated with all types of mental disorders including depression, anxiety and stress. the current findings are also consistent with other studies that found relations with specific types of child abuse and mental conditions (6, 24 - 30), although relationship between assessment of child abuse and mental disorders is not directly comparable because of diversity in instruments or methods. a review from canada reported that all types of abuse are damaging to children physically, emotionally, and psychologically and can cause long - term difficulties with behavior and mental health development (6). in a population - based survey of 1000 children and adolescents aged 10 - 17 years in the usa, a linear relationship between multiple victimization and other childhood disorders as well as adolescent depression and anger / aggression was shown (31). in the present study the highest correlation scores for all mental disorders (depression, anxiety and stress) were found in psychological domain. in a prospective cohort study childhood neglect another study emphasized the effect of sexual abuse on depression and chronic pain in adulthood and revealed that the more severe the abuse, the stronger was the association with poor outcomes in adulthood (32). our results similarly showed a dose - dependent relationship between psychological abuse with mental disorders so that the severe type predicted mental disorders 30 times more than the mild kind in logistic regression model. in the model other variables including sex, birth order and drug or alcohol abuse by parents were considered as predictors of mental disorders. the finding was similar to the results of other studies on iranian adolescents which showed that mental disorders, in particular depression and anxiety, were more prevalent in females (33, 34). among siblings, the lowest rank position appeared to be a protective factor against the development of mental disorders. many studies showed that a higher rank position in the birth order was positively associated with higher risk (35). one study concluded that being the middle child and living with both biological parents appeared to be protective factor against the development of emotional disorders or attention deficit hyperactivity disorders (adhd) (36). we found that alcohol and/or substance abuse by parents predicted depression, anxiety and stress symptoms in adolescents. this result is parallel with a lot of studies which mentioned the effect of parental addiction on psychological, emotional and behavioral disorders on children (37 - 39). our findings show that 93.5% of the adolescents in yazd, iran have experienced at least one mild type of child abuse and neglect and that child abuse has strong relationship with mental conditions. generally child abuse is an important public health disorder in iran as well as other parts of the world. it is notable that in some societies child abuse and neglect is perceived in terms of very harsh physical assault and sometimes this behavior is considered necessary for punishment of the child (40). so awareness of health care providers about child abuse and its association with mental disorders is needed. child abuse should be considered and explored by psychologists who encounter adolescents with mental disorders (i.e., depression, anxiety and stress). mandatory reporting of child abuse by all care givers e.g. those attending schools, in order to prevent or reduce its detrimental effects, is useful. in addition, success in preventing child abuse could lead to reductions in the prevalence of mental disorders. in the present study however, the data from this study were cross - sectional and retrospective in nature. it is probable that some reported types of abuse especially emotional and neglect were due to some mental disorders like depression. moreover many other important mental disorders (e.g., personality disorders) could be included in the study. our findings show that 93.5% of the adolescents in yazd, iran have experienced at least one mild type of child abuse and neglect and that child abuse has strong relationship with mental conditions. generally child abuse is an important public health disorder in iran as well as other parts of the world. it is notable that in some societies child abuse and neglect is perceived in terms of very harsh physical assault and sometimes this behavior is considered necessary for punishment of the child (40). so awareness of health care providers about child abuse and its association with mental disorders is needed. child abuse should be considered and explored by psychologists who encounter adolescents with mental disorders (i.e., depression, anxiety and stress). mandatory reporting of child abuse by all care givers e.g. those attending schools, in order to prevent or reduce its detrimental effects, is useful. in addition, success in preventing child abuse could lead to reductions in the prevalence of mental disorders. in the present study several types of child abuse and mental conditions were assessed. however, the data from this study were cross - sectional and retrospective in nature. it is probable that some reported types of abuse especially emotional and neglect were due to some mental disorders like depression. moreover many other important mental disorders (e.g., personality disorders) could be included in the study. | backgroundchild abuse is a serious social health problem all over the world with important adverse effects.objectivesthe aim of this study was to extend our understanding of the relation between mental disorders and child abuse.materials and methodsthe study was designed as a cross - sectional survey on 700 students in secondary schools using multiple cluster sampling in yazd, iran in 2013. we applied 2 self reported questionnaires : dass (depression anxiety stress scales)-42 for assessing mental disorders (anxiety, stress and depression) and a standard self - reported valid and reliable questionnaire for recording child abuse information in neglect, psychological, physical and sexual domains. the collected data was analyzed using spss software. p - values < 0.05 were considered as significant.resultsthere was a statically significant correlation between mental disorder and child abuse score (spearman rho : 0.2 ; p - value < 0.001). the highest correlations between mental disorders and child abuse were found in psychological domain, spearman s rho coefficients were 0.46, 0.41 and 0.36 for depression, anxiety and stress respectively (p - value < 0.001). based on the results of logistic regression for mental disorder, females, last born adolescents and subjects with drug or alcohol abuser parents had mental disorder odds of 3, 0.4 and 1.9 times compared to others ; and severe psychological abuse, being severely neglected and having sexual abuse had odds 90, 1.6 and 1.5 respectively in another model.conclusionsprogramming for mandatory reporting of child abuse by physicians and all health care givers e.g. those attending schools or health centers, in order to prevent or reduce its detrimental effects is useful and success in preventing child abuse could lead to reductions in the prevalence of mental disorders. |
subcuteneous swelling as metastases are an uncommon presenting feature for primary solid tumors like lung cancer usually in the range of 1.52.6%. it is important to distinguish such metastases from a soft - tissue mass as they may represent the first clinical sign of an occult tumor. in this report, we describe an unusual case of small - cell lung cancer metastasizing to his anterior chest, back and left arm as soft tissue nodule at the time of initial diagnosis ; an aggressive cancer which has a poor prognosis owing to its late presentation. a 64-year - old male, chronic smoker presented in the medicine outpatient department with complaints of breathlessness, loss of weight, multiple swellings on the chest, back and left arm since 2 months. there was no history of trauma, pulmonary tuberculosis, chronic obstructive pulmonary disease, bronchial asthma, ischemic heart disease, hypertension or diabetes. on examination, there were firm, variegated and no tender cystic swellings on the anterior chest, back and left arm (figure 1). the hemoglobin was 9.6 g%, total leukocyte count was 6300/cmm with a differential of 45% neutrophils, 37% lymphocytes, 17% monocytes and 1% eosinophils, in the peripheral smear. serum calcium, phosphorus and alkaline phosphatase were 13.2 mg%, 4.0 mg% and 7.2 bodansky unit, respectively. fine needle aspiration cytology of the swelling from chest showed small cell lung carcinoma seen as small round cells in rosettes and nests with high n / c and pepper salt chromatin (40, pap. computerized tomography of the chest showed pleural effusion, rib fracture with multiple small hypoechoic shadow suggestive of lung cancer (figure 3). he was referred to radio - oncology department for further management but he refused due to non - affordability. figure 2small cell lung carcinoma seen as small round cells in rosettes and nests with high n / c and pepper salt chromatin (40, pap. stain). small cell lung carcinoma seen as small round cells in rosettes and nests with high n / c and pepper salt chromatin (40, pap. figure 3computerized tomography of the chest showing pleural effusion and rib fracture with multiple small hypoechoic shadow, suggestive of lung cancer. computerized tomography of the chest showing pleural effusion and rib fracture with multiple small hypoechoic shadow, suggestive of lung cancer. small cell lung cancer results from bronchial epithelial cells, which are relatives of kultchitsky cells, a type of intestinal epithelial cell. it is much lower compared to adenocarcinomas (2.95%) and squamous cell carcinomas (1.16%) of the lung. the disease most frequently metastasizes to the central nervous system, bone marrow and suprarenal glands. small cell lung cancer may be accompanied by paraneoplastic syndromes, superior vena cava syndromes, compressions to the spinal cord and, very rarely, skin metastases. although they can occur in any part of the skin, most common sites for cutaneous metastases are chest, back, abdomen, and scalp. diagnosis may be delayed by several months, unless the skin lesion grows rapidly or other sites such as the lung or liver are affected by the tumor 's spread. early recognition of tumor from a suspicious skin lesion may lead to initiation of treatment before widespread metastases occur. in our case, the metastasis in the form of subcutaneous swelling was found simultaneously with the primary lung tumor, facilitating diagnosis. although at the time of initial presentation he also had pleural effusion and rib fracture. moreover in this case nature of swelling was not suspicious rather it looked like lipoma and on aspiration cytology it was metastasis from small cell lung cancer. the basic metastatic course can occur in the following steps : detachment from the primary tumor followed by invasion, intravasation into a vessel, circulation, stasis within a vessel, extravasation, invasion into recipient tissue bed, and proliferation. in conclusion, as seen in this case of rare unexpected skin metastasis of small cell lung carcinoma, which lack a pathognomonic physical appearance, like rapid enlargement, ulceration, tender, physicians should be vigilant about this entity. any skin lesions in the form of swelling should be evaluated meticulously and biopsies should be done to exclude metastases in the clinical scenario of breathlessness, chest pain and loss of weight especially in old age. because in this case though both primary and metastasis were detected at the same time, earlier diagnostic anticipation about this cuteneous swelling could have different and better prognosis. | subcutaneous swelling as first clinical presentation of small cell lung carcinoma is uncommon and rarely reported in literature. this case highlights a rare presentation in which subcutaneous swelling was the first clinical manifestation of a small cell carcinoma of lung which also had metastasis to rib bone, muscle and pleural involvement as pleural effusion. we describe the case of a 64-year - old male patient who presented with dyspnea, pleuritic pain, loss of weight and nodule on his anterior chest, back and left arm suspicious of lipoma. biopsies revealed small cell carcinoma of lung. this case demonstrates the meticulous work up of subcutaneous swelling in the clinical scenario of breathlessness, chest pain and loss of weight. |
the overall death rate has been significantly reduced in the last decades, but depending on subtype and stage, still a significant portion of patients will suffer from relapse or even die of the disease [1, 2 ]. while up to 70% of patients with breast cancer can be cured nowadays, a significant proportion of these patients is overtreated. it remains a challenge to identify those patients who will indeed profit from current treatment strategies and also to develop innovative concepts for patients currently at high - risk for relapse after treatment. for this reason, the identification of reliable prognostic biomarkers together with the development of clinically efficient therapies is urgently needed. today, the prognostic clustering of breast cancer in daily routine relies on the determination of a limited set of molecular markers (e.g. estrogen receptor (er), progesterone receptor (pr) and epidermal - growth - factor receptor 2 (her2, also referred to as her2/neu, erbb-2)) mostly by semi - quantitative assays e.g. by immunohistochemistry (fig. 1). clearly, some of these markers are first examples of personalized medicine and targeted treatment since for instance only the determination of er - expression by immunohistochemistry allows for a directed anti - hormonal therapy with receptor blockade or inhibition, or both.. moreover her2-overexpression has paved the way for anti - her2 treatment with the humanized monoclonal antibody trastuzumab [57 ] or the small - molecule inhibitor of the tyrosine kinase domains of her1 and her2, lapatinib [810 ]. the best her2-targeted treatment option together with chemotherapy in patients with metastasized but operable breast cancer is currently assessed in clinical trials. clinical parameters such as tumor size, lymph - node status and age as well as pathologic parameters such as histologic grading, hormone receptor status and her2-status are main factors for risk assignment in breast cancer therapy. this risk assignment results in allocation into a low risk group that may be properly treated with hormonal therapy only or other treatments and a high risk group mainly treated with chemotherapy if no patient specific contradictions apply (e.g. waiving of anthracycline - based chemotherapy in patients with existing heart failure). the intermediate risk group due to uncertain outcome is mainly treated with chemotherapy the best choice of therapy currently under intense clinical studies current clinocopathologic decision making. clinical parameters such as tumor size, lymph - node status and age as well as pathologic parameters such as histologic grading, hormone receptor status and her2-status are main factors for risk assignment in breast cancer therapy. this risk assignment results in allocation into a low risk group that may be properly treated with hormonal therapy only or other treatments and a high risk group mainly treated with chemotherapy if no patient specific contradictions apply (e.g. waiving of anthracycline - based chemotherapy in patients with existing heart failure). the intermediate risk group due to uncertain outcome is mainly treated with chemotherapy the best choice of therapy currently under intense clinical studies in addition to tissue based markers that have prognostic and predictive value, blood - based proteomic tests for early detection of breast cancer are emerging. consequently, non - invasive diagnostic approaches based on pathology - specific molecular - patterns in blood might identify breast cancer in an earlier phase of their disease [1214 ] and might be used to easily monitor therapy responses. nonetheless, breast cancer is clinically heterogeneous with varying response to treatment, even when taking into account the above mentioned therapeutic targets. the established methods that are suited to study one gene at a time do not seem to have the power to cover this clinical heterogeneity, which is likely to be due to a complex set of multiple somatic mutations, epigenetic changes and genomic rearrangements [16, 17 ]. to overcome the limitation of single gene or protein biomarkers, the implementation of dna microarray technology nearly a decade ago has enabled the quantitative measurement of complex gene expression - patterns (gene expression profiling) in breast and other cancers and has paved the way to new pattern - based biomarker strategies. dna array technology has been successfully used to identify subtypes in breast cancer based on their specific gene expression patterns. in general, a molecular taxonomy that allocates breast cancer samples into at least five subtypes, termed basal - like, erbb2, luminal a, luminal b and normal like breast cancer, has been reproduced by several independent groups and is generally accepted as gene - signature based molecular classification [1922 ]. interestingly, these molecular patterns seem to be remarkably stable between primary tumor and distant metastases. the identification of pattern - based biomarkers for prognosis is a major field of current clinical research in many cancer types including breast cancer. only a minority are likely to benefit from such therapy, but all of them will be affected by its toxicity. consequently, the identification of prognostic markers identifying the subset of patients eligible for a watchful waiting procedure and/ or adjuvant anti - hormonal/ anti - her2 therapy could help to minimize therapy - induced side effects. consecutively, expression - based outcome prediction by use of prognostic signatures has been explored in a variety of studies. the first breast cancer prognostic signature to be described has been a 70-gene signature by van it was developed based on the analysis of 78 young (25 (estimated 27% of the study population) receive chemotherapy. about 44% of patients are considered to fall into the primary study group that contains patients with a midrange recurrence score between 11 and 25. these patients, stratified into pre-, peri- or postmenopausal women, are randomized into a taxane - containing or a non - taxane containing chemotherapy to identify the best treatment option for this subset of patients. the tailorx trial has been designed to evaluate the role of intermediate rs in the assignment to adjuvant hormonal therapy alone in comparison to hormonal therapy in combination with chemotherapy. patients with er - positive ln - negative breast cancer are stratified according to their oncotypedx recurrence score. patients with a rs of 25 or higher are assigned to chemotherapy plus hormonal therapy. patients with a rs between 11 and 25 are randomly assigned to chemotherapy plus hormonal therapy (the standard treatment arm) or hormonal therapy alone (the experimental - treatment arm) overview of the tailorx trial. the tailorx trial has been designed to evaluate the role of intermediate rs in the assignment to adjuvant hormonal therapy alone in comparison to hormonal therapy in combination with chemotherapy. patients with er - positive ln - negative breast cancer are stratified according to their oncotypedx recurrence score. patients with a rs of 25 or higher are assigned to chemotherapy plus hormonal therapy. patients with a rs between 11 and 25 are randomly assigned to chemotherapy plus hormonal therapy (the standard treatment arm) or hormonal therapy alone (the experimental - treatment arm) response to 4 cycles of t / fac neoadjuvant chemotherapy might be predicted by a 74-gene profiler that was established in a small study on 42 patients receiving neoadjuvant paclitaxel and fluorouracil + doxorubicin + cyclophosphamide (t / fac) chemotherapy. the study cohort consisted of er - receptor positive and er - receptor negative patients with the majority of patients being her2-negative. 48% of patients had no lymph - node involvement and 52% of patients had n1 or n2 disease. among the 18 patients in the validation cohort the 74-gene profiler correctly predicted complete pathologic response in all three patients achieving complete remission thereby resulting in a positive predictive value of the positive 74-gene profile of 100%. however only a small proportion of patients with a negative 74-gene profile were correctly diagnosed resulting in negative predictive value of only 73% and also this study is to small to draw a conclusion. another pharmacogenomic predictor for complete pathologic response to neoadjuvant t / fac chemotherapy, the (diagonal linear discriminant analysis) gene profiler consisting of only 26 genes, was established based on the analysis of only 30 patients and was subsequently validated in a cohort of 51 patients. in addition, the dlda-30 predictor correctly identified 92% of those patients achieving a pathological complete response, but many patients that are predicted by the dlda-30 to have a cpr do not (ppv of 52%). taken together, all the aforementioned studies assessing response to chemotherapy have several important weaknesses : first, they are mainly based on much too small numbers of patients with too low statistical power considering the thousands of genes measured using array technology, resulting in overfitting of the data. this results in very good predictive values in initial studies, yet as these results are mostly validated by cross - validation within the same dataset they are unlikely to be validated in larger validation studies. the second weakness is the lack of sufficiently large validation studies. in principle, none of these approaches has been sufficiently validated, a prerequisite for clinical application. another critical issue is the inclusion of both patients with er - positive and er - negative tumors in these studies, although it is well known that these subtypes respond differentially to neoadjuvant chemotherapy [86, 87 ]. in an attempt to translate gene expression signatures generated in vitro using chemotherapy sensitive cell lines into clinical application, single - agent drug sensitivity signatures were combined with fec (fluorouracil, epirubicin and cyclophosphamide for six cycles) and t - et (docetaxel for three cycles followed by epirubicin and docetaxel for three cycles) regimen specific signatures. those were subsequently used to predict response in 212 patients with er - negative breast cancer treated with an epirubicin based therapy in the eortc 10994/big 00 - 01 trial. the fec predictor was validated in a set of 66 er - negative breast cancer patients (20% her2 positive) treated with fec from which 28 showed pathological complete responses. the fec predictor predicted a pathological complete response correctly in 27 of 40 patients (ppv : 68%) and accurately identified 25 of 26 patients that did not respond to fec chemotherapy (npv : 96%). in parallel, a tet predictor was validated in 59 patients with er - negative breast cancer (34% her2 positive) treated with the taxane regimen docetaxel for three cycles followed by epirubicin and docetaxel (tet), among which 27 patients showed a pathological complete response. this predictor showed a ppv of 71% and a npv of 92%. from this study, the authors concluded that selection of patients with expression of either the fec or the tet predictor would allow reasonable allocation to the particular treatment. allocation to fec or tet based chemotherapy according to the respective predictor could increase proportion of patients with pathological complete response significantly from 44% to around 70%. an attempt to increase the rate of pathological complete response (pcr) is the addition of gemcitabine and docetaxel to anthracyclin based chemotherapy, either combined (termed gedoc) or dose dense and sequential (termed gesdoc) with pcr rates in up to a quarter of patients [89, 90 ]. the use of gene expression profiling to predict benefit from this intensive chemotherapy was evaluated in 100 patients with er - receptor positive and negative primary non - metastatic breast cancer. using gene expression data from patients with cpr in the gedoc study as training set, a 512 gene expression profile that predicts a pcr after neoadjuvant systemic therapy containing gemcitabine, epirubicin and docetaxel was validated in patients undergoing gedoc treatment. the established predictor showed a 88% overall accuracy with a high sensitivity of 78% and a specificity of 90%. several studies have been reported describing the prediction of therapy response related to a certain chemotherapeutic regimen, e.g. the 74 gene profiler for response to t / fac neoadjuvant chemotherapy, the 512 gene signature for gemcitabine/ docetaxel/ anthracyclin based chemotherapy and the fec and tet predictor for epirubicin based chemotherapy combined with fluorouracil and cyclophosphamide or docetaxel, respectively [74, 83, 88, 91 ]. all of them have been identified as representative gene expression signatures that occur upon specific cytotoxic cell responses, and partially, it was demonstrated that the respective signature is specific for a certain combination of chemotherapeutic agents. when designing a study to search for a gene signature that could predict response to neoadjuvant fec treatment in patients with er - negative tumors treated in the eortc 10994/big 00 - 01 trial, farmer and colleagues found a stroma - related gene signature, the stromal metagene, whose expression was associated with significantly shorter relapse - free survival. interestingly, this signature could predict response not only to neoadjuvant fec treatment, but also to t - fac treatment (neoadjuvant chemotherapy with paclitaxel, 5-fluorouracil, doxorubicin and cyclophosphamide) from another independent cohort of er - negative breast tumors. this suggests that the underlying biological response of the tumor microenvironment might be important for chemotherapy - dependent tumor eradication and the importance of the tumor stroma in metastasis, prognosis and response towards therapy is increasingly recognized [9296 ]. as the stromal metagene showed its power in predicting treatment response, its prognostic power was assessed in an independent cohort of untreated patients from the nederlands kanker instituut (nki) and the erasmus medical center (emc). whereas higher expression of the stromal metagene was associated with a significantly shorter relapse - free survival in patients treated with chemotherapy, it was unrelated to survival in the untreated patients. this underlines the predictive power of the stromal metagene expression signature rather than its prognostic role in untreated patients. since this gene expression signature seemed to be predictive for outcome to more than one chemotherapeutic regimen, the question arises whether the breast cancer intrinsic subtypes themselves respond differentially to chemotherapy. this applies a fortiori, as it is already known that tumor intrinsic factors influence chemotherapy efficiency. the expression of er for example is negatively predictive for response to chemotherapy and the same might be suggested for its accompanying luminal a gene expression profile. indeed, in 82 patients treated with t / fac neoadjuvant chemotherapy basal - like and her2 subgroups were associated with high rates of pcr, 45% and 45%, respectively. in contrast and in line with er receptor status, luminal tumors had a pathological cr rate of only 7% and no pcr was observed in the normal - like subclass. the application of tumor intrinsic subtypes in predicting response to chemotherapy might be used in settings, where it is still difficult to find gene expression patterns that predict for therapy response [98, 99 ]. in conclusion, patients that harbor a prognostic high - risk gene expression profile according to fig. 3 and are assigned to undergo chemotherapy to minimize recurrence rate might be further allocated to tumor - specific chemotherapy. by extraction of gene expression profiles predictive for specific chemotherapies and merging to clinical patient parameters such as age, comorbidities, ecog status, patients might be allocated to the treatment they profit most of (fig. enormous efforts have been undertaken and a high number of reports exist on prognostic tumor markers. however, the number of markers that are clinically useful is still small irrespective whether genomic or proteomic technologies are applied. e.g. the lack of standardized technologies, study design with far too small patient size leading to overfitting thereby resulting in poor performance of established predictors in clinically meaningful validation studies, or even the lack of any meaningful validation studies. moreover, gene expression studies rely on the informative value of the whole specimen from which rna is extracted and expression profiling is conducted. at this point, it is important to emphasize that tumors are a heterogeneous mixture of cells including the tumor cells with varying degree of differentiation but also inflammatory immune cells, surrounding stromal tissue and blood vessels. the amount of the respective cell type varies significantly not only between different tumor stages and grades, but also between tissues of different patients with tumors of the same histological subtype and grade. this clearly influences the designation to a particular prognostic group and consecutively the designation to one or the other therapy. it seems reasonable to dissect the tumor and isolate pure - tumor cell populations prior to gene expression profiling, but evidence is emerging that the interaction of tumor with the stroma and cells of the immune system plays a critical role in tumor progression and response towards chemotherapy [101, 102 ]. furthermore, recent data emphasize the prognostic and predictive significance of stroma - related gene signatures. describe a sdpp (stroma - derived prognostic predictor), a 26-gene expression profile, which irrespective of standard clinical prognostic factors stratifies disease outcome and predicts response to neoadjuvant chemotherapy with two different anthracycline - based regimens, fec and t - fac, in a large cohort of patients. in addition to tumor intrinsic factors such as cellular composition and tumor differentiation, factors related to sample procurement also influence the overall gene expression profile. these include differences in sample preparation, selection of microarray platform and use of hybridization conditions. rna is unstable in tissue samples due to the high prevalence of rnases, requiring quick freezing und processing of the sample. when validating the 70-gene prognosis signature in node - negative breast cancer patients, bueno - de - mesquita and colleagues reported up to 1/3 of samples to be excluded due to bad sample and/ or rna quality. in this regard, rt - pcr based tests as the oncotypedx assay might be used in cases were only paraffin embedded tissue is available and if suited with comparable power regarding its prognostic and predictive capability. unfortunately, no study to date has systemically compared the same patient - samples to multiple test assays to answer this important question. pitfalls in rt - pcr based studies arise from other sources that might be as trivial as e.g the use of different primer pairs for detection. for the analysis of il17br expression, ma. applied a primer set in the 3 region revealed six time higher expression levels when directly comparing to a primer set in the 5 region used by reid. nonetheless, levels of both il17br and hoxb13 correlated and consequently, the il17br : hoxb13 ratio was comparable. when harmonizing studies on prognostic tumor markers, poor study design and analysis, assay variability and inadequate study reporting were identified as major barriers in the field of cancer diagnostics. this analysis lead to the development of the remark guidelines to encourage transparent and complete reporting on newly found prognostic markers. the remark guidelines range from accurate description of patient characteristics to illustration of study design and statistical methods that have been used. ideally, one should be able to compare the expression data obtained in any research facility at any time to any other data obtained in another facility at other time points using other microarray platforms. several approaches to deal with microarray data have been recently described, most of them being at the stage of translational research, but several ready to be implemented into clinical practice. in studies under the aegis of the transbig consortium, the predictive value of the 70-gene expression signature and the 76-gene expression signature are as good as the best validated clinical tool adjuvant!online [29, 45 ]. even more, due to their higher specificity, they both seem to identify low - risk patients better implying a potential to reduce unnecessary chemotherapy. in this regard, it is important to note that the oncotypedx recurrence score and the adjuvant ! online tool estimate different parameters. whereas both the mammaprint signature and the oncotypedx estimate only the risk for distant recurrence (risk of distant metastasis), the adjuvant ! online tool estimates risk for all causes of recurrence (local, regional, distant recurrence and contralateral breast cancer), making an exact comparison between mammaprint, oncotypedx and adjuvant ! online difficult, if not impossible. nonetheless, the majority of signatures are developed from distinct sets of mainly small patient populations with limited validation and follow - up, as obvious in the studies that have established the 92-gene and the 85-gene classifier predictive for docetaxel treatment, the 22-gene classifier predictive for doxorubicin - docetaxel treatment, the 3-gene classifier predictive for doxorubicin - cyclophosphamide treatment, the 28-gene classifier predictive for docetaxel - trastuzumab treatment, the 74-gene and the dlda-30 profile classifier predictive for t / fac chemotherapy, the fec- and tet- predictors, the gedoc predictor and finally both the stromal metagene and the sdpp. this holds partially true for the well established 70-gene profiler mammaprint, a signature superior to traditional clinical predictors within the follow - up of the initial study it was derived from. however, when applying this 70-gene profile to longer follow - up, the gene - expression signature show heterogeneous behavior, indicating that different mechanisms might be responsible for early (within 5 years) and late (beyond 5 years) distant metastasis. interestingly, and discordant with the fear of many researchers, it was shown that different chemotherapeutic agents can elicit similar response signatures. although this naturally has to be validated on larger cohorts, it reminds one that we, even with undirected chemotherapy such as anthracyclins, seem to target specific pathways rather than evenly administering cytotoxicity like with a watering can. other important conclusions can be drawn from the study by bonnefoi.. first, under certain circumstances, gene signatures calculated from cell line data can be recovered in the clinical in vivo setting and second, certain gene expression data obtained from different gene expression platforms might be integrated after biostatistical corrections are performed. besides developing classifiers for prognosis and prediction, gep also can be used to identify genes that e.g. mediate resistance to specific chemotherapeutic agents [105, 106 ]. moreover, gep might guide the pathologist, when correctly allocating histologically graded intermediate grade 2 breast carcinoma into genomic grade 1 or 3. gep has the potential to substantially refine cancer prognosis well beyond what is currently possible with the clinicopathologic indicators. in part, gep is ripe for introduction into the clinic and may guide systemic therapy in the future. this is especially true for the well validated 70-gene prognosis score (mammaprint) and the oncotype dx recurrence score, whereas caution has to be applied to the many genomics signatures assessed in only small subsets of patients likely not reflecting the whole spectrum of disease and therefore not being representative. moreover, beside allocation to the main 5 subtypes of breast cancer, already today gep can be used to identify the molecular basis of the disease. used gep to study seven spontaneous and 15 hereditary breast adenocarcinomas with mutations in either brca1 or brca2. they were able to identify a number of differentially expressed genes between brca1-mutated and brca2-mutated tumors and used these genes to accurately identify breast cancer samples that harbored these genetic mutations. the information deciphered by gep methods might not only accelerate identifying novel molecular targets, but might by providing the clinician a description of the tumors pathology and chemosensitivity accompany the patient through her exertive walk against the cancer. | breast cancer is a complex disease, whose heterogeneity is increasingly recognized. despite considerable improvement in breast cancer treatment and survival, a significant proportion of patients seems to be over- or undertreated. to date, single clinicopathological parameters show limited success in predicting the likelihood of survival or response to endocrine therapy and chemotherapy. consequently, new gene expression based prognostic and predictive tests are emerging that promise an improvement in predicting survival and therapy response. initial evidence has emerged that this leads to allocation of fewer patients into high - risk groups allowing a reduction of chemotherapy treatment. moreover, pattern - based approaches have also been developed to predict response to endocrine therapy or particular chemotherapy regimens. irrespective of current pitfalls such as lack of validation and standardization, these pattern - based biomarkers will prove useful for clinical decision making in the near future, especially if more patients get access to this form of personalized medicine. |
species of the genus leishmania are the causative agents of various parasitic infections which manifest itself in a variety of clinical forms depending upon the species of leishmania and the immunological status of the host. leishmania donovani is the causative agent of visceral leishmaniasis (vl) or kala - azar, which is fatal if patients are left untreated and is more common in less developed countries. the organism has a digenic life cycle residing as flagellated extracellular promastigotes in the gut of insect vector and as nonflagellated amastigotes in mammalian host macrophages. the drugs recommended for treatment of vl, namely, pentavalent antimonials, amphotericin b, and lipid formulations of amphotericin b, have many limitations like long course of treatment, toxic side effects and high costs. moreover, the occurrence of chemoresistance against classical drugs has worsened the situation further [3, 4 ]. thus, search for new drugs, new molecular targets, and novel therapeutic strategies are justified. in search of better leishmanicidal compounds, there has been considerable public and scientific interest in the use of plant derived compounds to combat human diseases. cryptolepine is an indoloquinoline alkaloid which was first isolated from the roots of cryptolepis triangularis collected in belgian congo and afterward from the roots of cryptolepis sanguinolenta from nigeria. this species has been used traditionally to treat malaria, hypertension, hyperglycemia, inflammation and cancer [7, 8 ]. although there are reports of antiparasitic activity of clp, its effect on leishmania donovani is yet to be evaluated. programmed cell death (pcd) appears to be the most preferred mechanism for mediating parasiticidal activity, as has been observed in kinetoplastids in response to diverse stimuli, for example, heat shock, chemotherapeutic agents such as pentostam, amphotericin b, camptothecin, oxidants such as h2o2 or even serum deprivation. apoptosis involves a series of morphological and biological changes including ros production, decrease in cellular gsh levels, and so forth, which ultimately results in dna fragmentation [3, 4, 13 ]. however, it has also been suggested that autophagy provides the front line of defense against oxidative stress and can actually protect cells by preventing them from undergoing apoptosis. autophagy is an evolutionarily conserved mechanism for the degradation of cellular components in the cytoplasm resulting in eventual breakdown and recycling of macromolecules. though autophagic cell death has been suggested to be involved in various systems, the precise role of this catabolic process in dying cells is not clear. in fact, autophagy may have originally arisen as a mechanism to protect unicellular organisms against any form of environmental stress. autophagy plays a role in lifespan extension and sir2 has been suggested to be involved in the process. however, role of autophagy as a survival mechanism in response to drug in leishmania remains to be elucidated. in the present study, we have evaluated the effect of naturally occurring indoloquinoline alkaloid cryptolepine (clp) on l. donovani ag83 promastigotes. we show that clp induces ros in the cells, ultimately resulting in dna fragmentation which is a hallmark of apoptosis. for the first time, we identified that the parasites try to combat against initial clp - induced stress response by initiating an autophagic response as a survival mechanism and activation of silent information regulator protein sir2 plays a role in the process. this study has a great potential in understanding the role of autophagy in the cell death mechanism of leishmania and will be helpful in identifying new drugs and newer therapeutic strategies to combat leishmaniasis in future. cryptolepine (figure 1) hydrochloride was synthesized using isatin and o, n - acetylindoxyl as described previously and was dissolved at 20 mm concentration in 100% dmso and stored at 20c. n - acetyl - l - cysteine (nac) was purchased from sigma - aldrich, was dissolved in 100% dmso at 50 mm and stored at 20c. fm4 - 64 and monodansylcadaverine (mdc) and monochlorobimane were purchased from molecular probes and stored at 20c and room temperature, respectively. the l. donovani strain ag83 promastigotes were grown at 22c in ray 's modified media and in m199 liquid media supplemented with 10% fetal calf serum as described previously. the effect of drug on the viability of l. donovani ag83 promastigote cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (mtt) assay [2, 23 ]. the cells at the exponential phase were collected and transferred into 24-well plate (approximately 4 10 cells / well). the cells were then incubated for various time periods in the presence of different concentrations of cryptolepine hydrochloride (clp). the cell pellet was washed with pbs (1x) twice and then finally suspended in 100 l of pbs (1x) in 96-well plates. ten microliters of mtt solution (10 g / ml) were added in each sample of 96-well plates and samples were incubated for 4 h. after incubation, 100 l of stop solution (stock : 4963 l of isopropanol and 17 l of concentrated hcl) was added and kept for 20 min at room temperature. the optical density was taken at a570 on an elisa reader (multiskan ex ; thermo fisher scientific, waltham, ma). transmission electron microscopy (tem) was carried out with both clp treated and untreated cells as described previously [4, 13 ]. sections were cut with a du - point diamond knife in an lkb ultramicrotome, stained on copper grids with uranyl acetate and lead acetate for 1015 min, respectively, and examined under jeol 100cx tem. l. donovani ag83 promastigotes (approximately 10 cells / ml) were cultured in 24-well plates with different treatments. fm4 - 64 (40 m) (excitation wavelength = 505 nm, emission wavelength = 725 nm) was added directly in the culture medium and kept for 90 min at room temperature. the cells were then washed twice with 1x pbs and stained with 50 m mdc (excitation wavelength = 335 nm, emission wavelength = 518 nm) for 10 min at room temperature. the cells were further washed twice with 1x pbs and live promastigotes were immobilized by mounting under poly - l - lysine coated coverslips as described previously. intracellular ros level was measured in clp - treated and untreated leishmanial cells as described previously. in brief, after treatment with clp and nac for different time periods, cells (approximately 10) were washed and resuspended in 500 l of medium 199 and were then loaded with a cell - permeate probe cm - h2dcfda for 1 h. this is a nonpolar compound that is hydrolyzed within the cell to form a nonfluorescent derivative, which in presence of a proper oxidant converted to a fluorescent product. fluorescence was measured through spectrofluorometer using 507 nm as excitation and 530 nm as emission wavelengths. gsh level was measured by monochlorobimane dye that gives a blue fluorescence when bound to glutathione [2, 3 ]. l. donovani promastigotes (approximately 10 cells) were treated with or without clp at different times. the cells were then pelleted down and lysed by cell lysis buffer according to the manufacturer 's protocol (apoalert glutathione assay kit ; clontech, mountain view, ca). cell lysates were incubated with monochlorobimane (2 mm) for 3 h at 37c. the decrease in glutathione levels in the extracts of nonapoptotic and apoptotic cells were detected by spectrofluorometer with 395-nm excitation and 480-nm emission wavelengths. clp - treated and -untreated l. donovani cells were pelleted down and washed twice with 1x pbs. the fluorescence intensities of the total fluorescent lipid peroxidation products were measured with excitation at 360 nm and emission at 430 nm as described previously [3, 4 ]. the l. donovani promastigotes were treated with clp at 20 m and 3-methyladenine (3-ma) at 10 mm for different times and washed twice with pbs. the cells were then resuspended in 100 l of binding buffer provided with the fluos - annexinv staining kit (roche diagnostics). the cells were stained with annexin v - fitc and pi as per instructions given by the manufacturer, and then they were scanned for fluorescence intensity of cell population in different quadrants. the fraction of cell population in different quadrants was analyzed using quadrant statistics [3, 13 ]. total rna was prepared from l. donovani ag83 promastigotes after different treatments for different times using the total rna isolation kit (roche biochemicals). cdna was synthesized from 60 ng of total rna using superscript ii rnaseh reverse transcriptase (invitrogen) and oligo (dt)1218 primers (invitrogen) following manufacturers instructions. for amplification of the desired cdna, gene - specific primers were designed from sequencing data bank website (table 1). real - time pcr was performed for atg 8, sir2, and gapdh genes. three separate reactions were carried out using three different rna preparations in 25 l volume using sybr - green super mix (applied biosystem) and same primer sets in a 7300 real - time pcr system (applied biosystem). reactions were carried out using the following profile : initial denaturation at 95c for 5 min followed by 35 cycles with denaturation at 95c for 45 s, annealing at 52c for 45 s and extension at 68c for 45 s. the pcr was followed by a melt curve analysis to ascertain that the expected products were amplified. values for each gene were normalized to expression levels of gapdh using the 2 method [25, 26 ]. the fold expression was calculated as described previously [25, 26 ] using the following equation : (1)fold expression=2ct. briefly, genomic dna was isolated from the parasites (approximately 10 cells / ml) after different treatments using an apoptotic dna ladder kit (roche diagnostics). the dna was quantified and equivalent amount of dna was electrophoresed in a 1.5% agarose gel at 75 v for 2 h and thereafter stained with etbr and photographed under uv illumination. l. donovani ag83 promastigotes (4 10 cells / ml) were incubated with five different concentrations of clp (2, 5, 10, 15, and 20 m) for 6, 12, and 24 h after which the cell viability was determined by mtt assay (figure 2(a)). at 12 h, 80% growth was inhibited by 20 m clp which was comparable with the inhibition achieved by 10 m clp at 24 h and 92% growth was inhibited by 20 m clp at 24 h. the effect of clp was to cause both time- and concentration - dependent decrease in cell viability of l. donovani promastigotes. the ic50 value of clp was calculated to be 8.2 m at 12 h in l. donovani ag83 promastigotes. as a positive control, cells were treated with different concentrations of camptothecin (cpt) (2, 5, and 10 m) for 6, 12, and 24 h and cell viability was determined by mtt assay (figure 2(b)). to understand the effect of clp on l. donovani promastigotes in detail, we carried out transmission electron microscopy (tem) with clp - treated and -untreated cells for different time points. dmso treated parasites (control cells) retain the normal nuclear architecture with a prominent central or slightly eccentrically localized nucleolus, while chromatin was usually distributed peripherally beneath the nuclear membrane (figure 3(a)). treatment with clp for 2 h revealed the appearance of multiple cytoplasmic vacuoles, but the nucleus appeared normal with minimum evidence of chromatin condensation. there is also one mitochondrion profile which is swollen, and the matrix appears to be lost (figure 3(b)). however, treatment with clp for 6 h causes extensive damage to the cells. the integrity of the plasma membrane was apparently maintained and membrane blebbing was also observed (figure 3(c)). taken together, these results suggest the involvement of initial autophagic response on treatment of l. donovani promastigotes with clp. however, at a later time period, cells exhibit features of apoptotic like cell death. to confirm the formation of autophagic vacuoles, we next carried out staining with monodansylcadaverine (mdc). mdc is an autofluorescent, autophagolysosome marker that specifically labels autophagic vacuoles in vivo and in vitro conditions [2730 ]. the autophagic machinery involves the fusion of the autophagic vacuoles with the lysosomal compartment for degradation. the lipophilic dye fm4 - 64 is a fluorescent endocytic marker which has been used in leishmania as a marker for the mvt - lysosome [24, 32 ]. fm4 - 64 was found to localize in a tubular compartment in control cells and no fluorescence of mdc was observed under these conditions. however, upon treatment with 20 m clp for 2 h, mdc labeled vesicles were observed which colocalized with fm4 - 64 labelled compartment (figure 4). moreover, pretreatment of cells with 3-methyladenine (3-ma), a specific inhibitor of autophagy [33, 34 ], caused disappearance of mdc labelled vesicles with no change in fm4 - 64 labelling pattern. altogether, these observations suggest the involvement of autophagy in response to clp treatment. the results of the em study suggested that apoptotic like cell death might be occurring in clp treated parasites at a later time point. a key regulator for induction of apoptosis is intracellular ros [3, 4 ]. so, next we wanted to see if clp causes generation of ros inside the cells. to measure the status of ros inside cells, we used a spectrofluorometric assay using cm - h2dcfda as described in section 2. dmso treated cells (control cells) contained a basal level of ros whereas treatment with 20 m clp caused a 4-fold increase in the ros levels in parasites at 3 h time period (figure 5). when cells were pretreated with nac (20 mm), the level of ros generation decreased and was nearly same as that of control cells. thus, it is conceivable from the above result that clp causes oxidative stress in leishmania parasites. one of the most important cellular defenses against intracellular oxidative stress is gsh, which plays a critical role in mediating apoptosis in eukaryotes, including leishmanial cells. gsh is an important molecule for protecting kinetoplastids from ros or toxic compounds. as shown in figure 6(a), clp causes a 49% decrease in gsh level after 3 h and the effect was more pronounced after 6 hrs treatment with clp. when cells were preincubated with nac (20 mm) for 1 h, followed by treatment with clp, gsh level was protected significantly and tends to become normal. lipid peroxidation was assessed by measuring the total fluorescent lipid peroxidation products in leishmanial cells after treatment with clp as described in section 2. clp treatment leads to an increase in lipid peroxides after 3 h of drug treatment and reached saturating level after 6 h. in the presence of 20 mm nac, the level of fluorescent products decreased significantly (figure 6(b)). although treatment of leishmania parasites with clp shows initial features of autophagy, apoptosis - like cell death does occur at the later stage. to understand the relationship, if any, of the autophagic features with the cell death mechanism, we first determined the cell viability after clp treatment when autophagy was inhibited by 3-ma. as evident from figure 7(a), treatment with 20 m clp for 2 h causes a 35% decrease in cell viability compared to control. however, when cells were pretreated with 3-ma, and then treated with 20 m clp for 2 h, there was a 58% decrease in cell viability compared to control. these results suggest that pretreatment of l. donovani ag83 cells with 3-ma makes them more sensitive to clp - induced cell death. cells were treated with 20 m clp for 2 and 6 h with or without pretreatment with 3-ma and the percentage of apoptotic cells was determined by flow cytometric analysis after staining with annexin v - fitc and pi (figure 7(b)). externalization of phosphatidyl serine (stained by annexin v) and presence of impermeant cell membrane (negative pi staining) are hallmarks of pcd. flow cytometric analysis with annexin v / pi staining showed that when cells were exposed to clp for 2 h, about 30.5% cells were annexin v positive (figure 7(b)) but when cells were pretreated with 3-ma and then treated with clp, about 46.2% cells were annexin v positive (figure 7(b)). this suggests that inhibition of autophagy by 3-ma causes an increase the number of apoptotic cells. after 6 h of clp treatment, 49% cells were annexin v positive and when pretreated with 3-ma before treatment with clp for 6 h, about 50.1% cells were annexin v positive (figure 7(b)). interestingly, pretreatment with 3-ma and then adding clp did not cause any formidable increase in the percentage of annexin v positive cells at 6 h time period. as inhibition of autophagy did not cause any significant increase in the cell death at 6 h time period, we surmise that the autophagic response may not influence the clp - induced cell death mechanism at a later time period probably due to the prolonged intracellular stress which commits the cells to die. to understand more clearly the role of the autophagic response in response to clp treatment, we performed rt - pcr analysis with the autophagic gene atg 8 and sir2. sir2 is a member of silent information regulator family of genes and has been implicated in lifespan extension along with autophagy. cytoplasmic sir2 overexpression has been reported to promote survival of leishmania parasites by preventing programmed cell death. thus, we investigated the effect on leishmania sir2 in the autophagic response induced by clp. treatment with clp for 2 h causes marked increase in the mrna level of atg 8 (figure 8(a)). clp caused about 3-fold increase in the level of atg 8 compared to untreated control at 2 h (table 2). however, cells pretreated with 3-ma before clp treatment showed no significant change in atg 8 mrna levels confirming the inhibition of autophagy by 3-ma. treatment with 3-ma only had no effect. interestingly, treatment with clp for 6 h did not show any significant change in the atg 8 mrna levels. this confirms the results of the flow cytometric analysis. level of sir2 was elevated after 2 h in response to clp treatment (figure 8(a)). clp caused about 2.7-fold increase in the mrna level of sir2 compared to untreated control at 2 h (table 2). however, pretreatment with 3-ma before addition of clp caused a slight decrease in the mrna level of sir2. when cells were treated with clp for 6 hrs, there was only 1.5-fold increase in the sir2 mrna level compared to untreated control suggesting the cells to be committed to death. pretreatment with 3-ma caused a decrease in the sir2 level compared to the untreated control cells. taken together, the above results suggest that treatment of l. donovani ag83 promastigotes with clp causes initial autophagic features as a survival mechanism which can be bypassed by employing specific inhibitor of autophagy (i.e., 3-ma). moreover, the results also suggest that the survival mechanism can not cope with the cellular stress at a later time period. the internucleosomal dna fragmentation by an endogenous nuclease (genomic dna fragmentation) is considered as a hallmark of apoptotic cell death [3, 4, 37 ]. we observed internucleosomal dna fragmentation in l. donovani ag83 cells in response to 20 m clp treatment (figure 8(b)). dna fragmentation was significantly enhanced by combined treatment of 3-ma and clp at 2 h compared to clp alone (figure 8(b), compare lanes 3 and 4). however, there was no significant difference at 6 h (figure 8(b), compare lanes 7 and 8). this confirms the involvement of apoptosis - like cell death in l. donovani ag83 cells in response to clp treatment which is augmented by inhibition of autophagy. treatment of leishmaniasis is unsatisfactory due to unavailability of effective vaccines and chemotherapy is still the mainstay for treating this dreaded disease. thus, there is an urgent need for new drug development and newer therapeutic strategies. cryptolepine is a naturally occurring indoloquinoline alkaloid which has been used as an antimalarial drug in central and western africa. cryptolepine has a broad spectrum of biological activity and has been reported to have anticancer activity. in the present study, we have investigated the effect of cryptolepine on l. donovani ag83 promastigotes in vitro. our results show that clp causes a decrease in the cell viability of l. donovani ag83 promastigotes in both time- and concentration - dependent manner. clp causes an increase in cellular ros production with concominant decrease in cellular gsh levels and increase in the level of lipid peroxidation. altogether, these observations suggest the involvement of apoptosis - like cell death in response to clp treatment. however, parasite ultra - structural studies by transmission electron microscopy led to some interesting observations. we observed multiple cytoplasmic vacuoles with normal nuclear architecture at an early stage after clp treatment. this type of vacuolization was suggestive of autophagy [38, 39 ]. to understand the mechanism in more detail, we carried out staining with mdc which specifically labels autophagic vacuoles. it has been suggested previously that leishmania contain a multivesicular tubule which is lysosomal in nature and constitutes the endocytic compartment which is intimately involved with the autophagic pathway. we observed clear mdc - positive vacuoles which colocalized with the multivesicular tubular compartment (fm4 - 64 positive) after treatment with clp. these results confirm the involvement of autophagy in l. donovani ag83 promastigotes in response to clp treatment. to understand the relationship between autophagy induction and apoptosis - like cell death in more detail, we next carried out our study with 3-ma which is a specific inhibitor of autophagy. mtt assay revealed that 3-ma and clp cotreatment causes further decrease in the number of viable cells compared to clp alone. this was further confirmed by flow cytometric analysis suggesting that autophagy serves as a survival mechanism and inhibition of autophagy can amplify the effect of clp on l. donovani ag83 promastigotes. however, this effect is true only at an initial time period (2 h) as we observed no significant changes by inhibiting autophagy at a later time period (6 h). we surmise that at 6 hrs, clp causes extensive damage to the cells which commits them to die rendering them unable to elicit the survival response. real - time pcr analysis revealed that there is a significant upregulation of atg 8 transcript level in response to clp treatment for 2 h though there was no significant change in the atg 8 transcript level at 6 h compared to control untreated cells. this again confirmed the involvement of autophagy in the initial phase of clp treatment. during past few years, the silent information regulator sir2 protein family has attracted great interest due to its implication in an organism 's life span extension. it has been reported previously that sir2 over expression promote survival of leishmania parasites by preventing programmed cell death. also, transient overexpression of sir2 has been clearly shown to stimulate the basal level of autophagy [20, 41 ]. thus, we anticipated a role of sir2 in clp - induced cell death of l. donovani promastigotes. rightfully, real - time pcr analysis revealed a significant upregulation in the sir2 transcript level at 2 h after clp treatment. this suggests that sir2 may signal the onset of autophagy in response to clp treatment. the fact that sir2 can form molecular complex with several atg genes and can deacetylate these proteins explains the importance of sir2 in the process. from an evolutionary perspective, autophagy has been suggested to have originally evolved as a protective mechanism for unicellular eukaryotes against starvation and other environmental stresses. though the connection between autophagy and apoptotic cell death is not clear, autophagy has been reported to promote or inhibit apoptosis in cancer cells. there are also reports of autophagic cell death (type ii cell death) in response to antimicrobial peptides in l. donovani and in response to naphthoimidazoles in t. cruzi. in the present study, we provide experimental evidence to show for the first time that autophagy represents a defense mechanism against clp - induced cell death in l. donovani ag83 promastigotes. we have also shown that the morphological and biochemical changes associated with autophagy precede the onset of apoptosis - like cell death in these unicellular kinetoplastid protozoan parasites. though dissection of the underlying molecular events is beyond the scope of this study, we surmise that sir2 is an important candidate in the regulation of the autophagic response. moreover, our findings also suggest that inhibition of autophagy by 3-ma can actually increase the effectivity of clp - mediated cell killing. this finding can lead to development of new therapeutic strategies to combat leishmaniasis in future. | leishmania donovani are the causative agents of visceral leishmaniasis worldwide. lack of vaccines and emergence of drug resistance warrants the need for improved drug therapy and newer therapeutic intervention strategies against leishmaniasis. in the present study, we have investigated the effect of the natural indoloquinoline alkaloid cryptolepine on l. donovani ag83 promastigotes. our results show that cryptolepine induces cellular dysfunction in l. donovani promastigotes, which leads to the death of this unicellular parasite. interestingly, our study suggest that cryptolepine - induced cell death of l. donovani is counteracted by initial autophagic features elicited by the cells. for the first time, we show that autophagy serves as a survival mechanism in response to cryptolepine treatment in l. donovani promastigotes and inhibition of autophagy causes an early increase in the amount of cell death. this study can be exploited for designing better drugs and better therapeutic strategies against leishmaniasis in future. |
paclitaxel (ptx) is one of the most effective antitumor drugs found in nature in the past few decades.1,2 it promotes polymerization of tubulin dimers, which results in the formation of highly stable nonfunctional microtubules in the absence of guanosine triphosphate and microtubule - associated proteins, ultimately preventing cell division.3,4 because of its unique mechanism of action against tumor cells, ptx has demonstrated excellent therapeutic efficacy for a wide range of cancers, although it works especially well for treatment of ovarian and breast cancers.1 paclitaxel is hydrophobic and is poorly soluble in water. currently, the ptx used clinically is in a vehicle composed of cremophor el and ethanol at a 50:50 (v / v) ratio. however, this solvent causes severe hypersensitive reactions and cytotoxicity and is incompatible with polyvinyl chloride, which is commonly used in injection systems.2,5 in addition, ptx can cause serious side effects at excessive doses, such as bone marrow suppression and cardiac rhythm disturbances.3 therefore, researchers have developed a variety of vehicles, including microspheres, nanoparticles, surgical pastes, polymeric micelles, and hydrogels, to control ptx release and to eliminate the toxicity of cremophor el to the body.1,3 hydrogels have been commonly used in drug delivery, and various natural polymers and synthetic copolymer hydrogels, such as chitosan, hyaluronic acid, alginate, polymethyl methacrylate, and polyethylene glycol, have been studied. these compounds usually form hydrogel - drug complexes outside of the body and are then delivered into the body.59 however, the formation of hydrogels frequently involves ultraviolet photopolymerization and various chemical cross - linking techniques, and there is a potential risk that toxic reagents may fail to be removed completely prior to hydrogel implantation. furthermore, some hydrogels have high elasticity, which generally prevents their extrusion through a syringe needle.6 therefore, an alternative vehicle is necessary. self - assembling peptides are a type of designed biomaterial and have been used in a number of biomedical applications, such as cell culture, tissue engineering, and drug delivery.1013 self - assembling peptides can spontaneously assemble to cross - linked nanofibers of 1020 nm in diameter and form a stable second structure of -sheet in aqueous solution. under physiological conditions, the peptide solution can rapidly self - assemble into hydrogels, which contain more than 99% (w / v) water.11,12,14 previous studies have used rada16 peptide to slow the release of proteins and localized therapies through injection into a particular tissue.1518 because the process of hydrogel formation is ionic - dependent and is irrelevant with ultraviolet or chemical cross - linking, it was supposed that rada16 may serve well as a potential drug delivery vehicle. the purpose of this study was to develop an injectable in situ gel drug delivery system based on a self - assembling peptide. the incorporation of peptide and drug was achieved simply by mix and magnetic stirring. to investigate the interaction of rada16-ptx, the release properties of hydrogel, and antitumor efficiency in the current study, we performed a morphology analysis, circular dichroism, dynamic light scattering, rheological test, in vitro release test, and a cell viability test. self - assembling peptide rada16 (n - radaradaradarada - c) was commercially synthesized and purified by shanghai biotech bioscience and technology co ltd (shanghai, china). paclitaxel was purchased from the national institute for the control of pharmaceutical and biological products (beijing, china). peptide solutions at concentrations of 5 and 10 mg / ml were prepared by dissolving rada16 peptide powder in milli - q water with 10 minutes of sonication. the peptide solution incorporated with ptx was prepared by adding peptide solution (5 or 10 mg / ml) into a glass vial with ptx at a final concentration of 1 mg / ml, followed by magnetic stirring for 48 hours, whereas the control sample was formed by adding milli - q water instead of peptide solution. all of the vials and solvents were sterilized, and the samples were prepared in a biological safety cabinet to avoid possible contamination for cell culture experiments. the morphology of samples was observed using an atomic force microscope (spi4000 probe station, seiko instruments inc, chiba, japan) with a tapping mode. for the preparation of the samples, rada16 solution or rada16-ptx suspension was diluted 50 times in milli - q water, and then 10 l of diluted solution was evenly placed on a freshly cleaved mica substrate. the mica surface was then rinsed with milli - q water to remove unattached peptide and air - dried. the measurement was performed at room temperature and all images were recorded with a resolution of 512 512 pixels. the typically scan parameters were set as follows : scan speed 1.00 hz, amplitude 11.2 v, and integral and proportional gains of 0.20.3 and 0.030.04, respectively. the particle size distribution of the rada16 solution and rada16-ptx suspension was investigated on a nano - particle size analyzer (lb-550, horiba ltd, kyoto, japan) with appropriate viscosity and refractive index settings, and the temperature was maintained at 25c during measurement. the rada16 solution and rada16-ptx suspension was diluted 20 times with milli - q water, and each sample was tested at least three times to generate the intensity - based size distribution plot report. the circular dichroism measurement was performed at 25c on an aviv model 400 (aviv biomedical inc, lakewood, nj) with a 2 mm path length quartz cuvette. all samples of rada16 solution and rada16-ptx suspension were diluted 50 times with milli - q water before the test, and then circular dichroism spectra were collected at 1 nm intervals and 1 nm bandwidths from 190 to 260 nm, with three time scans for the average. all spectra were corrected by subtracting the baseline, and the data were expressed as molar ellipticity [] with units of deg the secondary structure fractions of the peptide were calculated by free software cdpro using a modified contin method (continll program) and comparison with a set of reference proteins. the rheological properties of rada16-ptx suspension were measured on a rheometer (ar2000, ta instruments, new castle, de) with a 20 mm diameter and a 1 steel cone with 25 m truncation gap. a 70 l sample of rada16-ptx suspension was placed on the plate of the rheometer and a trap was placed around the cone to seal the liquid. the storage (g) and loss (g) modulus was measured at 37c, and the parameters used for the frequency sweep tests were strain 0.5% and frequency range 0.01100 rad / sec. for rheological testing of peptide - ptx hydrogel, rada16-ptx suspension was first induced to gel by adding 70 l of phosphatebuffered saline solution. after equilibrating for one minute, the excess solution was taken off, and the measurement was performed as described above. the release of ptx from rada16-ptx hydrogel was carried out in vitro in phosphate - buffered saline solution. rada16-ptx suspension 100 l was dripped to the bottom of a cuvette, then 10 ml 0.1 m phosphate - buffered saline (ph 7.4) with 0.3% (w / v) sodium dodecyl sulfate was added gently and incubated at 37c for an indicated time period. the concentration of ptx in phosphate - buffered saline was determined by reversed - phase high - pressure liquid chromatography, performed with a shimadzu lc-20 a high - pressure liquid chromatography system (shimadzu corporation, kyoto, japan). the mobile phase consisted of methanol and water (70:30). the injection volume was 20 l and the mobile phase flow rate was 1.0 ml / min. separation was achieved by a c-18 column (shim - pack vp - ods, 150 mm 4.6 mm, 5 m). a calibration curve of ptx was constructed to determine the percentage of ptx released from the peptide hydrogel. at the end of the release test, the human breast cancer cell line mda - mb-435s was used for the in vitro cell viability study. the cells were cultured in l-15 medium containing 10% fetal bovine serum at 5% co2. when cells reached 85% confluence, they were detached from cell culture flasks with trypsin and resuspended in cell culture medium at a concentration of 10 cells / ml. after 200 l of the cell suspension was added to each well of a flat - bottom 96-well plate and incubated overnight, the culture medium was removed. twenty microliters of diluted ptx (taiji pharmaceutical co ltd, sichuan, china), rada16, or rada16-ptx suspension was then pipetted into the central bottom of the well and equilibrated with 200 l of culture medium to initiate peptide gel formation. briefly, at different time points, 200 l of serum - free medium and 20 l of mtt solution (5 mg / ml in phosphate - buffered saline) were added to each sample, followed by incubation at 37c for four hours to allow mtt formazan formation. the medium was then removed, and the converted dye was dissolved with 200 l of 10% sodium dodecyl sulfate in 0.01 m hcl. after all crystals were solubilized, the optical density of the solution was determined at 570 nm against an sodium dodecyl sulfate solution blank using an enzyme - linked immunosorbent assay plate reader. cell viability was calculated by the following equation : in which abstest cells and abscontrol cells were the absorbencies of cells with different treatments and cells incubated with medium only, respectively. cell viability was also assessed by a live / dead assay. a work solution containing both 1 m calcein am and 2 m ethidium homodimer in phosphate - buffered saline were prepared according to the live - dead assay (molecular probes, invitrogen, carlsbad, ca) package instructions, and 200 l of work solution was added to each well. statistical analysis was performed using spss software (version 11.5, chicago, il) with one - way analysis of variance and a 95% confidence level. self - assembling peptide rada16 (n - radaradaradarada - c) was commercially synthesized and purified by shanghai biotech bioscience and technology co ltd (shanghai, china). paclitaxel was purchased from the national institute for the control of pharmaceutical and biological products (beijing, china). peptide solutions at concentrations of 5 and 10 mg / ml were prepared by dissolving rada16 peptide powder in milli - q water with 10 minutes of sonication. the peptide solution incorporated with ptx was prepared by adding peptide solution (5 or 10 mg / ml) into a glass vial with ptx at a final concentration of 1 mg / ml, followed by magnetic stirring for 48 hours, whereas the control sample was formed by adding milli - q water instead of peptide solution. all of the vials and solvents were sterilized, and the samples were prepared in a biological safety cabinet to avoid possible contamination for cell culture experiments. the morphology of samples was observed using an atomic force microscope (spi4000 probe station, seiko instruments inc, chiba, japan) with a tapping mode. for the preparation of the samples, rada16 solution or rada16-ptx suspension was diluted 50 times in milli - q water, and then 10 l of diluted solution was evenly placed on a freshly cleaved mica substrate. the mica surface was then rinsed with milli - q water to remove unattached peptide and air - dried. the measurement was performed at room temperature and all images were recorded with a resolution of 512 512 pixels. the typically scan parameters were set as follows : scan speed 1.00 hz, amplitude 11.2 v, and integral and proportional gains of 0.20.3 and 0.030.04, respectively. the particle size distribution of the rada16 solution and rada16-ptx suspension was investigated on a nano - particle size analyzer (lb-550, horiba ltd, kyoto, japan) with appropriate viscosity and refractive index settings, and the temperature was maintained at 25c during measurement. the rada16 solution and rada16-ptx suspension was diluted 20 times with milli - q water, and each sample was tested at least three times to generate the intensity - based size distribution plot report. the circular dichroism measurement was performed at 25c on an aviv model 400 (aviv biomedical inc, lakewood, nj) with a 2 mm path length quartz cuvette. all samples of rada16 solution and rada16-ptx suspension were diluted 50 times with milli - q water before the test, and then circular dichroism spectra were collected at 1 nm intervals and 1 nm bandwidths from 190 to 260 nm, with three time scans for the average. all spectra were corrected by subtracting the baseline, and the data were expressed as molar ellipticity [] with units of deg the secondary structure fractions of the peptide were calculated by free software cdpro using a modified contin method (continll program) and comparison with a set of reference proteins. the rheological properties of rada16-ptx suspension were measured on a rheometer (ar2000, ta instruments, new castle, de) with a 20 mm diameter and a 1 steel cone with 25 m truncation gap. a 70 l sample of rada16-ptx suspension was placed on the plate of the rheometer and a trap was placed around the cone to seal the liquid. the storage (g) and loss (g) modulus was measured at 37c, and the parameters used for the frequency sweep tests were strain 0.5% and frequency range 0.01100 rad / sec. for rheological testing of peptide - ptx hydrogel, rada16-ptx suspension was first induced to gel by adding 70 l of phosphatebuffered saline solution. after equilibrating for one minute, the excess solution was taken off, and the measurement was performed as described above. the release of ptx from rada16-ptx hydrogel was carried out in vitro in phosphate - buffered saline solution. rada16-ptx suspension 100 l was dripped to the bottom of a cuvette, then 10 ml 0.1 m phosphate - buffered saline (ph 7.4) with 0.3% (w / v) sodium dodecyl sulfate was added gently and incubated at 37c for an indicated time period. the concentration of ptx in phosphate - buffered saline was determined by reversed - phase high - pressure liquid chromatography, performed with a shimadzu lc-20 a high - pressure liquid chromatography system (shimadzu corporation, kyoto, japan). the mobile phase consisted of methanol and water (70:30). the injection volume was 20 l and the mobile phase flow rate was 1.0 ml / min. separation was achieved by a c-18 column (shim - pack vp - ods, 150 mm 4.6 mm, 5 m). a calibration curve of ptx was constructed to determine the percentage of ptx released from the peptide hydrogel. at the end of the release test, the human breast cancer cell line mda - mb-435s was used for the in vitro cell viability study. the cells were cultured in l-15 medium containing 10% fetal bovine serum at 5% co2. when cells reached 85% confluence, they were detached from cell culture flasks with trypsin and resuspended in cell culture medium at a concentration of 10 cells / ml. after 200 l of the cell suspension was added to each well of a flat - bottom 96-well plate and incubated overnight, the culture medium was removed. twenty microliters of diluted ptx (taiji pharmaceutical co ltd, sichuan, china), rada16, or rada16-ptx suspension was then pipetted into the central bottom of the well and equilibrated with 200 l of culture medium to initiate peptide gel formation. briefly, at different time points, 200 l of serum - free medium and 20 l of mtt solution (5 mg / ml in phosphate - buffered saline) were added to each sample, followed by incubation at 37c for four hours to allow mtt formazan formation. the medium was then removed, and the converted dye was dissolved with 200 l of 10% sodium dodecyl sulfate in 0.01 m hcl. after all crystals were solubilized, the optical density of the solution was determined at 570 nm against an sodium dodecyl sulfate solution blank using an enzyme - linked immunosorbent assay plate reader. cell viability was calculated by the following equation : in which abstest cells and abscontrol cells were the absorbencies of cells with different treatments and cells incubated with medium only, respectively. a work solution containing both 1 m calcein am and 2 m ethidium homodimer in phosphate - buffered saline were prepared according to the live - dead assay (molecular probes, invitrogen, carlsbad, ca) package instructions, and 200 l of work solution was added to each well. statistical analysis was performed using spss software (version 11.5, chicago, il) with one - way analysis of variance and a 95% confidence level. the self - assembling rada16 peptide is a designed nanoscale biomaterial, the sequence of which contains alternating hydrophilic and hydrophobic amino acid residues. paclitaxel is a hydrophobic antitumor drug that is poorly soluble in water. after stirring for 48 hours, ptx in rada16 solution was changed to a colloidal suspension, while the same component in pure water was still clear, and a significant amount of ptx powder was visible (figure 1). figure 2a and c showed the typical morphology of rada16, which consisted of multiple cross - linked nanofibers that were 21.5 3.3 nm in width and 2.2 0.2 nm in height. after stirring with ptx for 48 hours some bulged nanofibers and aggregates could be seen (figure 2b and d), and the width and height of those nanofibers were 45.3 10.6 nm and 5.6 2.3 nm, respectively. some ptx particles coated by peptide nanofibers were also observed (figure 2b and d). from the circular dichroism analysis, the rada16 peptide had a typical -sheet structure (minimum molar ellipticity at 215217 nm and maximum at 195206 nm), with 50.6% -sheet content (figure 3 and table 1, s (r) + s [d ]) according to the cdpro software analysis.19 however, when rada16 was stirred with ptx for 48 hours, there was an approximately 18.4% decrease in the -sheet content and a 19.8% increase in the unordered structure content. dynamic light scattering measurements showed that the diameter of nanofibers from a pure rada16 solution was in the range of 1050 nm. when ptx was stirred with 0.5% and 1% rada16 solution, the mean particle sizes were 2744.7 551.3 nm and 2569.4 434.9 nm, respectively, but there was no difference between the 0.5% and 1% rada16 solution groups (figure 4). when ptx was stirred with milli - q water, the mean particle size was over 6 m, and dynamic light scattering measurements could not be conducted. the circular dichroism data showed that the -sheet structure of rada16 was impaired to some extent by the addition of ptx, a result that was in accordance with the change in morphology on atomic force microscopy. the particle size of ptx was obviously decreased in the rada16 solution compared with that in the water. all of the data indicated that rada16 did not simply mix with ptx but rather interacted with it. as illustrated in figure 5, rada16 consists of repeated sequences of arg - ala - asp - ala, in which arg and asp are hydrophilic amino acid residues and ala is a hydrophobic amino acid residue ; the -sheet structure of the peptide therefore had both hydrophobic and hydrophilic regions.15 because ptx is a typical hydrophobic compound, the hydrophobic regions of peptide would be adsorbed to the hydrophobic surface of separated ptx particles under stirring, a phenomenon that resulted in the impaired -sheet structure of rada16 shown in the circular dichroism data. the atomic force microscopy images showed that there were many bulged rada16 nanofibers, which may have resulted from ptx molecules that were incorporated with nanofibers or from the aggregation of nanofibers induced by hydrophobic interactions. the data indicate that the self - assembling peptide and ptx could interact with each other and that the amphiphilic peptide was able to stabilize hydrophobic drugs in aqueous solution, a finding that was in agreement with previous reports.2022 this type of interaction also helps to increase the solubility of ptx and decrease the mean particle size of ptx in rada16 solution under stirring. therefore, the rada16-ptx suspension could potentially be injected into the body with a syringe needle. these results indicated that ptx molecules and particles can coexist in rada16- ptx suspension, and stabilized ptx can be obtained in this system. however, further investigation is necessary to determine the percentage of each state in the complexes. the rada16 solution was able to form a hydrogel in ionic environments, such as phosphate - buffered saline, physiological saline, and body fluid. however, could the rada16-ptx suspension gel under physiological conditions ? the test was performed at 37c, and phosphate - buffered saline was used to simulate body fluid. the storage modulus (g) and loss modulus (g) responded to the elasticity and viscosity of materials, respectively. as shown in figure 6, the frequency sweep indicated that the value of the storage modulus (g) in the rada16-ptx suspension was larger than that of the loss modulus (g) both in 0.5% and 1% rada16, and all of the low values (< 10 pa) for the storage and loss moduli were typical for low viscosity systems with a low degree of elasticity. with the addition of medium the storage modulus of 0.5% rada16 was over 300 pa and that of 1% rada16 was over 1000 pa, whereas the original values for both 0.5% and 1% rada16 were under 10 pa. the great increase in the storage modulus suggested the formation of a strong hydrogel. at the mean time, it was observed that the storage modulus of 0.5% rada16 hydrogel was much lower than that of the 1% rada16 hydrogel, which indicated that the concentration of peptide had a positive correlation with the elasticity of the hydrogel. the results demonstrated that the rada16-ptx suspension could form a highly elastic hydrogel under physiological conditions. as an in situ forming gel drug delivery system, the ideal vehicle should be an injectable solution in vitro and change to a gel in vivo. rada16 was able to form a hydrogel rapidly under physiological conditions without heating or chemical reaction, making it a favorable vehicle for drug delivery. paclitaxel is a hydrophobic drug that is poorly soluble in water. to maintain sink conditions throughout the release process, sodium dodecyl sulfate was added to the release medium to increase the solubility of ptx.5 the release was observed over a period of five days. as shown in figure 7b, the rada16 -ptx suspension could form a milky - white gel in phosphate - buffered saline, and the volume of hydrogel was obviously reduced over the release time. the resulting release profiles of ptx from the rada16-ptx hydrogels are shown in figure 7a. when a hydrogel was prepared with 1% rada16, approximately 40%45% of incorporated ptx was released from the hydrogel within the first day, with a half - release time of 2430 hours. in contrast, more than 60% of the ptx was released from the hydrogel within one day for the 0.5% rada16 hydrogel, and the half - release time was 810 hours. these results clearly demonstrated that a higher concentration of peptide in the hydrogel produced slower drug release rates. the initial burst effect was lower for the 1% rada16 hydrogel compared with the 0.5% rada16 hydrogel, and an increase in peptide concentration slowed the release rates of the loaded drug, resulting in a longer half - release time for the ptx. the atomic force microscopic image showed that many nanoparticles were present in the supernatant of the release medium (figure 8a), and the width and height of the nanoparticles were 83.1 30.3 nm and 4.9 2.9 nm, respectively. however, sodium dodecyl sulfate was also an amphiphilic molecule, which might form assemblies in aqueous solution. thus, an additional atomic force microscopy test with only sodium dodecyl sulfate in the release medium was performed to verify this result. the resulting images showed that sodium dodecyl sulfate was obviously accumulated on the surface of mica, and no similar size of nanoparticles formed (data not shown). these data suggest that ptx was encapsulated in peptide nanofibers and that rada16-ptx nanoparticles were formed in the release process. a schematic representation of a possible model for ptx release from the ptx - rada16 hydrogel is shown in figure 8b. considering the data from morphology and rheology analysis, it was found that the higher concentration of peptide solution had more cross - linked nanofibers and led to the formation of a more elastic hydrogel. because hydrogel formed from a higher concentration of peptide showed increased stability with a higher density of nanofibers and smaller sizes of pores, the probability of specific interactions between ptx and peptide nanofibers and diffusion hindrance was increased, yielding a decrease in the diffusivity of ptx and finally resulting in slower drug release rates.15,16 these results suggested that the release rates of ptx can be controlled by changing the peptide concentration, so the release time could be extended by adding a greater amount of peptide in future studies. in addition, the addition of sodium dodecyl sulfate as a surfactant can increase the release rates of ptx from the hydrogel, an effect that would be caused by the increased degradation rates of the rada16 peptide. as an antiproliferation drug, ptx is a potent inhibitor of angiogenesis, cell migration, and collagenase production. because it has been demonstrated that ptx has significant antitumor activity against various solid tumors, including breast cancer, the human breast cancer cell line mda - mb- 435s was used to study the antitumor efficiency of the rada16-ptx hydrogel. as in previous reports, the antiproliferative activity of the complex was dependent on the concentration of ptx.2 to simulate the in vivo situation and optimize the antitumor test, ptx10 g was administered to each well of the 96-well plate, and the culture medium was changed every day. in our study, the data showed that single use of rada16 produced no inhibition of tumor cell proliferation, while the rada16-ptx hydrogel inhibited growth of tumor cells effectively. as shown in figure 9, there was no significant difference in cell viability rates between the ptx - treated group and both rada16-ptx - treated groups at day 1. at day 3, there was a significant difference between the ptx group and the 1% rada16-ptx group, but there was no significant difference between the 0.5% and 1% rada16-ptx groups. at days 5 and 7, compared with the ptx group, both rada16-ptx groups demonstrated lower cell viability rates, and the cell viability rate in the 1% rada16-ptx group was significantly lower than that in the 0.5% rada16-ptx group. these data suggest that ptx and 0.5% rada16-ptx had poor ability to inhibit cell growth after days 3 and 5, respectively, whereas 1% rada16-ptx hydrogel maintained relatively high inhibitory activity even at day 7. it was concluded that the rada16 hydrogel could control release of ptx effectively and that a higher concentration of peptide hydrogel resulted in an increased release time for ptx. the fluorescence images showed that the tumor cells in the rada16 group continued to proliferate over the course of the observed days (figure 10). by contrast, in the ptx group and both rada16-ptx groups, only a few cells could be seen at day 3, which indicated that cell proliferation was inhibited by ptx. however, the number of tumor cells in the ptx group was slightly higher than that in both rada16-ptx groups. at day 5, in comparison with the 1% rada16-ptx group, more tumor cells were observed in the ptx and 0.5% rada16-ptx groups. at day 7, compared with previous days, an increased number of tumor cells was observed in the ptx group and both rada16-ptx groups, but fewer cells were seen in the 1% rada16-ptx group than those in the ptx group and the 0.5% rada16-ptx group. it was concluded that the 1% rada16-ptx hydrogel had a longer lasting inhibitory effect on proliferation of tumor cells than did the other tested groups. these results indicate that the self - assembling peptide system could be used to control the release of ptx. the release time of ptx from the hydrogel showed increased correlation with the concentration of peptide, and a more elastic hydrogel led to stronger interactions between the drug and the nanofibers, which decreased the diffusion rates of ptx and resulted in longer drug release times. the spontaneously gelling ability of rada16-ptx suspension in an ionic environment provided the controlled release of ptx, which would increase the contact time of drug at the site of action and enhance the efficiency of therapy.5,23 because ptx can not differentiate between cancer and normal cells, which may result in major toxicity to normal cells, the potential application of peptide hydrogel to the localized delivery of ptx might minimize the cytotoxicity and side effects. in addition, the self - assembling peptide was a biocompatible and biodegradable nanomaterial, which previous studies have reported to produce no toxic and immune response, and this material has been widely used for cell culture and injection into animals.2426 therefore, it suggested that this self - assembling peptide is a favorable vehicle for local ptx delivery as an in situ - forming hydrogel system. nevertheless, further experiments are still needed to estimate the antitumor efficiency of the peptide- ptx system in vivo. the self - assembling rada16 peptide is a designed nanoscale biomaterial, the sequence of which contains alternating hydrophilic and hydrophobic amino acid residues. paclitaxel is a hydrophobic antitumor drug that is poorly soluble in water. after stirring for 48 hours, ptx in rada16 solution was changed to a colloidal suspension, while the same component in pure water was still clear, and a significant amount of ptx powder was visible (figure 1). figure 2a and c showed the typical morphology of rada16, which consisted of multiple cross - linked nanofibers that were 21.5 3.3 nm in width and 2.2 0.2 nm in height. after stirring with ptx for 48 hours some bulged nanofibers and aggregates could be seen (figure 2b and d), and the width and height of those nanofibers were 45.3 10.6 nm and 5.6 2.3 nm, respectively. some ptx particles coated by peptide nanofibers were also observed (figure 2b and d). from the circular dichroism analysis, the rada16 peptide had a typical -sheet structure (minimum molar ellipticity at 215217 nm and maximum at 195206 nm), with 50.6% -sheet content (figure 3 and table 1, s (r) + s [d ]) according to the cdpro software analysis.19 however, when rada16 was stirred with ptx for 48 hours, there was an approximately 18.4% decrease in the -sheet content and a 19.8% increase in the unordered structure content. dynamic light scattering measurements showed that the diameter of nanofibers from a pure rada16 solution was in the range of 1050 nm. when ptx was stirred with 0.5% and 1% rada16 solution, the mean particle sizes were 2744.7 551.3 nm and 2569.4 434.9 nm, respectively, but there was no difference between the 0.5% and 1% rada16 solution groups (figure 4). when ptx was stirred with milli - q water, the mean particle size was over 6 m, and dynamic light scattering measurements could not be conducted. the circular dichroism data showed that the -sheet structure of rada16 was impaired to some extent by the addition of ptx, a result that was in accordance with the change in morphology on atomic force microscopy. the particle size of ptx was obviously decreased in the rada16 solution compared with that in the water. all of the data indicated that rada16 did not simply mix with ptx but rather interacted with it. as illustrated in figure 5, rada16 consists of repeated sequences of arg - ala - asp - ala, in which arg and asp are hydrophilic amino acid residues and ala is a hydrophobic amino acid residue ; the -sheet structure of the peptide therefore had both hydrophobic and hydrophilic regions.15 because ptx is a typical hydrophobic compound, the hydrophobic regions of peptide would be adsorbed to the hydrophobic surface of separated ptx particles under stirring, a phenomenon that resulted in the impaired -sheet structure of rada16 shown in the circular dichroism data. the atomic force microscopy images showed that there were many bulged rada16 nanofibers, which may have resulted from ptx molecules that were incorporated with nanofibers or from the aggregation of nanofibers induced by hydrophobic interactions. the data indicate that the self - assembling peptide and ptx could interact with each other and that the amphiphilic peptide was able to stabilize hydrophobic drugs in aqueous solution, a finding that was in agreement with previous reports.2022 this type of interaction also helps to increase the solubility of ptx and decrease the mean particle size of ptx in rada16 solution under stirring. therefore, the rada16-ptx suspension could potentially be injected into the body with a syringe needle. these results indicated that ptx molecules and particles can coexist in rada16- ptx suspension, and stabilized ptx can be obtained in this system. however, further investigation is necessary to determine the percentage of each state in the complexes. the rada16 solution was able to form a hydrogel in ionic environments, such as phosphate - buffered saline, physiological saline, and body fluid. however, could the rada16-ptx suspension gel under physiological conditions ? the test was performed at 37c, and phosphate - buffered saline was used to simulate body fluid. the storage modulus (g) and loss modulus (g) responded to the elasticity and viscosity of materials, respectively. as shown in figure 6, the frequency sweep indicated that the value of the storage modulus (g) in the rada16-ptx suspension was larger than that of the loss modulus (g) both in 0.5% and 1% rada16, and all of the low values (< 10 pa) for the storage and loss moduli were typical for low viscosity systems with a low degree of elasticity. with the addition of medium the storage modulus of 0.5% rada16 was over 300 pa and that of 1% rada16 was over 1000 pa, whereas the original values for both 0.5% and 1% rada16 were under 10 pa. the great increase in the storage modulus suggested the formation of a strong hydrogel. at the mean time, it was observed that the storage modulus of 0.5% rada16 hydrogel was much lower than that of the 1% rada16 hydrogel, which indicated that the concentration of peptide had a positive correlation with the elasticity of the hydrogel. the results demonstrated that the rada16-ptx suspension could form a highly elastic hydrogel under physiological conditions. as an in situ forming gel drug delivery system, the ideal vehicle should be an injectable solution in vitro and change to a gel in vivo. rada16 was able to form a hydrogel rapidly under physiological conditions without heating or chemical reaction, making it a favorable vehicle for drug delivery. paclitaxel is a hydrophobic drug that is poorly soluble in water. to maintain sink conditions throughout the release process, sodium dodecyl sulfate was added to the release medium to increase the solubility of ptx.5 the release was observed over a period of five days. as shown in figure 7b, the rada16 -ptx suspension could form a milky - white gel in phosphate - buffered saline, and the volume of hydrogel was obviously reduced over the release time. the resulting release profiles of ptx from the rada16-ptx hydrogels are shown in figure 7a. when a hydrogel was prepared with 1% rada16, approximately 40%45% of incorporated ptx was released from the hydrogel within the first day, with a half - release time of 2430 hours. in contrast, more than 60% of the ptx was released from the hydrogel within one day for the 0.5% rada16 hydrogel, and the half - release time was 810 hours. these results clearly demonstrated that a higher concentration of peptide in the hydrogel produced slower drug release rates. the initial burst effect was lower for the 1% rada16 hydrogel compared with the 0.5% rada16 hydrogel, and an increase in peptide concentration slowed the release rates of the loaded drug, resulting in a longer half - release time for the ptx. the atomic force microscopic image showed that many nanoparticles were present in the supernatant of the release medium (figure 8a), and the width and height of the nanoparticles were 83.1 30.3 nm and 4.9 2.9 nm, respectively. however, sodium dodecyl sulfate was also an amphiphilic molecule, which might form assemblies in aqueous solution. thus, an additional atomic force microscopy test with only sodium dodecyl sulfate in the release medium was performed to verify this result. the resulting images showed that sodium dodecyl sulfate was obviously accumulated on the surface of mica, and no similar size of nanoparticles formed (data not shown). these data suggest that ptx was encapsulated in peptide nanofibers and that rada16-ptx nanoparticles were formed in the release process. a schematic representation of a possible model for ptx release from the ptx - rada16 hydrogel is shown in figure 8b. considering the data from morphology and rheology analysis, it was found that the higher concentration of peptide solution had more cross - linked nanofibers and led to the formation of a more elastic hydrogel. because hydrogel formed from a higher concentration of peptide showed increased stability with a higher density of nanofibers and smaller sizes of pores, the probability of specific interactions between ptx and peptide nanofibers and diffusion hindrance was increased, yielding a decrease in the diffusivity of ptx and finally resulting in slower drug release rates.15,16 these results suggested that the release rates of ptx can be controlled by changing the peptide concentration, so the release time could be extended by adding a greater amount of peptide in future studies. in addition, the addition of sodium dodecyl sulfate as a surfactant can increase the release rates of ptx from the hydrogel, an effect that would be caused by the increased degradation rates of the rada16 peptide. as an antiproliferation drug, ptx is a potent inhibitor of angiogenesis, cell migration, and collagenase production. because it has been demonstrated that ptx has significant antitumor activity against various solid tumors, including breast cancer, the human breast cancer cell line mda - mb- 435s was used to study the antitumor efficiency of the rada16-ptx hydrogel. as in previous reports, the antiproliferative activity of the complex was dependent on the concentration of ptx.2 to simulate the in vivo situation and optimize the antitumor test, ptx10 g was administered to each well of the 96-well plate, and the culture medium was changed every day. in our study, the data showed that single use of rada16 produced no inhibition of tumor cell proliferation, while the rada16-ptx hydrogel inhibited growth of tumor cells effectively. as shown in figure 9, there was no significant difference in cell viability rates between the ptx - treated group and both rada16-ptx - treated groups at day 1. at day 3, there was a significant difference between the ptx group and the 1% rada16-ptx group, but there was no significant difference between the 0.5% and 1% rada16-ptx groups. at days 5 and 7, compared with the ptx group, both rada16-ptx groups demonstrated lower cell viability rates, and the cell viability rate in the 1% rada16-ptx group was significantly lower than that in the 0.5% rada16-ptx group. these data suggest that ptx and 0.5% rada16-ptx had poor ability to inhibit cell growth after days 3 and 5, respectively, whereas 1% rada16-ptx hydrogel maintained relatively high inhibitory activity even at day 7. it was concluded that the rada16 hydrogel could control release of ptx effectively and that a higher concentration of peptide hydrogel resulted in an increased release time for ptx. the fluorescence images showed that the tumor cells in the rada16 group continued to proliferate over the course of the observed days (figure 10). by contrast, in the ptx group and both rada16-ptx groups, only a few cells could be seen at day 3, which indicated that cell proliferation was inhibited by ptx. however, the number of tumor cells in the ptx group was slightly higher than that in both rada16-ptx groups. at day 5, in comparison with the 1% rada16-ptx group, more tumor cells were observed in the ptx and 0.5% rada16-ptx groups. at day 7, compared with previous days, an increased number of tumor cells was observed in the ptx group and both rada16-ptx groups, but fewer cells were seen in the 1% rada16-ptx group than those in the ptx group and the 0.5% rada16-ptx group. it was concluded that the 1% rada16-ptx hydrogel had a longer lasting inhibitory effect on proliferation of tumor cells than did the other tested groups. these results indicate that the self - assembling peptide system could be used to control the release of ptx. the release time of ptx from the hydrogel showed increased correlation with the concentration of peptide, and a more elastic hydrogel led to stronger interactions between the drug and the nanofibers, which decreased the diffusion rates of ptx and resulted in longer drug release times. the spontaneously gelling ability of rada16-ptx suspension in an ionic environment provided the controlled release of ptx, which would increase the contact time of drug at the site of action and enhance the efficiency of therapy.5,23 because ptx can not differentiate between cancer and normal cells, which may result in major toxicity to normal cells, the potential application of peptide hydrogel to the localized delivery of ptx might minimize the cytotoxicity and side effects. in addition, the self - assembling peptide was a biocompatible and biodegradable nanomaterial, which previous studies have reported to produce no toxic and immune response, and this material has been widely used for cell culture and injection into animals.2426 therefore, it suggested that this self - assembling peptide is a favorable vehicle for local ptx delivery as an in situ - forming hydrogel system. nevertheless, further experiments are still needed to estimate the antitumor efficiency of the peptide- ptx system in vivo. in conclusion, this study found that the rada16-ptx complex could form colloidal suspensions after magnetic stirring for 48 hours and that ptx was stabilized in rada16-containing aqueous solutions. the rada16 hydrogel prolonged the inhibitory effect of ptx on growth of breast cancer cells in vitro. this injectable, in situ - forming hydrogel system has great potential as a delivery vehicle for various hydrophobic drugs in future studies. | backgrounda nanoscale injectable in situ - forming hydrogel drug delivery system was developed in this study. the system was based on a self - assembling peptide rada16 solution, which can spontaneously form a hydrogel rapidly under physiological conditions. we used the rada16 hydrogel for the controlled release of paclitaxel (ptx), a hydrophobic antitumor drug.methodsthe rada16-ptx suspension was prepared simply by magnetic stirring, followed by atomic force microscopy, circular dichroism analysis, dynamic light scattering, rheological analysis, an in vitro release assay, and a cell viability test.resultsthe results indicated that rada16 and ptx can interact with each other and that the amphiphilic peptide was able to stabilize hydrophobic drugs in aqueous solution. the particle size of ptx was markedly decreased in the rada16 solution compared with its size in water. the rada16-ptx suspension could form a hydrogel in culture medium, and the elasticity of the hydrogel showed a positive correlation with peptide concentration. in vitro release measurements indicated that hydrogels with a higher peptide concentration had a longer half - release time. the rada16-ptx hydrogel could effectively inhibit the growth of the breast cancer cell line, mda - mb-435s, in vitro, and hydrogels with higher peptide concentrations were more effective at inhibiting tumor cell proliferation. the rada16-ptx hydrogel was effective at controlling the release of ptx and inhibiting tumor cell growth in vitro.conclusionself-assembling peptide hydrogels may work well as a system for drug delivery. |
type 1 diabetes (t1d) can cause significant stress in the affected children, due to its chronic nature and multipronged management ; involving multiple daily injections, blood glucose monitoring, dietary restrictions, and risk of complications. in developing countries, financial constraints, suboptimal health infrastructure, lack of universal health insurance, scarce school health facilities, poor health - seeking behavior in the community, and the social stigma attached to the disease are likely to further aggravate the psychosocial and cognitive profiles of children / adolescents with t1d, as compared to developed nations. there is, however, a paucity of reports on these aspects from developing countries. it is imperative to understand the extent of psychological comorbidity in children / adolescents with t1d, because this has a significant impact on the self - management of diabetes, and glycemic control in the patient. the study was undertaken with the objective of assessing the quality of life (qol), emotional well - being, and behavioral and cognitive profile of indian children and adolescents with t1d ; and to examine the influence of sociodemographic factors, glycemic control, age of onset, and duration of diabetes on these parameters. we enrolled 49 children / adolescents aged 6 - 18 years with t1d under follow - up at the pediatric endocrinology clinic of our tertiary care institute, for at least 6 months, after obtaining voluntary informed consent from parents and assent from the subjects. information regarding age of onset, duration of disease, socioeconomic status (based on modified kuppuswamy scale), and mother 's education were recorded. degree of glycemic control was assessed by last hba1c (considered normal if american diabetes association (ada) cut - offs, i.e., 8.5% for age 12 years), and number of episodes of perceived or documented hypoglycemia (blood glucose < 60 mg / dl or symptomatic) in past 1 month. the following instruments were used to assess qol, emotional adjustment, behavioral problems, and cognition in the subjects. this is a 22-item validated questionnaire to assess possible problems in the following six domains : impact of symptoms related to diabetes, impact of the treatment, impact on activities, parents issues, worry about the future, and perception of one 's own health. this was administered by the physician to subjects aged 10 - 18 years (n = 34). each question has five possible responses, ranging from 0 (never) to 4 (all the time), and the responses are added up to get the total score for the subscale., we considered the adverse impact on qol in a domain to be significant, if the score for that domain was in the upper half of the possible range. this was administered by the psychologist to subjects aged 10 - 18 years (n = 34). it comprises of five positively worded items ; related to positive mood, vitality, and general interests ; which are rated on a 6-point likert scale from 0 (not present) to 5 (constantly present). it has been shown to be a reliable tool to assess emotional well - being over the preceding 2 weeks. this was administered by the psychologist to parents of children aged 6 - 18 years (n = 49). this is a standardized measure of parent - reported behavioral problems, comprising of 120 questions, with responses graded on a likert scale, as 0 - 2. the responses are then summated as directed by the instrument to yield scores for nine behavior syndromes. the summation of the responses of some of the individual syndromes yields scores for internalizing behaviors (is), externalizing behaviors (es), and overall scores for total behavior problems (ts). internalizing behaviors include behaviors such as anxious / depressed, withdrawn / depressed, and somatic ; and externalizing behaviors include rule - breaking, aggression, and inattention. the range for each of the scores ts, es, and is is 0 - 100. this intelligence quotient (iq) scale is the indian adaptation of wechsler intelligence scale for children and provides scores for verbal iq (viq), performance iq (piq), and full scale iq (fsiq). this was administered by the psychologist to children aged 6 - 16 years (n = 41). statistical analysis was carried out using stata 9.0 (college station, texas, usa). bivariate analysis was carried out using student 's t - test (for categorical variables) and spearman 's rank correlation (for continuous variables) to assess the strength of association with potential predictors. this is a 22-item validated questionnaire to assess possible problems in the following six domains : impact of symptoms related to diabetes, impact of the treatment, impact on activities, parents issues, worry about the future, and perception of one 's own health. this was administered by the physician to subjects aged 10 - 18 years (n = 34). each question has five possible responses, ranging from 0 (never) to 4 (all the time), and the responses are added up to get the total score for the subscale., we considered the adverse impact on qol in a domain to be significant, if the score for that domain was in the upper half of the possible range. this was administered by the psychologist to subjects aged 10 - 18 years (n = 34). it comprises of five positively worded items ; related to positive mood, vitality, and general interests ; which are rated on a 6-point likert scale from 0 (not present) to 5 (constantly present). it has been shown to be a reliable tool to assess emotional well - being over the preceding 2 weeks. this was administered by the psychologist to parents of children aged 6 - 18 years (n = 49). this is a standardized measure of parent - reported behavioral problems, comprising of 120 questions, with responses graded on a likert scale, as 0 - 2. the responses are then summated as directed by the instrument to yield scores for nine behavior syndromes. the summation of the responses of some of the individual syndromes yields scores for internalizing behaviors (is), externalizing behaviors (es), and overall scores for total behavior problems (ts). internalizing behaviors include behaviors such as anxious / depressed, withdrawn / depressed, and somatic ; and externalizing behaviors include rule - breaking, aggression, and inattention. the range for each of the scores ts, es, and is is 0 - 100. this intelligence quotient (iq) scale is the indian adaptation of wechsler intelligence scale for children and provides scores for verbal iq (viq), performance iq (piq), and full scale iq (fsiq). this was administered by the psychologist to children aged 6 - 16 years (n = 41). statistical analysis was carried out using stata 9.0 (college station, texas, usa). bivariate analysis was carried out using student 's t - test (for categorical variables) and spearman 's rank correlation (for continuous variables) to assess the strength of association with potential predictors. the clinicodemographic features of the enrolled subjects are summarized in table 1. the mean age of subjects was 11.7 3.1 years. forty - three percent of subjects belonged to lower socioeconomic class, and the glycemic control was inadequate in a majority. the overall results of the qol, who-5, cbcl, and misic administered to the patients are summarized in table 2. nearly a third of the subjects had a significant adverse impact on qol due to their diabetes. amongst the subdomains, the maximally reported adverse impact was related to symptoms of diabetes and perception of health, while the minimally reported was the impact on activities. who-5 demographic and clinical characteristics of patients (n=49) results of tests for quality of life, emotional well - being, behavior, and cognition the results of bivariate analysis for the association of various potential predictors with the overall results are presented in table 3. early onset of t1d (< 5 years of age) was associated with significantly lower adverse impact on qol, and lower score on cbcl, suggestive of fewer behavioral problems. elevated hba1c level duration of disease was inversely associated with cbcl score, suggesting that behavioral problems decrease as the duration of diabetes increases. misic score was lower by 10.77 units in children from lower ses compared to children with higher ses. no significant association of gender, current age, presence of comorbid condition, and frequency of hypoglycemia was observed with the scores for any of the tests employed in the study, and hence their data is not presented in table 3. association of potential predictors with outcomes (qol, who-5, cbcl and misic) on bivariate regression analysis the association of the potential predictors with the subdomains of these tests is presented below. worries (p = 0.047), lesser negative impact on activities (p = 0.023), and better health perception (p = 0.019). worries were fewer (p = 0.033) among children whose mothers were educated beyond high school. there was a trend towards significance for the association between elevated hba1c and ' symptoms of diabetes (p = 0.092), and worries (p = 0.077). a significant inverse correlation was noted between the who and qol score (r = 0.518, p = 0.016), indicating that emotionally well - adjusted children perceived a lower adverse impact of the disease on qol. the duration of diabetes was inversely associated with es (p = 0.016), indicating that recently diagnosed children are more likely to have externalizing behaviors, and these become less common with increasing duration. children from lower ses, and those whose mothers educational level was below high school, had more withdrawn / depressed behaviors (p = 0.051 and 0.075, respectively). lower ses was associated with lower scores in information, arithmetic, analogies / similarities, general information, awareness, attention, concentration, and immediate memory and recall. onset of t1d before 5 years of age was associated with significantly lesser worries (p = 0.047), lesser negative impact on activities (p = 0.023), and better health perception (p = 0.019). worries were fewer (p = 0.033) among children whose mothers were educated beyond high school. there was a trend towards significance for the association between elevated hba1c and ' symptoms of diabetes (p = 0.092), and a significant inverse correlation was noted between the who and qol score (r = 0.518, p = 0.016), indicating that emotionally well - adjusted children perceived a lower adverse impact of the disease on qol. the duration of diabetes was inversely associated with es (p = 0.016), indicating that recently diagnosed children are more likely to have externalizing behaviors, and these become less common with increasing duration. children from lower ses, and those whose mothers educational level was below high school, had more withdrawn / depressed behaviors (p = 0.051 and 0.075, respectively). lower ses was associated with lower scores in information, arithmetic, analogies / similarities, general information, awareness, attention, concentration, and immediate memory and recall. being a life - long condition with significant burden of treatment, in terms of regular injections, monitoring blood sugar, restrictions on diet and lifestyle, planned and emergency hospital visits, and financial impact on the family ; compounded by the effect of fluctuating blood glucose on mood and cognition, t1d has a significant influence on the quality of life, emotional well - being, cognitive profile, and behavior of the affected children. shorter duration since diagnosis, poor glycemic control, low ses, and female gender have been noted to be associated with greater negative impact on these parameters, in studies reported from developed countries. the mean dawn qol score in our subjects was 29.3 (with maximum (worst) possible score of 84). this is comparable to the qol score of 97.5 (with maximum possible score of 255, using the diabetes quality of life for youth (dqoly) questionnaire) reported by matziou.. amongst the possible predictors studied, early onset of diabetes (before age 5 years) was associated with significantly lesser negative impact on the overall qol score as well as in most of the subdomains. in comparison to children who were diagnosed after 5 years of age, those with early onset were more optimistic about their life with diabetes, were less worried about the limitations imposed by diabetes on their current activities and future achievements, and had a more positive perception of their health. this may be related to the fact that being introduced to a way of life incorporating diabetes management at a tender age, they make fewer comparisons to their more carefree life before the diagnosis of diabetes, and hence adapt better. in contrast to previous reports (matziou., 2011, ingerski., 2010), we did not find an association of poorer glycemic control with either the overall qol score, or the perception of one 's own health status subdomain. this is indicative of a less robust understanding of the disease among our subjects, and suggests greater need for education and counseling of patients to help them prioritize their concerns. the who-5 instrument is highly specific for depression, and a lower score indicates a greater prevalence of / predilection for depression. the overall mean (standard deviation (sd)) who-5 score in our study population was 74.6 (19.4), which is higher (indicating better emotional adjustment) than that reported by de wit. the score was significantly lower among those with elevated hba1c compared to those with normal hba1c (71.1 (19.4) vs 90 (7.4), p = 0.004). similar to other studies, we found that lower who-5 scores correlated well with greater adverse impact on qol. using this simple instrument well - behaved and well - adjusted. children with suboptimal disease control should undergo regular psychological review, and counseling. with the cbcl questionnaire, the percentage of borderline clinical and clinical scores, and the mean total scores found in our patients are comparable to those previously reported. this is of special concern because the parents may not perceive such behaviors as abnormal and seek consultation. we observed a better level of adjustment and fewer behavioral problems in our patients with onset of t1d before the age of 5 years. also, the behavioral problems were inversely associated with the duration of disease, suggesting that there is greater adjustment to the diagnosis with time. we did not find an association of cbcl scores with the degree of glycemic control as measured by the hba1c. comparing with previous studies among diabetic children / adolescents, while studies by nardi., and akbas., also did not observe association between cbcl scores and hba1c, ohmann., reported significantly higher prevalence of somatic complaints and internalizing behaviors in children with suboptimal glycemic control. similarly, whereas some of the previous studies also suggest fewer behavioral problems with increasing duration of disease as in our study., nardi., reported higher prevalence of problems in the anxious / depressed, attention and hyperactivity and oppositional defiant scales as duration of disease increased. we found an association of maternal educational level below high school with greater prevalence of clinical range internalizing behaviors. among the domains of qol also, lower maternal education was associated with more worries about the future. the mother is the central caregiver in most families. her lower education level may affect her understanding of the disease and its management, and the ability to cope with psychological stress associated with diagnosis. this may translate into inadequate emotional support / care for the child, leading to more behavioral problems / stress in the child. a similar association of lower maternal education with greater stress and more frequent behavior problems in the child was observed in an indian study among caregivers of children with cerebral palsy. this finding also emphasizes the effect of the family on qol, and emotional adjustment of the diabetic child, and potential of the family unit as a target for psychological therapy and intervention. while we were limited by the lack of controls in our study, a full - scale iq of 105.1 (7.75) among controls in an indian study on children of alcoholic fathers has been reported. thus, the mean iq in our diabetic subjects is possibly lower than the normal population average. findings from studies from the west have variably suggested that children with early diabetes onset (ferguson., 2005, hannonen., 2010, naguib., 2009), poor glycemic control (ohmann., 2010, hannonen., 2010, naguib., 2009), and episodes of severe hypoglycemia (hannonen however, in our patients, the above factors or the scores on the qol and who-5 questionnaires did not have any significant association with cognitive performance. lower ses emerged as a consistently strong association with lower full scale iq score, as well as individual scores in specific components. (usa) have also reported that ses has a greater effect on academic performance in diabetic children than medical variables. the implication of this finding is that diabetic children from lower ses are at higher risk of cognitive impairment, and should be prioritized for iq assessment. we found a significant correlation between scores on the qol and the who questionnaires, and significantly poorer qol in patients with pathological who scores. the who questionnaire is shorter and quicker to administer, and may be used as the initial screen in set - ups with a crunch of time - people resources, before moving on to administering the qol on a regular basis. it is important to recognize psychological problems in children with t1d, as these may lead to poor motivation and inability to coordinate the multipronged therapeutic plan. the consequent poor metabolic control may further impair the psychological status, thus propitiating a vicious cycle, with progressive worsening of clinical and psychological situation, and even lesser impetus to control disease. psychological and behavioral interventions have been shown to have a beneficial effect on children and adolescents with diabetes evidenced in terms of better compliance to therapy, glycemic control as well as better relationships with family and peers and better coping capability. however, such interventions require time - people resources that are often missing in the setting of a developing nation. we hope that in resource - limited scenarios, where t1d is typically managed by a single physician, without the support of psychologists / social workers or diabetes counselors, our results will help in prioritizing children for behavioral monitoring and psychological evaluation. based on our findings, children with recent diagnosis, older age at onset, lower maternal educational level, elevated hba1c, or belonging to lower ses are recommended for more frequent / detailed behavioral and cognitive evaluation. | background and aims : the psychological stress associated with type 1 diabetes (t1d) may be higher in children from developing world due to limited health resources. the aims of the study were to assess the quality of life (qol), emotional well - being, behavioral, and cognitive profile of children / adolescents with t1d diagnosed at least 6 months prior.materials and methods : forty - nine children with t1d, aged 618 years were assessed using dawn youth qol questionnaire, who-5 well - being index, child behavior checklist (cbcl), and malin 's intelligence scale for indian children (misic). the association of the scores was studied with age, gender, socioeconomic status (ses), frequency of hypoglycemia, hba1c, and age of onset and duration of t1d.results:the mean (standard deviation (sd)) for dawn qol, who-5, cbcl, and misic scores was 24.7 (16.7), 74.6 (19.4), 52.6 (8.8), and 96.0 (11.2), respectively. the significant associations noted were : elevated hba1c with poorer emotional well - being ; higher negative impact on symptoms of disease and future prospects sub - areas of qol ; shorter duration of disease with more behavioral issues ; lower maternal education with more withdrawn / depressed behaviors and worry about future prospects ; and lower ses with lower misic scores. earlier onset (age < 5 years) was associated with fewer behavioral problems and less negative impact on qol.conclusion:children with recent diagnosis, older age at onset, lower maternal educational level, elevated hba1c, or belonging to lower ses were identified to have higher prevalence of various psychological and cognitive problems. in resource - limited settings, these children should be prioritized for behavioral and cognitive evaluation. |
nasopharyngeal angiofibromas are relatively rare tumors that represent 0.5% of all head and neck neoplasms and they predominantly occur in adolescent males (1). this type of tumor originates from the region of the sphenopalatine foramen. in most cases, imaging studies will demonstrate a widening of the pterygopalatine fossa and anterior bowing of the posterior wall of the maxillary sinus (2). i describe an extra - nasopharyngeal angiofibroma arising from the parapharyngeal space in a middle - aged woman without involvement of the sphenopalatine foramen or the nasopharynx. a 53-year - old woman presented with a diffuse anterior neck swelling of one - month duration. the patient had not undergone any previous surgery. ultrasonography (us) showed diffuse goiter with multiple thyroid nodules. a homogeneous and hypoechoic solid mass, adjacent to the upper margin of the right submandibular gland with focal flow signals on color doppler sonography within the mass was detected (figure 1). a computed tomography (ct) scan revealed a circumscribed mass, hypodense to the masseter muscle and hyperdense to the parotid gland in the right parapharyngeal space (figure 2 a - c). after intravenous contrast administration, the mass, 1.7 1.7 1.6 cm in size, demonstrated homogeneous enhancement without infiltration to the adjacent muscle. at that time enlargement of the bilateral thyroid lobes with multiple non - enhancing hypodense and calcified thyroid nodules was also noted. under general anesthesia after surgery, the patient revealed an uneventful postoperative course. at the three - year follow - up examination, angiofibromas are histologically benign, but potentially locally destructive fibro - vascular tumors that account for less than 0.5% of all head and neck neoplasms (3). they originate from the region of the sphenopalatine foramen and commonly spread to the sphenoid sinus, the nasal cavity, and the pterygopalatine foramen (1). angiofibromas that do not originate from the area around the sphenopalatine region are very rare (1). the primary site of extra - nasopharyngeal angiofibromas varies and can include the ethmoid and sphenoid sinuses, the nasal septum, the tonsils, the ears, the trachea, the larynx, and the infratemporal fossa (1). the most common site of extra - nasopharyngeal angiofiroma is the maxillary sinus. in the recent literature, there were five more cases of extra - nasopharyngeal angiofibromas reported in the literature since then (5 - 9). only one parapharyngeal angiofibroma was reported in a 25-year - old male in the english literature (10). they reported homogeneous enhancement on ct and hypervascularity on conventional angiogram in the left parapharyngeal mass. i report the very rare case of an extra - nasopharyngeal angiofibroma arising from the right parapharyngeal space in a middle - aged woman. to the best of our knowledge, this represents the second demonstrated case of an angiofibroma originating in such a location. extra - nasopharyngeal angiofibromas occur more commonly in females and tend to develop at an older age (11). the male - to - female ratio is roughly 3:1 and the mean age at presentation is 22 years. clinical manifestations of extra - nasopharyngeal angiofibromas are variable including headache, hoarseness, dysphagia, dyspnea, stridor, and pain according to their location (4, 11). extra - nasopharyngeal angiofibromas may have less specific and confusing clinical symptoms and diagnosis may be delayed (4). enlargement of the sphenopalatine foramen with an intensely enhancing mass is the typical pathognomonic radiological finding, especially in young boys (1). in the patient with characteristic radiological findings and typical clinical symptoms of nasopharyngeal angiofibromas, preoperative biopsy extra - nasopharyngeal angiofibromas are harder to diagnose radiologically due to their various locations (4). unlike nasopharyngeal angiofibromas, lack of hypervascularity on conventional angiograms does not exclude the diagnosis of extra - nasopharyngeal angiofibromas (2). an axial ct showed well enhancing mass in the right parapharyngeal space without perilesional infiltration. the mass was thought to be a minor salivary gland origin tumor or a paraganglioma, hemangiopericytoma, solitary fibrous tumor or neurogenic tumor, preoperatively. differential diagnoses of parapharyngeal masses may also include parapharyngeal space metastatic lymph node, lymphoma, inflammatory pseudotumor, tuberculous and fungal infections (12). the most acceptable treatment for angiofibroma is endovascular embolization and then surgical excision (13). nasopharyngeal angiofibroma seems to have a poorer prognosis than an extra - nasopharyngeal lesion due to inadequate surgical exposure ; consequently, it may result in a higher recurrence rate (3). in the present case, complete surgical resection was performed without preoperative embolization. on follow - up after a period of 3 years, there was no tumor recurrence. | nasopharyngeal angiofibroma is a relatively uncommon vascular tumor affecting adolescent males and it characteristically originates in the posterior lateral wall of the nasopharynx. primary extra - nasopharyngeal angiofibroma is very rare. here, i present a case of angiofibroma of the parapharyngeal space in a 53-year - old woman with ct and sonographic findings. |
icu = intensive care unit ; ifn = interferon ; il = interleukin ; lps = lipopolysaccharide ; pcr = polymerase chain reaction ; tlr = toll - like receptor ; tnf = tumour necrosis factor. hg, te, and sf planned, drafted, read, and approved the final manuscript. this article is part of a review series on infection, edited by steven opal. | international guidelines concerning the management of patients with sepsis, septic shock and multiple organ failure make no reference to the nature of the infecting organism. indeed, most clinical signs of sepsis are nonspecific. in contrast, in vitro data suggest that there are mechanistic differences between bacterial, viral and fungal sepsis, and imply that pathogenetic differences may exist between subclasses such as gram - negative and gram - positive bacteria. these differences are reflected in different cytokine profiles and mortality rates associated with gram - positive and gram - negative sepsis in humans. they also suggest that putative anti - mediator therapies may act differently according to the nature of an infecting organism. data from some clinical trials conducted in severe sepsis support this hypothesis. it is likely that potential new therapies targeting, for example, toll - like receptor pathways will require knowledge of the infecting organism. the advent of new technologies that accelerate the identification of infectious agents and their antimicrobial sensitivities may allow better tailored anti - mediator therapies and administration of antibiotics with narrow spectra and known efficacy. |
impaired insulin sensitivity (is) plays a crucial role in the development of type 2 diabetes (t2d), but its relevance for the occurrence of ischemic stroke still remains unclear. in that context, some studies suggested that decreased is, for example, insulin resistance, is an established risk factor for ischemic stroke, [24 ] while there are studies which could not demonstrate this relationship. moreover, it has been reported that decreased is is directly related to different subtypes of ischemic stroke in t2d patients and nondiabetics, measured by different metabolic tests, short insulin tolerance test, homeostasis model assessment for insulin resistance, and immunoreactive insulin after glucose loading in 2 h ogtt [2, 3 ]. simultaneously, it has been shown that higher levels of insulin resistance were found in nondiabetics with coexistence of intra- and extracranial atherosclerosis in contrast to those with only intra- or extracranial atherosclerosis. also, it has been suggested that hyperinsulinemia might be a risk factor for ischemic stroke [7, 8 ]. hyperinsulinemia represents a surrogate measure for insulin resistance in nondiabetics, as well as in t2d patients with significant residual insulin secretion capacity. simultaneously, higher levels of pai-1 have been found in blood from patients with t2d, in obese subjects, and in other conditions associated with insulin resistance [1113 ]. moreover, it has been shown that hypofibrinolysis due to higher pai-1 levels has been related to the insulin resistance [1417 ] and might be involved together with insulin resistance, in the pathogenesis of ischemic stroke. therefore, in this study we tried to determine the role of impaired is, together with the relevant changes in insulin and pai-1 levels in t2d patients as well as nondiabetics with ischemic stroke. we divided 62 t2d patients into two groups, with (n = 33) and without (n = 29) atherothrombotic ischemic stroke (atis), and 64 nondiabetics were assigned into group with atis (n = 34) and healthy subjects (n = 30), paired with the t2d patients with respect to sex and age. diagnosis of atis was done by a neurologist according to clinical signs and visualization methods, cranial computerized scan, and/or magnetic resonance imaging of the brain, repeated after the first 7 days from initial findings of ischemic stroke. only the patients with atis were included in the study, while the exclusion criteria involved patients with previously documented lacunar, cardioembolic, hemorrhagic cerebral infarction or coronary heart disease (history of myocardial infarction or coronary angiography). t2d patients were treated only with oral antihyperglycemic agents, while we excluded patients treated with insulin therapy or with other endocrine diseases, renal or hepatic insufficiency, previous and current infections, hematological or rheumatic diseases, uncontrolled hypertension, or severe alcohol consumption, during the last 4 weeks. at the time of metabolic evaluation, all the patients, irrespective of occurrence of the stroke, showed a uniform level of their physical activity. the patients were completely informed about the study, before they gave an informed consent to participate. the study was done at the clinic for endocrinology, diabetes and metabolic diseases and at the clinic for neurology, clinical centre of serbia, faculty of medicine, university of belgrade, and was approved by the institutional ethics committee. the obtaining of the medical history and physical examination, metabolic tests, and fibrinolytic activity evaluation were conducted in all the patients included the study within one - day visit. body mass index was calculated as weight in kilograms divided by the square of height in meters. arterial blood pressure was measured by sphygmomanometry and hypertension was diagnosed according to world health organisation criteria (systolic / diastolic blood pressure 140/90 mm hg) or by the use of antihypertensive agents. the metabolic test was performed after 6 months from occurrence of atis and following overnight fasting. is was tested by using ivgtt with frequently sampled plasma glucose (pg) and pi levels, followed by the minimal model analysis. the subjects were injected with glucose 0.3 g / kg body weight and the blood samples were taken immediately before intravenous glucose loading and sequentially every minute during the first 10 minutes and then 12, 14, 16, 20, 23, 24, 25, 27, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, and 180 minutes after intravenous glucose loading. insulin was added during the test intravenously (4 mu / kg / min), between minutes 20 and 25, in order to substitute potentially diminished insulin response. the insulin sensitivity index (si) was obtained from the data of pg and pi levels by computerized minimal model analysis (minmod program, kindly provided by dr. pg was obtained by glucose oxidase method using a beckman glucose analyser (beckman instruments). the continuous variables within each group of patients were analyzed with analysis of variance (anova) with post hoc tamhane test. the significance of the differences between correlation coefficients was analyzed by using fisher r - to - z transformation. data were analyzed using the statistical package for the social sciences (spss) software (advanced statistics, version 17.0), chicago, il. all three investigated groups of patients were matched according to the age, duration of t2d, and period of time from the onset of ischemic stroke. simultaneously, all patients were overweight and t2d patients had satisfactory metabolic control expressed as almost equal hba1c values, before metabolic investigation. the hypertension in patients with the stroke was significantly higher than in healthy controls, both in t2d patients and in nondiabetics. we did not find a significant difference in bmi between t2d patients and atis compared to t2d without atis and nondiabetics with atis compared to healthy subjects (table 1). when we analysed is, expressed as si index, the lowest si values were present in patients with t2d and atis and they were significantly lower in t2d patients with atis compared to t2d patients without atis (1.18 0.67 versus 2.82 0.61 moreover, si values were significantly lower in nondiabetics with atis in comparison to healthy subjects (3.18 0.93 versus 6.13 1.69 min / mu / l 10 ; p < 0.001) (figure 1). there is no difference in si between t2d patients without atis and nondiabetics with atis (p = ns). simultaneously, we found that pi levels were higher in t2d patients and atis compared to t2d patients without atis (19.61 4.08 versus 14.91 1.66 in addition, pi levels were higher in nondiabetics with atis in comparison to healthy subjects (15.14 2.20 versus 7.58 2.05 ; p < 0.001). moreover, t2d patients with atis showed significantly higher pi levels than nondiabetics with stroke (p < 0.001), but we did not document difference in pi levels between t2d patients without atis and nondiabetics with atis (figure 2). simultaneously, we found that pai-1 levels were significantly higher in t2d patients with atis compared to t2d patients without atis (7.75 1.04 versus 4.57 0.72 mu / l ; p < 0.001) and in nondiabetics with atis compared to healthy subjects (4.78 0.98 versus 3.49 1.04 in addition, pai-1 levels were not different among t2d patients without atis and nondiabetics with atis. moreover, we found that si levels correlated with pai-1 levels both in t2d (r = 0.690, p < 0.001) and in nondiabetic subjects (r = 0.456, p < 0.001) (table 2). by using fisher r - to - z transformation, we evaluated the difference between two correlations coefficients. the correlations of si and pai-1 in the t2d patients were stronger than si and pai-1 correlation in nondiabetics (p < 0.05). the use of binary logistic regression analysis has identified levels of pai-1, insulin, and si as independent predictors for atis both in t2d patients and in nondiabetics (tables 3 and 4). our study results revealed decreased is in patients with atis, whether they were t2d patients or nondiabetics, suggesting that insulin resistance may play a significant role in occurrence of the stroke. simultaneously, t2d patients with atis showed the lowest level of is, together with the fasting hyperinsulinemia. the documented hyperinsulinemia in these patients, in the settings of diminished is levels, suggests the existence of a significant residual insulin secretion capacity. moreover, both metabolic abnormalities, decreased is or insulin resistance and fasting hyperinsulinemia in nondiabetics with atis, have confirmed the importance of decreased is level in pathogenesis of ischemic stroke, which is consistent with the previous data. both experimental and human studies revealed the importance of insulin resistance for the occurrence of acute ischemic stroke [2326 ]. however, our results signify the persistence of compromised is and increased pi levels even 6 months after acute phase in t2d patients with ischemic stroke. we decided to evaluate is level in this study by using ivgtt with frequently sampled pg and pi levels with minimal model analysis, which correlates with hyperinsulinemic euglycemic clamp, previously implemented in the is studies [27, 28 ]. the existence of impaired is in different subtypes of ischemic stroke in patients with t2d has also been proposed. also, the novel study confirmed the highest values of two other parameters of insulin resistance, homeostasis model assessment for insulin resistance and insulin after glucose challenge in 2 h ogtt in patients with atherothrombotic cerebral infarction without previously documented abnormal glucose tolerance. the results from extensive atherosclerosis risk in communities (aric) study showed that fasting insulin levels, among the other investigated risk factors, are positively associated with occurrence of ischemic stroke in the general population, which highlights the influence of insulin resistance, consistent with results from the finnish cohort study that included elderly t2d patients and nondiabetics. novel data pointed out the important role of augmented insulin resistance and related metabolic abnormalities in the development of intracranial stenosis from its early stages even in nondiabetics. on the other hand, lots of data suggested that pai-1 plays a significant role in occurrence of coronary artery and cerebrovascular disease in t2d. recent studies shed new light on pai-1 as an important pathway for cardiovascular events, including ischemic stroke. despite those facts and the background of occurrence and progression of atherosclerosis, the link between insulin resistance, diminished fibrinolytic activity, and our results showed higher pai-1 levels in t2d and atis, in parallel with the absence of difference in pai-1 levels between t2d patients without atis and nondiabetics with atis. however, there are some conflicting results suggesting higher pai-1 levels in t2d without ischemic stroke compared to nondiabetics, implying the absence of further deterioration of fibrinolytic activity in ischemic stroke in t2d patients. moreover, there are indications of decreased is, hyperinsulinemia, and increased pai-1 in first - degree relatives of patients with ischemic stroke which is related to ethnicity, together with the findings supporting the hypothesis that diminished fibrinolytic activity may exist prior to ischemic stroke [3537 ]. a previous study demonstrated that the higher pai-1 activity in young adults with a first ischemic stroke was a consequence of acquired hypofibrinolysis, together with other investigations supporting the genetic control of decreased fibrinolysis. also, obese diabetics had higher pai-1 levels compared to nondiabetics, implying permanent impaired fibrinolytic activity, which potentiates thrombosis, based on effect of combination of hormonal (hyperinsulinemia) and metabolic (hyperglycemia) changes characteristic for t2d [13, 40 ]. in general, increased pai-1 level in patients with atis or t2d or nondiabetics could be present even 4 years after acute cerebral ischemic event. therefore, we speculate that disturbances in fibrinolysis may precede the occurrence of atis, having in mind higher pai-1 level in t2d patients without atis and almost equal pai-1 levels in nondiabetics with atis [14, 42 ]. we found that si levels correlated with pai-1 levels both in t2d and nondiabetic subjects, with stronger correlation in t2d than in nondiabetics. is has been independently related to pai-1 levels in our study and previously in patients with t2d and obese diabetic and nondiabetic subjects [43, 44 ]. in bari 2d trial it has been shown that diminishing of insulin resistance favorably changes the balance between fibrinolysis and thrombosis potentiating fibrinolysis during long term followup in t2d patients. in addition, it was suggested that patients who experience progression of symptomatic intracranial atherosclerosis are characterized by impaired endogenous fibrinolysis. pai-1 levels may influence stroke mechanisms in multiple ways, and they might differentiate between responders and nonresponders to reperfusion therapies, and they might represent potential target for stroke prevention. in order to minimize the previously confirmed harmful effect of glucose toxicity to the is level and fibrinolytic activity [13, 47 ], we selected t2d patients with or without ischemic stroke matched with respect to duration of disease, showing optimal metabolic control before the evaluation of insulin sensitivity level. previously described association of obesity, insulin resistance, and higher pai-1 levels, hypersecreted from adipocytes, was the reason to include overweight subjects in our investigation. since age is known to be strongly and independently correlated with the occurrence of ischemic stroke, investigated patients were younger than 65 years. measurements of is were not made until at least 6 months after the stroke, providing enough time for the patients to approach maximum recovery, showing similar level of the physical activity. in order to diminish the heterogeneity of stroke the binary logistic regression analysis applied to our data has demonstrated that insulin, pai-1, and si levels are independent predictors of the ischemic stroke occurrence, in t2d patients as well as in nondiabetics. in conclusion, the results demonstrated that decreased is levels together with fasting hyperinsulinemia are strongly associated with the onset of the atis, while this atherogenic effect might be strongly potentiated by increased level of pai-1. in this context, insulin resistance and | we analyzed (a) insulin sensitivity (is), (b) plasma insulin (pi), and (c) plasminogen activator inhibitor-1 (pai-1) in type 2 diabetes (t2d) patients with (group a) and without (group b) atherothrombotic ischemic stroke (atis), nondiabetics with atis (group c), and healthy controls (group d). is was determined by minimal model (si). si was lower in a versus b (1.18 0.67 versus 2.82 0.61 min1/mu / l 104 ; p < 0.001) and in c versus d (3.18 0.93 versus 6.13 1.69 min1/mu / l 104 ; p < 0.001). pi and pai-1 were higher in a versus b (pi : 19.61 4.08 versus 14.91 1.66 mu / l ; p < 0.001, pai-1 : 7.75 1.04 versus 4.57 0.72 mu / l ; p < 0.001) and in c versus d (pi : 15.14 2.20 versus 7.58 2.05 mu / l ; p < 0.001, pai-1 : 4.78 0.98 versus 3.49 1.04 mu / l ; p < 0.001). si correlated with pai-1 in t2d patients and nondiabetics, albeit stronger in t2d. binary logistic regression identified insulin, pai-1, and si as independent predictors for atis in t2d patients and nondiabetics. the results imply that insulin resistance and fasting hyperinsulinemia might exert their atherogenic impact through the impaired fibrinolysis. |
worry is conventionally studied within the anxiety disorders, but it has recently been given prominence in a theoretical account of persecutory delusions (freeman, 2007). worry brings more implausible paranoid ideas to mind, keeps them there, and escalates the distress. there is increasing evidence to support this view. in a national epidemiological survey, individuals reporting concerns of plots to harm them had almost ten times higher odds of reporting worry than individuals without paranoid fears (freeman., 2011). cross - sectional studies with patients with persecutory delusions have shown that high levels of worry are common, comparable to those seen in generalised anxiety disorder, and that higher levels of worry are associated with more distressing paranoia (freeman and garety, 1999 ; freeman., 2001 ; startup., 2007 ; morrison and wells, 2007 ; bassett., 2009 ; freeman., 2010). longitudinal studies have shown that worry predicts the later development and persistence of paranoid fears (startup., 2007 ; freeman., 2012b), and an experimental study has shown that higher levels of worry predict the occurrence of paranoid thoughts (freeman., 2008). in the strongest tests of the causal role of worry in persecutory delusions, two clinical intervention studies have shown that directly targeting the cognitive style of worry significantly lessens paranoia (foster., 2010 ; hepworth., this series of studies indicates that worry in psychosis may well require the level of attention it receives in the anxiety disorders. theoretical accounts of psychosis increasingly highlight both affective and cognitive routes to delusions (e.g. garety., 2001 ; myin - germeys., this is not meant to imply that affect has no cognitive mechanisms but to make the distinction between what have traditionally been considered as emotional and psychotic processes. non - affective cognitive disturbance in psychosis has had the greater focus (e.g. hemsley, 1993 ; frith, 1992 ; kapur, 2003 ; green., 2004), but the emotional route to delusions is gaining evidential support (e.g. lincoln. the study of worry in psychosis is an example of the latter affective route, since worry is traditionally considered central to the experience of anxiety problems and it is not listed in descriptions of psychosis, but is there an impact of worry on cognitive processes traditionally associated with psychosis ? is there an interaction between emotional and psychotic processes ? to answer this question we examined the immediate effects of a bout of worrying on three processes studied in psychosis : working memory, jumping to conclusions, and perceptual anomalies. meta - analyses have consistently shown that working memory performance is impaired in individuals with a diagnosis of schizophrenia (e.g. lee and park, 2005 ; forbes., 2009), leading to the view that it may be an endophenotype for the diagnosis (e.g. horan., 2008). jumping to conclusions, a tendency to seek less data before reaching a decision, has been specifically linked with delusional beliefs, and it has been reliably found in patients with psychosis (see garety., 2007 ; freeman, 2007 ; fine., 2007). particular cognitive dysfunctions in psychosis are considered to produce anomalies of experience (such as perceptual and attentional disturbances, altered experience of self, aberrant salience) that lead to delusional misinterpretations (e.g. maher, 1988 ; kapur, 2003 ; uhlhaas and mishara, 2007) ; the presence of such anomalies have been repeatedly found in patients with psychosis (e.g. chapman, 1966 ; phillipson and harris, 1985 ; bunney., 1999 ; parnas., 2003) and to be associated with delusional ideation (e.g. bell., 2006 ; we therefore studied worry in relation to three processes commonly considered important cognitive markers of psychosis. study of a bout of worry in patients with psychosis has not been reported before. however, a bout of worry in non - clinical worriers has been found to be associated with a reduction in working memory capacity (hayes., 2008 ; leigh and hirsch, 2011), and an anxious mood induction has been found to increase the jumping to conclusions bias in a non - clinical sample (lincoln., 2010a), although not in a delusions group (so., 2008). whether a mood induction increases perceptual anomalies is unknown, but levels of anxiety positively correlate with the presence of perceptual distortions (e.g. bell., 2011 ; tone., 2011). in the current study it was hypothesized that psychotic processes would be exacerbated when patients engage in a period of worry : that worry makes it harder to process information and reason and that it creates a more subjectively odd state. it was predicted that a decrease in working memory, an exacerbation of the jumping to conclusions reasoning style, and the occurrence of anomalous experiences would be greatest in a worry induction group and least in a worry reduction condition. in order to enhance the clinical relevance of the study, we chose to study a bout of worry in patients with persecutory delusions in which worry was identified as at a high level ; the effects of worry were studied in those who were prone to adopt this cognitive style. 67 patients with persecutory delusions completed the study during the baseline assessment, prior to randomisation, of a clinical trial (isrctn23197625) (freeman., 2012a). the participants were recruited from two mental health nhs trusts : oxford health nhs foundation trust, southern health nhs foundation trust. the inclusion criteria were : a current persecutory delusion as defined by freeman and garety (2000) ; scoring at least 3 on the conviction scale of the psyrats delusions scale (i.e. at least 50% conviction in the delusion) (haddock., 1999) ; that the delusion had persisted for at least three months ; a clinical diagnosis of schizophrenia, schizoaffective disorder or delusional disorder ; a clinically significant level of worry, as indicated by scores above 44 on the penn state worry questionnaire (see startup and erickson, 2006) ; aged between 18 and 65 ; and no changes to medication in the past month. criteria for exclusion were : a primary diagnosis of alcohol or substance dependency or personality disorder ; organic syndrome or learning disability ; a command of spoken english inadequate for engaging in therapy ; and currently having individual cbt. the psyrats delusions is a six item multidimensional measure. higher scores indicate greater severity. the panss is a 30-item rating instrument developed for the assessment of patients with schizophrenia. part a assesses ideas of reference (e.g. it was hard to stop thinking about people talking about me behind my back) and part b assesses ideas of persecution (e.g. i was convinced there was a conspiracy against me). each item is rated on a 5-point scale. the pswq is the most established measure of trait worry style and has been used in non - clinical and clinical populations (see review by startup and erickson, 2006). the vocabulary and matrix reasoning subtests were used to obtain an estimate of iq. in order to test the effects of the three conditions on worry and mood state, the participants marked on three 0 (not at all) to 100 (totally) visual analogue scales the degree to which three working memory tasks were used : digit span forwards, digit span backwards, and letter number sequencing. these are the most commonly used working memory tasks in schizophrenia research (see nuechterlein., 2004). data - gathering was assessed with a probabilistic reasoning task that has been extensively used with people with delusions. participants are asked to request as many pieces of evidence (coloured beads) as they would like before making a decision (from which of two hidden jars the beads are drawn). the colours of the beads in the jars were altered for the repeat of the task. the key variable is the number of beads requested before making a decision. in the current study the caps was adapted into a state measure by asking whether any of the perceptual anomalies had occurred in the past 5 min, with participants simply responding with a yes or no. the scale covers a variety of anomalies including changes in levels of sensory intensity (e.g. are sounds much louder than they normally would be?), distortion of own body or the external world (e.g. have you found the appearance of things or people seeming to change in a puzzling way e.g. distorted shapes or sizes or colour?), sensory flooding (e.g. have you found that sensations happened all at once and flooded you with information?), temporal lobe experiences (e.g. have you had the feeling that of being uplifted, as if driving or rolling over a road while sitting quietly?) and hallucinations (e.g. have you heard noises or sounds when there was nothing about to explain them?). in the current study we analysed the total score, but also tested separately the hallucination items and the non - hallucination perceptual anomalies. the cds was based on the view that depersonalisation constitutes a syndrome which, in addition to ineffable feelings of unreality, also includes emotional numbing, heightened self - observation, changes in body experience, distortions in the experiencing of time and space, changes in the feeling of agency, feelings of having the mind empty of thoughts, memories and/or images, and an inability to focus and sustain attention (sierra and berrios, 2000). two studies have shown that the scale assesses five distinct types of anomalous experiences (sierra., 2005 ; simeon., 2008). we used an adapted state version by asking about the occurrence of such experiences in the past few minutes. we used 19 items from the cds, since ten items were not suitable for a state version (e.g. previously familiar places look unfamiliar, as if i had never seen them before). we examined the total score but also used four factors from the largest study of the cds structure (simeon., 2008) : unreality of self (e.g. familiar voices (including my own) sound remote and unreal), perceptual alterations (e.g. i have the feeling that my hands or my feet have become larger or smaller), unreality of surroundings (e.g. what i see looks lifeless, as if i were looking at a picture), and temporal disintegration (e.g. it seems as if things that i have done had taken place a long time ago.). participants completed the baseline assessments and the tasks assessing working memory, jumping to conclusions, and anomalous experiences. they then either received a worry induction, worry reduction, or a neutral condition. the worry induction procedure was designed to encourage worry about topics that each participant already spent considerable time thinking about. it consisted of three stages. in the first stage the worry domains questionnaire (wdq ; tallis., 1992) was used to elicit common worries (that are not paranoid). in the second stage, participants took their two main worries identified from the wdq and completed the catastrophising procedure for each (vasey and borkovec, 1992). the catastrophising procedure produces a worry chain by the experimenter repeatedly responding to answers with what is it that worries you about x? participants were led to produce at least ten catastrophising steps for each worry. the third stage was 5 min of further engagement with worry, using instructions from mclaughlin. (2007) : during this period we would like you to engage with your worrisome thoughts. please refer to your list of worrisome topics. when you are asked to begin, please close your eyes and worry about your most worrisome topic in the way that you usually worry about it, but as intensely as you can, until the experimenter asks you to stop and open your eyes. if you normally worry about one topic at a time, please try to do the same during this period. however, if your thoughts change to another worry topic during this period feel free to allow these thoughts to continue. it is alright to change topics during this period if the changes occur naturally during the worry process. we have previously shown in non - clinical individuals that worry is increased using this procedure (southgate, 2009). the worry reduction procedure consisted of a 10 min mindfulness relaxation exercise (mindfulness of the breath) (kabat - zinn, 2006), which could be accompanied by positive music if the participant chose (delibes : copplia act 1 number 3 mazurka) (mayer., 1995). the neutral condition consisted of the person simply reading from a selection of magazines for ten minutes. participants who received the worry induction condition also received if they wished the worry reduction condition at the end of testing. all analyses were carried out using spss version 20 (ibm, 2011). to test change after the randomisation condition, analysis of covariance was used, with post - randomisation score as the dependent variable, group as a fixed factor, and baseline score as the covariate. when there was a main effect of group, least significant difference pairwise comparisons for the estimated marginal means were tested. basic demographic and clinical information for each of the randomisation groups is presented in table 1. the participants predominately had clinical diagnoses of schizophrenia, were unemployed, and were prescribed anti - psychotic medication. they had levels of paranoia as assessed by the gpts comparable to other studies of patients with persecutory delusions (e.g. freeman., 2010) and levels of worry as assessed by the pswq comparable to patients with generalised anxiety disorder (e.g. behar., 2003). it can be seen in table 2 that the worry induction condition significantly increased levels of worry compared with the other two conditions. the worry induction condition also produced an increase in anxiety and a decrease in happiness compared with the other two conditions. the post - randomisation performance of each group on the tasks assessing psychotic processes is summarized in table 3. it can be seen that there are no differences between the groups in performance on working memory or jumping to conclusions tasks (controlling for baseline scores). however there are increases in the occurrence of anomalies of experience in the worry induction condition compared with the other two conditions. the only exception for anomalous experiences is that the occurrence of hallucinations is not affected by randomisation condition. (the same significant effects are found if panss total score is added as a covariate.) previous research has shown that this is the most common presentation of paranoia (e.g. freeman and garety, 1999 ; morrison and wells, 2007 ; freeman., 2010). we examined the effects of a bout of worry in these individuals on processes customarily considered as markers of psychosis. given that worry was known to occur frequently in these patients we wanted to know whether it impacted on psychotic processes. the occurrence of worry did not affect working memory or jumping to conclusions, but it did lead to an increase in a range of low - level anomalous experiences. increases in sensations of the unreality of self and surroundings (e.g. feeling of being a detached observer), perceptual alterations (e.g. body feeling very light as it if were floating), and temporal disintegration (e.g. seeming as if things done recently had taken place a long time ago) were all more likely to occur after a period of worry. worrying less reduced the occurrence of anomalies of sensory intensity (e.g. lights or colours seeming brighter or more intense than usual) and sensory flooding (e.g. difficulty distinguishing one sensation from another). our assessment covered a wide variety of anomalous experiences and it was only the occurrence of hallucinations that was unaffected. the point of interest learned from the experiment is that a period of worry in these patients was creating a range of potentially confusing and puzzling perceptual experiences that are known to predict paranoid thoughts. this is an illustration of an interaction of affective with psychotic processes, and adds to our understanding of how worry may contribute to delusional experience. this information can be incorporated into the emerging therapies that target worry in psychosis (foster. 2011) ; clinicians can assess for and normalise the occurrence of such anomalies after a period of worry in patients with persecutory delusions. this fails to replicate the finding of worry impeding working memory in high non - clinical worriers (e.g. leigh and hirsch, 2011). however these previous studies assessed working memory and worry concurrently, while in the current study we simply assessed the effects after a period of encouraging worry. we do not know the extent to which individuals in the worry induction condition were continuing to worry, which is a limitation of the study. it is also clear that the worry induction was having effects more generally on mood ; this is to be expected but limits the precision with which the occurrence of anomalous experiences can be attributed specifically to worry. previous studies have had mixed findings on jumping to conclusions when manipulating levels of anxiety (so., 2008 ;, 2010a), and the current study does not support the idea that jtc changes with anxious mood state. we examined three key processes and did not alter significance levels for multiple testing, agreeing with the view that simply describing what tests of significance have been performed, and why, is generally the best method of dealing with multiple comparisons (perneger, 1998). the final key limitation is that we did not assess levels of paranoia before and after each randomisation condition which means that we could not test whether and how paranoia increased after worrying ; we simply tested the effects of worry on psychotic processes, and a larger experimental study that can examine mediation is now warranted (emsley., 2010). our contention is that the role of worry will receive increasing attention in the study and treatment of delusions. the study was supported by a grant (09/160/06) from the efficacy and mechanism evaluation (eme) programme, which is funded by the uk medical research council and uk nhs national institute for health research. the study was designed by daniel freeman, helen startup, graham dunn and david kingdon. | worry has traditionally been considered in the study of common emotional disorders such as anxiety and depression, but recent studies indicate that worry may be a causal factor in the occurrence and persistence of persecutory delusions. the effect of worry on processes traditionally associated with psychosis has not been tested. the aim of the study was to examine the short - term effects of a bout of worry on three cognitive processes typically considered markers of psychosis : working memory, jumping to conclusions, and anomalous internal experience. sixty - seven patients with persecutory delusions in the context of a non - affective psychotic disorder were randomised to a worry induction, a worry reduction, or a neutral control condition. they completed tests of the cognitive processes before and after the randomisation condition. the worry induction procedure led to a significant increase in worry. the induction of worry did not affect working memory or jumping to conclusions, but it did increase a range of mild anomalous experiences including feelings of unreality, perceptual alterations, and temporal disintegration. worry did not affect the occurrence of hallucinations. the study shows that a period of worry causes a range of subtle odd perceptual disturbances that are known to increase the likelihood of delusions. it demonstrates an interaction between affective and psychotic processes in patients with delusions. |
continuation of psychopharmacotherapy is essential for the clinical outcome of depressive and anxiety disorders ; however, factors that affect their discontinuation are not fully known. in clinical trials, discontinuation of antidepressants has been attributed to lack of clinical response, adverse effects, pharmacophobia in panic disorder (pd),1 and depression.2 genetic polymorphism, which is related to adverse effects induced by the selective serotonin reuptake inhibitors (ssris), has not been linked to antidepressant discontinuation thus far. however, perlis reported that the short (s) allele of serotonin transporter gene - linked polymorphic region (5-httlpr) might contribute to agitation and insomnia with the administration of fluoxetine. popp investigated 65 subjects who were administered antidepressants with a predominant serotonergic mechanism and reported that one - half of the subjects with the s / s genotype suffered significant adverse effects, 40% of subjects with s / long (l) genotype had side effects, but none of the subjects with the l / l genotype reported side effects.4 murphy reported that geriatric patients with s / s genotype of 5-httlpr showed more intense side effects with paroxetine (pax) than subjects with the l / l genotype, and subjects with the s / s genotype received a lower final dose of pax and they had lower adherence than subjects with l / l genotype.5 the 5-httlpr has been known as a functional polymorphism. in vitro, the activity of the l variant is more than doubled when compared to that of the s variant in the 5-htt mrna synthesis and 5-htt expression,6 which indicates that the 5-htt gene transcription is affected by the 5-httlpr genetic polymorphism.6 stahl previously reported that pd patients are more sensitive to ssris than depressed patients, given that the former can readily manifest short - term deterioration of their symptoms during the initial phase of pharmacotherapy.7 consequently, pd patients should be initiated from a lower dose than depressed patients.7 in addition, louie asserted that pd patients comorbid with major depressive disorder (mdd) showed lower tolerance to ssris compared to those with mdd alone.8 against this background, the object of the present study was to investigate the association between the discontinuation of the initial pax treatment and the genetic variants of 5-httlpr in japanese patients with pd or mdd, and then to investigate the difference between these two diseases in relation to the discontinuation. fifty - two japanese patients who were diagnosed as having pd, according to the diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm - iv - tr) criteria, and who were administered pax 10 mg / day (paroxetine hcl ; paxil ; glaxosmithkline kk, tokyo, japan), were subjected to analysis in this study. all pd patients (20 men, 32 women ; age, 33.98.3 years [mean standard deviation ] ; range of age, 2058 years) were outpatients at dokkyo medical university hospital (located in mibu, tochigi, japan) and the sakura la mental clinic (located in utsunomiya, tochigi, japan). body weight ranged from 4095 kg (57.911.4 kg). the pd patients in this study were part of the subject group (n=65) in our previous study.9 out of 65 patients, 52 subjects with pd without mdd were selected, and their raw data from the clinical evaluations and the genotyping of 5-httlpr were used and reanalyzed in the present study. patients were permitted to take low doses of lorazepam (< 2.0 mg / day) when they had panic attacks, and patients who reported insomnia were allowed to be prescribed brotizolam 0.25 or 0.5 mg at bedtime. the severity of pd was assessed by using the panic and agoraphobia scale observer - rated version10 before initiating pharmacotherapy with pax. patients were excluded from the present study if they had : 1) diagnosis of axis i other than pd ; 2) diagnosis of axis ii ; 3) severe general medical condition or major abnormal laboratory test ; 4) suicide risk ; 5) history of substance abuse ; 6) use of the 5-hydroxytryptamine (5-ht) agonist, benzodiazepines, antidepressants, antipsychotics before study entry ; or 7) pregnancy. written informed consent was obtained from all pd subjects. the present study was approved by the ethics committees of dokkyo medical university hospital and the sakura la mental clinic. in addition, 88 patients who were diagnosed as having mdd, according to the dsm - iv - tr criteria and who were treated with pax 20 mg / day for 2 weeks as initial treatment. all mdd subjects (29 men, 59 women ; age, 46.813.6 years ; range of age, 2069 years) were outpatients at hirosaki university hospital (located in hirosaki, aomri, japan). the 88 mdd patients were part of the subject group (n=120) in an earlier study.11 out of 120 subjects, 88 subjects with mdd whose genotype of 5-httlpr were selected, and their raw data were used and reanalyzed in the present study. body weight ranged from 4090 kg (56.310.0 kg). no other psychotropic drugs were administered other than diazepam (25 mg / day) for anxiety and brotizolam (0.25 mg / day) for insomnia. patients were excluded if they had : 1) diagnosis of axis i other than mdd ; 2) diagnosis of axis ii ; 3) severe general medical condition or major abnormal laboratory test ; 4) suicide risk ; 5) history of substance abuse ; 6) use of 5-ht agonist, benzodiazepines, antidepressants, antipsychotics, before study entry ; or 7) pregnancy. the present study was approved by the ethics committee of hirosaki university hospital. written informed consent patients who stopped taking pax due to adverse effects were categorized as discontinuation due to adverse effects. patients whose plasma pax concentration was below the limit of detection were categorized as discontinuation due to nonadherence. patients who did not visit the hospital 2 weeks after starting pax (ie, lost to follow - up) were categorized as discontinuation due to untraceability. pax plasma concentration was determined by high - performance liquid chromatography.13 the lowest limit of detection was 0.5 ng / ml, and the interassay coefficient of variation was < 5% at a pax concentration of 1 ng / ml. the l and s alleles of 5-httlpr were determined by polymerase chain reaction as reported by lesch and heils.6 to examine the relationship between the demographic variables and the discontinuation of pax treatment, multi - logistic regression analysis was conducted. for comparison of the discontinuation rates between mdd patients and pd patients, the chi - square test or the fisher s exact test (when data were sparse) was carried out. for comparison of the differences of demographic data between mdd patients and pd patients, the student s t - test, welch s t - test, and chi - square test, were used. multilogistic regression analysis, chi - square test, fisher s exact test, welch s t - test, and student s t - test were conducted with dr spss ii for windows (spss japan inc., tokyo, japan) and ibm spss statistics version 19.0 (japan ibm, tokyo, japan). fifty - two japanese patients who were diagnosed as having pd, according to the diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm - iv - tr) criteria, and who were administered pax 10 mg / day (paroxetine hcl ; paxil ; glaxosmithkline kk, tokyo, japan), were subjected to analysis in this study. all pd patients (20 men, 32 women ; age, 33.98.3 years [mean standard deviation ] ; range of age, 2058 years) were outpatients at dokkyo medical university hospital (located in mibu, tochigi, japan) and the sakura la mental clinic (located in utsunomiya, tochigi, japan). body weight ranged from 4095 kg (57.911.4 kg). the pd patients in this study were part of the subject group (n=65) in our previous study.9 out of 65 patients, 52 subjects with pd without mdd were selected, and their raw data from the clinical evaluations and the genotyping of 5-httlpr were used and reanalyzed in the present study. patients were permitted to take low doses of lorazepam (< 2.0 mg / day) when they had panic attacks, and patients who reported insomnia were allowed to be prescribed brotizolam 0.25 or 0.5 mg at bedtime. the severity of pd was assessed by using the panic and agoraphobia scale observer - rated version10 before initiating pharmacotherapy with pax. patients were excluded from the present study if they had : 1) diagnosis of axis i other than pd ; 2) diagnosis of axis ii ; 3) severe general medical condition or major abnormal laboratory test ; 4) suicide risk ; 5) history of substance abuse ; 6) use of the 5-hydroxytryptamine (5-ht) agonist, benzodiazepines, antidepressants, antipsychotics before study entry ; or 7) pregnancy. written informed consent was obtained from all pd subjects. the present study was approved by the ethics committees of dokkyo medical university hospital and the sakura la mental clinic. in addition, 88 patients who were diagnosed as having mdd, according to the dsm - iv - tr criteria and who were treated with pax 20 mg / day for 2 weeks as initial treatment. all mdd subjects (29 men, 59 women ; age, 46.813.6 years ; range of age, 2069 years) were outpatients at hirosaki university hospital (located in hirosaki, aomri, japan). the 88 mdd patients were part of the subject group (n=120) in an earlier study.11 out of 120 subjects, 88 subjects with mdd whose genotype of 5-httlpr were selected, and their raw data were used and reanalyzed in the present study. body weight ranged from 4090 kg (56.310.0 kg). no other psychotropic drugs were administered other than diazepam (25 mg / day) for anxiety and brotizolam (0.25 mg / day) for insomnia. patients were excluded if they had : 1) diagnosis of axis i other than mdd ; 2) diagnosis of axis ii ; 3) severe general medical condition or major abnormal laboratory test ; 4) suicide risk ; 5) history of substance abuse ; 6) use of 5-ht agonist, benzodiazepines, antidepressants, antipsychotics, before study entry ; or 7) pregnancy. the present study was approved by the ethics committee of hirosaki university hospital. written informed consent was obtained from the patients. patients who stopped taking pax due to adverse effects were categorized as discontinuation due to adverse effects. patients whose plasma pax concentration was below the limit of detection were categorized as discontinuation due to nonadherence. patients who did not visit the hospital 2 weeks after starting pax (ie, lost to follow - up) were categorized as discontinuation due to untraceability. pax plasma concentration was determined by high - performance liquid chromatography.13 the lowest limit of detection was 0.5 ng / ml, and the interassay coefficient of variation was < 5% at a pax concentration of 1 ng / ml. the l and s alleles of 5-httlpr were determined by polymerase chain reaction as reported by lesch and heils.6 to examine the relationship between the demographic variables and the discontinuation of pax treatment, multi - logistic regression analysis was conducted. for comparison of the discontinuation rates between mdd patients and pd patients, the chi - square test or the fisher s exact test (when data were sparse) was carried out. for comparison of the differences of demographic data between mdd patients and pd patients, the student s t - test, welch s t - test, and chi - square test, were used. multilogistic regression analysis, chi - square test, fisher s exact test, welch s t - test, and student s t - test were conducted with dr spss ii for windows (spss japan inc., tokyo, japan) and ibm spss statistics version 19.0 (japan ibm, tokyo, japan). to investigate the relationship between the discontinuation of pax treatment in all patients (ie, pd plus mdd) and demographic variables, multilogistic regression analysis was performed with total discontinuation due to all reasons, discontinuation due to adverse effects, discontinuation due to nonadherence, and discontinuation due to untraceability as the dependent variables and age, sex, body weight, diagnosis of pd or mdd, and l / s or s / s genotype of 5-httlpr as independent variables. no significant relationships were found between any of the independent variables and discontinuation due to adverse effects, discontinuation due to nonadherence, discontinuation due to untraceability, or total discontinuation (ie, nonadherence plus adverse effect plus untraceability). in the comparison between pd and mdd using the chi - square test and fisher s exact test (table 1), pd patients had a significant and higher discontinuation rate due to nonadherence than mdd patients (13.5% [7/52 ] versus 0% [0/88 ], respectively ; p<0.001). mdd patients had a significant and higher discontinuation rate due to untraceability than pd patients (12.5% [11/88 ] versus 1.9% [1/52 ] ; p=0.032). there was no significant difference between pd and mdd patients in the discontinuation rate due to adverse effects (13.5% [7/52 ] versus 6.8% [6/88 ] ; p=0.233) or discontinuation due to all reasons (28.8% [15/52 ] versus 19.3% [17/88 ] ; p=0.216). the mean body weight of patients with the l / s genotype was significant and higher compared to subjects with the s / s genotype of 5-httlpr (p=0.026). no significant differences were observed in sex, age, number of panic attacks / week, or the panic and agoraphobia scale score at baseline between patients with the l / s genotype and those with the s / s genotype. of the 52 pd patients enrolled, seven patients (13.5%) stopped taking pax due to adverse effects, such as dry mouth, nausea, and diarrhea (discontinuation due to adverse effects). these seven patients reported that they stopped taking pax because of adverse effects, but they consented to provide blood samples for determination of genotype. one patient did not come to the outpatient clinic (discontinuation due to untraceability). additionally, seven patients (13.5%) had pax plasma concentrations below the detection limit (discontinuation due to nonadherence). thus, 15 patients discontinued, and 37 patients continued with pax (table 3). multilogistic regression analysis was conducted to examine the relationship between demographic variables and discontinuation of pax treatment, with discontinuation due to nonadherence, discontinuation due to adverse effects, discontinuation due to untraceability, and total discontinuation as the dependent variables and age, sex, body weight, comorbid agoraphobia, and l / s or s / s genotype of 5-httlpr as the independent variables. no significant relationships were found between any of the independent variables and total discontinuation due to all reasons, discontinuation due to nonadherence, or discontinuation due to untraceability. the multilogistic regression analysis revealed a tendency for the l / s genotype to be associated with higher discontinuation rate due to adverse effects in pd patients compared to those with s / s genotype (25.0% versus 6.3%, respectively ; p=0.054). no significant differences were seen in age, sex, body weight, or initial montgomery sberg depression rating scale score between the patients with the l / s genotype and patients with s / s genotype before starting pax pharmacotherapy. none of the patients discontinued due to nonadherence (table 5), six discontinued due to adverse effects, including nausea and delirium. also, eleven did not come to the outpatient clinic (discontinuation due to untraceability). multilogistic regression analysis was conducted to investigate the relationship between discontinuation of pax, discontinuation due to adverse effects, discontinuation due to untraceability, and total discontinuation as the dependent variables and age, sex, body weight, and l / s or s / s genotype of 5-httlpr as the independent variables. to investigate the relationship between the discontinuation of pax treatment in all patients (ie, pd plus mdd) and demographic variables, multilogistic regression analysis was performed with total discontinuation due to all reasons, discontinuation due to adverse effects, discontinuation due to nonadherence, and discontinuation due to untraceability as the dependent variables and age, sex, body weight, diagnosis of pd or mdd, and l / s or s / s genotype of 5-httlpr as independent variables. no significant relationships were found between any of the independent variables and discontinuation due to adverse effects, discontinuation due to nonadherence, discontinuation due to untraceability, or total discontinuation (ie, nonadherence plus adverse effect plus untraceability). in the comparison between pd and mdd using the chi - square test and fisher s exact test (table 1), pd patients had a significant and higher discontinuation rate due to nonadherence than mdd patients (13.5% [7/52 ] versus 0% [0/88 ], respectively ; p<0.001). mdd patients had a significant and higher discontinuation rate due to untraceability than pd patients (12.5% [11/88 ] versus 1.9% [1/52 ] ; p=0.032). there was no significant difference between pd and mdd patients in the discontinuation rate due to adverse effects (13.5% [7/52 ] versus 6.8% [6/88 ] ; p=0.233) or discontinuation due to all reasons (28.8% [15/52 ] versus 19.3% [17/88 ] ; p=0.216). the mean body weight of patients with the l / s genotype was significant and higher compared to subjects with the s / s genotype of 5-httlpr (p=0.026). no significant differences were observed in sex, age, number of panic attacks / week, or the panic and agoraphobia scale score at baseline between patients with the l / s genotype and those with the s / s genotype. of the 52 pd patients enrolled, seven patients (13.5%) stopped taking pax due to adverse effects, such as dry mouth, nausea, and diarrhea (discontinuation due to adverse effects). these seven patients reported that they stopped taking pax because of adverse effects, but they consented to provide blood samples for determination of genotype. one patient did not come to the outpatient clinic (discontinuation due to untraceability). additionally, seven patients (13.5%) had pax plasma concentrations below the detection limit (discontinuation due to nonadherence). thus, 15 patients discontinued, and 37 patients continued with pax (table 3). multilogistic regression analysis was conducted to examine the relationship between demographic variables and discontinuation of pax treatment, with discontinuation due to nonadherence, discontinuation due to adverse effects, discontinuation due to untraceability, and total discontinuation as the dependent variables and age, sex, body weight, comorbid agoraphobia, and l / s or s / s genotype of 5-httlpr as the independent variables. no significant relationships were found between any of the independent variables and total discontinuation due to all reasons, discontinuation due to nonadherence, or discontinuation due to untraceability. the multilogistic regression analysis revealed a tendency for the l / s genotype to be associated with higher discontinuation rate due to adverse effects in pd patients compared to those with s / s genotype (25.0% versus 6.3%, respectively ; p=0.054). no significant differences were seen in age, sex, body weight, or initial montgomery sberg depression rating scale score between the patients with the l / s genotype and patients with s / s genotype before starting pax pharmacotherapy. none of the patients discontinued due to nonadherence (table 5), six discontinued due to adverse effects, including nausea and delirium. also, eleven did not come to the outpatient clinic (discontinuation due to untraceability). multilogistic regression analysis was conducted to investigate the relationship between discontinuation of pax, discontinuation due to adverse effects, discontinuation due to untraceability, and total discontinuation as the dependent variables and age, sex, body weight, and l / s or s / s genotype of 5-httlpr as the independent variables. discontinuation rate of ssris has been reported to be 16.8%31.0% in mdd.15 goethe reported that the discontinuation rate was 26.9%, and discontinuation rate did not differ among ssris.16 total discontinuation rate in pd and mdd in the present study was 22.8%, which is concordant with the previous report.15,16 stahl previously reported that patients with pd are typically initiated from a lower dose of ssris compared to depressed patients because their symptoms can easily worsen over the short term, worsening at the start of pharmacotherapy.7 toni reported a 10.6% discontinuation rate for antidepressants (pax, imipramine, and clomipramine) due to adverse effects in a 3-year longitudinal study of pd,1 which is very similar to that in pd (13.5%) in the present study. when taking the difference in daily dose of pax between pd (10 mg / day) and mdd (20 mg / day) patients into account, pd patients might be more intolerant of pax in the initial 2 weeks of treatment than mdd patients ; indeed, the present results showed that they can experience adverse effects easily in this initial phase of antidepressant treatment. freire evaluated and analyzed personality traits in subjects with : pd without mdd ; those with mdd without pd ; those with pd and mdd ; and control group by maudsley personality inventory.17 they reported that those with mdd without pd, and those with pd and mdd had significant lower extraversion score compared to control subjects, while a significant difference in extraversion score was not observed between subjects with pd without mdd and control subjects. such difference in extraversion score might explain the behavioral difference under pharmacotherapy with pax between two disorders in the present study. that is, when the treatment is not beneficial because of the lack of efficacy and/or adverse effects, mdd patients tend to stop the treatment without consultation with their doctor in charge (ie, untraceability) because of their introversion tendency ; however, pd patients tend to pretend to be adherent with pharmacotherapy without taking their drugs (ie, nonadherence). a meta - analysis of 26 studies reported the trend of association between the 5-httlpr s allele and the increased scores of anxiety - related personality trait (p=0.087).18 yamakawa found that among 264 women with metabolic disease, those with s / s genotype of 5-httlpr showed significantly reduced fasting blood glucose following nutritional intervention both over the short (11 weeks) and long (23 years) term,19 although they stopped short of confirming an association between adherence to the intervention and 5-httlpr polymorphism. thus, the s / s genotype of the 5-httlpr might be associated with anxiety - related personality trait, which leads patients to favorable adherence ; however, such association was not observed in the present study. whether 5-httlpr might contribute to the risk of adverse effects with ssris is uncertain ; however, perlis asserted that the s allele of 5-httlpr might contribute to the risk of insomnia and agitation with fluoxetine treatment in patients with mdd,3 while no such association was seen in mdd patients in the present study. one possible explanation is the difference of duration of observation (12 weeks in the report from perlis and 2 weeks in the present study). the results of the present study also indicate that discontinuation rate in the subjects with the l / s genotype of 5-httlpr showed a higher tendency of discontinuation rate due to adverse effects compared to those with s / s genotype in pd, while no such association was seen in mdd patients in the present study. of course, there are several limitations that should be mentioned in the present study. second, numbers of the subjects in both of pd and mdd group were relatively small. the power of each statistical analysis was calculated and yielded values ranging from 0.050.746, which suggests no statistical significant differences are associated with low statistical power, which might result in type ii error. additionally, the cyp2d6 genotype has significant impact on pharmacokinetics of pax even in asian population,20,21 which means activity of cyp2d6 is one of the confounding factors ; however, the cyp2d6 genotype has not been determined in the subjects in the present study. our preliminary findings should be replicated in a larger number of subjects and under longer duration of observation to draw firm conclusion. the findings of the present study indicate that 5-httlpr genotype might be one of the factors that are related to the discontinuation of initial pax treatment due to adverse effects in pd patients. the findings also indicate that pd patients may be more intolerant than mdd patients to pax treatment within the first 2 weeks. | objectivethe aims of the present study were to analyze the association between discontinuation of paroxetine (pax) and the genetic variants of the polymorphism in the serotonin transporter gene - linked polymorphic region (5-httlpr) in japanese patients with panic disorder (pd) and major depressive disorder (mdd).methodsthe 5-httlpr genotype was determined by polymerase chain reaction method. pax plasma concentration was measured by high - performance liquid chromatography to confirm adherence.resultswhen comparing between the pd and mdd patients with the chi - square test and fisher s exact test, the pd patients had a significant and higher discontinuation rate due to non - adherence than did the mdd patients (13.5% [7/52 ] versus 0% [0/88 ], respectively ; p<0.001). mdd patients had a significant and higher discontinuation rate due to untraceability than pd patients (12.5% [11/88 ] versus 1.9% [1/52 ] ; p=0.032). multilogistic regression revealed a tendency for the long / short and short / short genotypes to affect discontinuation due to adverse effects in pd patients (25.0% versus 6.3%, respectively ; p=0.054).conclusionthe results indicate that the 5-httlpr genotype might contribute to the discontinuation of initial pax treatment due to adverse effects in pd patients. |
in endodontics, we sometimes encounter complexities in the treatment of traumatized immature permanent teeth, particularly when the vital pulp exposure occurs in cases with incomplete root formation. traumatic injuries are the most common threats to the vitality and integrity of developing teeth since trauma may jeopardize the pulp vitality an important component responsible for root development is hertwig s epithelial root sheath (hers). these processes are controlled by specific basement membrane - mediated epithelial - mesenchymal interactions together with various enzymatic and biomolecular activities [35 ]. pulpal infection and inflammation can impair the differentiation and proliferation of cells between hers and dental pulp, or dental papilla. this impairment can hinder development of root, particularly in the developmental stages of root formation. hence, the primary aim of treatment in immature traumatized teeth should be maintaining the pulp vitality to allow natural root development or maturogenesis to occur. the term maturogenesis was first introduced by weisleder and benitez. it describes a vital pulp therapy procedure that triggers continuous deposition of dentin all along the root length and culminates complete physiological root formation together with natural root end closure. according to the glossary of american association of endodontists (aae), unlike apexogenesis, the definition of root development is not limited to the induction of apical end closure because dentinogenesis occurs throughout the root as well. at present, this procedure is the preferred treatment for immature and traumatized vital teeth because it induces healing via regeneration instead of repair. furthermore, it results in increased tooth thickness, root length, and fracture resistance [1013 ]. recently, several materials have been introduced for maturogenesis, including calcium hydroxide, mta, adhesive resins, biodentine, and bioaggregate (ba) [1417 ]. on the basis of findings of several in vitro and in vivo studies it induces minimal inflammation in the pulp tissue and significantly increases the secretion of il-1 in the pulp ; thus, providing favorable conditions for pulp regeneration [1416 ]. it seems that mta utilizes complex mechanisms to provide an appropriate surface (scaffold) for the adhesion of progenitor cells. it also activates cell interaction functions, ultimately stimulates continuous reparative dentinogenesis throughout the length of the root, and induces physiological root development. one important challenge of using gray and white mta in the anterior teeth is preventing the development of crown discoloration [1921 ]. some authors have elucidated that discoloration occurs due to a chemical reaction between the pulp and blood products. there are several techniques available to improve this esthetic problem ; for example, bleaching, laminate veneers and porcelain crowns. removal of discolored white mta using a high - speed bur with a water - cooling system under an operative microscope may cause considerable improvement in tooth color. however, internal bleaching of the crown has been recommended as the best procedure to attain desirable results. the aim of this case report was to describe the treatment of two immature traumatized permanent central maxillary incisors with complicated crown fracture using white mta. we discussed the biological basis of maturogenesis in this case, which was managed appropriately and followed - up for 10 years. a 7-year - old girl was referred to my private practice with complicated crown fracture in two permanent maxillary central incisors. her medical history was unremarkable. in the patient s dental history, she reported a traumatic accident in school as a result of falling down and hitting her front teeth on the table. inspection of the intraoral hard tissues revealed that both teeth had horizontal crown fractures and pulp exposure. periapical radiography showed incomplete root development without root or alveolar bone fractures (fig.1-a). (b) cervical pulpotomy was performed and white mta was placed as a conservative treatment procedure for inducing physiological continuous root development. the injured teeth responded to percussion with mild pain, and there was no pain in palpation. the periodontal evaluations and mobility tests were normal in all teeth and had results comparable to those of the control teeth. we administered local infiltration anesthesia using 2% lidocaine and 1:80000 epinephrine (darupakhsh, tehran, iran). on the basis of findings from a histological study by cvek, we performed cervical pulpotomy under a rubber dam using a high - speed size 2 round diamond bur (diatech, heerbrugg, switzerland) with copious water as a coolant. we removed the coronal pulp tissue to a level at which we could achieve adequate hemostasis. then, we rinsed the access cavity with 3 ml of 5.25% sodium hypochlorite (naocl) solution (golrange, tehran, iran) for up to three minutes to provide hemostasis. once hemostasis was achieved, we placed an approximately three millimeter - thick layer of proroot white mta (dentsply tulsa dental, tulsa, ok, usa), mixed according to the manufacturer s instructions, over the remaining pulp tissues. in order to set the mta, we placed a moist cotton pellet over the mta and covered it with coltosol (coltene / whaledent ag, altsttten, switzerland) as a temporary filling material and then took a final periapical radiograph (fig. finally, we restored the teeth with self - setting glass ionomer (fuji, tokyo, japan) and light - cure composite resin (3 m espe, st. (a) forty - eight hours later, crown restoration is complete with translucence self - cure gi cement and light cure composit resin. we re - evaluated the patient at one, three, six and 18 months, and then after 10 years. the patient returned on the ninth day with a slight gray discoloration of the teeth that was limited to the cervical area of the crowns. however, the teeth were asymptomatic, and there was radiographic evidence of hard tissue bridge formation and continuous root maturation. these findings were followed and finally we observed promising, successful, normal root development after one year (fig. 3). after we were confident that there was successful maturogenesis, we discussed the necessity of internal bleaching with the patient and her parents to improve tooth discoloration. (a) twelve months later, radiograph demonstrated complete root formation and apical closure. a longer root, greater crown - root ratio, smaller pulp space and thicker convergent dentinal walls are seen. (a) discoloration as a result of white mta application. (b) after four appointments of walking bleaching the teeth regained their original shade. cure composite resin restored esthetics after 12 months of follow - up. walking bleaching is considered to improve this type of esthetic problems. thus, after the administration of local anesthesia, we isolated the teeth with a rubber dam and accessed the pulp chamber again. we irrigated the pulp chamber with 5.25% naocl and dried it with a cotton pellet. considerable improvement in the color of the teeth was seen immediately after the removal of the discolored white mta. at the same session, we applied 10% carbamide peroxide gel (ph 6.62 ; nite white, beverly hills, ca, usa) as a bleaching agent in the cavity over the white mta. we placed a cotton pellet over the bleaching agent and finally sealed the cavity with coltosol. according to the manufacturer s instructions, we refreshed the bleaching material every three days. after four sessions, the color of the teeth had returned to normal and both teeth were clinically asymptomatic (fig. 4-b). under rubber dam isolation, we accessed the teeth again and irrigated them with sterile normal saline and then sealed them with coltosol for one week. after one week, we restored the access cavity with a layer of self - curing glass ionomer cement (fuji, tokyo, japan) as an orifice plug. we then restored the teeth and their crowns with light - cured composite resin (3 m espe, st. we also replaced the labial composite with translucent composite resin restoration to match the patient s improved esthetics (fig. after a five - year follow - up period, both teeth were clinically and radiographically asymptomatic, and the color of the crowns was normal (fig. after 10 years, radiographic findings showed that the teeth had developed satisfactorily and there was no evidence of internal or external resorption or other pathological changes (fig. follow - up radiograph five years after treatment showing hard - tissue barrier formation along the interface of white mta and pulp tissue. after 10 years, digital radiograph shows hard tissue barrier formation along the interface of white mta - pulp and root development is complete without pathological changes in pulp or periradicular tissue. clinical appearance at 10 years follow - up. (b) mandibular and maxillary incisors are control teeth to illustrate marked harmonic color change. preserving the pulp vitality of traumatized teeth with incomplete root formation is the most important criterion for tooth survival because a vital pulp can provide nutrition for metabolic and dentinogenesis activities and biosensory, and defense functions against noxious irritants and finally encourage continuous normal root development. in comparison with conventional root canal treatments, maintaining pulpal vitality in teeth with incomplete root formation requires preservation of the coronal cell - rich zone and radicular pulp tissues. these induce continuous physiological deposition of dentin in the coronal region and all along the length of the root canal with greater quality and structural integrity. this procedure results in increased resistance to vertical / horizontal root / crown fractures [10,11, 13 ]. additional advantages of pulp vitality preservation are : the prevention of the probable discoloration of tooth crown and apical periodontitis after current endodontic procedures and maintenance of a viable hers as a critical component in the induction of continuous physiological root formation with natural root end closure for obturation, if necessary [35 ]. the two vital pulp therapy procedures (defined as apexogenesis and maturogenesis) considered necessary to achieve these requirements include direct pulp capping and partial or cervical pulpotomy. pulp capping and partial pulpotomy are believed to have more favorable prognoses and many advantages as compared with cervical pulpotomy. however, in special situations as in our current cases, extensive crown fractures may not allow sufficient space for partial pulpotomy and coronal restoration materials. in addition, cervical pulpotomy is advocated in these cases due to the long interval between the accident and treatment. an additional important criterion is selection of a vital pulp therapy material that can initiate biologic responses in the pulp tissue. the preferred material must stimulate the production of biomolecules and markers of mineralization, while satisfactorily managing the various cellular regulatory and protective mechanisms. ultimately, it triggers complete root formation with higher strength and resistance to fracture and no pathological lesions. in order to achieve normal physiological activities of pulp cells, vital pulpal therapy materials should cause minimal inflammation and hyperemia, without pulpal necrosis or high alkalinity, and possess an optimal ability to seal the dental pulp tissue against bacterial invasion (i.e., microleakage). moreover, these materials should elevate extracellular ca concentration and enzymatic activities as dominant factors in bone metabolism. it is clear that pulp regeneration after injury is a complex process and its mechanisms remain to be fully elucidated. some studies have found a direct relationship between high ph and ca concentration in extracellular fluid and chemotactic, proliferative, and differentiation activities of hard tissue forming cells and alkaline phosphatase (alp) activity at mineralization foci. a high ph can increase levels of some specific calcification enzymes, such as alp ; enhance collagen synthesis by dental pulp cells and stimulate gene expression of bone - related proteins. also, the high concentrations of ca and the continuous release of ca from mta play important roles in reducing the permeability of newly formed capillaries in the healing tissue, intravascular fluid leakage, and the volume of intercellular fluid, as well as increasing the concentration of ca derived from the blood supply. these processes result in decreased inflammation in the pulp due to mta. also, high intracellular ca increases the activity of calcium - dependent pyrophosphatase and reduces the level of mineralization - inhibiting pyrophosphate ions within the tissues that also result in continuous mineralization process. these events can ultimately provide an appropriate scaffold for the migration, adhesion, differentiation, and proliferation of stem/ progenitor pulp cells. several other materials have been advocated for these purposes with success rates of 75100% in incisor teeth. the most common materials used are ca(oh)2, mta, adhesive systems, biodentine and bioaggregate (ba). many in vivo and in vitro studies have confirmed the superior physical and biological properties of mta [3638 ]. however, the mechanisms by which mta induces mineralization and manages cellular and molecular activities are unclear. it has been shown that mta may cause the secretion of proinflammatory cytokines in the pulp and may manage the activities of other biomolecules that induce several active markers of mineralization and growth factors in dentinogenesis [3946 ]. these findings elucidate how mta could play a protective role in dental pulp tissue just after tissue injury. in healthy pulp tissue, biomolecules and basement membrane act as natural scaffolds for the migration and adhesion of progenitor cells, differentiation of cells, and subsequent events. conversely, when the pulp is exposed after trauma and the basement membrane or dental epithelium (i.e., the natural scaffold) is absent, the hard - tissue - forming cells in the pulp need a suitable surface (scaffold) for adhesion and differentiation to commence dentinogenesis. we know that mta can act as a pulp - capping material in vital pulp therapy, and it has optimal interaction with pulp tissue ; therefore, it may be able to provide a suitable scaffold for reparative dentinogenesis. in addition, dental pulp has natural repair / regeneration capacity ; for example, pulp tissue fluid is rich in phosphate ions. when mta is placed on the exposed pulp tissue, a chemical reaction between the pulp tissue fluid and tricalcium oxide in mta results in the production of calcium hydroxide. calcium hydroxide releases a lot of ca ions, and both ca and phosphate ions are critical for bone metabolism. a continued reaction occurs between these two types of ions, which leads to the precipitation of hydroxyapatite crystals (hap). at first, there is probably continuous precipitation of hap crystals in the mta due to its porous structure. over time, with gradual dissolution of mta, hap crystals nucleate, grow, and deposit on the surface of mta. ultimately, a thick layer of dystrophic calcification with integrated structures (i.e., a dentinal bridge) is formed along the mta and pulp tissue according to the aforementioned mechanisms. this dentinal bridge is observable as a radiopaque zone on radiographs, as seen in this case report. subsequently, the pulp cells migrate, attach to hap crystals, and enable continuation of dentinogenesis with other biomolecules. in this article, two maxillary central incisors in a seven - year - old female patient with immature roots and complicated crown fracture were treated successfully using white mta after cervical vital pulpotomy. one of the complicating factors in the treatment of immature teeth is how to predict the depth of the inflammatory reaction in the traumatized pulp. of course, this depends on several factors, the most critical of which is the time interval between the accident and treatment. according to the study by cvek, the inflammatory reaction usually does not extend more than two millimeters from the exposed surface within 48 hours, but after seven days it can go deeper. therefore, cervical pulpotomy was the most suitable treatment choice in this case with a long interval between the accident and treatment. recheck examinations at all periodic follow - ups revealed that the treatment was successful in preserving pulpal vitality and continuation of root development in teeth. however, both teeth showed crown discoloration as reported in some other studies. this was likely due to a probable chemical reaction between the white mta and blood products or pulp tissue fluid [1922 ]. in this case, bleaching was postponed until the formation of a hard tissue bridge and confidence of successful maturogenesis. ultimately, after one year and complete maturogenesis, the teeth color was returned to normal with four bleaching sessions, and was maintained during the long - term follow - up. in the literature, there is controversy concerning whether root canal treatment is necessary after successful maturogenesis. some authors believe that severe calcification probably occurs over a long time ; thus, they advocate that it would be better to perform conventional root canal therapy after complete root formation and natural apical constriction. conversely, other studies report that there are no histological data based on pathological changes ; thus, they emphasize that root canal treatment in traumatized cases should be based on specific findings such as necrosis, infection, apical periodontitis and internal / external pathological root changes, not just pulp canal obliteration. in the current case, the teeth did not show any radiographic or clinical pathological changes ; therefore, we did not perform root canal therapy. in conclusion, this case report showed that successful maturogenesis can occur with long - term treatment of fractured crowns complicated with vital pulp exposure and incomplete root formation using white mta in the absence of pathological changes in the pulp and periapical tissues. in addition, endodontic treatment can be postponed until evidence of pathological changes is observed. | this case report describes the treatment of two immature maxillary central incisors in a 7-year - old female patient. she suffered complicated crown fracture because of trauma, and the root formation was incomplete. white mineral trioxide aggregate (mta) was selected as the pulp - capping material after cervical pulpotomy to preserve the pulp tissue vitality and achieve maturogenesis. follow - up evaluations showed successful treatment in terms of preservation of pulp vitality and demonstrated marked continuous physiological root development. during 10 years of follow - up, both teeth were clinically asymptomatic, and radiographic evaluations showed apparent root regeneration with apical root - end closure without pulp or periapical pathosis. |
the assembly of the human genome from whole - genome - shotgun sequence reads was once deemed impossible, and the probable outcome was dubbed the ' mad magazine version of the human genome '. this may have referred to mad 's fold - in back cover, where a drawing and the caption under it are folded twice to reveal a new picture and a new caption to go with it. this fold - in back cover is rather like alternative splicing, or, more likely, the intent was to imply production of a crazy - quilt genome structure caused by the incorrect joining of the millions of repeat sequences in the human genome. in spite of these concerns, paired - end sequencing of three sizes of clones largely avoided the misas - sembly problem and resulted in a faithful rendition of the genome, now with only 498 contigs in build 34 version 2. the spectre of the mad magazine version of the genome is coming back in a new guise, however : the corrupt annotation of pseudogenes. programs designed to scan genomic dna for genes are trained to look for gt and ag boundaries and some consensus regions around these boundaries. when they encounter a pseudogene that is nearly intact, they try mightily to make it code for an intact protein. the outcome is a frankenstein gene that never existed in reality, but was cobbled together from spare parts, beginning with the pseudogene carcass. although it is thought that the number of pseudogenes in the human genome is 20,000 or more, these genome features are not annotated as completely as intact genes. locus link has 21,382 protein coding genes, but only 2,592 pseudogenes are listed (as of 13th february, 2004). complete annotation of the human genome must also include identification and naming of all the pseudogenes. there are 57 intact and putatively functional huma cyp genes, as well as 58 pseudogenes. these examples illustrate the trouble that the pseudogenes present for gene prediction algorithms. in the first example, cyp2g2p encodes a nearly intact p450 (figure 1a) with two in - frame stop codons, one in exon 1 and one in exon 3. a frameshift or stop codon in exon 1 may trick the program into accepting the next downstream met as the starting met ; this happened in cyp2g2p, with a met immediately after the exon 1 stop codon being assigned as the start codon. if the phase of the two intron - exon junctions bracketing the defective exon is the same, then the exon may just be skipped. the result is a shortened protein (figure 1b), missing part of exon 1 and all of exon 3. the ensembl prediction for this same protein is even shorter ; it is missing all of exons 1, 3, 8 and 9. figure 1c shows the alignment of the lower - case portions of the pseudogene with the functional mouse cyp2g1 gene, to show what was missed by the prediction algorithm. the nine exons are shown, one per line, with the intron phase indicated in parenthese. lower - case letters are the parts of the pseudogene missed in the genscan prediction shown in panel b. (b) genscan prediction of the cyp2g2p pseudogene taken from the university of california santa cruz genome browser. the gene is shown in the same format as in panel a, with blank space for the missed regions. (c) alignments of the two regions left out of the genscan prediction against the mouse cyp2g1 functional gene. a frameshift or stop codon in an internal exon may trigger several possible solutions to be created by the program. figure 2 illustrates a second possibility not seen in figure 1. in the cyp2ab1p pseudogene, a bad gt boundary at the end of exon 2 and a stop codon in exon 3 the result shortens exon 2 by 13 amino acids and replaces the first 25 amino acids of exon 3 with 17 amino acids derived from an alternative frame translation from the first part of exon 3. later in the pseudogene, a defective exon boundary between exons 6 and 7 and a frameshift in exon 7 are avoided by creation of a second cryptic exon. this new exon replaces exon 6 and about half of exon 7 and bypasses both genetic defects. this pseudopseudogene is now taking on a new artificial look, like a car that has been in a wreck and now has a different coloured door. (a) the human cyp2ab1p pseudogene, using the same conventions as in figure 1. (c) alignments of the two regions left out of the genscan prediction against the mous cyp2ab1 putatively functional gene. the cyp51p1 pseudogene has only 130 of 508 amino acids correctly predicted (figure 3a, upper case). curiously, the genscan prediction adds one bogus exon 72,022 base pairs (bp) upstream ; this distant exon has no significant blast hits other than itself in the nr section, of genbank, and no hits in the dbest section. it is hard to imagine why this sequence is included as part of the cyp51p1 prediction. (a) the human cyp51p1 pseudogene. upper - case sequence (130/508 amino acids) is correctly predicted by genscan. (b) the underlined sequence is a new first exon, plus an upstream extension from the start codon, to find a phase 2 junction. most of the pseudogene is not predicted, although the pseudogene is 92 per cent identical to cyp51 over the entire length of the protein sequence. the prediction for this pseudogene is 937 amino acids long, about twice the size of any p450 protein (figure 4). investigation of the sequence shows that, after 211 p450 residues (about 42 per cent), most of the transporter add1 gene (major facilitator superfamily) nm_181785 has been added. the university of california santa cruz genome browser shows another gene in this region with multiple mrnas, but it is on the opposite strand. therefore, the genscan prediction for cyp51p2 fuses less than half of the true p450 pseudogene with another downstream gene and additional exon predictions that can not be real since they overlap a third gene on the opposite strand. this problem of gene fusions is not rare. while annotating p450 genes in the drosophila pseudoobscura genome, a predicted triple - gene fusion was discovered which was 2,182 amino acids long (contig3323_contig6140.179). this sequence sandwiched a p450 gene (cyp9f2 orthologue, minus the last seven amino acids) between a dna ligase iii homologue and an angiotensin - converting enzyme (acer) homologue. erroneous gene fusions of multiple cyp genes in gene clusters have been observed in caenorhabditis elegans, arabidopsis thaliana and takifugu rubripes (pufferfish). (a) the human cyp51p2 pseudogene. upper - case sequence (211/508 amino acids) is correctly predicted. this is a fusion of part of cyp51p2 with the add/ transporter gene and an additional sequence derived from the opposite strand of another gene. cyp2u1, a human p450 gene, has a verified gc boundary at the end of exon 3 (figure 5a). the genscan prediction misses the correct intron 1 gt boundary and jumps to the next one of the same phase. genscan skips the gc boundary at exon 3 and extends exon 3 by 11 amino acids. this gene is correctly annotated in genbank because the mrna sequence is known ; however, there are genes with no known mrna sequences that can be used to correct errors like this. (a) the human cyp2u1 gene known from full - length cdna has a gc intron boundary at the end of exon 3. the underlined sequence is not found in the true protein and the last two exons are missed after the gc boundary is read through. a recent re - annotation effort on the drosophila melanogaster genome has predicted approximately 2,000 more genes than is the case for the current berkeley drosophila genome project (bdgp) annotations. this result was obtained by relaxing the stringency of the fgenesh prediction algorithm ; 7,464 gene models were predicted above and beyond the bdgp annotations. validation by spotting the gene model exon sequences in a microarray and probing for mrna sequences that bound to the array did give support for a large percentage of these predictions. further analysis by reverse transcriptase polymerase chain reaction (rt - pcr) of a subset of the microarray - positive models confirmed that many of these genes are expressed and need to be added to the official gene count. first, the number of weakly supported or unsupported gene models was large, but a significant fraction of these weak predictions were verified by microarray and rt - pcr experiments. some of these genes had no blast hits in genbank and so represent new protein families ; this argues that the conservative estimates (approximately 25,000 genes) used by some human annotators are missing many real genes. there are an estimated 20,000 pseudogenes in the human genome and this number may be much higher. the collection of these defective genes may be called the human pseudogenome. as discussed above, what comes out of this set -- after genscan or ensembl gets through with it -- is pretty frightening. these are vivisected genes raised from the dead, as sure as shelley 's monster. another chromosome 6 gene, tcba1, contains an intron that is 479 kilobases long. znf451, a zinc finger protein, contains a single exon of 9,114 bp. these are all potential challenges for gene prediction programs. unfortunately, prediction algorithms are not written to cope with pseudogenes, sequence errors or gc boundaries, giving rise to the results shown in figures 1 - 5. even the detailed manual annotation of chromosome 6 found only 633 pseudogenes, as compared with 1,557 genes. other evidence suggests that the number of human pseudogenes should be closer to the number of intact genes. perhaps the working definition for the chromosome 6 annotation did not include some small solo exons or detritus exons scattered near documented genes. it is possible, however, that there might still be about 900 undocumented pseudogenes on chromosome 6. the genscan program is quite successful in predicting genes, especially the internal exons of real genes. it has been optimised for different g + c compositions and different species, but not for pseudogenes. in order to be able to annotate all of the human genome and other genomes, there is a need for a second program, which is optimised for detecting pseudogenes. these pseudogenes need to be named and documented on the sequence records and genome browsers in order to avoid the aforementioned problem of creating frankenstein genes. one program that may fulfil some of these criteria is gnomon, the national center for biotechnology information 's hidden markov model ab initio prediction program. this program assumes that very close exon predictions (less than 50 bp apart) are separated by a frameshift, because very short introns are rare (at least in vertebrates). the hidden markov model used by gnomon allows non - consensus splice sites such as the gc boundary in cyp2u1. stop codons in the middle of an otherwise strong alignment are disregarded and the stops are included in the model, which is treated as a pseudogene rather than as a gene. these features ought to allow the correct prediction of the pseudogenes in figures 1 - 4. in practice for example, all amino acids of cyp2g2p are correctly predicted, but 20 extra amino acids are added between exons 2 and 3. the cyp2ab1p sequence predicted by gnomon has 69 more correctly predicted amino acids than genscan, and includes two of the in - frame stops, but it misses exon 7 and adds the same cryptic exon as genscan did after exon 5. gnomon finds most of the c - terminal part of cyp51p1, but misses the first two - thirds. cyp51p2 coverage is improved, but both n- and c - terminal regions are missed, as well as two internal segments. in short, gnomon is better than genscan for these five genes but still misses the gold standard of hand curation. finally, once pseudogenes are correctly assembled, nomenclature groups must devise a suitable nomenclature for them this nomenclature could be applied to other gene families, or other proposals could be made, but this problem needs to be addressed. in the future, progress in genome annotation should make it increasingly hard to find the misleading and erroneous peptide predictions that currently abound, as these become replaced by accurate annotated and named pseudogene models. | annotation of the human genome is inching forward. seven human chromosomes have now been fully annotated, covering 17 per cent of the genome, and at least one chromosome has been re - annotated. the enormity of the task forces a dependence on automated tools for detecting and assembling the genes, followed by hand curation to correct errors and polish the gene models. the accuracy of gene prediction algorithms is very good for internal exons from intact genes, but these programs do peculiar and exasperating things to pseudogenes. these programs can actually resurrect pseudogenes from the dead, making them into viable gene models for intact proteins, albeit science - fictional proteins. this process is demonstrated for four human pseudogenes from the cytochrome p450 family and one putatively functional p450 gene, cyp2u1, having a non - consensus intron boundary. these examples are offered as a call - to - arms to improve pseudogene prediction as an art in itself, and not as a by - product of gene annotation. failure to do so will flood the databases with thousands of false - positive predictions. indeed, they are already there. |
chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an uncommon progressive or relapsing paralyzing disease caused by inflammation of the peripheral nerves. cidp is characterized by the occurrence of symmetrical weakness in both proximal and distal muscles, which progressively increases for more than 2 months1. sensory disturbances are usually slight, proximal and distal parts of the limbs are usually affected symmetrically. less common variants include widespread multifocal neuropathy, focal brachial plexus, and isolated limb nerve and initial cranial nerve presentations, ataxic sensory neuropathy, and generalized neuropathy associated with multifocal central nervous system. we report the clinical and brain single photon emission computed tomography (spect) and response to treatment in patients with atypical cidp2. a 32-year - old man experienced double vision around january, 2010, followed by weakness of his left upper and lower extremities. articulation disorders and loss of hearing in his left ear developed, and he was admitted to our hospital on february 14, 2010. physical examination was normal, and neurological examination showed clear consciousness with no impairment of cognitive function, but with articulation disorders. olfactory sensation was reduced. left ptosis and left gaze palsy, complete left facial palsy, perceptive deafness of the left ear, and muscle weakness of the left trapezius muscle were observed. paresis in the left upper and lower extremities was graded 4/5 through manual muscle testing. deep tendon reflexes were slightly diminished equally on both sides ; no pathologic reflex was seen. chest x - ray, electrocardiogram, thoracoabdominal computed tomography, abdominal ultrasound, and peripheral hematological values were normal. blood chemistry revealed glucose 174 mg / dl, hba1c 7.8%, ck 366 u / l, and no other abnormal findings. anti - acetylcholine antibody, anti - jo-1 antibody, and antiganglioside antibody were all negative. spinal fluid revealed a cell count of 3/l, protein 68 mg / dl, glucose 98 mg / dl, showing albumino - cytologic dissociation ; myelin basic proteins and oligoclonal bands were not detected. no gene mutation related to mitochondrial encephalomyopathy nerve conduction velocity test showed delay of conduction in the bilateral peroneal nerve and median nerve at motor conduction velocity with conduction blocks and only a slight delay in sensory conduction velocity. no abnormality of the brain parenchyma, cerebral nerves or cervico - thoracolumbar region was found on brain magnetic resonance imaging (mri). on electroencephalogram, alpha waves in the main frequency band of 8 to 9 hz were recorded and no obvious paroxysmal discharge was observed, indicating normal findings. brain spect scan showed reduced blood flow in the right inner frontal lobe and both occipital lobes (figure 1). nerve biopsy (left sural nerve) showed reduction of nerve density by 30%, with demyelination, but no adventitial thickening around capillaries, ruling out diabetic peripheral neuropathy. after high - dose intravenous gamma - globulin (25 g / day, 5 days) therapy, articulation disorder, ocular movement disturbance, hearing loss, and motor disturbance of the distal muscles and distal sensory disturbance in the left lower extremity resolved and the patient was able to walk, though mild motor disturbance of the distal muscles and distal sensory disturbance in the left upper extremity remained. figure 1brain single photon emission computed tomography scan showed reduced blood flow in the right inner frontal lobe and both occipital lobes. improvements in flow can be seen two weeks after intravenous gamma - globulin (25 g / day, 5 days). brain single photon emission computed tomography scan showed reduced blood flow in the right inner frontal lobe and both occipital lobes. improvements in flow can be seen two weeks after intravenous gamma - globulin (25 g / day, 5 days). tumors, vascular disease, trauma, tuberculosis, and bacterial infection are the most frequent major causes of multiple cranial nerve palsy. other causes include demyelination neuropathies such as guillain - barr syndrome, cidp, charcot - marie - tooth disease, granulomatous lesions such as tolosa - hunt syndrome and sarcoidosis, vasculitis such as behcet 's disease and sjgren syndrome, and diabetes mellitus. the sudden onset of double vision, articulation disorders, and left hemiplegia in our patient suggested cerebrovascular disorder, multiple sclerosis, or diabetic external ophthalmoplegia. the patient also showed manifestations of multiple cranial nerve disorder, i.e., of the trigeminal nerve, glossopharyngeal nerve, vagus nerve, and hypoglossal nerve. whole - body examination was negative. finally, based on ischemic brain spect images, spinal fluid findings and nerve biopsy results, high - dose intravenous gamma - globulin (25 g / day, 5 days) was initiated. after two weeks, articulation disorders, left ocular motility, deafness of the left ear, and left trapezius muscle weakness were improved. were improved after high - dose intravenous gamma - globulin therapy, i assumed that this case may be associated with some kind of inflammatory change. although this patient had disturbances of the central nervous system including the cranial nerves, no abnormal findings were found on brain mri. however, spect revealed a decrease in cerebral blood flow (cbf) in the right frontal lobe contralateral to the affected side of the body, extending to the region around the anterior cingulate gyrus and right cerebral cortex. the fab region of the immunoglobulin molecule binds to various antigens while the fc region binds to effector cells to regulate immune response. human blood components include 3 types of fc receptors (fcri, fcrii, and fcriii) whose ligand is the fc region of immunoglobulin, and most of these receptors activate inflammation. however, fcriiib is an exception and is known to enhance immunological tolerance and suppress inflammation. in cidp, it has been demonstrated that genetic polymorphism in the promoter region of fcriiib correlates with decrease in fcriiib expression on b cells. decrease in fcriiib is likely to lead to the breakdown in the immune suppression system and result in excessive activation of the inflammatory reaction. therefore, we believe that a similar condition was caused in this patient and inflammation spread to not only the peripheral nerves but also the central nerves, resulting in the decrease in cbf on spect. in addition, the decrease in cbf in this patient may be partially due to a transient ischemic condition arising from a change in the central process of the primary sensory nerves caused by a sensory disturbance through c fibers activated by inflammation. a published report on the therapeutic experience of 10 patients with cidp or its subtype notes that anti - tnf medications were effective in treating sensory disturbance. therefore, the inflammatory mediator that activated c fibers in our patient may be associated with tnf cytokines. that is, we think that, since the inflammatory response disappeared after high - dose intravenous immunoglobulin therapy, this accounts for the improvement of spect findings after therapy. cases of multiple cranial nerve palsy due to diabetes mellitus have been reported, though without complications of impairment of motion. but cidp with central nervous system symptoms as in this case is rare, and no study presenting the changes in spect before and after treatment has been reported. in addition, this case is important and valuable because it presents findings indicating that the central nervous system symptoms observed in a patient with cidp may be related to an ischemic condition caused by an inflammatory change in the central nervous system. the present case is a very unusual one of hemiplegic peripheral neuropathy accompanied with multiple cranial nerve palsy. | a 32-year - old man experienced double vision around january, 2010, followed by weakness of his left upper and lower extremities. articulation disorders and loss of hearing in his left ear developed, and he was admitted to our hospital on february 14, 2010. physical examination was normal, and neurological examination showed clear consciousness with no impairment of cognitive function, but with articulation disorders. olfactory sensation was reduced. left ptosis and left gaze palsy, complete left facial palsy, perceptive deafness of the left ear, and muscle weakness of the left trapezius muscle were observed. paresis in the left upper and lower extremities was graded 4/5 through manual muscle testing. sensory system evaluation revealed complete left - side palsy, including the face. deep tendon reflexes were slightly diminished equally on both sides ; no pathologic reflex was seen. no abnormality of the brain parenchyma, cerebral nerves or cervicothoracolumbar region was found on brain magnetic resonance imaging. on electroencephalogram, alpha waves in the main frequency band of 8 to 9 hz were recorded, indicating normal findings. brain single photon emission computed tomography (spect) scan showed reduced blood flow in the right inner frontal lobe and both occipital lobes. nerve biopsy (left sural nerve) showed reduction of nerve density by 30%, with demyelination. the patient also showed manifestations of multiple cranial nerve disorder, i.e., of the trigeminal nerve, glossopharyngeal nerve, vagus nerve, and hypoglos - sal nerve. whole - body examination was negative. finally, based on ischemic brain spect images, spinal fluid findings and nerve biopsy results, peripheral neuropathy accompanied with multiple cranial nerve palsy was diagnosed. |
one of the reasons for this could be the risks in delivery system, and sometime lawsuits could be held between obstetrician and pregnant women. it is debatable whether this shortage can be attributed to the decrease in the absolute number of obstetricians or a maldistribution. a theory proposed by newhouse jp insisted that an increase in the number of obstetricians will result in the uniformity of their distribution. the japanese ministry of health, labor, and welfare, the japan medical association, and the japan society of obstetrics and gynecology recommended the centralization of obstetric care facilities. as a result, many small obstetric care facilities have been closed in the rural areas. centralization is expected to increase the number of medical facilities with multiple full - time obstetricians and ensure safe childbearing care. previous studies have shown that obstetric care outcomes are favorable in communities with large obstetric care facilities [4, 5 ]. on the other hand, the closure of nearby medical facilities due to centralization is expected to result in pregnant women having to travel longer distances to seek the care they need in the event of an emergency. thus, the centralization of obstetric care facilities could make access to such facilities difficult for some residents. in a previous study, it was shown that pregnant women who live in rural areas experience more difficulty in accessing medical care than those living in urban areas. it has also been shown that poor access to obstetric care facilities is associated with poor outcomes [812 ]. thus, the obstetrician shortage is accompanied by a raging debate as to whether centralization is a useful measure. to the best of our knowledge, no study has evaluated the effects of the number and centralization of obstetricians and obstetric care facilities, respectively, on specific outcomes. the objective of the present study is to examine the effects of the centralization of obstetricians and obstetric care facilities on the perinatal mortality rate in japan. we used the gini coefficient to evaluate the effect of the centralization of obstetricians and obstetric care facilities. the gini coefficient, an economical concept developed by the italian statistician gini c. in 1936, represents the uniformity of incomes [13, 14 ]. several previous studies have used it to evaluate the distribution of medical care resources [1524 ]. data from a health facility census conducted by the ministry of health, labor, and welfare (concerning the numbers of obstetricians and obstetric care facilities) and a national census conducted by the ministry of internal affairs and communications (concerning municipality populations) were used to calculate the gini coefficients in the present study. secondary medical care zones (smczs) are decided according to the medical service law. an smcz is determined by the proportional level of medical care provided for situations requiring stays in hospitals and other facilities in a certain area. in addition to a community 's population and area, many other factors are taken into account, including geographic and other natural conditions, fulfillment of demands in daily life, and social conditions such as transportation. japan comprises 47 prefectures, each of which is divided, based on size, into a number of smczs. the gini coefficients were calculated using a lorenz curve. in order to determine the lorenz curve, the numbers of obstetricians and obstetric care facilities per smcz within each prefecture were calculated. the cumulative relative frequencies of smczs were plotted on the x - axis, while the respective cumulative relative frequencies of the number of obstetricians and obstetric care facilities were plotted on the y - axis. the area between the lorenz curve and the 45 line (also known as the line of perfect equality), multiplied by 2, is called the gini coefficient. the lorenz curve approaches a 45 line when the distribution is equal and shifts away from it, to the lower right, when it is unequal. a gini coefficient close to 0 represents a small disparity and that close to 1 a large disparity (i.e., a more unequal distribution). we then calculated the gini coefficients of obstetricians and obstetric care facilities in all prefectures. in order to investigate the effects of centralization of obstetricians and obstetric care facilities, we used perinatal mortality rates, obtained from the population dynamics survey conducted by the ministry of health, labor and welfare between 2006 and 2010, as the outcome. dependent variables included the total fertility rate (also sourced from the previously mentioned population dynamics survey), per capita numbers of obstetricians and obstetric care facilities for all prefectures (sourced from the census of health care facilities conducted by the ministry of health, labor, and welfare in 2008 and the national census conducted by the ministry of internal affairs and communications in 2010), and the calculated gini coefficients for obstetricians and obstetric care facilities in all prefectures. we performed multiple regression analyses in order to examine the effects of the centralization of obstetricians and obstetric care resources on the outcomes. statistical analysis was performed with spss for windows, version 17.0, with the level of statistical significance set at p < 0.05. the gini coefficients of per capita numbers of obstetricians and obstetric care facilities for all prefectures are shown in table 1. large disparities in the gini coefficients of obstetricians and obstetric care facilities were observed among prefectures (0.0510.382 for obstetric care facilities and 0.0760.513 for obstetricians). the results of the multiple regression analysis about the effects of the centralization of obstetricians and obstetric care facilities on the perinatal mortality rate are shown in table 2. a significant negative correlation was observed between the perinatal mortality rate and the per capita number of obstetricians. an increase in the number of obstetricians was shown to result in a decrease in the perinatal mortality rate. in addition, a significant negative correlation was observed between the perinatal mortality rate and the gini coefficient of obstetric care facilities. increasing centralization of obstetric care facilities was shown to result in a decrease in the perinatal mortality rate. the gini coefficient of obstetric care facilities had a slightly stronger influence on the perinatal mortality rate than the number of obstetricians. the perinatal mortality rate was not observed to be significantly related to either the number of obstetric care facilities or the gini coefficient of the obstetricians. there was a negative relation between the perinatal mortality rate and the number of obstetricians and the gini coefficient of obstetric care facilities. however, no significant correlation was observed between the perinatal mortality rate and the number of obstetric care facilities. the number of obstetricians might be more influential on the neonatal mortality rate compared to the number of obstetric care facilities. we propose that increasing the number of obstetricians can lower the perinatal mortality rate. a negative correlation between the number of obstetricians / gynecologists and when the number of obstetricians is reduced, perinatal mortality rate certainly worsens ; therefore, it is important to retain the number of obstetricians. improvements in the working environment and offering economic incentives are conceivable measures for increasing the number of obstetricians. the centralization of obstetric care facilities (increase in gini coefficient) has been suggested as a suitable approach to lower the perinatal mortality rate. the gini coefficient of obstetric care facilities had a slightly stronger effect on the perinatal mortality rate than the number of obstetricians. we suggest that centralizing obstetric care facilities can improve the perinatal mortality rate even when it is difficult to increase the number of obstetricians. possible measures for further centralization include improving emergency transport methods ; applying information technology ; standardizing medical care ; and using midwives. however, it would be skeptical to assume that further centralization will contribute to maintaining safety with regard to pregnancy and delivery. the first potential limitation of the present study is that we were unable to evaluate access to medical care. the degree of centralization of obstetricians and obstetric care facilities may not necessarily be related to access to medical care. secondly, we were unable to categorize obstetric care facilities by the levels of care they provide. in the future, it will be necessary to measure changes over time while accounting for access to medical care. lastly, because this study was officially announced and that everyone could access the data, it is also important and worth analyzing the effects of other factors on perinatal mortality rate in future. we demonstrated that increasing the number of obstetricians and centralizing obstetric care facilities can improve the perinatal mortality rate. we also proved that centralizing obstetric care facilities can improve the perinatal mortality rate, even when increasing the number of obstetricians is difficult. | objective. we investigated the effects of the centralization of obstetricians and obstetric care facilities on the perinatal mortality rate in japan. methods. we used the gini coefficient as an index to represent the centralization of obstetricians and obstetric care facilities. the gini coefficients were calculated for the number of obstetricians and obstetric care facilities of 47 prefectures using secondary medical care zones as units. to measure the effects of the centralization of obstetricians and obstetric care facilities on the outcomes (perinatal mortality rates), we performed multiple regression analysis using the perinatal mortality rate as the dependent variable. results. obstetric care facilities were more evenly distributed than obstetricians. the perinatal mortality rate was found to be significantly negatively correlated with the number of obstetricians per capita and the gini coefficient of obstetric care facilities. the latter had a slightly stronger effect on the perinatal mortality rate. conclusion. the centralization of obstetric care facilities can improve the perinatal mortality rate, even when increasing the number of obstetricians is difficult. |
it maintains the plasma calcium level by reabsorbing calcium from the bones and kidney and promoting the production of active vitamin d1). after thyroid surgery is a common cause of hypoparathyroidism, a congenital disorder is the most common cause in childhood2). digeorge syndrome, which is associated with deletion of chromosome 22q12, mutations in the glial cells missing homologue b (gcmb) gene, hdr syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia) caused by mutation of the gata3 gene, hrd syndrome (hypoparathyroidism, retardation, dysmorphism) caused by a defect in the tbce gene, familial x - linked recessive hypoparathyroidism, mutations of pth genes, and kearns - sayre syndrome resulting from mutations in mitochondrial dna are known causes of congenital hypoparathyroidism1,2). casr is a 1,078 amino acid g - protein - coupled receptor which consists of an extracellular domain of 612 amino acids, seven transmembrane domains of 250 amino acids and an intracellular domain of 216 amino acids. although calcium is the endogenous ligand of casr, other cations such as mg, ba, and gd can also bind casr4). the action of casr bound to ions other than ca is unknown3). casr is widely distributed, but the highest levels are found in the parathyroid gland and kidney tubular cells. its main function is to maintain calcium homeostasis5). in the parathyroid gland, parathyroid cells detect small perturbations of the extracellular calcium concentration and modulate the secretion of pth in an inhibitory manner. extracellular calcium concentration and pth secretion have an inverse sigmoidal relationship with each other5). in the kidney, casr is highly expressed on the basolateral side of the cortical thick ascending limb of the loop of henle (talh) in rats6). a high peritubular calcium concentration activates casr and activated casr inhibits transporters in the talh, which subsequently reduces ca and mg reabsorption7). inactivating and activating mutations of casr inactivating mutations lead to resistance to extracellular calcium, which causes hypercalcemic disorders including familial hypocalciuric hypercalcemia or neonatal severe hyperparathyroidism8). activating mutations in the casr lead to hyperresponsiveness to extracellular calcium, which causes hypocalcemia with hypercalciuria, a disorder called autosomal dominant hypocalcemia (adh)9). activating mutations of casr the association of adh with bartter syndrome (bs) has been previously described10,11). the present case report describes a patient with adh and type v bs due to an activating mutation in the transmembrane domain of casr. the patient was followed at the seoul national university children 's hospital, inje university busan paik hospital and maryknoll medical center for hypocalcemia, hypokalemia, and hypomagnesemia. she attended dong - a university hospital and laboratory results showed hypocalcemia, hypomagnesemia, and a low level of pth. oral calcium, magnesium, and vitamin d supplements were prescribed and the seizures were controlled. however, 1). at 14 years of age, she suffered weight loss from 48 kg to 43 kg and a loss of appetite. laboratory testing showed a serum calcium level of 16.9 mg / dl and serum phosphorus of 3.9 mg / dl. her serum creatinine was 1.4 mg / dl and her serum blood urea nitrogen 22.8 mg / dl (table 1). she had been prescribed calcium carbonate 1,000 mg / day and alfacalcidol 1.5 g / day before admission (table 2). medication was discontinued until serum calcium and phosphorus levels were below the normal range, after which the medications were restarted. at 17 years old, the patient visited hospital complaining of dizziness, nausea, and vomiting. her serum calcium level was 19.6 mg / dl and her serum potassium 2.8 mg / dl. calcification of the subcortical and basal ganglia of both frontal lobes was found on brain computed tomography (fig. her medication at admission was calcium acetate (4,000 mg / day), alfacalcidol (2.5 g / day), hydrochlorothiazide (75 mg / day), and amiloride (6.6 mg / day) (table 2). she had moved from maryknoll medical center to inje university busan paik hospital in busan a month before the second hypercalcemic episode and the dose of alfacalcidol had been increased from 1.25 g / day to 2.5 g / day at her initial visit to inje university busan paik hospital because her serum 25-hydroxy - vitamin d3 (25-ohvitd3) concentration was just above the lower limit of the normal range. all medication was discontinued and her serum calcium and potassium concentrations gradually normalized, but because of persistent dizziness she was transferred to seoul national university children 's hospital. on the day after transfer, she complained of paresthesia of her hand and her serum calcium concentration was 8.2 mg / dl. alfacalcidol and calcium carbonate were administered and the dose of medication was slowly increased to 2 g / day of alfacalcidol and 3,000 mg / day of calcium carbonate. alfacalcidol was changed to calcitriol 1.0 g / day and she was discharged with 1.0 g / day of calcitriol, 3,000 mg / day of calcium carbonate, and 50 mg / day of thiazide. in the outpatient clinic 2 days after discharge, her calcium carbonate was reduced to 2,000 mg / day because her serum calcium level was elevated to 10.5 mg / dl. ten days after her outpatient clinic visit, she was admitted again because of high calcium level. she did not complain of muscle weakness, nausea, or vomiting and she did not appear anxious or depressive. her height was 153.4 cm (5th-10th percentile) and weight 50 kg (25th-50th percentile). she was being treated with oral calcium carbonate (2,000 mg / day), calcitriol (1.0 g / day), and hydrochlorothiazide (50 mg / day) (table 2). her serum calcium concentration was 18.2 mg / dl (normal range, 8.8 - 10.5 mg / dl), serum phosphorus 3.9 mg / dl (normal range, 2.5 - 4.5 mg / dl), serum magnesium 1.7 meq / l (normal range, 1.2 - 5 meq / l), and serum potassium was 2.8 her pth level was below 5 pg / ml (normal range, 10 - 65 pg / ml) (table 1). all medications were withheld and intravenous hydration was started. medications were restarted when her calcium concentration reached 9.2 mg / dl and the calcium concentration was maintained at a stable level with 2,000 mg / day of calcium carbonate, 1.5 g / day of alfacalcidol, 12.5 mg / day of hydrochlorothiazide, and 1,800 mg of potassium chloride. after informed consent was obtained, direct sequencing of all coding exons of casr revealed that the patient had a heterozygous substitution of adenine (tat) for guanine (tgt) at codon 829, which would result in substitution of cysteine for tyrosine (y829c) in the protein (fig. this patient had adh with bs because of a y829c activating mutation of the casr gene. bs is a genetically heterogeneous disorder with a unifying pathophysiology of decreased salt absorption by the talh. about 30% of sodium and chloride filtered in the glomeruli is reabsorbed in the talh. defects in the function of proteins associated with sodium reabsorption in talh cause bs12). however, reabsorption not only of sodium, but also of water and of other ions, including calcium, magnesium, and potassium, is influenced. type 1 bs is caused by a loss - of - function mutation of the apical sodium - potassium - chloride cotransporter gene (nkcc2). type 2 bs is caused by mutations in the apical renal outer medullary potassium channel gene (romk). type iv bs results from a loss - of - function mutation of bsnd, which encodes barttin, the subunit of clc - k channels that is essential for their function. casr expressed in the talh is activated by a high extracellular concentration of calcium ions, and activated casr inhibits the nkcc2 and romk in the apical membrane and the sodium potassium atpase in the basolateral membrane of the talh12). in rat kidney, cytochrome p-450-dependent metabolites of arachidonic acid and cyclic amp are associated with the inhibitory effect of casr on romk13). calcium and magnesium in the talh are reabsorbed by a paracellular pathway driven by a positive luminal potential. potassium excretion through romk is essential to generate this positive luminal voltage relative to the basolateral side, so inhibition of romk blocks the generation of the higher voltage on the luminal side and increases urinary calcium and magnesium excretion10,12). the clinical presentations and onset timing of bartter 's phenotype differ according to the type of mutation10). another case reported a patient with k29e mutation, who presented with hypocalcemia and mild hypokalemia without alkalosis, hyperreninemia, and hyperaldosteronemia. in this patient, hypokalemia occurred at 22 years old and was corrected with small doses of oral potassium11). patients with a843e or c131w mutations show the phenotype of bs including nephrocalcinosis, hypokalemia, hypomagnesemia, metabolic alkalosis, hyperreninemia, and hyperaldosteronemia as well as hypocalcemia. in patients with a843e mutation, the extracellular calcium concentration at which casr shows half of its maximal activity (ec50) was 2.0 - 2.2 mmol / l (8.0 - 8.8 mg / dl) for casr with k47n or p221l mutations, 1.45 mmol / l (5.8 mg / dl) with the k29e mutation11), and below 0.5 mmol / l (2.0 mg / dl) for casr with a843e or c131w mutations. the ec50 of the wild - type casr is 3.4 mmol / l (13.6 mg / dl). this explains why patients with a843e or c131w mutations have the most severe symptoms of bs. the onset timing of phenotype of bs also correlates with ec50 ; the lower the ec50, the faster the onset of bartter 's phenotype10,11). the characteristics of bs were expressed mildly in this patient. serum potassium concentrations maintained in the normal range until hypokalemia appeared when the patient was about 17 years old. since then, the patient has been taking oral potassium. this phenotype is similar to that of casr with the k29e mutation, so it could be assumed that the ec50 of casr with the y829c mutation is similar to that of casr with the k29e mutation. however, this should be cautiously interpreted, as bs can differ between patients who have the same casr mutation11). the patient in this case had nephrocalcinosis and basal ganglia calcification. in one study of patients with casr mutation, basal ganglia calcification was found in 9 of 25 patients (36%) and nephrocalcinosis was present in 3 of 25 patients (12%)14). in another study, hypercalciuria was associated with nephrocalcinosis15), but basal ganglia calcification and nephrocalcinosis were not related to the severity of hypocalcemia14). the first episode occurred when she was 14 years old and the others when she was 17 years old. hypercalcemic episodes during the treatment of adh have been reported previously14,15), and occurred in patients who had taken a high dose of alfacalcidol. during her first hypercalcemic episode, our patient suffered fever and severe weight loss. the volume loss and the continuous medication might have caused the hypercalcemia. before the second event, her dose of alfacalcidol was increased because of low serum 25-ohvitd3, although her serum calcium concentration remained in the normal range. thus, an overdose of alfacalcidol might have induced the hypercalcemia. in the third hypercalcemic episode, the patient denied overdose of medication, infection, or over consumption of foods high in calcium. but, considering the increase of serum calcium concentration from 7.6 mg / dl to 10.5 mg / dl 2 days after discharge, the doses of calcium and vitamin d3 supplementation were probably too high for the patient. when symptoms disappear, medication doses should be adjusted to maintain serum calcium above the level that symptoms persisted. when increasing the doses of medications, special attention should be taken not to cause hypercalcemia. in conclusion, our paper reports a novel y829c mutation of casr. the patient with this casr gene mutation had a mild phenotype of bs in addition to adh. | the calcium sensing receptor (casr) plays an important role in calcium homeostasis. activating mutations of casr cause autosomal dominant hypocalcemia by affecting parathyroid hormone secretion in parathyroid gland and calcium resorption in kidney. they can also cause a type 5 bartter syndrome by inhibiting the apical potassium channel in the thick ascending limb of the loop of henle in the kidney. this study presents a patient who had autosomal dominant hypocalcemia with bartter syndrome due to an activating mutation y829c in the transmembrane domain of the casr. symptoms of hypocalcemia occurred 12 days after birth and medication was started immediately. medullary nephrocalcinosis and basal ganglia calcification were found at 7 years old and at 17 years old. three hypercalcemic episodes occurred, one at 14 years old and two at 17 years old. the bartter syndrome was not severe while the serum calcium concentration was controlled, but during hypercalcemic periods, the symptoms of bartter syndrome were aggravated. |
ethical approval was obtained from the university college london institute of child health / great ormond street hospital for children joint research ethics committee, and written consent was obtained from patients and/or parents. dna was extracted from 2 patients with hypopituitarism and features of charge and their parents, if available. an additional cohort of 100 patients with septo - optic dysplasia or hypopituitarism was screened ; 77 had septo - optic dysplasia with variable hormone deficiency (6 had ectopic posterior pituitary [epp ]), 21 had combined pituitary hormone deficiency without additional midline defects (6 with epp), and 2 had isolated ghd (normal posterior pituitary). the coding region of chd7 consisting of 38 exons (nm_017780) was sequenced in its entirety in all patients. exons were amplified by pcr on an eppendorf thermocycler over 35 cycles with exon - flanking primers, designed using primer3 (available at http://frodo.wi.mit.edu/primer3). pcr products were treated with microclean reagent (catalog no. 2mcl-10 ; web scientific ltd., crewe, united kingdom) according to manufacturer 's instructions and then sequenced using bigdye v1.1 sequencing chemistry (applied biosystems, foster city, california) and analyzed on a 3730 1 dna analyzer (catalog no. 625 - 0020 ; applied biosystems / hitachi, tokyo, japan). for sequence variants identified, we screened 190 ethnically matched control alleles at the same loci and screened the chd7 database describing chd7 mutations / variants from over 800 patients (www.chd7.org) (3), a control exome database of 1500 caucasian / african americans (http://snp.gs.washington.edu/evs), and 1000 genomes (www.1000genomes.org). two in silico splicing prediction models, human splicing finder, version 2.4.1, and netgene2 (www.cbs.dtu.dk/services/netgene2), were used to assess the effect of splicing variants. patients with chd7 variants were also screened for variants in hesx1, sox3, kal1, prokr2, fgfr1, fgf8, nelf, wdr11, sox2, and otx2. hormone measurements were performed in standard assays in the biochemistry laboratories of great ormond street hospital for children, kent and canterbury hospital (uk) and hospital universtari son dureta, palma de mallorca (spain). ethical approval was obtained from the university college london institute of child health / great ormond street hospital for children joint research ethics committee, and written consent was obtained from patients and/or parents. dna was extracted from 2 patients with hypopituitarism and features of charge and their parents, if available. an additional cohort of 100 patients with septo - optic dysplasia or hypopituitarism was screened ; 77 had septo - optic dysplasia with variable hormone deficiency (6 had ectopic posterior pituitary [epp ]), 21 had combined pituitary hormone deficiency without additional midline defects (6 with epp), and 2 had isolated ghd (normal posterior pituitary). the coding region of chd7 consisting of 38 exons (nm_017780) was sequenced in its entirety in all patients. exons were amplified by pcr on an eppendorf thermocycler over 35 cycles with exon - flanking primers, designed using primer3 (available at http://frodo.wi.mit.edu/primer3). pcr products were treated with microclean reagent (catalog no. 2mcl-10 ; web scientific ltd., crewe, united kingdom) according to manufacturer 's instructions and then sequenced using bigdye v1.1 sequencing chemistry (applied biosystems, foster city, california) and analyzed on a 3730 1 dna analyzer (catalog no. 625 - 0020 ; applied biosystems / hitachi, tokyo, japan). details of the pcr conditions and primers are available upon request. for sequence variants identified, we screened 190 ethnically matched control alleles at the same loci and screened the chd7 database describing chd7 mutations / variants from over 800 patients (www.chd7.org) (3), a control exome database of 1500 caucasian / african americans (http://snp.gs.washington.edu/evs), and 1000 genomes (www.1000genomes.org). two in silico splicing prediction models, human splicing finder, version 2.4.1, and netgene2 (www.cbs.dtu.dk/services/netgene2), were used to assess the effect of splicing variants. patients with chd7 variants were also screened for variants in hesx1, sox3, kal1, prokr2, fgfr1, fgf8, nelf, wdr11, sox2, and otx2. hormone measurements were performed in standard assays in the biochemistry laboratories of great ormond street hospital for children, kent and canterbury hospital (uk) and hospital universtari son dureta, palma de mallorca (spain). the first patient with features of charge syndrome and a chd7 variant was a caucasian male born at term with a birth weight of 3.72 kg (+ 0.34 sd score [sds ]). clinical features included tetralogy of fallot, sensorineural hearing loss, micropenis with bilateral undescended testes, abnormally shaped ears (figure 2c), a squint and hypermetropia, trigonocephaly, a small occipital meningocele, and severe developmental delay. he presented with short stature (height, 4.9 sds) at 7 years of age. developmental delay he was classified as probable / possible charge using blake criteria (1 major, 5 minor criteria), but not as charge using verloes criteria (0 major, 5 minor criteria). he was commenced on t4 and recombinant human gh (rhgh), and his growth velocity increased to 22 cm / y (figure 2b). magnetic resonance imaging (mri) revealed a small anterior pituitary, an absent pituitary stalk with an undescended or epp at the tuber cinereum, and a thin corpus callosum (figure 2a). his mother, who carried the same chd7 variant, was short (152 cm ; 1.67 sds) but had a normal igf - i concentration (table 1) and had received t4 for primary hypothyroidism from the age of 11 (tsh, 20 mu / l [normal 0.45.0 mu / l ] ; free t4, 9 pmol / l [normal, 919 she did not manifest features of charge syndrome. the sister (figure 2, b and c) (birth weight, 4.060 kg [+ 2.65 sds ] at 37 wk gestation), who did not carry the chd7 variant, had an atrial septal defect, an abnormally shaped ear, a squint, and developmental delay, but no hearing loss. she was short (height, 98.1 cm [2.92 sds ] at 5.75 y) and had ghd but normal cortisol, prolactin, and free t4 concentrations (table 1). her brain mri showed generalized white matter loss and midline abnormalities (cavum septum pellucidum and vergae, small anterior pituitary, a single anterior cerebral artery, thin optic chiasm), but a normal infundibulum and posterior pituitary. she currently has a normal height velocity of 8 cm / y at the chronological age of 7.59 years and has not yet been commenced on rhgh replacement. a, mri in patient 1 revealed aph, an absent pituitary stalk (aps) with an undescended / epp at the tuber cinereum and a thin corpus callosum. patient 1 presented with short stature at the age of 3 years and was commenced on gh at 7 years of age (left arrow). the sister of patient 1 was found to be gh deficient at 6 years of age. c, photographs of patient 1 and his sister. left, patient 1 with probable / possible charge syndrome according to blake criteria. right, the sister of patient 1 with an atrial septum defect (aps), abnormal ear, squint, developmental delay, and ghd. d, mri of patient 2 at 2 months (left) and 20 months of age (right) revealed aph and an aps with an epp at the tuber cinereum, bilateral colobomata, and underdeveloped frontal lobes. patient 2 presented with short stature at the age of 3 years and has recently been commenced on gh treatment at the age of 3 years (arrow). phenotypes in patients with chd7 variations abbreviations : tshd, tsh deficiency ; fshd, fsh deficiency ; ft4, free t4 ; sp, septum pellucidum ; cc, corpus callosum ; bl, bilateral ; hcg, human chorionic gonadotropin. endocrine deficits, charge features, and results of mri are shown in patients with chd7 variants. the proband carries a novel heterozygous missense variant, c.2194c > g (figure 1a), in exon 4 of chd7, resulting in substitution of a highly conserved proline residue by alanine, p.p732a. the variant was also detected in the unaffected mother, but not in the father or sister, in 1500 healthy caucasian / african americans (http://snp.gs.washington.edu/evs), in 1000 genomes, or in 190 control alleles. this variant is listed in the charge database (http://www.chd7.org), but it represents the same english patient and his mother that we describe here (prof. c. van ravenswaaij, university medical center groningen, personal communication). the second patient is a caucasian male (birth weight, 1.260 kg [+ 0.39 sds ] at 28.5 wk gestation), with a full spectrum of charge features (coloboma, mild choanal atresia, deafness, abnormal ear shape, growth and developmental delay, genital hypoplasia). he fulfilled both blake (3 major, 4 minor criteria) and verloes criteria (2 major, 4 minor criteria) for diagnosis of charge syndrome. his endocrinopathy (table 1) included ghd, with undetectable igf - i and a low igf binding protein 3 (igfbp3), gonadotropin deficiency (undetectable lh and fsh, and undetectable t on 3-day human chorionic gonadotropin test), severe cortisol deficiency, and central hypothyroidism, but with a normal prolactin concentration. mri at 2 months of age showed a thin corpus callosum and a cavum septum pellucidum, and at 20 months a small anterior pituitary and an absent pituitary stalk with an epp, located at the tuber cinereum (figure 2d). his inner ear structures and semicircular canals were normal ; however, his olfactory bulbs appeared small. he was commenced on t4, hydrocortisone, rhgh, and a 3-month course of i m t. his growth velocity increased from a pretreatment velocity of 6.6 cm / y to 15 cm / y (figure 2e), and there was a marked improvement in phallus size. genetic analysis revealed a novel heterozygous splice site variant in intron 35, c.ivs35+6t>c (figure 1b), in a region predicted to be important for splicing. netgene2 showed a decrease (0.86 to 0.32) in the confidence with which the splice donor site will be detected. the sequence variant was not identified in 190 control alleles, in the chd7 database (www.chd7.org), in a control database of 1500 caucasian / african americans, or in 1000 genomes. no chd7 variants were identified in our septo - optic dysplasia / hypopituitarism (without charge features) cohort. the first patient with features of charge syndrome and a chd7 variant was a caucasian male born at term with a birth weight of 3.72 kg (+ 0.34 sd score [sds ]). clinical features included tetralogy of fallot, sensorineural hearing loss, micropenis with bilateral undescended testes, abnormally shaped ears (figure 2c), a squint and hypermetropia, trigonocephaly, a small occipital meningocele, and severe developmental delay. he presented with short stature (height, 4.9 sds) at 7 years of age. developmental delay he was classified as probable / possible charge using blake criteria (1 major, 5 minor criteria), but not as charge using verloes criteria (0 major, 5 minor criteria). he was commenced on t4 and recombinant human gh (rhgh), and his growth velocity increased to 22 cm / y (figure 2b). magnetic resonance imaging (mri) revealed a small anterior pituitary, an absent pituitary stalk with an undescended or epp at the tuber cinereum, and a thin corpus callosum (figure 2a). his mother, who carried the same chd7 variant, was short (152 cm ; 1.67 sds) but had a normal igf - i concentration (table 1) and had received t4 for primary hypothyroidism from the age of 11 (tsh, 20 mu / l [normal 0.45.0 mu / l ] ; free t4, 9 pmol / l [normal, 919 she did not manifest features of charge syndrome. the sister (figure 2, b and c) (birth weight, 4.060 kg [+ 2.65 sds ] at 37 wk gestation), who did not carry the chd7 variant, had an atrial septal defect, an abnormally shaped ear, a squint, and developmental delay, but no hearing loss. she was short (height, 98.1 cm [2.92 sds ] at 5.75 y) and had ghd but normal cortisol, prolactin, and free t4 concentrations (table 1). her brain mri showed generalized white matter loss and midline abnormalities (cavum septum pellucidum and vergae, small anterior pituitary, a single anterior cerebral artery, thin optic chiasm), but a normal infundibulum and posterior pituitary. she currently has a normal height velocity of 8 cm / y at the chronological age of 7.59 years and has not yet been commenced on rhgh replacement. a, mri in patient 1 revealed aph, an absent pituitary stalk (aps) with an undescended / epp at the tuber cinereum and a thin corpus callosum. patient 1 presented with short stature at the age of 3 years and was commenced on gh at 7 years of age (left arrow). the sister of patient 1 was found to be gh deficient at 6 years of age. c, photographs of patient 1 and his sister. left, patient 1 with probable / possible charge syndrome according to blake criteria. right, the sister of patient 1 with an atrial septum defect (aps), abnormal ear, squint, developmental delay, and ghd. d, mri of patient 2 at 2 months (left) and 20 months of age (right) revealed aph and an aps with an epp at the tuber cinereum, bilateral colobomata, and underdeveloped frontal lobes. patient 2 presented with short stature at the age of 3 years and has recently been commenced on gh treatment at the age of 3 years (arrow). phenotypes in patients with chd7 variations abbreviations : tshd, tsh deficiency ; fshd, fsh deficiency ; ft4, free t4 ; sp, septum pellucidum ; cc, corpus callosum ; bl, bilateral ; hcg, human chorionic gonadotropin. endocrine deficits, charge features, and results of mri are shown in patients with chd7 variants. the proband carries a novel heterozygous missense variant, c.2194c > g (figure 1a), in exon 4 of chd7, resulting in substitution of a highly conserved proline residue by alanine, p.p732a. the variant was also detected in the unaffected mother, but not in the father or sister, in 1500 healthy caucasian / african americans (http://snp.gs.washington.edu/evs), in 1000 genomes, or in 190 control alleles. this variant is listed in the charge database (http://www.chd7.org), but it represents the same english patient and his mother that we describe here (prof. c. van ravenswaaij, university medical center groningen, personal communication). the second patient is a caucasian male (birth weight, 1.260 kg [+ 0.39 sds ] at 28.5 wk gestation), with a full spectrum of charge features (coloboma, mild choanal atresia, deafness, abnormal ear shape, growth and developmental delay, genital hypoplasia). he fulfilled both blake (3 major, 4 minor criteria) and verloes criteria (2 major, 4 minor criteria) for diagnosis of charge syndrome. his endocrinopathy (table 1) included ghd, with undetectable igf - i and a low igf binding protein 3 (igfbp3), gonadotropin deficiency (undetectable lh and fsh, and undetectable t on 3-day human chorionic gonadotropin test), severe cortisol deficiency, and central hypothyroidism, but with a normal prolactin concentration. mri at 2 months of age showed a thin corpus callosum and a cavum septum pellucidum, and at 20 months a small anterior pituitary and an absent pituitary stalk with an epp, located at the tuber cinereum (figure 2d). his inner ear structures and semicircular canals were normal ; however, his olfactory bulbs appeared small. he was commenced on t4, hydrocortisone, rhgh, and a 3-month course of i m t. his growth velocity increased from a pretreatment velocity of 6.6 cm / y to 15 cm / y (figure 2e), and there was a marked improvement in phallus size. genetic analysis revealed a novel heterozygous splice site variant in intron 35, c.ivs35+6t>c (figure 1b), in a region predicted to be important for splicing. netgene2 showed a decrease (0.86 to 0.32) in the confidence with which the splice donor site will be detected. the sequence variant was not identified in 190 control alleles, in the chd7 database (www.chd7.org), in a control database of 1500 caucasian / african americans, or in 1000 genomes. no chd7 variants were identified in our septo - optic dysplasia / hypopituitarism (without charge features) cohort. charge syndrome is a continuum of multiorgan disorders of variable severity, often caused by chd7 mutations. we describe 2 patients harboring rare sequence variants in chd7, both of whom have features of charge syndrome accompanied by multiple pituitary hormone deficiencies and structural abnormalities of the pituitary gland. charge syndrome is associated with hh and occasional ghd ; however, structural abnormalities of the pituitary gland have not previously been described. although charge syndrome is classically associated with hh, our data suggest that in some patients, congenital hypopituitarism may be associated with structural abnormalities of the pituitary gland and variations in chd7. a previous study has suggested that ghd occurs in 9% of charge patients ; 3 patients with ghd in this study had aph (19), showing that chd7 might have a role in anterior pituitary development. one patient from a cohort (n = 379) with a clinical diagnosis of charge has been reported to have tsh deficiency (1). acthd has been described in 1 patient with a clinical but not genetically confirmed diagnosis of charge syndrome (20). to summarize, abnormalities of pituitary function including ghd, tsh deficiency, and acthd, with a small anterior pituitary size, have rarely been associated with charge syndrome, but an undescended / epp has not (19, 21), and we describe this association for the first time. variations in other genes involved in pituitary development, for example hesx1, can also result in ghd with aph and either a normal or epp. our data support the hypothesis that chd7 variations may lead to a continuum of anatomical and functional hypothalamic / pituitary abnormalities. most chd7 mutations are heterozygous de novo, with a small number of inherited cases usually from an unaffected parent (9). no genotype - phenotype correlation has been identified, but missense mutations are associated with a milder phenotype (4). in both mice and humans, chd7/chd7 is expressed in the developing anterior pituitary, at lower levels in the intermediate and posterior lobes, and in the developing and mature hypothalamus (8), suggesting a potential role of chd7 in the development of the hypothalamo - pituitary axis. hurd (8) showed that homozygous mutant mice present with an olfactory pit and rathke 's pouch hypoplasia. chd7 was identified as a sox2 transcriptional cofactor ; both genes physically interact and have overlapping genome - wide binding sites that regulate a set of target genes, such as gli2 (14), which when mutated, can cause defects of pituitary development including an epp (22). chd7 and sox2 interaction may be further evidence of a potential role for chd7 in hypothalamo - pituitary development, given the role of sox2 here (23). layman (11) have shown a reduction in the expression of fgfr1, bmp4, and otx2 in the olfactory placode of chd7 mutant embryos, as well as a reduction of otx2 in the hypothalamus. chd7 is also thought to be necessary for access to otx2 target genes in the olfactory epithelium (24). because mutations in otx2 in humans are associated with variable hypopituitarism, often with an undescended / epp, this could be a further mechanism whereby chd7 mutations could lead to structural abnormalities of the pituitary gland. although chd7 mutations have been described in association with kallmann syndrome, usually additional charge features are present in kallmann syndrome patients with chd7 variants (25). our data parallel these findings ; the patients with congenital hypopituitarism without additional features of charge syndrome did not harbor chd7 variants, suggesting that features of charge may have to accompany the hypopituitary phenotype for chd7 to be implicated in the etiology. in patient 1, the p.p732a variant occurs at a highly conserved amino acid, which is not part of a domain with an established function (figure 1, c and d). the proband 's sister had abnormally shaped ears, abnormal facial features, and isolated ghd with a small anterior pituitary, but she did not carry the variant. the mother, who carries the variant, does not have features of charge syndrome or hypopituitarism, although an mri to assess her pituitary anatomy could not be performed. these data suggest that p.p732a may be a contributory factor to the etiology of the hypopituitary phenotype in our patient but is unlikely to be causative in isolation. recent evidence suggesting that the genetic basis in many cases of kallmann syndrome is digenic / oligogenic could be extrapolated to include overlapping syndromes such as charge or congenital hypopituitarism. a second mutation has not yet been identified in either patient, although we have screened other genes involved in hypothalamo - pituitary development and in kallmann syndrome (26). the ivs35 + 6t > c variant occurs in the intron / exon boundary of the exon encoding the first brk domain, suggesting that the final protein may be mistranslated, leading to disrupted brk and transcriptional activity mediated through the leucine zipper. because ivs35 + 6t > c is located in a proposed splicing region and is predicted to affect splicing using 2 in silico prediction models, it may cause transcription to proceed through into the intron, creating 63 new amino acids before an early stop codon. in this scenario, exons 3638 would not be translated, resulting in a truncated protein without a second brk domain or leucine zipper (refer to figure 1d). familial charge syndrome has been described, and in such families, offspring are often more severely affected than the parent (9). other family members occasionally seem to have milder associated clinical features without carrying the variant (27), as noted in the sister of the proband in pedigree 1, concluding that these features are unrelated to the variant in the proband and further supporting the possible presence of digenicity. we have for the first time identified an epp in combination with severe hypopituitarism in 2 unrelated patients with variable features of charge syndrome and chd7 variants, which are of unknown significance. our data suggest the need for pituitary function testing and mri in patients with charge syndrome. however, chd7 mutations are not common in pituitary insufficiency or septo - optic dysplasia without features of charge. we have for the first time identified an epp in combination with severe hypopituitarism in 2 unrelated patients with variable features of charge syndrome and chd7 variants, which are of unknown significance. our data suggest the need for pituitary function testing and mri in patients with charge syndrome. however, chd7 mutations are not common in pituitary insufficiency or septo - optic dysplasia without features of charge. | introduction : charge syndrome is a multisystem disorder that, in addition to kallmann syndrome / isolated hypogonadotrophic hypogonadism, has been associated with anterior pituitary hypoplasia (aph). however, structural abnormalities such as an ectopic posterior pituitary (epp) have not yet been described in such patients.objective:the aims of the study were : 1) to describe the association between charge syndrome and a structurally abnormal pituitary gland ; and 2) to investigate whether chd7 variants, which are identified in 65% of charge patients, are common in septo - optic dysplasia /hypopituitarism.methods : we describe 2 patients with features of charge and epp. chd7 was sequenced in these and other patients with septo - optic dysplasia / hypopituitarism.results : epp, aph, and gh, tsh, and probable lh / fsh deficiency were present in 1 patient, and epp and aph with gh, tsh, lh / fsh, and acth deficiency were present in another patient, both of whom had features of charge syndrome. both had variations in chd7 that were novel and undetected in control cohorts or in the international database of charge patients, but were also present in their unaffected mothers. no chd7 variants were detected in the patients with septo - optic dysplasia / hypopituitarism without additional charge features.conclusion:we report a novel association between charge syndrome and structural abnormalities of the pituitary gland in 2 patients with variations in chd7 that are of unknown significance. however, chd7 mutations are an uncommon cause of septo - optic dysplasia or hypopituitarism. our data suggest the need for evaluation of pituitary function / anatomy in patients with charge syndrome. |
surgical site infections (ssis), a significant postoperative complication, can lead to considerable patient morbidity and mortality. preventing ssis is an essential factor in improving the results of surgical procedures. various factors may be associated with frequency of ssis in laparoscopic cholecystectomies [lc ] like method of disinfection of the laparoscopic instruments, microdamage to the reusable instruments, bacteriobilia and gallbladder content spillages, use of antibiotics, and so forth. the rise in demand for minimally invasive surgery has resulted in increased frequency of gallbladder perforations. laparoscopy - attributable mortality reaches 0.5%, morbidity reaches 4%, and ssis rates following this procedure range between 0.1 and 2%. although a number of studies have focused on the implications of gallbladder content spillage during lc, little is known about the risk factors for ssis in lc. here we compare the clinical outcomes in patients with gallbladder content spillage during lc and the role of bacteriobilia in ssi after lc. the aim of this paper is to assess the occurrence of surgical site infections in patients with spillage of gallbladder contents and bacteriobilia during laparoscopic cholecystectomy. this study was conducted in 113 patients who underwent laparoscopic cholecystectomy (lc) during a period of 20 months at the department of surgery, maulana azad medical college and lok nayak hospital, new delhi. all patients more than 18 years with symptomatic cholelithiasis proven by ultrasonography were included in this study. patients 70 years of age, having evidence of cholangitis, pancreatitis, and previous biliary tract surgeries, and having immunocompromised state or diabetes mellitus were excluded. patients with acute cholecystitis were excluded from our study group as per the protocol in our institution to manage it conservatively, followed by interval cholecystectomy. the data were collected in all patients who underwent lc, to determine the risk factors and outcomes of gallbladder content spillage, that is, the type of content spillage, duration of surgery, pericholecystic adhesions, bile culture and antibiotic sensitivity, and wound culture if ssis occurred. all the patients in our study received a single dose of ceftriaxone 1 gm half an hour before the infraumbilical incision was made. standard conventional cholecystectomy was carried out in all patients by the same surgeon to prevent operator bias. if gallbladder rupture and gross spillage of bile or stones were encountered, spilled stones were retrieved in an endopouch and local peritoneal lavage with copious amounts of saline was performed. routinely the gallbladder was removed through the umbilical port. the decision to leave a closed system suction drain a sample of bile was taken from the gallbladder and sent for bacterial culture and sensitivity in all patients. after discharge the patient was called after 1 week for proper clinical assessment of the surgical site. thereafter patient was followed up weekly for 30 days. if local signs of inflammation or purulent discharge from the wound were present, stitches were removed and the pus was sent for culture and sensitivity. ultrasonography for collection was done in patients with clinical suspicion of deep infection, temperature > 38c (excluding postoperative day 1), not responding to 48 hrs of antibiotics, and increased pain and tenderness in abdomen. if collection was found therapeutic aspiration of collection was done, the quantity and type (pus / bile / blood) of content were noted, and the fluid was sent for culture and sensitivity. postoperative superficial or deep incisional soft tissue ssis and intra - abdominal abscess (organ / space ssi) were assessed by cdc defined criteria. statistical analysis of the data was carried out using spss 12.0 (spss, inc., descriptive statistics for continuous variables were calculated by student 's t - test and the mean standard deviation was also expressed. a p value of 50 years, that is, 57% of overall ssis. female : male ratio in our study was 6.57 : 1. organism profile of the culture from ssis showed monomicrobial infection : 58% staphylococcus aureus, 14% pseudomonas, 14% e. coli, and the rest of contaminants. bacteriobilia was present in 32 patients out of 113 [28% ], but only 5 patients developed ssis amongst these 32 patients. the occurrence of ssis in these patients with bacteriobilia was 16% as compared to 2% in patients without bacteriobilia. organisms found in culture of the patients with bacteriobilia were e. coli (75%), klebsiella (19%), pseudomonas (3%), and gram negative rods (3% species not specified). a total of 18 patients had gallbladder content spillage and of these 18 only 1 patient developed ssis ; that is, these spillages were associated with surgical site infection in only 14% of patients. statistically no significant difference in occurrence of ssis in patients with gallbladder content spillage was found (see tables 1 and 2). ssis are considered to be one of the most important surgical complications after any operative procedure. as per the study conducted by jawein. the laparoscopy - attributable mortality reaches 0.5%, morbidity reaches 4%, and ssis following this procedure range between 0.1 and 2%. laparoscopic surgeries in general are associated with a lesser number of infectious complications than open surgeries. in our study we evaluated the bile cultures and gallbladder content spillage as a possible cause for ssis in lc. the ssis rate in our study was around 6% of patients which was similar to ssis rate noted by den hoed.. rate of wound infections varies greatly from 1.08% to 14.5% in the studies conducted by jawien. and malatani. gaur and pujahari concluded that the umbilicus is the commonest site for sepsis following laparoscopic cholecystectomy. this may be because the deep umbilical depression is sometimes difficult to clean or it may be due to the routine protocol of our unit to extract the gallbladder through the umbilical port. none of the patients had deep or organ space infection, whereas jawein noted superficial infections in 60.6%, deep infections in 21.2%, and organ / space ssis in 18.2%. chang and koc. found that the overall rate of infection was 1.1% and 2.1%, respectively [7, 8 ]. various factors associated with ssis in lc need to be evaluated further and definitive measures taken to decrease the morbidity associated with this procedure. female : male ratio in our study is 6.57 : 1, which is high compared to studies conducted by assaff. and suri., 1.32 : 1 and 3 : 1, respectively [9, 10 ]. this may correlate with the demographic variation. in our study none of the male patients developed ssis. our data however did not reveal any significant association between the gender and ssis. similarly, chang. and also did not find any statically significant association between the ssi and gender of the patient [7, 11 ]. most of our patients belonged to the 3rd and 4th decades of life similar to study conducted by suri.. in our study a statistically significant association is found between the surgical site infection and age. this is low in comparison to the study conducted by koc. and assaff., that is, 34% and 41.3% of patients, respectively [8, 9 ]. noted trocar site infections developed more frequently in patients with gallbladder perforation (p = 0.004). in our study the bile cultures were positive in 32 patients out of 113, that is, 28%. the rate of bile culture positivity was similar to other studies ; that is, malatani. and mahafzah and daradkeh also noted positive bile culture in 17.3%, 20%, and 27% of patients, respectively [5, 10, 13 ]. in our study we found that the occurrence of ssis in patients with bacteriobilia was nearly 8 times more than in patients without bacteriobilia ; also we found statistically significant association between bacteriobilia and ssis. this may possibly be due to translocation of bacteria during retraction of the rectus and extraction of the gallbladder. mahafzah and daradkeh noted no significant difference in the postoperative complication between the patients with a positive bile culture or sterile culture [5, 13 ]. our organism profile in bile culture is similar to other studies such as suri. and valceanu., microorganism profile of the various ssis depends on various factors including the type of surgery. in our study staphylococcus aureus these results go in line with that of cantlon., who found that staphylococcus aureus was the most common pathogen followed by enterobacteriaceae that were the second most frequently isolated organisms. recorded that staphylococcus aureus had most frequency and the most common gram negative organisms were klebsiella, e. coli, and pseudomonas [16, 17 ]. the risk of ssis increases with age. antibiotics in case of spillage should be offered only to elderly patients. | aim. to assess the occurrence of ssis in patients with spillage of gallbladder contents and bacteriobilia during laparoscopic cholecystectomy. methods. we evaluated 113 patients who underwent laparoscopic cholecystectomy between september 2013 and april 2015. the ssis and their relationship with gallbladder rupture and bacteriobilia were assessed. results. the mean age of patients developing ssis was 45.57 8.89 years. 18 patients (16%) had spillage of bile from the gallbladder. percentage of ssis overall was 6%, while percentage of ssis in gallbladder content spillage was 5.5%. organism profile of the culture from surgical site showed monomicrobial infection : 58% staphylococcus aureus, 14% pseudomonas, and 14% e. coli. the occurrence of ssis in patients with bacteriobilia was 16% as compared to 2% in patients without bacteriobilia. conclusions. gallbladder content spillage is not a significant risk factor leading to increase in ssis. the occurrence of ssis is significantly higher in patients with bacteriobilia. |
when human participants are not engaged in any specific task a set of brain regions can be observed to be active (shulman., 1997). collectively this set of regions is now commonly referred to as the default mode network (dmn ; raichle., 2001 ; buckner., 2008). regions of the dmn often show a deactivation when participants perform cognitive tasks and this degree of deactivation can be predictive of subsequent performance (eichele., 2008). contributing to its popularity, the dmn is easy to identify in resting state functional mri data and its integrity seems to be compromised in a number of neurological and psychiatric syndromes, such as alzheimer 's disease (hafkemeijer., 2012) and autism (anderson., 2011). it is now becoming increasingly apparent that brain areas associated with the dmn are activated during the performance of certain types of tasks. indeed, the proposed anti - correlation between the dmn and task - related networks has been called into question (murphy., 2009 ; kelly., 2012). in this paper, we will focus on one of the task domains that has been suggested to activate the dmn, that of social cognition. it has been observed that there is strong overlap between the network of areas activated in social cognition and the dmn (corbetta., 2008 ; schilbach.,. we will explore the hypothesis that areas in the dmn are involved in certain types of social cognition, speculate on why this might be the case, and explore its implications for understanding social cognition not just in humans, but also in non - human primates. importantly, in this paper we attempt to take this discussion further by reviewing some recent anatomical data on the dmn and areas involved in social cognition, since a better understanding of the underlying anatomy is vital to understanding a brain network 's function. second, we discuss recent data on the anatomical relation between the dmn and social cognition networks in non - human primates providing independent evidence for a degree of overlap between dmn and brain areas mediating social cognition. we conclude by proposing some hypotheses on why certain aspects of social cognition might rely on the dmn and the consequences of this finding. the history of research into the dmn is described in detail by a number of papers (raichle and snyder, 2007 ; buckner., 2008), so we will only provide a brief overview here. the dmn was originally identified in block - design positron emission tomography (pet) studies by looking at brain areas that showed activity increases during passive or rest these papers argued that the dmn identified by task - negative contrasts is a specific anatomical network, distinguishable from task - negative effects in unattended sensory modalities related to attention (haxby., 1994). more recently, the dmn is often identified in pure resting state experiments. in this type of paradigm, participants ' brain activity is recorded while participants are not performing any task and are usually left undirected to think for themselves. by extracting the time course in a region of interest and correlating that with brain activity at each voxel, one can obtain a map of functional interactions between brain areas during rest. seeding, for instance, the posterior cingulate cortex (pcc) results in a map of dmn (greicius., 2002). alternatively, the data can be analyzed using model - free analysis techniques such as independent component analysis (ica), which allow one to characterize the spatio - temporal structure of the data (beckmann., 2005). this results in a number of independent components, each reflecting a distinct network of interacting brain regions, in which the dmn is often captured in a single, or very few, components. a number of areas are consistently found regardless of the method used, although some differences have been identified as well. areas consistently identified are the medial posterior cortex, specifically posterior cingulate cortex (pcc ; areas 23/31) and often the precuneus, the medial frontal cortex (mfc ; including areas 24/10-m/32), and bilateral inferior parietal and posterior temporal areas around the temporoparietal junction area (tpj). apart from these core nodes, other areas that are often reported to participate in the dmn are the hippocampal formation and medial temporal lobe and areas along the lateral temporal cortex extending toward the temporal pole. in the human brain diffusion - weighted imaging has been used to identify the cingulum bundle as an important white matter tract mediating the functional connectivity between two of the core hubs of the dmn, the posterior and anterior medial cortices (van den heuvel., 2008 ; greicius., it has been argued that some of these regions form part of the structural core of the neocortex, consisting of nodes linking all main major structural modules of the brain (hagmann., 2008). finally, based on a detailed analysis of the resting state functional connectivity patterns it has been suggested that the dmn comprises at least two subsystems, one including the lateral temporal cortex, temporal pole, and dorsomedial frontal cortex, and another one centered on the medial temporal lobe, hippocampal formation, posterior inferior parietal lobule (ipl), and ventral mfc (andrews - hanna., one of the first studies to explore the relationship between the dmn and the neural basis of social cognition was performed by schilbach and colleagues (schilbach., 2008). they performed a conjunction analysis on the data from 12 studies from their lab, defining the dmn by looking for areas that correlated negatively with the task - related regressors defined in these studies. their analysis revealed the left angular gyrus, the precuneus, and the ventral anterior cingulate cortex. the authors then noted that some of the activations were very similar to those observed in various aspects of social cognition from their lab and other groups, including the involvement of the precuneus in social interactions (schilbach., 2006), the left angular gyrus / tpj in differentiating between self and others (vogeley and fink, 2003), and anterior cingulate in action monitoring in self and others (amodio and frith, 2006). the authors proposed that the physiological baseline of the human brain is linked to the psychological baseline, the predisposition human beings have for social cognition as the default mode of thought. as outlined above, the dmn can also be identified, together with other functional networks, in fmri data collected at rest. recently, smith and colleagues compared resting state networks as defined by ica with networks of brain areas showing consistent co - activation during task performance using the activation maps of experiments included in the online brainmap database (www.brainmap.org). they showed close correspondence between the networks described by independent components in the resting state and networks of co - activating brain regions during experiments (smith. in a subsequent study they went on to use the meta - data associated with each study in their meta - analysis to investigate the type of tasks that commonly activate each network. a network highly reminiscent of the dmn, showing bilateral inferior parietal / tpj, precuneus / posterior cingulate, and medial frontal activation (their component 13, see also figure 1a) loaded strongly and exclusively on only one behavioral domain, that of social cognition (laird., 2011). overlap between the default mode network (dmn) and areas activated by social cognition paradigms. (a) dmn as found using model - free analysis of resting state fmri data (smith., 2009). (b, c) activation likelihood maps of activity during passive rest conditions (b), social cognition (c) and theory of mind (d). (e, f) conjunction maps of rest and social cognition (e) and of rest and theory of mind (f). to further investigate the overlap between the dmn and the activity during social cognition tasks, we performed an additional meta - analysis of fmri studies using the brainmap database and performed likelihood estimations (eickhoff., 2009) of functional brain activity associated with rest and associated with social cognition. the brainmap database was queried on jan 30th, 2012, when the database contained 2177 papers, 83 paradigm classes, 40934 participants, 10330 experiments, and 82135 locations. focusing only on studies in healthy participants (subjects : diagnosis first, we defined the network of regions active during rest by asking for activations during experiments were participants we instructed to remain passive (conditions : instruction is passive / rest and subjects : diagnosis is normals), which yielded 485 papers, with 1648 of 2417 experiments matching criteria. this analysis yielded foci in the posterior medial, anterior medial, and lateral temporoparietal cortices (figure 1b). this network of regions is very similar to the dmn defined by ica of resting state data by smith. second, we investigated activations related to the broad domain of social cognition (experiments : behavioral domain is cognition : social cognition and subjects : diagnosis is normals), which yielded 52 papers, with 186 of 216 experiments matching criteria. this analysis (figure 1c) yielded results very similar to those described above for the resting state, including medial frontal, posterior cingulate, and lateral temporoparietal foci. although the activation maps of the rest and the social cognition studies seem generally very similar, lateral temporoparieal activation seemed to be extending more dorsally during rest. in contrast, social cognition tended to activate a larger extend of medial frontal cortex. a conjunction between the dmn defined in the first analysis and these social cognition foci (thresholded > 100 voxels) showed significant overlap in the anterior (para)cingulate bilateral, left and right angular gyrus, left frontal operculum, and the posterior cingulate extending into the precuneus (figure 1e). the domain of social cognition is of course rather broad, comprising processes such as obtaining, retrieving, and processing information about the lifes, relationships, and mental states of the self and others. a network of dmn areas, including tpj and mfc has been attributed a role in mentalizing or theory of mind, i.e., the ability to understand and manipulates the beliefs of others (hagmann., 2008). this faculty is argued to be particularly well developed in humans as compared to other primates (saxe, 2006). therefore, in a final analysis, we looked more specifically at activations related to this domain of social cognition by searching for activations related to theory of mind (experiments : paradigm class is theory of mind task and subjects : diagnosis is normals), which yielded 24 papers, with 99 of 124 experiments matching criteria. again, a similar network was noticeable (figure 1d), although to a lesser extent. a conjunction between the dmn defined in the first analysis and the theory of mind network (thresholded > 100 voxels) showed significant overlap in the left angular gyrus and the posterior cingulate, again extending into the precuneus (figure 1f). having established that there is some global overlap between the networks identified as the dmn and those active during certain social cognitive tasks, we will now focus on the different nodes of these networks in a bit more detail. we will concentrate on the three nodes of the dmn most consistently reported : medial frontal cortex, medial posterior cortex, and lateral temporoparietal areas. increased activity in the medial posterior cortex was one of the first and most robust findings in the default mode literature. however, a recent study argued that only the pcc, area 23/31, has a connectivity pattern reminiscent of the dmn and that the precuneus, area 7-m, should therefore not be considered part of the default mode (margulies., 2009). this study showed functional interactions in the resting state of pcc with ventral and dorsal prefrontal regions, medial temporal cortex, and lateral inferior parietal and temporal cortex. in the domain of social cognition, pcc has been attributed a role in attributing mental states to others (saxe and powell, 2006). medial frontal regions belonging to the dmn have been less consistently characterized, with foci having been reported ventrally in the medial area 10 and dorsally in areas 32 and 24. however, the precise organization of the human medial frontal cortex and its similarity with the macaque mfc remains a topic of debate (beckmann. in contrast to the pcc, medial frontal regions have been argued to have more generalized roles in social cognition, beyond the specific attribution of mental states (saxe, 2006). the ventral part of the medial frontal cortex is commonly seen in tasks probing empathy and gray matter in this area correlates with mentalizing abilities and social network size (lewis., 2011). apart from these medial areas, the most commonly identified regions of the dmn are bilateral areas along the posterior ipl, often extending into posterior superior temporal cortex. this area includes a region that in the literature on the neural basis of social cognition is often referred to as the tpj. indeed, it has been argued that the tpj is the area most associated with theory of mind tasks or mentalizing (saxe, 2006). however, the precise locus of this social tpj remains a topic of debate, complicating any comparison of functional anatomies across domains. indeed, there is an ongoing debate on whether activations in the tpj related to theory of mind and those related to other cognitive processes, such as attentional switching are in the same or different cortical areas (decety and lamm, 2007 ; mitchell, 2008 ; scholz., 2009). we have recently performed two studies aimed at characterizing the lateral parietal cortex and tpj in the human brain. first, we used diffusion imaging, an mri technique aimed at characterizing the white matter pathways connecting areas of the brain (johansen - berg and rushworth, 2009), to parcellate these brain areas into subregions based on their structural connectivity with the whole brain. second, we used resting state fmri to investigate which larger cortical networks the resulting regions are part of. in the first study, we parcellated the human right lateral parietal cortex, focusing specifically on comparing its general organization with that of the macaque monkey (mars., 2011). we showed a general similarity in organization between the human and macaque parietal cortex, with some differences in the strength of connections with the prefrontal cortex. in this study, we subdivided the ipl into five separate regions, organized into a posterior - to - anterior arrangement. this organization was highly similar to that suggested by previous cytoarchitectonic parcellations in the human brain (caspers. in the second study, we focused on the right tpj in the human brain, an area whose exact anatomical location and connectivity have been poorly characterized. we parcellated a large area of interest incorporating all the locations that have been described as tpj in the literature and separated it into three components (mars. the dorsal area overlapped with the ipl regions in our parietal study (mars. ventrally, we identified two areas, one anterior (tpja) and one posterior (tpjp). we believe the posterior tpj region overlaps with the foci traditionally associated with social cognition. in order to investigate which larger cortical network each of the tpj clusters participates in, we next investigated the resting state functional connectivity of each of them. we observed that the tpjp showed coupling with regions along the medial surface, including posterior cingulate / precuneus and areas in the vicinity of the anterior cingulate cortex / paracingulate gyrus. these areas are strongly reminiscent of the dmn. to explore this issue, we used ica to identify the default mode in the same dataset and plotted some of the regions identified in the parietal and tpj studies on the same brain. as can be seen in figure 2, the border between the anterior and posterior ventral tpj subdivisons coincided with the extent of the dmn independent component. moreover, the dmn component overlapped with the most posterior ipl subdivisions, the posterior and anterior parts of the angular gyrus. in this respect, it is interesting to point out that some posterior ipl regions might be similar to macaque area opt, which has strong connections to the pcc and the limbic system (caspers., 2011). overlap between connectivity - based subregions of the inferior parietal lobule and temporoparietal junction area and the dmn. top : overlap between resting state function connectivity of the posterior tpj as defined by mars. (in press) in green and the dmn as defined using independent component analysis in pink. bottom : overlap between the dmn in pink and the anterior (tpja) and posterior (tpjp) areas from mars. (in press) and the anterior and posterior angular gyrus from mars. (2011). in summary, both at the level of the network and that of individual brain areas there seems to be a consistent overlap between the dmn and areas that are active during certain types of social cognitive tasks, most notably mentalizing tasks. although the precise anatomy of both the dmn and the social brain are only partially mapped out, the core nodes of the dmn have all received tentative labels in terms of their contribution to social cognition. the challenge for the future is to further identify and localize these nodes and to determine tractable computations that each area is performing. an exciting development which might prove beneficial to understanding the relationship between social cognition and the dmn is that the dmn has now been reported in a number of non - human primates. vincent and colleagues showed that macaque monkeys (macaca mulatta and macaca fascicularis) exhibit spontaneous brain activity similar to the human resting state while being scanned under light anesthesia (vincent., 2007). when they seeded an area in the medial posterior cortex, probably enclosing the posterior cingulate and parts of the precuneus, a network consisting of posterior lateral and medial frontal activity emerged, similar to the human dmn. following this result, kojima and colleagues used pet in awake monkeys to show task - related deactivations in macaque medial cortical areas (kojima., 2009). although some differences with the human dmn were apparent their results provided further indication that monkeys have a dmn similar to that in the human brain. building on this work, mantini and colleagues recently performed a meta - analysis of 15 fmri studies on awake macaques (mantini., 2011). similar to the approach taken in schilbach 's 2008 paper on humans, they looked at areas of the brain that were active during the rest periods as opposed to task blocks in these studies. the authors tentatively suggested that the macaque dmn included areas in the medial and lateral prefrontal cortex, posterior cingulate areas, and lateral inferior parietal and temporal - occipital regions. the authors also noted the possible existence of different subsystems within the macaque dmn, one consisting of the temporo - parietal - occipital cortex, mfc, and area 8b, and one consisting of posterior cingulate and inferior parietal areas. beyond these results in macaques, suggestions of brain activity associated with rest reminiscent of the human dmn in light of these results showing that networks reminiscent of the dmn can be found in primates other than humans, it is interesting to establish if there is any relationship between the dmn and areas involved in social cognition in these species. research into the neural basis of social cognition in non - human primates has generated considerable interest recently, amongst others due to the prominence of the so - called social brain hypothesis this hypothesis relates the relative size of the primate brain to challenges associated with living in complex social groups. in order to test the hypothesis that it is the dmn that mediates some of these animals ' social abilities, in would be necessary to relate differences in dmn organization with differences in social abilities between these species. unfortunately, studies comparing the neural basis of social abilities between species remain rare (rilling., some recent studies have focused on the effects of sociality on brain size between individuals of the same species. 2011) investigated whether there are areas in the macaque (macaca mulatta) brain that show structural differences in relationship to two factors describing the social life of captive macaques : the number of animals they are housed with and an animal 's position in the group 's social hierarchy. they reported a network of regions, including the rostral prefrontal cortex, amygdala, and anterior and middle superior temporal sulcus (sts), which showed increased gray matter when macaques were housed with more animals. some of these regions, including rostral prefrontal cortex and inferior temporal cortex also showed increased gray matter in animals that occupied a higher position in the social hierarchy., we can look at areas of the brain showing functional connectivity with the areas showing gray matter differences related to social network size. using the mid - sts region as a seed this results in posterior cingulate, anterior cingulate cortex, and lateral posterior areas (figure 3a). this network is very similar to vincent 's dmn in macaques, indicating that the overlap between the dmn and brain areas involved in social cognition potentially extends beyond the human brain. social brain in the macaque ? (a) areas in the macaque showing resting state functional connectivity with a region in the middle superior temporal sulcus that, in turn, showed increases in gray matter density in individuals living in larger social groups (data from sallet., 2011). (c) dual regression results showing a region of the medial frontal cortex (in blue) that in increasingly recruited into the dmn (in red) when animals live in bigger groups. we can then take this analysis a step further and see if any of these functional correlations with mid - sts are modulated by social network size. sallet and colleagues did precisely this, and reported increased functional coupling between mid - sts and the anterior cingulate with increasing network size (sallet., 2011), suggesting that the anterior cingulate is preferentially recruited into the dmn in participants with larger social networks. going even further, we can test this hypothesis of increased involvement of the anterior cingulate cortex in the dmn directly. using the recently established technique of dual - regression (filippini., 2009) it is possible to test for individual differences in recruitment of brain areas in any particular resting state network. we used this technique on resting state data obtained from 32 macaque monkeys including those in the study of sallet., we selected the component that best captured the dmn (figure 3b) and asked if there were any voxels in the brain that participate more in this component in participants housed with an increasing number of other animals. as can be seen in figure 3c, the medial frontal cortex is increasingly recruited in the dmn in these monkeys. these results thus provide direct evidence that the dmn differs in individuals as a function of social network size. it is informative to discuss some of the similarities and differences in the results obtained from humans and non - human primates. at first glance, both have a dmn consisting of medial frontal and parietal cores and lateral temporoparietal areas. moreover, effects of sociality, operationalized by social network size and mentalizing ability have been shown to correlate with gray matter density in areas of the human brain (bickart., 2011 ; lewis., 2011 ; dunbar, 2012), similar to the results obtained in macaques (sallet., 2011). however, there are also differences, both in the anatomy of the two brains and, of course, in the social abilities of the two species (passingham, 2008 ; cheney, 2011). some of the most notable anatomical differences are in regions reported in the gray matter density studies of social ability. (2011) noted that there is still uncertainty about the relationship between the mfc in humans and macaques. furthermore, activity around the tpj is commonly reported in the human dmn, but seed - based correlation analysis of the macaque dmn often show slightly more ventral areas in posterior lateral sts, which would be consistent with known connectivity of the macaque pcc (kobayashi and amaral, 2003). given the large relative expansion of the middle parts of both the ipl and the sts in the human as compared to the macaque brain (van essen and dierker, 2007) some changes in relative position of these lateral nodes of the dmn might be expected. in summary, we have investigated evidence for overlap between the dmn and areas involved in social cognition. we have shown that both at the network level and at the level of individual brain regions there is overlap between these two networks. we have highlighted the fact that the precise anatomical loci of areas involved in the dmn is not always known, particularly in the case of the area around the tpj. finally, we have investigated whether a similar relationship between brain areas involved in social skills and the dmn might be apparent in non - human primates. in what follows it is not the intention of this paper to claim that the purpose of the dmn is to do social cognition. rather, the goal is to highlight the proposed overlap between the social brain network and the dmn and to discuss the potential implications of this, both with reference to the human and the wider primate literature. although a number of authors have tried to recast social cognition in terms of underlying more basic processes, this has proven notoriously difficult (behrens., 2008). the activity shown in areas commonly attributed to the dmn during social cognition provides an interesting challenge to find a common computational function for these two seemly very different functions and some studies are currently proposing frameworks for addressing this issue (sadaghiani., 2010 ; yoshida., 2010). studies trying to find order in the variety of processes that might be present when participants are at rest appeared from the beginning of the research into the dmn. early studies already noted that rest consisted of a variety of functions (andreasen., 1995), suggesting that rest might best be characterized as random episodic silent thinking about one 's life and experiences. since then, more and more reports have emerged of processes that seem to engage the dmn and there is a growing number of proposals regarding what the common denominator of these processes, is ranging from mind wandering (mason., 2007) to the sense of self (qin and northoff, 2011). the first class refers to hypotheses that emphasize the fact that the dmn is active during situations in which there is no strong task constraint, when participants are allowed or even encouraged to broadly monitor the environment, in contrast to the narrow tunnel vision often associated with psychological laboratory tasks. consistent with this type of proposal, platt and colleagues have recently suggested a more computationally constrained role of the posterior cingulate in the detection of changes in the environment and subsequent changes in decision policy and behavior (pearson., 2011). the second class of hypotheses focuses on the involvement of the hippocampal and medial temporal structures in the dmn and attributes it a role more in mentation. in other words, processes which rely on episodic memory and mental simulations are prone to rely on the dmn. these hypotheses are broadly consistent with activity observed in the dmn during such processes as thinking about one 's future and constructing a mental representation based on autobiographical memory (spreng., 2009 ; andrews - hanna., 2010) and have also been cited as an important reason for the involvement of the dmn in social processes such as mentalizing. both of these theories would be consistent with a function of the dmn in social cognition. it has been suggested that social cognition relies on processes that might be distinct from other forms of intelligence. indeed, this social function of intellect hypothesis has been proposed repeatedly (jolly, 1966 ; humphrey, 1976) and forms the basis of the social brain hypothesis which states that our brains have expanded so much over the course of evolution precisely because of the challenges involved in living in large social groups (dunbar, 1998). the ecology of social cognition might provide some clues as to why the dmn might have properties that are beneficial to this type of mental faculty. the largely unconstrained nature of social decision making, including its reliance on potentially multiple instances of recursive thinking might be one reason why social cognition relies on a network such as the dmn. as noted above, the dmn is characterized by the presence of a number of very rich nodes, i.e., areas that form long - range connections to other brain regions (hagmann., 2008). furthermore, it has been argued that the core skill necessary to survive in our complex social environments is the ability to keep track of the complex and constantly changing social relationships, not only of oneself with the other group members, but also between the other group members among themselves (de waal, 1982 ; cheney and seyfarth, 2008). the presence of wide - range connections together with the subsystem involving areas associated with autobiographical memory might make the dmn a logical system to employ in social problem solving. if it is true that higher - order social cognition relies at least in part on the dmn in the human brain, the question is what the finding of a similar network in chimpanzees and macaques means. at the very least, a strong reliance of higher order social cognition on an already existent neural basis would be consistent with theories which propose that great apes, and by extension the common precursor of great apes and humans, may have (had) many of the relevant cognitive preconditions for uniquely human social cognition to evolve. the cooperative breeding hypothesis proposed by hrdy and colleagues (burkart., 2009 ; hrdy, 2009) suggests that it was the case, but that other apes and our common ancestor lacked the motivational preconditions that were required to developed full, it was the evolution of cooperative breeding, together with the existing ape - type brain, that lead to our complex social abilities (burkart., 2009). in conclusion, the overlap between the dmn and brain areas involved in social cognition deserves further attention. its precise anatomy and computational function still harbors many unknowns, but their solution might have implications far beyond the field of cognitive neuroscience. 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 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 default mode network (dmn) of the brain consists of areas that are typically more active during rest than during active task performance. recently however, this network has been shown to be activated by certain types of tasks. social cognition, particularly higher - order tasks such as attributing mental states to others, has been suggested to activate a network of areas at least partly overlapping with the dmn. here, we explore this claim, drawing on evidence from meta - analyses of functional mri data and recent studies investigating the structural and functional connectivity of the social brain. in addition, we discuss recent evidence for the existence of a dmn in non - human primates. we conclude by discussing some of the implications of these observations. |
muscle contraction produces force and change in the muscle s length, i.e., velocity. skeletal muscles are primarily classified into parallel muscles and pennate muscles, based on the muscle s form and structure. in parallel muscles, the shortening velocity is faster and the range of motion is larger because the muscle fibers are arranged longitudinally and directly reflect mechanical shortening. in contrast, in pennate muscles, the shortening velocity is slower and the range of motion is shorter because muscle fibers are arranged diagonally with a certain pennate angle. pennate muscles exert stronger force because they comprise a greater number of muscle fibers, with a larger cross - sectional area. generally, parallel muscles are located at joints, such as the elbow joint, and require exercise to change the joint angle significantly. conversely, many pennate muscles are located in the lower extremities and counteract the effects of gravity2, 3. the semitendinosus muscle (st) composes a part of the hamstrings and has a characteristic form (fig. 1.structure of the semitendinosus musclethe line of tendinous inscription (arrow 1) lies at the center of the muscle belly. the proximal compartment has the structure of a parallel muscle, whereas the distal compartment is considered a pennate muscle because it has a pennate angle (arrow 2 ; recorded at 65% from the proximal tendon).). the st has a tendinous inscription (ti) in the center of the muscle belly, and its muscle fibers are divided into a proximal compartment (pc) and a distal compartment (dc). the pc of the st is a parallel muscle, but the dc has a pennate structure with an average 12.9 pennate angle4. in other words, the st comprises both parallel and pennate muscles dependent on the existence of the ti. functional differences can therefore be assumed between the two compartments, because each has a unique innervation5, 6. structure of the semitendinosus muscle the line of tendinous inscription (arrow 1) lies at the center of the muscle belly. the proximal compartment has the structure of a parallel muscle, whereas the distal compartment is considered a pennate muscle because it has a pennate angle (arrow 2 ; recorded at 65% from the proximal tendon). reportedly, the st is susceptible to injury in the dc on the sports field7. it is believed that the muscle fibers are particularly easily injured during eccentric contraction because they can not withstand elongation due to the load3, 8. for example, the dc is often damaged during the swing phase of running. weakness of the hamstring muscles was predicted to underlie this damage9,10,11, and hamstring strengthening exercises have been proposed as a precaution12, 13. also, the increase in stiffness of the hamstrings may serve as a cause of shoulder and elbow pain14 and decrease of dynamic postural control15. although there are many reports on the injury of the dc and on the treatment of stiffness of the hamstrings16, 17, few studies have considered the difference between the pc and dc. in the context of our clinical rehabilitation for patients who have undergone total knee arthroplasty, it was observed that flexibility of the dc of the st is lost, but not that of the pc. it was also observed that muscle force or range of motion in the knee joint increases with improved flexibility of the dc. however, this is difficult to prove clinically because the st has a complex form. therefore, it is important to determine the functional differences between the pc and dc of the st. in this study, using an ultrasound device, muscle fiber lengths in each compartment of the st were measured during knee flexion and hip extension to determine the functional differences between the dc and pc as well as changes in joint angle. the left legs of seven men aged 2138 years, without orthopedic or neurological system disorders were examined. after the purpose of this study was explained to them, all of them provided written informed consent in order to participate. all procedures in this study were approved by our institutional ethics committee (no. 39 - 2). the participants performed knee flexion (kf) and hip extension (he) movements in the prone position, with 0 extension of the knee joint and 25 flexion of the hip joint. kf was defined as a flexion movement from about 0 to 80 knee flexion at 25 hip flexion. he was defined as an extension movement from about 25 to 0 hip flexion at 0 knee flexion. these movement angles were selected with reference to those that occur during normal walking and because of the ease of the movements. participants performed one movement every 4 seconds with the aid of an electric metronome. the ti of the st was recorded at least twice, and the pennate part was recorded at least three times (fig. there were two movements and two recorded parts. in each movement (knee flexion and hip extension), the tendinous inscription and pennate parts were recorded at different times.b. ultrasound images were recorded at the tendinous inscription part and distal pennate part, during two movements, for each subject (a). the knee and hip joints angles were recorded using a video camera, in synchrony with the ultrasound images (c). the ultrasound (a) and joint angle (b) images were analyzed using dartfish animation software (c). ; see below for further details)., the participants practiced the movements in order to maintain a fairly constant velocity and to avoid compensation caused by extension of the lumbar vertebrae during hip extension. experimental set - up and methods of measurement a. there were two movements and two recorded parts. in each movement (knee flexion and hip extension), the tendinous inscription and pennate parts were recorded at different times. b. ultrasound images were recorded at the tendinous inscription part and distal pennate part, during two movements, for each subject (a). the knee and hip joints angles were recorded using a video camera, in synchrony with the ultrasound images (c). the ultrasound (a) and joint angle (b) images were analyzed using dartfish animation software (c). the images of the ti and pennate parts of the st were captured using an ultrasound device (ssa-770a ; toshiba medical systems corporation, otawara, tochigi, japan). shift of the ti position was measured from images of the ti, while changes in distal muscle fiber length, longitudinal length of the dc muscle, pennate angle, and muscle thickness were measured from images of the pennate part. ultrasound images were converted to digital images at 60 frames per second by a personal computer. a probe with a linear form was used, taking care not to squeeze the muscle shape while lightly positioning the probe on the back of the thigh. one experimenter recorded all images, and two experimenters confirmed all images. an ultrasound image of the ti is shown in fig. 3afig. 3.ultrasound images(a) an example of the tendinous inscription part (see arrow 1 in fig. point (a) is the intersection of the tendinous inscription and superficial fascia. the muscle fiber on the left side of point (a) represents the proximal compartment. movement of point (a) is considered as a change in the muscle fiber length of the proximal compartment, as shown in c.(b) an example of the pennate part of the distal compartment is depicted (see arrow 2 in fig. we recorded the same muscle fiber and thickness values in a single movement (see fig. 4).(c, d) expressed models of the tendinous inscription part (c) and the pennate part (d) during movement of the knee and hip joints, respectively.sf : superficial fascia ; ti : tendinous inscription ; fas : fascia between the semitendinosus and semimembranosus muscles ; st : semitendinosus muscle ; sm : semimembranosus muscle ; mf : muscle fiber. muscle length in the pc was appreciated from the shift of the intersection of the ti and superficial fascia (point a in fig. it was inferred that the muscle length of the pc shortened when point a moved in a proximal direction and elongated when point a moved in a distal direction. this was because the part proximal to point a comprised muscle fibers of the pc. sometimes, the shift in ti position was longer than the width of the probe, and it was unable to be measured. therefore, sonic wave opacity tapes were affixed on the skin, and these tape points were considered a criteria point during repeated recordings. (a) an example of the tendinous inscription part (see arrow 1 in fig point (a) is the intersection of the tendinous inscription and superficial fascia. the muscle fiber on the left side of point (a) represents the proximal compartment. movement of point (a) is considered as a change in the muscle fiber length of the proximal compartment, as shown in c. (b) an example of the pennate part of the distal compartment is depicted (see arrow 2 in fig. we recorded the same muscle fiber and thickness values in a single movement (see fig. (c, d) expressed models of the tendinous inscription part (c) and the pennate part (d) during movement of the knee and hip joints, respectively. sf : superficial fascia ; ti : tendinous inscription ; fas : fascia between the semitendinosus and semimembranosus muscles ; st : semitendinosus muscle ; sm : semimembranosus muscle ; mf : muscle fiber an ultrasound image of the pennate part is shown in fig. the pennate part was captured at the clearest point (which is usually located 65% toward the tibial tuberosity from the ischial tuberosity ; fig. it was supposed that superficial fascia and muscle fibers that did not appear were in a straight line when the pennate part was measured (dotted line in fig. 4fig. 4.model of the pennate part of the semitendinosus musclethe superficial fascia and muscle fiber are composed of two planes with pennate angle,.sf : superficial fascia ; mf : muscle fiber ; : pennate angle ; ll : longitudinal length of the distal compartment muscles ; mfl : muscle fiber length ; t : muscle thickness). the length of muscle fiber in the dc and the longitudinal length of the dc muscle were calculated from the pennate angle and muscle thickness, because the muscle fiber was longer than that shown by the ultrasound image (fig. shortening and elongation of the muscle fiber length in the dc were determined relative to those during rest. model of the pennate part of the semitendinosus muscle the superficial fascia and muscle fiber are composed of two planes with pennate angle,. sf : superficial fascia ; mf : muscle fiber ; : pennate angle ; ll : longitudinal length of the distal compartment muscles ; mfl : muscle fiber length ; t : muscle thickness joint movement was recorded by a video camera (gz - hd300 ; victor company of japan, ltd., yokohama, kanagawa, japan) during all experiments, simultaneously with ultrasound images. joint angles were analyzed from video images and shift of the ti position, while muscle length of the dc, longitudinal length of the dc muscle, pennate angle of the dc, and muscle thickness of the dc were analyzed from ultrasound images. the joint angle was measured using the automatic tracking mode of dartfish (dartfish japan co., ltd. each time the joint angles changed by 1, the other data were measured manually because ultrasound images were too unclear to use an automatic mode. these were measured in both the ti and pennate parts during kf and he (one point for each part during each movement). in addition, the sum of the decreases in muscle length of the pc muscles and the longitudinal length of the dc muscles were obtained during kf and he. the measurement errors, errors arising owing to unclear images, and analysis errors for the dc were greater than those for the pc, because the length of the dc muscle fibers was calculated using two measurements (i.e., pennate angle and muscle thickness). thus, three data points were obtained for the pennate part in order to calculate the average of every three measurements and to decrease the effects of these errors. statistical computing r (version 3.0.1 ; the r foundation for statistical computing, vienna, austria) was used for statistical analysis. decreases in muscle fiber length and the joint angle in both compartments were correlated using the pearson s correlation coefficient. during kf, there were significant correlations in both compartments, with decreases in muscle fiber length and joint angle in both compartments showing correlations (pc, r=0.75, p<0.00001 ; dc, r=0.54, p<0.00001). during he, there were no significant correlations in either compartment (pc ; r=0.12, p=0.134, dc ; r=0.13, p=0.111). there were significant correlations in longitudinal length of the whole st and joint angle during kf and he (kf, r=0.86, p<0.00001 ; he, r=0.19, p=0.037). the decreases in muscle fiber length and joint angle are presented in fig. 5fig. 5.decreases in muscle fiber length during joint movementthe graphs depict decreases in muscle fiber length in (a) the proximal compartment (pc) during knee flexion ; (b) the proximal compartment during hip extension ; (c) the distal compartment (dc) during knee flexion ; (d) the distal compartment during hip extension ; and decreases in the sum of muscle fiber length of the proximal compartment and longitudinal length of the distal compartment (e) during knee flexion and (f) during hip extension. the gray lines indicate the standard deviation of the decreases in muscle fiber length.there was a strong correlation between joint angle and muscle fiber length during knee flexion (a and b). during hip extension (c and d), there was no correlation between joint angle and muscle fiber length.. during kf in both compartments (fig. 5a and 5c) however, during he in both compartments, the decreases in muscle fiber length were disconnected (fig. 5c and 5d). decreases in muscle fiber length during joint movement the graphs depict decreases in muscle fiber length in (a) the proximal compartment (pc) during knee flexion ; (b) the proximal compartment during hip extension ; (c) the distal compartment (dc) during knee flexion ; (d) the distal compartment during hip extension ; and decreases in the sum of muscle fiber length of the proximal compartment and longitudinal length of the distal compartment (e) during knee flexion and (f) during hip extension. the gray lines indicate the standard deviation of the decreases in muscle fiber length. there was a strong correlation between joint angle and muscle fiber length during knee flexion (a and b). during hip extension (c and d), there was no correlation between joint angle and muscle fiber length. only the decreases in muscle fiber length in the dc during he tended to be negative ; in other words, the muscle fibers were extended (fig. the other decreases in muscle fiber length tended to be positive ; in other words, the muscle fibers shortened. the mean and standard deviation of the changes in pennate angle and muscle thickness in the dc are shown in table 1table 1.changes in pennate angle of the distal compartments and muscle thickness of the distal compartments for all subjectsknee flexionhip extensionpennate angle(degrees)muscle thickness(cm)pennate angle(degrees)muscle thickness(cm)subject 14.6 3.40.7 3.12.1 2.22.2 2.3subject 23.7 2.00.9 1.34.2 4.05.1 5.2subject 36.6 4.71.3 2.02.1 1.92.9 1.9subject 44.8 3.33.1 1.51.7 2.04.4 2.1subject 59.4 4.23.2 3.14.7 3.84.0 2.6subject 65.0 2.41.1 1.24.2 2.44.5 2.2subject 75.0 3.50.1 1.90.7 3.84.9 2.8during knee flexion, the pennate angle increased and muscle thickness tended to increase. during hip extension, values are shown as mean standard deviation.. overall, the means increased. during knee flexion, the pennate angle increased and muscle thickness tended to increase. during hip extension, this study aimed to identify the functional differences between the pc and dc of the st during kf or he. decreases in muscle fiber length in the pc and dc were measured using ultrasound exam, and the correlation between decreases in muscle fiber length and joint angle were calculated. previous studies have reported differences in the examined muscles. these studies compared muscle activity in the medial and lateral sites in the biceps brachii longus muscle using wire electrodes and reported a difference in the activities of these muscles during elbow flexion, forearm supination, or shoulder exorotation19, 20. additionally, some studies compared muscle fiber and tendon length during walking using an ultrasound device and reported differences in the length of these fibers during each walking phase21, 22. however, the aforementioned studies did not examine in - series muscle fibers as well as the pc and dc of st. the present study hypothesized that the proximal muscle fiber and distal muscle fiber might have functional differences. it was found that decreases in muscle fiber length in the pc and dc correlated with the joint angle during kf. in addition, the longitudinal length of the whole st was correlated with the joint angle during kf. the difference in correlation was too small to determine the exact functional difference between the pc and dc, and it is possible that there are not functional differences between the pc and dc during kf. during he, there were no significant correlations between decreases in muscle fiber length in the pc and dc and joint angles. however, there were differences in the shortening or elongation of muscle fibers ; i.e., these displayed different fascicle behavior. these results differed from other results in that the muscle fiber of the pc shortened during he. the pennate muscle can control the change of muscle tendon complex length by changing the pennate angle23. this difference during he could be meaningful and was one of the functional differences. it is important to note that the measurement methods used for each compartment were different. measurement of decreases in muscle fiber length in the pc used the shift of the ti, recording all muscle fibers of the pc. conversely, measurement of the dc did not cover all muscle fibers of the dc, but only those in the pennate part. also, it is necessary to obtain three - dimensional measurements because the st is staggered5, and it is possible that this staggering differs between the pc and dc. next, it is necessary to consider that each compartment affects the tension of the other. when one compartment is undergoing contraction, afferent information from muscle spindles and golgi tendon organs in the other compartment changes. the response from afferent information was different even in the same muscle at every site, e.g. medial and lateral head of the gastrocnemius24. in st muscle, the afferent information of pc and dc may produce different responses. during kf, the correlation between the pc and dc did not determine those differences, which suggested that the pc and dc produced similar level of tension. during he, the muscle fiber of the dc might be eccentrically contracted, although it might be passively stretched by muscle activity of the pc. the muscle activity of the dc might be weaker than that of the pc, but would not be inactive. in addition, the longitudinal length of the whole st was correlated with joint angle during he. the fascicle behavior during he differed between the pc and dc, but both compartments shortened as a whole because the muscle activity was coordinated. it was unclear why the pc, parallel muscle, contracted stronger than did the dc during he. in parallel muscles, the shortening velocity is faster and the range of motion is larger than those in pennate muscles. during he, the change of muscle tendon complex length should be smaller, because few joint ranges were measured. during he, it was predicted that the pc played fewer roles and contracted weakly, but our results did not support this prediction. therefore, the dc that exerted stronger force might contract more weakly than does the pc. our results can be applied in clinical contexts. in the context of sports, it is thought that the st is prone to injury7, particularly during eccentric contraction3, 8. our results suggest that muscle fibers tend to elongate in the dc, which may be the cause of many injuries to the dc of the st. in addition, rapid hip extension may cause injury to the dc of the st, because hip extension caused elongation of the muscle fibers of the dc. first, the muscle fiber length of the pc was measured movement for the skin, but movement of the skin for the bone was not measured. the skin around the joint moved to a greater extent for the bone during joint movement25, which may have caused a skin effect. the proportion of muscle activity that each compartment contributed to a movement could not be determined and the muscle activity was not measured with, for example, electromyograms. in addition to the gluteus maximus, the gluteus medius, gluteus minimus, hamstrings, and adductors muscles act as hip extensors. additionally, the hamstrings and gracilis, sartorius, popliteus, and gastrocnemius muscles act as knee flexors. regarding the hip joint in particular, the contribution of the st may be minimal, because the correlation of the change in joint angle and decreases in muscle fiber length was not significant. finally, a single joint movement was examined by one method of force production and contraction velocity through a partial range of its motion. these components of movement were important with respect to the differences between parallel and pennate muscles. in addition, during walking and other activities, there are other effects, such as ground force reaction, and the activity of other muscles. the clinical implications of our findings may be limited, and it is necessary to perform additional investigation and to consider various other factors. | [purpose ] the tendinous inscription divides the semitendinosus muscle into the proximal and distal compartments. it was hypothesized that there are functional differences between those compartments. [subjects and methods ] seven adult males performed knee flexion and hip extension in the prone position. an ultrasound device measured the decrease in the length of muscle fibers in the two compartments during these movements. the knee and hip joint angles were concurrently measured using a video camera. pearson s correlation coefficients were calculated between the decrease in muscle fiber length in each compartment and joint angle. [results ] during knee flexion, decreased muscle fiber length was significantly correlated with increased knee flexion angle. during hip extension, there were no significant correlations for either compartment. only the decrease in muscle fiber length in the distal compartment during hip extension tended to be negative ; the other decreases in muscle fiber length tended to be positive. [conclusion ] correlations did not reveal any functional differences. however, only the distal compartment elongated during hip extension. this result might show a functional difference and could be applied in clinical contexts during hip extension. |
von hippel lindau (vhl) disease is a rare inherited condition affecting 1 in 36 000 births and is associated with the development of various multi - system tumours, including haemangioblastomas, phaeochromocytomas, renal cell carcinomas, endolymphatic sac tumours and papillary cystadenomas of the epididymis (in men) and broad uterine ligament (in women). although the association between vhl and primary epididymal papillary cystadenomas is well known, there is extremely limited information about its malignant counterpart (i.e. papillary cystadenocarcinomas) with only a few cases reported in the literature [26 ]. the few case reports available have not been associated with vhl disease. as a result, there is also limited information regarding their optimal long - term management. we present the first reported case of a 43-year - old gentleman with vhl disease presenting with azoospermia and subsequently diagnosed with a primary epididymal papillary cystadenocarcinoma, infiltrating the spermatic cord and accompanied by a metastatic deposit in the right testis. the patient was ultimately managed by a combination of radical orchidectomy and onco - microtese. a 43-year - old gentleman with known vhl disease and a history of multiple renal carcinomas presented with primary infertility and non - obstructive azoospermia. on examination, he had an atrophic left testis (secondary to a previous orchidopexy for cryptorchidism) and a firm mass in the right epididymis. he was referred for testicular sperm extraction. at the time of surgical sperm retrieval, a biopsy was taken from the epididymal mass, which was reported as a cystadenocarcinoma. a further biopsy was taken of a mass incidentally noted in the upper pole of the right testis, which on histology was suggestive of a metastatic deposit of renal cell carcinoma. an orchidectomy was advised, but declined by the patient as he was keen to start a family. after successfully having a child via in vitro fertilisation (ivf) treatment, he re - attended the urology department for definitive management of his epididymal and testicular tumours. he was asymptomatic and clinical examination revealed a palpable mass in the head of the epididymis measuring ~1.5 cm. a separate small mass was also palpable in the upper pole of the right testis. blood tests revealed normal testicular tumour markers (-fetoprotein, -human chorionic gonadotrophin and lactate dehydrogenase). follicle stimulating hormone and leutenizing hormone levels were 36 and 13 miu / ml respectively, with a testosterone level of 12.2 nmol / l. preoperatively, the optimal management strategy of his testicular and paratesticular lesions was complicated given the rarity of the case coupled with the challenge of multiple issues including : future fertility preservation.atrophic, previously undescended, contralateral testis.borderline testosterone levels.propensity to develop further tumours on a background of vhl disease. initially, a partial orchidectomy was considered to preserve future fertility. however, after review at the multidisciplinary team (mdt) meeting, it was felt that a radical orchidectomy was the safest oncologocial management option and was agreed by the patient. this was performed via an inguinal approach and an onco - microtese (microsurgical testicular sperm extraction in azoospermic cancer patients performed once the testicle has been removed) was carried out to allow for future family planning. isolated areas of dilated tubules were retrieved under 20 magnification with viable sperm cryopreserved for future ivf. subsequent histological review revealed a multinodular tumour in the head and body of the epididymis, which exhibited varying architectural patterns. in some areas, the tumour resembled that of a classical papillary cystadenoma with papillary structures projecting into cystic spaces lined by cuboidal to columnar cells with pale eosinophilic to clear cytoplasm. however, in other areas, the tumour was composed of cystic as well as solid sheets of polygonal cells with clear cytoplasm. there was also a separate deposit in the upper pole of the testis, which was composed of cystic spaces filled with eosinophilic secretions and lined by polygonal cells with clear cytoplasm. based on the immunohistochemical findings, it was felt that the intratesticular tumour deposit most likely represented a metastasis from the papillary cystadenocarcinoma of the epididymis (figs 1 and 2). the epididymal tumour resembled that of a classical papillary cystadenoma (4 magnification). figure 2:histology of separate tumour deposit in testis composed of cystic spaces filled with eosinophilic secretions and lined by polygonal cells with clear cytoplasm (4 magnification). histology of epididymal tumour showing varying architectural patterns. in some areas, the epididymal tumour resembled that of a classical papillary cystadenoma (4 magnification). histology of separate tumour deposit in testis composed of cystic spaces filled with eosinophilic secretions and lined by polygonal cells with clear cytoplasm (4 magnification). given the unusual nature of the case, the unknown prognosis of papillary cystadenocarcinomas and the lack of established treatment protocols post - orchidectomy, an mdt decision of close clinical surveillance with regular follow - up was made. papillary cystadenomas of the epididymis are known to occur in association with vhl disease. however, the development of a papillary cystadenocarcinoma, its malignant counterpart, is incredibly rare. there is a single case report, which has documented metastases from such tumours to the paraureteral region. however, metastasis to the testis has not been previously reported in the available published literature. the presentation on a background of infertility and atrophic undescended contralateral testis raises further dilemmas in management. surgical debulking remains as the cornerstone for the treatment of primary malignant epididymal tumours either via partial or radical orchidectomy. a radical orchidectomy was considered to be the optimal treatment in view of the nature and location of his tumour, but the limited functional potential of his contralateral atrophic testis made his management more challenging. in this case, fertility preservation was paramount to the patient. combining his radical orchidectomy with a simultaneous surgical sperm retrieval using an onco - microtese allowed safe oncological management and optimal fertility preservation. referral to specialist units with expertise in performing onco - microtese is important in such difficult cases. written consent was obtained from the patient for publication of this case report and the accompanying images. | papillary cystadenomas of the epididymis are known to occur in association with von hippel lindau (vhl) disease. the development of a papillary cystadenocarcinoma, its malignant counterpart, is rare with only a few sporadic cases reported in the literature. metastatic deposits are exceedingly uncommon ; in fact, only a single case report has documented metastases to the paraureteral region, but metastases to the testis have never been reported. a 43-year - old gentleman with vhl disease presented with non - obstructive azoospermia, a right epididymal mass, and an atrophic surgically corrected undescended left testis. the epididymal mass was reported as a papillary cystadenocarcinoma on biopsy. the patient was managed with a radical inguinal orchidectomy and bench microtese with successful sperm retrieval. metastatic papillary cystadenocarcinoma of the epididymis to the testis has never been previously reported. this case was managed by radical orchidectomy and subsequent onco - microtese, allowing safe oncological treatment and optimal fertility preservation. |
anterior cruciate ligament (acl) reconstruction improves stability and function of the knee and reduces the risk of chondral and meniscal injuries. however, it does not seem to protect the knee from degenerative arthritic changes in all patients1). hence, the techniques in acl reconstruction continue to evolve and many issues are still under debate. creation of a femoral tunnel by drilling through an anteromedial (am) portal has been a significant innovation in the recent years2). this technique allows independent creation of a femoral tunnel from a tibial tunnel and improves restoration of anatomy3) and stability in acl reconstruction2,4,5). on the other hand, short tunnels might result in inadequate early graft fixation strength and might cause difficulties in suspensory fixation9). previous cadaveric and clinical studies reported the mean femoral tunnel length drilled through an am portal as ranging from 15.7 - 34.2 mm6 - 8). we suspected that this wide variation could be due to different tunnel positions and orientations employed by different authors. we perform anatomic single bundle acl reconstruction with the limb draped free rather than fixed in a knee leg holder (fig. the hip flexion varies with knee flexion and studies in the available literature do not account for this fact. the available studies on this subject have analysed the tunnel length with respect to knee flexion during femoral tunnel drilling or axial angle of guidewire4,10,11). however, there is no study objectively analysing the three dimensional relationship between tunnel orientation and length. the purpose of this study was to investigate if the tunnel length varies significantly in different orientations and to determine the 3-dimensional (3d) optimal drilling angle of the trans - portal technique for appropriate femoral tunnel length. we hypothesized that tunnel length varies with orientation and there exists a tunnel orientation in which a tunnel of optimal length can be obtained. we conducted this study using computed tomography (ct) images stored in the hospital database. the inclusion criteria were age between 20 and 40 years, no pathology in the distal femur and no history of prior surgery. thirty continuous ct records satisfying the inclusion criteria were included in the study. height and weight of all patients though ct was indicated for only one knee, the ct image acquisition protocol of our hospital included both knees. for the purpose of this study, we did 3d ct reconstruction from the available database of both knees (sample size : 30 patients, 60 knees). the cts were acquired with seimens somatom scanner and images were analysed with synco software (seimens heathcare, malvern, pa, usa). an entry point was marked on the 3d reconstructed images as reported in the previous studies on single bundle anatomic reconstruction12). the medial condyle was subtracted from the image and the end face view of the lateral femoral condyle was obtained. orientation of the femoral tunnel position in a 3d plane is not easily measured per - operatively. hence, we resolved this problem by defining the tunnel orientation in a sagittal plane and an oblique axial plane (fig. 3). angles in these two planes, the sagittal inclination in the sagittal plane and axial angle in the oblique axial plane, could be measured intra - operatively and were used to define tunnel position on the reformatted ct images. we noted that the tunnel orientation along the sagittal plane (sagittal inclination) varies with the degree of hip flexion (fig. 1) and the sagittal angle of guidewire insertion (fig. we obtained oblique axial cuts along the various sagittal inclination planes in 5 increments (fig. 5) and virtual tunnels were created along the various axial angles (fig. 6) and the tunnel length was measured. the measurements were made in 64 different orientations, between 30 and 65, in 5 increments, in both sagittal and oblique axial planes. all measurements were performed on a picture archiving and communication system monitor (synco software ; seimens heathcare) by use of a mouse - point cursor and automated computer calculation for distance and angle. accepting an error of 0.1 mm for the groove depth and 10 for the angles, the formula derived from gaussian distribution (n=[zs / e]2, where z=1.96 for 5% level of significance, s is standard deviation of the sample and e is the accepted error)13), was used and the sample was found to be sufficient to draw conclusions among general population. inter - observer variation was determined by having two authors independently calculate the tunnel length on 10 ct images that were randomly selected. intra - observer variability was estimated by having one of the authors calculate the confidence interval of the same radiographs with an interval of 6 weeks. both were instructed on the use of the software by a musculoskeletal radiologist at the beginning of the study. we used the pearson correlation coefficient, with scores between 0.61 and 0.80 representing good correlation and those greater than 0.81 representing excellent correlation. we thus plotted an eight by eight table showing tunnel length in each orientation for all 60 knees and analysed the mean tunnel lengths in 64 orientations in those 60 knees. a one - way analysis of variance was used to identify the significance of differences between the tunnel lengths in various orientations and the simple linear co - efficient of correlation was calculated to derive the correlation between the tunnel length and orientation. posterior cortical breakage was identified when the virtual tunnel was not covered by cortical bone (fig. the inter - observer and intra - observer reliability was excellent (pearson correlation coefficient [r]=0.94 for inter - observer reliability and r=0.98 for intra - observer reliability). the mean lengths of femoral tunnels in the possible orientations are summarised in table 1 (fig. 7). we noted that higher sagittal inclinations resulted in longer tunnels but the difference was significant only when the axial angle of the tunnel was more than 40 (simple linear correlation, r0.9 for larger axial angles, r - simple linear correlation coefficient, t - test) (table 2). the tunnel length was observed to decrease with increasing axial angle, and though the results were statistically significant in all sagittal inclinations (r>0.85 for all sagittal inclinations) the correlation was more pronounced at low sagittal inclinations. tunnel length was more consistent at high sagittal inclinations compared to low sagittal inclinations (coefficient of variance [cv]=1.6 for 30 sagittal inclination and cv=1.03 for 65 sagittal inclination). the tunnel length was more than 35 mm in 57 out of 60 knees (95%) in all tunnel orientations between axial 30-45 angles and sagittal 50-65 inclinations and always more than 30 mm in these orientations. outside this range, posterior cortical breakage was noted only in sagittal inclinations of 35 or less. in this subgroup, axial angles of 35 or less resulted in posterior cortical breakage in all the knees. the principle finding of our research was that the tunnel length was positively correlated with the sagittal inclination and that the tunnel length was longer than 35 mm in 95% when the sagittal inclination was between 50 and 65 and the axial angle was between 30 and 45. am portal drilling of the femoral tunnel facilitates creation of a femoral tunnel at native acl insertion3,14) but results in shorter tunnels6 - 8) and increases the risk of posterior cortical breach6). a recent clinical trial has also found a tendency for shorter tunnels with use of the am portal technique15). in a study of post - operative ct scans following double bundle acl reconstruction, a femoral tunnel of less than 30 mm greis.9) reported that the length of a tendon placed within a bone tunnel influences tendon pull - out strength and advocated maximising tendon length inside the bone tunnel. a recent study by zantop.17) did not find any inferior deleterious outcome with insertion of a graft as little as 15 mm in a goat model. however, graft inset of 20 mm has been the general recommendation for acl reconstruction18). very short femoral tunnels would cause difficulties while using suspensory method of fixation that is considered as the most optimal method18 - 20). suspensory fixation devices, such as tight rope (arthrex inc, naples, fl, usa), have been attempted as a solution for short tunnels. however even with the use of such devices, short tunnels would result in either a thin cortical bridge or short graft inset length. a thin cortical bridge reduces the margin of error during tunnel drilling in spite of the risk of cortical breakage. earlier studies on the length of femoral tunnel in am portal drilling4,10,11) were cadaveric studies and consequently the number of parameters that can be analysed in a single study was limited and small variations were not reported. these studies have also been limited by lack of standardisation of the entry point of femoral tunnel. a ct - based study would allow an analysis of the influence of hip and knee flexion as well as axial plane orientation of guidewire simultaneously. we created virtual tunnels in 3d reconstructed ct scans and measured the tunnel length in many possible tunnel orientations. the technique of making virtual tunnels on ct scans was devised based on careful analysis of the surgical technique and knowledge of ct measurements. a femoral tunnel is seen as a line in 3d space and such a line is conventionally defined by its end points or its angular relation to the three planes. though the tunnel entry point on the medial wall of lateral condyle is fixed, its exit on the lateral cortex may vary. additionally, tunnel orientation in all three planes can not be measured unless we use computer assisted navigation. hence, we used a unique method of defining a line in space by defining inclination of the line 's projection in one plane (si) and then defining line by its angular relationship (aa) in the inclined plane along the line 's projection (fig. the orientation of a tunnel in three planes was resolved into two angles that can be measured surgically and reproduced on an analysis of ct. we can measure the sagittal plane projection of the tunnel and the real angle of the tunnel on the inclined plane i.e., the plane parallel to the ground when the knee is fully flexed and guide wire is parallel to the ground (fig. tunnel orientation in the sagittal plane depends on the hip flexion and the sagittal angle of guidewire insertion and knee flexion influences both the degree of hip flexion and angle of guidewire insertion (fig. 4). we noted that an increase in sagittal inclination of tunnel resulted in a corresponding increase in tunnel length only at the larger axial angles. at smaller axial angles, increasing the sagittal inclination this explains the current confusion in the literature regarding the influence of knee flexion on the tunnel length. basdekis.11) noted that increasing knee flexion increases the tunnel length and decreases the risk of posterior cortical breakage. however, the axial angle was not constant in both studies and its confounding effect was not commented. this is different from the position we used in surgery, wherein knee flexion caused hip flexion and thereby contributed to increased sagittal inclination of the tunnel. the analysis of the results of our study reveals that sagittal inclination of more than 50 results in tunnel length of more than 34 mm in most axial angles (table 1, fig. when tunnels have a high sagittal inclination, they have optimal tunnel length regardless of axial angle and the incidence of posterior cortical breach is less. in contrast, when sagittal inclination is less, tunnel length varies widely with axial angle (fig. hamilton reported that the length of femoral tunnel reduces with increased axial angle of drilling10). moreover, at smaller axial angles, the tunnel is consistent with various sagittal inclinations. however, the incidence of posterior cortical breakage was greater at smaller axial angles especially when sagittal inclination was also low. a combination of low sagittal and low axial angles results in posterior cortical breakage, while low sagittal and high axial angles result in very short tunnels. from our study, we can conclude that drilling the femoral tunnel with the knee maximally flexed and guidwire set at an axial angle of about 30-45 is the reliable method to safely achieve optimal tunnel length. this tunnel orientation results in an optimal tunnel length of 35 - 45 mm and minor errors in tunnel orientation will have less effect on the tunnel length because when the knee is maximally flexed and sagittal inclination of tunnel is about 60-65, the tunnel length is consistent. the results of our study concur with the study on the femoral tunnel length by tompkins.21). the authors employed maximal knee flexion while drilling the femoral tunnel through an am portal and reported an average tunnel length of 37 mm21). complex morphology of distal femur dictates that the tunnel orientation has a critical influence on the length of femoral tunnel. a < 40 of sagittal inclination was associated with wide variation of tunnel length and variations in axial angles and sagittal inclination affected the tunnel length only when axial angle was greater than 40. further studies on the relationship between the entry point and lateral cortex of the femur are needed to investigate this phenomenon. at a higher sagittal inclination, the tunnel length was longer regardless of the change in the axial angle. hence, the surgeon should strive to increase the sagittal inclination and maintain the axial angle at 30-45. our study has some limitations, this is a ct study based on observations during surgery and hence the influences of portal positioning and soft tissues on the tunnel orientation were not assessed. the surgeon who would follow our recommendations must be aware of the potential difficulties that might be encountered. two angles that must be considered are the sagittal inclination, which must be measured only on the sagittal plane and the axial angle that must be measured in the plane of guidewire, not just in the plane parallel to floor (fig. 3). care must be taken to identify the true sagittal line to measure the axial angle from. extremely low axial angles might result in posterior slipping of drill and consequent posterior drilling. use of a central medial visualisation portal or a 70 scope has been suggested by other authors to obviate this difficulty. tunnel orientation in the sagittal plane depends on the hip flexion and the sagittal angle of guidewire insertion and knee flexion influences both the degree of hip flexion and angle of guidewire insertion (fig. 4). we noted that an increase in sagittal inclination of tunnel resulted in a corresponding increase in tunnel length only at the larger axial angles. at smaller axial angles, increasing the sagittal inclination this explains the current confusion in the literature regarding the influence of knee flexion on the tunnel length. basdekis.11) noted that increasing knee flexion increases the tunnel length and decreases the risk of posterior cortical breakage. however, the axial angle was not constant in both studies and its confounding effect was not commented. this is different from the position we used in surgery, wherein knee flexion caused hip flexion and thereby contributed to increased sagittal inclination of the tunnel. the analysis of the results of our study reveals that sagittal inclination of more than 50 results in tunnel length of more than 34 mm in most axial angles (table 1, fig. when tunnels have a high sagittal inclination, they have optimal tunnel length regardless of axial angle and the incidence of posterior cortical breach is less. in contrast, when sagittal inclination is less, tunnel length varies widely with axial angle (fig. 7) hamilton reported that the length of femoral tunnel reduces with increased axial angle of drilling10). moreover, at smaller axial angles, the tunnel is consistent with various sagittal inclinations. however, the incidence of posterior cortical breakage was greater at smaller axial angles especially when sagittal inclination was also low. a combination of low sagittal and low axial angles results in posterior cortical breakage, while low sagittal and high axial angles result in very short tunnels. from our study, we can conclude that drilling the femoral tunnel with the knee maximally flexed and guidwire set at an axial angle of about 30-45 is the reliable method to safely achieve optimal tunnel length. this tunnel orientation results in an optimal tunnel length of 35 - 45 mm and minor errors in tunnel orientation will have less effect on the tunnel length because when the knee is maximally flexed and sagittal inclination of tunnel is about 60-65, the tunnel length is consistent. the results of our study concur with the study on the femoral tunnel length by tompkins.21). the authors employed maximal knee flexion while drilling the femoral tunnel through an am portal and reported an average tunnel length of 37 mm21). complex morphology of distal femur dictates that the tunnel orientation has a critical influence on the length of femoral tunnel. a < 40 of sagittal inclination was associated with wide variation of tunnel length and variations in axial angles and sagittal inclination affected the tunnel length only when axial angle was greater than 40. further studies on the relationship between the entry point and lateral cortex of the femur are needed to investigate this phenomenon. at a higher sagittal inclination, hence, the surgeon should strive to increase the sagittal inclination and maintain the axial angle at 30-45. our study has some limitations, this is a ct study based on observations during surgery and hence the influences of portal positioning and soft tissues on the tunnel orientation were not assessed. the surgeon who would follow our recommendations must be aware of the potential difficulties that might be encountered. two angles that must be considered are the sagittal inclination, which must be measured only on the sagittal plane and the axial angle that must be measured in the plane of guidewire, not just in the plane parallel to floor (fig. care must be taken to identify the true sagittal line to measure the axial angle from. extremely low axial angles might result in posterior slipping of drill and consequent posterior drilling. use of a central medial visualisation portal or a 70 scope has been suggested by other authors to obviate this difficulty. a tunnel length of more than 35 mm can be created by achieving a sagittal inclination of greater than 45 and an axial angle of 30-45. such an orientation can be obtained by flexing the knee fully so that the hip flexion increases so as to increase the sagittal inclination. | purposethe purpose of the study was to identify the femoral tunnel orientation that consistently results in a tunnel length of more than 35 mm in anterior cruciate ligament reconstruction.materials and methodscomputed tomography (ct) scans were obtained from 30 patients who did not have any pathology in the distal femur. virtual tunnels were marked on 3d (3-dimensional) reconstructed ct images after determining the orientation defined by sagittal inclination and axial angle. the length of a femoral tunnel in 64 different combinations of orientations (between 30 and 65 in 5 increments in two planes) was measured on 3d reconstructed images in both knees in 30 patients. reliability of measurement was assessed with correlation coefficient for intra - observer and inter - observer measurements. a one - way analysis of variance (anova) was used for statistical analysis.resultsthe mean femoral tunnel length varied significantly with each 10 change in orientation in both planes (p0.9) and reduced the incidence of posterior cortical breakage.conclusionsa tunnel orientation with an axial angle between 30-45 and a sagittal inclination of more than 50 can result in a tunnel length of more than 35 mm. |
targets of treatment in patients with st - elevation myocardial infarction (stemi) are alleviating the ischemic symptoms, preventing the complications, and restoring the coronary blood flow. fibrinolysis and primary percutaneous coronary intervention (pci) are the reperfusion therapies in stemi. primary pci is a safe, effective, and preferred treatment option for maintaining reperfusion. stent implantation is an important milestone in the treatment of ischemic heart disease in interventional cardiology era. stent implantation was first performed in 1986 and after this time elastic recoil, coronary dissection, and thrombosis due to coronary angioplasty have been prevented with stenting. however, it may cause new problems like occlusion of side branches, stent thrombosis, and restenosis. despite major advances in interventional techniques and anticoagulan - antiagregan therapies incidence of stent thrombosis has been reported between 1.4% and 4.4% in patients with acute myocardial infarction and undergoing pci with stenting. reasons of stent thrombosis are resistance to aspirin and/or clopidogrel, insufficient anticoagulation, type of stent use (bare metal, drug eluting, and long stents), presentation with acute coronary syndrome, characteristics of coronary lesions and vessel, procedural causes (stent apposition), and inadequate endothelization after stenting. red cell distribution width (rdw) is a quantitative measurement of variability and size of erythrocytes. higher rdw values have been reported with worse prognosis in patients with coronary artery disease, acute myocardial infarction, stroke, acute and chronic heart failure, pulmonary hypertension, and acute pulmonary embolism. pathophysiology of elevated rdw in cardiovascular, pulmonary, and thrombotic diseases has not clearly explained yet. however, it has been thought that increased cytokines may inhibit maturation of erythrocytes in bone morrow and may cause increased rdw values in cardiovascular diseases. in this study, we aimed to investigate the relationship between preprocedural rdw values in patients who underwent pci and stenting due to stemi and development of stent thrombosis during follow - up. we have retrospectively analyzed the data of 146 patients who previously underwent primary pci with stenting due to stemi and presented with acute coronary syndrome and detected stent thrombosis during coronary angiography (stent thrombosis group) between 2009 and 2013 in a high volume tertiary center. a total of 175 patients who underwent pci with stenting due to stemi before and underwent coronary angiography other than reason of acute coronary syndrome (refractory angina, abnormal treadmill stress test, etc.) and had similar procedural characteristics (type, diameter, and length of stent) but not stent thrombosis consisted the control group. patients with decompensated heart failure, cardiogenic shock, severe arrhythmia, chronic obstructive pulmonary disease, history of stent thrombosis, intervention to left main vessel and complex interventions, creatinine > 2 mg / dl, hemoglobin < 12 mg / dl, blood transfusion within 3 months, active infection, chronic inflammatory and rheumatic diseases, malignancy, and cirrhosis were excluded from the study. we have accessed the data of demographic and clinical features of patients via hospital records. hematological and biochemical data of patients were obtained from the results of preprocedural venous blood sample analyses retrospectively. rdw values were calculated with using an automatic analyzing machine (abbott cell - dyn 3700 ; abbott laboratory, abbott park, il). patients were divided into tertiles according to the admission rdw values (12.9 0.4, 14.2 0.4, and 16.3 1.5, respectively). angiographic data including type, diameter, length, and localization of stent were obtained from coronary angiography records. stemi is a clinical syndrome defined by characteristics symptoms of myocardial ischemia with persistent electrocardiographic st elevation and subsequent release of biomarkers of myocardial necrosis. diagnostic st elevation in the absence of left ventricular hypertrophy or left bundle - branch block is defined by the european society of cardiology / american heart association / world heart federation task force for the universal definition of myocardial infarction as new st elevation at the j point in at least 2 contiguous leads of 2 mm (0.2 mv) in men or 1.5 mm (0.15 mv) in women in leads v2v3 and/or of 1 mm (0.1 mv) in other contiguous chest leads or the limb leads. time between stent implantation and control coronary angiography due to acute coronary syndrome was calculated and stent thrombosis was defined as acute (between 24 hours), subacute (24 hours30 days), late (30 days1 year), and very late (after 1 year) thrombosis. stent thrombosis was defined as presence of thrombus inside of stent or 5 mm apart from stent whether it cause occlusion or not. chicago, il). continuous data with normal distribution were presented as mean and standard deviation, and categorical variables were expressed as percentages. the independent sample t test or the mann whitney u test was used for the continuous variables and the chi - square test for categorical variables. multivariate logistic regression analysis was used to determine the independent predictors of stent thrombosis. a receiver - operating characteristics (rocs) curve analysis was performed to identify the optimal cut - off point of rdw to predict stent thrombosis. results were presented with odds ratio (or), 95% confidence interval and p value. mean ages of patients in stent thrombosis group and control group were similar (56.3 10.7 vs 54.9 11.8, respectively ; p = 0.253). there were more hypertensive patients in stent thrombosis group (47% vs. 34%, respectively ; p = 0.025). however, total cholesterol (167 40 vs 180 35, respectively ; p = 0,002) and low - density lipoprotein (ldl) cholesterol levels (101 36 vs 113 30, respectively ; p = 0.002) were significantly lower in stent thrombosis group when compared to control group. there was no significant difference in terms of sex, diabetes mellitus, smoking, triglyceride, and high - density lipoprotein cholesterol levels between groups (table 1). figure 1 shows the distribution map of rdw levels of whole group, and fig. rdw levels were significantly higher in stent thrombosis group compared to control group (14.8 2.1 vs 14.1 1.2, respectively ; p = 0.007). similarly mean platelet count was also significantly higher in stent thrombosis group (281 80 vs 244 60, respectively ; p < 0.001). there were no significant difference in neutrophil to lymphocyte ratio, mean platelet volume, hemoglobin, and white blood cell count between groups (table 1). type of implanted stent (bare metal, drug eluting), location, diameter, and length of stent were similar between groups. distribution maps of the red cell distribution width (rdw) values of all patients in the study. distribution maps of the red cell distribution width (rdw) values of the patients according to presence of stent thrombosis. patients were divided into 3 tertiles according to baseline rdw values (12.9 0.4, 14.2 0.4, and 16.3 1.5, respectively). there was significant difference in terms of age and sex between tertiles (table 2). blood glucose levels were significantly higher in 3rd tertile when compared to other tertiles (52.5 10.5 vs 53.8 10.3 vs 60.7 10.5, respectively ; p = 0.018). hemoglobin (14.4 1.4 vs 14.5 1.2 vs 14.0 1.4, respectively ; p = 0.006) and mean platelet volume (8.7 1.4 vs 8.6 1.2 vs 8.2 1.2, respectively ; p rate of stent thrombosis in 3rd tertile was significantly higher than other tertiles (41% vs 38% vs 58%, respectively ; p = 0.006) (fig. 3). there was no significant difference about type of stent thrombosis between tertiles (acute, subacute, late, and very late). similarly there was no difference in terms of coronary artery where stent implanted, and diameter of stents between tertiles (table 2). however, there was significant difference in lengths of stents and types of stents. implanted stents in tertile 3 were more longer (18.5 5 vs 18 6 vs 22 7, respectively ; p < 0.001), and the rate of drug eluting stents was more higher (3% vs 6% vs 32%, respectively ; p < 0.001) (table 2). percentage of patients developing stent thrombosis stratified by tertile of preprocedural red cell distribution width (rdw). after multivariate logistic regression analysis, rdw and platelet count remained significant predictor of stent thrombosis (or : 1.397, 95% ci : 1.1771.657, p < 0.001 ; and or : 1.008, 95% ci : 1.0041.012, p < 0.001, respectively) (table 3). using a cut point of 13.9, preprocedural rdw predicted development of stent thrombosis with a sensitivity of 57% and specificity of 52% (roc area under curve : 0.59, 95% ci : 0.530.65, p = 0.007). our study has some fundamental results. first, rdw was significantly higher in stent thrombosis group compared to control group. second, rdw was found to be independent predictor of stent thrombosis in multivariate logistic regression analyses. third, rate of stent thrombosis was more higher in 3rd tertile where the patients had the highest mean rdw values. high rdw shows expected anisocytosis in nutritional insufficiency, iron, folic acid and vitamin b12 deficiency, chronic liver disease, and blood transfusion. it has been thought that chronic inflammation may shorten the half - life of erythrocytes, change the membrane characteristics, and cause to increase of rdw values. it has been demonstrated in several studies that there is a significant association between prognosis of acute and chronic ischemic heart diseases and rdw values. c - reactive protein is a well - known marker of cardiovascular diseases, and it has been shown that there was a significant correlation between rdw and c - reactive protein values. in a study conducted in 7556 healthy volunteers by zalawadiya, there was strong correlation between rdw values and 10 year framingham risk score. this correlation was valid after the adjustment of hemoglobin, vitamin b12, folic acid, ferritin, glomerular filtration rate, and body mass index. we have found significantly higher rdw values in stent thrombosis group, and rdw was found to be an independent predictor of stent thrombosis. sangoi showed that rdw was an independent predictor of in - hospital mortality in 109 patients with acute myocardial infarction. acet demonstrated that there was significant strong correlation between global registry of acute coronary events score and rdw values in 800 stemi patients. uyarel also showed an association between rdw values at presentation and in - hospital and long - term mortality in 2506 stemi patients who underwent primary pci. all of these studies have shown the relationship between rdw and worse prognosis in patients with cardiovascular diseases. however, the cause of worse prognosis is not explained yet. there was a significant correlation between rdw and stent thrombosis in our study. higher rdw values at presentation may be explained with increased ischemia, oxidative stress, neurohumoral activation, and inflammation, and all of these causes may be the reason of stent thrombosis in our study. there was no significant difference about type of stent, diameter, and length of stent, and these similarities exclude the procedural causes of early and late stent thrombosis. there were no differences in terms of age, diabetes, smoking, triglycerides, and high - density lipoprotein levels between stent thrombosis and control group. there were more hypertensives in stent thrombosis group compared to control group. however, total cholesterol and ldl cholesterol levels were significantly lower in stent thrombosis group, and this need to be explained. although we do not have the data about the use of medications, higher preprocedural total cholesterol, and ldl cholesterol levels in stent thrombosis group may be due to nonaggressive use of statins in this group. however, this issue does not go beyond the hypothesis. in analyses according to rdw tertiles, there were more older and female patients however lower hemoglobin levels in highest tertile. similar results about the relationship between higher rdw values and female predominancy were shown before. this may be explained by increased rate of anemia in female patients. anemia is known risk factor to cause increase in rdw values. although many parameters put into the logistic regression analyses, only rdw and platelet count emerged as an independent predictors of stent thrombosis. similarities of type, diameter, and length of stents between groups are explained by the study design before patient selection. the most important limitation of our study is the lack of data about medications that had been taken by patients during the follow - up. another limitation and a negative impact of our study is we could not find a high sensitivity and specificity cut - off value of rdw to predict stent thrombosis. this may restrict the use of rdw in daily clinical practice to predict stent thrombosis. higher rdw values may be a predictor of stent thrombosis in patients who underwent stent implantation due to stemi. | abstractstent thrombosis is a rare but potentially fatal complication of percutaneous coronary interventions (pcis). in recent years, the predictive and prognostic value of the red cell distribution width (rdw) as an indicator of inflammation has been shown in many cardiovascular diseases. aim of this study was to examine the predictive value of rdw for stent thrombosis in patients who underwent successful stent implantation for st - elevation myocardial infarction (stemi).in this retrospective study, 146 patients who underwent successful pci to native coronary artery due to stemi previously and presented with acute coronary syndrome with stent thrombosis were included (stent thrombosis group). a total of 175 patients who had similar procedural characteristics (type, diameter, and length of stent) and not had stent thrombosis were consisted control group.patients were divided into tertiles according to the admission rdw values (12.9 0.4, 14.2 0.4, and 16.3 1.5, respectively). stent thrombosis developed in 47 (40.9%) patients in the lowest tertile, 39 (37.9%) patients in mid tertile, and 60 (58.3%) patients in the highest tertile (p = 0.006). female gender ratio was statistically significantly higher in the 3rd tertile (13 [11.3% ], 8 [7.8% ], 24 [23.3% ], p = 0.003, respectively). rdw (or : 1.397 [95% ci 1.1771.657 ], p < 0.001) and platelet count (or : 1.008 [95% ci 1.0041.012 ], p < 0.001) remained independent predictors of stent thrombosis after multivariate logistic regression analysis. roc curve analysis demonstrated that, admission rdw values higher than 13.9 can predict the development of stent thrombosis with a sensitivity of 57% and a specificity of 52% (the area under the roc curve : 0.59 [95% ci 0.530.65 ] p = 0.007).high rdw values found to be independently associated with the development of stent thrombosis in patients with stemi. |
a 77yearold man complained of slight shortness of breath, and was referred to our hospital due to enlarging mediastinal / hilar lymphadenopathy with calcification and right pleural effusion on computed tomography (ct) (fig. chest ct on parenchymal (a) and mediastinal (b) window settings showed right pleural effusion and mediastinal / hilar lymphadenopathy with calcification. what is the appropriate procedure to approach this lesion, and what is the differential diagnosis ? endobronchial diagnosis at nagoya medical center was requested and endobronchial ultrasoundguided transbronchial needle aspiration (ebustbna) of lymph node station 7 was performed. pathological examination revealed amorphous eosinophilic tissue with few cellular components (figs. 2 and 3a, b). (if the plasma cell rate is more than 10%, myeloma can be diagnosed.) as a result, monoclonal gammopathy of undetermined significance (mgus) associated with mediastinal amyloidosis was diagnosed. echocardiography showed an ejection fraction of 45%, and there was brightness in part of the interventricular septum. cytology of the lymph node. a lump of amorphous light green material (papaniclaou staining, 200). (a) section showing amorphous eosinophilic materials with hematoxylin eosin staining (h&e, 200). (b) these materials stained positive for direct first scarlet staining (dfs, 200), indicating amyloid deposits. we are currently administering combination therapy of bortezomib, melphalan, and prednisolone to this patient. as a result, the serum igg has decreased and there is no systemic aggravation ; however, his cardiac function has not improved with this treatment. as jenkins. stated, amyloidosis should be considered in the differential diagnosis of a large mediastinal mass, especially if calcification and/or plasma cell neoplasm is present 1. | key clinical messagea 77yearold man was referred to our hospital due to enlarging mediastinal / hilar lymphadenopathy with calcification. endobronchial ultrasoundguided transbronchial needle aspiration (ebustbna) and bone marrow aspiration were performed. subsequently, monoclonal gammopathy of undetermined significance (mgus) associated with mediastinal amyloidosis was diagnosed. we hereby report a case in which ebustbna led to a successful diagnosis of amyloidosis. |
this study was approved by institutional review boards at baylor college of medicine (houston, tx, usa), the gulf coast regional blood center (houston), and the south texas tissue and blood center (san antonio, tx, usa). blood donors residing in the greater san antonio, texas, area who had t. cruzi antibodies detected by prism chemiluminescent immunoassay (abbott laboratories, chicago, il, usa) or ortho t. cruzi elisa (ortho - clinical diagnostics inc., raritan, nj, usa), and a positive result for a radioimmune precipitation assay (quest diagnostic laboratories, madison, nj, usa) or an esa chagas test (abbott laboratories) during january 1, 2008december 31, 2014, were invited to participate in the study. persons previously enrolled in a houston - based t. cruzi seropositive blood donor project were not eligible for this study (8). letters in english and spanish were sent to donors who had t. cruzi antibodies by the blood centers for this study. those who agreed to participate provided informed consent. we performed 3 procedures : 1) blood collection for additional serologic screening, 2) structured interview to assess potential transmission sources and health, and 3) 12-lead resting electrocardiogram (ecg) (8). we defined a case of t. cruzi infection if donor screening test results and > 2 serologic test results were positive. likely autochthonous t. cruzi infection was defined in a case - patient who had no major travel to a latin american country (lasting > 2 weeks or that included an overnight stay in a rural region), not having been born in latin america, and not having a mother born in latin america (4,810). congenital transmission from a maternal grandmother (2 contiguous congenital infections) can not be ruled out with this case definition but is unlikely given the low risk for congenital transmission (7). demographic information, likely autochthonous transmission, and concurrent conditions were determined through case - patient interview. ecg, electrocardiogam ; ind, indeterminate ; +, positive ;, negative. donors listed as showing autochthonous transmission (donors 1214) reported living in mexico or chile. esa, chagas test (abbott laboratories, chicago, il, usa) ; ortho, t. cruzi elisa (ortho - clinical diagnostics inc., raritan, nj, usa) ; prism, chemiluminescent immunoassay (abbott laboratories) ; ripa, radioimmune precipitation assay (quest diagnostic laboratories, madison, nj, usa). dpp, dual path platform immunochromatographic confirmation assay (chembio, medford, ny, usa) ; eia, chagatest recombinant v3.0 enzyme inmmunoassay (wiener, rosario, argentina) ; hemagen ; chagas eia kit (hemagen diagnostics, inc., columbia, md, usa) ; stat - pak, chagas immunochromatographic sssay (chembio, medford, ny, usa) ; tesa, trypomastigote excreted or secreted antigen immunoblot (biomrieux, marcy letoile, france). hemagen, stat - pak, and dpp were performed at baylor college of medicine, (houston, tx, usa), and eia and tesa were performed at the centers for disease control and prevention (atlanta, ga, usa). results were determined from readout of a resting 12-lead ecg and interpreted by a board - certified cardiologist. av, atrioventricular ; lad, left axis deviation ; lvh, left ventricular hypertrophy ; rbbb, right bundle branch block. for persons who donated blood in the greater san antonio area during the study period, we found that 61/256,801 donors had positive serologic results for t. cruzi infection (1/4,200 donors had positive serologic results for t. cruzi infection by 2 assays). seventeen (28%) of these were enrolled in the study ; additional serologic testing confirmed that 14 had antibodies against t. cruzi when the study began (table 1). these persons had a mean age of 47 years (range 1983 years) ; 50% were hispanic, 50% were non - hispanic white, and 50% were men. for 3 persons whose blood donor testing results were not confirmed by further serologic testing, 2 were non - hispanic and 1 was hispanic (2 women and 1 man) ; mean age was 51 years. because of the blinded nature of study recruitment, we can not identify demographic data for persons who received the letter and chose not to participate. likely autochthonous transmission of t. cruzi was suspected for 11 (79%) of 14 persons, as defined by study criteria. these 11 persons had a mean age of 50 years ; 7 were non - hispanic whites, and 6 were men. remaining data presented will concern only the 11 newly identified persons with likely autochthonous infections. a structured interview adapted from a questionnaire used by the centers for disease control and prevention (atlanta, ga, usa), the american red cross (washington, dc, usa), and blood systems, inc. (scottsdale, az, usa), was used to identify risk factors for t. cruzi infection (4). because of the lifelong nature of infection and antibody - based diagnostics used, a specific time of infection could not be established for each case - patient. most (91%) case - patients with likely autochthonous infection reported a history of living in a rural community (figure). residence in rural communities could pose a risk for t. cruzi transmission because this setting might lead to close proximity with sylvatic transmission cycles involving the vector and infected animals (11). current and previous residences of persons with likely autochthonous infection with trypanosoma cruzi, south central texas, usa, including 11 autochthonous donors with current residence and birthplace. previous residences in texas were chosen if the case - patient reported living in the location > 5 years. although recreational activities or occupations associated with outdoor exposure were reported among our cohort, we obtained evidence suggesting that opportunities for transmission might be occurring near homes in rural communities (table 2). specifically, patients with likely autochthonous infections reported seeing the vector around their current or previous residence (36%), and had animal housing near their homes (73%). an extensive history of outdoor recreational activities of hunting and camping, which has been suggested as a high - risk activity for t. cruzi transmission in the southern united states, was less common then expected (36%) (8,12). two of 11 case - patients reported agricultural jobs and staying in substandard housing during the harvest season, thereby introducing the potential for disease transmission from triatomines in the home. no risk (0) was defined as not living in a rural area and having no history of outdoor occupation or recreational activities. low risk (+) was defined as ever living in a rural area, having an outdoor occupation, or engaging in hunting or camping in an area with known triatomine activity. moderate risk (+ +) was defined as, in addition to low - risk activities, an extensive history of these activities (> 1 y), or having slept in a tent in a rural part of texas. high risk (+ + +) was defined as, in addition to moderate - risk activities, reporting 1 of the following : reported seeing triatomines, had collective animal housing around the property, or lived or slept in substandard housing. five case - patients reported a lack of knowledge of chagas disease by their primary care physicians. some case - patients were provided with misinformation, reporting having been told that their screening test result must be false positive because they had no travel history. furthermore, only 2 case - patients were offered treatment before enrollment in the study. one case - patient reported that, despite seeking treatment for > 1 year, he was unable to find a physician able and willing to help. this finding is particularly problematic given that a large proportion (6 of 11) of this cohort had abnormal ecg readings possibly attributable to chagasic cardiac disease. although precise cardiac etiologies could not be determined, prevalence of ecg abnormalities was higher than that for population - based studies (13,14) ; common findings included atrioventricular block and left axis deviation (table 1). a previous report also highlighted the same lack of physician awareness of chagas disease in texas, despite patients having positive serologic screening results and cardiac manifestations (8). given the low level of participation of seropositive blood donors, results of this study are limited to persons who participated and might not represent the larger texas blood donor population or general population. also, because a 7-year span separated initial screening and enrollment in this study, it is difficult to identify why 3 persons who were initially positive by blood bank screening had discordant results during the study. at follow - up, participating persons were tested with available centers for disease control and prevention assays, food and drug administration approved screening, or supplemental tests. our study adds 11 cases of likely domestically acquired t. cruzi infection to the increasing body of evidence for autochthonous chagas disease transmission in the southern united states. combined with previous studies indicating a high rate of t. cruzi infection in triatomine vectors and mammalian reservoirs in this area, our study shows that south central texas could be a focal point for endemic disease transmission (7,15). we also identified a major knowledge gap for chagas disease, which highlights the need for enhanced public health campaigns targeting clinicians and the general population in south central texas. | chagas disease, caused by trypanosoma cruzi, is a major neglected tropical disease affecting the americas. the epidemiology of this disease in the united states is incomplete. we report evidence of likely autochthonous vectorborne transmission of t. cruzi and health outcomes in t. cruzi seropositive blood donors in south central texas, usa. |
in most sexually reproducing species, anisogamy presents a dichotomy in reproductive investments, where smaller and more numerous male gametes must typically compete for the fewer and larger female gametes. males therefore typically compete for additional mating whereas females typically allocate their resources into offspring production and care. this provides opportunities for more intense selection on males [1, 2 ], and male variations enhancing their own fitness will be under selection, even if it is costly to females. in such cases, sex specific selection can lead to accumulation of mutations beneficial to the males but detrimental to females [36 ]. as a result, sexual conflict arises due to differences in reproductive interests and investments between the sexes and can translate into differences in fitness optima between sexes for a given trait. in addition, the nature of sexual conflict may depend on the extent to which one sex 's action pushes the other away from its fitness optimum. it has been known for a while now that the very act of mating, in many cases, is often associated with some form of physical and/or physiological harm to females, as a result of male reproductive strategies [7, 8 ]. there is evidence of male induced harm to female which ranges from forced mating in flies, butterflies, fish, frogs, birds, and primates [912 ] to traumatic insemination in bedbugs. studies from fruitflies, butterflies, and worms show that even if males do not directly (physically) harm females, molecular components of the male ejaculate contribute to the reduction of female 's lifespan [1416 ]. these observations reveal that males have evolved a variety of means to increase their own fitness. the widespread evidence of mating induced harm to females is compelling enough to expect, under the sexual antagonistic coevolution theory, that the most adversely affected sex (females) would counter - adapt to minimize the loss of fitness due to manipulations of the opposite sex [4, 17 ]. it would appear, however, that such expectations are not pervasive and depend not only on the extent of the costs and benefits of male manipulations, but also on the mechanisms available to female to counter respond (see for a discussion). a better appreciation of this issue therefore requires a closer inspection of the fitness consequences of male and female life history. in this paper we examine the sex specific costs of mating in closely related species of the d. melanogaster subgroup and further explore fitness contributions of the sexes within d. melanogaster, with a specific focus on male life history traits. drosophila melanogaster has played a key role as a model organism in which sexual conflict has been well studied. in d. melanogaster, males are quite aggressive and persistent in their attempts to court females enhancing their chances of securing multiple mates [2, 20, 21 ]. however, female fecundity and lifespan are reduced as a result of the aggressive and persistent courtship attempts of males [22, 23 ]. at the postmating stage, seminal fluid proteins are used by males to gain precedence over sperm from other males for fertilization [2426 ], effectively transferring a part of male - male competition to take place within the females ' reproductive tract. some of these seminal fluid proteins also influence female ovipositional behavior and physiology [27, 28 ], and the toxicity of some seminal fluid proteins involved in intrasexual competition also reduces a female 's lifespan [2932 ]. these negative effects of male reproductive life history on females are exacerbated in experimental manipulations where females are not allowed to coevolve with males. on the other hand, experimentally enforcement monogamy not only alleviated the costs but also increased fitness of the flies [6, 33 ]. although d. melanogaster females are known to remate, they do not appear to accrue much benefits of remating. costs of mating have also been explored to some extent in d. simulans, but absence of similar studies in d. mauritiana and d. sechellia precludes inferences of whether the pattern of mating costs is common across these closely related species. in other insect species, despite any fecundity benefits that females may receive from mating multiply [35, 37, 38 ], their reduced lifespan compared to males has obvious implications on sex specific lifetime reproductive success. essentially, since males typically live longer, they may stand to gain from additional mating and contribute relatively more to population fitness. by this measure it is possible therefore, in the long run, that the continual lower fitness of one sex can impose a load on population fitness [39, 40 ]. indeed, simulations [41, 42 ] and selection experiments indicate that sexual conflict and sexually antagonistic selection can reduce overall population fitness, thereby imposing additional costs on sexual reproduction. therefore, while sexual conflict may be responsible for the differential fitness optima of the sexes, the maintenance of such a system and the potential manifestation of sexually antagonistic coevolution may come into question, especially if the costs imposed by the manipulating sex outweigh any gains in the affected sex. one may expect that natural selection would favor a sexual system where both sexes contribute equally to population fitness or should at least resolve the conflict to minimize the difference in fitness optima between the sexes. therefore, how does natural selection deal with the reduction of female lifespan in every generation in drosophila ? with respect to population fitness, the loss of female fitness must either be invisible to natural selection or be compensated in some other way. one possibility is that, despite the potential of sexual conflict, males may not be pushing females beyond their threshold fitness optima, and any loss of fitness does not significantly affect population fitness. in this case, even though the potential for sexually antagonistic coevolution may exist, the fitness differential is not sufficient enough to elicit counter adaptations by females [17, 45 ]. the other possibility is that population fitness lost due to female deaths may be compensated or minimized in other ways. here, male life history may become important. males not only initiate sexual interactions ; they gain from multiple matings and they generally outlive females. as a consequence of reproductive longevity, males typically have more opportunities to mate compared to females. for instance, in species with overlapping generations like drosophila or humans, not only can males of any generation mate with surviving females of their own generation, but, due to their longevity, they can mate with females of successive generation as well. by this reasoning, males have more opportunities for fitness gains and therefore make greater fitness contributions to overall population fitness, relative to females. therefore we suspect that, despite any differences in fitness optima of the sexes in lifetime reproductive success, the overall population fitness may not be reduced beyond a threshold for natural selection to act against. we illustrate our argument through mating experiments and fitness assays in d. melanogaster, d. simulans, d. mauritiana, and d. sechellia species complex. these members of the melanogaster subgroup have radically different life histories and ecology [36, 46 ] which may influence the nature of sexual selection. we then conducted a series of assays in d. melanogaster in order to highlight the fitness consequences of mating for the sexes, with an emphasis on the consequences (costs imposed and fitness gained) of male life history. our results show that (1) mating induced reduction of female lifespan is common across d. melanogaster and simulans group ; (2) although exposure to multiple males is in general detrimental to females, it has little effect on males across species ; and (3) quite interestingly, in our assays with d. melanogaster, older males were competitive with younger males with respect to mating success. we discuss these results in the light of male driven sexual selection and its relative importance in the evolution of male traits, particularly those that are used to manipulate their mates. rather than proposing a mechanism for conflict resolution, our intention here is to bring to attention the importance of multiple ways in which males gain fitness compared to females and the observation that male reproductive longevity enables additional transgenerational contribution to population fitness. we used individuals from an outbred laboratory drosophila melanogaster strain that was established by crossing 6 different geographical strains. d. simulans (0251.2), d. mauritiana (0.248.1) strains were obtained from the tucson drosophila stock centre. virgin females and males were collected at emergence under light co2 anesthesia and housed separately and aged for 48 days on cornmeal and molasses medium. in experiments that involve several treatments and replicates, each treatment was done in separate vials that were carefully labeled and dated in order to facilitate tracking of flies through the experiments. in addition, flies were carefully aspirated into vials to ensure no injuries due to handling. this experiment was designed to highlight the potential deleterious effects of increasing male density on both female and male longevity, as well as on female fertility. we performed the experiment on d. melanogaster, d. simulans, d. mauritiana, and d. sechellia to test for a potential species effect. four different treatments consisting of one female housed with an increasing number of males (1, 3, 6, or 9 males) for her entire lifetime were initiated in separate vials. in all the treatments males were not renewed and therefore subject to ageing. a minimum of 20 replicates per species and treatments were initiated. every 5 days, for each treatment, and until all females and males died, living females and males were counted and transferred into new vials. the old vials were retained and the number of progeny that emerged from each vial was recorded. the aim of this experiment was to assess how female longevity and fertility are affected by male exposure for brief periods of time in d. melanogaster. treatment 1 consisted of females mated with males only once, and their exposure was restricted to only other mated females subsequently. treatment 2 consisted of females mated once and exposed to males once every seven days during their entire lives. control 1 was composed of virgin females that had never been exposed to males and control 2 included females housed with males for the entirety of their lifetimes. the females were exposed to the males for five hours. at the end of the exposure, any dead female was removed from the treatment vial and the treatment, replicate number, and date of death were recorded. all surviving females were transferred to new vials every five days to avoid larval overcrowding and vial contamination. to control for the deterioration of females during anaesthetization, all replicate vials for all treatments and controls were exposed to co2 as the start of each exposure. we observed that males outlived females to such an extent that it becomes possible for males to mate with females of successive generations. we therefore performed mate choice assays in d. melanogaster, to assess if aged males are competitive with younger males with respect to mating success. newly emerged males and females were collected from the base population and maintained in vials at a density of 3040 flies per vial with 50 : 50 ratio of males to females. two days prior to the mating experiments, males were separated from females and housed individually in separate vials. in this manner we collected males aged 5, 10, 15, 20, and 25 days that had mating experience of 3, 8, 13, 18, and 23 days, respectively. virgin females from the base population were collected within 3 hours after eclosion and aged for five days for mating experiments. pairs of 5-day - old versus 10-, 15-, 20-, and 25-day - old males were established. one male from each pair was marked with a notch on the wing to allow for identification. even though wing clipping has not been found to interfere with mating success in drosophila, we ensured that paired treatments within a set were reciprocally marked for half the treatments and tested for any significant effect of marking on male behavior. each male pair (young and old) was introduced into a vial with a female. a trial was retained for statistical analysis only if both males courted the females. in a separate experiment, we measured the number of progeny produced by 5-day - old males (mating experience of 3 days) versus 25-day - old males (mating experience of 20 days) in order to gain an idea of the differences in fitness contributions between young and old males. an analysis of deviance (survival package for r) was conducted to test for the effects of treatments, sex, and species on longevity. interactions between the different factors were also included using the following model : longevity ~ treatments + sex + species + treatments sex + treatment species + sex species + treatments sex species. we used the same model without the species factor to assess the effect of male exposure to female longevity. we used a chi square test with a single degree of freedom to test for deviation from random mating in the mating test experiments. in order to determine how increasing male / sex ratio (females exposed to increasing number of males) affects female fitness, we investigated the effect of increasing male density on both male and female viability (longevity) as well as on female fecundity across four closely related species of the drosophila melanogaster subgroup (d. melanogaster, d. simulans, d. mauritiana, and d. sechellia). we found a strong difference in longevity between the sexes (p = 8.378 10) across species (p = 5.109 10 ; figures 1 and 2, table 1). we also observed significant interactions between treatments (male density) and sex (p = 5.068 10 ; table 1) and between sex and species (p = 9.334 10), as well as between treatments, sex, and species (p = 8.888 10). interestingly we found no significant interactions between species and treatment (p = 0.2). in summary, these results show that while all four species respond similarly to increasing male density, the sexes across species appear to respond differently. we therefore separated the analysis of longevity of each sex in order to gain a better understanding of the effect of increased male density on the longevity of each sex. we found a significant treatment effect on female longevity ; the greater the male density the shorter the female longevity across all four species (p = 3.497 10 ; figure 1, table 1). in contrast, increasing male density did not affect male longevity at all (p = 0.2 ; table 1). however, we observed a species effect on male longevity as d. melanogaster and d. simulans males have shorter average life expectancies than d. mauritiana or d. sechellia males (33.34 24.64 days and 39.07 16.29 days versus 70.26 32.43 days and 60.09 20.96 days, resp.). this may reflect natural variation or laboratory selection for shorter lifespans in the former species. in this experiment however no new males were added to the vials in order to replace old or dead males ; it is therefore possible that the male / female ratio change across time could have affected female longevity. in order to test for such an effect, we performed the analysis of deviance again including the number of living males at female death within treatments ; these tests did not alter our conclusions (p = 5.680 10 and p = 1.874 10, resp.). due to the significant relationship between longevity and the amount of offspring per female (r = 0.503, p < 2 10), we used the residuals from the regression between longevity and number of offspring to test the effect of male density on offspring production. using such a correction, we found no significant effect of male density on female fecundity (f3,358 = 1.7538, p = 0.1557) but we did observe a significant difference in female fecundity between species (f3,358 = 10.5014, p = 1.231 10). the difference here lies mainly in the slightly higher fecundity in d. melanogaster relative to the other three species (figure 3). we conducted further tests of the effect of male exposure on female longevity and fecundity in d. melanogaster. in this experiment, rather than housing females and males together continuously, we assessed the effect of the frequency / periods of male exposure on female fitness. overall, we found a significant effect of treatment (p = 1.21 10 ; figure 4) and no difference between replicates within treatments (p = 0.1167). after bonferroni correction, we found that virgin females in control 1 outlived mated females from all the other treatments as well as control 2 (p < 0.05 ; figure 4). in contrast, females exposed only once to males had a shorter lifespan than virgin females but lived significantly longer than females exposed once every three days to males, or compared to females continuously housed with males (p < 0.01 in all comparisons). there is, however, no difference between females exposed only once to males in their lifetime and females exposed to males once a week (p = 0.493 ; figure 4). overall, these results show that multiple mating decreases female lifespans significantly but the effect is not linear with the number of males courting the female. although females mated once in their lifetime and never exposed to males afterwards live longer than females from most of the other treatments, we found a significantly lower number of offspring from this treatment compared to females with a greater exposure to males (treatments 2 and 3, p = 0.02162 and p = 0.02334, resp., after bonferroni correction) and females housed with males (control 2, p = 0.01246 after bonferroni correction, figure 5). in the first experiment above we observed that under conditions of biased sex ratio males outlived females to the extent that it raised the question of the propensity of males to mate with females from successive generations as they age. in order to test this hypothesis we performed mating competition assays in d. melanogaster, between 10-, 15-, 20-, and 25-day - old mated males and 5-day - old virgin males. within each group we found no significant difference in mating success between 5-day - old males versus 10- (p = 0.8, n = 60), 15- (p = 0.1, n = 65), 20- (p = 0.2, n = 62), or 25-day - old (p = 0.7, n = 61) males. males were marked with notched wings and of the 248 matings scored, 113 successful males had clipped wings and 135 were with nonclipped wings. these differences are not statistically significant (p = 0.16) according to fisher 's exact tests, confirming that notching had no significant effect on mating success. we also looked for any difference in the number of offspring produced by younger males (5 days old) or older males (25 days old), as a measure of their potential fitness contributions. although younger males produced, on average, a higher number of offspring compared to older males the latter did quite well (p = 0.013 ; mean 95% ci : young = 312.8 21.1, old = 228.7 14.3, n = 25). one of the consequences of male mating strategies in d. melanogaster is the reduction of female lifespan, either due to harassment during courtship, or physical trauma during mating, or as a result of toxic seminal fluid proteins transferred during copulation. whether or not these sex specific fitness consequences are common across drosophila species in a phylogenetic context has not been tested. such studies are useful to test the generality of any condition across species in a taxonomic group. our results show that sex specific fitness costs of mating are common and similar across species of the d. melanogaster, d. simulans clade ; females suffer lifespan reduction costs ; males are unaffected. while the evolution of such antagonistic traits has been attributed to female choice or sexual conflict [3, 15, 51 ], little has been discussed with respect to the consequences of unequal lifespans of the sexes, on net population fitness (but see [52, 53 ]). this issue may be relevant to determining whether or not sexually antagonistic coevolution will take place [17, 54 ]. our assays conducted with d. melanogaster suggest that, despite a reduction in their lifespan, females do benefit from a certain number of increased matings. quite importantly, our results suggest that male reproductive longevity provides opportunities for additional gains of fitness by males, which can benefit populations. darwin had noted that males, in a variety of animals, are almost always the active seekers and initiators of sexual interactions, using a variety of means, from song and dance, to coercion. these male behaviors are likely to influence the manner in which females respond to male sexual behaviors, thereby affecting the pattern, intensity, and direction of sexual selection or conflict in populations. indeed, recent research focusing on male life history [56, 57 ] is beginning to shed more light on the interrelationship between life history and sexual selection / conflict. drosophila melanogaster males seem to exercise choice by adjusting their ejaculate size based on female status ; they also adjust the nature of ejaculate to manipulate female behaviour and physiology [59, 60 ] and to compete with rival males. such studies and our present study exploring the role of male life history will be important in testing traditional ideas that males are indiscriminate and tend to live fast and die young [62, 63 ] and the assumption of the twofold costs of sex that males typically do not contribute more than their gametes. below, we examine our results in the perspective of male driven sexual selection (see [65, 66 ]) to highlight that not only is the fitness differential between sexes in lifetime reproductive success a male driven phenomenon but also the additional gains of lifetime reproductive fitness by males might render the loss of female lifetime reproductive fitness to evolve as a nearly neutral trait, preventing the expression of sexually antagonistic coevolution. our results extend what has previously been shown in d. melanogaster [23, 6769 ], and in d. simulans, to be common across all four related species of the melanogaster - simulans clade. this result is important in showing that, despite radically different ecologies and life histories, the mating strategies and costs of mating to females are similar (figure 1). d. mauritiana and d. sechellia are island endemics (to mauritius and seychelles, resp.). amongst the four species, d. sechellia has a distinctly different life history by having specific ecological and physiological adaptations to the morinda citrifolia plant. compounds found in the fruit of this plant are toxic to the other three species. apart from longevity differences between d. melanogaster and d. simulans compared to d. mauritiana or d. sechellia (see section 3) we found no major differences on mating outcomes of the sexes ; female longevity across species is significantly reduced with increasing and continuous exposures to males. however, intermittent exposure to fewer males was found to be less harmful to d. simulans female lifespan (figure 1, also see) as well as for d. melanogaster (this study). in fact, it is quite interesting that the results from assays using d. melanogaster alone (figure 4, see below) and taylor. 's study in d. simulans suggest that the optimal number of males and matings that is least harmful to female lifespan and maximizes female fitness (progeny produced) in these cosmopolitan species appears to be three (figures 4 and 5). females exposed to six or nine males suffered greater reduction of lifespan and produced less progeny. it is also quite interesting to see that the two cosmopolitan species appear to benefit somewhat from polyandry, in terms of fecundity (figure 3), but this is not true for the island endemics that appear to benefit more from monogamy (figure 3). these differences should however be treated with some caution because d. melanogaster was an outbred population compared to the isofemale lines of the members of the simulans complex (see section 2). further research, particularly in wild populations, will be useful in validating our results and speculations regarding species differences in fecundity. on the other hand our results show that increasing male density has no negative effects on male longevity across all species (figure 2). direct male - male interaction may be an important determinant of sexually selected traits in drosophila males and it may be expected that intense male - male competition is more likely in high male density conditions and is therefore detrimental to males (e.g., see). the lack of such a result is interestingly viewed in the light that male - male competition in drosophila may largely occur via sperm competition within the female 's reproductive tract [4, 30, 69 ]. one of the consequences of this male - specific evolution is the reduction of female lifespan due to toxicity of some seminal fluids. assays done in d. melanogaster to study the effect of temporal exposure of females to males show that the optimum number of matings in terms of number of offspring produced is greater than one. females mated only once produce a lower number of offspring compared to females exposed to males once a week, even though females in both treatments have comparable life expectancies (figure 5). these results are consistent with several other studies across insects, which indicate that, despite numerous deleterious effects that may be linked to remating, there are nonetheless many direct and indirect advantages to female fitness [37, 38, 7274 ]. females can benefit from remating through nuptial gifts, increased postcopulatory feeding, and increased resistance to starvation and desiccation which can in some cases outweigh male induced harm [7577 ]. in addition, it is noteworthy that male seminal fluids influence females ovipositional behavior and physiology in d. melanogaster, which may contribute indirectly to increasing female fitness despite the costs to lifespan. these results may warrant a speculation that difference in fitness optima between the sexes, with respect to lifetime reproductive success, may not be sufficiently large enough for natural selection to act against it, such that sexually antagonistic coevolution may not take effect. however, it is important to note that our results of a narrow range of matings (~3) that minimizes this fitness differential (figure 5) may be an underestimation, perhaps typical of laboratory conditions. studies in natural populations of d. melanogaster using microsatellite markers to determine paternity of progeny from females captured in the wild reveal that four to six remating occurrences may be more typical in the wild. in addition, there is some evidence that mated females may live longer than virgins in nature. this may be, as taylor. surmised, because females in nature are not continuously exposed to males, as in a laboratory setting. more studies in the typical number of matings that occur for males and females in natural populations will be needed to shed additional light on this issue. not only do our results show longer life expectancy of males in all species (figure 2), but, further exploring the relevance of this result in d. melanogaster, we found that males as old as 25 days have the similar probability of mating success compared with much younger males. longer male reproductive lifespan is quite interesting in the context of some other factors, which bring to light the reproductive potential of older males and their potential to contribute positively to population fitness. some lines of evidence suggest the propensity of older males to mate with younger females in drosophila. in the wild, males have been observed to patrol pupal sites in order to mate with teneral females. older males may benefit from the fact that younger d. melanogaster females are either less choosy or are not as efficient in rejection behaviors as older females [81, 82 ]. in fact, saleem. have shown that older males typically outcompete younger males in courtship and mating. in d. bipectinata all of these data, along with our results of comparable mating success between young and older males, suggest the potential of males to mate with females of different generations. however, edward and chapman found that the number of offspring sired by males declines with age. this is also true in our study ; younger males sired, on average, more offspring. however, males as old as 25 days are capable of producing offspring, and since they are likely to have mated (perhaps several times) in their earlier days, even a single or a couple of extra matings count as added contributions to population fitness. therefore, even if females typically die early as a result of mating (or multiple mating), older males can mate with younger females, many of whom are likely to belong to successive generations. this creates a bias in the effective population size of males relative to females, which can increase male reproductive variance relative to that of the females. indeed, observations on natural populations suggest the existence of such a reproductive asymmetry in nature. there is a well - described excess of polymorphism on the x chromosome relative to the autosomes among african populations [67, 85, 86 ]. aside from the effects of population expansion, which are less likely for the ancestral african populations [87, 88 ], increased reproductive variance of males relative to the females is the main explanation for such imbalance [89, 90 ]. our observations were made in controlled laboratory conditions and experiments simulating natural conditions need to be performed ; it is unlikely that the extent of female death in closed laboratory settings can be extended to nature, where females have ample opportunity to escape males. on the same note, it is also intriguing that selection against female death has not operated on laboratory drosophila populations. it is certainly not true that all mated females suffer similar mortality rates ; that is, there is genetic variation underlying the variation in female mortality. this variation represents a substrate that natural selection can operate on to select against factors reducing female fitness. intergenerational mating creates a unique situation that can offset at least some if not all costs due to early female mortality ; even though females ' lifespan is reduced due to mating, there are younger females that males can mate with. as a result, loss of fitness due to deaths of mated females will be inconsequential in the scheme of natural selection. this situation can have broad implications on the life expectancy of the sexes (see [52, 53 ]). for instance, bonduriansky has suggested that female life expectancy may play a major role in how the sexes coevolve, based on the intensity of sexual conflict and extrinsic factors that may affect female mortality. for instance, in high female mortality conditions (harsh environments or predation), females stand to benefit from male mating strategies, including those that are costly to them. on the other hand our study would imply that male mating strategy is also a factor that shapes female life expectancy, and early female demise obstructs the ability for females to evolve counter adaptations to male 's harmful effects. this would fit a prediction where the potential for sexually antagonistic coevolution may exist, but it is not realized. indeed, reproductive longevity of older males and preference for younger females have been suggested to have contributed to the increase in longevity in humans. the result of older male contributions will require further detailed study due to the broad implications of reproductive longevity of males and shortened life expectancy of females. all else being equal, the fisherian principle of sex ratio evolution would in theory predict that natural selection should favor a sexual system where both sexes contribute equally in every generation ; however everything is generally not equal in sexual organisms. our study addresses the fitness inequality between sexes that arises as a result of male - female competition in mating / remating and provides a possible explanation as to how many additional fitness contributions of males may be able to compensate or at least minimize the fitness lost due to early female deaths. as a result the fitness differential between sexes may not be sufficiently large enough to create a load on populations such that natural selection will act against it. the sexual disparity in fitness caused by individual male - female competition, wherever it occurs, would not matter as long as population fitness is not affected. additional studies on the relevance of male reproductive longevity and female benefits of remating will be useful in further tests of our proposition. | males have evolved a variety of behavioral, morphological, and physiological traits to manipulate their mates in order to maximize their chances of success. these traits are bound to influence how females respond to male behaviors and influence the nature of sexual selection / conflict. a common consequence of aggressive male mating strategies in drosophila melanogaster is the reduction of female lifespan. our study shows that this is common across members of the simulans clade. reduced life expectancy of females implies that female contribution to a population is less than that of males per generation. fitness differences between the sexes in every generation will invariably affect overall population fitness. how natural selection responds to the female deaths and thereby the unequal fitness of the sexes has rarely been addressed. we shed light on this issue and provide evidence, which suggests that additional gains of fitness by males due to their longevity and continued mating may provide one explanation as to why the loss of female fitness may be invisible (effectively neutral) to natural selection. male driven sexual selection and additional, transgenerational gains of male fitness can be an important force of evolutionary change and need to be tested with other organisms. |
molybdenum (mo) was discovered by swedish chemist peter jacob hjelm in 1781, and its radioactive isotopes are made using light - particle reactions, fusion - evaporation reactions, neutron- and charged particle - induced fission, and projectile fragmentation or fission (advameg 2015 ; parker and thoennessen 2012). typically generated radioactive isotopes include technetium (tc) in the isotopes of tc, tc (m + g), and tc, with tc being one of the most important. the letter m means that the isotope is metastable, indicating the short half - life of this isotope (e.g., tc has a half - life of approximately 6 h) (parker and thoennessen 2012). the short half - life prevents the isotope from being stored for a long period of time ; therefore, continued production is necessary. any difficulties in the supply due to the limited number of facilities would result in an unplanned interruption of patient care. some research has been conducted on the production of tc without the use of mo (stolarz. these processes use uranium, and the amount of radioactive waste produced is greatly reduced (barci - funel. 1993 ; pagdon. 2011). however, because this technique is under development, it is unlikely to occupy a significant share of the market in the short or medium term. the advantages of tc are the short biological and physical half - life that enables very fast clearance from the patient s body and the emission of a single gamma that is not accompanied by any beta emissions. today, tc is considered one of the most required medical isotopes ; 30 million patients per year use it worldwide. the approximate worldwide demand for mo used to produce tc is approximately 450 tbq weekly (aipes 2008). technetium-99 m (tc) is vital for certain diagnostic tests for cancers and heart diseases ; it is used in greater than 80% of nuclear medicine s diagnostic procedures across the world (wna 2015). examples of the types of diagnoses for which tc is used include the diagnoses of metabolic bone disease (fogelman. 1978), renal tubular acidosis (caglar and topalolu 2002), and methoxy isobutyl isonitrile (mibi) imaging in patients with malignant tumors (aktolun. 1992). the ministry of health (moh) in the state of kuwait currently imports this radioisotope (i.e., mo) to fulfill its demands and faces the risk of the shortage or even the stoppage of supply due to different scenarios ; i.e., the sources are shut down or an accident occurs with the cargo transferring the supply. the kuwait institute for scientific research (kisr) is currently supporting its national commitment to the international atomic energy agency (iaea) through a technical cooperation (tc) project with the iaea titled supporting the establishment of a nuclear research center, iaea - tc (kuw/1/006). as part of these collaborative efforts, it would be practical to assess the possibility of producing mo via different methods using a neutron - generating facility (ngf) and determining the best route of production that can meet the moh s demands in kuwait by considering future demands. the current research assists decision makers in kuwait s moh in predicting the future demands for tc by collecting and analyzing the consumption data of tc for different diagnostic applications from the moh s nuclear departments current and future change in tc demands is evaluated by collecting tc consumption data from all nuclear medicine departments in kuwait from 2012 to 2014 ; forecasting missing data using various methods, such as moving average and a linear trendline for backwards, mid - point, and forward data ; and forecasting kuwait s future total tc consumption using a logarithmic trendline with a seasonal index. the average percentage of error for the forecast data is calculated to determine the level of acceptance of the predicted results. the data were collected from 2012 to 2014 from nine moh nuclear medicine departments : al - amiri hospital, ibn sina hospital, kuwait cancer control center (kccc), al - farwaniya hospital, mubarak al - kabeer hospital, yiaco medical company, al - jahra hospital, jaber al - ahmad hospital, and chest diseases hospital. the consumption dosages from al - amiri hospital and mubarak al - kabeer hospital were provided as the total tc consumption of different imaging tests per day only. the imaging tests performed at these departments include tin colloid, thyroid, mibi, methylene - diphosphonate (mdp), microaggregated albulin (maa), testicular, dimercaptosuccinic acid (dmsa), myo view, hepatobiliary iminodiacetic acid (hida), mercaptoacetyltriglycine (mag3), hmpo, leukoscan, and diethylenetriamine - pentaacetic acid (dtpa). in addition to the two previous nuclear medicine departments, all data provided from the moh were in terms of the daily and weekly consumption dosage of tc. the collected data from al - amiri hospital contained many missing values for the year 2012 ; therefore, the year 2012 dosage was instead predicted using a linear backward trendline for all four quarters of the year. the data of the first quarter of 2013 for ibn sina hospital exhibited an outlier ; therefore, an average of the weekly consumption dose of the first quarter was used instead. for the same hospital, data from the fourth quarter were missing at the time the project was executed. the dosage for the fourth quarter of 2014 for kccc was not available at the time of data collection ; therefore, this information was predicted using the moving average method with four periods. the dosage for the last two months from the fourth quarter of 2014 for al - farwaniya hospital was not available ; therefore, this information was predicted using an exponential trendline with a seasonal index. the dosage for the last month of 2014 was not available for the mubarak al - kabeer hospital ; therefore, these data were forecast using the moving average method with two periods. due to unavailable data, the data for the third and fourth quarters of 2014 were forecast for the yiaco medical company using a linear trendline with a seasonal index with a 10% safety factor to reduce the error. al - jahra hospital had missing data for january and june 2014 ; these values were assumed to be the average of the summation of the previous and following months. the missing data for february and november 2013 were predicted using the moving average method with two periods. the data for the fourth quarter of 2014 were not available at the time, so they were forecast using the moving average method with four periods. the jaber al - ahmad hospital data for the first two quarters of 2012 were predicted using a backward linear trendline with a seasonal index to fill in the missing values. for the last two quarters of 2014, a linear trendline with a seasonal index was used to provide the missing values. finally, year 2014 data for the chest diseases hospital were forecast using a logarithmic trendline with a seasonal index because they were not available at the time. table 1 presents kuwait s overall tc consumption for the period from 2012 to 2014 based on the information mentioned previously and the data gathered from all stated nuclear medicine departments. the values represented in bold are the predicted values, and the remaining values represent the real data collected from moh. the consumption of tc in kuwait and predict future changes in consumption, the data obtained in table 1 were plotted and then forecast logarithmically with a seasonal index (fig. the average percentage of error for the forecast data is 2.4%, providing a good indication that the result obtained is acceptable. 1 that the consumption rate is decreasing slightly every year. because tc is used for patients, a 5% safety percentage was added to the forecast values to prevent a shortage of tc in the moh. table 2 presents the future predicted doses that are recommended for use by the moh. the data were collected from 2012 to 2014 from nine moh nuclear medicine departments : al - amiri hospital, ibn sina hospital, kuwait cancer control center (kccc), al - farwaniya hospital, mubarak al - kabeer hospital, yiaco medical company, al - jahra hospital, jaber al - ahmad hospital, and chest diseases hospital. the consumption dosages from al - amiri hospital and mubarak al - kabeer hospital were provided as the total tc consumption of different imaging tests per day only. the imaging tests performed at these departments include tin colloid, thyroid, mibi, methylene - diphosphonate (mdp), microaggregated albulin (maa), testicular, dimercaptosuccinic acid (dmsa), myo view, hepatobiliary iminodiacetic acid (hida), mercaptoacetyltriglycine (mag3), hmpo, leukoscan, and diethylenetriamine - pentaacetic acid (dtpa). in addition to the two previous nuclear medicine departments, all data provided from the moh were in terms of the daily and weekly consumption dosage of tc. the collected data from al - amiri hospital contained many missing values for the year 2012 ; therefore, the year 2012 dosage was instead predicted using a linear backward trendline for all four quarters of the year. the data of the first quarter of 2013 for ibn sina hospital exhibited an outlier ; therefore, an average of the weekly consumption dose of the first quarter was used instead. for the same hospital, data from the fourth quarter were missing at the time the project was executed. the dosage for the fourth quarter of 2014 for kccc was not available at the time of data collection ; therefore, this information was predicted using the moving average method with four periods. the dosage for the last two months from the fourth quarter of 2014 for al - farwaniya hospital was not available ; therefore, this information was predicted using an exponential trendline with a seasonal index. the dosage for the last month of 2014 was not available for the mubarak al - kabeer hospital ; therefore, these data were forecast using the moving average method with two periods. due to unavailable data, the data for the third and fourth quarters of 2014 were forecast for the yiaco medical company using a linear trendline with a seasonal index with a 10% safety factor to reduce the error. al - jahra hospital had missing data for january and june 2014 ; these values were assumed to be the average of the summation of the previous and following months. the missing data for february and november 2013 were predicted using the moving average method with two periods. the data for the fourth quarter of 2014 were not available at the time, so they were forecast using the moving average method with four periods. the jaber al - ahmad hospital data for the first two quarters of 2012 were predicted using a backward linear trendline with a seasonal index to fill in the missing values. for the last two quarters of 2014, finally, year 2014 data for the chest diseases hospital were forecast using a logarithmic trendline with a seasonal index because they were not available at the time. table 1 presents kuwait s overall tc consumption for the period from 2012 to 2014 based on the information mentioned previously and the data gathered from all stated nuclear medicine departments. the values represented in bold are the predicted values, and the remaining values represent the real data collected from moh. to further evaluate the consumption of tc in kuwait and predict future changes in consumption, the data obtained in table 1 were plotted and then forecast logarithmically with a seasonal index (fig. the average percentage of error for the forecast data is 2.4%, providing a good indication that the result obtained is acceptable. 1 that the consumption rate is decreasing slightly every year. because tc is used for patients, a 5% safety percentage was added to the forecast values to prevent a shortage of tc in the moh. table 2 presents the future predicted doses that are recommended for use by the moh. the moh in kuwait has recently enforced the recording of all dosages of tc used at its nuclear medicine departments. because this practice is new, data are available only from the year 2012 and beyond in the form of handwritten documents, which has led in some cases to missing data and lost documents. thus, to conduct this study, the missing data had to be predicted using different forecasting methods. although the overall result showed an error of only 2.4%, more data are required to obtain more realistic and reliable results. the overall result indicates that there will be a partial decrease of 1.012% in the overall total demand for tc from 2015 to 2018. this result therefore indicates that the consumption rate in kuwait is roughly constant or that the assumed values for the missing data and the high consumption rate in 2012 affected the results. the radioactive isotope tc is a radioactive isotope produced from the decay of mo, with a short half - life of 6 h. as such, it is useful for medical imaging for the diagnoses of various ailments. its emission can be detected by imaging equipment used by hospitals to produce clear and accurate images of defects within a human body. it is used in more than 80% of nuclear medicine s diagnostic procedures across the world, as it is vital for diagnostic tests for several cancers and heart diseases. the ministry of health in the state of kuwait currently depends on importing mo to meet its needs. as such this is done by gathering the consumption data of tc from all nuclear medicine departments in kuwait, predicting missing data using different statistical approaches, and predicting the future total consumption of tc. the predicted total tc consumption in kuwait has shown a reduction of 1.012% for the upcoming years with an average error of only 2.4%. this can be due to many reasons, mainly missing data, high consumption during the 2012 period, and human error. | abstractthe ministry of health (moh) in the state of kuwait currently depends on importing the radioisotope molybdenum (mo) in its isotopic form (99mo) to fulfill its demands. the present study was conducted on all nuclear medicine departments in the state of kuwait. daily, weekly, and monthly data were analyzed to statistically determine the current and future demands for the isotope 99mtc. this analysis was performed by collecting and analyzing data on moh consumption of 99mtc for different diagnostic applications. the overall results indicate a partial decrease of 1.012% in the overall total demand for 99mtc up to the year 2018 for the state of kuwait. |
hibernoma is a rare, benign, slow - growing tumor of brown fat origin the majority occurring in the extremities and the neck. the presacral location is rare, although a few cases in the retroperitoneum have been described in the literature.[25 ] we describe a case of presacral hibernoma with radiologic - pathologic correlation. the imaging findings are nonspecific but, given the rarity of this tumor, when there is a large well - defined heterogeneous retroperitoneal mass with dilated intratumoral vessels, the possibility of a hibernoma should be considered in the appropriate clinical setting. a 36-year - old female presented with abdominal distention and dull, vague, left lower quadrant pain since 2 years. a large solid pelvic mass was found and pelvic ultrasound was requested. on ultrasound, there was an ill - defined large heterogenous mass with increased vascularity seen posterior and superior to the uterus, with poor demarcation from the uterus and the left ovary. the patient being unwilling for mri, ct scan of the pelvis was performed for further evaluation. ct scan revealed a large presacral, heterogeneous, hypervascular mass, measuring approximately 10.6 10.0 5.8 cm and causing compression and displacement of the rectum anteriorly [figure 1a c ]. this mass had multiple tortuous arterial and venous channels, with the dominant arterial supply being from branches of the inferior mesenteric artery (i m a). on microscopy, there were numerous multivacuolated adipose cells with eosinophilic cytoplasm and eccentric nuclei, consistent with hibernoma [figure 3 ]. noncontrast (a) and contrast - enhanced (b) axial ct scans through the pelvis demonstrate a large well - defined, heterogeneous, enhancing presacral mass (arrows), with dilated tortuous vessels (arrowheads). sagittal reformatted contrast - enhanced image (c) demonstrates the mass pushing the rectum (r) and uterus (ut) anteriorly. ub = urinary bladder prone, axial, plain ct scan through the pelvis, with the biopsy needle (arrow) in the presacral mass photomicroscopic image of the biopsy specimen shows numerous multivacuolated adipose cells, with eosinophilic cytoplasm and eccentric nuclei as the mass was hypervascular, preoperative embolization of this mass was clinically requested. on selective i m a catheter angiogram, there were dilated vascular branches feeding this mass, with an early tumor blush, but no arteriovenous communication was seen [figure 4a ]. selective catheter angiogram of the inferior mesenteric artery (i m a), (a) shows a feeding vessel (arrow) and early tumor blush (arrowhead). selective i m a angiogram, (b) after particle embolization of the feeding branch of the i m a shows the embolized feeding branch (arrow) with near complete loss of tumor blush (arrowhead) on gross examination, the mass was lobulated and well encapsulated, measuring 13.1 12.6 4.2 cm. the cut surface was brownish yellow in color, with multiple coursing vessels and without any areas of hemorrhage or necrosis [figure 5 ]. cut surface of the gross specimen shows the brownish - yellow color of the mass, with multiple vessels but without any areas of hemorrhage or necrosis hibernoma is a rare, benign, slow - growing tumor of brown fat origin. the tumor is named hibernoma due to its similarity to brown fat, which has a thermogenic role in hibernating animals. the tumor was first termed as pseudolipoma by merkel in 1906 and subsequently renamed hibernoma by gery in 1914. the imaging features of this rare tumor have been mostly described for lesions occurring the extremities and neck region, with only six cases of retroperitoneal hibernoma described in the english literature.[125 ] to the best of our knowledge, this is the first case report of a presacral hibernoma, describing cross - sectional ct imaging and conventional catheter angiographic feature of this tumor with preoperative vascular embolization. hibernoma is seen in the 20- to 50-year age - group, with a female preponderance. however, a large series by the armed forces institute of pathology (afip) did show a slight male preponderance. the intra - abdominal hibernoma, by itself, does not produce symptoms and as a result the tumor is often large at initial presentation, the large size of the tumor causing abdominal discomfort and mass effect on adjacent structures. the usg findings of intra - abdominal hibernoma have not been described, but subcutaneous and intramuscular hibernomas are seen as well - defined heterogenous masses with variable echogenicity ; some may appear as hypoechoic masses, with increased vascularity on color doppler flow. on ct scan, the hibernoma is seen as a large heterogeneous, well - circumscribed mass, with attenuation ranging between fat and muscle. the heterogeneity of the hibernoma is attributed to the presence of varying amount of fat, muscle, and vessels. in our case, the mass demonstrated soft - tissue attenuation, without any definite fat attenuation or necrosis. following intravenous contrast administration, the hibernoma shows heterogeneous enhancement, with hypertrophic branching intratumoral vessels as was seen in our case. retroperitoneal hibernoma causes displacement of the adjacent organs and structures but no infiltration. on mri, hibernoma demonstrates high signal intensity on t1w and t2w images ; however, the intensity is less than that of subcutaneous fat and there is incomplete fat suppression on fat - saturated pulse sequences. the differential diagnosis of a large well - defined heterogenous retroperitoneal and/or presacral mass containing large intratumoral vessels could include liposarcoma, angiolipoma, and hemangiopericytoma. liposarcoma are heterogenous soft - tissue masses with areas of necrosis, hemorrhage, and/or calcification, but without large intratumoral vessels. also, in contrast to the slow - growing well - defined hibernoma, the liposarcoma has a poor margin, with infiltration into adjoining structures. a mature teratoma classically presents as a complex mass containing fat, calcification, rarely a fat - fluid level, without intralesional vascularity. angiolipoma and hemangiopericytoma may have increased vascularity ; however, none of these lesions show both increased signal intensity on t1w and t2w images and large branching intratumoral vessels. on angiography, hibernoma demonstrates dilated feeding vessels, typical tumor blush, and early venous return, as was seen in our case. in the past, angiography was performed for evaluating presurgical vascular anatomy and also to differentiate hibernoma from hypovascular lesions like lipomas and fibromas. these days, ct and mri have superseded the role of conventional angiography in the diagnosis of such lesions. given the nonspecific features on imaging, histological diagnosis is usually required for appropriate planning before surgery. despite the increased vascularity of this tumor, image - guided core biopsy microscopically, hibernoma is composed of multivacuolated adipocytes with granular eosinophilic cytoplasm and eccentric nuclei, univacuolated cells with peripheral nuclei, and abundant capillaries. | hibernoma is a rare benign tumor of brown fat origin. it is found in locations where brown adipose tissue is present in the fetus. these locations include the neck, axilla, thorax, and extremities. retroperitoneal hibernoma is an extremely rare site of presentation. we present a rare case of presacral hibernoma. preoperative embolization of this highly vascular tumor was performed. to the best of our knowledge, this is the first reported case of a presacral hibernoma in the english literature. |
cardiovascular diseases are among the leading causes of death worldwide, with more than 2150 deaths each day. many studies have found a direct relationship between levels of low - density lipoprotein (ldl) cholesterol (or total cholesterol) and the rate of new - onset coronary heart disease (chd) in men and women who were initially free of chd. when other risk factors are present, including diabetes and hypertension, this risk increases even more. this has led to large numbers of clinical trials providing strong evidence that chd incidence is reduced in cholesterol - lowering therapy. a meta - analysis involving 10 trials of 79,494 subjects found that statins reduce coronary events, strokes, and all - cause mortality without increasing non - coronary mortality. high cholesterol is one of the major controllable risk factors for coronary heart disease, heart attack, and stroke, consequently increasing the use of statins throughout the years. it is evident that statin use has a clear benefit on stroke and total mortality by greatly reducing cholesterol. despite the positive results linking statin use and reduction in chd, studies have shown poor adherence to these medications. a systematic review and meta - analysis of 22 cohorts determined several sociodemographic, medical, and healthcare utilization characteristics these factors are useful guides for targeting statin adherence interventions, especially since adherence is an essential factor to receiving the full benefits of these medications. although there have been studies identifying predictors of adherence and non - adherence to statins, there is less evidence identifying reliable predictive variables of mortality among new statin users. in particular, the veteran population is of importance due to the prevalence of risk factors within this group. over 90% are male and more than 40% are over 65 years of age, both of which are risk factors of chd - related deaths. this study aims to identify variables in the first year of statin therapy that are predictive of long - term mortality. this retrospective cohort study used data from the veterans affairs facilities within the veterans integrated service network 22, including sites from loma linda, long beach, los angeles, san diego, and las vegas. data were collected on study subjects identified as new statin users between december 1, 2006, and november 30, 2007. socioeconomic status was determined by zip code median household income and divided into five quintiles from lowest to highest income. mpr was calculated as the number of days of statin drug supply the patient filled divided by the number of days in the specific time period. inclusion criteria included age 18 years or older, diagnosis of dyslipidemia, new statin user per the dates listed above, enrollment in the va system for at least 2 years before the study period, and eligibility to receive prescription benefits from the va. patients were excluded if they had missing baseline or follow - up body mass index (bmi), ldl, and total cholesterol (tc) laboratory data, or if they were missing zip code income data (figure 1). baseline comparisons between the mortality group and the survival group were performed using t - test and chi - square test. the primary outcome measure of mortality predictors at 6 years follow - up for new statin users were analyzed using a logistic regression model. the independent variables assessed included race, age, ethnicity, bmi, socioeconomic status, ldl outcomes at 1 year, and baseline comorbidities. all analyses were performed using spss version 18 (spss inc, chicago, il). baseline comparisons between the mortality group and the survival group were performed using t - test and chi - square test. the primary outcome measure of mortality predictors at 6 years follow - up for new statin users were analyzed using a logistic regression model. the independent variables assessed included race, age, ethnicity, bmi, socioeconomic status, ldl outcomes at 1 year, and baseline comorbidities. all analyses were performed using spss version 18 (spss inc, chicago, il). there were a total of 7735 patients who met inclusion criteria, and 4566 patients were included in the final analysis after applying the exclusion criteria. patients in the mortality group were older (p < 0.001), on more medications (p < 0.001), had higher total cholesterol levels (p 0.001) and had higher incidence of hypertension (p < 0.001), vascular disease (p < 0.001), chronic obstructive pulmonary disease (p = 0.001), congestive heart failure (p < 0.001), history of myocardial infarction (p < 0.001), and angina (p = 0.008). patients in the survival group were more likely to be hispanic (p < 0.001), with higher bmi (p < 0.001) and ldl cholesterol (p < 0.001) (table 1). age, medication count, ethnicity, and bmi were predictors of long - term mortality. in addition, patients with comorbidities such as hypertension (or = 1.46, 95% ci = 1.081.98, p = 0.015), diabetes (or = 1.36, 95% ci = 1.071.72, p = 0.012), tobacco use (or = 1.52, 95% ci = 1.161.99, p = 0.002), copd (or = 1.88, 95% ci = 1.402.53, p 0.001), and congestive heart failure (or = 1.80, 95% ci = 1.232.63, p = 0.002) all showed an increased risk of mortality. there were no significant associations between mortality and race, ldl outcomes at 1 year, and annual income level, as shown in table 2. there were a total of 7735 patients who met inclusion criteria, and 4566 patients were included in the final analysis after applying the exclusion criteria. patients in the mortality group were older (p < 0.001), on more medications (p < 0.001), had higher total cholesterol levels (p 0.001) and had higher incidence of hypertension (p < 0.001), vascular disease (p < 0.001), chronic obstructive pulmonary disease (p = 0.001), congestive heart failure (p < 0.001), history of myocardial infarction (p < 0.001), and angina (p = 0.008). patients in the survival group were more likely to be hispanic (p < 0.001), with higher bmi (p < 0.001) and ldl cholesterol (p < age, medication count, ethnicity, and bmi were predictors of long - term mortality. in addition, patients with comorbidities such as hypertension (or = 1.46, 95% ci = 1.081.98, p = 0.015), diabetes (or = 1.36, 95% ci = 1.071.72, p = 0.012), tobacco use (or = 1.52, 95% ci = 1.161.99, p = 0.002), copd (or = 1.88, 95% ci = 1.402.53, p 0.001), and congestive heart failure (or = 1.80, 95% ci = 1.232.63, p = 0.002) all showed an increased risk of mortality. there were no significant associations between mortality and race, ldl outcomes at 1 year, and annual income level, as shown in table 2. this study showed that different lipid outcomes after the first year of statin therapy were not indicative of long - term mortality. other significant predictors of mortality in new start statin users were seen after a 6 year follow - up period. age, race, number of medications, comorbidities, and bmi were shown to be predictive of long - term mortality in a veteran population, much of which was expected. statin users in the mortality groups were more likely to be taking more medications, an indicator of their underlying diseases, also predictive of mortality. previous studies have shown a highly significant negative association between statin use and mortality. in an overview of 16 individual trials including 29,000 subjects, patients assigned to statin drugs experienced significant reductions in risks of stroke and total mortality. however, the results of our study showed that actual differences in ldl levels at a given point in time among statin users appeared to confer no predictive effect on long - term mortality outcomes. patient stratification between high potent (rosuvastatin and atorvastatin) vs non - potent statins did not affect the outcome. sub - groups including hdl, tg, and tc further showed no predictive value of long - term mortality. statin doses and whether the treatment goal of ldl was attained at 1 year follow - up were not assessed in this study. the study by blackburn. reported a detectable excess of cardiovascular morbidity associated with non - adherence to statin therapy. numerous other studies have shown that a reduction in ldl is associated with a decreased risk in cardiovascular events and total mortality. these findings suggest that occurrences of cardiovascular events can be prevented with improvements in maintaining adherence in statin users. although our analysis showed no significant difference in adherence as a predictor of long - term mortality, it should be mentioned that cardiovascular outcomes were not assessed. in addition, ldl values in both survival and mortality groups suggest uncontrolled ldl levels at 1 year follow - up, which may be due to the low adherence rates to statin therapy in both groups. therefore, risk factors for cardiovascular events and total mortality including male gender, older age, vascular disease, copd, hypertension, and diabetes took the lead in the risk factor evaluation in the multiple regression analysis, despite the great differences in their baseline rates. in addition, some degree of time immortal bias may be present because no patient s died in the first year (as per inclusion criteria requiring ldl outcomes at 1 year). in addition, follow - up ldl outcomes were performed after 1 year only, extrapolation of values beyond that time point are not possible. the study was performed in a veteran population primarily comprised of male patients, limiting generalizability of the analysis. although this group is of particular interest when looking at the risk factors associated with coronary heart disease, associations between lipid variables and mortality in a broader patient population may vary. in addition, homeless veterans without a zip code were excluded from this study. according to the annual homeless assessment report (ahar) to congress from the us department of housing and urban development, there were an estimated 75,609 homeless veterans on a single night in the us in 2009. the exclusion of this population omitted patients with significant health risks not accounted for in this study. while many studies have examined the risks and benefits between statin and non - statin users, this study attempts to recognize the predictors of mortality in new start statin users. multiple independent variables have been identified as predictors of mortality in statin users after a 6 year follow - up. different lipid outcomes after the first year of statin therapy were not indicative of long - term mortality. future studies should further assess optimal lipid - lowering therapies in the veteran population, especially in the later stages of life to improve clinical outcomes. skaggs school of pharmacy and pharmaceutical sciences provided funding for the research assistant, as part of their summer research internship program. declaration of financial / other interests the authors have no relevant financial or other relationships to disclose. jda peer reviewers on this manuscript have received an honorarium from jda for their review work, but have no other relevant financial relationships to disclose. skaggs school of pharmacy and pharmaceutical sciences provided funding for the research assistant, as part of their summer research internship program. declaration of financial / other interests the authors have no relevant financial or other relationships to disclose. jda peer reviewers on this manuscript have received an honorarium from jda for their review work, but have no other relevant financial relationships to disclose. | backgroundcardiovascular diseases are among the leading causes of death worldwide and studies have found a direct relationship between levels of low - density lipoprotein cholesterol and coronary heart disease. statins are the most commonly prescribed medications to lower cholesterol, a major controllable risk factor for coronary heart disease.objectivethis study aims to find what factors in the first year of statin therapy are predictive of long - term all - cause mortality.methodsdata for this retrospective cohort study were collected on patients identified as new statin users between december 1, 2006 and november 30, 2007 at five veterans affairs healthcare systems from southern california and nevada. multiple independent variables were assessed utilizing a logistic regression model assessing for all cause mortality at 6 years follow - up. the independent variables included race, age, ethnicity, body mass index, socioeconomic status, and baseline comorbidities. secondary analysis analyzed high - density lipoprotein levels, adherence, total cholesterol, and triglycerides.resultsincreased age, increased medication count, hypertension, diabetes, tobacco use, chronic obstructive pulmonary disease, and congestive heart failure were all associated with an increased risk of mortality. hispanic ethnicity, asian race, and increased body mass index were associated with decreased risk of mortality. there were no significant associations between mortality and race, ldl outcomes at 1 year, or annual income level.conclusionthere is clear evidence that statin use is associated with decreased events in cardiovascular disease and total mortality. this study found multiple independent variables as predictors of mortality in new start statin users after a 6 year follow - up, but differences in lipid groups after 1 year were not predictive of long - term mortality in the cohort studied. |
recurrent lateral dislocation of the patella is difficult to treat because of many complex contributing factors such as generalized ligamentous laxity, inadequate medial retinacular tissue, insufficient trochlear groove restraint, patellar tendon length abnormalities, limb alignment torsional abnormalities, and others. over 130 different surgical methods have been described which can be classified as proximal realignment, distal realignment, proximal and distal realignment, lateral retinacular release, and medial retinacular placation. the medial patellofemoral ligament (mpfl) was first described by kaplan. since warren and marshall introduced the concept of a three - layered pattern of the medial capsular ligament of the knee and described the fibers of the mpfl existing within layer ii, and biomechanical studies, had demonstrated that the mpfl accounts for approximately 50% to 60% of the total lateral restraint ; thus being the primary medial stabilizer of the patella, mpfl reconstruction has became an accepted technique to treat patellofemoral instability. recently, numerous techniques for reconstruction of the medial patellofemoral complex have been described with promising clinical results. many authors have described the method of making the patellar tunnel and using the wire anchors to fix the side of the patella. however, drilling tunnels transversely across the patella creates a stress riser and can lead to fracture. the use of wire anchors increase the economic burden of patients and also the patellar bone may be too soft for a secure anchoring. here, we present a report on mpfl reconstruction for recurrent patellar dislocation with bone - fascia tunnel fixation at the medial margin of the patella. in this article, we discuss a surgical technique for mpfl reconstruction along with some discussion of the anatomic and clinical rationale. the aim of the study is to evaluate the results of mpfl reconstruction with bone - fascia tunnel fixation at the medial margin of the patella after a 6-year - minimum follow - up. we hypothesized that this procedure is equal with other techniques for mpfl reconstruction, improving patellofemoral joint function and quality of life and giving pain relief. from june 2005 to may 2006, all patients (72 patients) with lateral patellofemoral joint instability had been treated at the third hospital of hebei medical university. five patients preferred to conventional treatment, where two of them had combined multiple - ligament injuries. sixty - five patients who underwent a primary mpfl reconstruction were included in the study. this study was approved by the ethics committee of the third hospital of hebei medical university. inclusion criteria included patients with a diagnosis of recurrent patellar dislocation, confirmed with history and physical and radiographic examinations. exclusion criteria for this operation were a history of previous knee surgery ; multiple ligament injury ; significant patellofemoral articular cartilage degenerative changes according to the outerbridge classification (grades iii iv) ; significant patellar malalignment, wherein the tibial tubercle - to - trochlear groove distance (tt - tg) is greater than 20 mm ; and severe trochlear dysplasia (table 1). demographics of the patients demographic data, physical examination, kujala scale, lysholm score, and tegner activity score were completed preoperatively and at each follow - up evaluation. clinical data were prospectively collected preoperatively and at 3, 6, 12, 24, 36, 48, 60, and 72 months after surgery. when the follow - up was longer than 6 years, the last data available were used for statistical analysis. examination under anesthesia is performed on both knees to assess for increased lateral translation of the patellar and for a tight lateral retinaculum. a lateral release was performed if the patella was unable to be everted to neutral. a tourniquet is applied to the thigh and then diagnostic arthroscopy is performed before reconstruction of the mpfl. the cartilaginous situation, tracking of the patella through a range of flexion, and trochlear shape are assessed. a longitudinal incision (23 cm) is performed on the anteromedial side of the patella, then the soft tissue is separated to the bone surface and the periosteum is peeled back. a bone groove which extends approximately from the superomedial corner of the patella to the midpoint of the medial margin of the patella the groove must be deep enough, about 3 mm, so that the allograft can be completely embedded. the two ends of the graft are fixed with a non - absorbable braided suture. the middle of the allograft is put into the groove, with the patellar fascia overlying. the absorbable sutures are used to fix the graft, so the bone - fascia tunnel is performed (figure 2). the method of suture is mainly divided into three needles (figure 3). bone - fascia tunnel is performed (a) and reconstruction of mpfl with proximal and distal bundle (b). the middle suture needle passes through the tendon graft to limit movement (a, b). a 2-cm incision is made over the femoral attachment of the mpfl which is just distal to the adductor tubercle and superoposterior to the medial femoral epicondyle. then, the second and the third layers of the medial patellofemoral complex, where the mpfl is anatomically situated, are separated from each other down to the femoral insertion side. the free ends of the graft are then pulled through with the help of a vascular clamp. a guide pin with an eyelet is placed at the anatomical femoral insertion and directed slightly anterior and superior to avoid penetration of the condyle posteriorly. the guide pin is then overdrilled with an appropriately sized cannulated acorn reamer based on sizing of the doubled graft to a depth adequate to allow full seating of the free ends of the graft within the femur. the sutures are then placed through the eyelet of the guide pin which is then advanced out the contralateral cortex of the distal femur. next, the grafts are pulled into the femoral tunnel, and they are free and not bottomed out. the knee is cycled for several times from full flexion to full extension with the graft under tension. insufficient tension will result in a lack of correction of the lateral instability, whereas excessive tension will cause increased pressure in the patellofemoral joint and may restrict knee range of motion. therefore, it is essential to find a tolerated tension ; the surgeon can arthroscopically adjust the tension and see normal patellar tracking restored during the whole range of knee motion. once sufficient tension has been obtained, the femoral attachment of the graft is fixed with a bioabsorbable interference screw of the same size as the drill which is used in 30 of knee flexion. this is because biomechanical studies, have shown that the mpfl has its maximal restraint against patella lateralization in 30 of knee flexion. the wound is closed in layers, and routine dressings, bandages, and a knee brace are applied. at the beginning, all patients followed the same rehabilitation protocol after mpfl reconstruction. postoperatively, the patients were partially weight bearing in full extension with a knee brace. two weeks after surgery, the patients were allowed to progress from partial to full weight bearing. in 65 patients, 6 patients (9.2%) were athletes who returned to sporting activity routine such as weight bearing with the contralateral limb and cycling at 4 weeks after surgery. then, they started running, and sport - specific rehabilitation programs at 12 weeks after surgery. they return to high - risk sports such as soccer or basketball at 6 months after surgery. fifty - nine patients (90.8%) were non - athletes who were allowed to remove the knee brace and began to do aggressive quadriceps, hamstring, and hip muscle strengthening exercises after 4 weeks. the time patients returned to daily activity routine or sports was based on their individual level of performance. statistical analysis was performed with spss software (version 11.0 ; spss, chicago, il, usa). demographic data, physical examination, kujala scale, lysholm score, and tegner activity score were completed preoperatively and at each follow - up evaluation. clinical data were prospectively collected preoperatively and at 3, 6, 12, 24, 36, 48, 60, and 72 months after surgery. when the follow - up was longer than 6 years, the last data available were used for statistical analysis. examination under anesthesia is performed on both knees to assess for increased lateral translation of the patellar and for a tight lateral retinaculum. a lateral release was performed if the patella was unable to be everted to neutral. a tourniquet is applied to the thigh and then diagnostic arthroscopy is performed before reconstruction of the mpfl. the cartilaginous situation, tracking of the patella through a range of flexion, and trochlear shape are assessed. a longitudinal incision (23 cm) is performed on the anteromedial side of the patella, then the soft tissue is separated to the bone surface and the periosteum is peeled back. a bone groove which extends approximately from the superomedial corner of the patella to the midpoint of the medial margin of the patella the groove must be deep enough, about 3 mm, so that the allograft can be completely embedded. the two ends of the graft are fixed with a non - absorbable braided suture. then, the diameter of the folded graft is measured with a sizing guide. the middle of the allograft is put into the groove, with the patellar fascia overlying. the absorbable sutures are used to fix the graft, so the bone - fascia tunnel is performed (figure 2). the method of suture is mainly divided into three needles (figure 3). bone - fascia tunnel is performed (a) and reconstruction of mpfl with proximal and distal bundle (b). the middle suture needle passes through the tendon graft to limit movement (a, b). a 2-cm incision is made over the femoral attachment of the mpfl which is just distal to the adductor tubercle and superoposterior to the medial femoral epicondyle. then, the second and the third layers of the medial patellofemoral complex, where the mpfl is anatomically situated, are separated from each other down to the femoral insertion side. the free ends of the graft are then pulled through with the help of a vascular clamp. a guide pin with an eyelet is placed at the anatomical femoral insertion and directed slightly anterior and superior to avoid penetration of the condyle posteriorly. the guide pin is then overdrilled with an appropriately sized cannulated acorn reamer based on sizing of the doubled graft to a depth adequate to allow full seating of the free ends of the graft within the femur. the sutures are then placed through the eyelet of the guide pin which is then advanced out the contralateral cortex of the distal femur. next, the grafts are pulled into the femoral tunnel, and they are free and not bottomed out. the knee is cycled for several times from full flexion to full extension with the graft under tension. insufficient tension will result in a lack of correction of the lateral instability, whereas excessive tension will cause increased pressure in the patellofemoral joint and may restrict knee range of motion. therefore, it is essential to find a tolerated tension ; the surgeon can arthroscopically adjust the tension and see normal patellar tracking restored during the whole range of knee motion. once sufficient tension has been obtained, the femoral attachment of the graft is fixed with a bioabsorbable interference screw of the same size as the drill which is used in 30 of knee flexion. this is because biomechanical studies, have shown that the mpfl has its maximal restraint against patella lateralization in 30 of knee flexion. the wound is closed in layers, and routine dressings, bandages, and a knee brace are applied. postoperatively, the patients were partially weight bearing in full extension with a knee brace. two weeks after surgery, the patients were allowed to progress from partial to full weight bearing. in 65 patients, 6 patients (9.2%) were athletes who returned to sporting activity routine such as weight bearing with the contralateral limb and cycling at 4 weeks after surgery. then, they started running, and sport - specific rehabilitation programs at 12 weeks after surgery. they return to high - risk sports such as soccer or basketball at 6 months after surgery. fifty - nine patients (90.8%) were non - athletes who were allowed to remove the knee brace and began to do aggressive quadriceps, hamstring, and hip muscle strengthening exercises after 4 weeks. the time patients returned to daily activity routine or sports was based on their individual level of performance. furthermore, the function of athletes was acceptable after 6 years. statistical analysis was performed with spss software (version 11.0 ; spss, chicago, il, usa). at the last follow - up, all the patients demonstrated a significant improvement compared to the preoperative status. the congruence angle had significant improvement from 19.2 6.3 before surgery to 6.0 0.5 at the last follow - up. the lateral patellar angle had significant improvement from 6.9 3.5 before surgery to 5.1 2.4 at the last follow - up. the patellar tilt angle had significant improvement from 24.5 5.2 before surgery to 12.3 1.9 at the last follow - up (table 2). patellofemoral measurements on computed tomography the kujala score was significantly increased from 52.9 3.2 points preoperatively to 90.1 5.8 points postoperatively (p < 0.05). the mean lysholm score was significantly increased from 47.2 5.2 to 92.5 6.2 points postoperatively (p < 0.05). the tegner activity score improved overall from 3.1 0.6 points to 5.8 0.9 points at follow - up (table 3). the graft of the femoral side is almost secured in the femoral tunnel with an absorbable interference screw, but for the fixation of the patellar side, it includes a few techniques for the reconstruction of this ligament, making the patellar tunnel ; and using the wire anchors is described by many authors. nomura and inoue described the patellar tunnel which was drilled from the proximal third of the medial margin of the patella to the center of the anterior aspect of the patella. in 2007, carmont and maffulli described a similar technique of drilling the bone tunnels that traverse the entire patella ; and in 2009, papp and cosgarea described the blind patellar tunnel which was drilled from medial to lateral at the midpoint of the mpfl insertion. gomes warned against damaging the cartilage surface of the patella by creating a bone tunnel. dobbs. was able to show that drilling tunnels transversely across the patella creates a stress riser and can lead to fracture. this complication has been seen using a previously described technique where fithian and gupta reported a 305-lb 17-year - old male who fell 1 year after reconstruction of mpfl by drilling two tunnels on the patella which enter at the medial articular margin and exit at the anterior (ventral) patellar surface. mikashima. reported that no significant difference after a 2-year follow - up was observed in the quality of fixation between suturing to the periosteum and the fibrous tissue overlying the patella and fixation in a bone tunnel. they had two patella fractures whose reconstructed mpfl was fixed into a bone tunnel in the patella. in 2008, schottle. described their technique of securing the graft to the medial patella with two suture anchors and to the femur with a biodegradable interference screw. the pull of the reconstructed mpfl on the patellar side is over the femoral drill tunnel and extended to the proximal suture anchor, where it is fixed by knots. the fixation strength is mainly between the tendon and suture. as a result of the suture anchor, the tension on the tendon - to - bone contact increased which did not help tendon - to - bone healing, as compared with the pressure contact. the use of the suture anchor also increased the economic burden of patients and also the patellar bone may be too soft for a secure anchoring. the cancellous bone of the bone groove makes up 2/3 of the tunnel, and the remaining 1/3 is formed by the covered fascia. in addition to the medially directed force to the patella, the reconstructed mpfl also provides a posterior force ; therefore, it is a posteromedial force on the patella. such a force may limit the graft in the bone groove to be well associated with the firmly sutured fascia. so, the fixation of the graft at the patellar side by the bone - fascia tunnel technique permits secure fixation and direct pull on the patella, which has mechanical characteristics similar to those of the bone tunnel technique. the graft is also permitted to heal in a bone tunnel, allowing for increased surface area for graft - to - bone healing. this eliminates the risks of patellar fracture and violation of the patellar articular surface. in 2004, steensen. described that the average width of the patellar insertion was larger than that of the femoral insertion. in 2008, parker. evaluated and compared the patellofemoral kinematics of a single - stranded isometric medial patellofemoral ligament reconstruction technique with that of a double - stranded anatomic technique that more closely recreates the normal anatomy of the medial patellofemoral ligament. in our technique for mpfl reconstruction, the making of the bone groove at the patellar side and the use of allograft allows us to place the graft at the anatomical insertion and recreate the double - bundle structure of the mpfl and a sail - like triangular shape of the graft, which is comparable to the original anatomy,. reconstruction of the mpfl using the double - bundle anatomic technique creates a proximal and a distal bundle and reproduces the anatomy of the native mpfl. this seems to provide a higher stability during flexion and decreases the patella rotation in contrast to a single - bundle isometric technique. there were several limitations to our study, including the non - randomized and retrospective study design. the greatest limitations were the small number of patients and the lack of a control group. in the future study, we will enroll more patients and compare different mpfl reconstruction methods to observe which method is associated with lower morbidity and more rapid rehabilitation, which might be beneficial for such patients. there were several limitations to our study, including the non - randomized and retrospective study design. the greatest limitations were the small number of patients and the lack of a control group. in the future study, we will enroll more patients and compare different mpfl reconstruction methods to observe which method is associated with lower morbidity and more rapid rehabilitation, which might be beneficial for such patients. this method for mpfl reconstruction is a relatively easy and safe procedure that provides enough graft strength comparable to previous techniques. this is a simple technique where the mpfl is reconstructed safely to avoid patella fracture, anatomically to restore physiological kinematics and stability, and economically to reduce costs with bone - fascia tunnel fixation at the medial margin of the patella. jl and yl carried out the conception and design of the study, acquisition and interpretation of data, and drafted the manuscript. ys revised the manuscript critically for important intellectual content and gave final approval of the version to be published. yingze zhang for his support to obtain the approval of the ethics committee for this study. | backgroundmedial patellofemoral ligament (mpfl) reconstruction has become an accepted technique to treat patellofemoral instability, and numerous surgical techniques have been described to reconstruct the mpfl. we describe a mpfl reconstruction procedure where bone - fascia tunnel fixation occurs at the medial margin of the patella for recurrent patellar dislocation.objectivempfl reconstruction is the preferred operative treatment for recurrent patellar dislocation. the purpose of this study was to report a simple technique for reconstruction of medial patellofemoral ligament with bone - fascia tunnel fixation at the medial margin of the patella for recurrent patellar dislocation and to evaluate the results at 6-year - minimum follow-up.methodsthe study included 65 patients (28 males, 37 females ; mean age, 29.4 5.6 years) who underwent mpfl reconstruction using the bone - fascia tunnel fixation at the medial margin of the patella technique and who were followed for a mean duration of 78.5 3.8 months. objective assessment, kujala scale, lysholm score, and tegner activity score were obtained preoperatively and at the time of final follow-up.resultsthere were no patellar complications, including redislocation, in the present study. the congruence angle had significant improvement from 19.2 6.3 before surgery to 6.03 0.50 at the last follow - up. the lateral patellar angle had significant improvement from 6.9 3.5 before surgery to 5.1 2.4 at the last follow - up. the patellar tilt angle had significant improvement from 24.5 5.2 before surgery to 12.30 1.90 at the last follow - up. the kujala score was significantly increased from 52.9 3.2 points preoperatively to 90.1 5.8 points postoperatively (p < 0.05). the mean lysholm score was significantly increased from 47.2 5.2 to 92.5 6.2 points postoperatively (p < 0.05). the tegner activity score improved overall from 3.1 0.6 points to 5.8 0.9 points at follow-up.conclusionwe have done a simple technique where the mpfl is reconstructed safely to avoid patella fracture, anatomically to restore physiological kinematics and stability, and economically to reduce costs with bone - fascia tunnel fixation at the medial margin of the patella. |
the development of inexpensive and efficient photovoltaic devices is still a topic of high scientific importance. the vast majority of today s commercially available photovoltaic elements are based on inorganic semiconductors. the impressive advancements in the field of organic semiconductors within the last two decades have introduced the realistic potential for a much cheaper way to produce electrical energy from light. organic semiconductors combine the general properties of semiconductors with the easy processability of organic molecules, while manufacturing of solar cells based on inorganic semiconductors still requires high vacuum - based coating processes. thin - film photovoltaic devices based on organic semiconductors can be easily printed on lightweight, rugged, and flexible substrates. another clear advantage of organic semiconductors is the possibility to chemically modify the material properties. additionally, the electronic structure of organic semiconductors can be relatively easy modified by changing the molecular structure via chemical synthesis. in contrast to all these advantages, the initial synthesis of new organic semiconductors is a time - consuming and expensive process. a large number of physical and chemical properties of organic semiconductors can be investigated by electrochemical and photoelectrochemical measurements. the degree of electrochemical doping may be monitored via electrochemical impedance spectroscopy (eis). cyclic voltammetry offers an appropriate way to determine the position of homo and lumo levels of organic semiconductors. various other in situ spectroscopic methods have been developed to study optical and electronic changes induced by electrochemical processes. however, for all of them, individual samples need to be prepared which is a rather time and material - consuming process. in addition, concurrent or subsequent processes such as photodegradation or photodoping can be investigated using photoelectrochemistry. even when only partially studying the electrochemical and photoelectrochemical properties, different samples and overall relatively big amounts of material are required. therefore, finding a way of drastically decreasing the amount of material required for investigation is highly relevant. one attempt is based on a strong miniaturization of the area addressed by the electrochemical cell, which automatically leads to a drastic reduction of the amount of material to be consumed. this approach can be realized by, for example, photoelectrochemical scanning droplet cell microscopy (pe - sdcm), as it is capable of performing all common photoelectrochemical and electrochemical techniques on a single substrate. the central idea behind pe - sdcm is that only a small electrolyte droplet released from the tip of a capillary with a small inner diameter comes into contact with the sample surface that is acting as the working electrode (we). in this paper, a modified version of a pe - sdcm adapted for use with nonaqueous electrolyte solutions the photoelectrochemical properties of a thin film of poly[2-methoxy-5-(3,7-dimethyloctyloxy)-1,4-phenylenevinylene ] (mdmo - ppv), which is a model ppv - based donor organic semiconductor, was studied in detail. recently, the doping effect on the optical properties of mdmo - ppv was reported. although its applicability in photovoltaic devices was already shown, no detailed photoelectrochemical characterization of this material was done up to now. using the pe - sdcm, all common electrochemical and photoelectrochemical experiments were performed on spot sizes of less than 0.04 mm. the user can easily switch between electrochemical and photoelectrochemical experiments without having to change the cell or the substrate. using pe - sdcm, more than 100 electrochemical experiments on individual spots could be performed on a single 15 15 mm substrate consuming less than 2 ml of electrolyte. performing such a large number of experiments on a single substrate drastically reduces the amount of consumed material, reduces the time for sample preparation, and eliminates possible variations between different samples. also, different light sources like lasers, leds, or halogen lamps can be used to meet various experimental requirements. to demonstrate the measurement capabilities of pe - sdcm for electrochemical and photoelectrochemical characterization of organic semiconductors under nonaqueous conditions, a thin film of poly[2-methoxy-5-(3,7-dimethyloctyloxy)-1,4-phenylenevinylene ] (mdmo - ppv ; covion gmbh, frankfurt, germany) was used. the structure of the polymer is presented in figure 1. at first, the organic semiconductor was dissolved in pyridine (99+%, alfa aesar) with a concentration of 10 g l. the thin film used for electrochemical characterization was prepared by spin - casting the dissolved polymer molecules onto a precleaned 15 15 mm ito / glass slide (15 sq, kintec co.) the glass / ito substrate was cleaned by consecutive sonication in isopropanol, acetone and deionized water. the film thickness was measured using a dektak xt stylus profilometer (bruker corp. all electrochemical and photoelectrochemical measurements shown in this publication were carried out using a special photoelectrochemical scanning droplet cell microscope adapted for the use of nonaqueous, organic - based electrolytes. the main body of the cell was made from an acrylic block (22 mm 22 mm 12 mm) into which three connected channels (3.3 mm in diameter) were drilled (see figure 2). each channel was sealed using polypropylene (pp) screws with an o - ring underneath the head of each screw. a -ag / agcl system adapted for use in organic - based solvents was used as a micro quasi reference electrode (-qre). the potential sensitive part of the -qre was prepared by electrodeposition of agcl on the first 10 mm of a longer 100 m in diameter ag wire in 1 m hcl. the partially coated wire was inserted into a small glass capillary for increased mechanical stability, leaving the potential sensing part exposed outside it. to avoid contact between the uncoated part of the ag wire and electrolyte, the glass capillary was sealed on both ends using two component epoxy resin (uhu gmbh, germany). this leads to increased mechanical stability of the -qre and avoids possible cross contaminations. the potential of this electrode was 0.211 v versus standard hydrogen electrode (she). the counter electrode (ce) used for the pe - sdcm was made from a flattened 100 m in diameter au wire (99.999%, wielandt dentaltechnik, germany). flattening of the au wire increases the surface area of the ce and results in a high ce / we surface ratio. scheme of the photoelectrochemical scanning droplet cell microscope (pe - sdcm). the lower part of the pe - sdcm, from which the droplet is released, was made from a borosilicate glass capillary with an outer diameter of 2.5 mm. it was tailored using a commercial capillary puller (pc-10, narishige, tokyo, japan). the size of the tip was adjusted to the final desired size using an in - house developed capillary polisher equipped with 2400 and 4000 grade sic sandpaper (struers a / s, ballerup, denmark). after polishing, the glass tube was thoroughly cleaned with acetone and isopropanol, followed by deionized water, and blown dry using nitrogen. a silicone seal was formed at the rim of the capillary by dipping it into liquid silicone (rtv, momentive, albany, u.s.a.) and dried under constant nitrogen flow for several hours. this operation of the pe - sdcm in contact mode, where the tip of the cell is pressed against the surface of the sample. this approach leads to a very high reproducibility of the area addressed by the pe - sdcm due to confinement of the electrolyte droplet within the inner volume defined by the silicone sealing. additionally, such a sealing strongly reduces contamination of the electrolyte (e.g., water) by eliminating direct exposure of the electrolyte to the atmosphere during measurements. next, this capillary was mounted in a screw allowing an easy way of fixing it to the acrylic block. in order to locally illuminate the area addressed by the pe - sdcm the length of the fiber, re and ce were chosen in such a way that all three, when installed inside the tip capillary, are in close proximity to the surface (see figure 2b). this ensures homogeneous illumination of the wetted area and minimizes potential drop between re and sample surface (we). inside a third screw a 800 m in diameter stainless steel capillary was installed using a two - component epoxy glue (uhu gmbh, germany). this capillary was used as electrolyte inlet and was connected via a teflon tube to a high - precision syringe pump (world precision instruments, u.s.a.). the pump served as an electrolyte reservoir and was additionally used to precisely control the size of the electrolyte droplet formed at the tip of the capillary. to determine the area wetted by the pe - sdcm, a sputter deposited ti thin film was anodized in an aqueous 0.1 m na2so4 solution. the area of the colored oxide spot was determined using an automated optical pattern recognition software (nis elements d, nikon, japan). all photoelectrochemical experiments presented in this work were performed using a 532 nm, diode pumped solid state laser module with built - in auto power control. this specific wavelength was chosen to match the absorption maximum of mdmo - ppv, which was determined by spectroscopic ellipsometry (se). various different light sources, for example, high - power leds, uv / vis continuum emitters, monochromators with optical fiber output, and different kinds of lasers have already been successfully used with pe - sdcm. the 532 nm laser was coupled externally into the fiber using an in - house developed fiber port. to avoid any disturbance of the measurements by ambient light sources, the intensity of the light source was adjusted by using a gray - shade filter (thorlabs, u.s.a.) positioned midway between laser source and fiber port. blockage of the laser beam was done by means of a manually operated shutter. to determine the optical power density on the area addressed by the pe - sdcm, a fully assembled pe - sdcm was positioned on the detector window of an optical power meter (coherent lasermate q). the pe - sdcm was positioned on the sample surface in a fully automated fashion using a gantry robot build from three linear stages. for maximum reproducibility of the wetted area the applied force was continuously monitored using a small force sensor (me - messsysteme, germany) and, if necessary, readjusted by feedback to the z - axis. the complete setup was controlled by an in - house developed labview program. as electrolyte, a 0.1 m solution of tetrabutylammonium hexafluorophosphate (tbapf6, 99%, fluka analytical, u.s.a) dissolved in propylene carbonate (pc, 99.7%, sigma - aldrich) was used. the electrolyte was prepared and stored in a glovebox to avoid possible contaminations caused by, for example, oxygen or water. all experiments were carried out in a three - electrode configuration using a potentiostat with a built - in frequency response analyzer for electrochemical impedance spectroscopy (eis) measurements (compactstat, ivium technologies, the netherlands). in order to characterize the electrochemical properties of the mdmo - ppv, cyclic voltammetry was performed. using this technique, detailed information about the oxidation / reduction processes as well as their kinetics can be obtained. first, cyclic voltammetry with different scan rates was done. to avoid any interaction, for each measurement, the pe - sdcm was moved to a different location, and measurements with scan rates of 1, 3, 10, 30, and 100 mv s were performed on sequentially addressed spots. all these cycles are presented together in figure 3 to allow a direct comparison. as can be seen, the observed maximum of the current density increases when increasing the scan rate, which can be easily explained by increased mass transport at higher scan rates. due to the strong overlapping of the experimental data, the curves corresponding to the first three potential scan rates (up to 10 mv s) are displayed separately in the inlet of figure 3. interesting observations can be done, if looking at the shape of the current voltage curves as a function of the scan rate. for scan rates up to 10 mv s two oxidation peaks are found (see inlet figure 3). for the first experiment with a scan rate of 1 mv s, the oxidation peaks are found at 0.8 and 1.3 v. when increasing the speed of change of polarization up to 10 mv s, the oxidation peaks are shifted to higher potentials. the lower potential oxidation peak shifts to 0.83 v, while the higher potential oxidation peak shifts to 1.44 v. the scan rate dependent peak position suggests a kinetic hindrance of the electrochemical oxidation process. as a result, higher potentials are needed for the same oxidation process to occur. moreover, in all three experiments, no clear reduction peak could be observed, but instead, a very broad negative current distribution is noticeable. when comparing current voltage characteristics measured with different polarization speeds, the different behavior of the reduction part of the voltammograms could be explained by a dissolution process of the previously oxidized mdmo - ppv, which becomes more significant at lower polarization speeds where a second oxidation peak was detected. for higher scan rates (30 and 100 mv s), only one clear oxidation peak is found with a maximum at 0.86 and 0.9 v, respectively. as compared to the previously discussed case of low polarization speeds, the second oxidation peak is absent probably due to kinetic hindrance of the oxidation process. additionally, for the high scan rate cases, a broad reduction peak centered at 0.8 v is observed. in order to get a better understanding of the polymer oxidation and reduction processes, a series of cyclic voltammograms was measured with a scan rate of 10 mv s on a single spot. in this experiment, the maximum potential was incrementally increased in steps of 0.05 v up to the final potential of 1.5 v. the results are plotted in figure 4. during experiments, all curves with the maximum potential up to 0.8 v, showed only background current. this is observable in the inlet of figure 4, where only the first five experiments (with final potential up to 0.95 v) are presented. in the scan with a final potential of 0.85 v, a sharp and well - defined peak with maximum at 0.84 v, was found. in the next scan, with a final potential of 0.9 v the intensity of this peak reduces and a broadening can be observed. additionally, a second anodic peak can now be observed at 0.68 v. the presence of this new peak is related to an initial oxidation of the mdmo - ppv. this peak was not measured before, probably due to the formation of an interfacial barrier on the polymer surface causing the previously discussed peak at 0.84 v. the next scan with a final potential of 0.95 v has only the initial oxidation peak centered at 0.68 v with a slightly higher current density than in the previous scan. for final potentials above 0.95 v, the peak characterizing the first oxidation step shifts toward higher potentials. the current density value at the peak maximum increases gradually up to 0.03 ma cm in the case of the scan with the highest final potential. for the last three measured scans (1.41.5 v) the position of the first oxidation peak is shifted for about 50 mv up to 0.8 v for each additional experiment. for cyclic voltammograms with the final potential above 1 v, a strong increase in the current can be observed at the end of the anodic sweep. all performed cyclic voltammograms that show a clear faradaic current have a rather undefined broad reduction peak. when increasing the maximum potentials of the scans, the maximum current density of the reductive current is shifted toward more positive potentials. all scans with reverse potentials up to 1.5 v and scans with reverse potentials up to 0.95 v (inset). besides cyclic voltammetry, a series of potentiostatic experiments were performed. in this study the constant potential applied was increased stepwise. within the entire measurement series, the addressed area of the mdmo - ppv layer was kept at the corresponding potential, while the resulting current was measured for 70 s. the obtained results are displayed as a 3d graph in figure 5. in order to obtain a more detailed description of the electrochemical processes caused by small changes in the applied potential, sufficiently small potential steps were used. a potential increase of 50 mv for each step was previously found to be suitable for investigating electrochemical doping of polymers and was used also for this study. time - dependent potentiostatic measurements on mdmo - ppv at different applied potentials. for applied potentials below 0.75 v, only a background current density in the order of 10 ma cm could be measured. the recorded current stays constant within the 70 s time frame of each measurement. in the potential range between 0.750.85 v the increase in the current density is related to the oxidation of mdmo - ppv and to the potential corresponding to the interfacial barrier previously discussed in the cyclic voltammetry studies. during the first 5 s of each potentiostatic measurement, a significant decrease in the current density can be observed, caused by the occurring electrochemical processes. at a later stage, when increasing the applied potential even further (above 0.85 v), the final current density measured after 70 s increases from one curve to the next. this effect can be explained by an increased conductivity of the oxidized organic semiconductor due to doping. as previously found, a significant decrease in current density within the first 5 s is observed. the magnitude of the onset current decrease (during the first 5 s) depends on the applied potential and gets significantly lower for the highest potentials. a numerical integration of the potentiostatic curves presented in figure 5 was performed in order to quantify the total charge taking part in the electrochemical processes at the semiconductor / electrolyte interface. this total charge is plotted as a function of the applied potential and the results are plotted in figure 6 to allow for a further discussion of the electrochemical processes. up to 0.75 v, a constant charge density of about 3 c cm is observed. this charge density is directly related to the background electrochemical current of the system. similar to the previously discussed current transient experiments, at 0.85 v, a small oxidation is evidenced by a charge density increase. when increasing the potential further up to about 1.1 v, the charge density slowly increases up to about 0.6 mc cm. for potentials between 1.1 and 1.4 v, an almost exponential growth of the charge density is observed. this charge density increase is mainly related to the second oxidation step of the mdmo - ppv, as observable in the fast decaying (within the first 5 s) current density curves plotted in figure 5. as the applied potential increases from 1.4 to 1.5 v, the charge density increase can be also related to degradation processes. additionally, the mentioned strong charge density increase is confirming the previously discussed electrochemical processes, which results in a conductivity increase of the mdmo - ppv due to the electrochemical oxidation. overall, in the charge density dependence on the applied potential presented in figure 6, no abnormal behavior of the system could be identified. this indicates long - term reliability of the pe - sdcm since the coulometric data is only confirming the electrochemical processes previously evidenced in the potentiostatic investigations from figure 5. total charge density measured on mdmo - ppv as a function of applied potential in the potentiostatic study. in an attempt to study the electrical properties of the mdmo - ppv / electrolyte system, electrochemical impedance spectroscopy was performed using the pe - sdcm. usually, conjugated polymers in their undoped form have insulating properties. upon doping, generally, at low doping levels they are considered as semiconductors. with increasing the doping level, their electrical resistance further decreases and they can show metal - like behavior. during the eis study shown here, the frequency dependent changes of the impedance are monitored as a function of the applied dc bias (offset). before each eis experiment, a potentiostatic pretreatment was performed for 70 s in order to equilibrate the electrochemical processes. the used equilibration time of 70 s was chosen in agreement to the previous potentiostatic experiments, which showed a current density stabilization after this time interval (see figure 5). all impedance spectra were recorded at a single addressed spot using sequentially increasing biases up to 1.4 v. after each spectrum, the bias was increased by 0.2 v and the frequency dependence of the impedance was determined again. in figure 7, the corresponding bode plots are shown in part (a) together with the associated phase shifts in part (b). as can be noticed, for biases below 0.6 v, there is no change in the shape and value of the impedance. in this bias range, the high frequency impedance suggests an electrolyte resistance of approximately 10. similarly, the impedance value observable at the lowest frequency indicates a working electrode resistance in the order of 10. starting with the applied bias of 0.6 v, a deviation of both the impedance and phase shift at low frequencies appears. this decrease is related to the oxidation of mdmo - ppv, resulting in an expected insulator - to - metal transition. this decrease is continuing up to a bias of 1 v, where the working electrode impedance decreases by almost 2 orders of magnitude. in the same time, the phase shift changes substantially, finally reaching a value under 25, observable at the middle of the investigated frequency range. at even higher applied potentials, an unexpected increase in the impedance can be observed. this effect can be related to dissolution of the oxidized mdmo - ppv layer, resulting in a change in the working electrode geometry. when a semiconductor is illuminated with light having an energy larger than the semiconductor bandgap, the energy of the photons can be absorbed and excited electrons and holes are generated in the conduction and valence band, respectively. when these photoexcited electrons or holes are in the space charge region they migrate toward the electrode surface where they participate in charge transfer reactions giving a photocurrent. to demonstrate the functionality of pe - sdcm for photoelectrochemical measurements on organic semiconductors, photoinduced phenomena were studied on the mdmo - ppv film. the area addressed by the pe - sdcm was illuminated by the built - in optical fiber with a radiation of 532 nm wavelength (green) provided by an external laser. the wavelength of the radiation was chosen to match the maximum absorption of the polymer, as reported in a previous study. photocurrents caused by photogenerated electrons (cathodic currents) or holes (anodic current) are negligible when they are the majority charge carriers. under these conditions, however, their photocurrent becomes significant when the reacting electrons or holes are minority charge carriers. in this case their concentration is greatly increased by photoexcitation as compared with the concentration of the minority charge carriers in the absence of radiation (in the dark). therefore, photocurrents can only be observed with reactions in which the participating carriers are minority charge carriers. this means transfer of holes at n - type electrodes and electrons at p - type electrodes leading to anodic and cathodic photocurrents, respectively. in figure 8, photocurrent transients measured on mdmo - ppv under chopped light at different applied potentials are displayed. after approximately 10 s in the dark, the polymer was irradiated for another 10 s, defining one exposure cycle. in total, four exposure cycles were performed for applied potentials of 0.0, 0.2, and 0.4 v. for all potentials, the material is acting as a photocathode under illumination, which is a clear proof of the p - type nature of this polymer. also, the measured photocurrents are overall decreasing with time due to the photodegradation of the material. within each illumination cycle, the measured photocurrent is highest at the beginning and is decreasing toward the end of each irradiation period. the strongest decay in photocurrent within each cycle is found for the first exposure cycle and is continuously getting smaller for the following cycles. the dark current after each illumination cycle returns to its initial background value. when increasing the applied potential toward more positive values, the measured photocurrent becomes smaller for all experiments. this can be explained by the system being brought closer to flat - band conditions, thereby decreasing the electric field inside the space - charge region. photocurrent transients measured under intensity modulated green laser light. to study the long - term stability of the polymer under irradiation, photocurrent transients were measured for 600 s during illumination with the green laser light at different potentials. the same potentials of 0.0, 0.2, and 0.4 v used in the previous irradiation / dark sequence analysis were also applied here to enable a direct comparison. the measured current transients are shown in figure 9, and the 600 s exposure time interval is indicated in the figure. the initial dark current densities are shown in the figure before opening the optical shutter in order to indicate the current density background (dark) level. immediately after opening, the shutter allowing laser irradiation of the mdmo - ppv / electrolyte interface, the maximum in photocurrent is observed for all applied potentials. within the next 200 s, after 600 s of illumination, no clear distinction can be done anymore between photo- and dark current. therefore, it may be concluded that this polymer suffers from severe photodegradation when being in contact with tbapf6 electrolyte. as already previously observed in figure 8, this can be explained by a decrease in the space charge region allowing a less efficient exciton separation at the electrochemical interface. in addition to photocurrent measurements, the photopotential of mdmo - ppv in tbapf6 electrolyte was measured for 300 s, and the results are shown in figure 10, as obtained from the open circuit potentials measured versus the reference electrode. when a space charge region is present at the semiconducting polymer interface, the photoexcited electrons and holes are separated and each one is moving in opposite direction under the influence of the electric field inside the space charge region. this field - assisted migration of the photoexcited charge carriers induces an opposite potential in the electrode which decreases the potential difference across the space charge region. this reduction further leads to shifting of the fermi level by a certain energy which can be detected by measuring the photopotential. the electrode potential in the dark continuously decreases from 0.13 v down to 0.05 v. this time variation can be related to the porous nature of the electrode most likely being penetrated more and more by the electrolyte molecules with time. the photopotential measured under green light irradiation stays constant at about 0.46 v for about 50 s and then gradually decreases to 0.22 v within the remaining 250 s of measurement time. however, this decrease is stronger than the decrease of the potential in the absence of irradiation a modified version of a photoelectrochemical scanning droplet cell microscope adapted for use with nonaqueous electrolytes is presented. the possibility for localized measurements on single addressed small areas (0.04 mm) on a polymer surface is exploited. the small dimension of the cell allows considerably lower consumption of both electrolyte and studied material, as compared to using conventional electrochemical cells. this advantage is evidenced by a detailed electrochemical and photoelectrochemical characterization of a model organic semiconductor (mdmo - ppv). two oxidation peaks were found during the cyclic voltammetry studies, which were confirmed by potentiostatic investigations. the characteristics of the oxidation processes were studied using scan rate dependent experiments as well as variable maximum potential applied during the potential scan. electrochemical impedance spectroscopy was successfully performed at the micrometer scale revealing the changes in the film resistance as a function of the applied potential. the photovoltaic response of the mdmo - ppv was studied using the described pe - sdcm under irradiation with 532 nm wavelength. high signal - to - noise ratios (> 1000) were obtained during the photocurrent measurements on the addressed spot in a reproducible manner. the photopotential of the electrode was also successfully characterized in order to test the applicability of pe - sdcm for photovoltaic studies. | a model organic semiconductor (mdmo - ppv) was used for testing a modified version of a photoelectrochemical scanning droplet cell microscope (pe - sdcm) adapted for use with nonaqueous electrolytes and containing an optical fiber for localized illumination. the most attractive features of the pe - sdcm are represented by the possibility of addressing small areas on the investigated substrate and the need of small amounts of electrolyte. a very small amount (ng) of the material under study is sufficient for a complete electrochemical and photoelectrochemical characterization due to the scanning capability of the cell. the electrochemical behavior of the polymer was studied in detail using potentiostatic and potentiodynamic investigations as well as electrochemical impedance spectroscopy. additionally, the photoelectrochemical properties were investigated under illumination conditions, and the photocurrents found were at least 3 orders of magnitude higher than the dark (background) current, revealing the usefulness of this compact microcell for photovoltaic characterizations. |
all experimental procedures were in accordance with the guidelines of the institutional animal ethical committee. animal maintenance and handling were in accordance with the indian national institute of nutrition guidelines. all diets were formulated according to american institute of nutrition (ain)-93 g standards, which have been recommended for pregnant and lactating female rats. the composition of the c diet was the same as ain-93 g and contained 18 g protein/100 g diet (table 1). we formulated two iso - energetic treatment diets low in protein (9 g/100 g diet) by increasing the amount of maize starch to 488 g/100 g diet. of these diets, one (low protein (lp)) had soyabean oil (7 %) while the other (low protein dha + ara supplemented (lps)) had a combination of single cell oils containing 4045 % of dha and ara (martek biosciences) with soyabean as the basal oil. the percentages of these single cell oils were calculated based on oral diet safety studies. special attention was given while processing the lps diet, which was dried at room temperature (30c) to avoid the oxidation of added oils. the dry pellets were stored at refrigeration temperature (4c) and used within 1 week. table 1.composition (g/100 g) of control and experimental diets (mean values and standard deviations)diets clplpsmean sd mean sd mean sd diet compositioncarbohydrate603046890568904protein1820294039403fatsoybean oil750274025602arasco00000000081200dhasco0000000000056000vitamin mix100010001000mineral mix||350035003500fatty acid composition (%) 16 : 01101117618 : 042412818 : 1n-922222015018 : 2n-654054130618 : 3n-377754220 : 4n-600003522 : 6n-3000024n-6:n-37272509c, control diet ; lp, low - protein diet ; lps, low - protein diet supplemented with dha and arachidonic acid. proximate analysis (ten batches of diets per group). arasco, arachidonic acid single cell oil (martek biosciences). dhasco, docosahexaenoic acid single cell oil (martek biosciences). mineral mixture (g / kg mix) : calcium carbonate 357, potassium phosphate 196, potassium citrate 7078, sodium chloride 78, potassium sulfate 466, magnesium oxide 24, ferric citrate 606, zinc carbonate 165, manganous carbonate 063, cupric carbonate 03, potassium iodate 001, sodium selenate 001, ammonium paramolybdate 0007, sodium metasilicate 145, chromium potassium sulfate 0275, lithium chloride 001, boric acid 008, sodium fluoride 006, nickel carbonate 003, ammonium vandate 0006, sucrose 22102|| vitamin mixture (g / kg mix) : nicotinic acid 3, calcium pantothenate 16, pyridoxine - hcl 07, thiamin - hcl 06, riboflavin 06, folic acid 02, d - biotin 002, vitamin b12 (01% in mannitol) 25, vitamin e15, vitamin a 08, vitamin d 025, vitamin k 0075, sucrose 974655. fatty acid composition of extracted fat was analysed by gas chromatography, expressed as median values. composition (g/100 g) of control and experimental diets (mean values and standard deviations) c, control diet ; lp, low - protein diet ; lps, low - protein diet supplemented with dha and arachidonic acid. dhasco, docosahexaenoic acid single cell oil (martek biosciences). mineral mixture (g / kg mix) : calcium carbonate 357, potassium phosphate 196, potassium citrate 7078, sodium chloride 78, potassium sulfate 466, magnesium oxide 24, ferric citrate 606, zinc carbonate 165, manganous carbonate 063, cupric carbonate 03, potassium iodate 001, sodium selenate 001, ammonium paramolybdate 0007, sodium metasilicate 145, chromium potassium sulfate 0275, lithium chloride 001, boric acid 008, sodium fluoride 006, nickel carbonate 003, ammonium vandate 0006, sucrose 22102 || vitamin mixture (g / kg mix) : nicotinic acid 3, calcium pantothenate 16, pyridoxine - hcl 07, thiamin - hcl 06, riboflavin 06, folic acid 02, d - biotin 002, vitamin b12 (01% in mannitol) 25, vitamin e15, vitamin a 08, vitamin d 025, vitamin k 0075, sucrose 974655. fatty acid composition of extracted fat was analysed by gas chromatography, expressed as median values. the animal house at agharkar research institute (pune, india) is registered under the committee for the purpose of control and supervision of experiment on animals, india, and follows vigorously all instructions for adequate animal care. nulliparous female wistar rats weighing 200250 g were obtained from the in - house colony of the institute. the animals were maintained at 22c on a controlled 12 h light and 12 h dark cycle with an appropriate ventilation system. rice husk was used as bedding material and was non - nutritive and non - toxic. when they reached body weights of 200 g they were kept for breeding. mating was confirmed by sperm - positive vaginal smear. on confirmation of pregnancy, i.e. 0 d of gestation, they were randomly allocated to one of the three diet groups (ten per group). in both the c and lp groups, the dams received the dietary treatment during gestation (21 d) and lactation (21 d). daily food intake during pregnancy and weekly body weights were recorded for dams in all three groups. dam weights after parturition, litter size and litter weights at birth were recorded. large litters were reduced to eight pups on day 1 to ensure a standard litter size per dam. brain fatty acids were analysed for a sub - sample of pups at postnatal day 1 and during lactation (day 14). fresh whole brains were collected from anaesthetised pups and cryo - frozen at 80c. hydrolysis and transesterification of lipids were carried out according to the method of o'fallon.. heptanoic acid (17 : 0 ; sigma) was used as an internal standard. the methyl esters were extracted with hexane and the fatty acid composition of the extract was analysed by gc using shimadzu model gc-17a version 3 fitted with capillary column spb 35. the analysis conditions were as follows : capillary column, 30 m 032 mm inner diameter, film thickness, 0.25 m. the temperature programme was as follows : the initial temperature 100c (5 min hold), 100220 c (10c per min). gc parameters were : injector temperature 200c, detector temperature 220c, flame ionisation detector, carrier gas he ; flow rate 18 ml / min ; split ratio 33:1. for milk collection, pups were separated from their dams after providing a sufficient time for nursing the young ones. on day 1 of lactation, two pups / litter / diet group were randomly selected for collection of gastric milk samples. on day 14 of lactation too, the gastric milk sample from one pup / litter / diet group was randomly selected. before collection of milk, pups were exposed to anaesthetic ether and then milk was obtained from the stomach and the collected samples were cryo - frozen at 80c. the milk lipids were extracted and transesterified according to the method of christopherson & glass and analysed by gc using shimadzu model gc-17a version 3 fitted with capillary column spb 35. the analysis conditions were as follows : capillary column, 30 m 032 mm inner diameter, film thickness, 0.25 m. the temperature programme was as follows : initial temperature 40c (5 min hold), 40100c (6c / min) and 100220c (10c / min). the carrier gas was he with a flow rate of 18 ml / min and a split ratio of 33:1. fatty acid concentrations were calculated from their relative peak areas as compared with the internal standard. individual fatty acids were identified by comparing the retention time of the peak with that of standard fatty acid methyl esters (37-component fame mix ; sigma). the treatment groups were compared with the c group by one - way anova and the post hoc bonferroni test was applied to make pair - wise comparisons. the kruskal wallis independent sample non - parametric analysis was also applied to test significance in the case of small sample size (n < 30), for the comparison of brain and milk fatty acids profiles. the data were analysed using the spss / pc+ package (version 11.0 ; spss inc.). despite differences in protein levels, there was no difference in food intake during pregnancy (table 2) and lactation among the dams from the c and treatment groups. the total weight gain during pregnancy was the highest in the dams from the c group ; however, it did not differ significantly from those in the lp and lps groups (table 2). table 2.effect of dietary treatment on food consumption, body weight gain and reproductive performance of control diet - fed (c) and experimental dams (mean values and standard deviations)diet group clplpsmean sd mean sd mean sd p no. of dams9910food consumption (g / week)gestation week 1140525511981761228254nsgestation week 2135532511112101096199nsgestation week 310491541037151931274nspre - pregnancy weight (g)219315520591722280277nsweight gain (g)gestation week 1176321502215432nsgestation week 2194461593015528nsgestation week 3150221482113339nstotal gain875185836102819214nsreproductive outcomelitter size10623103369435nslitter weight (g)564143550156483142nsbirth weight (g)540555055307nslp, low - protein diet ; lps, low - protein diet supplemented with dha + arachidonic acid. effect of dietary treatment on food consumption, body weight gain and reproductive performance of control diet - fed (c) and experimental dams (mean values and standard deviations) lp, low - protein diet ; lps, low - protein diet supplemented with dha + arachidonic acid. diet had no effect on litter size and litter weights at birth (table 2). the fatty acid composition of pups ' brains on postnatal day 1 and during lactation (day 14) is shown in table 3. at birth, there was no difference in the level of sfa between groups ; however, the level of mufa was significantly higher in brains of pups from the lp group compared to pups from the c and lps groups. among the essential fatty acids, the level of la was similar between groups but that of ala was significantly higher in pups of the lps group. among the supplemented fatty acids, the level of ara in brain lipids did not differ between groups, but dha was significantly higher in the pups from the lps group. in addition, pups from this group had a significantly higher amount of epa (20 : 5n-3) and total n-3 and lower level of n-6 docosapentaenoic acid (22 : 5) as compared with the lp group. consequently, the n-6:n-3 ratio for brain fatty acids from lps pups was significantly lower than for pups from the c and lp groups. table 3.brain lipid fatty acids expressed as percentage total fatty acids of neonatal offspring of dams fed the control diet (c) and experimental diets (mean values and standard deviations)diet group clplpsmean sd mean sd mean sd postnatal day 1no. of pups6910male355female345total sfa453494393644949total mufa341433913535733la (18 : 2n-6)180610061007ala (18 : 3n-3)110605031306gla (18 : 3n-6)170811060504ara (20 : 4n-6)911787158818epa (20 : 5n-3)080306061508dpa (22 : 5n-6)0505080400701dha (22 : 6n-3)550941106210total n-6131201172110424total n-3740552188916total pufa205211693719335n-6:n-3170324051203postnatal day 14no. of pups667male334female333total sfa368124626838034total mufa348243266432225la (18 : 2n-6)160709021607ala (18 : 3n-3)07090010041709gla (18 : 3n-6)160708031006ara (20 : 4n-6)135041181413513epa (20 : 5n-3)200808062209dpa (22 : 5n-6)040208020202dha (22 : 6n-3)840561079509total n-6171201431416324total n-311113691213417total pufa283162121229740n-6:n-3150221061208lp, low - protein diet ; lps, low - protein diet supplemented with dha + arachidonic acid ; la, linoleic acid ; ala, -linolenic acid ; gla, -linolenic acid ; ara, arachidonic acid ; dpa, docosapentaenoic acid. mean values within a row with unlike superscript letters were significantly different (p<005 ; preplanned comparisons using post hoc tests). brain lipid fatty acids expressed as percentage total fatty acids of neonatal offspring of dams fed the control diet (c) and experimental diets (mean values and standard deviations) lp, low - protein diet ; lps, low - protein diet supplemented with dha + arachidonic acid ; la, linoleic acid ; ala, -linolenic acid ; gla, -linolenic acid ; ara, arachidonic acid ; dpa, docosapentaenoic acid mean values within a row with unlike superscript letters were significantly different (p<005 ; preplanned comparisons using post hoc tests). by mid - lactation (day 14), the percentage of ara and dha in brain lipids of pups increased significantly in all diet groups (fig. however, compared with pups from the c and lps groups, brain lipids of pups from the lp group not only had significantly higher sfa (mainly palmitic ; 16 : 0 2973 % and stearic ; 18 : 0 582 % compared with 2618 and 507 % in pups from the c group) but also had markedly higher levels of docosapentaenoic acid, which is a marker of n-3 fatty acid deficiency. on the other hand, with regard to essential fatty acids, lps pups continued to accrue higher levels of ala, ara, epa, dha and total n-3 fatty acids as compared with lp pups. 1.effect of dietary treatment on accretion of arachidonic acid (ara) (a) and dha (b) in whole brain phospholipids of offspring during the early postnatal period (///, day 1 ;, day 14). values are means, with standard errors represented by vertical bars (six to ten rats per group). mean value was significantly different from that at day 1 (p < 005 ; preplanned comparisons). lp, low - protein diet ; lps, low - protein diet supplemented with dha + ara. effect of dietary treatment on accretion of arachidonic acid (ara) (a) and dha (b) in whole brain phospholipids of offspring during the early postnatal period (///, day 1 ;, day 14). values are means, with standard errors represented by vertical bars (six to ten rats per group). mean value was significantly different from that at day 1 (p < 005 ; preplanned comparisons). lp, low - protein diet ; lps, low - protein diet supplemented with dha + ara. the fatty acid composition of dams ' milk at the beginning of lactation (day 1) and mid - lactation (day 14) is shown in table 4. on day 1, there was no difference in the levels of sfa, mufa or essential fatty acids, namely la and ala, between groups. however, the level of scfa and medium - chain fatty acids (c4-c12) was significantly lower while the levels of both supplemented fatty acids (ara and dha) were significantly higher, resulting in a higher amount of total n-3 and pufa in the milk samples of dams from the lps group than those from the c and lp groups. the n-6:n-3 ratio of milk was the highest in the dams from the lp group. table 4.milk fatty acids expressed as percentage total fatty acids during early and late lactation in dams fed the control diet (c) and experimental diets (mean values and standard deviations)diet group clplpsmean sd mean sd mean sd lactation day 1no. of pups666male333female333total scfa + mcfa14346133434124total mufa278262896230329total sfa494464986343635la (18 : 2n-6)186241832518923ala (18 : 3n-3)190514042007ara (20 : 4n-6)180513033407dha (22 : 6n-3)050402021303total n-6204261972122422total n-3240616043511total pufa228242132126128n-6:n-39231134417030lactation day 14no. of pups555male333female223total scfa178131903218337total mufa189132012620016total sfa508055261250415la (18 : 2n-6)164101550714806ala (18 : 3n-3)200512012905ara (20 : 4n-6)1304040042310dha (22 : 6n-3)0602006011708total n-6177071590617216total n-3260513014612total pufa203051720621825n-6:n-37118124133909lp, low - protein diet ; lps, low - protein diet supplemented with dha + arachidonic acid ; mcfa, medium - chain fatty acids ; la, linoleic acid ; ala, -linolenic acid ; ara, arachidonic acid. mean values within a row with unlike superscript letters were significantly different (p<005 ; preplanned comparisons using post hoc tests). post - nursing gastric milk samples collected from pup stomachs. milk fatty acids expressed as percentage total fatty acids during early and late lactation in dams fed the control diet (c) and experimental diets (mean values and standard deviations) lp, low - protein diet ; lps, low - protein diet supplemented with dha + arachidonic acid ; mcfa, medium - chain fatty acids ; la, linoleic acid ; ala, -linolenic acid ; ara, arachidonic acid. mean values within a row with unlike superscript letters were significantly different (p<005 ; preplanned comparisons using post hoc tests). post - nursing gastric milk samples collected from pup stomachs. by mid - lactation (day 14) the percentage of scfa and medium - chain fatty acids increased significantly in the milk of dams from the lps group (41183 ; p = 000) and nullified the between - group differences observed on day 1 of lactation. among the essential fatty acids, ala, ara, dha, total n-3 and pufa content was the highest in the milk of dams from the lps group, but was the lowest in the case of dams from the lp group. within each dietary group, a reduction in dha content of milk from day 1 to day 14 was observed but was maximum in the case of the lp group (02006). a similar reduction was observed in the n-6:n-3 ratio, for both the c and lps groups, but more sharply in the case of the latter while it remained constant in the lp group. correlations between dams ' milk and pup brain fatty acids were computed for day 14 of lactation, by pooling the data for all diet groups (table 5). we observed significant positive associations for the n-3 essential fatty acids, i.e. ala but not for the n-6 essential fatty acids, namely la. further, a strong direct association was also noted for the respective n-3 lc - pufa, i.e. dha, but not for the n-6 lc - pufa, i.e. ara. however, the total n-3 and n-6 levels in milk and brain lipids showed significant positive associations as did the total pufa content. one interesting observation was the significant inverse association between total sfa in maternal milk and total n-3 content of pup brains (r 0612 ; p < 005). table 5.pearson correlation coefficients for dam milk and offspring brain fatty acids during mid - lactation (day 14)fatty acidcorrelation coefficient (r)total sfa0.435total mufa0.021la (18 : 2n-6)0.133ala (18 : 3n-3)0.728ara (20 : 4n-6)0.462dha (22 : 6n-3)0.692total n-60.595total n-30.773total pufa0.755n-6:n-30.724la, linoleic acid ; ala, -linolenic acid ; ara, arachidonic acid.significant one - tailed correlation : p < 005, p < 001. pooled sample for all diet groups : n 15. pearson correlation coefficients for dam milk and offspring brain fatty acids during mid - lactation (day 14) la, linoleic acid ; ala, -linolenic acid ; ara, arachidonic acid. significant one - tailed correlation : p < 005, p < 001. pooled sample for all diet groups : n 15. maternal low protein intake during pregnancy and lactation has been shown to considerably perturb the normal metabolism of essential fatty acids, namely la and ala, resulting in poor ara and dha accumulation in the brain of growing offspring. yet, there is paucity of studies investigating the effects of supplementation of lc - pufa at low protein levels on changes in brain fatty acid content of the offspring and none have examined the consequent alterations in maternal milk profile. we observed that in conditions of protein restriction, supplemental dha and ara fed to dams led to their higher incorporation into pup brain lipids by altering the fatty acid profile of dams ' milk. before discussing the important findings of the study it may be worthwhile to mention some of the points. firstly, although the number of dams allocated to different diet groups was small, it was similar to those reported in earlier studies. secondly, the number of pups dissected at day 0 and day 14 was modest as it was necessary to maintain a sizeable number of pups / group till adulthood, being a long - term study. nevertheless, non - parametric tests were used for comparing differences in fatty acid profiles. we observed that even at the low protein level of 9 %, with or without supplementation, litter size or litter weight did not differ significantly from that of the c group. our observations are in agreement with reports from earlier studies that used diets containing menhaden fish oil or used a large range of la / ala ratios (1071035) and did not find differences in length of gestation, litter size or pup weights between c and treatment groups. even a protein - restricted diet with supplemental fish oil fed to pregnant dams did not show adverse effects on gestation, litter size or pup weight. this has been attributed to the fact that in rats, a considerable part of growth and development occurs after birth than in utero, making them less susceptible to unfavourable nutrient situations while in the womb. the rate of accumulation of dha and ara in the rat brain is the highest during early postnatal life and supply of these fatty acids during brain growth can boost the rate of brain mass expansion. we observed a significant increment in total brain ara and dha content from birth to 14 d of postnatal life in pups from all dietary groups ; however, the highest accretion was noted in pups of lps dams. further, we also noted a strong positive correlation between maternal milk ala, dha, total n-3 content and brain n-3 fatty acid profiles, corroborating the hypothesis that maternal milk fatty acid content indeed determines pup brain fatty acid composition. two important and independent changes observed in milk fatty acid composition of dams from the lp and lps groups are in need of attention. firstly, in the case of dams from the lp group, despite comparable levels of n-6 la in their milk, the milk was deficient in its long - chain metabolite, namely ara as compared with its proportion in milk from dams in the c group. as mentioned earlier, it has been shown that mammary glands actively participate in lc - pufa synthesis during lactation, and so the protein - restricted diet may have down - regulated enzymes involved in desaturation and the elongation pathway of n-6 la causing a reduction in the synthesis of ara. have reported similar effects of feeding 10 % protein diets to pregnant and lactating rats on the metabolism of essential fatty acids in mammary glands. the second notable observation was in the case of the lps group which showed a significantly low proportion of scfa and medium - chain fatty acids in dams ' milk during initiation (day 1) of lactation. however, by mid - lactation (day 14) a significant increase in these fatty acids made the levels comparable with those of the c group, indicating metabolic adaptations. for example, even in human subjects it has been shown that milk fatty acid composition undergoes changes during the course of lactation, which may be a feedback mechanism in order to meet the requirements for postnatal growth of the offspring. our observation therefore suggests that examining alterations in milk fatty acid composition in the light of pup growth during lactation would be of immense importance. provision of preformed dha and ara to the pups through milk is crucial for optimal accretion of lc - pufa in the rapidly growing brain of pups, as they are incapable of synthesising them from precursors. we observed that supplementation of preformed lc - pufa to protein - restricted dams resulted in higher dha and total n-3 fatty acids in their milk, unlike their non - supplemented lp counterparts. this observation thus calls attention to the significance of lc - pufa supplementation at low protein levels in order to overcome the resulting deficiency due to poor inter - conversion of ala to epa and dha and also to optimise the n-6:n-3 ratio of milk. providing lc - pufa during protein deficiency also served to conserve maternal ala pools, possibly due to a lower amount of ala entering the biosynthetic pathway, and enhanced epa levels in milk by retro - conversion of dha. this brought about a significant change in the total n-3 pools and n-6:n-3 ratio of lps milk and led to superior accretion of dha in the brains of lactating pups. a large body of work suggests that an adequate supply of dha during early life is essential for optimal growth and brain development. the present findings clearly demonstrate that low maternal dietary protein levels can profoundly affect dha accretion in developing offspring due to n-3-deficient milk, and supplementing dha and ara in such a scenario can positively alter both maternal milk and pup brain fatty acid composition. the effect of the observed biochemical change on the functional capacity of the brain in the offspring needs to be evaluated. however, evidence from several supplementation studies in situations of adequate protein suggests that learning and cognitive behaviour are augmented with improvement in brain dha status. therefore, our findings in a protein - deficient condition assume greater importance owing to the fact that the effects of supplementation are more pronounced in conditions of deficiency. they emphasise the need for supplying adequate food sources of dha to mothers during pregnancy and lactation to offset the negative effects of maternal protein deficiency on accretion of fetal brain dha and subsequently brain growth. | long - chain pufa (lc - pufa) are important for fetal and neonatal brain development. however, their accretion in the brain is compromised during maternal protein restriction. hence, we investigated the effect of maternal supplementation with n-3 dha plus n-6 arachidonic acid (ara) at a low protein level (9 %) on offspring brain fatty acid accretion using wistar rats (nine rats per group) randomly fed a control (c), a low - protein (lp) or a low - protein dha + ara - supplemented (lps) diet during gestation and lactation. at birth, pups from the lps group had the highest brain dha and n-3 fatty acid levels (p = 0001), whereas pups from the lp group had the highest mufa (p = 005) but the lowest dha and total n-3 pufa levels (p = 0000). during lactation, pups from the lps group accrued significantly more -linolenic acid (p = 0003), epa (p = 002) and dha (p = 0000) in brain lipids than pups from the lp group, whereas brain lipids of pups from the lp group had markedly increased levels of the n-3 deficiency marker docosapentaenoic acid and n-6:n-3 ratio (p = 0000). owing to supplementation, milk from lps dams had the highest dha and ara, but lower scfa and medium - chain fatty acids as compared with milk from c and lp dams during early lactation, but normalised by mid - lactation. to conclude, adverse effects of restricted maternal protein intake on lc - pufa accretion in the brain of offspring were ameliorated by alterations in maternal milk fatty acid profile due to supplementation. results underscore the importance of lc - pufa for protein - deficient mothers during gestation as well as lactation to achieve the optimum brain lc - pufa status of progeny. |
the concept of mental health is a state of health in which a person knows his abilities, can deal with normal life pressures, can be fruitful to the community, and can also have the power of decision - making and participation. self - esteem means the acceptance and a value that a person feels in him. psychologists also believe that self - esteem is a portion of mental health, and they consider the enjoyment of self - esteem as a basic and central cause of the social emotional adjustment of the people. self - esteem involves all the daily activities of humans, so it is one of the most important aspects of one 's personality and a determinant of the actualization and behavioral characteristics of human beings, and most of the experts know it as an important and fundamental factor in social and emotional adjustment. this characteristic is a general feature in all human beings and is not limited or transient, but is fixed and permanent. people with a low level of self - esteem should be diagnosed, to improve their self - esteem, with appropriate educational programs. students are constantly exposed to stressors and experience numerous problem due to the nature of their life during immigration to other places far from home. the precede model is a model for health education and promotion planning, which is used for changing behavior. green and kreuter believe that the precede model has been considered successful in the application of clinical and field trials. this model provides a framework in which the factors affecting behavior, such as : the predisposing, enabling, and reinforcing factors in the educational and assessment phases are determined. the precede model can be used as a guideline to analyze health problems or health behaviors, and also to determine knowledge, attitudes, and those beliefs that are effective in changes. several studies, particularly in the last 15 years, suggest that self - esteem is considered as a major psychological factor influencing health and quality of life. it has also been determined that a sense of empowerment and value trace is created for every person with increasing his / her self - esteem, and positive changes flourish in them to gain success, such as : academic achievement, more efforts for success, having a high self - esteem, and a great desire to preserve and promote health. a research on self - esteem and mental health in canada showed that the most important mental need in 87.4% of the adolescents was self - esteem. a significant relationship was found between low self - esteem and behavioral problems, such as, lack of confidence in relation to others, loss of social relations, and anxiety and depression. wright,. in a large interventional study, in australia, tried to improve and evaluate community awareness and participation in mental health and mental disorders of individuals, 12 to 18 years of age, using the precede model, while the researchers survey and effort did not show any study based on the precede model for promoting both self - esteem and mental health among students in iran. subsequently, this study was performed to evaluate the effect of an educational - participatory program, based on the precede model, for promoting the self - esteem and mental health of students in iran. this is a pre - post experimental study with the control group, conducted during 2006 2007. the studied population was the first year students (the freshman students) of medical sciences from two universities in the north east of iran. sampling was done initially, based on a pilot study, among universities of medical sciences in the north east of iran. two universities were selected that showed a high prevalence of psychological problems among their students and they also had similar academic conditions and situations. next, one of the two universities was randomly assigned as the case group and the other was identified as the control group. considering p1 = 0.27, p2 = 0.05, z = 1.96, z = 1.64, and with confidence interval values of 95%, the sample size was determined to be more than 134 students. the students were randomly selected based on a stratified random sampling method. following this, both the groups were matched for age, sex, discipline, and educational degree. the inclusion criteria were, studying in medical science subjects, passing at least one university educational term, and voluntary participation in the study. exclusion criteria were, having a psychiatric disorder and not volunteering for participation in the study. the following tools were used for data collection. in order to prepare this section of instruments, after a literature review on information resources, a questionnaire was designed. for assessing the predisposing factors, the questions were designed in three parts : (a) demographic data ; (b) knowledge including a 10-item, three - choice option questions with somewhat answers, with a possible range score from 0 to 20 (example : do you know the conflicts of the situations you encounter ?) ; and (c) attitude including an 18-item scale (six points that included, disagree a little, disagree, completely disagree, agree a little, agree, completely agree) with a possible range score of 6 to 36. (example : the best way to prevent getting sick, i see the physician regularly). for assessing the enabling factors a 10-item, three - choice option questions with somewhat answers were designed, related to economic, religious, and environmental factors, with a possible range of score from 0 to 20 (example : do you think economic conditions are involved in predicting health behaviors, particularly in your mental health ?). for assessing the reinforcing factors yes, no, and somewhat as answers (example : have you been encouraged by others such as family, teachers, classmates, friends, etc ?) for determining the content validity of the tools, the questionnaires were given to seven faculty members in the related subjects. after the announcement of their viewpoints and after resolving the problems and reforming the faults, they were submitted to 50 medical students, for reviewing and verifying from the sentences, matching points of view, with implementation, purposes, and aims of the scale producer, and resolving any objection or challenge regarding the understanding of the same concept from each question, by the students. after reviewing and correction of the cases, the reliability of the questionnaire was estimated for 188 students, using the internal consistency and test retest. the cronbach 's alpha coefficients for internal consistency of the scales were 0.74, 0.78, and 0.78, respectively, and after three weeks of test retest, the reliability coefficients were 0.80, 0.77, and 0.77, respectively. this known and standard tool, which was developed by rosenberg (1989), includes 10 questions, and each question is scored based on a four - choice options scale, from quite agree to disagree, with divers scoring for question numbers 9, 6, 5, and 2. hemmati has reported a cronbach 's alpha coefficient of 0.78 for the farsi translation of this questionnaire. the general health questionnaire-28 is the most recognized screening tool in psychiatry and mental health status, and has had dramatic effects on the development of researches. the 28-item questionnaire was made in order to increase the variance rate, and was based on factor analysis, copied from the main form. it has four scales, with seven questions in each scale, including physical symptoms, anxiety, depression, and disorder in social function. the ghq 's non - traditional method of scoring is the same as the likert method in which scoring of each test is with four degrees, 0, 1, 2, and 3, and thus the total score of each individual varies from 0 to 84. in this study the cut off point was considered to be 22 for the student population and for both sexes, so, persons with scores less than 22 were considered to have optimal mental health. several studies conducted on this questionnaire indicated and showed the high validity and reliability of the questionnaires. for conducting the educational - participatory program based on the precede model, focus group discussion (fgd) intervention was designed and performed on 12-person subgroups during 12 thirty - to - forty minute sessions for each one, by using life skills training separately, such as, problem - solving, confidence, decision - making, and critical thinking, by one of the researchers. after three months from the pre - test, the questionnaires were completed by the case and control groups. the data were analyzed separately by the spss-11.5 software and anova, regression, pearson correlation coefficient, paired - t, and independent - t - test. in order to prepare this section of instruments, after a literature review on information resources, a questionnaire was designed. for assessing the predisposing factors, the questions were designed in three parts : (a) demographic data ; (b) knowledge including a 10-item, three - choice option questions with yes, no, and somewhat answers, with a possible range score from 0 to 20 (example : do you know the conflicts of the situations you encounter ?) ; and (c) attitude including an 18-item scale (six points that included, disagree a little, disagree, agree, completely agree) with a possible range score of 6 to 36. (example : the best way to prevent getting sick, i see the physician regularly). for assessing the enabling factors a 10-item, three - choice option questions with somewhat answers were designed, related to economic, religious, and environmental factors, with a possible range of score from 0 to 20 (example : do you think economic conditions are involved in predicting health behaviors, particularly in your mental health ?). for assessing the reinforcing factors somewhat as answers (example : have you been encouraged by others such as family, teachers, classmates, friends, etc ?) for determining the content validity of the tools, the questionnaires were given to seven faculty members in the related subjects. after the announcement of their viewpoints and after resolving the problems and reforming the faults, they were submitted to 50 medical students, for reviewing and verifying from the sentences, matching points of view, with implementation, purposes, and aims of the scale producer, and resolving any objection or challenge regarding the understanding of the same concept from each question, by the students. after reviewing and correction of the cases, the reliability of the questionnaire was estimated for 188 students, using the internal consistency and test retest. the cronbach 's alpha coefficients for internal consistency of the scales were 0.74, 0.78, and 0.78, respectively, and after three weeks of test retest, the reliability coefficients were 0.80, 0.77, and 0.77, respectively. this known and standard tool, which was developed by rosenberg (1989), includes 10 questions, and each question is scored based on a four - choice options scale, from quite agree to disagree, with divers scoring for question numbers 9, 6, 5, and 2. hemmati has reported a cronbach 's alpha coefficient of 0.78 for the farsi translation of this questionnaire. the general health questionnaire-28 is the most recognized screening tool in psychiatry and mental health status, and has had dramatic effects on the development of researches. the 28-item questionnaire was made in order to increase the variance rate, and was based on factor analysis, copied from the main form. it has four scales, with seven questions in each scale, including physical symptoms, anxiety, depression, and disorder in social function. the ghq 's non - traditional method of scoring is the same as the likert method in which scoring of each test is with four degrees, 0, 1, 2, and 3, and thus the total score of each individual varies from 0 to 84. in this study the cut off point was considered to be 22 for the student population and for both sexes, so, persons with scores less than 22 were considered to have optimal mental health. several studies conducted on this questionnaire indicated and showed the high validity and reliability of the questionnaires. for conducting the educational - participatory program based on the precede model, focus group discussion (fgd) intervention was designed and performed on 12-person subgroups during 12 thirty - to - forty minute sessions for each one, by using life skills training separately, such as, problem - solving, confidence, decision - making, and critical thinking, by one of the researchers. after three months from the pre - test, the questionnaires were completed by the case and control groups. the data were analyzed separately by the spss-11.5 software and anova, regression, pearson correlation coefficient, paired - t, and independent - t - test. in this study, in both groups, 69.8% were girls and 30.2% were boys, with a mean age of 20.6 1.95 years. before performing the program on the case group, the variables affecting the mental health of the students, including the predisposing, enabling, and reinforcing factors, and particularly self - esteem were analyzed by the linear regression test, with controlling of the confounding factors in which self - esteem, examination times, and knowledge and attitude of significant variables were introduced [table 1 ]. the statistical test showed predictors, or on the other hand, variables affecting the mental health of the students, to fully explain the process and for effective intervention in accordance with the educational - ecological models level, for the researchers. multiple regression of self - esteem, knowledge, attitude, examination, and family on the mental health one - way anova analysis showed no significant relationship between the demographic characteristics, including marital or single status of the top students, or even sex with self - esteem and / or mental health, before their performances. also, according to table 2, a significant correlation was obtained based on the polychoric correlation coefficient before intervention in the experimental (case) group, between the predisposing and enabling factors and self - esteem and mental health, and it was significant even after intervention, so that the correlation between mental health and self - esteem (p < 0.0001), and mental health and knowledge (p < 0.008) was at a significant level of 0.01, and also between self - esteem and knowledge (p < 0.02), self - esteem and attitude (p < 0.01), and mental health and attitude (p < 0.03) was obtained at a significant level of 0.05, respectively, even when such a status was not observed in the samples of the control group. matrix of polychoric correlation coefficient of variables in experimental group before intervention according to table 3, an independent t - test showed that there were no significant differences between mental health, self - esteem, predisposing, enabling, or reinforcing factors in the case and control groups before the intervention (participatory - educational program based on the precede model). comparison of study variables based on independent t- test before intervention after intervention using an independent t - test, a significant difference had emerged between the mental health, self - esteem, predisposing, enabling, and reinforcing factors in the case and control groups [table 4 ]. comparing tables 3 and 4, the findings indicated the program efficacy on the students of the experimental group rather than the control group. the aim of the research was to determine the effect of a participatory - educational program based on the precede model to promote the self - esteem and mental health of the students. in short, the research findings introduced self - esteem, examination times, knowledge, and attitude as the significant and affecting variables on the mental health of students. there was also a significant difference between the predisposing, enabling, and reinforcing factors, self - esteem, and ultimately the students mental health, in the case group and the control group, after the intervention. a significant correlation was observed between mental health and self - esteem, mental health and knowledge, self - esteem and knowledge, self - esteem and attitude, and mental health and attitude in the case group after intervention, while such a correlation was not seen in the control group. in the study, the mental health status showed no significant difference between single and married students, but in fakhraei 's study, the psychological disorder status was reported to be significant in single students. fakhraei, ziaei and his colleagues, and faraji did not show any relationship between marital states and psychological disorders. ildarabadi,. reported that the depression in married students was higher than in single students. it is also important to note that the length of the education period in the university, especially in medical sciences, varies in different subjects and this can cause variations in the prevalence rate of mental disorders in the students of those fields. the results before intervention indicated the influence and close relationship of knowledge, attitude, and self - esteem with mental health. meanwhile, the regression analysis test introduced the self - esteem, knowledge, and attitude variables as significant variables affecting the students mental health. the results were considered to be the base and foundation of the design and implementation of the intervention programs based on the model, hence, they were highlighted in the focus group discussions (fgd) among students, and its effectiveness was identified in the intervention program process after implementation on the case group, rather than on the control group. it also showed significant differences in the correlation between self - esteem and mental health, correlation of mental health with knowledge and attitude, and correlation of self - esteem with knowledge and attitude ; so self - esteem and the mental health status of the samples were better with increase in knowledge and a positive attitude level. the promotion of students mental health was observed as their self - esteem increased and that coordinated with the results of this research and many other texts, such as, those by mann and colleagues, mohair, and moshki. particularly, the significance of these factors, after performing the program, indicated a confirmation of compliance with the study findings and reports of pope, botvin, and talebpour,. the influence of knowledge and attitude status from the predisposing factors has been emphasized on mental health and self - esteem, and also a significant correlation between the variables of knowledge, attitude, economic, religious, and environmental factors has been confirmed in the related literature, with self - esteem and their mental health. meanwhile, the role of self - esteem as a variable of an impressive predictor correlated with mental health has been explained in the studies of mann and his colleagues, quoted by tudor, furnham and cheng zimmermann. even krueter and green have noted and expressed the influencing role of self - esteem based on the model and have mentioned it specifically and implicationally in the training - ecological stage of the model. its efficacy can show the power of the model, which can facilitate the exact determining of the study aims, through community participation and empowerment. it can not be achieved, except through the process of community assessment, using evidence based on community - based participatory approaches, capacity building, and correcting the contributing factors during a review, and regular and continuous evaluation in the form of this model. it is possible that the significant increasing factor of self - esteem and especially mental health, is the use of the educational - participatory program based on the precede model in the case group rather than in the control group. using this model as a framework of an intervention, to reduce mental health problems, confirms the study findings in relation to the predisposing factors (knowledge and attitudes), enabling factors (educational resources and skills), and reinforcing factors (social support), with the results of other related researches, including the use of the precede model in anxiety reduction of the tehran fire fighters, the impact of education on depression in bypass heart surgery patients, and in accordance with the precede model in isfahan, the development and evaluation of community mental health with public awareness based on the precede proceed model in australia. phoenix and winnie utilized the predisposing, reinforcing, and enabling factors in the educational diagnosis of the precede model, to examine their effects on the mental well - being and quality of life among adults in hong kong. structural equation modeling showed that the sense of coherence (predisposing factor), social support (reinforcing factor), and daily hassles (enabling factor) were significantly related to mental health promoting behaviors, which were associated with the mental well - being. we also found a significant relationship between mental health and the mentioned factors of the precede model in this study. wright and colleagues showed that mental health campaigns were differently higher in the target region. it meant that the object of the enabling factor which was to make mental health information more available was accessed. they also stated that suicide beliefs associated with depression and psychosis increased or stabilized in the target region, while it declined in the comparison region. skarsater,. indicated that if mental health was well - designed as an intervention it could empower individuals to take responsibility for adopting mental health promoting behaviors. they also recommended the precede model as a useful framework for understanding factors involved with mental health promoting behaviors, among a large number of their samples. considering the effect of the predisposing, enabling, and reinforcing factors of the educational ecological model on self - esteem and the students mental health at first glance, and as the ultimate goal, the cumulative effect of related variables with each of the factors, and during implementation of the educational - participatory programs is matched and has conformity with the precede model. this model provides some steps for policy development, implementation, and evaluation of an educational program process, because a health education program is a process that is presented in regular consecutive stages. it is clear that the samples of this study can not be a representative of the country 's student population because of the wide extent and the different distribution of cultural, social, and ethnic characteristics. on the other hand the study samples are limited to the study subjects in two universities of medical sciences, and certainly more studies are needed among students in other subjects across the country, iran, as also in the state universities, non - profit, and even non - governmental universities. however, may different factors such as individual ethnicity, customs and habits of the students that are effective on students self - esteem and their mental health and similar researches should be conducted and expanded in different parts of the country to observe how they work and impact. this study is a part of an extensive and innovative project, using internationally recognized standards of a health promotion program model to promote self - esteem and mental health in the iran students community. the study also offers confidence to researchers in this manner to use approved skills and training methods with student empowerment and participation based on the precede model, which finally leads to health promotion in a part of the community. in fact, this pattern can provide a clear concept of designing an effective program through community awareness about mental health, so that it can have the ability to comply with all areas, problems, and mental disorders. in the same manner, and with the help of health education, we should step on the path to promote personal and social confidence and empower people to participate in health promotion. to promote mental health in fact, this strategy shows that even educated people of the society should strive to rely on their own resources. to achieve such self - confidence, people should be informed adequately about having the accurate attitude and sufficient skill to participate in the planning, implementation, and evaluation of health interventions. the goal will be effectively achieved only through appropriate educational activities intending to empower people to protect their health, family, and community in which they live. | background : the students vulnerability to different problems can have an impact on their mental health. regarding the lack of evidence on the effectiveness of interventional programs based on health education planning models in this area in developing countries, an educational - participatory program based on the precede model was used, to promote the medical science students self - esteem and mental health status, in iran.methods:in this experimental study, 154 students from the universities of medical sciences in the north east of iran were selected by stratified random sampling method. then, they were randomly assigned to two groups of case and control. the questionnaires, including the enabling, reinforcing, and predisposing factors, the rosenberg self - esteem scale, and the ghq-28 were used for data collection. then, an intervention plan, including focus group discussions and training of selected life skills, based on the precede model, was conducted for the case group.results:the predisposing, reinforcing, and enabling factors, and the self - esteem and mental health of the students showed a significant difference between the case and control groups. the pearson correlation coefficient showed that there was a correlation between mental health and knowledge (p = 0.008), between self - esteem and knowledge (p = 0.02), self - esteem and attitude (p = 0.01), and mental health and attitude (p = 0.03).conclusion : health promotion planning by using life skills training based on the precede model can result in participation and empowerment, in order to promote the self - esteem and mental health of the students. |
this study was initiated in july 2000 as a prospective survey of patients with type 2 diabetes who were in long - term follow - up in a single diabetes clinic at austin health, melbourne, australia. austin health is a major university teaching hospital and a tertiary referral center, serving a population of approximately 700,000. long - term follow - up was defined by the patients having at least three previous estimations of urinary albumin excretion rate (aer) performed on 24-h urine collections with at least one aer having been performed within the year 2000. patients with type 1 diabetes or diabetes secondary to medication or pancreatitis were specifically excluded from the current study. using these criteria, 665 patients with type 2 informed consent was obtained from participating patients as approved by the austin health human research ethics committee. baseline clinical and biochemical characteristics of all participants were ascertained including a full clinical history, medication use, anthropometric data, and smoking habits. the presence of preexisting cvd was defined on the basis of a clinical history of myocardial infarction, unstable angina requiring hospitalization, coronary revascularization (including coronary artery bypass grafting, angioplasty, or coronary stenting), heart failure, stroke, carotid artery surgery, peripheral revascularization (including bypass grafting, angioplasty, or stenting), and amputation for critical limb ischemia. fasting blood samples were taken for glucose, hba1c, lipid profiles, and creatinine. the glomerular filtration rate was estimated (egfr) using the chronic kidney disease - epidemiology collaboration (ckd - epi) formula. all regularly attending patients at the austin health diabetes clinics perform a 24-h urine collection before each visit for the determination of urinary sodium, potassium, and albumin excretion using standard methods in the department of laboratory medicine at austin health. baseline levels for each participant were determined as the mean excretion rates across all urine samples performed during 2001. data on 24huna were available in 638 participants (96%) who formed the study cohort (supplementary figure 1). the clinical characteristics of these patients were not different from those in whom 24huna was not measured. patients were given general dietary advice as part of their routine care at an initial assessment by a dietitian. however detailed assessment of dietary salt intake was not performed. during follow - up, all patients continued to have standard medical care including antihypertensive, lipid - lowering, and antidiabetic medications according to recommended guidelines. timing and cause of death were identified from a search of hospital patients records and the australian institute of health and welfare death registry. to evaluate the independent predictors of all - cause mortality in patients with type 2 diabetes the final model variables were determined by sequential penalized likelihood (akaike information criterion) (7). the functional form (in particular, nonlinearity) of continuous variables in the final model categorical variables were parameterized as simple indicator variables ; the potential for multicollinearity was assessed using the variance inflation factor and condition number. overall cox model fit was assessed by approximation of cumulative cox - snell residuals to (-log) kaplan - meier estimates, residual plots, and testing of the proportional hazards assumption and goodness - of - fit test (8). the cumulative hazard of all - cause mortality was graphically displayed using nelson - aalen estimates stratified by percentiles of covariate(s) of interest (9). to identify the independent predictors of the cumulative incidence of cardiovascular mortality, we used the fine and gray model, which extends the cox proportional hazards model to competing risks data (10) by considering the subdistribution hazard while adjusting for the competing risk of noncardiovascular deaths. the strength of the association between each variable and the outcome was assessed using the sub - hazard ratio, which is the ratio of hazards associated with the cumulative incidence function in the presence and absence of a prognostic factor. the fine and gray model was implemented in stata statistical software (v11, 2009 ; college station, tx) using the stcrreg module. this study was initiated in july 2000 as a prospective survey of patients with type 2 diabetes who were in long - term follow - up in a single diabetes clinic at austin health, melbourne, australia. austin health is a major university teaching hospital and a tertiary referral center, serving a population of approximately 700,000. long - term follow - up was defined by the patients having at least three previous estimations of urinary albumin excretion rate (aer) performed on 24-h urine collections with at least one aer having been performed within the year 2000. patients with type 1 diabetes or diabetes secondary to medication or pancreatitis were specifically excluded from the current study. using these criteria, 665 patients with type 2 informed consent was obtained from participating patients as approved by the austin health human research ethics committee. baseline clinical and biochemical characteristics of all participants were ascertained including a full clinical history, medication use, anthropometric data, and smoking habits. the presence of preexisting cvd was defined on the basis of a clinical history of myocardial infarction, unstable angina requiring hospitalization, coronary revascularization (including coronary artery bypass grafting, angioplasty, or coronary stenting), heart failure, stroke, carotid artery surgery, peripheral revascularization (including bypass grafting, angioplasty, or stenting), and amputation for critical limb ischemia. fasting blood samples were taken for glucose, hba1c, lipid profiles, and creatinine. the glomerular filtration rate was estimated (egfr) using the chronic kidney disease - epidemiology collaboration (ckd - epi) formula. all regularly attending patients at the austin health diabetes clinics perform a 24-h urine collection before each visit for the determination of urinary sodium, potassium, and albumin excretion using standard methods in the department of laboratory medicine at austin health. baseline levels for each participant were determined as the mean excretion rates across all urine samples performed during 2001. data on 24huna were available in 638 participants (96%) who formed the study cohort (supplementary figure 1). the clinical characteristics of these patients were not different from those in whom 24huna was not measured. patients were given general dietary advice as part of their routine care at an initial assessment by a dietitian. however detailed assessment of dietary salt intake was not performed. during follow - up, all patients continued to have standard medical care including antihypertensive, lipid - lowering, and antidiabetic medications according to recommended guidelines. timing and cause of death were identified from a search of hospital patients records and the australian institute of health and welfare death registry. to evaluate the independent predictors of all - cause mortality in patients with type 2 diabetes the final model variables were determined by sequential penalized likelihood (akaike information criterion) (7). the functional form (in particular, nonlinearity) of continuous variables in the final model categorical variables were parameterized as simple indicator variables ; the potential for multicollinearity was assessed using the variance inflation factor and condition number. overall cox model fit was assessed by approximation of cumulative cox - snell residuals to (-log) kaplan - meier estimates, residual plots, and testing of the proportional hazards assumption and goodness - of - fit test (8). the cumulative hazard of all - cause mortality was graphically displayed using nelson - aalen estimates stratified by percentiles of covariate(s) of interest (9). to identify the independent predictors of the cumulative incidence of cardiovascular mortality, we used the fine and gray model, which extends the cox proportional hazards model to competing risks data (10) by considering the subdistribution hazard while adjusting for the competing risk of noncardiovascular deaths. the strength of the association between each variable and the outcome was assessed using the sub - hazard ratio, which is the ratio of hazards associated with the cumulative incidence function in the presence and absence of a prognostic factor. the fine and gray model was implemented in stata statistical software (v11, 2009 ; college station, tx) using the stcrreg module. the mean age of participants was 64 years, 56% were men, the median duration of diabetes was 11 years, and 47% were obese (bmi > 30 kg / m). complications of diabetes were common with 45% of participants having previously experienced a cardiovascular event, including 35% who had a myocardial infarction. one - third had background retinopathy on ophthalmological review (33%), 30% had an egfr 20 g / min), including 13% with macroalbuminuria (> 200 g / min). eighty - five percent of patients had hypertension (defined by the use of antihypertensives and/or blood pressure > 140/90). achievement of therapeutic targets was low, reflecting the tertiary referral nature of the clinic, with two - thirds of patients having an hba1c > 7.0%, 55% with an ldl cholesterol level > 2.5 mm, and 44% with a systolic blood pressure > 140 mmhg. baseline urinary sodium excretion was estimated in 638 patients from a median of two collections performed during the year 2001 (range 15). the mean urinary sodium excretion was 184 mmol/24 h, similar to previous global population surveys (11). one - third of participants had a sodium excretion 208 mmol/24 h (highest tertile) with the middle tertile falling between 150 and 208 mmol/24 h. patients with urinary sodium excretion in the highest tertile were younger, with a shorter duration of diabetes, more likely to be obese, but with better renal function, and hemoglobin, when compared with patients in tertile 2 (table 1). sex was also a strong predictor of sodium excretion, consistent with the higher sodium intake described in men in global population surveys (11). on multivariate analysis, 24huna (as a continuous variable) was positively correlated with egfr, bmi, male sex, and the use of ace inhibitors and was negatively correlated with age (full regression model [supplementary table 1 ]). the presence or absence of heart failure, atrial fibrillation, and the use of diuretics or -blockers was not associated with 24huna after adjusting for age and sex. baseline characteristics of patients with type 2 diabetes, stratified according to tertiles (t) of 24-h urinary sodium excretion data are mean sd unless otherwise indicated. baseline 24huna excretion was strongly correlated with the yearly mean 24huna during follow - up. over two - thirds of individuals with 24huna in similarly, over two - thirds of individuals with 24huna in the lowest tertile remained in the lowest tertile in subsequent years (data not shown). less than 5% of individuals changed from the lowest to the highest tertile or the highest to the lowest tertile on an annual basis. overall, the intraindividual cv of mean annual 24huna was 23 11% over 8 years. vital status was determined in 620 patients with the remaining 18 (3%) lost to follow - up. the median follow - up for survival analysis was 9.9 years (5,475 patient - years) during which 175 deaths were recorded (28%). cvd was listed as a major contributing cause in 75 deaths (43% of all deaths) with the other major causes of death including cancer (n = 36) and infection (n = 19). in 17 patients, although death was verified, no cause of death could be ascertained. all - cause mortality was inversely associated with 24huna such that individuals with the lowest 24huna had the highest cumulative hazard for mortality (fig. 1). after adjusting for parameters associated with 24huna, as well as other factors independently associated with all - cause mortality (including age, sex, duration of diabetes, atrial fibrillation, the presence and severity of ckd (egfr and log aer) for every 100 mmol rise in 24huna, all - cause mortality was 28% lower (95% ci 645%, p = 0.02) (table 2). overall, 24huna explained 7% of the variability (r = 0.07, 95% ci 0.020.14) in all - cause mortality in this cohort. no significant interactions between categorical variables or between categorical and continuous variables were demonstrated in the model implying the relative hazard associated with sodium intake was uniform regardless of the presence and severity of hypertension, cvd, ckd, and other risk factors. moreover, its effect was stronger than for other reversible risk factors including ldl cholesterol and hba1c, and was equivalent to that of systolic blood pressure (table 2). interestingly, blood pressure levels were inversely correlated with all - cause mortality with no convincing evidence of nonlinear effects including j - curve associations. cumulative hazard (nelson - aalen) of all - cause mortality, stratified by percentiles (5th, 25th, 75th, and 95th) of 24-h urinary sodium excretion. independent associations with all - cause mortality and cumulative incidence of cardiovascular mortality in individuals with type 2 diabetes all - cause mortality : independent associations with all - cause mortality in individuals with type 2 diabetes in a multivariate cox model. the model explained 52% of the variation in all - cause mortality (95% ci 0.42 0.64) and was well specified (harrell s c : 0.79 ; ph test : p = 0.136 ; goodness - of - fit test : p 0.37). cardiovascular mortality : independent associations with the cumulative incidence of cardiovascular mortality in individuals with type 2 diabetes in the fine and gray (proportional hazards) model after accounting for the competing risk of noncardiovascular death. 2) after adjusting for the competing risk of noncardiovascular death and other predictors (table 2). no association was observed between 24huna and noncardiac deaths (data not shown). when modeled as a continuous variable, the association between 24huna and all - cause and cardiovascular mortality was linear. no convincing evidence of nonlinear effects, including j - shaped associations, was demonstrated as judged by residual - by - time analysis, fractional polynomials, and (cubic) regression splines (7). again, no significant interactions between categorical variables or between categorical and continuous variables were demonstrated, implying the relationship between 24huna and mortality was independent of bmi, age, sex, and the type of antihypertensive therapy or achieved level of blood pressure control. furthermore, the association between 24huna and mortality was independent of 24-h potassium excretion (data not shown). the cumulative incidence (fine and gray) of cardiovascular mortality over the 5th, 25th, 75th, and 95th percentile (a d, respectively) of 24-h urinary sodium excretion in men and women (solid line and dotted line, respectively), adjusted for other covariate predictors (table 2) and accounting for noncardiovascular mortality as the competing risk. the other predictors are set at : egfr = 76.6 ml / min/1.73 m (median) ; atrial fibrillation = yes ; preexisting cardiovascular disease = yes ; log10 aer = 1.2 (median) ; systolic blood pressure = 140 mmhg (mean) ; diabetes duration = 10.4 years (median). the mean age of participants was 64 years, 56% were men, the median duration of diabetes was 11 years, and 47% were obese (bmi > 30 kg / m). complications of diabetes were common with 45% of participants having previously experienced a cardiovascular event, including 35% who had a myocardial infarction. one - third had background retinopathy on ophthalmological review (33%), 30% had an egfr 20 g / min), including 13% with macroalbuminuria (> 200 g / min). eighty - five percent of patients had hypertension (defined by the use of antihypertensives and/or blood pressure > 140/90). achievement of therapeutic targets was low, reflecting the tertiary referral nature of the clinic, with two - thirds of patients having an hba1c > 7.0%, 55% with an ldl cholesterol level > 2.5 mm, and 44% with a systolic blood pressure > 140 mmhg. baseline urinary sodium excretion was estimated in 638 patients from a median of two collections performed during the year 2001 (range 15). the mean urinary sodium excretion was 184 mmol/24 h, similar to previous global population surveys (11). one - third of participants had a sodium excretion 208 mmol/24 h (highest tertile) with the middle tertile falling between 150 and 208 mmol/24 h. patients with urinary sodium excretion in the highest tertile were younger, with a shorter duration of diabetes, more likely to be obese, but with better renal function, and hemoglobin, when compared with patients in tertile 2 (table 1). sex was also a strong predictor of sodium excretion, consistent with the higher sodium intake described in men in global population surveys (11). on multivariate analysis, 24huna (as a continuous variable) was positively correlated with egfr, bmi, male sex, and the use of ace inhibitors and was negatively correlated with age (full regression model [supplementary table 1 ]). the presence or absence of heart failure, atrial fibrillation, and the use of diuretics or -blockers was not associated with 24huna after adjusting for age and sex. baseline characteristics of patients with type 2 diabetes, stratified according to tertiles (t) of 24-h urinary sodium excretion data are mean sd unless otherwise indicated. p < 0.01 vs. middle tertile (t2). baseline 24huna excretion was strongly correlated with the yearly mean 24huna during follow - up. over two - thirds of individuals with 24huna in similarly, over two - thirds of individuals with 24huna in the lowest tertile remained in the lowest tertile in subsequent years (data not shown). less than 5% of individuals changed from the lowest to the highest tertile or the highest to the lowest tertile on an annual basis. overall, the intraindividual cv of mean annual 24huna was 23 11% over 8 years. vital status was determined in 620 patients with the remaining 18 (3%) lost to follow - up. the median follow - up for survival analysis was 9.9 years (5,475 patient - years) during which 175 deaths were recorded (28%). cvd was listed as a major contributing cause in 75 deaths (43% of all deaths) with the other major causes of death including cancer (n = 36) and infection (n = 19). in 17 patients, although death was verified, no cause of death could be ascertained. all - cause mortality was inversely associated with 24huna such that individuals with the lowest 24huna had the highest cumulative hazard for mortality (fig. 1). after adjusting for parameters associated with 24huna, as well as other factors independently associated with all - cause mortality (including age, sex, duration of diabetes, atrial fibrillation, the presence and severity of ckd (egfr and log aer) for every 100 mmol rise in 24huna, all - cause mortality was 28% lower (95% ci 645%, p = 0.02) (table 2). overall, 24huna explained 7% of the variability (r = 0.07, 95% ci 0.020.14) in all - cause mortality in this cohort. no significant interactions between categorical variables or between categorical and continuous variables were demonstrated in the model implying the relative hazard associated with sodium intake was uniform regardless of the presence and severity of hypertension, cvd, ckd, and other risk factors. moreover, its effect was stronger than for other reversible risk factors including ldl cholesterol and hba1c, and was equivalent to that of systolic blood pressure (table 2). interestingly, blood pressure levels were inversely correlated with all - cause mortality with no convincing evidence of nonlinear effects including j - curve associations. cumulative hazard (nelson - aalen) of all - cause mortality, stratified by percentiles (5th, 25th, 75th, and 95th) of 24-h urinary sodium excretion. independent associations with all - cause mortality and cumulative incidence of cardiovascular mortality in individuals with type 2 diabetes all - cause mortality : independent associations with all - cause mortality in individuals with type 2 diabetes in a multivariate cox model. the model explained 52% of the variation in all - cause mortality (95% ci 0.42 0.64) and was well specified (harrell s c : 0.79 ; ph test : p = 0.136 ; goodness - of - fit test : p 0.37). cardiovascular mortality : independent associations with the cumulative incidence of cardiovascular mortality in individuals with type 2 diabetes in the fine and gray (proportional hazards) model after accounting for the competing risk of noncardiovascular death. 2) after adjusting for the competing risk of noncardiovascular death and other predictors (table 2). no association was observed between 24huna and noncardiac deaths (data not shown). when modeled as a continuous variable, the association between 24huna and all - cause and cardiovascular mortality was linear. no convincing evidence of nonlinear effects, including j - shaped associations, was demonstrated as judged by residual - by - time analysis, fractional polynomials, and (cubic) regression splines (7). again, no significant interactions between categorical variables or between categorical and continuous variables were demonstrated, implying the relationship between 24huna and mortality was independent of bmi, age, sex, and the type of antihypertensive therapy or achieved level of blood pressure control. furthermore, the association between 24huna and mortality was independent of 24-h potassium excretion (data not shown). the cumulative incidence (fine and gray) of cardiovascular mortality over the 5th, 25th, 75th, and 95th percentile (a d, respectively) of 24-h urinary sodium excretion in men and women (solid line and dotted line, respectively), adjusted for other covariate predictors (table 2) and accounting for noncardiovascular mortality as the competing risk. the other predictors are set at : egfr = 76.6 ml / min/1.73 m (median) ; atrial fibrillation = yes ; preexisting cardiovascular disease = yes ; log10 aer = 1.2 (median) ; systolic blood pressure = 140 mmhg (mean) ; diabetes duration = 10.4 years (median). advice to reduce dietary salt intake is a key element of many lifestyle intervention programs for type 2 diabetes. however, the association between dietary salt intake and mortality outcomes has not been previously studied specifically in the context of type 2 diabetes. in this article, we show that 24huna was independently associated with all - cause and cardiovascular mortality in patients with type 2 diabetes such that the highest mortality risks were observed in individuals with the lowest sodium intake and vice versa. although such data may seem contrary to established dogma, in fact previous observational studies, both in the general population and in hypertensive patients, have failed to give consistent results regarding the association of salt and mortality. in some studies, higher salt intake has been associated with an increased risk of cardiovascular events (1215), while in others including the national health and nutrition examination surveys (nhanes) i, ii, and iii lower salt intake has been associated with an increased risk of cardiovascular events and or mortality (16,17). other studies in the general community have failed to demonstrate any association of dietary salt intake with cardiovascular events (1820). although hypertension and cvd are common in patients with type 2 diabetes, the issue of whether dietary salt intake influences mortality and morbidity has not been studied specifically in the context of type 2 diabetes, and any inference from outcomes in other populations should be viewed with caution. the major strength of the current study is the use of multiple 24-h urine collections to estimate dietary sodium intake. with few exceptions (13,16), the majority of previous studies that have examined the association between salt and mortality, including nhanes (17), have relied on dietary recall, which can underestimate dietary salt intake by up to half (21). all patients were trained in urine collection and were experienced in performing 24-h urine collections at the start of the study. moreover, in this population, the annual intraindividual cv of 24huna was low (23% over an 8-year period). in addition, the urinary sodium excretion observed was very similar to previous global population surveys (11), confirming that the patients were not sodium - restricted, which may otherwise confound analysis by indication. the chief limitations of our data are that they are context - specific. the findings of our study specifically detail outcomes in a high - risk subpopulation of patients with longstanding type 2 diabetes attending a tertiary referral center. nonetheless, these are precisely the patients in whom more aggressive lifestyle interventions are often applied. our patients were mostly obese, hypertensive, with a history of poor metabolic control, and multiple comorbidities, including a significant burden of renal impairment and preexisting cvd at baseline. in all patients, it is possible that in this clinical setting, nontraditional risk factors have a stronger relationship with outcomes or that paradoxical associations are observed (22). for example, most of our patients were hypertensive despite treatment, and it has been previously shown in treated hypertensive men that low 24huna is associated with an increased risk of cardiovascular morbidity and mortality (16) and that tight blood pressure control in diabetic patients is associated with increased all - cause mortality (23). however, in our study, the relationship between urinary sodium excretion and mortality was independent of the presence and severity of hypertension. in our statistical analysis, we specifically assessed the nonlinear effects of predictive variables modeled for potential interactions and, in the case of cardiovascular mortality, modeled within the paradigm of a formal competing risks (fine and gray) model, which estimated the cumulative incidence of cardiovascular deaths while taking into consideration the competing risk of other causes of death, which may otherwise confound results (24). this strategy is especially important in patients with diabetes, as diabetes is also associated with increased noncardiovascular mortality (25), which may potentially confound cause - specific analysis. although such strategies have a number of advantages, it is nevertheless possible that associations demonstrated in this study may be because of confounding by unmeasured factors or those that are difficult to quantify. variability in dietary sodium intake may be associated with a range of differences in diet composition, processing, and preparation that may themselves impact on adverse outcomes in diabetic individuals. for example, sodium intake is dominated by sodium added in manufactured foods in western diets, (75% of intake) (11). in addition, we can not exclude the possibility that salt appetite and cardiovascular risk are linked to a common unidentified etiological factor. any pathophysiological mechanisms that may underlie our observed association between mortality and salt intake in patients with type 2 diabetes remain to be established. certainly, raas activity (4), insulin resistance (5), catecholamine levels (4), and lipids (4) may be influenced by sodium intake, and each of these potential mediators may be of particular relevance in the setting of type 2 diabetes and/or established atherosclerosis. for example, dietary sodium restriction leads to increased levels of angiotensin ii and aldosterone, chiefly via an increase in plasma renin activity. given the primacy of raas in the development and progression of diabetes complications, as adjudged by the efficacy of raas blockade, it is perhaps not surprising that activation of raas by reducing sodium intake may also be associated with adverse outcomes. the same may also be said for increased sympathetic activity, insulin resistance, and dyslipidemia associated with sodium restriction. in summary, we show that 24huna, the best marker of dietary sodium intake, was negatively associated with all - cause mortality specifically in the setting of type 2 diabetes, after adjusting for baseline risk factors. this may reflect the special status of dietary salt and the pathways it regulates in diabetic pathophysiology. such data call into question universal recommendations that all adults should endeavor to reduce their salt intake (12). ultimately, the explanation for our findings needs to be tested experimentally in clinical trials performed specifically in diabetic patients. | objectivemany guidelines recommend that patients with type 2 diabetes should aim to reduce their intake of salt. however, the precise relationship between dietary salt intake and mortality in patients with type 2 diabetes has not been previously explored.research design and methodssix hundred and thirty - eight patients attending a single diabetes clinic were followed in a prospective cohort study. baseline sodium excretion was estimated from 24-h urinary collections (24huna). the predictors of all - cause and cardiovascular mortality were determined by cox regression and competing risk modeling, respectively.resultsthe mean baseline 24huna was 184 73 mmol/24 h, which remained consistent throughout the follow - up (intraindividual coefficient of variation [cv ] 23 11%). over a median of 9.9 years, there were 175 deaths, 75 (43%) of which were secondary to cardiovascular events. all - cause mortality was inversely associated with 24huna, after adjusting for other baseline risk factors (p < 0.001). for every 100 mmol rise in 24huna, all - cause mortality was 28% lower (95% ci 645%, p = 0.02). after adjusting for the competing risk of noncardiovascular death and other predictors, 24huna was also significantly associated with cardiovascular mortality (sub - hazard ratio 0.65 [95% ci 0.440.95 ] ; p = 0.03).conclusionsin patients with type 2 diabetes, lower 24-h urinary sodium excretion was paradoxically associated with increased all - cause and cardiovascular mortality. interventional studies are necessary to determine if dietary salt has a causative role in determining adverse outcomes in patients with type 2 diabetes and the appropriateness of guidelines advocating salt restriction in this setting. |
proteomics - grade porcine trypsin, ethanol, high - performance liquid chromatography (hplc)-grade acetonitrile, formic acid, urea, phosphate buffered saline (pbs), sodium hydroxide, tris(hydroxymethyl)aminomethane (tris), and xylenes were purchased from sigma - aldrich corp. (st. louis, mo, usa). acetic acid, ammonium bicarbonate, dithiothreitol (dtt), chloroacetamide (claa), hplc - grade h2o with 0.1% formic acid, hplc - grade h2o, and hplc - grade acetonitrile with 0.1% formic acid were from fisher scientific (pittsburgh, pa, usa). fresh human lenses were obtained from the national disease research interchange (philadelphia, pa, usa). three lenses were analyzed in total : 21-year (m), 22-year (m), and 27-year (f). lenses were stored on wet ice during shipment and fixed immediately upon arrival. a solution of 75:25 ethanol : acetic acid was used to lightly fix tissue while avoiding traditional formaldehyde crosslinking that can interfere with mass spectrometry experiments. whole lenses were fixed for 12 hours, manually cut in half across the equator using a razor blade, and fixed for an additional 12 hours. eight micron sections were cut and collected using a microm hm 325 microtome (thermo scientific, waltham, ma, usa) and allowed to dry on glass slides overnight at 37c. sections were deparaffinized prior to labeling, using a series of xylene and alcohol washes (xylenes 3, 100% ethanol, 95% ethanol, 70% ethanol, pbs 5 minutes each). to visualize the rz, lenses were incubated in wheat germ agglutinin - tritc (life technologies, eugene, or, usa), using a 1 mg / ml stock solution at 1:100 dilution for 2 hours, followed by three, 5-minute pbs washes. sections used for confocal imaging were covered with vectashield mounting medium (vector laboratories, burlingame, ca, usa) and sealed with a coverslip. laser capture microdissection was performed on a palm microbeam system (carl zeiss microscopy, thornwood, ny, usa). uncovered sections were visualized at 20 magnification using the rhodamine fluorescence setting (540/600 nm excitation / emission). regions of interest were manually selected in the software, based on cellular morphology and measured distance from lens capsule. tissue was cut in brightfield mode and cells from each selected region were catapulted into an eppendorf cap containing 25-l hplc - grade h2o. microdissection was carried out until 2 10 m tissue was collected for each region to ensure approximately equal starting material. in total, tissue from four regions was collected : df (outer fiber cell layers, 0100 m from lens capsule) ; rz (100140 m from lens capsule) ; tz (140240 m from lens capsule) ; ic (240 m and beyond). tissue was then spun down into the tube and frozen for side - by - side membrane preparation of all four regions. for each lens, a total of three replicates of df, rz, tz, and ic were collected for initial experiments. collected cells were washed to prepare membranes and to remove soluble proteins, as described previously. samples were first reduced (25 mm dtt, 56c for 1 hour) and alkylated (55 mm claa, room temperature, 45 minutes in dark) before a series of washing and centrifugation steps to remove soluble proteins. the sample was vortexed and incubated for 30 minutes in homogenizing buffer containing 8 m urea (50 mm ammonium bicarbonate, 5 mm edta, 10 mm naf, 1 mm dtt, ph 8), followed by 20-minute centrifugation at 100,000 g (sorvall mtx150 micro ultracentrifuge, thermo scientific). the supernatant was removed and the pellet was saved and washed further using the following procedure : homogenizing buffer containing 8 m urea (three times total), 0.1 m naoh (one time), hplc - grade h2o (one time), 95% ethanol (one time), and hplc - grade h2o (three times). prior to trypsin digestion, the pellet was resuspended in 50% tfe in 50 mm ammonium bicarbonate, then diluted to 5% tfe before addition of 1 l of 0.1 g/l trypsin (pierce) in a volume of 100 l. digestion proceeded overnight at 37c and was stopped with the addition of 0.2 l neat formic acid. for each tissue region, the entire sample was bomb - loaded onto a reverse - phase 360 m outer diameter (o.d.) 100 m inner diameter (i.d.) capillary trap column (3 cm length/5 m jupiter c18 beads, 300, phenomenex) in - line with a 360 m o.d. reverse - phase analytical column packed with 20 cm jupiter c18 beads (3 m, 300, phenomenex) and equipped with a laser - pulled emitter tip. using an eksigent nanolc - ultra hplc system, peptides were eluted at a flow rate of 500 nl / min over a 120-minute gradient of 0.1% formic acid in water (solvent a) and 0.1% formic acid in acetonitrile (solvent b). the gradient consisted of 2% to 10% b in 20 minutes, 10% to 30% b in 30 minutes, 30% to 95% b in 15 minutes, 95% b for 15 minutes, followed by equilibration at 2% b. gradient - eluted peptides were mass analyzed on an ltq velos pro linear ion trap mass spectrometer with a nanoelectrospray ionization source (thermo scientific). full scan (m / z 3002000) spectra were acquired and the top 10 most abundant ions in each ms scan were selected for fragmentation via collision - induced dissociation (cid). tandem mass spectra were converted into dta files using scansifter and searched using a custom version of sequest (thermo fisher scientific) operating on the vanderbilt accre computing cluster. tandem mass (ms / ms) spectra were searched against a concatenated forward and reverse (decoy) database containing the homo sapiens subset of uniprotkb sprot protein database (www.uniprot.org in the public domain). additional search parameters included : trypsin enzyme specificity, monoisotopic masses were used for searching product ions, and oxidation of methionine, carbamidomethylation of cysteine, and phosphorylation of serine, threonine and tyrosine were allowed as variable modifications. scaffold 4.3.4 (proteome software, portland, or, usa) was used to summarize and validate search results, where a minimum probability threshold of 95% was required for peptide identifications and data were filtered to a false - discovery rate (fdr) of < 1% at the protein level. peptide abundance in each region was compared using normalized spectral counts and a student 's t - test was used to determine whether differences between the df and rz were statistically significant (p < 0.05). proteins with statistically significant changes near the rz were selected for further quantitation using mrm. representative peptides for each protein were selected based on their appearance across multiple samples in the discovery phase of analysis described above. heavy - labeled peptide standards were synthesized by jpt (spiketides tql peptides, jpt, berlin, germany). peptides contained isotopically labeled terminal arginine or lysine residues (c and n) and a trypsin - cleavable c - terminal tag. peptide standards were used to generate a calibration curve, which confirmed a linear response between 1.5 and 100 fmol/l (data not shown). after membrane enrichment and before enzymatic digestion, isotopically labeled peptides were spiked into samples at approximately endogenous levels. skyline software (university of washington, maccoss lab) was used to set up scheduled, targeted mrm methods and four to five ms / ms transitions were monitored per peptide. for targeted proteomics, 6 10 m tissue was collected and membranes were prepared as described above. prior to trypsin digestion, the pellet was resuspended in 8 m urea (100 mm tris buffer, ph 8), then diluted with 100 mm tris to 2 m urea before addition of 1 l of 0.1 g/l trypsin (mass spectrometry - grade, pierce) in a total volume of 10 l. digestion proceeded overnight at 37c and was stopped with the addition of a small amount of neat formic acid. the 10 l digest was transferred to a reduced - volume autosampler vial and 2.5 l sample per run was injected via autosampler (nanoacuity hplc system, waters) onto a vented column setup utilizing a 40 mm by 0.1 mm (jupiter 5 micron, 300a) kasil fritted trap followed by a 200 mm by 0.1 mm (jupiter 3 micron, 300a), self - packed analytical column coupled directly to a tsq - vantage (thermo scientific) via a nanoelectrospray source. after trapping and equilibration, peptides were resolved using a 90-minute aqueous to organic gradient (solvent a = 0.1% fa in water and b = 0.1% fa in acn) operating at 400 nl / min. a series of unscheduled runs determined retention times and the most useful transitions to monitor and then a scheduled instrument method encompassing a 6-minute window around the measured retention time along with calculated collision energies was created using skyline. q1 peak width resolution was set to 0.7, collision gas pressure was 1 mtorr, and utilized an ez method cycle time of 3 seconds. the resulting raw instrument files were imported into skyline for peak - picking and quantitation. transition or fragment ion peak areas were summed to represent the intensity of endogenous peptides, which were normalized to the internal standard. for statistical analysis, a 1-way anova was performed with a post hoc multiple comparison tukey test using spss software (ibm, armonk, new york, usa). proteomics - grade porcine trypsin, ethanol, high - performance liquid chromatography (hplc)-grade acetonitrile, formic acid, urea, phosphate buffered saline (pbs), sodium hydroxide, tris(hydroxymethyl)aminomethane (tris), and xylenes were purchased from sigma - aldrich corp. (st. louis, mo, usa). acetic acid, ammonium bicarbonate, dithiothreitol (dtt), chloroacetamide (claa), hplc - grade h2o with 0.1% formic acid, hplc - grade h2o, and hplc - grade acetonitrile with 0.1% formic acid were from fisher scientific (pittsburgh, pa, usa). fresh human lenses were obtained from the national disease research interchange (philadelphia, pa, usa). three lenses were analyzed in total : 21-year (m), 22-year (m), and 27-year (f). lenses were stored on wet ice during shipment and fixed immediately upon arrival. a solution of 75:25 ethanol : acetic acid was used to lightly fix tissue while avoiding traditional formaldehyde crosslinking that can interfere with mass spectrometry experiments. whole lenses were fixed for 12 hours, manually cut in half across the equator using a razor blade, and fixed for an additional 12 hours. eight micron sections were cut and collected using a microm hm 325 microtome (thermo scientific, waltham, ma, usa) and allowed to dry on glass slides overnight at 37c. sections were deparaffinized prior to labeling, using a series of xylene and alcohol washes (xylenes 3, 100% ethanol, 95% ethanol, 70% ethanol, pbs 5 minutes each). to visualize the rz, lenses were incubated in wheat germ agglutinin - tritc (life technologies, eugene, or, usa), using a 1 mg / ml stock solution at 1:100 dilution for 2 hours, followed by three, 5-minute pbs washes. sections used for confocal imaging were covered with vectashield mounting medium (vector laboratories, burlingame, ca, usa) and sealed with a coverslip. laser capture microdissection was performed on a palm microbeam system (carl zeiss microscopy, thornwood, ny, usa). uncovered sections were visualized at 20 magnification using the rhodamine fluorescence setting (540/600 nm excitation / emission). regions of interest were manually selected in the software, based on cellular morphology and measured distance from lens capsule. tissue was cut in brightfield mode and cells from each selected region were catapulted into an eppendorf cap containing 25-l hplc - grade h2o. microdissection was carried out until 2 10 m tissue was collected for each region to ensure approximately equal starting material. in total, tissue from four regions was collected : df (outer fiber cell layers, 0100 m from lens capsule) ; rz (100140 m from lens capsule) ; tz (140240 m from lens capsule) ; ic (240 m and beyond). tissue was then spun down into the tube and frozen for side - by - side membrane preparation of all four regions. for each lens, a total of three replicates of df, rz, tz, and ic were collected for initial experiments. collected cells were washed to prepare membranes and to remove soluble proteins, as described previously. samples were first reduced (25 mm dtt, 56c for 1 hour) and alkylated (55 mm claa, room temperature, 45 minutes in dark) before a series of washing and centrifugation steps to remove soluble proteins. the sample was vortexed and incubated for 30 minutes in homogenizing buffer containing 8 m urea (50 mm ammonium bicarbonate, 5 mm edta, 10 mm naf, 1 mm dtt, ph 8), followed by 20-minute centrifugation at 100,000 g (sorvall mtx150 micro ultracentrifuge, thermo scientific). the supernatant was removed and the pellet was saved and washed further using the following procedure : homogenizing buffer containing 8 m urea (three times total), 0.1 m naoh (one time), hplc - grade h2o (one time), 95% ethanol (one time), and hplc - grade h2o (three times). prior to trypsin digestion, the pellet was resuspended in 50% tfe in 50 mm ammonium bicarbonate, then diluted to 5% tfe before addition of 1 l of 0.1 g/l trypsin (pierce) in a volume of 100 l. digestion proceeded overnight at 37c and was stopped with the addition of 0.2 l neat formic acid. for each tissue region, the entire sample was bomb - loaded onto a reverse - phase 360 m outer diameter (o.d.) 100 m inner diameter (i.d.) capillary trap column (3 cm length/5 m jupiter c18 beads, 300, phenomenex) in - line with a 360 m o.d. reverse - phase analytical column packed with 20 cm jupiter c18 beads (3 m, 300, phenomenex) and equipped with a laser - pulled emitter tip. using an eksigent nanolc - ultra hplc system, peptides were eluted at a flow rate of 500 nl / min over a 120-minute gradient of 0.1% formic acid in water (solvent a) and 0.1% formic acid in acetonitrile (solvent b). the gradient consisted of 2% to 10% b in 20 minutes, 10% to 30% b in 30 minutes, 30% to 95% b in 15 minutes, 95% b for 15 minutes, followed by equilibration at 2% b. gradient - eluted peptides were mass analyzed on an ltq velos pro linear ion trap mass spectrometer with a nanoelectrospray ionization source (thermo scientific). the instrument was operated using a data - dependent method with dynamic exclusion enabled. full scan (m / z 3002000) spectra were acquired and the top 10 most abundant ions in each ms scan were selected for fragmentation via collision - induced dissociation (cid). tandem mass spectra were converted into dta files using scansifter and searched using a custom version of sequest (thermo fisher scientific) operating on the vanderbilt accre computing cluster. tandem mass (ms / ms) spectra were searched against a concatenated forward and reverse (decoy) database containing the homo sapiens subset of uniprotkb sprot protein database (www.uniprot.org in the public domain). additional search parameters included : trypsin enzyme specificity, monoisotopic masses were used for searching product ions, and oxidation of methionine, carbamidomethylation of cysteine, and phosphorylation of serine, threonine and tyrosine were allowed as variable modifications. scaffold 4.3.4 (proteome software, portland, or, usa) was used to summarize and validate search results, where a minimum probability threshold of 95% was required for peptide identifications and data were filtered to a false - discovery rate (fdr) of < 1% at the protein level. peptide abundance in each region was compared using normalized spectral counts and a student 's t - test was used to determine whether differences between the df and rz were statistically significant (p < 0.05). proteins with statistically significant changes near the rz were selected for further quantitation using mrm. representative peptides for each protein were selected based on their appearance across multiple samples in the discovery phase of analysis described above. heavy - labeled peptide standards were synthesized by jpt (spiketides tql peptides, jpt, berlin, germany). peptides contained isotopically labeled terminal arginine or lysine residues (c and n) and a trypsin - cleavable c - terminal tag. peptide standards were used to generate a calibration curve, which confirmed a linear response between 1.5 and 100 fmol/l (data not shown). after membrane enrichment and before enzymatic digestion, isotopically labeled peptides were spiked into samples at approximately endogenous levels. skyline software (university of washington, maccoss lab) was used to set up scheduled, targeted mrm methods and four to five ms / ms transitions were monitored per peptide. for targeted proteomics, prior to trypsin digestion, the pellet was resuspended in 8 m urea (100 mm tris buffer, ph 8), then diluted with 100 mm tris to 2 m urea before addition of 1 l of 0.1 g/l trypsin (mass spectrometry - grade, pierce) in a total volume of 10 l. digestion proceeded overnight at 37c and was stopped with the addition of a small amount of neat formic acid. the 10 l digest was transferred to a reduced - volume autosampler vial and 2.5 l sample per run was injected via autosampler (nanoacuity hplc system, waters) onto a vented column setup utilizing a 40 mm by 0.1 mm (jupiter 5 micron, 300a) kasil fritted trap followed by a 200 mm by 0.1 mm (jupiter 3 micron, 300a), self - packed analytical column coupled directly to a tsq - vantage (thermo scientific) via a nanoelectrospray source. after trapping and equilibration, peptides were resolved using a 90-minute aqueous to organic gradient (solvent a = 0.1% fa in water and b = 0.1% fa in acn) operating at 400 nl / min. a series of unscheduled runs determined retention times and the most useful transitions to monitor and then a scheduled instrument method encompassing a 6-minute window around the measured retention time along with calculated collision energies was created using skyline. q1 peak width resolution was set to 0.7, collision gas pressure was 1 mtorr, and utilized an ez method cycle time of 3 seconds. the resulting raw instrument files were imported into skyline for peak - picking and quantitation. transition or fragment ion peak areas were summed to represent the intensity of endogenous peptides, which were normalized to the internal standard. for statistical analysis, a 1-way anova was performed with a post hoc multiple comparison tukey test using spss software (ibm, armonk, new york, usa). although traditional fixation enables excellent antibody and lectin labeling of lens fiber cells, it causes protein - protein crosslinking that can interfere with traditional mass spectrometry proteomic analysis that uses trypsin to cleave at basic residues. thus, we attempted alternative mass spectrometry - compatible fixation methods that could preserve cellular structure, including the lens capsule and outer cell layers. fresh human lenses were lightly fixed in 75:25 ethanol : acetic acid for a total of 24 hours. wheat germ agglutinin - labeled paraffin sections showed the outer cell layers remained intact and cellular morphology could be visualized. confocal fluorescence microscopy images confirmed the presence of the rz approximately 100 m beneath the lens capsule (fig. a return to more ordered cellular organization is visible in the tz (140240 m from lens capsule) where cellular compaction begins. a test section was processed and analyzed by lc - ms / ms to ensure this fixation did not interfere with trypsin digestion and mass spectrometry (data not shown). lens was ethanol - fixed and sectioned, followed by labeling with wga - tritc to highlight cell membranes. the ordered differentiating fiber cells (df) rapidly become morphologically more complex in the rz before radial cell columns are again visible in the tz. after light fixation and wga labeling, lens sections were visualized on the lcm instrument using the fluorescence microscope setting. regions and approximate measurements included differentiating fiber cells (df, outer fiber cell layers, 0100 m from lens capsule) ; rz (100140 m from lens capsule) ; tz (140240 m from lens capsule) ; ic (240 m and beyond) ; 2 10 m of tissue was collected per region, which is equivalent to approximately 80,000 fiber cell cross - sections based on average fiber cell diameter. after each sample was prepared to isolate integral membrane and membrane - associated proteins, the proteins were digested with trypsin and were analyzed by lc - ms / ms. shotgun proteomics results identified expected lens proteins, ranging from crystallins to cytoskeletal proteins (full protein list for 22y lens in supplementary table s1). compared to conventional tissue homogenization proteomics experiments, total spectral counts from our lcm samples are quite low due to the very small amount of tissue captured and prepared for proteomic analysis. the small amount of tissue is also likely why naturally abundant lens fiber major intrinsic protein (aqp0) only appears in the rz region at low spectral counts given that trypsin efficiency is likely low for this integral membrane protein. to identify proteins that were changing in the rz, we initially performed the student 's t - test to compare peptide spectral counts between the df and rz regions. statistically significant changes were detected for the if protein vimentin, which decreases by approximately 80% from the df to the rz (fig. 2) and decreases further in the tz and ic. a concurrent increase in the beaded filament proteins filensin and phakinin is observed at the rz, suggesting a switch of ifs occurs near the rz. other cytoskeletal proteins including periplakin also change significantly, while spectrins and several crystallins remained fairly constant across the regions analyzed. these results were used to develop a targeted quantitative mass spectrometry assay for more accurate quantitation of proteins of interest with significant changes at the rz. indicates a statistically significant difference detected between the df and rz regions (p < 0.05). to more accurately quantify protein changes around the rz, we adopted a targeted proteomics strategy, using mrm for protein quantitation. in this experiment, known concentrations of heavy - labeled isotopes for each peptide of interest that differ in mass from the endogenous peptide but share physiochemical properties and behave similarly in the instrument were spiked into samples. absolute endogenous peptide concentrations can be calculated from the measured ratio of the endogenous peptide to its heavy - labeled counterpart and the known concentration of the heavy internal standard. using shotgun proteomics data, one to three representative peptides were chosen for each protein of interest (supplementary table s2). peptides were selected based on several criteria, including identification across several mass spectrometry runs and biological samples, absence of labile or modifiable residues like methionine or cysteine, and ideal peptide length (720 amino acids) for detection by mass spectrometry. standard curves were measured to determine the quantifiable range of the internal standards (data not shown) and the concentration of each internal peptide standard was optimized to approximate endogenous peptide levels. for the final analysis, the 4 regions of interest were isolated from 3 separate lenses (21-, 22-, and 27-year). a larger amount of tissue (6 10 m) was pooled for each region so technical replicates could be run for each sample. an example of the mrm signals observed for an endogenous and heavy - labeled filensin peptide is shown in figure 3. transitions, or peptide fragmentation patterns, selected for mrm of a filensin tryptic peptide (77lgelagpedalar89). precursor masses for endogenous (m / z 656.35, + 2) and heavy (m / z 661.35, + 2) peptides were monitored along with characteristic fragment transitions shown above. relative intensity and retention time of heavy peptide standard transitions (a) are consistent with endogenous peptide (b). targeted, quantitative mrm results generally agreed with shotgun proteomics results, providing more confidence and better quantitation of small abundance changes between the four regions. figure 4 shows the relative intensity of peptides quantified from 3 different lenses (two technical replicates per lens). intensities are normalized to the region with the highest peptide concentration ; other regions are expressed as a percentage of the highest value. statistically significant changes were determined using a 1-way anova with tukey test independently for each peptide. both vimentin peptides decreased 80% from the df to the rz, in agreement with shotgun proteomics data, and continued to decrease in the tz and ic (fig. 4a). periplakin and periaxin follow similar trends, decreasing by 30% to 40% from df to rz and even further in the tz (figs. 4b, 4c). filensin and phakinin, which make up the beaded filament of the lens, increase at least 2-fold at the rz and show slightly variable but decreasing abundance in the tz with a trend toward increasing abundance in the ic (figs. brain acid soluble protein 1 (basp1) follows a similar trend where increasing abundance was observed at the rz compared to the df zone with slightly less protein detected in the tz and ic. supplementary table s3 shows the absolute quantitation of peptides in each region from three separate lenses, where peptide abundances (fmol) calculated represent the peptide quantity in approximately 2 10 m tissue collected. despite some discrepancies in the absolute fmol calculated per lens, which could be due to differences in tissue thickness differences in the absolute amount of peptides from the same protein could be due to differing digestion efficiency, ionization efficiency, peptide solubility, or the presence of posttranslational modifications that affect peptide m / z and therefore signal for targeted peptide analysis. each bar represents six measurements (two technical replicates per lens, three lenses total : 21-, 22-, and 27-year). after calculating fmol values for each representative peptide, the region with the highest signal was set to 1 and other regions expressed as a fraction of that value. a 1-way anova with tukey test was used to determine statistically significant differences between regions. although traditional fixation enables excellent antibody and lectin labeling of lens fiber cells, it causes protein - protein crosslinking that can interfere with traditional mass spectrometry proteomic analysis that uses trypsin to cleave at basic residues. thus, we attempted alternative mass spectrometry - compatible fixation methods that could preserve cellular structure, including the lens capsule and outer cell layers. fresh human lenses were lightly fixed in 75:25 ethanol : acetic acid for a total of 24 hours. wheat germ agglutinin - labeled paraffin sections showed the outer cell layers remained intact and cellular morphology could be visualized. confocal fluorescence microscopy images confirmed the presence of the rz approximately 100 m beneath the lens capsule (fig. a return to more ordered cellular organization is visible in the tz (140240 m from lens capsule) where cellular compaction begins. a test section was processed and analyzed by lc - ms / ms to ensure this fixation did not interfere with trypsin digestion and mass spectrometry (data not shown). lens was ethanol - fixed and sectioned, followed by labeling with wga - tritc to highlight cell membranes. the ordered differentiating fiber cells (df) rapidly become morphologically more complex in the rz before radial cell columns are again visible in the tz. after light fixation and wga labeling, lens sections were visualized on the lcm instrument using the fluorescence microscope setting. regions and approximate measurements included differentiating fiber cells (df, outer fiber cell layers, 0100 m from lens capsule) ; rz (100140 m from lens capsule) ; tz (140240 m from lens capsule) ; ic (240 m and beyond) ; 2 10 m of tissue was collected per region, which is equivalent to approximately 80,000 fiber cell cross - sections based on average fiber cell diameter. after each sample was prepared to isolate integral membrane and membrane - associated proteins, the proteins were digested with trypsin and were analyzed by lc - ms / ms. shotgun proteomics results identified expected lens proteins, ranging from crystallins to cytoskeletal proteins (full protein list for 22y lens in supplementary table s1). compared to conventional tissue homogenization proteomics experiments, total spectral counts from our lcm samples are quite low due to the very small amount of tissue captured and prepared for proteomic analysis. the small amount of tissue is also likely why naturally abundant lens fiber major intrinsic protein (aqp0) only appears in the rz region at low spectral counts given that trypsin efficiency is likely low for this integral membrane protein. to identify proteins that were changing in the rz, we initially performed the student 's t - test to compare peptide spectral counts between the df and rz regions. statistically significant changes were detected for the if protein vimentin, which decreases by approximately 80% from the df to the rz (fig. a concurrent increase in the beaded filament proteins filensin and phakinin is observed at the rz, suggesting a switch of ifs occurs near the rz. other cytoskeletal proteins including periplakin also change significantly, while spectrins and several crystallins remained fairly constant across the regions analyzed. these results were used to develop a targeted quantitative mass spectrometry assay for more accurate quantitation of proteins of interest with significant changes at the rz. indicates a statistically significant difference detected between the df and rz regions (p < 0.05). to more accurately quantify protein changes around the rz, we adopted a targeted proteomics strategy, using mrm for protein quantitation. in this experiment, known concentrations of heavy - labeled isotopes for each peptide of interest that differ in mass from the endogenous peptide but share physiochemical properties and behave similarly in the instrument were spiked into samples. absolute endogenous peptide concentrations can be calculated from the measured ratio of the endogenous peptide to its heavy - labeled counterpart and the known concentration of the heavy internal standard. using shotgun proteomics data, one to three representative peptides were chosen for each protein of interest (supplementary table s2). peptides were selected based on several criteria, including identification across several mass spectrometry runs and biological samples, absence of labile or modifiable residues like methionine or cysteine, and ideal peptide length (720 amino acids) for detection by mass spectrometry. standard curves were measured to determine the quantifiable range of the internal standards (data not shown) and the concentration of each internal peptide standard was optimized to approximate endogenous peptide levels. for the final analysis, the 4 regions of interest were isolated from 3 separate lenses (21-, 22-, and 27-year). a larger amount of tissue (6 10 m) was pooled for each region so technical replicates could be run for each sample. an example of the mrm signals observed for an endogenous and heavy - labeled filensin peptide is shown in figure 3. transitions, or peptide fragmentation patterns, selected for mrm of a filensin tryptic peptide (77lgelagpedalar89). precursor masses for endogenous (m / z 656.35, + 2) and heavy (m / z 661.35, + 2) peptides were monitored along with characteristic fragment transitions shown above. relative intensity and retention time of heavy peptide standard transitions (a) are consistent with endogenous peptide (b). targeted, quantitative mrm results generally agreed with shotgun proteomics results, providing more confidence and better quantitation of small abundance changes between the four regions. figure 4 shows the relative intensity of peptides quantified from 3 different lenses (two technical replicates per lens). intensities are normalized to the region with the highest peptide concentration ; other regions are expressed as a percentage of the highest value. statistically significant changes were determined using a 1-way anova with tukey test independently for each peptide. both vimentin peptides decreased 80% from the df to the rz, in agreement with shotgun proteomics data, and continued to decrease in the tz and ic (fig. 4a). periplakin and periaxin follow similar trends, decreasing by 30% to 40% from df to rz and even further in the tz (figs. 4b, 4c). filensin and phakinin, which make up the beaded filament of the lens, increase at least 2-fold at the rz and show slightly variable but decreasing abundance in the tz with a trend toward increasing abundance in the ic (figs., brain acid soluble protein 1 (basp1) follows a similar trend where increasing abundance was observed at the rz compared to the df zone with slightly less protein detected in the tz and ic. supplementary table s3 shows the absolute quantitation of peptides in each region from three separate lenses, where peptide abundances (fmol) calculated represent the peptide quantity in approximately 2 10 m tissue collected. despite some discrepancies in the absolute fmol calculated per lens, which could be due to differences in tissue thickness, the trends match very well among the three ages studied. differences in the absolute amount of peptides from the same protein could be due to differing digestion efficiency, ionization efficiency, peptide solubility, or the presence of posttranslational modifications that affect peptide m / z and therefore signal for targeted peptide analysis. each bar represents six measurements (two technical replicates per lens, three lenses total : 21-, 22-, and 27-year). after calculating fmol values for each representative peptide, the region with the highest signal was set to 1 and other regions expressed as a fraction of that value. a 1-way anova with tukey test was used to determine statistically significant differences between regions. several different methods have been used to study changes in membrane - associated proteins in different lens fiber cell populations. after gross dissection of whole human lenses, the quantitative proteomics method of itraq was used to assess binding of cytosolic proteins to the plasma membrane with age. the combination of lcm and lc - ms / ms has also been used previously to analyze differences in the bovine cortical and nuclear lens membrane proteome. here, we isolated extremely narrow regions (2030 m) of tissue for quantitative proteomic analysis of the human lens outer cortex to determine molecular changes associated with the extreme morphologic changes of the plasma membrane in the rz. these techniques enable spatially resolved proteomics of regions that can not be separated by manual dissection. although we enriched for the plasma membrane fraction, the majority of proteins detected by proteomic analysis were crystallins and cytoskeletal proteins, likely due to their high abundance and reported association with the plasma membrane, even after treatment with urea. based on these results, we could not assess membrane protein changes and instead focused on cytoskeletal proteins. targeted proteomics analysis enabled relative quantitation of peptides between different lens regions, even those present at low levels. intermediate filaments are a highly conserved family of cytoskeletal proteins that can be organized into several classes. immunohistochemical studies indicate vimentin, a major type iii if protein, is expressed in the lens epithelium and fiber cells, although the expression pattern changes based on lens age and region. our results indicate vimentin is most abundant in the very outer fiber cells of the df region and its abundance decreases in the rz and further into the lens. because we analyzed the plasma membrane fraction, this decrease in vimentin signal at the rz could be due to either reduced vimentin expression, increased solubility of the protein, or decreased association with the plasma membrane. it is possible that loss of vimentin in the rz is due to a shift from membrane to cytoplasm, which has been documented in the literature. studies of adult mouse lens show membrane - specific vimentin localization in outer cortical fibers, while vimentin antibody labeling is both cytoplasmic and membranous further into the lens before signal is lost around the region of nuclei loss. similar labeling patterns have also been documented in bovine lens. furthermore, loss of vimentin antibody labeling has been correlated with nuclei loss in mouse lens. however, we observe a decrease in vimentin signal in the rz, before nuclei are completely lost. it is possible that the drop in vimentin signal in our experiments could be due to changes in vimentin solubility or membrane association. deeper into the lens cortex, vimentin is known to be cleaved, which could affect solubility and detection by both immunohistochemistry and mass spectrometry. phosphorylation of vimentin is known to induce depolymerization of ifs, although this modification was not detected in our studies. although vimentin knockout does not result in any notable phenotype, vimentin overexpression causes cataract, suggesting that proper expression and regulation of vimentin is critical for lens clarity. filensin and phakinin are lens - specific proteins that assemble to make 6 to 8 nm beaded filaments. our results indicate that filensin and phakinin levels increase significantly from the df to the rz, while vimentin decreases. the switch from vimentin ifs to lens - specific beaded filaments containing filensin and phakinin has been documented in several species including bovine and mouse. the control of these two if systems appears to be independently modulated during fiber cell differentiation, as beaded filament expression is detected before vimentin labeling is lost. adult mouse lenses labeled with anti - filensin antibody reveal membraneous labeling in the outer cortex, followed by membranous and cytoplasmic labeling further into the nucleated lens fiber cells. however, filensin is not present in developing embryonic fiber cells until after elongation has progressed, suggesting it plays a role in lens organization after cells have begun differentiation. the decrease in signal from rz to tz could be due to several factors, including altered solubility or membrane association. the functional consequences of beaded filament increase in the rz have not been established. however, tem studies indicate ball and socket joints arise in the rz, and knockout studies indicate beaded filaments are critical for maintaining interdigitations and long - range order of lens fiber cells. therefore, filensin and phakinin may become more associated with the membrane to establish interlocking domains and a beaded filament system that is maintained further into the lens cortex. the switch from ifs to beaded filaments at the rz suggests an important role for lens - specific ifs. periaxin is localized to the plasma membrane, concentrated mostly at tricellular junctions where beaded filaments and vimentin labeling also occur. periaxin is critical for hexagonal geometry and membrane stabilization, perhaps functioning as a scaffolding protein in lens. periplakin, an if - associated protein (ifap), has been shown to bind both ifs and beaded filaments, directly interacting with vimentin and phakinin. periplakin and periaxin are also part of the eppd (ezrin, periaxin, periplakin, desmoyokin) junction complex in lens. although their roles are not well understood, both periaxin and periplakin could anchor ifs to the plasma membrane through protein - protein interactions. aqp0 has been demonstrated to interact with beaded filaments, but aqp0 did not pull down with periplakin - bf complexes in previous studies, suggesting there may be another protein anchor connecting periplakin and bf to the plasma membrane. brain acid soluble protein 1 has been detected in lens but little is known about its function in this tissue. in the brain, the growth - associated protein basp1 is localized to the plasma membrane at the tip of elongating axons. although the role of basp1 in lens has not been established, the increase in basp1 levels at the rz is not surprising given its function in regulating the actin cytoskeleton in brain. changes in protein - protein interaction, that is, between periplakin and vimentin, could alter if attachment to the plasma membrane, serving as a way to modulate the lens cytoskeleton and thus influence membrane shape in the rz. actin polymerization could also be driving the formation of cellular interdigitations that originate in the rz, as branching f - actin and the clathrin / ap-2 complex were shown to associate with interlocking domains in rat and monkey lenses. although we do not detect major changes in actin or clathrin abundance in our studies, their involvement in rz morphology can not be ruled out. it is possible that changes in actin polymerization or interactions with other proteins, in addition to basp1, are important but remain undetected in our quantitative proteomics studies. additional work is required to understand how these proteins might be involved in membrane reorganization in the rz. although the switch from vimentin to beaded filaments has been documented in lens, this work is the first to show a rapid if change associated with the human lens rz. our proteomics results do not definitively prove that the if switch is responsible for the morphologic changes of the rz, but suggest the involvement of beaded filaments in establishing the ball and socket joints that originate in the rz. this notion is supported by the loss of flap structures and long - range fiber cell stacking in the deep cortex of beaded filament knockout mice. changes to the if system could allow remodeling of the plasma membrane before the fiber cells return to a more ordered arrangement in the tz. this phenomenon has only been observed in human and macaque lenses, suggesting that accommodation in long - lived primates could require more extensive preparation of interdigitations between lens fiber cells. future work should examine human lenses with cataract - causing mutations in beaded filament genes to determine whether the formation of the rz is affected. using spatially resolved proteomics, we characterized protein changes in the narrow rz and surrounding regions of the human lens outer cortex. membrane - associated ifs switch from vimentin to beaded filaments in this region, with changes in other ifaps, suggesting involvement of the cytoskeleton and associated proteins in the dramatic membrane remodeling in the rz. consistent quantitative results between three separate lenses suggest the rz region is part of a tightly - regulated differentiation process in human lens. future work is necessary to characterize the function and necessity of the rz for normal human lens physiology. | purposeto quantify protein changes in the morphologically distinct remodeling zone (rz) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics.methodslightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (wga - tritc). morphology directed laser capture microdissection (lcm) was used to isolate tissue from four distinct regions of human lens outer cortex : differentiating zone (df), rz, transition zone (tz), and inner cortex (ic). liquid chromatography - tandem mass spectrometry (lc - ms / ms) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. peptides from proteins of interest were quantified using multiple reaction monitoring (mrm) mass spectrometry and isotopically - labeled internal peptide standards.resultsresults revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the rz. several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the df to the ic, while filensin, phakinin, and brain acid soluble protein 1 (basp1) increase significantly at the rz.conclusionsmass spectrometry - compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. results reveal dramatic cytoskeletal protein changes associated with the rz, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human rz. |
antitumor necrosis factor - alpha (tnf-) agents are widely used for effective treatment of autoimmune rheumatic and dermatological diseases such as rheumatoid arthritis (ra), ankylosing spondylitis (sa), psoriasis (ps), or psoriatic arthritis (psa). nevertheless, anti - tnf- agents have been associated with growing number of adverse events, particularly infections [1, 2 ] of which some can be life threatening. tnf- is an important proinflammatory cytokine in the host defense mechanism against many intracellular pathogens. it suppresses hepatitis b virus (hbv) replication and promotes hbv eradication by stimulating hbv - specific cytotoxic t - cell response [35 ]. it has been reported that reactivation of hbv infection may occur directly due to lack of tnf- or indirectly via diminishing t - cell activation [6, 7 ]. tnf- inhibitors are therefore likely to induce hbv replication and reactivation in cases when chronic infection is present. hbv is regarded as a leading cause of acute hepatitis, cirrhosis, and hepatocellular carcinoma, being responsible for about 600000 deaths every year. chronic hbv infection is defined as an overt when hepatitis b surface antigen (hbsag) is detectable in the serum. patients who present antibodies to hepatitis b core antigen (anti - hbc) with concurrent hbsag negativity do not have chronic hepatitis but only experienced hbv infection and were able to clear it. nevertheless, some of these patients may be occult carriers, harboring intrahepatic hbv replication, and therefore can be susceptible to hbv reactivation. hepatitis b virus (hbv) reactivation in patients treated with anti - tnf- agents has been frequently reported in the last decade, with inconsistent results. considering the high socioeconomic burden of hbv infection related conditions, as well as increasing role of anti - tnf- agents in treatment of autoimmune rheumatic and dermatological diseases, it is highly important to estimate the impact of anti - tnf- agents to hbv reactivation in these patients. we have conducted a systematic review and meta - analysis in order to assess the prevalence of hbv reactivation among patients treated with anti - tnf- agents because of ra, sa, ps, and psa. we conducted comprehensive literature search of medline, scopus, and isi web of knowledge databases using the following search : ([infliximab ] or [rituximab ] or [etanercept ] or [adalimumab ] or [abatacept ] or [anti - tnf ]) and [hbv reactivation ]. the search was limited to human subjects with language restriction to english studies until 1st september 2013. the snowball strategy, including manual search of the references listed by studies retrieved from the online databases and from previously published systematic reviews, was also performed to identify potential additional studies. the eligibility criteria for inclusion in the review implied that (i) patients should be affected by at least one of the following diseases : rheumatoid arthritis (ra), ankylosing spondylitis (sa), psoriasis (ps), or psoriatic arthritis (psa) ; (ii) study must refer to treatment with one or more of the following biologic agents : infliximab, rituximab, etanercept, adalimumab, and abatacept ; (iii) the hbv serological status of patients prior to the pharmacological treatment and the prevalence of hbv reactivation after the treatment should be reported. studies were excluded if it included only hepatitis c virus (hcv) infected patients (other than those also coinfected with hbv). data from the included studies were independently extracted by two investigators (np and el) and entered into an excel 2010 (microsoft corp., any discrepancies regarding individual study inclusion, data extraction, and interpretation were resolved by consulting a third investigator (sb). we extracted the following data : first author name, year of publication, number of patients, mean age, and gender of patients. further we extracted data on the hbv serological status of patients, medical conditions for which patients were treated, the biologic agent used, the presence of other disease modifying antirheumatic drugs (dmards), and antiviral prophylaxis. the main outcome used in the meta - analysis was the prevalence of hbv reactivation reported as prevalence proportion. the studies with sample size less than 15 were not included in meta - analysis. additionally, when available, we collected individual - level data on hbv reactivation, namely, age, gender, condition treated for, anti - tnf agent used, and, if any, antiviral prophylaxis. based on this data we performed additional meta - analyses according to diseases and biologic agents when possible. based on the serological status, we stratified results of the meta - analysis according to two main subgroups of patients expected to have different prevalence hbv reactivation : patients presenting with overt chronic hbv infection (being hbsag positive) and patients presenting with occult hbv infection (being hbsag negative and anti - hbc positive). based on information reported in the article full text and tables, it was always possible to obtain the data on the subgroups. to determine pooled proportions, the variances of the raw proportions (r / n) were stabilized using a freeman - tukey - type arcsine square root transformation : y = arcsine[(r/(n + 1)) ] + arcsine[(r + 1)/(n + 1) ], with a variance of 1/(n + 1), where n is the denominator for population size. pooled proportions from all studies were calculated as the back transform of the weighted mean of the transformed proportions, using a random - effects model. we assessed heterogeneity among studies using the cochran q test and quantified inconsistencies across studies and their impact on the analysis by using the i statistic [24, 25 ]. as we anticipated large heterogeneity considering the very different clinical presentation of patients, we considered statistically significant heterogeneity when pheterogeneity < 0.1. the robustness of pooled proportions was explored by conducting sensitivity and subgroup analyses. the results of the literature search are reported in a flow chart (figure 1). after searching 3 databases we identified 632 relevant articles. additionally, 4 more relevant papers were identified through reference search of relevant systematic reviews. after removing duplicates, 226 remaining abstracts were examined, and further 161 were excluded because of being unrelated to the subject, 34 were excluded because they referred to medical conditions other than those named in inclusion criteria, and 5 were systematic reviews or meta - analyses. finally when we retrieved full text of the remaining 26 papers, 5 were excluded as they were case reports. the characteristics of the 21 studies included in the systematic review are reported in table 1 [1120, 2636 ]. six studies included patients with overt chronic hbv infection and 9 included patients with occult hbv infection, while 6 studies addressed both groups of patients. fourteen studies included patients treated with biologic agents because of rheumatoid arthritis (66.7%), 6 (28.6%) patients treated because of ankylosing spondylitis, 5 (23.8%) because of psoriasis, and 4 (19.0%) because of psoriatic arthritis. in 18 (85.7%) studies patients were treated with etanercept, in 16 (76.2%) with infliximab, in 17 (81.0%) with adalimumab, and in 3 (14.3%) with rituximab. in eleven (52.4%) studies antiviral prophylaxis eleven studies (57.1%) out of 21 were excluded because of the small sample size (< 15 subjects), so the analysis was restricted to 10 studies. the result of the meta - analysis is reported in table 2, with a pooled estimate of hbv reactivation being 4.2% (95% ci : 1.48.2%, i : 74.7%), with significant heterogeneity among studies. in the subgroup analyses, the pooled prevalence of hbv reactivation among patients with occult infection was 3.0% (95% ci : 0.67.2, i : 77.1%) and the pooled prevalence of hbv reactivation among patients with overt hbv infection was 15.4% (95% ci : 1.241.2%, i : 79.9%). in both subgroups, high heterogeneity was present (table 2). by restricting the meta - analysis to patients with rheumatoid arthritis the pooled prevalence of hbv reactivation was 3.3% (95% ci : 0.77.5%, i : 62.6%), with significant heterogeneity among studies (table 3). when these results were stratified according to occult or overt infection, results show that the hbv reactivation for patients with occult infection was 2.6% (95% ci : 0.46.6%, i : 59.2%), compared with 10.7% (95% ci : 1.450.2%, we also addressed the prevalence of hbv reactivation in relation to the anti - tnf agent used. the pooled prevalence of hbv reactivation among patients treated with etanercept was 3.9% (95% ci : 1.18.4%, i : 51.1%) and 3.0% (95% ci : 0.57.6%, i : 49.6%) for those with occult hbv infection (table 4). patients treated with adalimumab showed a pooled prevalence of hbv reactivation of 4.6% (95% ci : 0.512.5%, i : 28.7%) (table 5). no case of hbv reactivation was recorded in studies eligible for meta - analysis among 81 patients treated with infliximab (80 occult carriers and 1 overt). finally we pooled data on hbv reactivation rate in relation to usage of antiviral prophylaxis. pooled hbv reactivation rate was 4.0% (95% ci : 1.28.3%, i : 75.6%) for patients without any antiviral prophylaxis (table 6). analysis on patients submitted to antiviral prophylaxis as well as stratification on occult and overt carriers was not possible because small numbers of cases were included. based on meta - analysis we conducted, we report relatively low pooled prevalence of hbv reactivation in patients treated with anti - tnf- agents for rheumatic and dermatological conditions. the pooled reactivation rate for all patients included, as well as for those with ra, was several times higher in chronic overt hbv carriers compared to occult carriers. the pooled reactivations rates for those treated with etanercept and adalimumab were similar and also similar to overall pooled reactivation rate. the pooled reactivation rate for patients with no antiviral prophylaxis did not differ from overall pooled reactivation rate. several studies addressed the issue whether anti - tnf- agent induced hbv reactivation, with inconsistent results. we report considerably low overall pooled hbv reactivation rate of 4.2% compared to some authors before, who included both overt and occult patients with reported reactivation rate up to 6.8%. having this in mind, it could be said that some of previous studies overestimated the risk of hbv reactivation in patients treated for rheumatic and dermatological conditions. this is especially true for the patients with occult infection as kim. and urata. previously reported hbv reactivation rate among occult carriers of 15.6% and 9.6%, respectively, while pooled rate for occult carriers from our analysis is only 3.0%. however, when interpreting these results, substantial heterogeneity among the studies should be taken into account. for example, study by kim. used a 2-fold or greater increase in liver function test as the criteria for reactivation, while most of the others tried to detect hbv dna. furthermore regional differences in hbv infection prevalence could also influence the observed reactivation rates because of small samples size. it is possible that presence of certain comorbidities, such as diabetes mellitus or obesity, or some other clinical parameters mediate the risk for hbv reactivation. however no study reported these data so we hope that studies to come will be more informative. we observed a higher prevalence of hbv reactivation among patients with chronic infection compared to those with the occult one. this is reasonable and expected, as patients positive with anti hbc antibodies and negative with hbsag do not necessary harbor undetected intracellular hbv replication. however, the high pooled reactivation rate among overt carriers should be having implication on clinical guidelines, as we consider patients with detectable hbsag to be eligible for antiviral prophylaxis in order to prevent anti - tnf- induced hbv reactivation. unfortunately, we were not able to confirm this in our meta - analysis as numbers of patients included were too few to distinguish between the overt carriers subjected and not subjected to antiviral prophylaxis. instead we were able to calculate only the pooled reactivation rate for all patients nontaking antiviral prophylaxis, irrespective to their hbv infection status. this rate did not differ significantly from the overall pooled one, but the vast majority of cases included were the occult carriers. the future studies including considerable numbers are needed in order to clearly define the criteria for antiviral prophylaxis in patients treated with anti - tnf- agents at risk for hbv reactivation. we also tried to estimate whether hbv reactivation rate is dependent on anti - tnf- agents used, as well as on underlying condition. however it was possible only to conduct subanalysis on patients treated with etanercept and adalimumab, as on those treated for ra. as observed pooled reactivation rates did not show considerable difference among the subgroups ; we did not find any of these to determine patients ' susceptibility towards hbv reactivation. still, if such an association exists it needs to be confirmed in studies to come. we have included only patients treated with anti - tnf- agents for rheumatic and dermatological conditions. this limits external validity of our findings, as anti - tnf- agents are also used for treatment of other autoimmune conditions, such as inflammatory bowel diseases, and are part of chemotherapeutic protocols in treatment of b - cell lymphomas. however, with limiting the analysis on specific subgroup of patients we aimed to decrease heterogeneity and therefore make the results more reliable. further, we believe that certain comorbidities, such as diabetes and obesity, could mediate a risk for hbv reactivation among patients treated with anti - tnf- agents. unfortunately none of the studies reported them, so we were not able to include them in the analysis. we were also unable to conduct subanalysis on patients treated with infliximab and rituximab and to distinguish the pooled reactivation rates among overt and occult carriers treated with antiviral prophylaxis. finally, there is a question of heterogeneity among studies in relation to criteria defining hbv reactivation, which could lead to differences in interpretation of the collected data. nevertheless, we find our results valuable to the clinicians encountering patients in risk for hbv reactivation being in need for anti - tnf- agents in everyday practice. in conclusion, although hbv reactivation rate appears to be relatively low in patients treated with anti - tnf- agents for rheumatic and dermatological conditions, the antiviral prophylaxis is recommended in patients with overt chronic hbv infection. more informative studies including large number of cases are needed in order to identify if any patient or treatment related factor mediates the reactivation risk. the individual approach and close monitoring of each patient could be an answer in balancing the need for therapy with hazard associated with hbv reactivation. | introduction. antitumor necrosis factor - alpha (tnf-) agents are widely used for treatment of rheumatic and dermatological diseases. we conducted the systematic review and meta - analysis to assess the prevalence of hbv reactivation among patients treated with anti - tnf-. methods and findings. a comprehensive literature search of medline, scopus, and isi web of knowledge databases was conducted. from 21 studies included in the systematic review, 9 included patients with occult chronic hbv infection and 6 included patients with overt infection while 6 addressed both groups. based on 10 studies eligible for meta - analysis we report pooled estimate of hbv reactivation of 4.2% (95% ci : 1.48.2%, i2 : 74.7%). the pooled prevalence of reactivation was 3.0% (95% ci : 0.67.2, i2 : 77.1%) for patients with occult infection, and 15.4% (95% ci : 1.241.2%, i2 : 79.9%) for overt infection. the prevalence of reactivation was 3.9% (95% ci : 1.18.4%, i2 : 51.1%) for treatment with etanercept and 4.6% (95% ci : 0.512.5%, i2 : 28.7%) for adalimumab. for subgroup of patients without any antiviral prophylaxis the pooled reactivation was 4.0% (95% ci : 1.28.3%, i2 : 75.6%). conclusion. although hbv reactivation rate is relatively low in patients treated with anti - tnf- for rheumatic and dermatological conditions, the antiviral prophylaxis would be recommended in patients with overt chronic hbv infection. |
there have been multiple reported cases of bilateral quadriceps tendon ruptures (qtr) in the literature. these injuries frequently associated with delayed diagnosis, which results in delayed surgical treatment. in very unusual cases, bilateral qtrs can be associated with other simultaneous tendon ruptures. we present a rare case of bilateral qtr with a simultaneous achilles tendon rupture involving a 31 years old caucasian man who is a semi - professional body builder taking anabolic steroids. to date bilateral qtr with additional ta rupture has only been reported once in the literature and to our knowledge this is the first reported case of bilateral qtr and simultaneous ta rupture in a young, fit and healthy individual. the diagnosis of bilateral qtr alone can sometimes be challenging and the possibility of even further tendon injuries should be carefully assessed. a delay in diagnosis could result in delay in treatment and potentially worse outcome for the patient. there have been multiple reported cases of bilateral quadriceps tendon ruptures (qtr) in the literature. the diagnosis of bilateral qtr can sometimes be challenging and the possibility of any further tendon injuries should be carefully assessed. to date bilateral qtr with additional ta rupture has only been reported once in the literature and to our knowledge this is the first reported case of bilateral qtr and simultaneous ta rupture in a young, fit and healthy individual. a 31 years old male patient attended the accident and emergency department after he had fallen directly onto both knees the day before. he complained of pain in both knees and examination revealed effusion and pain over bilateral quadriceps tendons. there was a tender palpable suprapatellar gap on the left and to a lesser extent on the right side. there were bilateral lack of active extension and the patient was not able to straight leg raise bilaterally. (figure 1 and 2) ap radiographs of the knees lateral radiographs of both knees the clinical diagnosis of bilateral quadriceps tendon rupture was confirmed by urgent ultrasonography and the patient was admitted for surgical repair the next day. (fig 3 and 4) the patient did not complain of any other symptoms at the time. intraoperative findings consisted of a musculo - tendinous junction tear on the right and an insertional tear of the quadriceps tendon on the left which were surgically repaired with sutures and suture anchors respectively. both knees were immobilised in extension splints and the patient was allowed to fully weight bear with the support of crutches. ultrasound scan confirming left quadriceps tendon rupture ultrasound scan confirming right quadriceps tendon rupture at the first follow up appointment the patient admitted to non compliance to the post operative plan of strict immobilisation and he had taken the splints off to see whether he could move his knees on numerous occasions over the last two weeks. furthermore he mentioned pain in his left calf, which he apparently recognised on the day of discharge from hospital. calf squeeze test as described by simmonds revealed no plantar flexion of the foot on compressing the calf muscles. an urgent ultrasound scan of the left ta was organised and confirmed a ta rupture. (fig 5) the patient declined admission to the hospital and the left leg was immobilised in an extended above knee equinus cast. the patient did not attend the urgent follow up appointment after the ultrasound scan confirming a ta rupture, but only re - resented six weeks later. there have been more than a hundred cases of bilateral simultaneous quadriceps tendon ruptures reported in the literature. qtr commonly occurs as a result of rapid, eccentric contraction of the muscle with a planted foot and knee partially flexed. patients with bilateral qtr are more likely to present with some predisposing risk factors.[1,6 - 8 ] younger patients are more likely to have one or more risk factors, whereas older patients are more likely to be obese or hypertensive. clinical examination reveals classically the triads of symptoms of acute painful knee swelling, functional loss and a palpable suprapatellar gap. plain radiographs, ultrasound and magnetic resonance imaging are further diagnostic tools which can help the clinician to establish the diagnosis. despite this bilateral qtr there was delay of more than 2 weeks in 51.9% of these patients and delay of more than 3 months in 33.3% of patients. patients have been investigated for various different possible pathologies and an initial misdiagnosis was made. this included guillain - barre syndrome, stroke, neurologic condition, disc prolapsed and psychiatric disease.[11 - 13 ] acute achilles tendon ruptures are also associated with a high incidence of misdiagnosis and values between 20 30 % are mentioned in the literature. although the diagnosis of ta rupture is not difficult and almost solely based on the history and clinical findings, misdiagnosis can be the result of inconsistence of clinical findings and the insignificance of presenting symptoms, but also due to failure of the patient to seek medical care following injury. a literature search on the ovid database including the keywords : quadriceps tendon, achilles tendon, rupture, simultaneous, and bilateral identified only one report of simultaneous bilateral avulsion of the quadriceps and achilles tendons in 1 limb and of the patellar tendon in the other. this patient had previous bilateral nephrectomies performed, was on chronic haemodialysis and underwent a parathyroidectomy six days after sustaining the tendon ruptures. to our knowledge this is the first reported case of bilateral qtr and simultaneous ta rupture in a young, fit and healthy individual with the only risk factor being the use of oral anabolic steroids. tendon ruptures commonly occur amongst the athletes as a result of overuse inflammation and partial tears within the tendons, which predispose to failure. however, pathogenesis of tendon ruptures in patients on anabolic steroids differs from regular failures. anabolic steroid usage results in reduction of collagen cross - linkage in musculo - tendinous units thereby reducing the ultimate threshold for failure of the tendon. [16 - 17 ] overall, early surgical treatment does result in satisfactory functional outcome for patients. neubauer in their meta - analysis of bilateral qtr identified that any patients who were operated on after 2 weeks following injuries has a lower percentage of success rate in achieving full recovery. scuderi in their series of 20 patients with quadriceps tendon rupture also identified the benefits of early repair. hence, it is advisable that these patients be operated on within 2 weeks of onset of injuries. this made prompt diagnosis of the condition crucial in determining final outcome of these patients. the diagnosis of bilateral quadriceps tendon injuries should therefore lead to a high clinical suspicion of further tendon injuries, even if the patient do not report any symptoms. although rare, patients should be assessed thoroughly with good complete history taking and clinical examination. | introduction : there have been multiple reported cases of bilateral quadriceps tendon ruptures (qtr) in the literature. these injuries frequently associated with delayed diagnosis, which results in delayed surgical treatment. in very unusual cases, bilateral qtrs can be associated with other simultaneous tendon ruptures.case report : we present a rare case of bilateral qtr with a simultaneous achilles tendon rupture involving a 31 years old caucasian man who is a semi - professional body builder taking anabolic steroids. to date bilateral qtr with additional ta rupture has only been reported once in the literature and to our knowledge this is the first reported case of bilateral qtr and simultaneous ta rupture in a young, fit and healthy individual.conclusion:the diagnosis of bilateral qtr alone can sometimes be challenging and the possibility of even further tendon injuries should be carefully assessed. a delay in diagnosis could result in delay in treatment and potentially worse outcome for the patient. |
the oral administration of red ginseng root (p. ginseng) extracts has long been used to treat various diseases, including liver and kidney dysfunction, hypertension, non - insulin - dependent diabetes mellitus, and postmenopausal disorders, in china, korea, and japan. topical applications have also been used for atopic suppurative dermatitis, wounds, and skin inflammation. the materials for korean red ginseng products are selected from among ginseng roots (panax ginseng ca meyer) carefully cultivated in well - fertilized field for 6 years and then steamed and dried in the sun six times. the red ginseng extract produced by korea ginseng corporation (taejon, korea) is dried and powdered by freezing prior to use. in this paper, we introduce the biological and pharmacological effects of ginsenoside rb1 isolated from red ginseng roots on skin damage in mice. macrophages migrate to an injured area to kill invading organisms and produce cytokines that recruit other inflammatory cells responsible for the diverse effects of inflammation [1, 2 ]. moreover, growth factors and cytokines are central to the wound - healing process [46 ]. thus, the burn wound - healing process is complex, involving inflammatory factors, including monocyte migration and cytokine production, and growth factors and angiogenesis during reepithelialization. vascular endothelial growth factor (vegf) plays an important role in skin tissue repair through angiogenesis during the healing of burn wounds [4, 7, 8 ]. furthermore, it has been demonstrated that chemokines including macrophage inflammatory protein-1(mip-1) and monocyte chemoattractant protein-1 (mcp-1) are expressed at high levels in murine full - thickness dermal wounds at times preceding and coinciding with maximal macrophage infiltration [912 ]. interleukin 1- (il-1) is also known to be released from monocyte - derived macrophages during inflammation and stimulates vegf expression in endothelial cells, keratinocytes, synovial fibroblasts, and colorectal carcinoma cells [1316 ].. found that the expression of mcp-1 of macrophage and keratinocyte origin correlated with the accumulation of mast cells during wound healing. weller. reported that mast cell activation and histamine release were required for wound healing. numata. showed that the accelerated wound - repair activity of histamine was mediated by the activity of basic fibroblast growth factor (bfgf), which leads to angiogenesis, and macrophage recruitment in the wound - healing process. thus, the process of wound repair is thought to be closely associated with the network systems among various cells such as keratinocytes, fibroblasts, macrophages and mast cells, and might be modulated by interactions among chemokines, cytokines, growth factors, and related biofactors secreted from these cells. the genus panax derives its name from the greek words pan (all) and akos (healing). in 1988, kanzaki. reported that an orally administered red ginseng root extract stimulated the repair of intractable skin ulcers in patients with diabetes mellitus and werner 's syndrome in clinical trials.. showed that the local administration of ginseng saponins markedly improved wound healing in diabetic and aging rats. reported that ginsenoside rg1 promoted functional angiogenesis into a polymer scaffold (in vivo) and the proliferation and chemoinvasion of tube - like capillary formation by human umbilical vein endothelial cells (huvecs) through enhanced expression of nitric oxide synthetase, phosphatidylinositol-3 kinase, and the akt pathway (in vitro). furthermore, choi reported that ginsenoside rb2 improved wound healing through its facilitating effects on epidermal cell proliferation, by upregulating the expression of proliferation - related factors. however, sato. found that the intravenous administration of ginsenoside rb2 inhibited metastasis to the lung by inhibiting tumor - induced angiogenesis in b16-bl6 melanoma - bearing mice. thus, there are perplexing contradictions in the reported effects of various ginseng saponins on angiogenic activity as shown in table 1. to clarify these differing effects, we first attempted to examine the effects of various ginseng saponins on wound healing. among six ginseng saponins (ginsenoside rb1, rb2, rc, rd, re, and rg1) (figure 1), we found that ginsenoside rb1 enhanced burn - wound healing most strongly. in summary, we reported the promotion of burn - wound healing by the topical application of ginsenoside rb1 at low doses (100 fg, 10 pg, and 1 ng per wound) to be due to the promotion of angiogenesis during skin wound repair through stimulation of vegf production and an increase in hypoxia - inducible factor (hif-) 1 expression in keratinocytes and the elevation of interleukin (il-) 1 from macrophage accumulation in the burn wound area. furthermore, we found the facilitating effects of ginsenoside rb1 at low doses (100 fg, 10 pg, and 1 ng per wound) to be due to the promotion of angiogenesis via the activation of basic fibroblast growth factor (bfgf) through an increase in histamine released from mast cells recruited by the stimulation of monocyte chemoattractant protein-1 (mcp-1) as another mechanism. we will explain our experiments regarding the facilitating effects of ginsenoside rb1 on burn - wound healing in detail. the burn area in mice treated with a topical application of ginsenoside rb1 in the range of 10% to 10% was significantly reduced on days 820 compared to that in vehicle - treated burn - wound control mice (figure 2). to clarify the mechanism behind the facilitating effect of ginsenoside rb1 on wound healing, we examined levels of il-1 and vegf in exudates of the burn. the levels increased with time over 9 days. at 1 ng of ginsenoside rb1 per wound, the level of il-1 was increased on days 1, 3, and 5 but significantly decreased on day 9 compared to that in vehicle - treated control mice (figure 3). the topical application of bfgf (2.5 g per wound) also increased il-1 production on day 3. the vegf level in the exudates from the wound increased until day 5 and then decreased. the application of ginsenoside rb1 increased vegf levels on days 1 and 9 (figure 4). the application of bfgf (2.5 g per wound) or ginsenoside rb1 (100 fg, 10 pg, and 1 ng per wound) for 9 days increased the length of blood vessels by 3- to 3.5-fold and the corresponding area by 3.5- to 5.0-fold, compared to the control (figure 5 and table 2). ginsenoside rb1 at concentrations from 100 fg / ml to 1 ng / ml enhanced the vegf production and hif-1 expression induced by il-1 in the human keratinocyte cell line hacat (figure 6). these findings suggest the enhancement of wound healing by ginsenoside rb1 to be due to the promotion of angiogenesis during the repair process as a result of the stimulation of vegf production caused by the increase in hif-1 expression in keratinocytes. furthermore, the mcp-1 level in the exudates of vehicle - treated (control) mice reached a maximum 1 day after the burn treatment and declined rapidly from day 3. ginsenoside rb1 (1 ng per wound) and bfgf (2.5 g per wound) significantly increased the level of mcp-1 on day 1 compared to that in control mice (figure 7). ginsenoside rb1 (1 ng per wound) significantly increased the histamine level on day 5 compared to that in control mice (figure 7). furthermore, ginsenoside rb1 (100 fg, 10 pg, and 1 ng per wound) and bfgf (2.5 g per wound) significantly increased histamine production on day 7 (figure 7). the facilitating effects of ginsenoside rb1 may be due to the promotion of angiogenesis via the activation of bfgf through the increase in histamine released from mast cells recruited by the stimulation of mcp-1 production. based on these experimental results, the enhancing effects of ginsenoside rb1 on burn wound healing are summarized in figure 8. it has been reported that ginsenoside rb2 as well as ginsenoside rb1 promotes wound healing. the symptoms of cutaneous aging, such as wrinkles and pigmentation, for example, develop earlier in sun - exposed skin than in unexposed skin, a phenomenon referred to as photoaging. ultraviolet b (uvb) radiation is one of the most important environmental factors because of its hazardous effects, which include the generation of skin cancer, suppression of the immune system, and premature skin aging. as shown in table 3, it has been reported that red ginseng extract prevents skin aging induced by uvb irradiation. we found that ginsenoside rb1 isolated from red ginseng roots inhibited the increases in skin thickness, epidermis, and wrinkle formation induced by chronic uvb irradiation. in this paper, we will introduce the effects of ginsenoside rb1 on chronic uvb irradiation - induced cutaneous aging in hairless mice. the topical application of ginsenoside rb1 at lower doses, 100 fg, 10 pg, and 1 ng / mouse, significantly inhibited the increase in skin thickness induced by uvb irradiation during weeks 2 to 12 compared to the skin thickness of vehicle - treated uvb - irradiated mice (control) (figure 9). the reduction in skin elasticity induced by uvb irradiation was significantly inhibited by the topical application of ginsenoside rb1 (100 fg, 10 pg, and 1 ng / mouse) during weeks 6 to 12 compared to that of control mice (figure 10). wrinkling induced by uvb irradiation at week 9 was inhibited by the topical application of ginsenoside rb1 (100 fg, 10 pg, and 1 ng / mouse) (figure 11). the topical application of ginsenoside rb1 (100 fg, 10 pg, and 1 ng / mouse) inhibited the increase in epidermal thickness induced by uvb irradiation but had no effect on the increase in the extracellular matrix of the dermis (table 4). the occurrence of apoptotic cells was localized to the stratum granulosum of the epidermis and was increased by uvb irradiation. the increase in apoptotic cell levels induced uvb irradiation furthermore, 8-hydroxy-2-deoxyguanosine (8-ohdg, a marker of oxidative dna damage) was also localized to the stratum basale and dermis, and its level was increased by uvb irradiation. the increase in 8-ohdg - positive cells induced by uvb irradiation was inhibited by ginsenoside rb1 (table 5). uvb (20 mj / cm) irradiation reduced the level of bcl-2 expression in human primary keratinocytes. ginsenoside rb1 increased the bcl-2 levels in uvb - treated human primary keratinocytes at the lower concentrations of 100 fg, 10 pg, and 1 ng / ml (figure 12). uvb exposure of skin cells results in several types of dna damage such as the formation of the cyclobutane pyrimidine dimer, pyrimidine pyrimidone photodimers and 8-ohdg [3335 ], and consequently dna damage induced by long - term uv exposure leads to skin carcinogenesis. furthermore, there are many reports that apoptotic stimuli such as uv radiation and tumor necrosis factor- induce cell death by activating caspases. bcl-2 is a member of the large bcl-2 family and protects cells from apoptosis. on the other hand, it has been reported that bax and bak appear to permeabilize the outer mitochondrial membrane, allowing the efflux of apoptogenic proteins [3739 ]. the protective effect of ginsenoside rb1 on uvb - mediated apoptosis may be partly due to the upregulation of bcl-2 expression in human keratinocytes. thus, the protective effect of ginsenoside rb1 on skin photoaging induced by chronic uvb exposure may be due to the increase in collagen synthesis and/or the inhibition of metalloproteinases expression in dermal fibroblast and the inhibition of epidermal hyperplasia. further research is needed to clarify the mechanism of the protective effect of ginsenoside rb1 on photoaging induced by chronic uvb irradiation of the skin. the topical application of ginsenoside rb1 isolated from red ginseng roots enhances burn wound healing, and ginsenoside rb1 prevents chronic uvb - induced skin photoaging, at very low doses. further studies will be needed to clarify the clinical significance of these findings for skin damage induced by burn wounds or uv irradiation. | ginseng roots (panax ginseng ca meyer) have been used traditionally for the treatment, especially prevention, of various diseases in china, korea, and japan. both experimental and clinical studies suggest ginseng roots to have pharmacological effects in patients with life - style - related diseases such as non - insulin - dependent diabetic mellitus, atherosclerosis, hyperlipidemia, and hypertension. the topical use of ginseng roots to treat skin complaints including atopic suppurative dermatitis, wounds, and inflammation is also described in ancient chinese texts ; however, there have been relatively few studies in this area. in the present paper, we describe introduce the biological and pharmacological effects of ginsenoside rb1 isolated from red ginseng roots on skin damage caused by burn - wounds using male balb / c mice (in vivo) and by ultraviolet b irradiation using male c57bl/6j and albino hairless (hr-1) mice (in vivo). furthermore, to clarify the mechanisms behind these pharmacological actions, human primary keratinocytes and the human keratinocyte cell line hacat were used in experiments in vitro. |
in the absence of a vaccine and in lieu of a cure, antiretroviral combination therapy has been the main form of treatment for individuals infected with hiv. as is the case with treatment of most rapidly evolving viruses / diseases, drug resistance decreases the effectiveness of treatment. the high replicative capacity of hiv and the infidelity of the reverse transcriptase quickly lead to a heterogeneous population of viruses within patients, from which resistance has emerged to all 30 of the currently used antiviral drugs. hiv-1 protease inhibitors (pis) have recently emerged as the most effective drugs in the treatment of hiv. pis are competitive active site inhibitors that mimic the transition state of the enzyme and are the most potent antiretroviral drugs for the treatment of hiv / aids. these drugs are ideal for therapy as they target the viral protease responsible for viral maturation and thus the spread of the virus. unfortunately, the rapid evolution of hiv-1, coupled with the selective pressure of therapy, results in many viable multidrug resistant variants. in fact, mutations at 45 of the 99 residues that make up hiv-1 protease have been implicated in drug resistance. while resistance due to mutations at 11 of these 45 residues can be explained as direct changes within the active site, the resistance mechanisms for the majority of the remaining mutations outside the active site of the enzyme mostly remain elusive. drug resistance mutations in hiv-1 protease allow the enzyme to become less susceptible to inhibition while retaining enzymatic activity. points of inhibitor protease contact at residues within the active site where the inhibitor protrudes beyond the substrate envelope are sites selected for resistance, as their interactions are more critical for inhibitor binding than substrate turnover. while mutations at some active site residues, such as 82 and 84, lead to resistance to all pis, other mutations are signatures of specific inhibitors, such as d30n for nelfinavir and i47a for lopinavir. these mutations directly impact inhibitor binding by altering or reducing contacts necessary for inhibiting the enzyme, but can also simultaneously decrease the catalytic efficiency or enzymatic fitness. the mutations at the remaining 34 of the 45 residues associated with drug resistance occur outside the active site. these changes have often been considered secondary or accessory mutations, and are thought to indirectly impact inhibitor binding while assisting in enzyme fitness or stability. structural studies on the effect of several hiv-1 protease secondary mutations have provided insights into how inhibitor binding may be affected. however, for the most part, their specific role in protease inhibitor resistance or mechanism of action has not been elucidated. darunavir (drv) is the most potent of the united states food and drug administration (fda) approved hiv-1 protease inhibitors. this high potency combined with the inhibitor s fit within the substrate envelope appears to account for drv s robustness against drug resistance. drug resistance to drv usually occurs only in patients who have high levels of pre - existing pi resistance, requiring at least seven mutations to simultaneously occur for therapeutic failure. in fact, drv is being investigated as a potential monotherapy in treatment - nave patients. in drv - resistant hiv variants, single site mutations can not confer high levels of resistance to drv, and a combination of multiple mutations including those outside the active site are needed to decrease potency. however, the role of these mutations in conferring resistance is not well understood : some may be enhancing enzymatic activity, while others may directly confer drug resistance and still others may be residual mutations from previous therapy history. in this study, we examine some of the most common of these mutations, v32i, l33f, l76v, and l90 m (as a control ; not a signature of drv resistance but frequent in multidrug resistance), for their impact on drv inhibition. using a combination of static and dynamic structural analyses, by determining crystal structures of complexes and performing molecular dynamics simulations, we elucidate the possible roles of these secondary mutations both independently (l76v, l90 m, v32i) and in combination (v32i / l33f) in conferring resistance. we find how mutations at residues with no direct contact with the inhibitor can alter the structure and dynamics of the protease to affect inhibitor binding through common mechanisms, which we define through a network hypothesis. to determine how mutations remote from the active site contribute to drv resistance in hiv-1 protease, the impact of four mutations (l76v, l90 m, v32i, and v32i / l33f ; figure 1) in a subtype b background was investigated in terms of enzyme inhibition, inhibitor - bound crystal structures, and molecular dynamics simulations. crystal structures of mutant protease variants superimposed with the wt protease complex structure in blue. the enzyme inhibition constant for drv was measured against each of the protease mutants, in addition to wt subtypes b and c for comparison (table 1). drv is highly potent against wt subtype b protease with a ki of 2 pm, as we previously reported. the level of inhibition for the mutants varied from 2 pm to 45 pm, with the l90 m mutant being inhibited as potently as the wt protease and the v32i / l33f double mutant exhibiting the greatest decrease in susceptibility to drv with a fold - change greater than 20. hence, single mutations are not enough to confer high levels of drv resistance, as expected, and the mutations had varying degrees of effects on drv susceptibility. drv inhibition constants (ki) of hiv-1 protease variants, with fold - changes relative to subtype b wt protease in parentheses. the overall vdw interaction energy between the inhibitor and protease was determined from crystal structures. to structurally characterize the effects of the mutations on drv binding, we determined the crystal structures of variants l76v, l90 m, v32i, and v32i / l33f, which diffracted to resolutions of 1.51.9 in the p212121 space group (table 2). alignment of the four complex structures on our previously determined structure of the wt protease drv complex (1t3r) showed that the variants had only minor backbone variations, mainly in the 20s loop likely due to crystal packing differences (figure 1). therefore, the mutations had very little impact on the overall backbone structure of the protease. from surleraux the high - resolution cocrystal structures enabled detailed analysis of protease drv contacts in each of the five complexes. the wt complex had the most extensive van der waals (vdw) contacts with the inhibitor with a favorable energy of 44.5 kcal / mol, similar to v32i and l90 m variants (table 1). the l76v variant and v32i / l33f double mutant lost more than 1 kcal / mol in vdw contact energy with drv relative to the wt complex. thus, despite no large - scale changes in the protease backbone, subtle changes in repacking occurred around drv in these two complexes to weaken protease interactions with the inhibitor. however, the extent of contacts lost with drv in the mutant crystal structures with respect to wt protease does not correlate completely with the fold - change loses in ki values (table 1). contacts involving specific drv moieties (figure 2) and protease active site residues (figure 3) were analyzed in detail. in general, the impact of mutations on drv contacts are larger at the p2 and p2 than the central p1 and p1 moieties. the bis - thf group of drv p2 moiety forms the most extensive contacts in all of the complexes (figure 2), but also loses considerable contacts due to the mutations, except in the v32i structure. in the case of v32i, drv contacts are retained as in the wt complex, consistent with no significant change in total vdw or ki values (table 1). when this mutation occurs together with l33f in the double mutant though, contacts are lost in all three of p2, p1, and p2 moieties. in the l90 m variant, although interactions get weaker at the p2 position, gain of contacts at p1 compensate for this loss yielding comparable total vdw contacts and susceptibility to drv as wt protease. (a) chemical structure of drv (tmc114) with the inhibitor moieties p2p2 indicated. (b) vdw interaction energy (kcal / mol) of drv moieties for contacts with the protease active site in the crystal structures, and changes in vdw interaction energy in mutant structures relative to the wt complex. (a) the two monomers of wt protease in surface representation with the bound drv displayed as sticks. active site residues are colored from blue to red for increasing vdw contacts with the inhibitor. the monomer that interacts mostly with the p2p1 moieties of drv is on the left, and the primed - side monomer is on the right. (b) the vdw interaction energy of active site residues in crystal structures (top), and changes in mutant complexes relative to the wt structure (bottom). only the residues displaying considerable changes relative to wt are included for both monomers. see figures s1 and s2 in the supporting information for all active site residues. while the apo form of the protease is a symmetric homodimer, drv induces asymmetry to the complex and thus despite identical residues mutating in both monomers, the effect of these mutations on protease specifically, l76v and l90 m mutations cause considerable loss of contacts at i47, but to a lesser extent at i47. other active site residues whose contacts are altered in mutant structures include i50 at the tip of the flaps, and 818284 at the 80s loop. contrary to previous reports, we do not see any major enhancement of drv contacts with the catalytic d25 in the l90 m mutant, or any of the other 3 variants. residue 32 is at the periphery of the active site, and v32i mutation causes a unique pattern of rearrangement of inhibitor contacts than the other variants studied. unlike l76v and l90 m, contacts with 47 are retained in v32i. although the backbone is not shifted significantly, the proximity of residue 32 to the 80s loop causes subtle rearrangements to result in repositioning of the drv away from i84 s and more toward i50s at the tip of the flaps. as a result, drv contacts with residues 84, 81, and 84 are lost but those with 50 and 50 are enhanced. the larger isoleucine also forms additional contacts with the inhibitor in the unprimed - side monomer. in the v32i / l33f variant, which loses an additional 7-fold in binding affinity relative to v32i (table 1), in contrast with v32i alone, additional loss of interactions at residues 47 and 50 are observed. these losses of contacts are similar to the alterations observed in the l76v and l90 m variants. thus, the double mutant v32i / l33f variant alters the active site in a synergistic manner, leveraging both alterations similar to l76v and l90 m, and some changes from v32i. the change in variants affinity is not simply due to a loss of van der waals contacts, but an interdependent change in optimal contacts. in the wt complex, drv forms a network of hydrogen bonds within the active site involving both backbone and side chains. most of these bonds, including the two water - mediated ones with i50, are conserved in the variant complexes. two exceptions occurred in the l76v and v32i complexes : consistent with the loss of vdw contacts, in the l76v complex a hydrogen bond to the backbone of d30 is lengthened from 2.0 to 3.0. in the v32i variant, an additional water - mediated hydrogen bond with the side chain of d30 is formed. nevertheless, overall the hydrogen bonds with drv within the various complexes are conserved. analysis of crystal structures above revealed that the mutations away from the active site are able to influence interactions of drv - contacting residues at the active site. the alterations in vdw contacts or hydrogen bonds lost, however, only partly correlate with the experimentally determined enzyme inhibition constants. another possible mechanism by which these secondary mutations could alter inhibitor binding is by influencing the dynamic ensemble of the enzyme. starting from the crystal structures of the drv complexes, three replicates of fully hydrated 10 ns md simulations of each drv complex were performed and analyzed. root - mean - square deviations (rmsd) of c atoms during the simulation and the average root - mean - square fluctuations (rmsf) about their mean positions readily reveal that the secondary mutations alter the overall enzyme dynamics (figure 4a). the l90 m and v32i / l33f variants display larger fluctuations throughout the enzyme compared to the other variants, although the catalytic d25 stays relatively rigid in both monomers. these altered fluctuations are not restricted to the sites of mutation, but propagate throughout the enzyme. (a) rmsd of c atoms from the initial positions, and rms fluctuations of residues averaged over three 10 ns trajectories. (b) significantly altered change in distance between residue pairs around the active site relative to wt complex, sampled during the md simulations ; increased and decreased distances are indicated by blue and red, respectively. see figure s3 for the distance distributions. to further analyze the impact of mutations on the dynamic ensemble sampled by the protease, the distance distributions were calculated across the active site at a variety of positions (figure 4b and figure s3). in all the variant complexes, the dynamic ensemble sampled by the protease many of the distances displaying a significant change are longer than the wt distance, indicating a widening of the active site. in the l76v complex, the changes are highly asymmetric, with one side of the active site constricting and the other widening (figure 4b). in all variant complexes, the 80s loops in both monomers form the side walls of the active site. relative to the wt complex, the distance between residues 81 in the two monomers are shorter, and that between 8484 are longer in all variants. part of the side walls are closer and the lower part is farther away in the mutant complexes compared to the wt. in addition to the 80s loop, certain distances involving residue 50 at the tip of the flaps, and even the catalytic d25 are altered in the variant complexes. the catalytic site is the most invariant and dynamically restricted region of the protease, both when different crystal structures are compared and dynamics analyzed by simulations and nmr experiments. therefore, widening of the d25d25 distance in the v32i / l33f double mutant is an unusually profound impact of remote changes on the catalytic region. the md simulations also permitted a detailed analysis of the interaction network both for direct interactions within the active site to drv and the internal hydrogen - bonding network throughout the enzyme (figure 5, figures s4s6). throughout the md simulations the wt complex maintains a network of stable hydrogen bonds. starting from the bottom of the enzyme the c - terminal -helix forms a network of hydrogen bonds that links the termini of the protein to the flap regions. the backbone of residue 95 links to residue 90 which in turn contacts residue 86, residue 88 bridges to residues 29, 31, and 74, and residue 76 bonds to both residues 31 and 33 which is bonded to residue 78. the hydrogen bonds linking residues 47 and 54 within the flaps stay tightly hydrogen bonded throughout the simulation. thus, as we previously observed, the hydrogen bonding network is stably retained within the wt md simulation. (a) crystal structure of drv bound to the active site, and only one monomer of the protease is shown for clarity. the sites of mutation (l76, l90, v32, and l33) have colored side chains. (b) histograms of the changes of the percentage time hydrogen bonds are formed relative to the wt simulation for each of the complexes. (c) schematic hydrogen bond network of the hiv-1 protease dimer with the percentage time hydrogen bonds are formed during the wt simulation (figure s4). (d) schematic representation of the v32i_l33f complex simulation with the change in hydrogen bonding relative to the wt simulations. the remaining variants schematic are shown in figure s5. in comparing the simulations of the variant drv complexes with the wt, subtle changes are seen in the vdw contacts within the active site (figure s6), similar to what was observed in the crystal structures. however, in each of the four variants, with the notable exception of the 4754 linkages, the hydrogen bond network is disrupted to a greater or lesser extent asymmetrically, including the direct hydrogen bonds with drv (figure 5b, s4, s5). the v32i / l33f variant is the most disrupted with 12 hydrogen bonds changing by greater than 20% relative to the wt complex throughout the dimer, with 11 being weakened (figure 5d) including most dramatically the interactions of the side chain of asn 88. eight of these changes are within the monomer that coordinates the highly rigid bis - thf moiety including weakened interactions at points of contact between the protein and drv. thus, mutations distal to the active site often weaken the strength of the hydrogen bonds in the network, which is propagated through to the active site including altering vdw packing, pushing the flaps, and thereby the contact of i47 with drv. taken together, one can decipher how contacts between protein and inhibitor are affected by these changes even for residues that are packed through vdw contacts or covalently linked along the backbone and are not directly involved in the hydrogen bonding network (figure 5a and c). hiv-1 protease evolves in complex combinations to evade inhibition, but still maintains biological function. however, in highly resistant variants, active site mutations often coexist with mutations outside the active site. this is particularly necessary when resistance is achieved to the highly potent inhibitor drv, which fits well within the substrate envelope. however, the role of these changes outside the active site has long been thought to be only in recovering viral fitness, or protease stability. in the current study, we have primarily chosen enzyme variants that are associated with drv resistance : l76v, v32i, and l33f. although l76v causes only a 1.5-fold decrease in drv binding affinity (table 1), this mutation is often observed in highly mutated drv - resistant variants, as well as variants with hyper - susceptibility to other pis. l33f is a highly networked mutation co - occurring with many others in highly drug - resistant patient isolates, often together with v32i. therefore, comparison of v32i and the v32i / l33f double mutant permits the context - dependence of mutational effects in drug resistance. while not directly associated with drv resistance, l90 m is a canonical highly networked mutation that typically arises in multidrug resistant proteases. the large and rigid p1/p1 moieties in nfv and sqv have been implicated in susceptibility to l90 m, a feature lacking in drv. l90 m has been found in more than half of patient isolates with at least one pi resistance substitution, and hence is often present in patients needing drv - based salvage therapy. thus, elucidating the physical impact of these secondary mutations on drv binding provides a detailed perspective on how the enzyme accommodates such frequently observed changes. specifically, we find that mutations outside the active site impact inhibitor binding thereby playing a direct role in conferring drug resistance. compared to the wt complex, the overall structure and backbone conformations are very similar in the cocrystal structures of the variant complexes. however, the mutations cause subtle but significant rearrangements in the structure to cause altered interactions with the bound inhibitor, as well as impacting the dynamic ensembles of these complexes. we had previously hypothesized and tested that alterations in the hydrophobic core of the enzyme could alter the conformational dynamic ensemble through changes in the hydrophobic sliding of internal residues potentially impacting drug resistance. this impact on dynamics is not localized to the points of mutation but would propagate throughout the enzyme. in the present study we hypothesize these mutations outside the active site share a common pathway of altering the overall enzyme dynamics and propagating their effects to the active site. although the resistance - associated mutations are located at a variety of positions in the protease and away from the active site, they all may utilize a common mechanism or pathway of altering the protease the mutations cause subtle changes through the repacking of the active site ; in particular, these are observed in the crystal structures at residues 47, 50, and 84 in both monomers, and also observed in the md analysis (figure s6). within the crystal structures of both the l76v and l90 m complexes residue i47, which is located in the flap, loses contact with drv. in contrast, in the v32i complex i47 contacts are retained, while this loss is restored when l33f occurs in v32i / l33f (these changes are also observed in subtle differences in the md simulation figure s6). interestingly, v32i and i47v are the second most frequent pair of residues often found to coevolve, thus compensating for each other. mutations at i47, together with i54, which its backbone is hydrogen bonded with, is a major drv resistance site. among about 30 total active site residues that contact drv, i47 is consistently the residue whose contacts are affected the most in l76v, l90 m, and v32i / l33f variants (figure s3). these results suggest that the interactions of residue 47 with inhibitors within the active site may represent a pivotal site in conferring drug resistance to pis, and these interactions can be altered by changes propagated through the enzyme from remote sites. in addition to repacking around the inhibitor in the crystal structures, the secondary mutations share a common pattern of altering the dynamic ensemble sampled by the protease, and the shape of the active site. overall in the dynamic ensemble of the v32i and the v32i / l33f variants the active site is expanded, with the double mutant expanding the active site more, while l76v active site contracts and the l90 m active site displays asymmetric changes. hence, even though not located at the active site, mutations at all these remote sites affect the shape of the active site in the dynamic ensemble. how are single mutations at a remote site able to alter interactions and dynamics of the active site with highly common molecular mechanisms ? we propose a network hypothesis where the perturbation introduced by mutation of a distal residue is propagated to the active site through a network of interactions within the protease structure (figure 5). the distal mutation sites we studied are all part of a hydrogen - bonded network connecting to the active site where the inhibitor binds. our network hypothesis postulates that the mutations have similar effects and common mechanisms as they all cause a rearrangement of this same network. this hypothesis is supported by the alterations observed during the md simulations in the stability of the hydrogen bonding networks (figure 5), where changes propagate from residues 7478 and 8790, through 2833, to 8485 and 25. this altered interaction network includes repacking of the vdw contacts with residues 47 and 54, which are pivotal in linking the networked residues to the rearrangement of the flaps, residues 29 and 30 that directly hydrogen bond to drv, and 82 and 84 that are key sites within the active site cavity. we hypothesize that all these are the active site residues where the impact of distal mutations is propagated as a common mechanism of resistance in all variants and their subtle rearrangements can cause inhibitor specific resistant changes. these common mechanisms provide an explanation for why some mutations are redundant and thus are not observed together in patient sequences, while others are synergistic and occur together to confer higher levels of drug resistance as they impact one another at pivotal sites that confer resistance often through expanding the active site. this hypothesis does not exclude the possibility that some changes may still provide additional stability, increasing the combined fitness of the variants. most significantly, our findings show that all of the mutations we have studied, although outside the active site, still directly alter the shape and flexibility of the active site, thus likely play a direct role in conferring resistance. each of the four protease mutants was constructed using a standard site - directed mutagenesis protocol on a wt - hxb2 protease gene with a codon sequence optimized for e. coli expression. the wt pr gene contained the amino acid substitution q7k to minimize the enzyme s autoproteolytic activity. briefly, the mutant hiv-1 protease gene was cloned into the pxc-35 plasmid, which was then transformed into the tap56 strain of escherichia coli. transformed cells were grown in 6 1 l cultures from which cell pellets were harvested 3 h after induction. the cell pellets were lysed and the protease was retrieved from inclusion bodies with 100% glacial acetic acid. the protease was separated from higher molecular weight proteins by size - exclusion chromatography on a sephadex g-75 column. the purified protein was refolded by rapid dilution into a 10-fold volume of 0.05 m sodium acetate buffer at ph 5.5, containing 10% glycerol, 5% ethylene glycol, and 5 mm dithiothreitol (refolding buffer). the protease solution was concentrated, followed by dialysis to remove any remaining acetic acid. protease used for crystallization was further purified with a pharmacia superdex 75 fast - performance liquid chromatography column equilibrated with refolding buffer. crystals were set up with a 5-fold molar excess of inhibitor to protease, which ensures ubiquitous binding. the final protein concentration ranged from 0.8 to 1.6 mg / ml in refolding buffer. the hanging - drop method was used for crystallization as previously described. for the l76v, l90 m, and v32i mutants, the reservoir solution consisted of 126 mm phosphate buffer at ph 6.2, 63 mm sodium citrate, and ammonium sulfate at a range of 2429%. for the v32i / l33f double mutant, the reservoir solution consisted of 0.1 m citrate - phosphate buffer, 7% dmso, and 2530% ammonium sulfate. a substrate peptide mimicking the ma - ca (p17-p24) cleavage site labeled with k - e(edans)-s - q - n - y - p - i - v - q - k(dabcyl)-r (0.5 m, final concentration) was added just prior to the reading to each well containing 50 nm of pr and varying concentrations of inhibitor. the fret pair (edans, the donor and dabcyl, the quencher) was attached to the indicated amino acids of the peptide (molecular probes). fluorescence intensity increase upon hydrolysis of the fluorogenic substrate was monitored at 490 nm (emission of edans) from the highest inhibitor concentration to the lowest, as well as the no inhibitor control well. initial velocities were obtained from the progress curves and plotted against inhibitor concentration to get inhibition curves. resulting curves were globally fitted to morrison s equation to obtain the ki value, as described previously. the maestro component of the schredinger software suite was used to analyze the hydrogen bonds between the inhibitor and the protease residues and neighboring waters after optimization of the complex structure. briefly, a hydrogen bond was defined by a distance between donor and acceptor of 120. the vdw contacts between the inhibitor and protease were calculated using a simplified lennard the md simulations were performed using the program sander in the amber 8 package, as previously described. a set of three simulations was run for each of the four mutants and the wt - pr yielding a total of 15 trajectories for analysis. each simulation was assigned initial velocities according to the maxwellian distribution and random seeds were assigned with five different values for each pr. an in - house script was used to determine the intra and intermonomeric c distances between various residues using the trajectories. to calculate the hydrogen bond duration between various residues within the network from the simulations, the visual molecular dynamics (vmd) version 1.9.1 vmd was used to write out the trajectory in a pdb format using the coordinates and trajectory files generated by ptraj from the amber simulation software. vmd was also used to generate the trajectory pdb files to determine the vdw contact energies over the simulations. the in - house vdw script was then modified to assess vdw contacts from the simulations. once the robustness of the system was assessed, the three trajectories for each system were concatenated into one file containing 1500 frames and the total vdw contacts were analyzed. | hiv-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of hiv infection and aids. darunavir (drv) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. with few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. through a series of drv protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with drv resistance alter the structure and dynamic ensemble of hiv-1 protease active site. these alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance. |
in the past few decades, there has been a growing interest in the development of artificial nano - materials. one class of material such as this is photonic crystals (pcs), which are periodic dielectric or semiconductor structures that display a photonic band gap (pbg) in their electromagnetic wave transmission characteristics [1 - 4 ]. the existence of the pbg has inspired the design of various nano - optical and opto - electronic devices. since the formation of the pbg is due to the multiple bragg reflections within a pc, ordinary pcs are a combination of lossless materials for which the stop band has a small width, almost less than 25% of the central frequency. this is due to the fact that a significant percentage of incident radiation passes through the periodic structure cores so that the reflected radiation is weaker than the transmitted radiation. because of this, bragg scattering comes into play when there are periodicities along the direction of propagation. to exhibit a noticeable pbg with fewer periodicities metals would be one of the best options since they are more reflective than dielectric or semiconductor materials over a broad range of frequencies, due to their imaginary dielectric component that exists in even the optical and near infrared regions, where metals are dispersive and absorptive in these frequency regions [6 - 8 ]. recently, considerable progress has been made in constructing these periodic arrangements which are called metallic or metallodielectric photonic crystals (mpc / mdpc) in one-, two-, and three - dimensional systems. for instance, one - dimensional (1d) mdpcs with metal thicknesses on the order of hundreds of nanometres has been proved transparent to visible light while blocking ultraviolet and infrared. this characteristic can be used for example in laser safety glasses, uv protective films and flat panel displays. two - dimensional (2d) mpc structures are usually made of metallic rods or nanodisks that are periodically arranged on a waveguide layer. waveguided mpcs have exhibited unique optical properties because of the strong coupling that exists between the particle plasmon resonance and waveguide mode. such structures have a number of potential applications in biosensors and all - optical switching, among others. in the case of three - dimensional (3d) structures, there are also several applications such as high - efficiency light sources and thermal photovoltaic power generation. besides these advantages, pcs can be used for radiation suppression and emission enhancement below the electronic band gap and near a photonic band edge, respectively, where a pc functions as a reservoir for an exited light emitter or active medium such as an atom, a molecule or a quantum dot (qd). according to fermi s golden rule, the decay rate is proportional to the local density of states (ldos) that counts the number of electromagnetic modes available to the photons for emission into the environment. thus, any modification in the ldos would lead to manipulation of the decay rate. recently, the inhibition, enhancement and quantum interference (qi) effects of spontaneous emission (se) from the qds doped in 3d pcs have been widely studied, both experimentally and theoretically [15 - 19 ]. controlling spontaneous emission by using quantum optics would lead to several interesting effects, such as optical gain enhancement and photoluminescence enhancement, optical switching, quantum information processing and electromagnetically induced transparency. qi in a three- or multi - level atomic system can arise from the superposition of ses when electron transitions take place between the upper and lower levels. under certain circumstances the initially excited atomic system may not decay to its ground level due to a cancellation of se by qi between atomic transition levels. due to this, dark states with zero absorption amplitude would appear causing the multi - level atomic system to act like a transparent medium, which has potential applications for optical switches and photonic devices. in this paper, the effects of electronic qi on the absorption coefficient of qds have been investigated for small and large concentration of the dopants (qds). we consider that the qds are four energy level systems where the two upper levels are very close, coupled to a lower one via the same and single field continuum and damped by the mpc interaction. recently, theoretical and experimental studies have shown that it is possible to make 3d mpcs that contain nano - sized metallic spheres which are transparent to visible and near infrared light. in this paper, we have used an ideal 3d isotropic mpc model made from metallic spheres of radius a with a frequency dependent refractive index n1(), which are arranged periodically in a background dielectric material with a constant refractive index (n2). the dispersion relation and photonic band structure of this idealized theoretical 3d model was developed by s. john in the following references. although many simplifying approximations have been used in this model, it is sufficient for our purpose as it leads to qualitatively correct physics and exhibits many of the observed and computed characteristics of 3d mpc which opens a band gap in the rage of visible frequencies. in many photonic band structure calculations related to mpcs, the refractive index function for metallic materials is derived using the drude model, which offers an excellent fit to measured data over a wide frequency range. using this model, the refractive index for a metallic material is expressed as where p and are the frequency of the incident laser beam, the plasma frequency and the damping factor of the conduction - band electrons, respectively. the plasma frequency is defined by, where n0 is the electron density, meff is the effective mass of the electron, e is the electron charge and 0 is the permittivity of the free space. the damping rate (which is also called the electron collision rate) is the inverse of the mean electron collision time. the parameter is frequency independent and therefore absorption can be neglected at optical frequencies, since / = 1. (1), for frequencies below p, the local wave vector is imaginary and the metal behaves as a dispersive and absorptive environment. nonetheless, if the diameter of the metallic spheres in our crystal structures can be chosen close to or smaller than the relevant skin depth of the corresponding metal, the em wave can be transmitted by tunnelling through the structure. the position of the pbg can be determined by selection of proper thicknesses and refractive indices. since we are interested in optical frequencies, the radius of the metallic spheres and lattice constant of the pc have been chosen in reduced units as a = 0.25c/p and l = 10.5c/p, respectively. in these parameters, is the reduced planck constant and p is the plasmon energy, i.e. p = p. a plot of the band structure of the pc consisting of spherical metal nanoparticles in a dielectric background (n2 = 1.5) has been illustrated in fig. plot of the bloch wave vector k as a function of the normalized photon energy for a metallic pbg. the vertical dashed lines show v/p and c/p which are the maximum normalized energy of the lower energy band and the minimum normalized energy of the upper energy band, respectively. the nano - sized active medium in the 3d mpc considered here is assumed to be four - level qds, with diameters ranging from 2 to 10 nanometres, and two upper levels |c and |b which are close to one another. the remaining two lower levels are denoted as |a and |d. the two upper levels are dipole coupled to the |a state via the same single field continuum. we also assume that se is allowed from the excited states (upper levels) to the |a state and from |a state to |d state, whereas the transition |b |d, |c |d and |c |b are inhibited in the electric dipole approximation. there are many potential qds suitable for our theoretical model such as cdse / zns or inas / gaas core shell qds. four - level qd with the two upper levels |c and |b which are near one another, and two lower levels |a and |d. here, is the probe field frequency while ca and ba are the transition frequencies.c and b are the decay rates from the exited states to the |a state. the decay rate from |a to |d is given as a, whereas c = ca and b = ba are the detunings of the atomic transition energies.k is the detuning of the probe field, which has a central frequency at the middle point of the two upper levels in this section, the interaction between qds doped within the pbg reservoir and a probe field with slowly varying amplitude is investigated. the total semiclassical hamiltonian of the system can be written as here, hq, hqf, hr, hqr and hqq are the hamiltonians of the four - level qd, the qd (2), the equation of motion of the density matrix elements can be written as follows : in eqs. (3 - 9), ij (ij = a, b, c or d) are density matrix elements (coherences), p is the strength of quantum interference and is defined by p = acab/acab. in this paper, the maximum quantum interface has been considered, which corresponds to a dipole transition moment ac that is parallel to ab. this gives p = 1. here, ac and ab are the electric dipole moments induced by the transitions since the two upper energy levels are very close (bc = 0.03 ev), it is reasonable to consider ab = ab =. is the rabi frequency of the probe field, defined as = e/2, where the dipolar transition moments and external field e are parallel. the parameters and are related to the interaction of qds when the mpc is densely doped. this interaction is called dipole dipole interaction (ddi), and its effect was calculated using mean - field theory. the dependency of all decay rates to energy and local density of states can be written as here, the function z() is called the form factor, which contains the information about the electron photon interaction and is obtained in reference [39 - 41 ]. for simplicity, all parameters have been normalized with respect to (bc)/2, which gives a constant value for the resonant energies ab and ac. here, 0 is the decay rate (line - width) for an excited electron in a qd when it is located in a vacuum. the expression of the absorption coefficient is written in terms of density matrix coherence as : here, n is the concentration of quantum dots and is the energy of the incident laser beam. in order to study the linear response of the system, we have calculated the normalized absorption coefficient given in eq. (11) (/0) by using a very low driving field (= 0.01). we consider that the metallic spheres are made of silver with p = 9 ev. the two upper resonant energies (ab = 2.78 ev and ac= 2.783 ev) are considered to be far away from the upper edge band gap in the first brillouin zone where b c= 1.570. the value of a can be set by changing the resonant energy ad. this decay rate (a) can be totally suppressed if the resonant energy lies within the band gap. the system of equations in (38) has been solved numerically for cases where ddi was neglected and taken into account while the system approaches a steady state configuration. the results have been shown in figs. 3 and 4 where the normalized absorption coefficient versus the detuning parameter has been drawn for different values of lower decay rate (a = 0.000, 0.005 and 0.100). as one can see, in both cases this means that when the lower resonance state (i.e. |a-|d) lies within the band gap, the normalized absorption coefficient is zero, and when it goes further from the band gap it is nonzero. this behaviour demonstrates the switching between absorption and nonabsorption states that can be used to make optical switches. numerical plots of the normalized absorption coefficient (/0) versus dimensionless detuning parameter (k) for different values of lower decay rate (a = 0.0, 0.005 and 0.1) when the ddi is zero numerical plots of the normalized absorption coefficient (/0) versus dimensionless detuning parameter (k) for different values of lower decay rate (a = 0.0, 0.005 and 0.1) when the ddi has been taken into account (= = 2) in conclusion, we have studied the effect the quantum interference and ddi on the absorption of a mpc doped with an ensemble of four - level qds, for both cases where ddi was neglected or accounted for, while the system approached a steady state. a single driving laser field which induces a dipole moment in each qd was applied to measure the absorption. the density matrix method and linear - response theory have been used to calculate the absorption. it is found that when the resonance energy of the lower levels is within the band gap, the system is in an absorbing state. however, when the resonance energy of the lower levels is outside the band gap, the system is in a nonabsorption state. we anticipate that the results described here will be useful for developing new types of optical switching devices. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. | in this work, the absorption coefficient of a metallic photonic crystal doped with nanoparticles has been obtained using numerical simulation techniques. the effects of quantum interference and the concentration of doped particles on the absorption coefficient of the system have been investigated. the nanoparticles have been considered as semiconductor quantum dots which behave as a four - level quantum system and are driven by a single coherent laser field. the results show that changing the position of the photonic band gap about the resonant energy of the two lower levels directly affects the decay rate, and the system can be switched between transparent and opaque states if the probe laser field is tuned to the resonance frequency. these results provide an application for metallic nanostructures in the fabrication of new optical switches and photonic devices. |
oral submucous fibrosis (osf) is a chronic condition almost exclusively occurring among indians and to a lesser extent in the other asiatic people. however, with the increase in immigration of people from the indian subcontinent, dental professionals in many developed countries will encounter this disease in the near future. a wide range of treatment including drug management, surgical therapy, and physiotherapy have been attempted till date, with varying degrees of benefit, but none have been able to cure this disease. this is mainly due to the fact that the etiology of the disease is not fully understood and the disease is progressive in nature. instead of continuing the limited available modes of therapy, the idiopathic nature of this condition indicates new avenues for its management. colchicine is an alkaloid found in the crocus like plant, colchicum autumnale, named for the land of colchis at the eastern tip of black sea. chemically, it is colchicinum - n-(5, 6, 7, 9-tetrahydro-1, 2, 3, 10-tetramethoxy-9-oxobenzo [a ] heptalen-7-yl) acetamide. colchicine is an ancient drug - at least 2,000 years old, which is attracting renewed interest because of its actions at a subcellular level. many studies during the past years have elucidated a variety of previously unsuspected drug actions and have demonstrated the varying effectiveness of colchicine therapy for a surprisingly broad array of diseases including recurrent aphthous stomatitis, behcet 's disease, familial mediterranean fever, polymyositis, and scleroderma. the pharmacodynamics of colchicine as an anti - fibrotic agent is well - established by various in vitro and in vivo studies [table 1 ] warranting its use in the treatment of various diseases associated with fibrosis. the long held view that colchicine 's anti - inflammatory actions are specific for gout is no longer tenable. perhaps the most important anti - inflammatory properties of colchicine are related to the drug 's effect on polymorphonuclear leukocytes and monocyte chemotaxis, leukocyte adhesiveness, the drug 's action in potentiating factors that increase leukocyte cyclic adenosine monophosphate (camp) levels, thereby, inhibiting lysosomal degranulation that accompanies phagocytosis and its effect on the release of prostaglandin e, which suppresses the leukocyte function. effects of colchicine on fibroblasts and collagen fibers hence, the exciting combination of an anti - fibrotic agent along with anti - inflammatory properties of a drug that is surprisingly well tolerated, easily available, and cost effective, prompted us to embark on this study. this study was planned to compare the effectiveness of colchicine and intralesional hyaluronidase with intralesional corticosteroid and hyaluronidase in the management of osf. this study was conducted in government dental college, bangalore, between 2002 and 2004. a formal ethical clearance to conduct this study was given by the ethical committee of the institute. an informed consent was obtained from the patients before including them in the study. a detailed case history of the patient with emphasis on their habits (chewing betel nut, pan parag, etc.) and a thorough clinical examination was recorded on a standard proforma. a clinical diagnosis of osf was made based on the world health organization (who) criteria and the patients were graded clinically according to gupta dinesh chandra s.. hematoxylin and eosin and van gieson 's stains were used to analyze the staining intensity of the inflammatory infiltrate and the density of collagen fibrils, respectively, in the pre and post treatment biopsy specimens. fifty patients (41 males and nine females) in the age group of 15 years to 55 years, thus diagnosed as having osf, were included in the study and were divided randomly into two groups for the purpose of treatment. group 1-patients were administered orally tablet colchicine (tablet zycolchine, zydus cadila healthcare ltd., 0.5 ml of this solution was injected intralesionally in each buccal mucosa once a week. baseline liver function tests, serum urea, and creatinine were done for these patients to rule out any existing hepatic pathology and the tests were repeated once every month during the study period and one month following cessation of treatment. group 2-patients were administered intralesional injection of hyaluronidase 1,500 iu as in group 1 and 0.5 ml of injection hydrocortisone acetate 25 mg / ml in each buccal mucosa once a week alternatively. patients in both the groups were asked to discontinue all betel nut chewing habits during the entire study period. post - treatment punch biopsy was taken one week after the cessation of treatment for histopathological evaluation. patients were asked to observe for any local allergic symptoms as itching, redness or ulcerations at the site of injection or if any constitutional symptoms developed and to report the same immediately. treatment was discontinued in such patients. during the subsequent visits the patient 's response to the treatment procedures was recorded with emphasis on ascertaining the amelioration of specific symptoms like burning sensation, trismus, and intolerance to hot and spicy foods. clinical examination of the oral mucosa, site of lesion, margins, extension, color and surface texture and presence of fibrotic bands was recorded. the interincisal opening of the mouth was recorded during each visit [figures 1 and 2 ]. pre and post treatment histopathological specimens were compared for the inflammatory cells and fibrous tissue [figures 3a, 3b, 4a, and 4b ]. student 's t - test and analysis of variance (anova) were used to compare pre and post treatment results. mouth opening recorded before treatment in a patient belonging to group i clinical improvement of mouth opening recorded in the same patient after treatment of 12 weeks (a) photomicrograph of tissue section taken before treatment showing atrophic epithelium, relatively avascular hyalinized collagen with abundant inflammatory infiltrate (low - power view : h and e 40), (b) photomicrograph of the same tissue specimen stained with van gieson 's showing dense avascular hyalinized collagen (low - power view : 40) (a) photomicrograph of tissue section taken after treatment showing reduced density of collagen and inflammatory infiltrate (low - power view : h and e 40), (b) photomicrograph of the same tissue specimen stained with van gieson 's showing reduced density of collagen (low - power view : 40) the mean age of the patients in the study was 28.40 8.6 (mean sd) years, with 68 per cent in the age group of 20 - 29 years. sixty - four percent of the patients complained of both trismus and burning sensation. all the patients had an areca nut chewing habit, either in the form of betel nut or other commercially available products like pan parag, pan masala, gutka etc. the buccal mucosae were affected in all the patients, however, only five showed tongue involvement. the clinical grading of the patients and the inter incisal distance measured in them are shown in tables 2 and 3, respectively. distribution of the clinical grading of the patients in both the groups distribution of inter - incisal distance measured in patients in the study groups almost all the patients in both the groups were relieved from burning sensation after treatment. however, 33% in group 1 got relief in the second week itself as against 21% in group 2 [table 4 ]. for inter group comparison of the effectiveness of treatment in increasing the mouth opening parameters, student 's t test was done that resulted in a value p 0.05 for grade 4 which indicated that clinical grade 3 responded better than grade 4. to compare the effectiveness of treatment in reducing the histological parameters in between the two groups after treatment, student 's t test was done which resulted in a value of p 0.05 for grade 4 which indicates that clinical grade 3 responded better than grade 4. osf is a morbid, crippling, and a premalignant condition of the oral mucosa associated with the areca nut chewing habit. it is commonplace in various indian states to use pan quid with tobacco and lime. several medical and surgical approaches have been tried for the management of osf over the decades. the results are not predictable with some therapies and none has been consistently successful. in 2001, haque., successfully treated 29 osf patients with recombinant interferon gamma (rhifn-) and concluded that ifn- may reverse osf. they tried ifn- in osf based on the studies which showed that systemic recombinant ifn- improved the mouth opening, musculoskeletal, and pulmonary efficiency in patients with scleroderma. in view of the female predilection, its presentation in middle life and histological similarities, the analogy that osf is an idiopathic scleroderma of mouth collagen fibrils in osf are more embryonic in nature with a defective maturation similar to scleroderma. canniff., showed that osf and scleroderma were similar with an increased frequency of hla dr3 and haplotypic pairs b 8/dr3 in the latter and hla dr3 a10 and b 7 in the former. however, there is difficulty in the availability of ifn- and the cost of this agent is high in developing countries. considering the shortcomings of all previous treatment modalities for osf and the positive observations of the study by haque. there was also a significant improvement in the mouth opening and in the movement of the tongue (clinical grade 5). the histopathological findings also showed a marked reduction in the inflammatory cell infiltrate and density of collagen fibrils. the mechanism by which colchicine improved the clinical status of osf patients in our study was difficult to ascertain since the drug was used in combination with hyaluronidase. also since this was the first study where cochicine had been used in the treatment of osf, we could not compare our results. however, we attributed them to the effect of colchicine which is both an anti - fibrotic and anti - inflammatory agent. like ifn-, colchicine also reduces collagen synthesis, down regulates fibroblast proliferation and upregulates anti - fibrotic cytokine and collagenase synthesis in the basal layer of the epithelium and lamina propria. one of our patients, however, showed relapse of the restricted mouth opening during the follow - up of 6 months after the cessation of treatment. the cause of the relapse could be due to a number of factors like the dosage being less for that particular individual or the non - compliance of treatment by the patient. colchicine has been reported to be beneficial in the treatment of diseases associated with fibrosis in animals and human beings. the short and long term administration of colchicine therapy in moderate dosages is surprisingly well tolerated. none of our patients reported any local or systemic adverse reactions during treatment and also after the cessation of drug intake. the most common toxic side effect reported reflect the drug 's action on rapidly proliferating gastrointestinal tract epithelial cells and include nausea, vomiting, diarrhea, and abdominal pain. these symptoms are especially frequent at dosage levels 2 - 3 mg / day although they are rapidly and completely reversible. it is possible that the dosage we used in our study, i.e. 0.5 mg colchicine administered orally twice a day, is an acceptable dosage, because at this dosage the patients achieved significant therapeutic results with no adverse reaction. large amounts of the drug and its metabolites enter the liver and then the bile and, hence, should not be administered to patients with hepatic disease. follow - up blood cell count should also be performed periodically and medication stopped if the toxic effects develop. tests to determine baseline serum urea nitrogen, creatinine levels, a complete blood cell count, and liver function test, performed prior to initiating the treatment and during follow - up of our patients also recorded values within the normal range. the encouraging results should prompt a clinical trial on more number of osf patients to broaden the therapeutic usefulness and applications of one of our most ancient treatment agents. this baseline study gives scope for further studies with the systemic use of colchicine alone in the treatment of osf, and, also for research in the use of the drug as a formulation that can be administered locally into the fibrous bands to confirm the above results. | background : oral submucous fibrosis (osf) is a precancerous condition of the oral mucosa. existing treatments give only temporary symptomatic relief. colchicine is an ancient drug with anti - fibrotic and anti - inflammatory properties. we planned to study the effects of colchicine in the management of oral submucous fibrosis.materials and methods : fifty osf patients were divided randomly into two groups and treated for 12 weeks. group 1-patients were administered tablet colchicine orally, 0.5 mg twice daily and 0.5 ml intralesional injection hyaluronidase 1,500 iu into each buccal mucosa once a week. group 2-patients were administered 0.5 ml intralesional injection hyaluronidase 1,500 iu and 0.5 ml intralesional injection hydrocortisone acetate 25 mg / ml in each buccal mucosa once a week alternatively. student 's t test and analysis of variance (anova) were used to compare pre and post treatment results. p<0.05 was considered as significant.results:thirty-three percent in group 1 got relief from burning sensation in the second week. inter group comparisons of increase in mouth opening and reduction in histological parameters indicated that group 1 patients responded better than group 2.conclusion:these encouraging results should prompt further clinical trials with colchicine alone on a larger sample size to broaden the therapeutic usefulness of the drug in the management of osf. |
professionals athlete must exercise at high intensity with restricted rest times between attempts ; thus recovery methods in sport can aid athletes to improve performance in competitions. in order to enhancement function, a kind of post - exercise recovery interventions are frequently utilized to improve recovery from training time. the composition and timing of nutrient intake can considerably impact recovery from severe exercise.[36 ] some appearance of recovery, including reduced muscle damage and boosted performance can be magnified by nutritional intervention, carbohydrate (cho)-electrolyte drink beverage intake. the beverages are generally utilized throughout and after exercise, ranging from bottled water to cho - electrolyte replacement drinks. beverages can aid in quick restoration of performance, blood sugar and muscle glycogen if used instantly after exercise. post - exercise recovery drink have underlined on timing, type of beverage, macro- and micro - nutrient content (calories, cho and protein (pro), mineral, and vitamins).[914 ] recent research suggests that a cho - pro) beverage consumed after exercise may satisfyingly affect the exercise recovery. therefore, composition of pro to cho in beverages can enhance performance and recovery between exercise sessions with a short recovery period. chocolate milk is as a popular and easily available drink among children and adults and is proposed to be effective. karp. have shown that chocolate milk is an effective recovery drink after exercise in comparison with cho replacement drink and fluid replacement drink. another study, reported that post - exercise chocolate milk consumption is as effective as a cho beverage. examined effects of chocolate milk consumption on markers of pro turnover, muscle glycogen, and performance during recovery from endurance exercise, and observed beneficial effects of chocolate milk compared to the cho only beverage. dough, a yogurt - based, salty drink popular and non - alcoholic beer are two other famous drinks in iranian diet. dough is a low - cho high - pro drink, which has a high amount of electrolytes like sodium (usually 0.8 - 1 g/100 g) and high calcium while non - alcoholic beer is a high - cho low - pro with vitamin b, c, and mineral. although these beverages are highly consumed by professional athletes, their effect on post - exercise recovery is not assessed yet. in present study, we tried to evaluate the effects of different beverage consumption on recovery time, performance, lipids profile, inflammatory biomarkers after rast test in iranian professional taekwondo athletes. this study was conducted as repeated measures crossover design with 22 men taekwondo player (mean sd : age 23.8 2.7 years ; stature 173.7 4.3 cm ; bmi 24.02 0.92)., all subjects were informed of all procedures of the study and signed an informed consent. participants were asked to refrain from exercise 24 h before trial initiation and to sustain their customary physical activity and dietary patterns. participants were asked to fill a food recall for 1 day before intervention. subjects completed standard protocol running - based anaerobic sprint test (r.a.s.t), after a self - determined warm - up for 10 min. participants concluded after protocol blood lactate was determined by lactometer (scout) so that immediately and 1 h posterior r.a.s.t protocol received no.1 beverage. subjects spend 2 h recovery periods. finally, after 2 h recovery time, blood sample were obtained. second and third sessions trial were similar to prior trial, separated by at least 4 days, instead of no. 1 beverage, participants received no.2 and no.3 beverages. the subjects were also asked to replicate their diet and activity in during study period. experimental trial data were analyzed using a simple repeated - measures analysis of variance (anova). simple contrasts were planned a priori in the case of a significant main effect, and dough was the reference category. within group comparisons analyses were performed with the spss version 16 (spss inc, chicago, il) statistical package. experimental trial data were analyzed using a simple repeated - measures analysis of variance (anova). simple contrasts were planned a priori in the case of a significant main effect, and dough was the reference category. within group comparisons analyses were performed with the spss version 16 (spss inc, chicago, il) statistical package. results showed that average pre- and post - recovery in crp for dough has significantly decreased (p 0.05). about mean pre- and post - recovery in ldl and hdl were no significant differences in all three beverages. however, there were no other within - subject differences for any of the other variables measured, including hdl, ldl, vo2max [table 1 ]. plasma concentrations of inflammatory, profile lipids and performance markers before and after ingestion of beverage in addition, no significant different (p > 0.05) in dietary intake (kcal, cho, pro, fat) were observed between three treatment periods [table 2 ]. mean composition of subjects diets (total kilo - calories), for the 24-h period prior to each trial the aim of this study was to assess the effects of three beverages consumed during recovery from rast test on inflammatory biomarkers, lipids profile and performance in iranian professional taekwondo player athletes. in this study, the serum crp levels in dough treatment was reduced more than non - alcoholic beer, whereas, in cho fluid treatment was increased. however, there was no difference in lipids profile, among groups. on the other hand, vo2max increased in dough and non - alcoholic beer treatments, but not in cho fluid group. on the one hand, in a study carried out by agerbaek. on a total of 58 healthy, non - fat men, it was concluded that 6-week intervention with fermentative product of milk caused a significant decrease in ldl ; however, hdl in the both groups of placebo and intervention showed no changes. on the other hand, in a time - frame study carried out by demosthenes., on a total of 1514 men and 1528 women who use dairy products including milk, cheese, and yogurt, it was figured out that the diet containing dairy products has no relation to the level of hdl. furthermore, in another study, 34 women were using yogurt or fermentative milk during 4 weeks and the results showed that dairy products cause a decrease in the level of ldl. in fact, the observations were parallel with the results of the current study and it showed that using yogurt drink causes a significant decrease in the level of ldl. out of the possible reasons for the resemblance in these results, it can be pointed that there was no differences in the ingredients of yogurt and yogurt drink of which calcium and vitamin d cause a decrease in ldl. whereas, other studies showed different results ; for instance, in a cross 3-period, 7-week study done upon 29 healthy women, intervention with yogurt caused an increase in hdl but upon ldl no impact was noticed. in another study carried out by mcnamara., the 3-week effect of yogurt intervention on 18 men were investigated and the results showed that yogurt had no effect on lipids profile. the period of study and the subjects were the possible reasons for conflicting results of above mentioned studies with the current study. in cross - sectional study done on 3042 men and women using dairy products, the results showed that dairy intake causes a significant decrease in the level of crp. the study had the same results as those of the present study and its possible reasons are : the presence of pro of high - quality, riboflavin, genjoged linolenic acid that might be effective on biomarkers. whereas, in a study, van meijl investigated the impact of dairy intake on 35 fat or overweight volunteers during 8 weeks, and he noticed no impacts upon the prominent factors. supplementation with drinking milk during 12 weeks had no significant impacts on crp and il-6 in those suffering from high blood pressure. it must be taken into account that these studies have not been planned to show the effect of dairy products on prominent biomarkers. in a study done by jimenez - flores on 35 athletic university students, the effects of two supplements namely milk of high - pro and trade supplement of high - cho were compared and the results showed that the athletic performance of those using milk supplement is more than that of those using cho supplement. in another study, cacao milk intake by 9 athletes in 2 competitions as far as 1 week caused significant decrease in their athletic performance and recovery. in a cross - sectional study done by choi on 9948 subjects, it was figured out that cho has a reverse relation with the amount of hdl plasma without taking fat intake and energy into account. additionally, in another cross - sectional study upon 2157 american teenagers, the reverse relation of cho with the level of hdl and the positive relation with the level of plasma were not seen. in a study done by aeberli., the results obtained from a 6-period, 3 week interference upon 29 volunteers showed that cocas sweeten cause an increase in ldl plasma. these results are different from the findings of the current study and the period of study and ingredients present in non - alcoholic beer containing vitamin b are effective upon the results. studied seven athletic persons and no differences was seen in the level of crp plasma between two groups using cho drink and coca based pro drink after their exercises. in another study done upon 18 healthy non - sports men, the results obtained from two groups using placebo and cho showed no changes in the level of crp however, in another study the levels of crp during using coca sweeten showed a significant increase. in a study done by fallowfield upon 12 athletic men and 4 athletic women, the group using electrolyte - cho solution noticed a significant progress in its performance during 4 h after recovery. in another study done upon 18 athletic men and athletic women during 2 weeks, the solution containing cho and sodium showed a more significant increase in their performance during the activity of resisting exercise than the other solutions selected by athletes themselves. concerning the results of abovementioned studies, the similar ingredients present in non - alcoholic beer, the period as well as the plan of study, and the subjects might have been effective on the sameness of the results. however, in a study done by price and cripps, 9 athletic men used glucose, bicarbonate sodium and placebo and no differences in their athletic performance was seen.[3840 ] this study suggests that dough was effective in reducing ldl and reducing inflammatory biomarkers including crp with little effect on performance in subjects. these findings further support the effectiveness of dough as a potential recovery aid for athletes between intense workouts. the strength of this study is since no study has been conducted on the effects dough consumption. on the other hand, in this study, the limitation of this study is not measuring other factors especially electrolyte and mineral contents. one another limitation of this study was as a result, further research is needed to clarify these findings. this study suggests that dough was effective in reducing ldl and reducing inflammatory biomarkers including crp with little effect on performance in subjects. these findings further support the effectiveness of dough as a potential recovery aid for athletes between intense workouts. | background : after exercise, recovery is very essential in professional sport. athletes use sport beverages to enhance endurance and physical performance. the purpose of this study was to examine the effects of dough versus non - alcoholic beer and carbohydrate (cho) fluid on performance, lipids profile, inflammatory biomarkers after running - based anaerobic sprint test (r.a.s.t) in taekwondo players.methods:this study was conducted as repeated measures crossover design with 22 men taekwondo player. subjects completed standard protocol r.a.s.t so that immediately and 1 h posterior r.a.s.t protocol received number 1 beverage. subjects spend 2 h recovery periods. second and third sessions trial were similar to prior trial, separated by at least 4 days, instead of number 1 beverage, participants received number 2 and number 3 beverage.results:data showed that average pre- and post - recovery in c - reactive protein (crp) or dough significantly decreased (p 0.05). about mean pre- and post - recovery in low density lipoprotein (ldl) and high density lipoprotein (hdl) there were no significant differences in all three beverages. besides, amount of crp was significant between three beverages (p 0.05) in dietary intake were observed between three treatment periods.conclusions:dough was effective in reducing ldl and reducing inflammatory biomarkers including crp with little effect on performance in subjects. |
nephrectomy is a common operation that is performed for neoplasms, trauma, living organ donation, and other disease conditions that make retention of the organ undesirable. in 2013, estimates of 45,610 unilateral total nephrectomies and 19,610 partial nephrectomies were performed in the united states. the outcomes of nephrectomy care are reported primarily for inpatient results, with complication rates being highly variable. the lack of consistent and reproducible metrics for measuring outcomes in inpatient surgical care has led our group to adopt 4 objective measurements for the surgical adverse outcome (ao) : inpatient mortality, risk - adjusted postoperative length of stay, 90-day postdischarge death without readmission, and 90-day readmission to an acute care hospital. each of these 4 components can be independently risk - adjusted. by using the medicare data set, the full scope of postdischarge events can be captured in the analysis. the medicare population only constitutes one - third of the total / partial nephrectomies annually in the united states, but it provides a high - risk population for definition of important comorbid conditions and provides a benchmark for care redesign and improvement. thus, this study will report a risk - adjusted national prediction model for nephrectomy surgery across the full continuum of inpatient and 90-days of postdischarge outcomes. this design can be used for comparing provider performance and measurement of care improvement efforts within hospitals across a wide array of different inpatient episodes of care. the centers for medicare and medicaid services (cms) inpatient limited data set for 2010 to 2012 was used to identify all elective nephrectomy patients with an international classification of diseases 9th revision - clinical modification (icd-9) procedure code of 55.4 (partial nephrectomy) or 55.51 (nephroureterectomy). the use of the cms inpatient limited data set for research does not require institutional review board review, but the authors are expected to comply with federal policy of not reporting data cells of less than 11 observations to preserve patient confidentiality. nephroureterectomy is an all - inclusive term used in icd-9 coding to identify all total nephrectomy operations regardless of the length of ureter that may have been resected. all qualifying cases were required to have an icd-9 principal diagnosis of 189.0 to 189.2, 223.0, or 223.1. procedures were only included if performed on days 0, 1, or 2 of hospitalization and patients were 65 years of age or older. missing data, transfers from another acute care hospital, and all discharges against medical advice were excluded. the developmental database was used for the patient - level design of risk - factors. it included all cases meeting the above criteria that were from hospitals with 20 or more qualifying cases for the study period, but only for those hospitals meeting accurate coding criteria that we have previously developed. a minimum of 20 cases for each hospital is required for the control chart methods detailed below, and the use of good coding hospitals for model development is to optimize accuracy in final prediction equations. final predictive models were then applied to all hospitals and patients in the study database independent of coding accuracy, but only for hospitals that had 4.5 predicted aos to avoid excessively small numbers in statistical evaluation. an extensive group of candidate risk factors including medical comorbidities identified as present - on - admission, and specific diagnoses (e.g., cancer) were used in model development. hospital dummy variables were used in each predictive model to account for hospital effects. in predictive modeling, hospital effects can have a large influence on the coefficients of final risk factors. hospital dummy variables eliminate hospital effects so that variables in final models are not affected by extreme hospital performances and were then removed to avoid hospital influences and bias on final risk factor coefficients. stepwise logistic regression was used to develop final prediction models for each of the 4 aos (fig. second, all live discharges were evaluated for risk - adjusted prolonged length - of stay (prlos). in surgical care standardized definitions, surveillance, and reporting for complications of surgical care are not present. because specific coded complications are numerous and inconsistent following major operations with many having no measurable impact on ultimate outcomes, we have developed prlos as a surrogate marker for severe inpatient complications. this necessitates only a single prediction model as a composite representation of significant inpatient morbidity among live discharges. the prlos patients are identified by developing a linear prediction model for length - of - stay among patients without coded complications, identification of excess lengths of stay against prediction values in patients without coded complications, and identification of those cases where observed minus predict values exceed the upper control limit by 3- using a moving - range control chart. the 3- outliers have major complications, have dramatic increases in costs of care, and are strong predictors of postdischarge aos. once prlos patients are identified, they become the dependent variable in a logistic risk equation to predict this surrogate event as a marker of severe inpatient complications. a schematic representation of how each segment of the nephrectomy population of study patients was used in the development of each of the final 4 prediction models used in this study. the third model is for 90-day deaths following discharge of nephrectomy patients that were not readmitted to an acute care hospital (pd-90). the fourth prediction model is for 90-day readmissions (ra-90) among patients who survived the entire 90-day postdischarge period of time. candidate risk factors for the postdischarge events were the same as for inpatient adverse outcomes except for the addition of prlos from the index hospitalization. readmissions for medical diagnostic categories (mdcs) 2 (eye diseases), 17 (myeloproliferative diseases), 22/24 (burns / major trauma), and all medicare severity - diagnosis related groups (ms - drgs) related to the management of trauma or cancer regardless of mdc were excluded as readmissions not associated with the index hospitalization. the 90-day postdischarge window has been selected since it is consistent with model 2 of the cms bundled payment for care improvement initiative that is currently being conducted by medicare and it is consistent with the comprehensive care of joint replacement program that is being launched by medicare, which makes 90-days the likely interval for accountability in pending medicare bundled payment initiatives. our prior studies in other major surgical areas have identified over 40% of readmissions that are associated with the index hospitalization occur between 30 and 90 days after discharge. all analyses were performed with sas software (version 9.4, sas institute, cary, nc). only hospitals with 25 or more cases in the study dataset met criteria of 4.5 predicted aos and were used to measure comparative performance. the final dataset had total predicted aos set equal to total observed aos to account for the 1.4% difference from the application of the developmental dataset. total observed aos for each hospital were determined by only identifying the first qualifying ao for each patient to avoid double - counting. total predicted aos were identified by respective prediction models using only those cases that were eligible at each stage. the standard deviation (sd) for predicted cases in each hospital was computed by the formula :, where p is the probability of an ao and n is the number of total cases within each hospital. negative z - scores indicated performance that was better than predicted and positive z - scores indicated suboptimal performance. the risk - adjusted ao rate for each hospital was computed by multiplication of the overall population ao rate by the observed / predicted ao rate of that specific hospital. the risk - adjusted ao rate expresses the overall outcome of the hospital on a linear scale that would be expected in the entire population of patients. performance that demonstrates the observed - to - predicted ratio of 1.0 for an overall population ao rate of 15% would have a risk - adjusted rate of 15%. if the observed - to - predicted ratio was 2.0, then the risk - adjusted rate would be 15% multiplied by 2.0, or 30%. if the observed to predicted ratio was 0.5, then the risk - adjusted rate would be 7.5%. the method adjusts all hospital performance to that which would be expected by multiplying each hospitals ratio of observe - to - predicted rates against the performance of the national performance. regression analysis was performed by hospital volume of cases against hospital ao rates to assess the influence of case load upon outcome results. the centers for medicare and medicaid services (cms) inpatient limited data set for 2010 to 2012 was used to identify all elective nephrectomy patients with an international classification of diseases 9th revision - clinical modification (icd-9) procedure code of 55.4 (partial nephrectomy) or 55.51 (nephroureterectomy). the use of the cms inpatient limited data set for research does not require institutional review board review, but the authors are expected to comply with federal policy of not reporting data cells of less than 11 observations to preserve patient confidentiality. nephroureterectomy is an all - inclusive term used in icd-9 coding to identify all total nephrectomy operations regardless of the length of ureter that may have been resected. all qualifying cases were required to have an icd-9 principal diagnosis of 189.0 to 189.2, 223.0, or 223.1. procedures were only included if performed on days 0, 1, or 2 of hospitalization and patients were 65 years of age or older. missing data, transfers from another acute care hospital, and all discharges against medical advice were excluded. the developmental database was used for the patient - level design of risk - factors. it included all cases meeting the above criteria that were from hospitals with 20 or more qualifying cases for the study period, but only for those hospitals meeting accurate coding criteria that we have previously developed. a minimum of 20 cases for each hospital is required for the control chart methods detailed below, and the use of good coding hospitals for model development is to optimize accuracy in final prediction equations. final predictive models were then applied to all hospitals and patients in the study database independent of coding accuracy, but only for hospitals that had 4.5 predicted aos to avoid excessively small numbers in statistical evaluation. an extensive group of candidate risk factors including medical comorbidities identified as present - on - admission, and specific diagnoses (e.g., cancer) were used in model development. hospital dummy variables were used in each predictive model to account for hospital effects. in predictive modeling, hospital effects can have a large influence on the coefficients of final risk factors. hospital dummy variables eliminate hospital effects so that variables in final models are not affected by extreme hospital performances and were then removed to avoid hospital influences and bias on final risk factor coefficients. stepwise logistic regression was used to develop final prediction models for each of the 4 aos (fig. second, all live discharges were evaluated for risk - adjusted prolonged length - of stay (prlos). in surgical care standardized definitions, surveillance, and reporting for complications of surgical care are not present. because specific coded complications are numerous and inconsistent following major operations with many having no measurable impact on ultimate outcomes, we have developed prlos as a surrogate marker for severe inpatient complications. this necessitates only a single prediction model as a composite representation of significant inpatient morbidity among live discharges. the prlos patients are identified by developing a linear prediction model for length - of - stay among patients without coded complications, identification of excess lengths of stay against prediction values in patients without coded complications, and identification of those cases where observed minus predict values exceed the upper control limit by 3- using a moving - range control chart. the 3- outliers have major complications, have dramatic increases in costs of care, and are strong predictors of postdischarge aos. once prlos patients are identified, they become the dependent variable in a logistic risk equation to predict this surrogate event as a marker of severe inpatient complications. a schematic representation of how each segment of the nephrectomy population of study patients was used in the development of each of the final 4 prediction models used in this study. the third model is for 90-day deaths following discharge of nephrectomy patients that were not readmitted to an acute care hospital (pd-90). the fourth prediction model is for 90-day readmissions (ra-90) among patients who survived the entire 90-day postdischarge period of time. candidate risk factors for the postdischarge events were the same as for inpatient adverse outcomes except for the addition of prlos from the index hospitalization. readmissions for medical diagnostic categories (mdcs) 2 (eye diseases), 17 (myeloproliferative diseases), 22/24 (burns / major trauma), and all medicare severity - diagnosis related groups (ms - drgs) related to the management of trauma or cancer regardless of mdc were excluded as readmissions not associated with the index hospitalization. the 90-day postdischarge window has been selected since it is consistent with model 2 of the cms bundled payment for care improvement initiative that is currently being conducted by medicare and it is consistent with the comprehensive care of joint replacement program that is being launched by medicare, which makes 90-days the likely interval for accountability in pending medicare bundled payment initiatives. our prior studies in other major surgical areas have identified over 40% of readmissions that are associated with the index hospitalization occur between 30 and 90 days after discharge. all analyses were performed with sas software (version 9.4, sas institute, cary, nc). only hospitals with 25 or more cases in the study dataset met criteria of 4.5 predicted aos and were used to measure comparative performance. the final dataset had total predicted aos set equal to total observed aos to account for the 1.4% difference from the application of the developmental dataset. total observed aos for each hospital were determined by only identifying the first qualifying ao for each patient to avoid double - counting. total predicted aos were identified by respective prediction models using only those cases that were eligible at each stage. the standard deviation (sd) for predicted cases in each hospital was computed by the formula :, where p is the probability of an ao and n is the number of total cases within each hospital. negative z - scores indicated performance that was better than predicted and positive z - scores indicated suboptimal performance. the risk - adjusted ao rate for each hospital was computed by multiplication of the overall population ao rate by the observed / predicted ao rate of that specific hospital. the risk - adjusted ao rate expresses the overall outcome of the hospital on a linear scale that would be expected in the entire population of patients. performance that demonstrates the observed - to - predicted ratio of 1.0 for an overall population ao rate of 15% would have a risk - adjusted rate of 15%. if the observed - to - predicted ratio was 2.0, then the risk - adjusted rate would be 15% multiplied by 2.0, or 30%. if the observed to predicted ratio was 0.5, then the risk - adjusted rate would be 7.5%. the method adjusts all hospital performance to that which would be expected by multiplying each hospitals ratio of observe - to - predicted rates against the performance of the national performance. regression analysis was performed by hospital volume of cases against hospital ao rates to assess the influence of case load upon outcome results. a total of 23,193 patients (87.7%) were in the developmental database for ipd model design that came from good coding hospitals. there were 10 significant risk factors in the ipd model, with a c - statistic of 0.790. for prlos, there were 1471 length - of - stay outliers (6.3%), 25 risk factors, and a c - statistic of 0.696 in the final model. there were 206 (0.9% of total cases) deaths in pd-90, the risk model had 7 risk factors, and the c - statistic was 0.811. a total of 3400 patients (14.7% of total cases) in the developmental database were 90-day readmissions following exclusion of nonassociated ms - drgs. there were 22 significant risk factors in the ra-90 model with a c - statistic of 0.660. the odds ratios of significant risk factors in prediction models are identified in table 1. a total of 5123 patients (22.1%) underwent robotic - assisted nephrectomy or partial nephrectomy. robotic - assisted procedures were associated with significantly lower rates of prlos and ra-90, but no benefits in mortality rates. the risk factors and odds ratios (standard error) for predictive models in nephrectomy. the ms - drgs of readmissions in the developmental database are presented in table 2. a total of 3399 patients (14.7%) were readmitted 4382 times during the 90 days following discharge. a total of 2339 readmissions (53.4%) were in the first 30 days, 1130 (25.8%) were between days 31 and 60, and 913 (20.8%) were from day 61 to 90. readmissions were from cardiac, infectious, gastrointestinal, kidney / urinary tract, and other causes. there were 23,477 patients from 439 hospitals in the study database that had 25 or more cases and predicted aos of 4.5 or more. the addition of patients from all hospitals in the study database was then reduced by nearly an equivalent amount by the increased in the threshold for hospital evaluation to 25 instead of 20 cases. a total of 176 patients (0.75%) died during inpatient care and 1561 (6.7%) were live discharges with prlos. an additional 307 patients (1.3%) died within 90 days following discharge without readmission, and 3458 live discharged patients (14.7%) were readmitted 1 or more times. among readmitted patients, another 208 patients died within 90 days for a total mortality rate including inpatient and 90-day postdischarge deaths of 2.9% (691 patients). there were 4891 patients (20.8%) who had 1 or more aos including inpatient and the 90-day postdischarge period. a total of 8 hospitals (1.8%) had outcomes that were better than 2 sds from the average and 17 hospitals (3.9 %) were 2 sds poorer than average. in fig. 3, the risk - adjusted median ao rates of hospitals are presented by decile of performance. the best performing decile had a median risk - adjusted ao rate of 10.2% while the poorest performing decile of hospitals had a risk - adjusted ao rate of 32.1%. a weak correlation coefficient of 0.08 favored smaller hospitals with better outcomes, but no statistical significance was seen (p = 0.16) with analysis of variance. there were 23,477 patients from 439 hospitals in the study database that had 25 or more cases and predicted aos of 4.5 or more. the addition of patients from all hospitals in the study database was then reduced by nearly an equivalent amount by the increased in the threshold for hospital evaluation to 25 instead of 20 cases. a total of 176 patients (0.75%) died during inpatient care and 1561 (6.7%) were live discharges with prlos. an additional 307 patients (1.3%) died within 90 days following discharge without readmission, and 3458 live discharged patients (14.7%) were readmitted 1 or more times. among readmitted patients, another 208 patients died within 90 days for a total mortality rate including inpatient and 90-day postdischarge deaths of 2.9% (691 patients). there were 4891 patients (20.8%) who had 1 or more aos including inpatient and the 90-day postdischarge period. a total of 8 hospitals (1.8%) had outcomes that were better than 2 sds from the average and 17 hospitals (3.9 %) were 2 sds poorer than average. in fig. 3, the risk - adjusted median ao rates of hospitals are presented by decile of performance. the best performing decile had a median risk - adjusted ao rate of 10.2% while the poorest performing decile of hospitals had a risk - adjusted ao rate of 32.1%. the hospitals in the largest volume decile had more than 105 cases. a weak correlation coefficient of 0.08 favored smaller hospitals with better outcomes, but no statistical significance was seen (p = 0.16) with analysis of variance we have identified a dramatic difference in performance by hospitals using the 4 risk - adjusted metrics in nephrectomy. best performing hospitals had adverse outcome rates approaching 10% while poorest performing hospitals exceeded 30%. it is likely that hospitals and urologic surgeons are not completely aware of postdischarge aos among their patients given the high rates of readmissions to other facilities. improvement of outcomes must begin with better methods for tracking patient outcomes than has previously existed. this will result in better care for patients and reduce penalties from medicare for excessive readmission rates. predictive modeling for readmissions has been difficult and likely relates to socioeconomic, geographic, and patient compliance issues being major factors.. the prediction models of table 1 identify those risk factors that are associated with the 4 categories of aos. these predictive models can be of use in describing the patient conditions that require increased attention by clinicians caring for nephrectomy patients. cardiac, pulmonary, and biliary disease when present predict adverse outcomes across the inpatient and postdischarge period and require focused attention for prevention. better postdischarge follow up and improved patient contact after discharge can improve readmission rates. of particular interest, patients who have major inpatient complications as identified by prolonged length - of - stay predict postdischarge death and readmission in nephrectomy patients and in surgical patients in general. special follow - up and surveillance strategies are necessary to improve outcomes for this population in particular. the trends in hospitalized surgical care over recent decades have resulted in shorter lengths - of - inpatient care. patients are rapidly discharged and the consequences have been that more complications of care are not identified until after discharge. outcomes of nephrectomy and other operations can not be accurately made without inclusion of postdischarge events. postdischarge deaths and readmissions may not be captured by quality review initiatives or even by the operating surgeon. a total of 20% to 40% of discharged surgical cases is readmitted to hospitals other than the primary institution. the use of medicare administrative claims data permits tracking the patients over time and accurate identification of all aos across the continuum of care, including postdischarge deaths. while the medicare database may be criticized for having only the elderly nephrectomy patient, alternative patient samples across all age groups (e.g., national inpatient sample from the healthcare cost and utilization project) do not have encrypted patient identifiers that permit identification of postdischarge deaths and readmissions. while most clinicians recognize that postdischarge events need to be included in outcome assessments, the duration of postdischarge time included in this measurement remains controversial. traditional mortality and complication rates that are reported for urology and other surgical care have been inclusive of 30-days following the procedure. many have used this 30-day interval for reporting postdischarge readmissions. however, as is illustrated in table 2, significant readmissions occur from days 31 to 90. the comprehensive care for joint replacement initiative by medicare will be inclusive of 90-days following discharge, and it can be expected that other bundled understanding why patients are readmitted as is illustrated in table 2, and developing care redesign strategies to avoid unnecessary readmissions (and emergency department visits) will be essential. discussion has surrounded whether better outcomes are a function of hospital volume for specific operations. some have argued that better outcomes follow larger volumes, while others have not identified that relationship. in this study, it does not appear that better results are associated with larger volumes of cases particularly when postdischarge adverse outcomes are included. gore studied all - payer radical nephrectomy over 5 years in the state of washington. they identified highly variable rates of prolonged length of stay (75th percentile) among hospitals using methodology different from our study. overall mortality rates were similar to our report, but 30-day readmission rates demonstrated little variability among hospitals as opposed to the highly variable rates that we identified. they concluded that hospital variation was a major issue in urologic care including radical cystectomy, prostatectomy, and nephrectomy. similarly, hwang demonstrated hospital length - of - stay for radical nephrectomy was a significant predictor of deaths and readmissions. leow identified increased 30-day readmissions following inpatient complications from the operation, which is an observation that is similar to our prolonged length of stay in the index hospitalization being a predictor of 90-day postdischarge deaths and readmissions. our study identified a favorable effect of robotic surgery on prolonged length - of - stay outliers and upon 90-day readmissions. unfortunately, icd-9 coding convention does not permit the identification of laparoscopic - assisted nephrectomy. consistent with our findings, schmid found improved 30-day readmissions with minimally invasive approaches to nephrectomy. the analytic methods used in the current study underscore that major new technology can be evaluated for its impact on outcomes by including a risk factor among candidate variables for new methods that are employed. we have used screens to identify only quality coding hospitals for model development, but appreciate that comprehensive clinical abstraction of cases is potentially best. clinical data would permit identification of the stage of cancer and would also identify those cases with extension of the primary cancers into the vena cava. knowledge of the stage of primary renal cell carcinoma will affect the selection of minimally invasive surgical decisions versus open nephrectomy. medical centers receiving more advanced stage cancers will have a risk profile that is not currently captured, and may partially explain the absence of enhanced outcomes in large volume facilities in this study. however, clinical data is self - reported, expensive to gather, and is often deficient in accurate postdischarge information. enhancement of administrative data with readily available clinical information, such as admission laboratory data from electronic medical records, has been shown to enhance discrimination of administrative data mortality models and should be useful for the future. enhancement of administrative data with numerical laboratory data at admission has improved model performance for risk - adjusted prolonged length of stay in cardiac inpatient episodes of care. it can only be hoped that the electronic medical record will enhance the retrieval of pertinent clinical information (e.g., stage of cancer or laparoscopic - assisted procedures) for model development without requiring full chart abstraction. this was necessary because all - payer databases other than medicare are generally not available. state - based, all - payer databases that have encrypted patient identification will permit studies that evaluate outcomes for all patients and provide the necessary postdischarge information about readmissions. these databases with patient identifiers are present in only about 10% of states at present. it should be emphasized that bundled payment strategies by medicare will be using the same database that we have used in this study. a final limitation is that additional exclusions need to be defined for the evaluation of hospital performance. we have excluded ophthalmology, cancer, and trauma readmissions. we have identified that the overwhelming majority of readmissions of table 2 are linked either to the index hospitalization or represent decompensation of underlying medical illnesses. a small number of additional exclusions need to be made to bring our readmission criteria in compliance with that being used by medicare. in conclusion, the risk - adjusted ao rates in nephrectomy and partial nephrectomy are highly variable among acute care hospitals. observed ao rates that can be compared with risk - adjusted predicted results allow individual hospitals and clinicians to benchmark their performance. thus, the methods for outcome modeling and measurement are applicable for other surgical conditions and more comprehensive populations of patients. in an era of increased public reporting of outcomes and value - based purchasing of healthcare services, it is very important for hospitals and surgeons to know what are their results of care that are inclusive of the postdischarge period of time. risk - adjusted results permit the evaluation of specific areas that need improvement (e.g., infections), provide focus for care redesign, and serve as a method of actually tracking of improved outcomes from efforts to modify hospital processes and clinician practice patterns. | abstractwithout risk - adjusted outcomes of surgical care across both the inpatient and postacute period of time, hospitals and surgeons can not evaluate the effectiveness of current performance in nephrectomy and other operations, and will not have objective metrics to gauge improvements from care redesign efforts.we compared risk - adjusted hospital outcomes following elective total and partial nephrectomy to demonstrate differences that can be used to improve care. we used the medicare limited dataset for 2010 to 2012 for total and partial nephrectomy for benign and malignant neoplasms to create prediction models for the adverse outcomes (aos) of inpatient deaths, prolonged length - of - stay outliers, 90-day postdischarge deaths without readmission, and 90-day relevant readmissions. from the 4 prediction models, total predicted adverse outcomes were determined for each hospital in the dataset that met a minimum of 25 evaluable cases for the study period. standard deviations (sds) for each hospital were used to identify specific z - scores. risk - adjusted adverse outcomes rates were computed to permit benchmarking each hospital 's performance against the national standard. differences between best and suboptimal performing hospitals defined the potential margin of preventable adverse outcomes for this operation.a total of 449 hospitals with 23,477 patients were evaluated. overall ao rate was 20.8% ; 17 hospitals had risk - adjusted ao rates that were 2 sds poorer than predicted and 8 were 2 sds better. the top performing decile of hospitals had a risk - adjusted ao rate of 10.2% while the lowest performing decile had 32.1%. with a minimum of 25 cases for each study hospital, no statistically valid improvement in outcomes was seen with increased case volume.inpatient and 90-day postdischarge risk - adjusted adverse outcomes demonstrated marked variability among study hospitals and illustrate the opportunities for care improvement. this analytic design is applicable for comparing provider performance across a wide array of different inpatient episodes. |
the cytoplasmic and nuclear compartments of eukaryotic cells are separated by the nuclear envelope (ne). the double membrane of the ne is perforated by nuclear pore complexes (npcs), which are large multiprotein complexes that support passive diffusion of small molecules, and facilitate receptor - mediated translocation of proteins and ribonucleoprotein complexes. overall, the vertebrate npc is a 120 mda protein complex made of 30 different proteins called nucleoporins (or nups) that are repetitively arranged as distinct subcomplexes to form the npc (cronshaw. 2002 ; lim and fahrenkrog 2006 ; rout. 2000 ; schwartz 2005 ; tran and wente 2006). in the plane of the ne, the eightfold symmetric central framework of the npc, also known as the spoke complex, encloses a central pore that is 50 nm long and is narrowest (40 nm) at the ne midplane (beck. 2004, 2007 ; stoffler. 2003). attached to the central framework are cytoplasmic filaments and a nuclear basket (figs. 1, 2). fig. a cytoplasmic face of negatively stained and b a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from triturus alpestrus. c cytoplasmic face of negatively stained and d a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from xenopus laevis. view of the nuclear basket of isolated nuclei from xenopus oocytes prepared by e critical point drying and field emission scanning electron microscopy, f quick - freeze / freeze - drying / rotary metal shadowing and g thin - sectioning and transmission electron microscopy. a reproduced with permission from gall (1967). three - dimensional reconstruction of the central framework of negatively stained npcs after detergent treatment a from xenopus oocytes, and b its adaptation for a consensus model of the npc architecture and c from yeast nuclei and d its adaptation into a model. e consensus model of the npc based on a reconstruction of native npcs embedded in thick amorphous ice. f reconstruction of npcs from intact nuclei of dictyostelium discoideum examined by cryo - electron tomography. (2007) electron micrographs of npcs over time. a cytoplasmic face of negatively stained and b a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from triturus alpestrus. c cytoplasmic face of negatively stained and d a stretch along a nuclear envelope of embedded and thin - sectioned nuclei from xenopus laevis. view of the nuclear basket of isolated nuclei from xenopus oocytes prepared by e critical point drying and field emission scanning electron microscopy, f quick - freeze / freeze - drying / rotary metal shadowing and g thin - sectioning and transmission electron microscopy. three - dimensional reconstruction of the central framework of negatively stained npcs after detergent treatment a from xenopus oocytes, and b its adaptation for a consensus model of the npc architecture and c from yeast nuclei and d its adaptation into a model. e consensus model of the npc based on a reconstruction of native npcs embedded in thick amorphous ice. f reconstruction of npcs from intact nuclei of dictyostelium discoideum examined by cryo - electron tomography. a reprinted with permission from hinshaw. (2007) we begin, here, with a retrospective that summarizes the analysis and elucidation of npc architecture by electron microscopy (em) techniques. we will discuss how the dissection of npc structure by electron tomography and x - ray crystallography has in a methodical and progressive manner reached the ultrastructural, molecular and atomic scale. in parallel, we will overview the progress that has contributed to the contemporary understanding of how the npc functions as a selective gate - barrier. setting our sights on the future, we will close by highlighting the importance of reconciling both npc structure and function, as we strive towards converging on a single conceptual, mechanistic understanding of the npc. npc structure was initially dissected using transmission em (tem), but later expanded to scanning transmission as well as to scanning em and, most recently, to cryo - electron tomography (cet). the first em study on the ne in 1950 revealed that it is perforated by pores (callan and tomlin 1950). gall (1967) showed for the first time that npcs exhibit an octagonal structure (fig. this was later confirmed by a number of studies in the following years (aaronson and blobel 1974 ; franke and scheer 1970 ; maul 1971), whereby its overall architecture, particularly its eightfold symmetry, appeared to be evolutionarily conserved (franke and scheer 1970). although its functional significance was ambiguous at that time, it was already evident from these early studies that the npc is composed of a membranous structure, specifically with a part residing within the ne, and nonmembranous, filamentous structures being attached to the cytoplasmic and the nuclear face of the npc (fig. 1b) (franke and scheer 1970 ; kessel 1969). according to today s consensus, the npc consists of an approximately cylindrical central framework, eight cytoplasmic filaments and a nuclear basket (fig. 1c g) that is composed of eight filaments that join into a distal ring (fig. refinement in em techniques using negatively stained and frozen - hydrated npcs from xenopus laevis oocyte nes (akey and radermacher 1993 ; hinshaw. 2c) (yang. 1998) provided early 3d reconstructions of the central framework. the central framework of the npc (also known as the spoke complex) resides within the ne, and is anchored to the region where the inner and outer nuclear membranes fuse. early structural studies showed that the cytoplasmic and nuclear ring moieties are integral to the central framework. 2003) and intact, transport - competent nuclei isolated from dictyostelium discoideum (fig. 2004, 2007) have improved the resolution of the central framework to 6 nm and revealed the first reconstructions of peripheral, flexible components of the npc, i.e. the cytoplasmic filaments and the nuclear basket. in dictyostelium, the cytoplasmic filaments are about 35 nm in length and the nuclear basket is about 60 nm long. together with the 50 nm central framework, the npc has an overall length of about 150 nm and an outer diameter of 125 nm (beck. although the overall linear dimensions of the npc vary between species, the overall 3d architecture appears to be evolutionarily conserved (fahrenkrog. 1998 ; kiseleva. 2004 ; yang. 1998). enclosed by the central framework is the hourglass - shaped central pore of the npc, which has a diameter of 6070 nm at its cytoplasmic and nuclear periphery and is 45 nm in the midplane of the npc / ne (beck. 2004, 2007 ; pante and kann 2002 ; stoffler. 2003). this central pore mediates the traffic between the cytoplasm and the nucleus, i.e. diffusion of small molecules and ions, as well as the selective transport of signal - carrying macromolecular cargo with diameters up to 39 nm (pante and kann 2002) (see later section). the peripheral channels of the npc have a diameter of about 8 nm and have been implicated in the diffusion of small molecules and ions (feldherr and akin 1997 ; hinshaw. 1992) and/or in trafficking of integral membrane proteins to the inner nuclear membrane (soullam and worman 1995). structurally, the cytoplasmic and the nuclear openings of the peripheral channels are not topologically continuous (stoffler. however, since biochemical studies suggest that passive and facilitated transport across the npc proceed via routes that are sterically nonoverlapping (naim. 2007), it remains debated if (1) there are two routes existing in the central pore [i.e., facilitated transport along the walls of the central pore and passive diffusion through a narrow diffusion tube located at the pore center (peters 2005) ], or (2) whether passive diffusion might somehow also utilize the peripheral channels (akey and radermacher 1993 ; beck. other potential roles for the peripheral channels have been proposed, such as in maintaining the ne electrical conductance (danker. 1999 ; enss. 2003 ; mazzanti. 2001 ; shahin. 2001) or as mechanical buffer zones that accommodate deformations of the central framework upon translocation of large cargoes (stoffler. yeast cells have 200 npcs / nucleus, proliferating human cells have 1020 npcs/m (i.e. 2,0005,000 npcs / nucleus) and a mature xenopus oocyte has about 60 npcs/m yielding 5 10 npcs / nucleus (gerace and burke 1988 ; gorlich and kutay 1999). a comprehensive ultrastructural study using freeze - fracture em of yeast cells in combination with 3d reconstruction has shown that the distribution of yeast npcs in the ne is not equidistant, but rather cluster into regions of higher density (winey. this observation is not limited to yeast and is present in other cell types as well (franke 1974). in yeast, the number of npcs was found to increase steadily, beginning in the g1-phase and peaking in the s - phase of the cell cycle, suggesting that npc assembly occurs continuously throughout the cell cycle (winey. similarly, the density of npcs increases throughout the cell cycle in hela s3 cells (maeshima. 2006). the molecular building blocks of the npc comprise 30 different proteins known as nucleoporins or nups, which are present in at least eight copies per npc (cronshaw. the transmembrane group, which contains transmembrane -helices and a cadherin fold, comprises the outermost features of the npc central framework and is thought to assist in anchoring the npc to the ne. the second group of nucleoporins contain -propeller and -solenoid folds, which localizes towards the inside of the npc, whereas the third class harbors the conserved sequence motif of phenylalanine glycine (fg)-repeats in combination with a coiled - coil fold and likely contributes to the formation of the npc s inner central framework and the peripheral structures (devos. 2006 ; schwartz 2005 ; tran and wente 2006). other less frequent structural motifs found in nucleoporins are zinc - finger domains as in nup153 and ranbp2/nup358 (higa. 2007) or rna - recognition motifs as in nup35 (handa. -propellers are predicted in the third class of the nucleoporins, and in fact seven - bladed -propellers have been resolved from the n - terminal domains (ntd) of the human nucleoporins nup133 and nup214 as well as from the ntd of nup159p in yeast by x - ray crystallography (berke. proteins with -propeller folds participate in diverse cellular functions and serve as platforms for multiple dynamic protein protein interactions. along this line, yeast nup133p and nup159p both play roles in mrna export from the nucleus, given that deletion or mutations in their ntds impair their functions in mrna export, probably by preventing the association of multiple mrna export factors with the npc (berke. the ntd of human nup133 furthermore contains an amphipathic -helical motif capable of sensing membrane curvature (drin. this motif corresponds to an exposed loop, which connects two blades of the -propeller and folds to an -helix upon interacting with small liposomes. whether the -helical motif in nup133 serves to recognize the curved topology of the nuclear pore membrane to anchor the npc during interphase or to recognize small vesicles containing ne fragments critical for ne reassembly after mitosis, or both, remains to be clarified (drin. the ntd of human nup214, in comparison to its yeast homolog nup159p, consists of two distinct structural elements : the -propeller and a 30-residue c - terminal extended peptide segment (napetschnig. this extension binds to the bottom of the -propeller with low affinity and has been suggested to play an auto - inhibitory role in npc assembly. the first crystal structure obtained for a nucleoporin was the npc targeting domain of human nup98 (hodel. this domain, similar to the nuclear pore - targeting domain of its yeast homolog nup116p, consists of a six - stranded -sheet sandwiched against a two - stranded -sheet and is flanked by two -helical regions (hodel. this domain exhibits multiple conformations and is stabilized only when bound to a ligand, i.e. nup96 and nup145p - c in the case of nup98 and nup116p, respectively (robinson. 2005). conformational diversity may allow nup98 and nup116p to bind to multiple targets within the npc or to associate and dissociate quickly from the npc to increase the mobility of the nucleoporins, as described for nup98, which shuttles in a transcription - dependent manner (griffis. an attempt to crystallize the first subcomplex of the npc, the nup62 complex, yielded the structure of the -helical coiled - coil domain of rat nup58/45 (melcak. the intradimer interface is hydrophobic, whereas dimer - dimer interactions occur through large hydrophilic residues. the tetramer can adopt various conformations leading to a lateral displacement between tetramers suggesting an intermolecular sliding mechanism (melcak. the nup62 complex has recently been mapped to the cytoplasmic periphery of the npc s central pore (schwarz - herion. 2007), so that sliding of nup58/45, and possibly of nup62 and nup54 as well, could contribute in modulating the diameter of the central pore in response to transport activity (melcak. fg - repeat domains (also known as fg - domains) are found in about one - third of the nucleoporins and mediate the interaction between soluble transport receptors loaded with signal - bearing cargo and the npc. these fg - domains constitute the key components of the selective gate - barrier that limits the diffusion of cargoes through the npc (see following section). atomic structures of short fg - repeat peptides in complex with, for example, the import receptor importin (bayliss. 2002a) or the putative mrna export factor tap / nxf1 (grant. 2002, 2003), have consistently shown that the interaction between fg - repeats and the different transport receptors involves primarily the phenylalanine ring of the fg - repeat core and hydrophobic residues on the surface of the receptor (isgro and schulten 2005, 2007a, b). hydrophilic linker regions between individual fg - motifs, which constitute the majority of amino acid mass in the overall fg - domain, appear to influence the strength of the binding and allow simultaneous binding of several fg - cores to the receptor (liu and stewart 2005). based on biophysical measurements, the fg - domains of yeast nucleoporins have been found to be natively unfolded (denning. similarly, fg - domains of human, fly, worm and other yeast species are most likely disordered based on their amino acid composition (denning and rexach 2007). this notion is further supported by immuno - em studies of vertebrate fg - repeat nucleoporins, which suggest that the fg - domains are flexible and mobile within the npc (fahrenkrog. single molecule studies using atomic force microscopy (afm) on the recombinantly expressed fg - domain of human nup153 further revealed that this 700 residue domain is in fact an 180 nm long unfolded molecule when adsorbed on the surface of mica (lim., it is now estimated that each npc is populated by at least 128 fg - domains together displaying 3,500 fg - repeats (strawn. 2004). nup153 and nup214 are both known to play roles in distinct nucleocytoplasmic transport (nct) pathways and interact with a number of nuclear transport receptors via their fg - repeats (ball and ullman 2005 ; bernad. 2006 ; hutten and kehlenbach 2006 ; sabri. 2007 ; van deursen. 1996). the location of the fg - domains of nup153 and nup214 shifts in a transport - dependent manner in the npc, further supporting their role in nct (paulillo. 2005). systematic deletion of fg - repeat regions in yeast nucleoporins revealed, however, that yeast npcs suffer from only slight changes in distinct nuclear transport pathways, but otherwise remain functional despite having removed up to 50% of their fg - repeats (strawn. this suggests that the fg - repeats exhibit a functional redundancy in bulk nct, except for specific nuclear transport pathways, which may require individual fg - nucleoporins, and/or that other interaction sites for transport receptors exist within the npc (terry and wente 2007). besides playing important roles in nct, the crystal structure of the rrm domain of mouse nup35 revealed that all three fg - sequences of this nucleoporin are in ordered secondary structure elements and that these fg - sequences do not interact with transport receptors, such as importin, but rather with, for example, the integral membrane protein ndc1. thus, the fg - sequences of nup35 may contribute to the formation of the npc s central framework (handa. npcs are porous to small molecules (e.g. water and ions), which freely diffuse through the npc, while more massive cargoes (i.e. > 40 kda in size) [a recent study has showed that this limit may extend to 100 kda (wang and brattain 2007) ] require the assistance of soluble transport receptor proteins to be effectively chaperoned through the npc. a large number of these receptor proteins, known collectively as karyopherins (kaps) or more specifically as importins (imp) and exportins (exp), were discovered around the 1990s (rexach and blobel 1995 ; wozniak. today, the biochemical role of the receptors in unlocking the npc barrier as part of the nuclear trafficking machinery is relatively well understood. as illustrated in fig. 3, appropriate cargoes are identified through a short sequence of residues known as nuclear localization / export signals (i.e. nls / nes) for import and export, respectively, which exhibit binding interactions with the karyopherins [sometimes using an adaptor such as, for example, importin- (gorlich. 1994) ]. otherwise, passage through the npc is obstructed for large, non - nls / nes harboring molecules that do not bind to the karyopherins (i.e. passive) (paine. the directionality of nct is driven by an asymmetric distribution of the two nucleotide states of the small gtpase ran (melchior. being predominant in the nucleus, rangtp functions to release the cargo from its import receptor by binding to the receptor itself (gorlich. exportins bind to their cargo in the presence of rangtp, which ferries the complex back into the cytoplasm. once in the cytoplasm, gtp hydrolysis causes the disassembly of the export complex, thereby recycling the export receptor and fueling the cytoplasmic rangdp pool. passage through the npc is restricted to transport receptors, which bind and chaperone nls - cargo through the npc, while access is prohibited for passive (non - nls) molecules of similar size. binding of rangtp to the transport receptor releases the nls - cargo into the nucleus. the gray shaded area emphasizes the location of the fg - domains and the question mark highlights the uncertainty with regard to the biophysical aspects of the selective gating mechanism within the npc, which operates to restrict or promote cargo translocation schematic representation identifying the main biochemical constituents of the nuclear import machinery. passage through the npc is restricted to transport receptors, which bind and chaperone nls - cargo through the npc, while access is prohibited for passive (non - nls) molecules of similar size. binding of rangtp to the transport receptor releases the nls - cargo into the nucleus. the gray shaded area emphasizes the location of the fg - domains and the question mark highlights the uncertainty with regard to the biophysical aspects of the selective gating mechanism within the npc, which operates to restrict or promote cargo translocation early models of the npc gating mechanism were derived from initial em studies, which linked the biophysical origin of the selective gate to the presence of a central plug or central transporter the central transporter was suggested to consist of an iris - like mechanism that is hinged to the central channel (akey 1990) (fig. although recent evidence indicates that this feature represents to a large extent cargo complexes arrested during translocation (beck. 2004, 2007 ; stoffler. 2003), this model suggested a translocation mechanism that has largely defined the criteria for subsequent npc models in the field (akey and goldfarb 1989). these steps include a peripheral binding to the npc, followed by a docking step, and then a translocation step (e.g. by a dilation of the transporter). fig. a the central transporter consisting of an iris - like mechanism that is hinged to the central channel. b being natively unfolded and located at the nuclear / cytoplasmic peripheries of the npc, the virtual gating model suggests that the entropic movements of the fg - domains can act as a barrier to inert cargo. c the affinity gradient model proposes that receptor - cargo complexes traverse a pathway that is lined by fg - domains of increasing affinity. d the oily - spaghetti model predicts that the fg - domains can only be pushed aside by receptor - cargo complexes. e the undersaturated and subsequently the saturated hydrogel model purport that the fg - domains crosslink via a dense number of inter - fg interactions to form a highly organized three - dimensional network within the npc. f the nanomechanical reversible collapse model asserts that the selective gating mechanism consists of a stochastic flux of collapsing and distending fg - domains that is regulated by binding and unbinding interactions between the fg - domain and the transport receptors. a the central transporter consisting of an iris - like mechanism that is hinged to the central channel. b being natively unfolded and located at the nuclear / cytoplasmic peripheries of the npc, the virtual gating model suggests that the entropic movements of the fg - domains can act as a barrier to inert cargo. c the affinity gradient model proposes that receptor - cargo complexes traverse a pathway that is lined by fg - domains of increasing affinity. d the oily - spaghetti model predicts that the fg - domains can only be pushed aside by receptor - cargo complexes. e the undersaturated and subsequently the saturated hydrogel model purport that the fg - domains crosslink via a dense number of inter - fg interactions to form a highly organized three - dimensional network within the npc. model asserts that the selective gating mechanism consists of a stochastic flux of collapsing and distending fg - domains that is regulated by binding and unbinding interactions between the fg - domain and the transport receptors. (1995b) recognized that the fg - domains represent the key constituents of the npc selective gate by determining that the fg - repeats act as docking sites for kaps. by identifying and binding to the fg - domains, the kaps (2000) showed that each npc consists of up to 12 different fg - domains (i.e. nucleoporins), which are located near the nuclear and cytoplasmic peripheries of the npc. to further emphasize the functional redundancy between the various fg - domains in the npc (1) the asymmetric fg - nups have been shown to be dispensable for nct (zeitler and weis 2004) ; (2) the direction of transport through the npc can be inverted (nachury and weis 1999) ; (3) active transport is able to proceed in npcs lacking cytoplasmic filaments (i.e. fg - rich nup358) (walther. 2002) ; (4) the selective gating mechanism has been found to remain functional even after 50% of the fg - repeats had been depleted (strawn., it is generally agreed that the fg - domains act as the physical constituents of the npc barrier (ben - efraim and gerace 2001 ; macara 2001 ; ribbeck and gorlich 2002 ; rout. 2000) (fig. 3). however, the mechanistic manner in which the fg - domains contribute to the selective gating of the npc is widely speculated and has been the subject of several reviews (fahrenkrog and aebi 2003 ; lim. 2006a ; stewart 2007 ; suntharalingam and wente 2003 ; weis 2003). akin to the entropic fluctuations of unstructured microtubule - associated proteins (maps) (mukhopadhyay and hoh 2001) and neurofilament sidearms (brown and hoh 1997), the brownian affinity gating model (rout. 2000), later called virtual gating (rout. 2003), proposes that the entropic behavior of peripheral fg - domains acts as a substantial barrier to inert cargo (fig. translocation is anticipated for receptor - mediated cargoes due to interactions between the fg - repeats and the transport receptors, which increases the residence time and probability of entry into the npc. in a similar manner, the oily - spaghetti model (macara 2001) postulates that noninteracting fg - domains are pushed aside by cargo complexes but otherwise obstruct the passage of passive cargo (fig., the dualistic ability of each npc to restrict or promote cargo translocation (i.e. to simultaneously act as a barrier and a vectorial transport facilitator) is only figuratively understood. cargo complex movement through the npc has been compared to stepping from one fg - repeat to the next (rexach and blobel 1995), or sliding over a surface comprised of fg - repeats (peters 2005). alternatively, the affinity gradient model (ben - efraim and gerace 2001) suggests that transport complexes step through npcs lined with fg - nucleoporins exhibiting increasing binding affinities with the cargo complexes (fig. finally, the selective phase model (ribbeck and gorlich 2002) predicts that fg - domains attract each other via hydrophobic inter - fg - repeat interactions to form a hydrophobic gel or meshwork (fig. this interpretation is based on experiments, which show that the addition of hydrophobic solvents disrupts the meshwork and triggers a nonselective opening of the pore (ribbeck and gorlich 2002 ; shulga and goldfarb 2003). hence, it is predicted that passive, more hydrophilic material is obstructed while hydrophobic cargo complexes are able to dissolve through the sieve - like meshwork. frey. (2006) showed that the yeast fg - nup, nsp1, can be cast in the form of a macroscopic hydrogel to lend support to the selective phase model (ribbeck and gorlich 2002). however, the same authors reported that the nsp1 hydrogel lacked any discriminatory effects between inert, non - kap binding proteins and kap - complexed proteins unless the fg concentration within the gel was raised (50 mm) to the point where it formed a saturated hydrogel (frey and gorlich 2007). in this manner, they explained that an efficient permeability barrier was recovered due to the formation of a highly ordered meshwork consisting of cohesive inter - fg repeats (fig. in addition to preventing the translocation of inert molecules, it was found that such a saturated hydrogel could reproduce the diffusion rates of receptor - driven transport in the npc (kubitscheck. (2007) reported evidence that refutes the gel - forming quality of nsp1. to investigate the cohesiveness of different fg - domains, patel. devised a low affinity assay, which could detect the binding of cfp - nups to gst - nups immobilized on sepharose beads. by avoiding the non - physiological conditions used to form the hydrogels (frey. 2006 ; frey and gorlich 2007), they found that only glfg - domains show weak cohesive interactions, whereas fxfg - domains (such as nsp1) do not bind together. based on their findings, the authors suggested that the fxfg domains on both cytoplasmic and nuclear peripheries act as an entropic repulsive barrier, while the glfg - domains form a cohesive meshwork in the central npc channel (patel. therefore, they proposed a two - gate model that combines elements of both brownian gating and the selective phase (patel. 2007). such a two - gate model, however, is likely to be more appropriate to yeast npcs, because vertebrate npcs are known to be composed almost entirely of fxfg - domains (except for the glfg - domain of nup98) (suntharalingam and wente 2003). furthermore, by a systematic depletion of fg - domains in yeast, the authors showed that the npcs displayed similar qualitative leakiness in all the cases studied, which indicates that all the fg - domains exhibit a functional redundancy in that both peripherally and centrally anchored fg - nups play equal roles in maintaining the selective gating mechanism. (2006b, 2007a) developed an experimental platform that allowed for the collective behavior of surface - tethered fg - domains to be probed at the nanoscopic length scale. by reproducing the physical dimensions of the npc, they found that the fxfg - domains of the vertebrate nup153 resembles a polymer brush (halperin. this is caused by packing constraints between the entropy - dominated fg - domains, which force the surface - tethered fg - domains to extend in a net directionality away from the surface (i.e. entropic barrier). this creates a significant repulsive barrier that can repel large macromolecules while allowing small molecules such as ions and water to diffuse more easily through. this may explain why a reduced number of fg - domains is enough to maintain an effective barrier in the npc. by showing that the extended brush - like fg - domains collapsed in hexanediol, they were further able to explain why the npcs could reversibly open and close when similar reagents were added / removed in nuclear transport assays (patel. 2007 ; ribbeck and gorlich 2002 ; shulga and goldfarb 2003). this was further substantiated with atomic force microscope (afm) single molecule force spectroscopy (smfs) analysis, which showed that individual nup153 fg - domain molecules (1) lack intra - fg interactions ; (2) are natively unfolded ; (3) can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, i.e. resembling a worm - like chain (wlc) (bustamante. smfs analysis revealed complex plateau - like (un)binding topologies between kap1 (importin) and nup153 when kap1-modified afm tips were used (lim. 2001 ; tompa 2002) in fg - receptor interactions and is in agreement with the fact that kap1 consists of five experimentally verified hydrophobic fg - binding sites (bayliss. ; liu and stewart 2005), with additional five binding sites predicted by molecular dynamics (md) simulations that can be simultaneously occupied (isgro and schulten 2005). to further correlate the governing biochemical interactions to the biophysical behavior of the fg - domains, lim. (2007a) studied the nanomechanical response of the brush - like fg - domains under the influence of transport factors such as kap1, rangtp and rangdp. consequently, they found that the entropic barrier collapsed in vitro due to kap1-fg binding interactions (i.e. causing a reduction in conformational entropy of the fg - domains) and was reversed by the sequestration of kap1 by rangtp (but not rangdp). by validating similar effects in situ using an immunogold - em labeling assay in xenopus oocytes nuclei, they hypothesized that the selective gating mechanism consists of a rapid, stochastic flux of collapsing and distending fg - domains that regulates passage through the npc (fig. such seamlessness may explain the reversibility of nct and apparent open communication between the cytoplasm and nucleus (kopito and elbaum 2007). besides resolving the overall structure of the npc, an accurate picture of how selective gating is achieved by the (structureless) fg - domains remains unclear due to a general lack of understanding with regard to their behavior and function within the npc. so far, only the reversible collapse of the fg - domains has been directly observed to occur in the npc (lim. the source of this ambiguity stems in part from the difficulty in trying to visualize the fg - domains in vivo, which is evident given the lack of resolution even when using state - of - the - art structural techniques such as cryo - tomography (cryo - et) to detect the fg - domains (beck. although immunogold - em techniques can provide positional information of the fg - domains within the npc, this is limited to static views of fg - domain behavior (fahrenkrog. 2002 ; paulillo. 2005, 2006 ; schwarz - herion. new microscopic techniques such as the afm is also usually limited in resolution and chemical sensitivity due to the complexity of the npc and its cellular environment (jaggi. 2002, 2003), the fg - domains themselves have so far only been directly visualized as individual bioploymers by afm (lim. in combination with biochemical efforts to identify whether different transport receptors use preferential fg - domains during nct (terry and wente 2007), it may be beneficial to use bottom - up strategies to investigate how the fg - domains behave on a biophysical level to give rise to the selective gating of the npc. this is because biochemical approaches can only provide a marginal mechanistic description of the selective gating process. moreover, because nct occurs over tens of nanometers in and around the npc, i.e. a near - field effect, this in fact necessitates a consideration of such physical details, including (1) the geometry and nanoscopic dimensions of the npc, (2) the fg - domain anchoring sites within the npc and (3) the limited number of fg - domains located within the npc. thus, it will be essential to establish fg - domain conformations at the npc - relevant length scale. to critically underscore this point, can macroscopic assays that address the characteristics of enormously large numbers of fg - domains be interpreted to represent and describe the biophysical behavior of 100150 natively unfolded fg - domains anchored within the npc ? how does one reconcile the notion of having extended fg - domains in the npc when the diameter of the central pore is about ten times larger than the size of a fg - domain at dynamic equilibrium [stokes radius typically a few nanometers (denning. what physical basis can allow for the fg - domains on opposing sides of the central pore to extend so far past their stokes radius so as to touch each other, much less to form an inter - fg crosslinked meshwork spanning the diameter of the central pore (frey and gorlich 2007) (fig. fg binding effects prevent the formation of such a meshwork by causing individual fg - domains to collapse or conversely, if a brush - like conformation is a valid prerequisite for the extension of the fg - domains to form a barrier in the npc (lim. 2006b), does this necessarily preclude the possibility of inter - fg interactions occurring between neighboring fg - domains ? bearing in mind that the fg - domains are anchored to the npc instead of free floating in solution, whether or not fxfg- and glfg - domains cohere in the npc (patel. 2007) will not only depend on how strongly (and dynamically, i.e. frequently) they interact with each other, but also on their physical proximity within the npc (i.e. anchoring site). therefore, other contextual effects that require consideration include the following : (1) how confinement effects based on the hourglass - like geometry affect the entropic behavior of the fg - domains (e.g. preferring to remain in the central pore or outside) ? (2) the strength of fg fg interactions in the midst of competing effects such as the steric hindrance between the hydrophilic regions of neighboring fg - domains ; (3) how nonspecific interactions can bias their behavior (paradise. 2007 ; timney. 2006), e.g. macromolecular crowding (zimmerman and minton 1993) ? (4) more generally, how does the complex cellular environment (exhibiting a multitude of specific / nonspecific interactions) in and around the npc near - field affect fg - domain conformations ? (5) finally, how do these effects influence and affect the interactions between the fg - domains and transport receptors ? given these arguments, the modus operandi of the npc selective gating mechanism requires careful substantiation at the near - field, mesoscopic scale (i.e. the length scale relevant to the properties of a material or phenomenon). to emphasize its relevance, the outermost surface of a hydrogel at the gel liquid interface has been shown to consist of noncrosslinked polymer chains (kim. moreover, fg - domain behavior will have to be assessed at the relevant time scales, since selective gating appears to be a rapid kinetic process over a time scale of the order of 5 ms (kubitscheck. this emphasizes the need to move beyond conventional biological methods and to adopt more interdisciplinary experimental approaches (dutta and belfort 2007). in addition, the use of molecular dynamics (md) simulations (isgro and schulten 2005, 2007a, b) and other theoretical frameworks (zilman. 2007) may be able to illuminate aspects of fg - domain behavior and the nuclear trafficking machinery that experimentalists can then look out for and validate. perhaps most importantly, the desire to conceptually reconcile and reach a single objective understanding of npc function and structure will require that such aspects of nuclear transport processes be scrutinized in individual npcs in vivo. however, beyond the technical challenges involved, a difficulty remains as to how a ground - state can be defined in the npc so as to monitor subsequent changes in the nebulous haze of fg - domains without any quantitative information regarding the endogenous receptors and cargo already bound to the fg - domains, which will influence fg - domain behavior. in any case, such attention to detail will be key to defining a unified picture of the npc. as noted by paine. (1975) in nature more than 30 years ago, as solute size approaches the dimensions of the pore, solute pore wall interactions become increasingly important. | the spatial separation between the cytoplasm and the cell nucleus necessitates the continuous exchange of macromolecular cargo across the double - membraned nuclear envelope. being the only passageway in and out of the nucleus, the nuclear pore complex (npc) has the principal function of regulating the high throughput of nucleocytoplasmic transport in a highly selective manner so as to maintain cellular order and function. here, we present a retrospective review of the evidence that has led to the current understanding of both npc structure and function. looking towards the future, we contemplate on how various outstanding effects and nanoscopic characteristics ought to be addressed, with the goal of reconciling structure and function into a single unified picture of the npc. |
colorectal cancer (crc) is a common malignancy and a leading cause of cancer death worldwide, and the incidence of crc is increasing rapidly in eastern countries including korea as a result of recent changes in diet and lifestyle. the primary treatment of crc is a complete resection in stage i - iii patients followed by adjuvant chemotherapy in stage iii and high - risk stage ii patients. in the considerable number of patients with distant metastases (i.e., stage iv disease) at the time of diagnosis, complete resection of colorectal liver metastases can provide a chance for long - term survival in approximately 20% of these patients. oxaliplatin plus fluoropyrimidine combination chemotherapy is widely used in both the adjuvant and palliative setting to improve survival of crc patients. with increasing incorporation of liver metastasectomy in the treatment strategy, sinusoidal obstruction syndrome (sos) in the liver is a long - term toxicity of oxaliplatin reported in patients who underwent hepatic resection after oxaliplatin - based chemotherapy [6 - 10 ]. sos, previously known as hepatic veno - occlusive disease, was most commonly reported to occur after bone marrow transplantation for hematologic malignancies. sos has been reported in 19%-79% of crc patients who underwent hepatic resection after oxaliplatin - based chemotherapy. toxic effect of oxaliplatin on sinusoidal endothelial cells (sec) causes disruption of the sinusoidal wall, subsequently causing congestive obstruction with impairment of sinusoidal blood flow. as a result, diffuse sinusoidal injury leads to portal hypertension, hepatomegaly, and hyperbilirubinemia with severe complications such as ascites and variceal bleeding in rare cases. in addition, development of sos may be associated with increased morbidity and mortality following hepatic resection in patients treated with preoperative oxaliplatin - based regimens. prediction of sos development is important in selection of proper candidates for hepatic resection and preoperative management of patients at risk of sos. increase in spleen size, elevated aspartate aminotransferase to platelet ratio index, and hyaluronic acid levels have been reported as reliable indicators of sos. however, there is no reliable biomarker for prediction of sos before oxaliplatin treatment that could help in the decision of oxaliplatin use. in addition, association of the susceptibility to sos with antitumor efficacy in the adjuvant setting has not been studied. many recent efforts have been made to clarify the pathogenesis of sos at the molecular level and to use this as a molecular marker of sos. this model also suggested that additional mechanisms such as an increase in expression of matrix metalloproteinase-9 (mmp-9) (and to a lesser extent mmp-2), reduced synthesis in nitric oxide, and oxidative stress contribute to sec injury. vascular endothelial growth factor (vegf) is known to regulate mmp-9 activation by inducing its expression, and the degree of the increase in vegf serum level parallels the clinical severity of sos. therefore, vegf blockade may attenuate sinusoidal injury by down - regulating mmp-9 production. in the liver, nitric oxide is produced by the nitric oxide synthase 3 (nos3) expressed in the sec. decreased activity of glutathione s - transferase (gst) leads to a decrease of adduct formation between glutathione and platinum, consequently attenuating a defense mechanism against oxaliplatin. in this study, we have chosen several genes related to the pathogenesis of sos (vegfa, mmp9, nos3, and gstp1), as shown in the previous model, and studied the association between their genetic polymorphisms and sos following oxaliplatin treatment using spleen size as a surrogate of sos. crc patients receiving adjuvant 5-fluorouracil, leucovorin, and oxaliplatin (folfox) were selected in order to eliminate the potential bias caused by the extent of liver metastasis. we also analyzed the association of sos with disease - free survival (dfs) following adjuvant folfox. this retrospective analysis included stage iii or high - risk stage ii crc patients who received adjuvant folfox chemotherapy after complete resection of crc from september 2005 to december 2009 at seoul national university hospital (snuh) and participated in pharmacogenomics study of chemotherapy among cancer patients. other eligibility criteria were age over 18 years, adenocarcinoma histology, complete resection of primary tumor with negative margin, adequate organ function, completion of at least six cycles of the planned 12 cycles of chemotherapy, and computed tomography (ct) images obtained before and after chemotherapy adequate for measurement of spleen size. for adequate measurement, contrast - enhanced ct images were obtained with at least 5 mm slice thickness or less. patients with underlying severe liver disease such as active viral hepatitis, severe steatohepatitis, or liver cirrhosis before chemotherapy were excluded. each cycle of folfox-4 consisted of oxaliplatin (85 mg / m) on day 1 and leucovorin (200 mg / m) and a bolus of 5-fluorouracil (5-fu ; 400 mg / m) followed by a 22-hour infusion of 5-fu (600 mg / m) on days 1 and 2, which was repeated every 2 weeks. modified folfox-6 consisted of oxaliplatin (85 mg / m), leucovorin (400 mg / m), and a bolus of 5-fu (400 mg / m) followed by a 46-hour infusion of 5-fu (2,400 mg / m) repeated every 2 weeks. the study protocol was reviewed and approved by the institutional review board of snuh and was conducted in accordance with the declaration of helsinki. genomic dna was extracted from peripheral blood mononuclear cells using a qiaamp dna kit (qiagen, valencia, ca), and each polymorphism was determined using a pyrosequencing method (pyromark q96 i d, qiagen). primer sequence and biological effect of each polymorphism ten polymorphisms in 4 potentially sos - related genes were analyzed : 2578c / a, 1154g / a, 634g / c, and 936c / t in vegfa ; 1562c / t, 836a / g, and 2003g / a in mmp9 ; 786t / c and 894g / t in nos3, and ile105val in gstp1. spleen size was measured by loading the ct images into a 3d workstation (rapidia ver. the outline of the spleen on each axial image of ct scans was traced using an electronic free - curve in the software, cross - sectional areas were calculated, and then the sum of the areas was multiplied by slice thickness for calculation of spleen volume. spleen sizes measured from ct images obtained before chemotherapy and after completion of chemotherapy were compared. association between change in spleen size and each genetic polymorphism was analyzed using student s t test. the relationship between splenomegaly and each polymorphism was also analyzed using pearson s chi - square or fisher s exact test. splenomegaly was functionally defined as a 50% increase in spleen size after oxaliplatin - based chemotherapy. multivariate logistic regression analysis was performed to evaluate the effect of other clinicopathologic factors on splenomegaly. in this analysis, the backward stepwise regression model including only variables with a p - value 65 years vs. 65 years ], lowest platelet count during chemotherapy [65 years vs. 65 years ], lowest platelet count during chemotherapy [< 75,000/mm vs. 75,000/mm ] and the presence of chronic liver disease). in multivariate logistic regression analysis performed using variables with p - values < 0.10 in univariate analysis, the cumulative dose of oxaliplatin and the lowest platelet count during the chemotherapy period were associated with splenomegaly (table 3). during adjuvant folfox chemotherapy and the follow - up period after completion of chemotherapy, patients with splenomegaly had lower values of mean platelet count compared to patients without splenomegaly. this difference in mean platelet count was the most prominent from 3 months to 6 months after initiation of folfox (p < 0.05, by student s t test), and the difference was gradually reduced after completion of chemotherapy (fig., patients with splenomegaly had more severe thrombocytopenia compared to those without splenomegaly during the chemotherapy period (mean lowest platelet count, 85,00028,000/mm vs. 115, 00042,000/mm ; p < 0.001). we also analyzed the association of splenomegaly, a surrogate marker of sos, with treatment outcome of adjuvant folfox chemotherapy. the 3-year dfs rate was 89.1% (95% confidence interval [ci ], 79.9% to 98.3%) in patients who developed splenomegaly and 85.7% (95% ci, 77.7% to 93.7%) in patients without splenomegaly (p=0.42 by log - rank test) (fig. in the current study, splenomegaly, a surrogate of sos, was frequently observed after adjuvant folfox chemotherapy in crc patients. an increase in spleen size compared with baseline size before starting oxaliplatin - based chemotherapy was observed in 109 patients (87.9%), with a median increase in spleen size of 31%. although the direct comparison was difficult due to the difference in the cumulative dose of oxaliplatin and treatment duration every study, this change in spleen size is comparable to that reported in another study. the cumulative dose of oxaliplatin and the lowest platelet count during chemotherapy were clinical factors associated with splenomegaly. patients with splenomegaly showed more severe thrombocytopenia than patients without splenomegaly during or after oxaliplatin - based chemotherapy. this is in line with a previous study reporting on the relationship between splenomegaly and thrombocytopenia, which suggested splenic sequestration induced by portal hypertension as a possible mechanism of thrombocytopenia in patients with sos. thrombocytopenia related to sos is common, but usually not severe. in this study, thrombocytopenia less than 50,000 mm was only observed in five cases and there were no significant bleeding events. thrombocytopenia due to oxaliplatin - induced sos can be prolonged until 2 - 3 years after completion of oxaliplatin treatment. oxaliplatin - induced sos is infrequently presented with ascites, jaundice, and hepatomegaly suggestive of portal hypertension. in our study, none of the patients showed clinically apparent symptoms and signs of portal hypertension except splenomegaly. a number of studies have been conducted for discovery of molecular predictive biomarkers of sos based on its pathogenesis. rubbia - brandt., who examined gene expression profiles in livers with oxaliplatin - induced sos and matched normal controls, found 913 differentially expressed genes. results of pathway analysis showed significant upregulation of expression in six pathways : acute phase response, coagulation system, hepatic fibrosis / hepatic stellate cell activation, and oxidative stress. in addition, angiogenic and hypoxic factors including vegfc and hypoxia - inducible factor 1-alpha (hif1a) were upregulated. a similar study conducted by a french group also confirmed the upregulation of genes involved in angiogenesis and coagulation in oxaliplatin - induced sinusoidal injuries. glutathione forms an adduct with platinum by gst, which leads to detoxification of oxaliplatin. in another study evaluating the role of gst polymorphism as a risk factor for sos, gstm1-null genotype we have analyzed the association between splenomegaly and genetic polymorphisms in four sos - related genes. we hypothesized that these genes might be relevant to the pathogenesis of sos, as suggested in an animal model. in this model, upregulation of mmp-9 and subsequent decrease of nitric oxide contributed to sec injury, as well as glutathione depletion followed by production of reactive oxygen species. further studies are needed to determine the role of other genes or polymorphisms as a risk factor of sos. some studies have suggested a negative impact of oxaliplatin - related sos on long term outcomes with early recurrence and decreased overall survival in patients with colorectal liver metastases. in addition, more severe grade of sos was correlated with lower histopathological tumor regression. in our study, splenomegaly, used as a biomarker of sos, showed no association with survival outcome. a possible explanation is that, unlike previous studies, our study was conducted in patients in the adjuvant setting without distant metastasis. in addition, the frequency of hepatic recurrence was not significantly different according to the development of splenomegaly (2.1% [1/47 ] in patients with splenomegaly vs. 5.2% [4/77 ] in patients without splenomegaly, p=0.65). the current study has some limitations including the retrospective nature of the study and no histopathological confirmation of sos. however, the homogeneity of the study population including only patients in the adjuvant setting is the strength of the study. in summary, we show that splenomegaly occurred in 87.9% of crc patients receiving adjuvant folfox treatment and it was also associated with more severe thrombocytopenia. as we found no association between the genetic polymorphisms analyzed herein and development of splenomegaly, future studies investigating other biomarker candidates are warranted for prediction of sos. | purposesplenomegaly is a clinical surrogate of oxaliplatin - induced sinusoidal obstruction syndrome (sos). we investigated development of splenomegaly and its association with treatment outcome and genetic polymorphisms following adjuvant 5-fluorouracil, leucovorin, and oxaliplatin (folfox) in colorectal cancer (crc) patients.materials and methodssplenomegaly was determined by spleen volumetry using computed tomography images obtained before initiation of chemotherapy and after completion of adjuvant folfox in crc patients. ten genetic polymorphisms in 4 sos - related genes (vegfa, mmp9, nos3, and gstp1) were analyzed using dna from peripheral blood mononuclear cells.resultsof 124 patients included, increase in spleen size was observed in 109 (87.9%). median change was 31% (range, 42% to 168%). patients with splenomegaly had more severe thrombocytopenia compared to patients without splenomegaly during the chemotherapy period (p < 0.0001). the cumulative dose of oxaliplatin and the lowest platelet count during the chemotherapy period were clinical factors associated with splenomegaly. however, no significant associations were found between genetic polymorphisms and development of splenomegaly. disease - free survival was similar regardless of the development of splenomegaly.conclusionsplenomegaly was frequently observed in patients receiving adjuvant folfox and resulted in more severe thrombocytopenia but did not influence treatment outcome. examined genetic polymorphisms did not predict development of splenomegaly. |
resistance to insulin is characteristic of metabolic syndrome (mets), which is defined as a cluster of the following cardiovascular risk factors : central obesity, impaired glucose tolerance, dyslipidemia, and hypertension. mets constitutes a major health problem, as it is strongly associated with type 2 diabetes mellitus (t2d) and cardiovascular disease [13 ]. furthermore, insulin resistance is a consistent finding in t2d and appears to contribute to the development of t2d. however, t2d develops only if there is dysfunction of beta - cells. in the absence of beta - cell dysfunction individuals therefore, many people with remarkable resistance to insulin may never develop t2d [5, 6 ]. lifestyle factors clearly underlie mets incidence, but genetic susceptibility may be important as well. for example, specific ethnic groups are more susceptible to mets than others [8, 9 ]. in particular, south asians are predisposed to develop mets and subsequently t2d and cardiovascular disease at a younger age [1012 ]. they also have a higher prevalence of abdominal obesity, are less sensitive to insulin, and have a lower glucose disposal rate than europeans [1214 ]. in addition, south asians have lower plasma levels of hdl and adiponectin and higher levels of glucose, insulin, leptin, complement c3, plasminogen activator inhibitor-1, fibrinogen, and tissue plasminogen activator compared to europeans [1520 ]. however, traditional risk factors such as smoking, hypertension, and dyslipidemia do not explain the increased risk for cardiovascular disease in south asians [21, 22 ]. insulin resistance itself has been held responsible for the high rates of t2d and cardiovascular disease in this ethnic group [10, 12 ]. oral minimal modeling is a pharmacokinetic / pharmacodynamic algorithm developed to estimate beta - cell function and insulin sensitivity index (isi) from dynamic data. in the present study, we used this oral minimal model (omm) to investigate the relationship between mets traits and beta - cell function in south asian and european families with prevalent t2d. the recruitment of patients with t2d and their relatives at our university outpatient clinic has been described in detail previously. in brief, 48 south asians and 54 europeans that are residing in the netherlands were initially recruited for the present study and we used an oral glucose tolerance test (ogtt) to group the subjects in t2d or not2d according to the who criteria. in addition, the international diabetes federation (idf) criteria were used to define mets. four south asians had t2d but not mets and were excluded from the study as the number of subjects was too small for meaningful analyses and there were no european counterparts for comparison. hence, 98 subjects were included in our analyses (44 south asians and 54 europeans) from 25 families (25 patients with t2d but not on insulin therapy and 73 relatives) and they were distributed among 3 groups ; no metabolic syndrome / no type 2 diabetes mellitus (nomets / not2d), metabolic syndrome / no type 2 diabetes mellitus (mets / not2d), and metabolic syndrome / type 2 diabetes mellitus (mets / t2d). body height and weight were measured in light clothing without shoes and were used to estimate body mass index. waist circumference was measured halfway between the lowest rib and the iliac crest while the maximum circumference of the hips was measured in the standing position ; from these measurements the waist - to - hip ratio was calculated. systolic and diastolic blood pressure were measured in the sitting position with an electronic blood pressure monitor (datascope accutorr plus inc., a 75 g glucose load was administered (t = 0), after an overnight fast, and 11 venous blood samples were acquired at prespecified time intervals (60 min, 15 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, 180 min, and 210 min) for the measurement of plasma glucose, insulin, and c - peptide levels. baseline blood samples were obtained in order to estimate glucose, insulin, c - peptide plasma concentrations, and the lipid profile. plasma glucose was estimated using a hexokinase - based method (gluco - quant ; roche diagnostics, mannheim, germany). plasma insulin and c - peptide were measured separately by a competitive chemiluminescent immunoassay, supplied by euro / dpc. the assay was performed on a dpc immulite 2000 analyzer (euro / dpc) according to the manufacturer 's recommended protocol. serum total cholesterol, hdl, ldl, and triglycerides were determined with an automatic enzymatic procedure by roche diagnostics (mannheim, germany). the omm, which consists of the glucose omm and the c - peptide omm, was used to describe changes of plasma glucose, insulin, and c - peptide concentrations during an oral glucose stimulus [2628 ]. glucose, c - peptide, and insulin concentrations were measured at 11 time points before and after intake of 75 g glucose. the glucose omm estimated isi with plasma glucose and insulin concentrations measured during the ogtt. in addition the c - peptide omm indices were calculated during the oral glucose tolerance test and in terms of insulin secretion can be interpreted as follows : (1) the basal (basal) that gives a basal nonstimulated measurement of insulin secretion, (2) the dynamic (dynamic) that provides the amount of insulin secreted during the dynamic phase (first - phase secretion by beta - cells), (3) the static (static) that assesses the release of insulin that occurs after a time delay (second - phase secretion by beta - cells) and represents a beta - cell response according to the prevailing glucose concentration, and (4) the total (total) overall secretion which is the sum of the dynamic and static phase release of insulin from beta - cells. the parameters of glucose omm and c - peptide omm were multiplied to obtain the disposition indices (dis), which is a measure for beta - cell function corrected for insulin sensitivity. it can be considered a measure of the functionality of the pancreas in the intact individual : dibasal = basal isi, didynamic = dynamic isi, distatic = static isi, and ditotal = total isi. anova test was used to compare the mean of raw data presented in table 1 and the figures of different subgroups within the two ethnicities. adjusted analyses were performed with multiple linear regression models in which all covariables were entered synchronously. all statistical tests were conducted with spss software, version 20 (spss inc., chicago, il). 32 out of 44 south asians (72.7%, 95% ci 58.0 to 83.8%) and 23 out of 54 europeans (42.6%, 95% ci 30.3 to 55.9%) had mets according to the idf criteria. in table 1, the systolic blood pressure, fasting, and 2 h plasma glucose levels were increased in south asians with mets and t2d compared to the nomets / not2d group. south asians with mets and t2d were on average 10.4 years younger than europeans with mets and t2d. compared to the analyses of the south asian subgroups, we observed a larger heterogeneity among the 3 subgroups of the europeans in clinical and biochemical characteristics with significant differences in age, weight, body mass index, waist, waist - to - hip ratio, blood pressure, and fasting and 2 h plasma glucose levels. adjusted for sex and age, the europeans had 7.18 10 dl / kg / min per u / ml (95% ci 0.58 to 13.78, p = 0.033) higher isi than the south asians. this was fully explained by the differences between the two subgroups without mets (after excluding this subgroup to the analysis the effect of ethnicity disappeared, p = 0.367). the fitting of both c - peptide and glucose omm was satisfactory as the average weighted residuals did not deviate systematically from the zero value (supplementary figures 1 and 2 in supplementary material available online at http://dx.doi.org/10.1155/2016/9286303). figure 2 shows the beta - cell responsivity indices estimated by the c - peptide omm during the ogtt. adjusted for sex and age, ethnicity was significantly related to the basal responsivity index, = 0.086 10/min, 95% ci 0.17 to 0.155, and p = 0.016, but not to the other responsivity indices, p > 0.7. within the south asian group adjusted for sex and age, the mets / not2d group had 0.164 10/min (95% ci 0.045 to 0.282, the dynamic, the static, and the total responsivity indices showed all the same trend of a lower beta - cell response (in effect less insulin secretion) in the mets / t2d groups in both ethnicities : 191.9 10 (95% ci 282.2 to 101.6, p = 0.001), 12.1 10/min (95% ci 16.1 to 8.0, p = 0.001), and 14.3 10/min (95% ci 18.9 to 9.6, p = 0.001), respectively. the south asian nomets / not2d and mets / not2d had similar values in the dynamic, the static, and the total responsivity indices, whereas in the europeans there was a clear trend to gradually lower values over these groups in the direction of the mets / t2d subgroup : pfor trend = 0.005, 0.001, and 0.001, respectively. the dis according to subgroup and ethnicity are shown in figure 3. adjusted for sex and age the four dis did not differ significantly between the ethnicities (p > 0.11). both ethnicities showed the same gradual course of the different di from high to intermediate and low over the nomets / not2d, mets / not2d, and mets / t2d subgroups, respectively (pfor trend < 0.002). there were no clear differences between the ethnicities, although the european nomets / not2d subgroup had relatively high di compared to the same south asian subgroup. the relationships between the mets traits and omm indices adjusted for sex and age according to ethnicity are shown in table 2. therefore, each trait was analyzed separately in a multiple linear regression model adjusted for sex and age. waist - to - hip - ratio was strongly associated with isi in both ethnicities, but it may have a more detrimental effect of the glucose disposition in europeans than in the south asians. the dyslipidemia characterized by high triglycerides and low hdl was related to isi and the dis solely in the south asians, whereas blood pressure, both systolic and diastolic, was solely related to isi and the dis in the europeans. as expected, fasting glucose was associated with isi and dis as these indices are calculated with glucose values. in the present study, we found a clear trend for increasing insulin resistance from nomets / not2d to mets / not2d and to mets / t2d. we also found that south asians without mets were more resistant to insulin than a corresponding group of europeans. in the european families, the dynamic, static, and total beta - cell responsivity indices followed the trend of isi in the same direction over the three subgroups towards decreasing insulin secretion by the beta - cells in the t2d group. the south asian nomets / not2d and mets / not2d subgroups both had high beta - cell responsivity indices and the mets / t2d subgroup had low values. the dis are the product of isi and beta - cell responsivity indices and independent of ethnicity followed a decreasing trend over the subgroups in the direction of t2d. notably, a number of separate mets traits had different associations in the two ethnicities : dyslipidemia had a strong relationship with isi and di in the south asians, whereas blood pressure was associated with isi and di in the europeans. over the last years several studies have examined the effect of lifestyle, dietary habits, and genetic polymorphisms on the development of mets in different ethnicities, in order to identify high - risk populations such as the south asians, who appear to have an increased predisposition for t2d [3032 ]. most of these reports focused on epidemiological analyses and evaluations of the impact of biochemical and physical characteristics (e.g., total abdominal fat and intra - abdominal adipose tissue) on the development of mets. in a number of studies, isi and beta - cell function were assessed in different ethnicities using fasting blood glucose and plasma insulin concentrations (for instance, homeostatic model assessment (homa)) [3335 ]. these methods do not assess the dynamic response of beta - cells to glucose stimuli [36, 37 ]. our study is the first to employ omm in order to examine the relationships between mets traits and isi and beta - cell response. the estimations of isi and insulin secretion by omm are reasonably well correlated with those assessed by the hyperinsulinemic euglycemic clamp method. we demonstrated for the first time, to our knowledge, that in south asians and europeans both isi and beta - cell function are lower in subjects with mets and in subjects with mets and t2d compared to nomets / not2d subjects. in addition, we confirmed an increased insulin resistance in nomets / not2d south asians compared to nomets / not2d europeans [39, 40 ]. moreover, in south asians with nomets / not2d the disposition indices were lower comparing to the disposition indices in europeans and not statistically different from the disposition indices in south asians with mets / not2d. these findings indicate that south asians are a vulnerable population and have an increased risk to develop chronic diseases such as mets and t2d, in which insulin resistance and beta - cell function are key pathogenetic factors. our study showed that omm could be used to evaluate glucose homeostasis in different populations and to examine associations between insulin resistance, abnormal beta - cell function, and mets traits. waist - to - hip ratio reflects the association between visceral obesity and the causal path from insulin resistance to t2d [4143 ]. hypertriglyceridemia and increased enzymes involved in triglyceride transport and metabolism may have a toxic effect on beta - cells, but hypertriglyceridemia can be a consequence of insulin resistance and t2d as well. unfortunately, we can not infer from our data whether dyslipidemia was the cause or the consequence of insulin resistance and reduced dis. in the european families, insulin resistance and reduced di this suggests that disorders of carbohydrate metabolism were complicated by higher levels of blood pressure. a possible explanation is an impaired insulin signaling through pi3k signaling pathway and the increased vascular inflammation noted in insulin resistance which can decrease nitric oxide (no) production and increase endothelin-1 (et-1) secretion leading to raised blood pressure [45, 46 ]. the differences in the associations between metabolic syndrome traits, beta - cell function, and insulin sensitivity in different ethnicities have not been fully explained yet. a possible etiology is the different genetic profile of europeans and south asians ; however, further research is required towards this direction in order to understand the metabolic pathways and the effect of the genome on these associations. in the present analysis, we investigated associations between metabolic syndrome traits and insulin sensitivity and beta - cell function after adjusting for age and sex. of note the results did not change significantly when we adjusted also for bmi. the only differences that we noticed were in europeans where there was no longer an association between isi, waist - to - hip ratio, and diastolic blood pressure as well as a correlation between fasting glucose and isi and distatic (supplementary file table 1). family studies avoid a number of serious selection biases, but the findings can not easily be generalized to the general population. moreover, we prefer to analyze family data using a family matrix, for instance, solar, but unfortunately there were too many small cells. adjustment for family ties in regression analyses suffered from the same problem ; therefore, we restricted the adjustment of all primary analyses to age, which correlates with generation in kindreds. we did not analyze genetic variation, and transmission was not a topic of our research. moreover, separate analyses of small and large families did not change the results (data not shown). we chose the comparison between the mets / t2d status groups and not between the two ethnic groups because south asians and europeans differ in many aspects and therefore we tried to avoid bias due to the ethnicity differences. we have used the idf criteria for mets, because they take ethnic specific cut - off points for central obesity, in effect waist circumference, into account. south asians residing in the western world differ in lifestyle and phenotype from the south asians living in south asia, and therefore we tested the necessity of such ethnicity - specific cut - off points : if the european cut - off points were used for all subjects, only three male south asians would have not been classified as mets. still a number of arguments could be made to include analyses using a single cut - off point, but the effects on our results were negligible (data not shown) and therefore we decided to follow the idf recommendations. a limitation of the study was the relatively small sample size and for this reason these findings can not be generalized to the broader community. furthermore, we excluded the nomets / t2d subgroup, because meaningful analysis was not possible, as there were only 4 south asians and no europeans counterparts. in our study, we observed low isis in the mets / t2d group relative to the nomets / not2d group in both ethnicities, a finding that indicates an association between insulin resistance and a pathologic metabolic state (t2d). the pathogenic mechanisms that lead to insulin resistance appear to differ among ethnicities, as isi and dis were associated with different metabolic traits in each ethnicity. the simultaneous assessment of insulin sensitivity and beta - cell function allowed us to study metabolic profiles that promote t2d and glucose intolerance. we used this primarily to explore the underlying mechanisms of disturbances in glucose metabolism, but also to explore the potential of designing ethnicity - specific risk models for early identification of subjects that are at risk for developing diabetes mellitus. in this study, south asians, in contrast to europeans, appear to be predisposed to insulin resistance and exhibit impaired beta - cell function at an earlier age. dyslipidemia related to insulin resistance and pancreatic dysfunction may underlie or enhance this susceptibility of south asians. | background. there are different metabolic syndrome traits among patients with different ethnicities. methods. we investigated this by studying 44 south asians and 54 europeans and classified them in three groups according to the occurrence of metabolic syndrome (mets) and type 2 diabetes (t2d). insulin sensitivity index (isi), static, dynamic, and total beta - cell responsivity indices (), and disposition indices (dis) were calculated with the use of oral minimal model (omm). results. in both ethnicities, isi was lower in the subgroup with mets and t2d as compared to the subgroup without mets nor t2d (p < 0.004). south asians without mets were more insulin resistant than europeans without mets (p = 0.033). in the south asians, isi, dynamic di, and static di were associated significantly (p < 0.006) with high - density lipoprotein cholesterol and triglycerides. in the europeans, isi was associated with waist - to - hip ratio (p = 0.005) and systolic and diastolic blood pressure (p < 0.005), while static di was related to the systolic blood pressure (p = 0.005). conclusions. mets was linked with insulin resistance and reduced capacity to handle glucose regardless of ethnicity. isi and dis were associated with lipid traits in south asians and with blood pressure in europeans suggesting that insulin resistance enhances different metabolic syndrome traits among different ethnicities. |
host genetic factors are major determinants of susceptibility to infectious disease in humans (1). htlv - i is a persistent virus, infecting 10 - 20 million people worldwide. most infected people remain healthy, but 1 - 2% develops a progressive paralytic myelopathy (htlv - i - associated myelopathy ; ham / tsp) and a further 2 - 3% develops an aggressive t cell leukemia / lymphoma. ham / tsp is a chronic debilitating inflammatory disease of the central nervous system, characterized by axonal damage and demyelination, most pronounced in the midthoracic spinal cord. furthermore some of the self - immune diseases such as uveitis and polymiositis are found to be resulted from this virus (2 - 5). the endemic regions affected with this virus include the south - west of japan, caribbean islands, and some regions of the usa (6). the other parts with less prevalence of the virus are taiwan and some regions of africa and israel. mashhad in the north of iran has been recognized as a new endemic region of the virus (10, 11). the symptoms include weakness or dryness in one or both feet, backache and no control on urine. one - third of the patients become paralyzed after 10 years and one half of them are not able to walk (12). on the contrary to alt in which the number of the male patients is a little more, ham affects the female disproportionately. high proviral load and being female are two factors of high risk of ham / tsp and the relative risk of ham / tsp increases when the provrial load becomes clearly more than one copy in any pbmcs (13, 14). both the class i mhc proteins (hla - a, -b, and -c), which present viral peptides for recognition by virus - specific ctl, and class ii mhc proteins (hla - dr and -dq), which present peptides to cd4 t cells, are likely to be important in the immune response to htlv - i infection (15, 16 and 17). the results of several previous studies have suggested associations between various hla class i or class ii alleles and susceptibility to t cell leukemia / lymphoma or ham / tsp (18 - 22). however, small sample size, mixed ethnicity, lack of adequate controls, or lack of staging in these studies precluded a definite conclusion. if the relationship between htlv-1 infection and class ii is considered as the factor of susceptibility to disease, it may help in understanding pathogenesis of ham / tsp. the supplied peptides of axon antigens bonded to the products of mhc class ii activate cd4+on the surface of t lymphocytes and the activated cd4+can cross the blood - cerebral obstacle and identify the cell affected with virus. the activated cd4 + causes apoptosis through producing cytokins and damage to the cell affected with virus and the peripheral cells may play a role in cns (23, 24). both mhc class i and class ii molecules are important in determining the recognition of htlv - i peptides and for the generation of an effective immune response. the predominant cytotoxic t lymphocyte response (ctl) against tax protein, which is necessary for viral expression, can be restricted by a wide variety of mhc class i molecules in htlv - i infected subjects. it has been suggested that no single mhc class i allele is associated with ham / tsp (15, 25). in japanese htlv - i - infected subjects has been demonstrated that expression of hla - a02 and hla cw08 alleles reduced the proviral load of htlv-1 and consequently the risk of ham / tsp, while hla - b5401 allele increased the proviral load and the risk of ham / tsp in htlv - i carriers. furthermore, hla - drb1 0101 increased the risk of ham / tsp in carriers in the absence of hla - a02 (18, 21). the aim of this study was to investigate the frequency of hla - dqb1 alleles in htlv - i - infected individuals and healthy controls resident in mashhad. the studied subjects were 66, including 16 ham / ts patients, 20 asymptomatic carriers and 30 healthy controls. sixteen of 20 ham / tsp patients were female (60%) and 72% of htlv - i carriers were female. the mean age of patients was 44.1414.62 and the mean age of htlv - i carriers was 51 10.22. all patients fulfilled established criteria for ham / tsp when they examined by neurologist and htlv - i carriers had symptoms of htlv - i - associated diseases. all of the ham / tsp patients, htlv - i carriers and healthy controls had the same ethnic background. the study protocol was approved by the ethics committee of mashhad university of medical sciences. 10 cc blood was taken from all the subjects for dna extraction and serum preparation. serum samples were screened for htlv - i antibody by enzyme linked immunosorbent assay (elisa) according to the manufactures ' instruction (diapro, italy) and reactive samples were confirmed by western blotting (genelab diagnostic, singapore). dna was extracted by non - enzyme salting out method using biogen kit (mashhad - iran) according to manufacturer 's instruction. polymerase chain reaction with sequence - specific primers (pcr - ssp) method was carried out to determine hla - dqb1 alleles according to the previously published method (26). briefly, seven alleles of hladqb1 were detected using specific primers in eight pcr reactions. this procedure currently detected dqb1 alleles including 0501 - 0504, 0601 - 0609, 0201, 0301 - 0305, and 0401 - 0402 which corresponding to the serological specificities dq4, dq5 and dq6 alleles, whereas the dq2, dq7, dq8 and dq9 specificities were amplified by two primer mixes. we used at least 2 primers (primm - italy) for each reaction (table 1, 2). the studied subjects were 66, including 16 ham / ts patients, 20 asymptomatic carriers and 30 healthy controls. sixteen of 20 ham / tsp patients were female (60%) and 72% of htlv - i carriers were female. the mean age of patients was 44.1414.62 and the mean age of htlv - i carriers was 51 10.22. all patients fulfilled established criteria for ham / tsp when they examined by neurologist and htlv - i carriers had symptoms of htlv - i - associated diseases. all of the ham / tsp patients, htlv - i carriers and healthy controls had the same ethnic background. the study protocol was approved by the ethics committee of mashhad university of medical sciences. 10 cc blood was taken from all the subjects for dna extraction and serum preparation. serum samples were screened for htlv - i antibody by enzyme linked immunosorbent assay (elisa) according to the manufactures ' instruction (diapro, italy) and reactive samples were confirmed by western blotting (genelab diagnostic, singapore). dna was extracted by non - enzyme salting out method using biogen kit (mashhad - iran) according to manufacturer 's instruction. polymerase chain reaction with sequence - specific primers (pcr - ssp) method was carried out to determine hla - dqb1 alleles according to the previously published method (26). briefly, seven alleles of hladqb1 were detected using specific primers in eight pcr reactions. this procedure currently detected dqb1 alleles including 0501 - 0504, 0601 - 0609, 0201, 0301 - 0305, and 0401 - 0402 which corresponding to the serological specificities dq4, dq5 and dq6 alleles, whereas the dq2, dq7, dq8 and dq9 specificities were amplified by two primer mixes. we used at least 2 primers (primm - italy) for each reaction (table 1, 2). the frequency of hla - dqb1 alleles was calculated in ham / tsp patients, asymptomatic carriers and healthy control groups. the frequency of hla - dqb1 07 was significantly higher in patients with ham / tsp (75%) compared with healthy controls (30%) (p = 0.004, or=8.52) (table 1). the hla - dqb1 02 and dqb1 05 was positive in 7 ham / tsp patients, while in healthy control group 24 subjects were positive. the frequency of both hla - dqb1 07 and hla - dqb1 06 alleles in ham / tsp patients was more than the ones if they infected with more than the ones free of it if they are infected with htlv-1 (p- value=0.0009 and rr=10.95) (table 3). in the present study we examined the frequency of hla - dqb1 allele in ham / tsp patients, asymptomatic carriers and healthy controls. our results indicated that the individuals with dqb1 07 allele are susceptible to be affected with this disease 7 times more than the ones lacking this allele, suggesting hla genes play a significant role in the susceptibility to ham / tsp disease. within african population, two antigens determined dr15 and dq1, occurred at significantly increased frequency among htlv - i carriers compared with seronegative control subjects (42% versus 22% for dr15 [odds ratio { or } = 2.7 ; 95% confidence interval { ci } = 1.0- 7.2 ] and 78% versus 53% for dq1 [or = 3.1 ; 95% ci = 1.2 - 8.5 ]). asymptomatic carriers were shown to have an hla class ii allele distribution similar to that of patients with atl, and the frequencies of the alleles drb1 1501, drb1 1101, and dqb1 0602 were significantly greater in patients with atl and asymptomatic carriers than in patients with ham / tsp. in addition, haplotypes drb1 1101-dqb1 0301 and drb1 1501-dqb1 0602 were significantly increased among patients with atl compared with patients with ham / tsp (28). we also showed that the individuals with hla - dqb1 - 2 and hla - dqb-5 are 1.5 times more susceptible to develop ham / tsp compared with the subjects who lacked these alleles, therefore, these two alleles may be considered as the protective factors. rafatpanah (19, 20) demonstrated that the frequency of hla - drb1 01 was significantly increased in ham / tsp patients compared with carriers (p 0.028 ; or=9.4). the frequency of hla - cw08 was also significantly increased in ham / tsp patients compared with controls (p=0.03 ; or=13.5). it has been showed that there is an association between hla class i, hla - a30 allele and htlv - i infection (15, 20). kitze showed that the ham / tsp patients with particular hla haplotypes (a24cw7b7dr1dq5, a2cw7b7dr1dq5, a24cw - b52dr15dq6, a11cw1b54dr4dq4, and a24cw1b54dr4dq4) express more frequently intrathecal synthesis of antibodies against htlv-1 synthetic peptides, especially against htlv-1 env gp21 synthetic peptides (21). an association of hla - drb1 0101 with disease susceptibility also was identified, which doubled the odds of ham / tsp in the absence of the protective effect of hla - a02 (22). in the present study, the frequency of hla - dqb1 07 in ham / tsp patients was high. it seems that there is a correlation between hla - dqb1 07 and disease susceptibility. the genotype of hla - dqb1 06 and 07 was more frequent in patients with ham / tsp compared with control group (26). this results is different from the results obtained kitze (25) who reported that there is a higher risk to develop ham / tsp among the individuals with hla - dq-0105 haplotype. this discrepancy may be associated with the difference in the genetic background of studied population (27). although we find an association between hla - dqb1 alleles and ham / tsp, however the study should be repeated with more sample size. furthermore, the study of other hla class i and class ii alleles should be taken into account to understand a more comprehensive role of hla alleles in disease susceptibility. | objective(s) : htlvi-1 is the first human retrovirus with limited endemic regions in the world. the epidemiological studies have shown that the genetic background and immune response to the virus have a significant role in htlv - i - associated diseases. among the genes are involved in htlv - i infection, the role of human leukocytes antigen (hla) have been studied in different population. in the present study we examined the association between hla - dqb1 alleles and htlv - i infection in ham / tsp patients, htlv - i carriers and healthy controls in north east of iran, mashhad.materials and methods : the blood samples of 16 patients with ham / tsp, 20 htlv-1 carriers, and 30 healthy individuals were taken and dna was extracted by salting out method. hla - dqb1 typing was performed using pcr - ssp method and the frequency of hla - dqb1 alleles were compared by fischer exact test.results : there was a significant difference between ham / tsp patients and healthy controls in the frequency of hla - dqb1 07 (p=0.004, rr=7). furthermore, we found that possession of hla- dqb1 02 or hla - dqb1 05 increased the risk of disease 1.5 times.conclusion : the data presented here suggest that both hla - dqb1 07 and hla - dqb1 06 are associated with disease development. |
it is characterized by acute inflammatory response which causes coronary plaque rupture with subsequent thrombosis. accumulating evidence showed that inflammation and immune cell activation play a key role in collagen loss in the fibrous cap which is a prelude to fibrous cap rupture [3, 4 ]. the extent of cardiac damage due to this inflammatory response is reflected by peripheral levels of inflammatory biomarkers involved in the process of plaque instability. moreover, markers of plaque destabilization and plaque rupture such as high sensitivity c - reactive protein (hscrp) may be used to predict future cardiovascular events not only in apparently healthy subjects, but also in patients with acs. granzyme b (gzb) is a serine protease released from cytotoxic t lymphocytes (ctls) and natural killer (nk) cells, playing an important role in cellular apoptosis by activating intracellular caspases. furthermore, gzb is also identified as an extracellular protease degrading specific extracellular substrates such as fibronectin, vitronectin, and laminin, which implies the role of gzb in extracellular matrix (ecm) remodeling. therefore, the mechanisms by which gzb may contribute to plaque instability may include ecm degradation and/or induction of macrophage or smooth muscle cells apoptosis in the fibrous cap. interleukin 18 (il-18), previously known as interferon - gamma (ifn-) inducing factor, is a proinflammatory member of il-1 superfamily which plays a role in the initiation and progression of atherosclerosis. both clinical and experimental studies have supported its role in atherosclerotic plaque progression and destabilization [11, 12 ]. nevertheless, it was described as an independent predictor of future adverse events in patients with acs. another contributing factor to the formation of atherosclerotic plaques and may participate in their destabilization is chemokines. in this regard, fractalkine (fkn) or cx3cl1 is a unique dual function chemokine that exists in two forms ; a soluble form which acts as a chemoattractant and a membrane bound form acting as an adhesion molecule. fkn was recently identified as an independent key factor in the pathogenesis of plaque vulnerability and subsequent plaque rupture. hence, the current study was designed to determine the circulating levels of gzb in patients with acs being compared with healthy control subjects. furthermore, the associations between gzb with markers of inflammation and plaque destabilization (hscrp, il-18, and fkn) as well as other metabolic, anthropometric, and risk factors are being evaluated. a total of 48 subjects (36 men and 12 postmenopausal women) were enrolled in the study, of which 36 nondiabetic acs patients, being compared to 12 age- and sex - matched apparently healthy subjects as the control group. twenty - two patients (61%) were diagnosed with st segment elevation myocardial infarction (stemi) and 14 (39%) with non st segment elevation myocardial infarction (nstemi) or unstable angina (ua). stemi, nstemi, and ua were diagnosed according to criteria stated by the consensus document of the joint european society of cardiology / american college of cardiology committee for the redefinition of myocardial infarction. patients were recruited from the intensive care unit, cardiology department, el - demerdash hospital, ain shams university educational hospitals, cairo, egypt. subjects with fasting plasma glucose 7.0 mmol / l or with a previous history of diabetes mellitus, inflammatory diseases, autoimmune disorders, malignancy, hematological diseases, hepatic or renal diseases, acute or chronic infections, or administration of immunosuppressive drugs were excluded from this study. the study was carried out in accordance with the regulations and recommendations of the declaration of helsinki, being approved by the committee on medical ethics of el - demerdash hospital as well as faculty of pharmacy, ain shams university ethical committee and informed consent was obtained from all participants. a detailed family and medical history and drug treatment(s) systolic blood pressure (sbp), diastolic blood pressure (dbp), heart rate (hr), left ventricular ejection fraction (lvef), and the incidence of prior acs were obtained from medical records. routine serum samples for creatine kinase (ck) and ck - mb isoenzyme were obtained at admission and at 6- to 8-hour intervals. hypertension (htn) was defined as sbp > 140 mm hg and/or dbp > 90 mm hg and/or initiation of antihypertensive medication. demographic data and biochemical parameters of stemi and nstemi / ua groups are demonstrated in table 2. moreover, beta blockers, calcium channel blockers, angiotensin converting enzyme inhibitors, diuretics, nitrates, and statins were described according to the current guidelines. peripheral blood samples (5 ml) were taken from acs patients on day 1 (day of onset) and day 3 after onset. samples were divided into 2 aliquots ; the first part was collected on plain vacutainer tubes for serum preparation used for lipids profile, hscrp, and il-18 assay. both plasma and serum were divided into several aliquots, being stored at 80c for subsequent assay. lipids profile was measured by enzymatic method according to kits provided by hannover, germany. serum hscrp and il-18 were quantified by enzyme linked immunosorbent assay (elisa) technique using commercial available kit (hscrp : accubind, momobind inc., ny, usa elisa kit and plasma fkn elisa kit was supplied by r&d systems, inc, minneapolis, mn, usa. all elisa procedures were done by hyprep automated elisa system (hyperion inc, miami, fl) according to the manufacturer 's instructions. the ibm statistical package for social sciences (spss) statistics (v.19.0, ibm corp. continuous variables were presented as mean sem and categorical ones as actual numbers and percentages. comparisons between two independent normally distributed mean groups were done using independent - samples t - test. changes in the serum or plasma levels of the inflammatory markers were evaluated with paired t - test or wilcoxon matched pairs test. correlations between markers were ascertained with ranked spearman test. multiple stepwise regression analysis was done to identify risk factors that contribute to plasma gzb levels. serum levels of hscrp, gzb, il-18, and fkn showed significant elevation in acs group when compared to the healthy control group at p < 0.001 (table 1). stepwise in the nstemi / ua and stemi groups, hscrp and gzb were significantly increased in the later group in comparison to the former (table 2). comparisons between hscrp, gzb, il-18, and fkn levels on day 1 and day 3 being compared to the control group levels are given in table 3. both stemi and nstemi / ua groups showed the same dynamic changes in the measured biomarkers but with different degrees. in comparison to day 1, hscrp levels significantly decreased on day 3 in either nstemi / ua or stemi groups to 74% and 65%, respectively. moreover, plasma gzb levels significantly increased to 118% in nstemi / ua and to 126% in the stemi group in day 3 compared to day 1 in both groups, respectively. however, plasma fkn decreased significantly on day 3 by 8% in nstemi / ua and by 11% in stemi group in comparison to its level in day 1. as shown in figure 1, a significant positive correlation is observed between gzb and fkn (r = 0.58, p < 0.001). furthermore, il-18 levels displayed a significant positive correlation with fkn (r = 0.345, p < 0.05) and gzb (r = 0.416, p < 0.01). for the clinical parameters of acs patients, as depicted in table 4, correlation analyses also revealed significant correlations between gzb and sbp, dbp, peak ck, and peak ck - mb. however, serum il-18 did not show significant correlation with any of anthropometric or clinical parameters in the acs patient group. on the other hand, fkn levels showed significant positive correlations with bmi and peak ck - mb only. next, we conducted multivariate regression analysis to examine the cardiovascular risk factors contributing to plasma granzyme b levels. we set plasma granzyme b levels as a dependent variable and age, gender, bmi, htn, hypercholesterolemia, current smoker, and patient history of previous acs as independent variables. as depicted in table 5, htn and patient history were independent risk factors that significantly affect plasma granzyme b levels in patients with acs. increased systemic inflammatory mediators in patients with stable atherosclerotic plaques are a major driving force for plaque disruption and hence the incidence of acs syndrome. however, other inflammatory conditions such as diabetes mellitus may affect circulating levels of those biomarkers [19, 20 ]. therefore, the need for assessment of the inflammatory state in patients with acs in absence of such interfering condition has been rising. since extracellular gzb was discovered, several in vitro studies have been done to verify its mechanisms of action but only a limited number of clinical studies evaluated its role as a circulating biomarker for cardiovascular diseases [2123 ]. these studies attributed the rise in gzb levels to its nature as proapoptotic protein not as a novel proinflammatory marker. accordingly, our study evaluated the association between the blood levels of known inflammatory cytokines like hscrp, il-18, and fkn with gzb in nondiabetic patients with acute cardiac events. we also focused on the temporal changes in levels of those biomarkers in patients with stemi versus nstemi / ua to highlight the differential inflammatory response in different types of acs and moreover to explain the impact of sample timing (day 1 and 3) on the recorded results. the present study demonstrated a significant increase in blood levels of hscrp, gzb, il-18, and fkn in acs patients compared to the control group. elevated levels of gzb were detected in the plasma of patients with atherosclerosis, with the highest levels detected in patients with unstable plaques, lending support to the hypothesis that granzyme b influences plaque instability. in addition, the consequences of elevated granzyme b levels may extend beyond plaque rupture. had described the novel role of elevated levels of gzb at day 14 after acute myocardial infarction (ami) in postinfarct ventricular remodeling. moreover, the authors found that gzb protein expression was increased at the infarcted myocardium from day 3 to 17 after ami. within the atherosclerotic plaques, macrophages, foam cells, and smooth muscle cells (smcs) also express gzb as well as it also can be detected extracellularly [9, 25 ]. interestingly, gzb expression in the atherosclerotic plaque was found to be increased with disease severity. recently, hendel. also identified that the reduced expression of the gzb endogenous inhibitor, proteinase inhibitor 9, in vascular smcs is associated with the disease progression and that may increase smcs susceptibility to gzb - induced apoptosis within the plaque. therefore, we found that gzb levels were increased significantly in stemi group in comparison to nstemi / ua group. although kondo and coworkers have proved that plasma gzb is elevated in patients with ami, however, tsuru. could not evaluate changes in plasma gzb concentration in ua patients where only few patients showed detectable results. this may also support the significant difference in our plasma gzb levels between stemi and nstemi / ua which probably may be due to proapoptotic nature of gzb associated with the significantly higher degree of cardiac necrosis, reflected by significant peak ck and peak ck - mb, and inflammation (hscrp) observed in stemi compared to nstemi / ua group. there is no expression of fkn in the normal coronary artery ; however, fkn and its receptor cx3cr1 are highly expressed in atherosclerotic lesions. as was the case in our study, li. found higher fkn levels in patients with acs when compared to control subjects or even when compared to patients with stable angina. ziakas. identified higher peak values of acute phase reactants in patients with stemi versus nstemi / ua. for hscrp, previous studies demonstrated significantly higher admission and peak values in subjects with stemi compared to nstemi and in ami compared to ua [31, 32 ]. this comes in line with our finding where hscrp levels were significantly higher in stemi patients compared to nstemi / ua patients. for il-18, our finding was confirmed by brunetti. who reported that serum levels of il-18 are not affected by acs diagnosis. furthermore, a recent study revealed that the circulating levels of fkn were not statistically different between ami and ua groups. an in vitro study identified that the production of gzb from cultured peripheral blood mononuclear cells is time dependent, where gzb levels in culture media gradually increased up to 48 hours after incubation. moreover, gzb showed peak plasma levels on day 7 after ami. this comes in agreement with our results, where plasma gzb levels significantly increased on day 3 compared to day 1 in both stemi and nstemi / ua groups. in the study conducted by ghanavatian. our study hscrp decreased after 3 days in comparison to day 1 in both groups. to the best of our knowledge, this is the first study to compare plasma fkn levels in timed samples after acs. on the other hand, a study of inflammatory cytokines imbalance in patients with acs revealed that il-18 levels were significantly high on admission and remained unchanged after 12 and 24 hours. this was the case in our study where serum il-18 did not change significantly from day 1 to day 3 in both groups. this association could be explained in context of the results of kyaw and coworkers who concluded that cd8 + t lymphocytes, the most abundant inflammatory cells in advanced atherosclerotic lesions, promote plaque vulnerability via gzb - mediated apoptosis of macrophages, smcs, and endothelial cells. previously, it was shown that the interaction between fkn and its receptor is the main player in trafficking such gzb producing cd8 + t lymphocytes into the vascular lesions that suggests a strong relationship between fkn and gzb levels as was demonstrated in our results. the extracellular role of gzb indicates that gzb may have an indirect mechanism for eliciting cytokine release. recently, it was demonstrated that granzyme b processes il-1 into a significantly more potent proinflammatory fragment. additionally, a direct relationship between il-18 and gzb was identified by omoto. who found that gzb as a serine protease cleaves inactive pro - il-18 at the same residue cleaved by its documented activator caspase-1 ; hence gzb could be considered as a novel activator of the proinflammatory il-18. as a result, accumulation of the extracellular gzb may result in elevation of serum il-18 levels which is consistent with our results. to the best of our knowledge however, previous studies showed that ifn- stimulates fkn expression in vascular endothelial cells [39, 40 ]. moreover, other members of il-1 superfamily such as il-1 were also involved in fkn induction. these relationships may illustrate the positive significant correlation between fkn and il-18 in our study. there might be some sort of interplay between gzb, il-18, and fkn in the pathogenesis of acs. the current study also confirmed the different inflammatory response in stemi patients with respect to nstemi / ua patients. indeed, further large scale prospective studies are required to demonstrate the possible use of plasma gzb in risk stratification after myocardial ischemia. | objective. the proapoptotic protein, granzyme b (gzb), was identified as a contributor to the atherosclerotic plaque instability and recently as inflammatory activator. we studied the release kinetics of gzb and other markers of inflammation such as high sensitivity c reactive protein (hscrp), interleukin 18 (il-18), and fractalkine (fkn) in the early phase after acute cardiac events in different acs subgroups. methods. thirty - six nondiabetic patients with acs were compared to 12 control subjects. according to acs diagnosis, the patients were classified into 22 patients with st elevation myocardial infarction (stemi) and 14 patients with non - st elevation myocardial infarction or unstable angina (nstemi / ua). blood samples were taken on day 1 (day of onset) and day 3 to measure hscrp, il-18, fkn, and gzb by elisa. results. patients with acs showed significantly higher gzb, il-18, and fkn levels than the controls. stemi group showed significantly higher gzb levels than nstemi / ua group. on day 3, fkn levels displayed a significant decrease, while gzb levels were significantly increased. il-18 levels were more or less constant. gzb levels were positively correlated with il-18 (r = 0.416, p < 0.01) and fkn (r = 0.58, p < 0.001). conclusions. unlike il-18 and fkn, plasma gzb may be a marker of acs disease severity. |
cilj ove multicentrine studije bio je da se rezultati sedam automatizovanih komercijalnih imunoeseja uporede sa referentnom tehnikom hplc. je, podeljeno u alikvote, smrznuto i poslato laboratorijama koje su uestvovale u istraivanju. je pomou referentnog sistema hplc i pomou sedam automatizovanih komercijalnih imunoeseja (roche cobas e601, beckman coulter unicel dxi 800, ortho vitros es, diasorin liaison, siemens advia centaur, abbott architect i system i ids isys). u poreenju sa referentnim metodom, koeficijenti regresije kretali su se izmeu 0,923 i 0,961 (svuda p 75 nmol / l (figure 1), and hence with analyte concentrations greater than the conventional threshold of moderate 25oh - d deficiency. this may be attributable to the specific immunoreactivity of the antibodies against the samples tested, but also to the fact that this is a 2-step chemiluminescent method, whereas the other immunoassays are based on direct (i.e. 1-step) competitive binding against 25oh - d or vitamin d binding protein (vdbp) (table i). interestingly, the percentage bias calculated on samples with 25oh - d < 75 nmol / l was much lower (n=56 ; 12.8% ; 95% ci 21.1 to 4.5%). no previous studies have investigated the performance of this assay, to the best of our knowledge, and thereby comparison with results of other analytical investigations is impossible. along with cobas e601, this method is also calibrated against the nist - srm 972, so that these results are not unexpected. it has been recently shown that the values of 25oh - d are typically low - biased in methods traceable to nist - srm 972, especially when measuring samples containing prevalently 25oh - d3 (20) such as those obtained from our study population of unselected outpatients (supplementation with 25oh - d2 is not prescribed in our area). the results of other assays were globally satisfactory, with excellent correlations and significant agreement at the cut - off of 25oh - d deficiency (table ii and iii). a negative bias was also observed for the abbott architect, in agreement with that previously reported by jovii. (18), who also used an hplc technique as the gold standard. in conclusion, the excellent correlations with the reference hplc technique found in this study attest that all seven automated immunoassays may be reliably used for routine assessment of 25oh - d in clinical laboratories. the differences observed in this study are at least in part attributable to the different test design. more specifically, the roche cobas e601 electrochemiluminescent immunoassay is a competitive protein binding method which uses a specific protein that binds to vdbp, whereas the other methods are based on the competitive binding of antibodies to 25oh - d (table i). it is also noteworthy that direct methods, in which 25oh - d and vdbp are not completely separated, may also display heterogeneous immunoreactivity compared to 2-step immunoassays (10). regardless of these differences, only one method slightly exceeded the minimum performance goal for bias as compared with hplc, and another one displayed significant disagreement at the cut - off of 25oh - d deficiency. in both cases, however, the correlation with the reference method was excellent, thus emphasizing the current issue of poor standardization of vitamin d testing (table i). this hypothesis is also supported by the rather heterogeneous value of bias (from 14.5 to 8.5 although this should be regarded as a potential confounding factor when assessing 25oh - d according to the conventional recommendations to maintain the concentration of this vitamin above a certain threshold (2, 3), the objective of harmonization, however, may be achieved with relatively modest efforts. due to the high reliability of individual results against the reference technique (table ii and iii), it is reasonable to hypothesize that extension of traceability to common standards (e.g. nist - srm 2972) across different methods and platforms should be effective to consistently reduce the bias and improve comparability among the various automated 25oh - d immunoassays available on the market. | summarybackgroundthe measurement of 25oh vitamin d continues to grow in clinical laboratories. the aim of this multi - center study was to compare the results of seven automated commercial immunoassays with a reference hplc technique.methodsone hundred and twenty consecutive outpatient serum samples were centrifuged, divided in aliquots, frozen and shipped to the participating laboratories. 25oh vitamin d was measured with a reference hplc system and with seven automated commercial immunoassays (roche cobas e601, beckman coulter unicel dxi 800, ortho vitros es, diasorin liaison, siemens advia centaur, abbott architect i system and ids isys).resultscompared to the reference method, the regression coefficients ranged from 0.923 to 0.961 (all p<0.001). the slope of deming fit ranged from 0.95 to 1.06, whereas the intercept was comprised between 15.2 and 9.2 nmol / l. the bias from the reference hplc technique varied from 14.5 to 8.7 nmol / l. the minimum performance goal for bias was slightly exceeded by only one immunoassay. the agreement between hplc and the different immunoassays at 50 nmol / l 25oh vitamin d varied between 0.61 and 0.85 (all p<0.001). the percentage of samples below this cut - off was significantly different with only one immunoassay.conclusionsthe excellent correlation with the reference hplc technique attests that all seven automated immunoassays may be reliably used for routine assessment of 25oh - d in clinical laboratories. the significant bias among the different methods seems mostly attributable to the lack of standardization and calls for additional efforts for improving harmonization of 25oh - d immunoassays. |
activation of the cellular arm of the immune system is seen as the first biological event after administration of immunotherapy, and consequently, measurement of such a response (mostly t - cell response) is of great interest. a variety of bioassays exist for immune monitoring, including the enzyme - linked immunosorbent spot (elispot) assay and cytometry - based tests such as intracellular cytokine staining, hla - peptide multimer staining, and the carboxyfluorescein succinimidyl ester assay (2427). even though the fundamentals of these assays have been well established, a plethora of different laboratory protocols is used (24,27,28). substantial variability in results among laboratories exists in multicenter trials, which hampers data reproducibility and prevents meaningful comparisons among studies (28). for example, in the first cic - cri elispot proficiency panel, 36 laboratories used the elispot assay to analyze the same donor peripheral blood mononuclear cell sample for a t - cell immune response, resulting in spot counts ranging from nondetectable to strongly positive (figure 1). the challenge is the absence of a quality control measure for t cell based assays that can be used as a gold standard (29), which has prevented the field from establishing t - cell response as a biomarker for immunotherapy trials and conducting reliable investigations of its relationship with clinical outcomes (30). high variability of results for the enzyme - linked immunosorbent spot (elispot) immune response assay. identical peripheral blood mononuclear cell samples from the same patient were sent to 36 different laboratories experienced with elispot methodology. the image shows the spot count results in microtiter plates in which each well represents the result of one laboratory. each spot in this assay represents a single t - cell capable of reacting against a defined target antigen. these results reflect the outcome of the first elispot proficiency panel, which identified sources of variability among laboratories. under the auspices of cic - cri and c - imt, two large international immune monitoring proficiency panel programs were initiated in 2005 (2022). their goals were to provide an external quality assurance process for laboratories conducting immune monitoring in clinical trials and to harmonize assay performance. harmonization of clinical trial conduct and data collection as established through the international conference on harmonization for good clinical practice had previously proven to be a highly successful model for improving clinical development procedures (31). proficiency panels are quality control experiments in which the same patient samples are tested across multiple laboratories, using their respective protocols, and the results are centrally evaluated (32). the cic - cri proficiency panel program is led by a scientific steering committee and involves a central laboratory for accrual of peripheral blood mononuclear cells, shipping logistics, and independent central data analysis. peripheral blood mononuclear cell samples and antigens, pretested and defined for their response status, are sent to all participating laboratories, where they are tested for response under the local conditions. more than 80 laboratories from 14 countries have participated, encompassing the academic, nonprofit, biotech, and pharmaceutical sectors, and the united states department of defense (20,22). the objective of the first elispot panel was to identify sources of variability among assay procedures. the second panel adjusted these sources of variability while keeping the respective laboratory protocols intact. this adjustment led to a substantial reduction in variability : the percentage of participants unable to detect all responders (six responders among eight samples) was reduced from 47% to 14%, and the percentage of participants unable to detect 50% or more of all responders (outliers) dropped from 11% to 0% (figure 2). the combined panel results led to initial elispot harmonization guidelines (table 2) (20), which synchronize key variables across laboratories and substantially influence assay outcome but do not impose standardization of assays on individual laboratories. the introduction of these guidelines is central to this harmonization to provide general assistance for the conduct of the individual assay protocol in the context of standard operating procedures (eg, exclusion of apoptotic cells, use of pretested serum for background reduction, and quality control during the computerized spot evaluation) (table 2) (20). confirmatory findings from a parallel experiment were published by the c - imt proficiency panels (21). as the use of serum is a crucial variable for elispot assays, a separate elispot proficiency panel focused on serum use and showed that serum - free medium for incubation of cells can be as effective as qualified serum - supplemented medium, thus addressing this crucial variable for assay protocols as part of the harmonization guidelines (33). initial harmonization guidelines for the enzyme - linked immunosorbent spot (elispot) immune response assay harmonization guidelines can be used by each individual laboratory performing an immune response assay in the context of standard operating procedures (sops) and without adopting a standard assay protocol. through general steps such as use of pretested serum [or serum - free media (33) ], exclusion of apoptotic cells from the analysis, human auditing of the computerized assay read out procedure, and training of operators on the laboratory sops, quality of assays can be substantially improved. effect of assay harmonization on data variability of the enzyme - linked immunosorbent spot (elispot) assay. in the cancer immunotherapy consortium of the cancer research institute elispot proficiency panel, grey bars represent the first panel round and stippled bars the second panel round. in the first panel round, 47% of panelists missed detection of at least one response correctly, and 11% of panelists failed to detect at least three responses correctly (characterized as an outlier based on the first panel results, harmonization criteria were given to panelists, and the testing was repeated in the second panel (stippled bars). elispot performance improved, with only 14% of panelists missing at least one responder and zero outliers. the first round of the hla - peptide multimer - staining panel of cic - cri allowed the formulation of initial harmonization guidelines (22), which will likely reduce assay variability. these recommendations are 1) the use of more than two colors for staining, 2) the collection of more than 100 000 cd8 t cells, and 3) the use of a background control sample to set appropriate analytical gates (data points of interest) (22). the second cic - cri panel round focused on confirmation of these guidelines, as well as assessment of the influence of specific protocol steps on final results. however, the results demonstrated that the use of standardized reagents and protocols alone does not lead to the desired performance among panelists. the ongoing second panel allows free choice of reagents and protocols but requires compliance with evolving minimum guidelines based on the results of the first panel. the clinical trial application of immune assays was addressed by an international working group, the cancer vaccine clinical trial working group (8), which proposed that immunoassays in clinical trials should be performed at least at three different time points throughout any study one baseline and two follow - up time points. assays should be established, reproducible, and technically (not clinically) validated in the respective laboratories (according to the harmonization criteria for respective assays). at least two assays should be used in parallel to demonstrate the same findings (eg, elispot and hla - peptide multimer staining), which is particularly important if developmental decisions (eg, moving a new agent into more advanced trials) would be based on immune response data from such assays. moreover, the cutoff values for an immune response should be identified prospectively to define the change necessary for a response and to define the proportion of study patients needed for a positive study outcome. in addition, a separate initiative is under way to provide a publication framework for the results from immune monitoring trials. this project, named miata (minimal information about t - cell assays), is currently undergoing a public consultation process to obtain community feedback (28). overall, only when the issue of current assay variability is adequately addressed can cellular immune response become a reliable parameter in clinical trials and may be more reliably studied for its relationship with clinical outcomes. for decades, investigators have relied on modified who criteria (34) or, more recently, recist (35,36) to assess clinical activity of anticancer agents. these standard criteria were designed to capture effects of cytotoxic agents and depend on tumor shrinkage to demonstrate activity. however, the response patterns seen with immunotherapeutic agents extend beyond those of cytotoxic agents and can manifest, for example, after a period of stable disease in which there is no tumor shrinkage or after initial tumor burden increase or appearance of new lesions (eg, tumor infiltrating lymphocytes) (15,3740). this potential delayed detection of clinical activity on radiographic assessment may reflect the dynamics of the immune system the time required for t - cell expansion followed by infiltration of the tumor and a subsequent measurable antitumor effect. for example, some reported clinical experiences with cancer vaccines (3739) demonstrated that patients with stable disease or progressive disease may have subsequent tumor regression, whereas others may show initial mixed responses, with regression in some lesions while other lesions remain stable, progress, or first appear. in these studies, patients with measurable tumor burden decrease had more responses that did not fit currently accepted response criteria than conventional responses (eg, 5/6 and 4/5 unconventional out of all recorded responses) (3739). such patterns have been noted by many investigators ; however, they were inconsistently included in publications or were not systematically captured because of the absence of suitable response criteria (8), which, in turn, did not allow for their clinical significance to be adequately studied (8). it has become evident that recist or who criteria may not offer a complete description of the response to immunotherapeutic agents, and either adjusted or new criteria are needed (8). similar to the immune response assays discussed above, the cancer vaccine clinical trial working group addressed the topic of clinical endpoints and potential delayed detection of clinical activity in a series of workshops. the main conclusions were that the appearance of measurable clinical activity at the tumor site may take longer for immunotherapies than for cytotoxic therapies ; responses to immunotherapies may occur after conventional progressive disease (tumor burden increase) ; discontinuation of immune therapy may not be appropriate in some cases, unless progressive disease is confirmed (as is usually done for recist - based response) ; there should be allowance for clinically insignificant progressive disease (eg, small new lesions in the presence of other responsive lesions) ; and durable stable disease may represent clinical benefit. such elements might be included in new antitumor response criteria that adapt standard criteria to the unique characteristics of immunotherapy (8). building on these recommendations, a novel set of response criteria based on who and recist were evaluated in a series of large multinational studies (17,41,42), including 487 patients with advanced melanoma participating in the bristol - myers squibb and medarex clinical trial program for ipilimumab, a fully human monoclonal antibody that blocks ctla-4. in these studies, four distinct response patterns were detected : immediate response, durable stable disease, response after tumor burden increase, and response in the presence of new lesions (figure 3). the first two patterns are conventional (figure 3, a and b), whereas the latter two (figure 3, c and e) are novel and specifically recognized with immunotherapeutic agents. photographs of a case study of the first novel tumor response pattern (figure 3, d) show that tumor burden initially increases (day 84) and then decreases (day 112) to a complete response (day 503). importantly, all patterns (conventional and new) seem to be associated with favorable survival compared with patients with progressive disease by who criteria (43,44). to create a process, which systematically captures all observed response patterns, clinical response patterns observed with anti - cytotoxic t lymphocyte associated protein 4 immunotherapy (ipilimumab). immunotherapy patterns of response depicted as a continuous variable of relative change of tumor burden (%) over time. tumor burden is described through the sum of the perpendicular diameters (spd) of all measurable lesions (baseline and new) at each time point. a and b) conventional response patterns : (a) immediate response ; (b) durable stable disease with possible slow decline in tumor burden. c and e) novel immunotherapy response patterns : (c) increase in total tumor burden followed by response. (d) clinical images corresponding to pattern (c) : tumor burden on the skin at baseline (day 0) is increased at first follow - up (day 84) and subsequently declines (day 112) to a complete response (day 503) (courtesy of dr k. harmankaya). e) the second novel pattern shows a response in the presence of new lesions ; existing lesions present at baseline (blue) and new lesions (red) are added to define the total tumor burden (green). despite new lesions yellow triangles indicate dosing with immunotherapy ; horizontal lines indicate standard thresholds for response or progression. in particular, they allow for the assessment of tumor burden as a continuous variable, which considers index lesions identified at baseline together with new lesions as they may occur after treatment start. measurability is defined as 5 5 mm or more on helical computer tomography scans. the sum of the perpendicular diameters (spd) of index lesions at baseline is added to that of new lesions to calculate total tumor burden according to the following formula : thus, percentage changes in tumor burden between assessment time points describe the size and growth kinetics of total measurable tumor burden over time. response categories under irrc are defined as immune - related complete response (ircr), immune - related partial response (irpr), immune - related stable disease (irsd), and immune - related progressive disease (irpd) using the same thresholds to distinguish between categories as defined under standard who criteria (table 3). decrease in total measurable tumor burden is assessed relative to the baseline tumor burden, that is, spd of all index lesions at baseline. the response category irpd should be confirmed at two consecutive time points as already done for irpr or ircr. overall, immune - related response based on two or more tumor assessments is derived as shown in table 3. derivation of overall immune - related response in solid tumors after wolchok. ircr = immune - related compete response complete disappearance of all index and new measurable lesions ; irpr = immune - related partial response decrease in tumor volume 50% relative to baseline ; irsd = immune - related stable disease not meeting criteria for ircr or irpr, in absence of irpd ; irpd = immune - related progressive disease index lesions are measurable (> 5 5 mm), and non - index lesions are not measurable (5 5 mm). assuming response and progression are confirmed by a second assessment at least 4 weeks apart. using irrc, the appearance of new lesions alone does not constitute irpd if they do not add to the tumor burden by at least 25%. patients with new lesions but an overall tumor burden decrease qualifying for partial response (50% decrease) or qualifying for stable disease (25% increase) are considered to have irpr or irsd, respectively (same percentage changes including new lesions) (table 3). these new patterns are considered clinically meaningful because they appear to be associated with favorable survival (43,44). importantly, early increase in the size of lesions, which may be attributable to the infiltration of lymphocytes, does not preclude an ircr, irpr, or irsd from being obtained at the next consecutive time point. if a patient is classified as having irpd, confirmation by a second scan in the absence of rapid clinical deterioration is required. thus, the definition of confirmation of progression represents an increase in tumor burden of at least 25% compared with baseline at two consecutive time points at least 4 weeks apart. it is recommended that this confirmation be done at the discretion of the investigator in the context of the patient s tumor type, disease stage, and clinical status because awaiting a response after tumor burden increase may not be appropriate for patients with rapid symptomatic progression accompanied by a decline in performance status. in summary, the development of irrc may provide a novel and long - needed tool for clinical trials to assess signs of activity of immunotherapies. because data obtained with ipilimumab (43) suggest an association of immune - related response patterns with favorable patient survival, these criteria may identify important clinical patterns otherwise characterized as progressive disease by who criteria. further prospective evaluation of irrc in immunotherapy trials is required to confirm their clinical utility. in contrast to chemotherapy, for which an early clinical effect is possible, immunotherapies often demonstrate delayed clinical effects (12,14,17,4649). for example, figure 4 shows the overall survival outcome for a study of sipuleucel - t immunotherapy vs placebo in advanced prostate cancer (12), where the separation of kaplan meier curves occurs after approximately 8 months after random assignment. in randomized trials, in which immunotherapies are compared with either placebo or inactive controls, kaplan meier survival curves may be superimposable for a time before separation is observed. generally, if there is such a delayed separation, the statistical power to differentiate the entire curves is reduced (1,9,50). delayed separation of survival curves of sipuleucel - t immunotherapy vs placebo in advanced prostate cancer, where the separation of kaplan meier curves occurred after approximately 8 months after random assignment. all rights reserved. to better understand possible lessons from existing datasets of phase iii immunotherapy trials, a workshop of the cic - cri in 2006 reviewed all publicly accessible data from such trials. this analysis suggested the possibility of a delayed separation of kaplan meier curves for immunotherapeutic agents (mostly cancer vaccines) (1), including melacine in stage ii melanoma (46), vitespen in advanced melanoma (47), vitespen in stage ii / iii renal cell carcinoma (48), sipuleucel - t (12) (figure 4), prostvac - vf (14) in advanced prostate cancer, and the anti - ctla-4 antibody ipilimumab in advanced melanoma (17,49). a separation of curves in these trials was observed after 48 months or later following random assignment. however, such kinetics may differ among agents and disease settings, thus requiring individual investigation. the survival analysis methods commonly used to design and analyze two - arm randomized clinical trials do not have a provision for data demonstrating a delay in the separation of kaplan meier curves. these methods assume that the ratio of the arm - specific hazard rates (risks for patients to experience events) is a constant over time (proportional hazards assumption) and is necessary when using the standard formula for computing the number of events required for the final analysis of a trial (51). furthermore, the log - rank test has optimal properties under proportional hazards, and cox regression models assume proportional hazards. under these assumptions, the hypothesized hazard ratio (hr), describing the difference in survival between the study arms, applies immediately after random assignment, and the event rates at all times will have the same ratio to differentiate between the arms. initially, the hazard rates are equal, and therefore, the hazard ratio is 1.0, and the kaplan meier curve estimates will be indistinguishable (if an inactive comparator is used). subsequently, the hazard rates become unequal and the curves will separate. for practical purposes, it is assumed for this discussion that the hazard rates after the delayed clinical effect eventually become proportional. thus, the events occurring before the time when the hazard rates become unequal will not differentiate the study arms. as a consequence, computation of the required number of events for final analysis under proportional hazards assumptions will lead to a statistical power insufficient for a trial with a delayed separation. depending on the timing of the delay alternative methods should be considered to compute the required number of events for final analysis when delayed separation is expected (1,9). a variety of methods including simulation and numerical integration of a proposed theoretical hazard function can be used. simulation is attractive because it may also account for other deviations from usual trial planning assumptions. however, any method used to compute the required number of events will require careful scrutiny and validation. another critical element for the computation of the number of events is the timing (quantification) of the delayed effect. ideally, the quantification used to compute the required events will come from previous randomized trials, possibly in the phase ii setting (9). the following example illustrates the practical implications of the delayed separation on study statistical power, number of events, and study duration. figure 5 illustrates the mathematical form of a delayed separation, with a hazard ratio of 0.7 after the separation. the control arm has an exponential survival distribution with median survival of 18 months (red dashed curve). the form of the delayed separation is specified by a hazard ratio function (hr(t), solid grey line) that has value 1.0 for the first 3 months and then decreases linearly between 3 and 6 months to become 0.7 after 6 months. the experimental arm survival distribution (solid blue line) is the result of mathematically blending the control arm survival distribution function and the hazard ratio function. the survival distributions in figure 5 can be used to illustrate the consequences of delayed separation. the following additional specifications are made : 1:1 random assignment, n = 600, accrual time 18 months, two - sided type i error probability.05, statistical power of 90%, and use of the log - rank statistic. under proportional hazards (no delayed separation), 331 events are required for final analysis and are projected to occur at 2.85 years. with a delayed separation to 3 months as specified, 331 events would result in a statistical power estimate of only 62.7%. using simulations that take into account delayed separation, it is found that 90% statistical power requires 510 events and would not be realized until 5.66 years. the control arm has an exponential survival distribution with median survival of 18 months (red dashed curve). the form of the delayed separation is specified by a hazard ratio function (hr(t), solid gray line) that has the value 1.0 for 3 months and then decreases linearly between 3 and 6 months to become 0.7 at 6 months and then remains constant. the experimental arm survival distribution (solid blue line) is the consequence of mathematically blending the control arm survival distribution function and the hazard ratio function. it is therefore recommended that the computations of the required events be based on a plausible specification of the timing of the delayed separation, the desired statistical power, use of the log - rank statistic, and the understanding that the trial will be overpowered if delayed separation is not observed or is less than that specified. the log - rank test is recommended for study planning and final analysis to avoid deviation from conventional methods, to avoid prespecification of parameters describing the delay (52,53), and as a hedge against absence of a delay. the cost of using the log - rank statistic is some loss of optimal conditions if a delay is present. because immunotherapeutic agents may differ regarding presence and timing of delayed separation, randomized phase ii trials these must be carefully considered because the presence of a delayed separation will inflate the chances of a negative early interim analysis and of concluding futility because of projected results without a delayed separation. as our knowledge of immunologic and clinical science has evolved (47), we have begun to address the unique characteristics of immunotherapeutic agents in clinical trials and to use more appropriate endpoints (8,9,10,43). thus, they may induce cellular immune responses before influencing tumor burden or patient survival (8,9). for adequate investigation of immunotherapies in clinical trials, a new development paradigm is needed, including adjustment of established endpoints to address this biology. the challenges around adequate clinical endpoint use in immunotherapy trials were addressed through community - wide workshops paired with comprehensive laboratory and clinical programs providing large datasets (8,9,17,2022,41,42). the inability to use cellular (t - cell) immune response assays to define biomarkers and to investigate their relationship with clinical outcomes has its roots in highly variable and often nonreproducible assay results in multicenter trials (28,30). as was demonstrated by several large international proficiency panels, the use of harmonized assays can reduce this variability (2022) and may help to build a general framework for assay use in multicenter clinical trials similar to what the international conference on harmonization / good clinical practice did for clinical protocols. furthermore, harmonized assays may allow investigation of the relationship between immune responses and clinical outcomes. correlation of immune response and clinical outcomes would certainly accelerate the learning process regarding the biological activity of immunotherapeutic agents in the clinic and help the early selection of promising candidates for advanced trials. as observed in many clinical trials, immunotherapy can induce novel patterns of antitumor responses distinct from those of chemotherapy, which are consequently not captured by the who or recist criteria. in a 5-year collaborative effort between academia and industry (8,44), clinical observations were translated into new response criteria (43), which more comprehensively capture all observed response patterns. the irrc provide a systematic description for phenomena such as mixed responses, in which new lesions appear while existing lesions shrink or in which some lesions shrink while others grow. through the new irrc system, immunotherapy patterns of response are described as tumor burden over time where tumor burden is the spd of all measurable lesions (baseline and new). the irrc are generally based on the who and recist criteria and do not require a substantial departure from standard oncology practice. the novelty of the irrc lies in the measurement of new lesions, which are included in the overall tumor burden, allowing for it to be described as a continuous variable (before and after conventional progression) (9,44). this response analysis bears similarities to a concept described for the investigation of cytostatic agents such as sorafenib, in which tumor size was described as a continuous variable (54). histological evidence in cases in which biopsy material was available suggests that the appearance of new lesions or increase in the size of existing lesions may be the result of lymphocytic infiltration and may not represent true disease progression (40,55). in these cases, the irrc provide a means of accounting for delayed changes in tumor burden through confirmation of progression at subsequent time points. this new confirmation of progression is similar to the confirmation routinely done for response and may enable the detection of a change in kinetics when initial tumor burden increase through lymphocytic infiltration is followed by a lymphocyte - induced tumor response (delayed response). importantly, initial observations in advanced melanoma with anti - clta-4 antibody indicate an association of immune - related responses with favorable survival, suggesting that these criteria may identify patients with previously unrecognized benefit (43,44). the clinical applicability of these criteria lies principally in the more comprehensive description of clinical signals of activity in early trials. consequently, the irrc offer an additional tool for investigating immunotherapies and are undergoing prospective validation. considering the time of translation of immunologic responses into clinical activity, the survival of patients may not be affected until some months after treatment start compared with chemotherapy. a resulting delayed separation of kaplan meier curves was observed in multiple randomized immunotherapy trials and was often not apparent until 48 months or more after random assignment (12,14,17,4649). in patient populations with advanced cancers, a substantial number of events can occur in the time window before separation and thus lead to substantial loss of statistical power (1,9,50). whereas it remains unclear whether past studies would have had a different outcome if delayed separation had been considered during trial planning, its consideration for future evaluations appears important. knowledge of survival kinetics may also allow better planning of interim analyses because if they are performed too early, they may give misleading results and conclude futility prematurely. in addition, immunotherapeutic agents may differ in the timing of delayed separation and should be tested in randomized phase ii trials to improve the planning for phase iii trials. use of modified statistical methods to characterize the hazard ratios before and after separation of survival curves allows improved planning of randomized trials in which a delayed separation of curves is expected. in summary, the described recommendations for improved clinical endpoints have the potential to positively alter the clinical investigation of cancer immunotherapy. funding for scientific meetings, workshops, and the immune monitoring proficiency panels was provided by the cancer immunotherapy consortium of the cancer research institute, a nonprofit organization. | unlike chemotherapy, which acts directly on the tumor, cancer immunotherapies exert their effects on the immune system and demonstrate new kinetics that involve building a cellular immune response, followed by changes in tumor burden or patient survival. thus, adequate design and evaluation of some immunotherapy clinical trials require a new development paradigm that includes reconsideration of established endpoints. between 2004 and 2009, several initiatives facilitated by the cancer immunotherapy consortium of the cancer research institute and partner organizations systematically evaluated an immunotherapy - focused clinical development paradigm and created the principles for redefining trial endpoints. on this basis, a body of clinical and laboratory data was generated that supports three novel endpoint recommendations. first, cellular immune response assays generate highly variable results. assay harmonization in multicenter trials may minimize variability and help to establish cellular immune response as a reproducible biomarker, thus allowing investigation of its relationship with clinical outcomes. second, immunotherapy may induce novel patterns of antitumor response not captured by response evaluation criteria in solid tumors or world health organization criteria. new immune - related response criteria were defined to more comprehensively capture all response patterns. third, delayed separation of kaplan meier curves in randomized immunotherapy trials can affect results. altered statistical models describing hazard ratios as a function of time and recognizing differences before and after separation of curves may allow improved planning of phase iii trials. these recommendations may improve our tools for cancer immunotherapy trials and may offer a more realistic and useful model for clinical investigation. |
newly synthesized proteins in the endoplasmic reticulum (er), are transported via a series of membrane - bound shuttles or vesicles to and between the golgi apparatus, where their content proteins undergo various post - translational modifications such as glycosylation, to mature. specialized transport vesicles containing mature cargo originate from the golgi and destined for secretion, dock and transiently fuse at specialized plasma membrane structures called [porosomes ] to expel intravesicular contents. this unique process of transport and secretion, is universal to living cells.the discovery of proteins involved in the budding and fusion of such transport vesicles, the discovery of the porosome[2, and the regulated release of vesicular contents from cells, are landmark discoveries. while an elegant mammalian cell - free system was used in the discovery of the various players involved in vesicle budding and fusion, a new and powerful microscope (atomic force microscope or afm) was brilliantly employed in discovery of the [porosome ] in live cells, in unraveling the interaction, assembly, and disassembly of membrane proteins involved in membrane fusion, and in deciphering the regulated release of intravesicular contents during cell secretion.these seminal and landmark discoveries are summarized. a preparation of isolated golgi stacks, atp, and a 100,000 g cytosolic fraction from mammalian cells, was found to reconstitute transport between golgi cisternae. for the first time, this assay system provided the tool for discovery of proteins involved in the fission and fusion of transport vesicles within cells [46 ]. golgi population containing vsv - encoded g protein (the cargo) but lacking the glycosylating enzyme, is incubated with an acceptor golgi population containing the enzyme, the transfer of g protein from donor to acceptor is determined as a measure of radiolabled n - acetylglucosamine in the oligosaccharide chain of g proteins in the acceptor compartments. (non - clathrin coat) and containing the vsv g protein, appear only in presence of the 100,000 g cytosol and atp. it was further demonstrated that transport was blocked in the presence of n - ethyl - maleimide (nem), and in the presence of non - hydrolyzable gtp analogues such as gtps. in presence of gtps the 70 nm vesicles that accumulated were coated, however, in the presence of nem, uncoated vesicles accumulated. since the uncoated vesicles bound to golgi cisternae, coat had to be removed following budding to allow fusion of vesicles with the target membrane.these results were key to the discovery of the vesicle budding and fusion machinery. immediately thereafter, coated vesicles were purified following their accumulation in presence of gtps. eight polypeptides were found to form the coat, which included a previously identified and characterized protein the adp ribosylation factor (arf), and seven other proteins termed cops. in the vesicle budding process, cytosolic gtp - bound arf binds the golgi membrane, initiating recruitment of coat proteins from the cytosol, leading to the formation of the bud. hydrolysis of arf - bound gtp, releases the coat from vesicles, making them fusion ready with the target membrane. hence arf is the master molecule that controls coating and uncoating. since in presence of nem, uncoated vesicles accumulate and are bound to acceptor golgi cisternae without fusing, implied the requirement of a nem - sensitive factor in membrane fusion. addition of fresh cytosol to the nem treated reaction mixture, restored fusion, demonstrating that the nem - sensitive factor was present in the cytosol, and thereby guided the purification of the nem - sensitive factor (nsf). using nsf as bait, a 20s membrane fusion particle protein sequencing of the resolved particle demonstrated the presence of both a membrane protein found in synaptic vesicles, and two proteins found at the presynaptic membrane, syntaxin and snap-25. vamp and syntaxin are both integral membrane proteins, consisting of an amino terminal cytoplasmic domain, a membrane - spanning segment, and polar residues at their carboxy terminus. in the mid 1990s, an atomic force microscope (afm) trained on isolated live pancreatic acinar cells in near physiological buffer solution, provided for the first time, the structure, organization, and dynamics of the cell plasma membrane at nanometer resolution and in real time. organized circular structures measuring on the average 400500 nm in diameter termed pits, containing on the average 34, 100180 nm in diameter and 2545 nm in depth depressions, were observed for the first time at the apical plasma membrane in live pancreatic acinar cells using high - resolution afm. when cells were stimulated to secrete, the exposure of live pancreatic acinar cells to the fungal toxin cytochalasin, a known inhibitor of actin polymerization and cell secretion, resulted in the collapse of depressions and a concomitant loss in secretion. depressions to be the long sought - after fusion pores, however, there was the need for direct evidence of secretory products exiting the pits and depression structures. it took almost 5 years to finally demonstrate using immuno - afm, that indeed secretory products are discharged through depressions during cell secretion. subsequent studies using growth hormone secreting cells of the pituitary gland, also demonstrated the presence of depressions, and the release of growth hormone through them. these findings finally demonstrated depressions to indeed be the long - sought fusion pores in cells. further studies on chromaffin cells, mast cells [2, 20 ], -cells of the endocrine pancreas [2, 20 ], and neurons [2, 21 ], all demonstrate the presence of depressions and their function as the cells secretion machinery.the universal presence of depressions as the secretory apparatus in cells, probably led to coining of the term it had been speculated, that such a structure may exist at the cell plasma membrane where secretory vesicles transiently associate and fuse to release their contents from the cell. therefore the discovery of the porosome is a landmark in biology, ending both speculations for its presence as well as the race for its discovery. unlike what had previously been postulated, afm studies on live cells demonstrated porosomes to be permanent structures at the cell plasma membrane, and not arising following a secretory stimulus. the subsequent imaging of porosomes, and porosome - associated secretory vesicles in situ using conventional transmission electron microscopy (tem) [21, 23, 24 ], has further confirmed their presence and determined their structure and function. porosomes are cup - shaped basket - like - designed lipoprotein structures at the cell plasma membrane [21, 23, 24 ], typically 100180 nm in exocrine and neuroendocrine cells, and 1015 nm at the nerve terminal. there appears to be a direct correlation between secretory vesicle size and the size of porosomes in cells. the molecular structure and chemistry of the porosome was determined following its immuno - isolation, and both its structural and functional reconstitution into artificial lipid membrane [21, 23, 24 ]. the composition of the porosome was further assessed, and the distribution of various porosome proteins determined using porosome dynamics [16, 18 ], immuno - afm studies, yeast 2-hybrid analysis, and cholesterol - depletion experiments. demonstration that the fungal toxin cytochalasin, a known inhibitor of actin polymerization and cell secretion, resulted in the collapse of porosomes, clearly demonstrated actin to be a major component of the porosome complex. similarly, since membrane - bound secretory vesicles dock and fuse at porosomes to release vesicular contents, it was demonstrated that plasma membrane - associated t - snares are part of the porosome complex. immunochemical analysis of isolated porosomes, demonstrated the association of snap-23/25, syntaxin, cytoskeletal proteins actin, -fodrin, and vimentin, and the calcium channels 3 and 1c, the chloride channel clc-3, synaptotagmin, the alpha subunit of the heterotrimeric gtp - binding protein go, together with the snare regulatory protein nsf [21, 23, 24 ]. additional yeast 2-hybrid analysis, further demonstrated the direct interaction between snap-23 and the l - type calcium channel in porosomes of the exocrine pancreas. studies further demonstrate that syntaxin-1 co - localizes with cholesterol, in a triton / lubrol - solubilized synaptosomal membrane preparation. depletion of cholesterol from the triton / lubrol - solubilized synaptosomal membrane preparation using saponin, results in the dissociation of both syntaxin-1 and n - type calcium channel from neuronal porosomes. these studies demonstrate that cholesterol is an integral component of the porosome complex in neurons, and is required for its stability. the size and shape of the isolated porosome complex has been determined using both negative staining tem and afm [21, 23, 24 ]. electron micrographs of isolated porosomes are identical to the afm micrographs of hydrated porosome preparation. porosomes isolated from exocrine pancreas and reconstituted into artificial liposomes, demonstrate a similar basket - like morphology in tem micrographs. when purified porosomes were reconstituted into lipid membranes in an electrophysiological bilayer setup (epc9) and exposed to isolated secretory vesicles, both the electrical activity of the reconstituted membrane as well as the transport of vesicular contents from the cis to the trans compartment, was observed [21, 24 ]. results of these experiments demonstrate that isolated lipid - reconstituted porosomes are functional supramolecular complexes [21, 24 ]. isolated secretory vesicles fuse with the porosome - reconstituted bilayer, as demonstrated by an increase in capacitance and conductance, and in a time - dependent release of secretory products from the cis to the trans compartment of the bilayer chamber. in the exocrine pancreas, amylase transport from the cis to the trans compartment of the bilayer chamber is detected using immunoblot analysis of the buffer in the trans compartment. furthermore, the chloride channel inhibitor dids, was found to inhibit current activity in the porosome - reconstituted bilayer, demonstrating its function in the porosome complex. these studies demonstrate that porosomes in the exocrine pancreas and neurons are 100150 nm and 1015 nm in diameter, respectively, are supramolecular cup - shaped lipoprotein basket - structures at the cell plasma membrane, where membrane - bounded secretory vesicles dock and fuse to release intravesicular contents. the discovery of the porosome, the universal secretory machinery in cells, has finally provided a molecular understanding of the secretory process in cells. neurotransmission, and the release of neuroen - docrine and exocrine substances require certain specialized cells to secrete. altered or loss in cellular secretions is causal to numerous diseases [2, 20 ], and has therefore been the focus of continued scientific investigations for over six decades. it was previously believed that during cell secretion, secretory vesicles merge with the cell plasma membrane, leading to passive diffusion of vesicle contents and subsequent retrieval of excess membrane (vesicle membrane) through endocytosis [27, 28 ]. this view however, was in stark contrast to the universal findings that during cell secretion, empty and partially empty vesicles accumulate with no appreciable loss in vesicle number. unable to account for this discrepancy was therefore frustrating, as previously echoed by professor erwin neher in a 1993 article in the journal nature : it seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later. this cloud of frustration has finally lifted with the discovery of the porosome, a universal new cellular structure at the cell plasma membrane, where secretory vesicles transiently dock and fuse without complete merger, to expel intravesicular contents from cells. since this discovery, a large body of evidence has accumulated demonstrating transient fusion of secretory vesicles at the cell plasma membrane instead of the dogmatic view of complete merger of secretory vesicles at the cell plasma membrane during cell secretion. studies now demonstrate that secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells, that single synaptic vesicles fuse transiently and successively without the loss of identity, and that zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity, and similarly the number of secretory vesicles in growth hormone cells of the pituitary remain unchanged after secretion. numerous articles have been written on these landmark discoveries, three of which [3436 ] would complement and provide a further indepth view on the subject. | abstractcellular protein transport and secretion is fundamental to the very existence of an organism, regulating important physiological functions such as reproduction, digestion, energy production, growth, neurotransmission, hormone release, water and ion transport, etc., all required for the survival and maintenance of homeostasis within an organism. molecular understanding of transport and secretion of intracellular product has therefore been of paramount importance and aggressively investigated for over six decades. only in the last 20 years, the general molecular mechanism of the process has come to light, following discovery of key proteins involved in er - golgi transport, and discovery of the porosome the universal secretion machinery in cells. |
the quality and commercial value of american cranberry fruit (vaccinium macrocarpon ait) are determined by their color. anthocyanins have important therapeutic values, including antitumor [2, 3 ], antiulcer, antioxidant, and anti - inflammatory activities. six anthocyanins have been reported in cranberries based on the high - performance liquid chromatography (hplc) analysis of acid - alcohol fruit extracts on reversed - phase c18 column. these are cyanidin 3-galactoside, cyanidin 3-glucoside, cyanidin 3-arabinoside, peonidin 3-galactoside, peonidin 3-glucoside, and peonidin 3-arabinoside [6, 7 ]. the proportions of individual anthocyanins in cranberry fruit may affect the color stability of cranberry products such as juice and sauce [8, 9 ]. yan reported that cyanidin 3-galactoside showed antioxidant activity superior to six other monoglycosides of quercetin and myricetin isolated from cranberry fruit as well as vitamin e by evaluating compounds for 1,1-diphenyl-2-picrylhydrazyl radical - scavenging activity and ability to inhibit low - density lipoprotein oxidation in vitro. light has been shown to be the most important environmental factor influencing anthocyanin biosynthesis in plants, although in some species, such as vitis vinifera cv. phytochromes are among the most extensively researched photoreceptors which sense light, and are known to be involved in anthocyanin biosynthesis [13, 14, 15 ]. phytochromes respond to red (660 nm) and far - red (730 nm) light, and direct plant gene expression by switching between the red - absorbing form (pr) and the far - red absorbing form (pfr). previously, we have examined the effect of various wavelengths of light on the development of the cranberry plant and anthocyanin biosynthesis in cranberries which were still attached to the plant. we have observed that red light stimulates flowering and anthocyanin biosynthesis in cranberry plant and fruit, respectively. in general, leaves and stems decrease light exposure for berries lower on the plant. preharvest anthocyanin content of cranberries at the bottom and the top of the plant varies significantly, primarily due to the differences in light accessibility. water - harvesting has become a common practice in the cranberry industry, and it is accomplished by flooding the cranberry bog with water to float the buoyant fruit for easy collection. however, potential effects on the berries due to the water - harvest technique have not been studied systematically. one study did show that prolonged fruit immersion increases fungal rot of the berries. during the water - harvest, cranberries on the surface of the water receive the same or more light than the fruit still attached to the plant. in this paper, we evaluate the effects of natural light, red light, and far - red light on individual as well as total anthocyanin levels in cranberry fruit under conditions that mimic water - harvesting. cranberries were exposed to different light conditions and individual anthocyanin content was analyzed. different light conditions, natural light (48 h), natural light (24 h), dark, red light, and far - red light, are indicated in the bottom. percentage of anthocyanin increased in submerged, harvested cranberries exposed to different light conditions in comparison with the control, which was kept in the dark. p 98% confidence level (p<.02)) when the submerged, harvested cranberries were exposed to various light conditions. the total anthocyanin content of berries before exposure to any experimental light conditions was 18.95 0.88, and was the same as the control that was kept in the dark. compared to the control, cranberries exposed to one 24-hour day - night cycle had 75.3% higher anthocyanin content, and berries exposed to a 48-hour day - night cycle posted only a small further increase (87.2%). red and far - red light had substantially less effect on total anthocyanin biosynthesis than natural light (7587% vs. 3542%). red light increased total anthocyanin content (41.5%) more than far - red light (34.7%). separation of cranberry anthocyanins by reverse - phase hplc revealed six anthocyanins which were assumed to be cyanidin 3-galactoside, cyanidin 3-glucoside, cyanidin 3-arabinoside, peonidin 3-galactoside, peonidin 3-glucoside, and peonidin 3-arabinoside (table 2) according to previous reports [6, 24 ]. the relative amounts of the six anthocyanins in the control samples which were kept in the dark (figure 1) are consistent with earlier reports [6, 24 ]. variation in different individual anthocyanins under different light conditions was also subjected to the statistical analysis (student - test), which showed significant differences except for the cyanidin 3-glucoside under each light condition, and for cyanidin 3-galactoside under red light and far - red light conditions, as shown in table 2. compared with the dark conditions, the natural light conditions enhanced all the anthocyanins substantially, 72%100% (in the 48-hour cycle), except for the cyanidin 3-glucoside, which increased by 54% (table 2), whereas the red and far - red light had the most prominent effect on cyanidin 3-glucoside and peonidin 3-arabinoside, showing 7092% increase (table 2). the biosynthesis of cyanidin 3-galactoside was least affected by red and far - red light, showing only 29 and 17% increase, respectively (table 2). although experimental conditions in our previous study were different (30 minutes of light treatments per day for eight days), results had shown that red light and sunlight increased anthocyanin biosynthesis more than the far - red light did, consistently with the conclusions of the present study. however, in the above two cases cranberry fruit that were still attached to the plant and cranberry fruit that were no longer attached to the plant, the effect of far - red illumination appeared to be close to the effect of red light, not similar to the dark control. exposure of etiolated normal seedlings of brassica rapa to red light and far - red light showed that far - red illumination enhanced more anthocyanin synthesis than red light. study of different phytochromes in arabidopsis photomorphogenesis has shown that phytochrome a regulates plant responses to far - red light irradiation, whereas phytochrome b plays a predominant role in responses to red light irradiation. therefore, it is considered that coactions between different photoreceptors involved in the effects of red light and far - red light on anthocyanin content in cranberry fruit are as coactions between the photoreceptors involved in flavonoid biosynthesis. in addition, anthocyanins contain two parts in their structures : aglycones and sugars. the biosynthesis of anthocyanins was catalyzed by several enzymes from pla (phenylalanine ammonia - lyase), c4h (cinnamic acid 4-hydroxylase), 4cl (4-coumarate : coa ligase), chs (chalcone synthase), chi (chalcone isomerase), f3h (flavanone 3 -hydroxylase), dfr (dihydroflavonol 4-reductase), as (anthocyanin synthase) through 3gt (udp - glucose : flavonoid 3-o - glycosyl transferase). chs is the first committed enzyme of flavonoid biosynthesis. as is the first committed enzyme of anthocyanin biosynthesis. the expressions of the above enzymes are regulated differently by environments such as light and temperature. this results in the disproportional increase of different anthocyanins such as peonidin 3-glucoside compared to cyanidin 3-glucoside, due to the different aglycones although same sugar ; or cyanidin 3-galactoside compared to cyanidin-3-glucoside, due to the different sugars although same aglycon. this study demonstrates that during water - harvesting conditions, where the berries are no longer attached to the plant, exposure of the berries to light still results in increased anthocyanin levels. this study also shows that levels of individual anthocyanins increase differently following different light exposure such as natural light, red light, and far - red light. the variation in composition of anthocyanin this study also contributes to our understanding of cranberry anthocyanin biosynthesis under water - harvesting conditions. | anthocyanins are a group of plant antioxidants known for their therapeutic use. the effects of natural light, red light, and far - red light on individual as well as total anthocyanin content in cranberry fruit (vaccinium macrocarpon ait) were examined in an experimental setting designed to mimic water - harvesting conditions. the reversed - phase high - performance liquid chromatography (hplc) method was used to separate and analyze the anthocyanins. in contrast to the case of the control sample that was kept in the dark, natural light increased the total anthocyanin level by 75.3% and 87.2% after 24 and 48 hours of water immersion, respectively. red light and far - red light increased the total anthocyanin level by 41.5% and 34.7%, respectively. the amount of each individual anthocyanin increased differently under natural light, red light, and far - red light, suggesting that expressions of enzymes that catalyze the anthocyanin biosynthesis are regulated differently by environments. |
soft tissue infection often occurs after injuries, which, if not treated appropriately, could slow wound healing and further deteriorate the patient s condition [13 ]. thus, prevention and treatment of infection remains a major challenge for clinical surgeons. vacuum sealing drainage (vsd) has been increasingly recognized as a valid treatment approach in the management of various types of tissue injuries. it facilitates complete suction of seepage, necrotic tissues, and pus from the wound area by using a negative - pressure device. emerging studies have demonstrated that vsd treatment can speed wound healing and reduce incidence of infection [68 ] recent evidence indicates that if antibiotic is added to bone cement, infected joint arthroplasties can be prevented and treated. it has been suggested that prophylactic use of antibiotic - impregnated bone cement decreases the deep infection rate in total joint arthroplasty. these findings suggest that antibiotic - impregnated bone cement may be a promising therapy for prevention of soft tissue infections. to the best of our knowledge, the effect of antibiotic - impregnated bone cement on soft tissue defects and infection has not been reported before. we conducted a prospective randomized study to compare the combination of vsd therapy and antibiotic - impregnated bone cement versus the single vsd therapy for the therapeutic effect in patients with soft tissue defects and infection. to the best of our knowledge, this is the first study to test whether open flesh wounds benefit from the combined treatment of vsd therapy and antibiotic - impregnated bone cement. the treatment appeared to speed wound healing and prevent infection. the study might shed new light on clinical treatment of soft tissue defects and infection. this prospective randomized non - blinded study enrolled 46 cases with soft tissue defects and infection in our hospital between january 2010 and may 2014. the study included 24 males and 22 females, who were aged 3572 years with a mean age of 53 years. their wound causes were : diabetic foot in 16 cases, postoperative infection following fracture internal fixation in 6 cases, sacral decubitus in 10 cases, and post - traumatic limb infection in 14 cases. these patients were randomly divided into 2 groups by random numbers generated by computer : the experimental group was treated with vsd combined with antibiotic bone cement, while the control group received vsd treatment only. in order to avoid bias due to different injury causes, the cases with the same injury cause and similar size of tissue defect were randomly divided into 2 groups. therefore, both groups (n=23) included 8 cases with diabetic foot, 3 cases with postoperative infection following fracture internal fixation, 5 cases with sacral decubitus, and 7 cases with post - traumatic limb infection. bacterial culture results were pseudomonas aeruginosa - positive in 13 cases (sensitive to cefoperazone plus sulbactam), escherichia coli - positive in 6 cases (sensitive to gentamicin), and staphylococcus aureus - positive in 27 cases (sensitive to vancomycin) (table 1). the following antibiotic - laden polymethyl - methacrylate bone cements (heraeus, beijing landmover medical company, beijing, china.) were used in the study based on our previous experiences : vancomycin bone cement (15% vancomycin), cefoperazone bone cement (10% cefoperazone), and gentamicin bone cement (1.6% gentamicin). other materials used in our study were : vsd sponge, seaweed salt hydration alcohol polyethylene foam with pore diameter of 0.21.0 mm, and built - in multi - side holes and drainage pipes were purchased from beijing hongren ningrui technological company (beijing, china) ; semipermeable membranes comprised of polyurethane and acrylic acid were provided by beijing hongren ningrui technological company (beijing, china). after thorough debridement, the wound was veiled by the tailored vsd sponge sutured with surrounding natural skin in the control group. in case of a deep wound, vsd dressing was used to fill the wound cavity, leaving no dead space. it was appropriate that the vsd dressing was attached within 2 cm of the drainage tube fringe. when treating large wounds, drainage tubes in cascading series were required. then, the skin around the wound was cleaned with 75% alcohol and a semipermeable membrane was used to seal the wound and the vsd dressing. all the drainage tubes were connected to a 3-way stopcock connected to a negative - pressure device (beijing hongren ningrui technological company, beijing, china). presence of sunken vsd dressing and absence of fluid accumulation under the membrane was a reflection of effective negative pressure status. prior to the vsd treatment, prepared pillar- or pie - shaped antibiotic - laden bone cement was placed in the bone or soft tissue cavities in the experimental group. in case of large cavities, bone cement beads were used. the vsd treatment was performed again until a clean, red, granulating wound bed was achieved (ready for surgery). if much scar tissue remained and suturing was not suitable, free flap transplantation or skin grafting was chosen. the following variables of the experimental and control groups were compared to evaluate the outcome of different treatment measures : length of hospital stay, the time needed until the wound was ready for surgery, speed of wound closure, duration of antibiotic administration, and the number of vsd dressing renewals. student s t - test was used to compare the difference of each variable between the 2 groups. this prospective randomized non - blinded study enrolled 46 cases with soft tissue defects and infection in our hospital between january 2010 and may 2014. the study included 24 males and 22 females, who were aged 3572 years with a mean age of 53 years. their wound causes were : diabetic foot in 16 cases, postoperative infection following fracture internal fixation in 6 cases, sacral decubitus in 10 cases, and post - traumatic limb infection in 14 cases. these patients were randomly divided into 2 groups by random numbers generated by computer : the experimental group was treated with vsd combined with antibiotic bone cement, while the control group received vsd treatment only. in order to avoid bias due to different injury causes, the cases with the same injury cause and similar size of tissue defect were randomly divided into 2 groups. therefore, both groups (n=23) included 8 cases with diabetic foot, 3 cases with postoperative infection following fracture internal fixation, 5 cases with sacral decubitus, and 7 cases with post - traumatic limb infection. bacterial culture results were pseudomonas aeruginosa - positive in 13 cases (sensitive to cefoperazone plus sulbactam), escherichia coli - positive in 6 cases (sensitive to gentamicin), and staphylococcus aureus - positive in 27 cases (sensitive to vancomycin) (table 1). the following antibiotic - laden polymethyl - methacrylate bone cements (heraeus, beijing landmover medical company, beijing, china.) were used in the study based on our previous experiences : vancomycin bone cement (15% vancomycin), cefoperazone bone cement (10% cefoperazone), and gentamicin bone cement (1.6% gentamicin). other materials used in our study were : vsd sponge, seaweed salt hydration alcohol polyethylene foam with pore diameter of 0.21.0 mm, and built - in multi - side holes and drainage pipes were purchased from beijing hongren ningrui technological company (beijing, china) ; semipermeable membranes comprised of polyurethane and acrylic acid were provided by beijing hongren ningrui technological company (beijing, china). the vsd dressing was tailored according to the size of the wound. after thorough debridement, the wound was veiled by the tailored vsd sponge sutured with surrounding natural skin in the control group. in case of a deep wound, it was appropriate that the vsd dressing was attached within 2 cm of the drainage tube fringe. when treating large wounds, drainage tubes in cascading series were required. then, the skin around the wound was cleaned with 75% alcohol and a semipermeable membrane was used to seal the wound and the vsd dressing. all the drainage tubes were connected to a 3-way stopcock connected to a negative - pressure device (beijing hongren ningrui technological company, beijing, china). presence of sunken vsd dressing and absence of fluid accumulation under the membrane was a reflection of effective negative pressure status. prior to the vsd treatment, prepared pillar- or pie - shaped antibiotic - laden bone cement was placed in the bone or soft tissue cavities in the experimental group. in case of large cavities, bone cement beads were used. the vsd treatment was performed again until a clean, red, granulating wound bed was achieved (ready for surgery). if much scar tissue remained and suturing was not suitable, free flap transplantation or skin grafting was chosen. the following variables of the experimental and control groups were compared to evaluate the outcome of different treatment measures : length of hospital stay, the time needed until the wound was ready for surgery, speed of wound closure, duration of antibiotic administration, and the number of vsd dressing renewals. student s t - test was used to compare the difference of each variable between the 2 groups. there was no significant difference between the 2 groups in gender, age, average area of tissue defect before treatment, or the delay between infection and treatment (p>0.05). in the experimental group, the wound was healed in 23 cases within 4 weeks postoperatively, of which direct suture was performed in 12 cases, and additional free flap transplantation or skin grafting was performed in 6 cases and 5 cases, respectively. notably, vsd treatment was performed only once in 6 cases in the experimental group and the drainage tube remained unobstructed for 14 days. function of the injured limbs of the patients and their vocational abilities recovered well (typical cases : figures 1, 2). for the control group (n=23), the wound was healed in 15 cases within 6 weeks postoperatively. among the 15 cases, the wound was directly sutured in 8 cases, and additional free flap transplantation or skin grafting was performed in 3 cases and 4 cases, respectively. the wound was ruptured in 3 cases within 2 months following vsd treatment, and these were re - admitted to the hospital. the experimental group was compared with the control group for the number of vsd dressing renewals (times), the time needed until the wound was ready for surgery (days), the speed of wound closure (cm / week), the duration of antibiotic administration (days), and the length of hospital stay (days) (table 3). statistical analyses revealed that the experimental group had significantly fewer vsd renewals, shorter time needed until the wound was ready for surgery, shorter duration of antibiotic administration, faster speed of wound closure, and shorter hospital stay compared with the control group (p0.05). in the experimental group, the wound was healed in 23 cases within 4 weeks postoperatively, of which direct suture was performed in 12 cases, and additional free flap transplantation or skin grafting was performed in 6 cases and 5 cases, respectively. notably, vsd treatment was performed only once in 6 cases in the experimental group and the drainage tube remained unobstructed for 14 days. function of the injured limbs of the patients and their vocational abilities recovered well (typical cases : figures 1, 2). for the control group (n=23), the wound was healed in 15 cases within 6 weeks postoperatively. among the 15 cases, the wound was directly sutured in 8 cases, and additional free flap transplantation or skin grafting was performed in 3 cases and 4 cases, respectively. the wound was ruptured in 3 cases within 2 months following vsd treatment, and these were re - admitted to the hospital. the experimental group was compared with the control group for the number of vsd dressing renewals (times), the time needed until the wound was ready for surgery (days), the speed of wound closure (cm / week), the duration of antibiotic administration (days), and the length of hospital stay (days) (table 3). statistical analyses revealed that the experimental group had significantly fewer vsd renewals, shorter time needed until the wound was ready for surgery, shorter duration of antibiotic administration, faster speed of wound closure, and shorter hospital stay compared with the control group (p<0.01). this study compared the combination of vsd and antibiotic - impregnated bone cement with vsd only in management of soft tissue defects and infection. to the best of our knowledge, this is the first study to evaluate the impact of the combined treatment on soft tissue defects and infection. our results showed that 23 cases in the experimental group were healed within 4 weeks postoperatively, and no infection recurrence was reported in 1-year follow - up. in contrast, only 15 of the 23 cases in the control group were healed within 6 weeks postoperatively and infection recurrence was reported in 3 cases in the follow - up. we found that the combined treatment might be useful for accelerating wound healing and preventing infection recurrence in patients with soft tissue defects and infection. vsd is a negative - pressure technique that drains out seepage, pus and necrotic tissues. it has been widely used for treatment of skin defects, soft tissue defects, and complicated wounds [1517 ]. however, the vsd sponge could not kill microorganisms by itself. in case of severe infection, large amounts of pus might be produced, which might clog the pores of the vsd and inhibit its normal functioning. polymethyl methacrylate bone cement has been widely recognized as one of most stable antibiotic carriers. it aids in maintaining long - term and high concentrations of antibiotics for infected wounds. topical antibiotics have been increasingly used for prevention and treatment of various types of infections. it has been suggested that perioperative topical prophylaxis is beneficial for several different surgical procedures. topical use of antibiotics has several potential advantages over systemic administration of antibiotics, such as limiting the possibility of systemic toxicity, sustaining high concentrations of antimicrobials locally, and reducing the potential development of antibiotic resistance. antibiotic - impregnated cement has been revealed to be similar to systemically administered antibiotics, and is independent and additive in combination with other prophylactic measures. in the present study, the combined use of antibiotic - impregnated cement and vsd achieved better outcome than the vsd treatment only, as evidenced by significantly fewer vsd dress renewals, shorter time needed until the wound was ready for surgery, short duration of antibiotic administration, faster wound healing, shorter hospital stay, and lower incidence of infection recurrence in the experimental group. the better outcome may be explained by strong postoperative local resistance of the antibiotic - impregnated cement to infection because of elution of antibiotics. notably, the vsd dressing was used only once in 6 cases of the experimental group, and the drainage pipeline remained unobstructed for 14 days. one possible explanation for this is that topical antimicrobials from the antibiotic - impregnated bone cement might kill microorganisms or inhibit their growth, thereby leading to reduced production of secretions. it reveals that use of antibiotic - laden bone cement might a complementary measure to vsd in the prevention and treatment of soft issue defects and infection. moreover, a sealed, effective and negative pressure status, and unobstructed pipelines are required during the surgery. topical application of antibiotics might cause adverse effects such as local hypersensitivity, contact dermatitis reactions, and possible disturbance of the wound - healing process. there are also concerns with regard to higher cost of antibiotic - laden bone cement than plain bone cement and possible development of antibiotics resistance [2832 ]. unlike systemic antibiotics, rigorous trials to accurately determine the dose of topical antibiotics are not available, thus the dose of topical antibiotics is difficult to determine. in this study, based on the results of bacterial culture and drug sensitivity test, vancomycin bone cement (15%), cefoperazone bone cement (10%), and gentamicin bone cement (1.6%) were used. the ratios of antibiotics in bone cement were determined based on previous clinical experiences of our surgeons in the present study. this is a preliminary study with some limitations. the small sample size and short - term follow - up limit the power of the study. more studies are required to address these concerns and to validate the findings regarding the 2 treatments. compared to application of vsd only, combination of vsd and antibiotic - laden bone cement accelerated wound healing, shortened hospital stay, and decreased infection recurrence in the management of soft tissue defects and infection. this combined therapy might be a better approach to speed wound healing and prevent infection. | backgroundthis study aimed to evaluate the combined effect of vacuum sealing drainage (vsd) and antibiotic - loaded bone cement on soft tissue defects and infection.material/methodsthis prospective non - blinded study recruited 46 patients with soft tissue defects and infection from january 2010 to may 2014 and randomly divided them into experimental and control groups (n=23). patients in the experimental group were treated with vsd and antibiotic - loaded bone cement, while the patients in the control group were treated with vsd only.resultsin the experimental group, the wound was healed in 23 cases at 4 weeks postoperatively, of which direct suture was performed in 12 cases, and additional free flap transplantation or skin grafting was performed in 6 cases and 5 cases, respectively. no infection reoccurred in 1-year follow - up. in the control group, the wound was healed in 15 cases at 6 weeks postoperatively, of which direct suture was performed in 8 cases, and additional free flap transplantation or skin grafting was performed in 3 cases and 4 cases, respectively. in the other 8 cases the wound was healed at 8 weeks postoperatively. infection reoccurred in 3 cases during the follow - up. the experimental group had significantly fewer vsd dressing renewals, shorter time needed until the wound was ready for surgery, shorter duration of antibiotic administration, faster wound healing, and shorter hospital stay than the control group (p<0.01).conclusionsthe combination of vsd and antibiotic bone cement might be a better method for treatment of soft tissue defects and infection. |
computed tomography (ct) and magnetic resonance imaging (mri) are excellent modalities for the localization of mediastinal masses and there are often features that may allow the correct diagnosis to be made. however, ct and mr can not usually assess the aggressiveness of masses or identify viable tumour in residual masses after chemotherapy. metabolic imaging using [f]fluorodeoxyglucose (fdg)-positron emission tomography (pet)/ct, although not required in many cases, may be helpful for further characterization of masses and to guide the most appropriate site for biopsy. the thymus arises from the 3rd branchial pouch on each side, which fuse and migrate from the pharynx to the anterior mediastinum. a normal thymus may be seen anywhere along this path, and is usually seen anterior to the ascending arch. the thymus reaches its maximum weight at puberty and then undergoes fatty replacement with residual thymus identified in only 5% of people aged 40 years. ct identifies the normal thymus in virtually all children under 13 years of age and in most people under the age of 30 years. physiologic uptake of fdg is seen in the thymus in 28% of normal people aged less than 40 years, with the incidence dependent on the age of the patient, being identified in up to 73% of children under 13 years, decreasing to 12% of 2040 year olds and up to 8% of people in their fourth decade. there does appear to be a correlation between the degree of uptake and the attenuation of the gland suggesting that the disappearance of thymic uptake is related to fatty infiltration of the gland. in children, the thymus is quadrilateral in shape initially and then becomes arrowhead shaped or bilobed and this is the pattern of uptake seen on fdg - pet / ct in 60% of cases. unilateral extension to the right has been described in 25% and focal uptake in the midline in 16% of cases. changes in thymic size occur in response to sepsis, congenital heart disease, use of steroids and following chemotherapy and radiotherapy with re - growth occurring after recovery or following the termination of chemotherapy or steroids.. 1) and is thought to be a rebound phenomenon with the thymus infiltrated by lymph follicles with large nuclei and plasma cells. it is important to differentiate thymic hyperplasia from ongoing or recurrent disease, especially in patients with lymphoma. the uptake in the normal thymus is variable with reported standardized uptake values (suvs) ranging from 1.8 to 3.6 ; values greater than 4 are of concern. however, in a recent study 44% of patients with thymic hyperplasia following chemotherapy had an suvmax of greater than 4. they usually occur in adults between 45 and 60 years of age and are often found incidentally and symptoms occur late. in 3050% thymoma is also associated with autoimmune disorders such as systemic lupus, ulcerative colitis and hypogammaglobulinaemia. in 20% they may be classified as non - invasive when they are encapsulated, or invasive (1537%) if there is extension beyond the capsule with spread along pleural and pericardial surfaces. they can be staged using the masoaka classification (table 1). however, in 1999 the world health organization proposed a histological classification, revised in 2004, which is widely accepted. in this classification thymomas are classified as a, ab, b1, b2 or b3 based on morphology and atypia of cells. stratified thymic tumours into 3 subgroups based on their prognosis with type a, ab and b1 low - risk thymomas, types b2 and b3 high - risk thymomas and thymic carcinoma. table 1masaoka clinical staging system.stage5-year survival (%) 1encapsulated tumour with no gross or microscopic invasion961002macroscopic invasion into the mediastinal fat or pleura86953invasion into pericardium, great vessels or lung56694apleural or pericardial metastatic spreadup to 504blymphatic or haematogenous spread masaoka clinical staging system. thymic tumours are diagnosed with ct or mri, and both modalities can define the anatomic extent of tumour but can not always differentiate the subtypes. most thymomas show mild uniform enhancement, with low - grade thymomas usually well defined and surrounded by clear fat planes and calcification, haemorrhage or necrosis may occur. in 34% thymic carcinomas may appear similar to thymomas but are aggressive and tend to metastasize to the lungs, liver, nodes and bone. the prognosis for low - risk (a, ab and b1) thymomas is very good following complete resection with a 100% 20-year survival. b3 thymomas have a complete resection rate of 92% with a 29% recurrence rate and a 36% 20-year survival. in thymic carcinoma complete resection is often not possible and debulking with adjuvant chemo-/radiotherapy is undertaken but the recurrence rate is high (3677%). fdg - pet reflects glucose metabolism and may indicate tumour malignancy and therefore may be helpful in differentiation of thymoma types based on the suv. sung. found that although there was a significant difference in uptake between the low - risk a, ab, b1 subtypes (suvmax 4) and thymic carcinoma (suvmax 10), there was no significant difference in uptake between low - risk and high - risk b2 and b3 subgroups (suvmax 5.6). these authors noted that the uptake was homogeneous in 75% of thymic cancers and in 22% of high - risk tumours but not in low - risk tumours. luzzi. in a small study found the suv could differentiate low - risk (a, ab, b1) from high - risk (b2, b3 and carcinoma) tumours and that the suv in low - risk tumours was significantly less than that found in thymic lymphoma. using a tumour / mediastinum ratio, endo. could differentiate the 3 groups and these authors suggested the degree of uptake in thymomas was related to the degree of atypia rather than the lymphocyte infiltration. found that the mean suv was helpful in distinguishing thymic hyperplasia (suv 1.890.58) from thymomas (suv 4.750.88). lee. in a study of pet in the management of thymic tumours found that management was changed appropriately in only 7% of patients, with pet not influencing management in 88%. the effect of pet appears to be much less than in other tumours and this may be partly related to the comparative rarity of distant metastases with thymic tumours and also the variable, rather low uptake within thymic tumours making assessment of small volume disease difficult. in this study the negative predictive value (npv) was 64.3%, which the authors suggested may limit the usefulness of pet in the follow - up of residual masses although the high positive predictive value (ppv) may allow for modification of management in a small group of patients. ct and mri are used to determine the site and extent of neurogenic tumours and their relationship to the spinal canal. in patients with neurofibromatosis type 1 (nf1), ct and mri most patients have multiple neurofibromas but 30% also develop plexiform neurofibroma, which although composed of the same cell type as dermal neurofibromas grow along nerve roots and extend into surrounding structures. in 10% the plexiform neurofibromas undergo malignant transformation into malignant peripheral nerve sheath tumours (mpnst). the overall survival of patients with mpnst is poor (30% 5-year survival) with a high recurrence rate and metastases to lung, liver, nodes and the retroperitoneum. clinically, malignant degeneration is suggested by increasing pain and deformity, and on imaging malignant transformation is suggested by interval growth or by the development of necrosis, haemorrhage or growth into adjacent structures. biopsy may be difficult with sampling errors and complete surgical resection may not be possible in malignant transformation because of the local extent of the tumour. fdg - pet has been found to be useful in other sarcomas with the suv used for risk assessment and as an independent predictor of survival and disease - free progression. in mpnst, fdg - pet is not only sensitive in the detection of malignant transformation (95% sensitivity, 72% specificity) but also provides prognostic information with tumours with an suv > 3 having a shortened survival time compared with those with an suv <3 (13 months vs 52 months). studied 69 patients with fdg - pet / ct with imaging performed at 90 and 240 min after injection. using an suvmax of 3.5 on delayed imaging for malignancy, these authors reported a sensitivity of 97% with a specificity of 87%. lesions with an suvmax < 2.5 should be considered benign and lesions between 2.5 and 3.5 should be kept under review. these authors also found that the suvmax of mpnsts increased significantly with time unlike benign lesions, and that there was a correlation between suvmax and grade of tumour (fig. 2). figure 2mpnst. non - homogeneous uptake on pet / ct. mediastinal germ cell tumours are usually benign and do not require imaging with fdg - pet. germ cell tumours are classified based on cell type into (a) benign teratomas, (b) seminomas and (c) non - seminomatous germ cell tumours (nsgct). a residual mediastinal mass may remain and fdg - pet has been used to identify residual viable disease. found pet predicted residual viable tumour with a sensitivity of 80% and a specificity of 100% in masses greater than 3 cm, whereas for ct the sensitivity and specificity were 73%. in another multicentre trial these comprise a heterogeneous group of tumours and have a poorer prognosis than mediastinal seminomas (5-year survival 48% compared with 88%). again these tumours show high uptake on fdg - pet, but it is not usually required for staging (fig. residual masses remain in approximately 40% of patients consisting of necrosis in 40%, mature teratoma in 40% and mature teratoma is chemotherapy resistant and surgical resection is required. in a large study of nsgcts with residual masses and histological confirmation the sensitivity and specificity of fdg - pet for viable tumour was 70% and 48% with a ppv of 59%, which was not dissimilar to ct, and fdg - pet / ct appears to offer no advantage over conventional imaging or markers. a residual mediastinal mass may remain and fdg - pet has been used to identify residual viable disease. however, becherer. found pet predicted residual viable tumour with a sensitivity of 80% and a specificity of 100% in masses greater than 3 cm, whereas for ct the sensitivity and specificity were 73%. in another multicentre trial these comprise a heterogeneous group of tumours and have a poorer prognosis than mediastinal seminomas (5-year survival 48% compared with 88%). again these tumours show high uptake on fdg - pet, but it is not usually required for staging (fig. residual masses remain in approximately 40% of patients consisting of necrosis in 40%, mature teratoma in 40% and viable tumour in 20%. mature teratoma is chemotherapy resistant and surgical resection is required. in a large study of nsgcts with residual masses and histological confirmation the sensitivity and specificity of fdg - pet for viable tumour was 70% and 48% with a ppv of 59%, which was not dissimilar to ct, and fdg - pet / ct appears to offer no advantage over conventional imaging or markers. fdg - pet / ct is not routinely used in mediastinal masses but is of value in assessing high - risk thymoma and thymic carcinomas. | abstractcomputed tomography (ct) and magnetic resonance imaging (mri) are excellent modalities for the localization of mediastinal masses and there are often features that may allow the correct diagnosis to be made. however, ct and mri can not usually assess the aggressiveness of masses or identify viable tumour in residual masses after chemotherapy. metabolic imaging using [18f]fluorodeoxyglucose (fdg)-positron emission tomography / ct, although not required in many cases, may be helpful for further characterization of masses and to guide the most appropriate site for biopsy. |
the term is a misnomer as the lesion does not contain pus nor it is granulomatous. it is considered as a capillary haemangioma of lobular subtype as suggested by mills, cooper, and fechner, which is the reason they are often quite prone to bleeding. the most common intraoral site is marginal gingiva, but lesions have been reported on palate, buccal mucosa, tongue, and lips. extraoral sites commonly involve the skin of face, neck, upper and lower extremities, and mucous membrane of nose and eyelids. being a non - neoplastic growth, excisional therapy is the treatment of choice but some alternative approaches such as cryosurgery, excision by nd : yag laser, flash lamp pulsed dye laser, injection of corticosteroid or ethanol, and sodium tetradecyl sulfate sclerotherapy have been reported to be effective. there are only anecdotal reports of successful treatment of mucosal pyogenic granulomas with diode laser. in this report, we seek to highlight the therapeutic success achieved with diode laser in the treatment of intraoral pyogenic granuloma. a 50-year - old female patient reported to the out patient department of oral medicine and radiology, with a complain of localized gingival growth for 3 months. the outgrowing mass was not painful but often bled while eating, rinsing, or sometimes spontaneously. patient reported a similar growth in the gingiva about 3 years ago, which was treated in the same institute by surgical excision. intraoral examination revealed an oval - shaped, pedunculated mass - like growth seen in relation to the buccal aspect of gingiva with respect to 23, 24 region, measuring approximately 2.5 3.5 cm. this discrete nodular, erythematous enlargement was covering almost two - third of the crown of 24 [figure 1 ]. on palpation, the mass was soft to firm in consistency and readily bled on probing. pyogenic granuloma. based on the clinical findings and the previous biopsy report, the case was provisionally diagnosed as recurrent pyogenic granuloma. a discrete nodular, erythematous enlargement covering almost two - third of the crown of 24 no bony abnormalities were seen on intraoral periapical radiographic examination. the lesion was treated by soft - tissue diode laser manufactured by picasso (kavo, usa), with following specifications : wavelength 808 nm (10), output energy 0.17.0 w, and input power 300 va. we used 810-nm wavelength and 7 w power, keeping it in continuous / interrupted pulse mode. the tip was kept at a distance of about 1 mm from the soft tissue throughout the procedure, and it took 45 min to completely excise the mass. the diode laser provided an optimum combination of clean cutting of the tissue and haemostasis [figures 2 and 3 ]. she was not prescribed any antibiotics, analgesics, or anti - inflammatory medication and was subjected to routine scaling and curettage. she revisited after 5 days for follow - up visit, but failed to keep further appointments. diode laser (picasso - kavo) tip was kept almost at a distance of 1 mm from the soft tissue. also, there was minimal bleeding during the procedure the excised mass [figure 4 ] was sent for histopathologic evaluation which showed hyperplastic stratified squamous parakeratotic epithelium with an underlying fibrovascular stroma. the stroma consisted of a large number of budding and dilated capillaries, plump fibroblasts and areas of extravasated blood, and a dense chronic inflammatory cell infiltrate. although pyogenic granuloma may appear at any age, 60% cases are observed between the ages of 10 and 40 ; incidence peaks during the third decade of life and women are twice as likely to be affected. the clinical presentation is generally of a dull red, sessile, or pedunculated smooth surfaced nodule that may easily bleed, crust, or ulcerate.. they may typically begin as small, red papules that rapidly enlarge to become pedunculated raspberry - like nodules. rarely, patient may develop multiple satellite angiomatous lesions after excision of a solitary pyogenic granuloma. oral pyogenic granulomas show a striking predilection for the gingiva as seen in the present case, which accounts for 75% of all cases. gingival irritation and inflammation that result from poor oral hygiene may be a precipitating factor in many patients. the lip, tongue, and buccal mucosa are the next most common sites. in majority of cases, minor trauma and/or chronic irritation recently, angiopoietin-1,2 and ephrin b2 agents in other vascular tumours such as bartonella henselae, b. quintana, and human herpes virus 8 have been postulated to play a part in recurrent pyogenic granuloma. multiple pyogenic granulomas with satellite lesions may occur as a complication of tumour removal or trauma. viral oncogenes, hormonal influences, microscopic arteriovenous malformation along with inclusion bodies and gene depression in fibroblasts have all been implicated. differential diagnosis of pyogenic granuloma includes haemangioma, peripheral giant cell granuloma, peripheral ossifying fibroma and metastatic carcinoma, and amelanotic melanoma. although the conventional treatment for pyogenic granuloma is surgical excision, a recurrence rate of 16% has been reported. other methods used by various workers include cauterization with silver nitrate, sclerotherapy with sodium tetra decyl sulfate and monoethanolamineoleate ligation, absolute ethanol injection dye, laser therapy using continuous and pulsed co2 and nd : yag systems have been used for a variety of intraoral soft tissue lesions such as haemangioma, lymphangioma, squamous papilloma, lichen planus, focal melanosis, and pyogenic granuloma, because they carry the advantage of being less invasive and sutureless procedures that produce only minimal postoperative pain. rapid healing can be observed within a few days of treatment, and as blood vessels are sealed, there are both a reduced need for post - surgical dressings and improved haemostasis and coagulation. it also depolarizes nerves, thus reducing post - operative pain, and also destroys many bacteria and viral colonies that may potentially cause infection. reduced post - operative discomfort, oedema, scarring, and shrinkage have all been associated with its use. white proposed that laser excision is well tolerated by patients with no adverse effects. they also stated that co2 and nd : yag laser irradiation is successful in surgical treatment. meffert used the flash lamp pulsed dye laser on a mass of granulation tissue and concluded that previously resolute tissue responded well to the series of treatments with pulsed dye laser. diode laser has shown excellent results in cutaneous pyogenic granulomas with only minimal pigmentary and textural complications. gonzales demonstrated both symptomatic and clinical clearing of the lesions with excellent cosmetic results in 16 of 18 treated patients. we achieved complete resolution of this lesion located on the upper gingiva with diode laser without producing any complications. hence, diode laser may be a good therapeutic option for intraoral pyogenic granulomas. in conclusion, the use of laser offers a new tool that can change the way in which existing treatments are performed, or serve to compliment them. modern medicine needs to explore and take advantage of current trends to derive maximum benefit in terms of technology, patient 's acceptance and, post - operative management. | lasers have opened a new door for the treatment of various disorders. treatment of soft tissue intraoral mucosal growth by laser has profound effect on the patient acceptability taking the functional and aesthetic factor into consideration. the patient is able to get the outdoor treatment without the phobia of local anaesthetic and is out of the clinic in few minutes in contrast to the traditional method of surgical excision. very few cases have been reported in literature regarding treatment of mucosal growth by soft tissue lasers. we present a case of recurrent pyogenic granuloma in a patient treated with an alternative approach, that is, diode laser, without the use of anaesthesia, sutures, anti - inflammatory drugs, or analgesics. the diagnosis of this lesion is equally important for correct treatment planning. |
pd-1 was shown to deliver a negative signal after binding to either of its two ligands, pd - l1 (b7-h1) or pd - l2 (b7-dc) in immune responses [14 ]. meanwhile, pd-1 and its ligands have been reported to play an important role in the etiopathogenesis of murine autoimmune diseases. spontaneous autoimmune diseases including lupus - like glomerulonephritis, arthritis, and fatal autoimmune dilated cardiomyopathy developed in pd-1 deficient mice of different genetic backgrounds [57 ]. in addition, blockade of pd-1/pd - l pathway could exaggerate or accelerate the development of autoimmune diseases such as diabetes in prediabetic nod mice and experimental autoimmune encephalitis [8, 9 ]. recently, up - regulated expression of pd-1 molecule and/or its ligands was demonstrated in human diseases including rheumatoid arthritis (ra) and inflammatory colitis, indicating that pd-1 and pd-1 ligands were actively participating in the pathogenetic process in human autoimmune diseases [10, 11 ]. systemic lupus erythematosus (sle) is a prototype of human systemic autoimmune disease, characterized by dysregulated activation of both t and b lymphocytes and development of many autoantibodies. accordingly, pd-1 and pd-1 ligands may play a critical role in the pathogenesis of sle. the present study thus aimed to investigate the expression and function of pd-1 and its two ligands on peripheral mononuclear cells (pbmcs) in patients with sle. twenty - eight patients who fulfilled at least 4 criteria for the diagnosis of sle set by the american college of rheumatology were enrolled in the study. all enrolled patients were not taking corticosteroid and immunosuppressant agents for more than 4 weeks prior to the study. their peripheral blood was drawn, and pbmc were immediately isolated for flowcytometric analysis and in vitro culture. flowcytometry with dual staining was performed using fluorescece - conjugatd mabs including fitc - conjugated anti - cd3, anti - cd19, and anti - cd14 (bd biosciences, sna diego ca, usa) and pe - conjugated anti - pd-1, anti - pdl1, and anti - pdl2 (mih clones, e bioscience, sna diego, ca, usa). briefly, pbmc were prepared from fresh heparinized blood using ficoll - hypaque density gradient separation and adjusted to 5 10 cells / ml. two hundred microliters of cell suspension were incubated simultaneously with 20 l fitc - conjugated mab and 20 l pe - conjugated mab in different combinations on ice in the dark for 30 minutes. cells incubated with fitc- and pe - conjugated mouse igg were used as isotype controls. the stained cells were analyzed with a facscalibur (becton dickinson) flow cytometer and associated software programs (cellquest). in selected sle patients and normal individuals, their pbmcs (300 l) at the concentration of 1 10 cells / ml were cultured in 96-well microtiter plates under the stimulation with pma (5 ng / ml) plus ionomycin (1 g / ml). for blocking pd-1/pd-1 ligands interaction, we added mab against pd-1 (mih4 clone) or pd - l1 (mih1 clone) or pd - l2 (mih18 clone) in the cultured cell system. their endotoxin levels were all less than 0.001 ng/g antibody as determined by the lal assay. the different groups include addition of anti - pd-1 mab (1 g / well), anti - pd - l1 mab (1 g / well), anti - pd - l2 mab (0.5 g / well), or anti - pd - l1 plus anti - pd - l2. after 48 hours ' culture, the supernatants were collected, and cytokines including ifn-, il-2, il-4, and il-10 were assayed by elisa kits. the concentrations of different cytokines in cultured supernatants were measured by commercially available elisa kits (bender medsystems, vienna, austria). the assay employed the quantitative sandwich enzyme immunoassay technique and was performed as manufacturer 's instruction. standard or samples were added to react with specific anticytokine mab precoated onto a microplate for 2 hours at room temperature. after washing, horseradish peroxidase - conjugated polyclonal antibodies against cytokines were added to react with the bound cytokines for 1 hour. then, substrate solution was added and incubated for 20 minutes, followed by addition of stop solution. the optical density of each well was immediately determined by microplate reader set to wavelength 450 nm. fisher 's lsd method was applied to compare the levels of pd-1 and pd-1 ligands - expressing cells between sle and normal controls. wilcoxon rank sum test was used to compare the increase percentages of cytokine production between patients with sle and normal subjects. the expression of pd-1 and its two ligands on freshly prepared pbmc from 26 normal controls and 28 patients with sle were shown in table 1. one representative facs dot - plot data of pd-1 and pd-1 ligands on t cells, b cells, and monocytes was depicted in figure 1. in normal controls, pd-1 was barely detectable on t cells (< 1%). only a very minority of b cells (mean 2.14%) and monocytes (mean 5.78%) expressed a low level of pd-1. in contrast, pd - l1 expression was readily detected on t cells, b cells, and monocytes. different to pd - l1, pd - l2 expression was mainly noted on monocytes (mean 72.12%) with only a minority of t cells and b cells (mean < 4%) expressed pd - l2. similar expression pattern of pd-1 and pd-1 ligands on each mononuclear cell subset was also observed in patients with sle. compared with normal controls, patients with sle had significantly increased percentages of pd-1-expressing cd3 t cells (1.51 1.12% versus 0.64 0.53%, p <.001) and pd-1-expressing cd19b cells (5.11 3.91% versus 2.14 1.67%, p <.005). regarding pd-1 ligands, sle patients had more pd - l1-expressing cd19b cells (20.59 10.24% versus 13.21 1.67%, p <.005) and pd - l2-expressing cd14monocytes (84.78 12.82% versus 72.12 26.92%, p <.005) than normal controls. despite slightly increased frequencies of pd-1 and pd-1 ligands expression in some cell populations from sle patients, the mean fluorescence intensities of pd-1 and pd-1 ligands expression on the positive cells were not significantly different between patients with sle and normal controls (data not shown). the result suggests that the expression of pd-1/pd-1 ligands was not impaired in human sle. we thus designed to investigate the functional effects of blocking pd-1 or pd-1 ligands on the production of various cytokines produced by in vitro cultured pbmc. the cytokines examined in the study included th1-derived cytokines (il-2 and ifn-) and th2-derived cytokines (il-4, il-10). in selected patients and normal individuals, 8 in each, their freshly prepared pbmcs were cultured and stimulated with pma plus ionomycin in the absence or presence of anti - pd-1 mab, anti - pd - l1 mab, anti - pd - l2 mab, or anti - pd - l1 plus anti - pd - l2 mabs. we found that blockage of either pd-1 or pd-1 ligands substantially increased the production of il-2, ifn-, and il-10 in all cases of pbmc from both sle and normal individuals but has no any effect on il-4. as expected, the concentration of cytokines produced by stimulated pbmc in vitro varied widely among different sle patients and normal controls. for further comparison between sle and normal subjects, the amplitudes of enhancement in cytokine production were expressed as percentages of increase, which was calculated by the increased amount of cytokine in the presence of blocking ab divided by baseline cytokine concentration in isotype igg control. among the experiments, a few samples of stimulated pbmc had no detectable baseline cytokine. they were omitted in comparison due to difficulty in calculation of percentages of increase. as shown in table 2, blockage of pd-1/pd-1 liagnds pathway greatly increased production of ifn-, which ranged averagely from 134% to 160% in normal controls and from 153% to 203% in sle. similar to ifn-, il-2 levels were much increased, in which the amplitudes of increased percentages ranged averagely from 156% to 243% in normal control and from 189% to 397% in patient with sle (table 3). the enhancing effect on il-10 was readily seen too, which ranged averagely from 97% to 126% in normal controls and from 73% to 132% in sle (table 4). there were also no significant differences in the increased production of il-2 and il-10 between sle patients and normal subjects. in addition, it appeared that anti - pd-1 possessed similar effects as anti - pd - l1 or anti - pd - l2. meanwhile, there were no synergic effects by anti - pd - l1 plus anti - pd - l2 compared with either alone. there was no any effect on il-4 effect by blockage of pd-1/pd-1 ligands pathway (data not shown). the expression of pd-1 and its two ligands on pbmc of normal individuals and patients with sle in the present study was in concordance with previous observation [1315 ]., pd - l1 is constitutively expressed on t cells, b cells macrophages, and dentritic cells (dcs) and further upregulated upon activation. in contrast, the expression of pd - l2 was regulated more tightly and observed on activated macrophage and dcs. the expression of pd - l1 is also detected on a wide variety of nonlymphoid cells including vascular endothelial cells, epithelial cells, muscle cells, hepatocytes, cells in pancreas, astrocytes in the brain, as well as at sites of immune privilege including placenta and eye [1618 ]. the expression of pd - l1 on nonlymphoid tissue was shown to play a crucial role in the control and maintenance of peripheral t cell tolerance. compared with normal controls, generally patients with sle had increased expression of pd-1 and pd-1 ligands on t cells, b cells, and monocytes, some of which reached statistically significant differences, because pd-1 could be induced on t cells after activation, and increased activated t cells have already been well documented in patients with sle. thus the increased numbers of pd-1-expressing cd3 t cells in sle patients could be due to increased numbers of activated t cells in the periphery of sle patients. increased expression of pd-1 and/or pd-1 ligands had also been reported in other human autoimmune diseases. hatachi. demonstrated that pd-1 t cells were enriched in ra synovial fluid, and phenotypic analysis suggested that these cells a unique anergic cell subset. kobayashi. showed that the expression of pd-1 in salivary lymphocytes and pd - l1 on ductal and acinar epithelial cells in salivary glands was enhanced in patients with sjogren 's syndrome. however, decreased pd-1 expression on peripheral cd4 t cells was found in patients with type-1 diabetes in one study. probably, pd-1/pd-1 ligands pathway plays different roles in systemic versus organ - specific autoimmune diseases. in addition, one study in lichen planus, a t cell - mediated chronic inflammatory mucocutaneous disease, revealed abundant expression of pd-1 and pd - l1 in infiltrating t cells and macrophages in the subepithelium and substantial pd - l1 on kerotinocytes. the inhibitory function of pd-1 molecule was initially suggested by the studies on pd-1 deficient mice, which exhibited hyperactivation of the immune system and subsequently developed autoimmune diseases. interestingly, pd-1 deficient mice developed different kinds of autoimmune diseases depending on the genetic background [57 ]. on c57bl/6 background, pd-1 deficient mice developed lupus - like glomerulonephritis and arthritis with deposition of igg, and c3 in the glomeruli, whereas pd-1 deficient mice suffered a fatal dilated cardiomyopathy on balb / c background. the result of our functional study was in concordance with the concept of the immunosuppressive role of pd-1/pd-1 ligands. we showed that blockage of pd-1/pd-1 ligands pathway augmented the production of cytokines in stimulated pbmc in vitro, with predominant effects on th-1 derived cytokines (il-2 and ifn-). regarding th-2 cytokines, we also observed enhancing effect on il-10 though weaker compared with those on il-2 and ifn-. however, there was no effect on il-4 production. blocking pd-1 molecule or either of the two pd-1 ligands demonstrated similar effects on the production of various cytokines. as previously reported, blockage with both anti - pd - l1 and anti - pd - l2 did not show a significantly synergic effect in our study. ansari. reported that blockade of pd-1 or pd - l1 but not pd - l2 exaggerated diabetes in prediabetic nod mice. they observed the expression of pd - l1 but not pd - l2 on the cells, which might explain the reason why the anti - pd - l1 but not anti - pd - l2 antibody exaggerated diabetes, whereas, salama. observed the acceleration of eae by the blockade of pd-1 and pd - l2 but not pd - l1. since pd - l1 but not pd - l2 was highly expressed in the central nervous system, the expression of ligands in target organs might not determine the efficient of each blocking antibody. blockade of pd-1/pd - l pathway might have an opposite effect on autoimmune diseases in certain condition. examined the effect of blocking antibodies against pd - l1 and pd - l2 on the colitis model. antibodies against pd - l1 but not pd - l2 suppressed the development of colitis, indicating the costimulatory function of pd - l1 in the inflammatory response in the gut. according to the report by loke and allison, the expression of pd - l1 and pd - l2 was differentially regulated by th1 and th2 cytokines. since the recent analyses using blocking antibodies against pd - l1 and pd - l2 revealed nonoverlapping functions of these ligands, the differential regulation of pd - l1 and pd - l2 expression might have some biological significance. in conclusion, the present study found that there were no intrinsically defective expression of pd-1 and pd-1 ligands on pbmc in patients with sle. on the contrary, we noted increased expression of pd-1 and/or its ligands in some subsets of pbmc from patients with sle. since pd-1 and pd-1 ligands play a role in regulation of both activation and tolerance of lymphocytes, the findings of our study most likely reflect the activation status of pbmc in lupus patients and they do not rule out a defect in tolerance mediated by pd-1. the exact function of increased pd-1 and pd-1 ligands in some subsets of pbmc in the pathogenesis of human sle needs further study. | programmed death-1 (pd-1) was shown to deliver an inhibitory signal after binding to its ligands, pd - l1 (b7-h1) or pd - l2 (b7-dc). recently, up - regulated expression of pd-1 molecule and/or its ligands was demonstrated in human diseases including rheumatoid arthritis and inflammatory colitis. the study aimed to investigate the expression and function of pd-1 and pd-1 ligands on circulating t cells, b cells and monocytes from patient with systemic lupus erythematosus (sle). the results showed that patients with sle had significantly increased percentages of pd-1-expressing cd3+t cells and cd19+b cells, pd - l1-expressing cd19+b cells and pd - l2-expressing cd14+b monocytes. in selected sle patients and normal subjects, functional study of pd-1/ pd-1 ligands pathway on the production of cytokines by stimulated pbmc was examined. blockages of pd-1 or pd-1 ligands substantially increased the production of il-2, ifn- and il-10, the amplitude of increase roughly ranged from one to three times. there were no significant differences of the enhancing effects on cytokine production by blockage of pd-1/pdl pathway between sle patients and normal subjects. the study indicates that there are no intrinsically defective expression and function of pd-1 and pd-1 ligands on pbmc in patients with sle. |
in the past, mothers and clinicians spent the 9 months of pregnancy in more or less blissful ignorance of what was happening inside the womb. ultrasound was first used in obstetrics in the late 1950s, and it quickly became apparent that it could be used to track an entire pregnancy. there was an upsurge in the use of the technique, and it has now become part of the routine management of pregnancy.6 initially, ultrasound simply provided a basic image of the fetus, allowing clinicians to identify, for example, whether it was in the breach position. visualising a fetus may reveal its gender, whether it is likely to have down syndrome, and whether the umbilical cord is threatening its circulation. ultrasound is often described as offering reassurance to mothers, but this is likely to be the case only if the fetus is deemed normal. it is precisely because ultrasound has the capacity to identify abnormalities that it is used in medicine.7 8 as soon as it becomes feasible to identify anomalies at the prenatal stage, questions are raised about what can and what should be done. the urge to intervene is a natural corollary of the ability to present a problem in a way that preserves its visual immediacy. people feel differently about things they can see from the way they feel about things they merely know.9 10 feeling differently means that one is likely to act differently. a powerful example of this is the ethiopian famine in the 1980s, and the uk 's response to it. but when pictures of the famine were aired, when the suffering of the people who we had previously known about only in the abstract, became visible, there was a sudden upsurge in public response. as we could see, we now felt obliged to act.11 12 ultrasound technology has made the fetus visible and created a corresponding urge to intervene. it is for this reason that it is important to recognise the connection between fetal surgery and ultrasound, but also the more general connection between the acquisition of data and the compulsion to act on that data. so although an imaging technology may in isolation be a non - invasive means of observation, it may become part of a technological process that results in increased interventions. the question then to be asked is whether this systematic process of increased surveillance, diagnosis and intervention is beneficial, and if so, to whom ? the most obvious beneficiaries of fetal surgery are the fetuses that are diagnosed and treated in utero, and i will discuss this shortly. however, it is important to note that fetal surgery may also serve the interests of people other than the fetus. it could be argued that these benefits are vicarious that clinicians and parents benefit only insofar as the fetus does. however, there are additional ways in which parents and clinicians may benefit, which are not simply derived from the effects on the fetus. doyal and ward4 note the advantages that may accrue to clinicians who make a name for themselves in this pioneering area. there is not scope in this paper to explore the ethics of professional self - interest in this context, but doyal and ward4 are certainly right to identify the existence of such interests. benefits to parents and clinicians may also be derived from the fact that surgery offers a course of action doing, rather than simply waiting. as i have suggested, it may be difficult to maintain a non - interventionist attitude to problems that have been identified, especially if they have been made visible. there is evidence to suggest that acting is psychologically easier than simply waiting in a number of contexts. for example, among patients waiting for heart surgery, the waiting itself is a significant cause of psychological distress, independently of any symptom - based factors or specific fears of outcomes being worsened by the passage of time. rather, it is the waiting in and of itself that is psychologically harmful.13 14 of course, it might be argued that patients awaiting surgery are likely to be anxious anyway. the looming prospect of surgery together with the existence of a known medical problem may be stressful. prospective parents, on the other hand, may be apprehensive about what the future holds, but are not necessarily ill themselves. therefore, it might be thought that the analogy does not hold, and that the anxiety experienced by patients waiting for treatment is linked specifically with their underlying medical condition. however, research indicates that waiting for surgery for someone else is also highly psychologically distressing, for example in the case of spouses of patients with heart problems,15 and also in the case of men whose partners are waiting for in - vitro fertilisation treatment.16 the stress of waiting for treatment is not specifically linked to the person who has the illness, but is shared between all those who may be affected by the outcome of the anticipated event. the psychological difficulty of waiting for the birth of a fetus with known medical problems is a source of distress to parents and health professionals too. turning this around, regardless of whether fetal surgery benefits the fetus, it may provide a means of resolving the psychological stress caused by the anxiety of waiting until the birth. need for intervention, but this need in itself is derived from the routine use of the diagnostic technology. that is to say, the use of ultrasound creates new needs that it, in turn, provides the means of fulfilling. if parents and clinicians did not see the images, their need for the psychological comfort of surgical intervention would not arise it is often assumed that medical progress and the development of new medical technologies necessarily serve to reduce the sum of human need, but there is another way of looking at this : technology identifies new problems, which in turn generate new needs that must be met through further developments in medical technology. according to bioethicist daniel callahan,17 this constitutes an ongoing cycle, or arms race. he believes that this endless push towards further technologies and interventions places a strain on resources that may ultimately serve to increase suffering rather than diminish it. i would suggest that callahan17 is right to recognise the role of new technologies in creating new medical needs. fetal surgery is a medical intervention whose function is, at least partly, to relieve the psychological stress that adverse ultrasound findings cause to parents and clinicians. once the technology has been developed, however, it is very hard to suggest that it should be disregarded, or that the individuals who might seek to use it should be prevented from doing so. for this reason, i will not go further into callahan 's argument here. in the next section, i will focus on the ethical management of fetal surgery in the context of individuals who may be directly affected by it. the motivations for fetal surgery that i have discussed above apply to parents and clinicians, but what are the advantages, if any, for the fetus in being operated upon while still in utero ? there are some medical problems that, without surgery, will almost inevitably cause the death of the fetus before, or shortly after, birth. among the conditions that might fall into this category are congenital diaphragmatic hernia, in which the abdominal organs protrude into the chest, impeding lung development.18 teratomas that develop at the base of the coccyx are also highly risky for fetuses, as they can grow so large as to place a strain on the heart. when this has happened, surgery may be the only way of preserving the life of the fetus.19 these benefits come at a cost to the mother. the first at the time of the fetal intervention, the second at the time the baby is born. mothers will need to spend 57 days in hospital.20 any future children will have to be delivered by caesarean. open fetal surgery very frequently involves premature labour and delivery on average babies are born approximately 8 weeks after surgery. mean gestational age at delivery according to one study was 32.5 weeks.20 the risks of endoscopic and percutaneous procedures are lower, but still significant. doyal and ward4 argue that only the most serious conditions can justify fetal surgery. a similar stance the risks imposed on women whose fetuses undergo surgery are thought to be justified only if the life of the fetus is in danger. this might seem to offer a relatively straightforward ethical blueprint for the management of fetal surgery. other conditions non - lethal tumours, for example, or cleft palate could be addressed after the birth of the child without going through the mother, as it were. however, if we are to countenance fetal surgery at all, why should we restrict it to conditions that are life - threatening to the fetus ? women should not have to undergo surgery unless it is absolutely necessary. yet who should be charged with making this judgement as to whether the risks to women are acceptable in balance against the benefits to the fetus ? there are in fact some persuasive arguments in favour of fetal surgery for non - life - threatening conditions. if a problem has been identified by ultrasound, fetal surgery may offer the possibility of remedying the condition there and then without the need for postnatal intervention. giving birth to a healthy child and taking it home straight away women may be eager to secure this benefit for their child even if its life is not at risk. when defects such as cleft palate or teratoma have been diagnosed prenatally, scarless fetal surgery will be a major advantage over postnatal treatment. it is plausible that some patients might want to spare their offspring the risk of disfiguring scars. this raises the question of whether the adult 's willingness to undergo such procedures is sufficient justification to undertake them. it may be difficult to define the circumstances under which fetal surgery can take place without lapsing into paternalism : restricting patients ' options for their own benefit, against their wishes. is it ethically acceptable to impose the risks of surgery on someone who stands to derive no clinical benefit ? surely a large part of the answer depends on the ability to consent. a variation of this question has been explored by paris and harris21 in the context of operating on a twin in utero. therefore, they argue that both become patients. however, the healthy twin 's interests are not served by being exposed to the risks of surgery. for this reason, in fetal surgery, mother and fetus become patients, and their interests might also seem to conflict in a similar way. yet paris and harris21 do not rule out fetal surgery entirely, only in circumstances in which a healthy twin may be put at risk. the difference here is that the mother has the ability to consent to the intervention, whereas the healthy twin does not, but harris and paris'21 recognition of fetuses as patients in their own right seems to raise some problems. if so, women 's autonomy and bodily integrity might be threatened by the development of fetal surgery. doyal and ward4 discuss the risk of coercion in this context, arguing that women 's autonomy should be regarded as paramount, and surgery should only ever be performed with the mother 's informed consent. the legal and moral status of the fetus, in their analysis, is kept within very restricted parameters it may be special, but its interests are explicitly and necessarily subordinate to those of the mother. just as fetal surgery stretches the notion of what constitutes a patient, so the concept of informed consent seems to have become strangely distorted in this context. according to at least one practitioner : there will be very few patients for whom it is an appropriate intervention [] we are going to deal with the unusual patient who can give an informed consent [] these patients are women who are carrying fetuses with life - threatening conditions.22 here, in a strange inversion of what one might normally understand by the term, informed consent is deemed valid not by virtue of the wishes and understanding of the patient, but by virtue of the condition experienced by the fetus. similarly odd ideas about consent and patient choice are implicit in the following quote : the key to choosing appropriate patients for fetal intervention is to identify those that would not survive with postnatal therapy alone.23 here again, it is not the mother who chooses whether to undergo the treatment. rather, the medical establishment chooses her as an acceptable vehicle through which the treatment can be offered. it is no longer clear here just what role informed consent and respect for autonomy are playing in this undoubtedly complex ethical situation. although doyal and ward4 take the mother 's consent to be of fundamental importance, they too make some problematic assumptions. given the high rate of failure for open fetal surgery in its early days, they suggest that the women involved must have been inadequately informed. the implication here is that there are some risks that no one would consent to if fully informed, but this does not harmonise with what is known about decisions related to reproduction and fertility. it has been repeatedly shown that fertility patients are willing to undergo procedures that they know to be highly unlikely to succeed and that are risky and highly expensive. women in this context frequently describe themselves as having had no choice. in an article published in the sunday times, a woman who underwent fetal surgery in the uk says the only option for me was to have the surgery.24 this language of compulsion is not easily assimilated into conventional ethical frameworks centred on autonomy and choice. this does not mean that women have been misinformed, as doyal and ward4 assume. it means that decisions made in such situations are not based on self - protection or risk avoidance. even if the risks of fetal surgery were still greater, the benefits more marginal, and women had access to every possible relevant fact and statistic, they might still be willing to undergo fetal surgery. crucially, pregnant women 's altruism is not necessarily or ordinarily limited to life - saving interventions. most of the sacrifices that pregnant women make are designed give the child the best start possible, rather than save its life per se. there are conditions for which fetal surgery might offer relatively modest benefits (eg, reduced scarring) but a woman may still wish to confer these benefits on her child. our society expects and demands that pregnant women and mothers should be altruistic.25 pregnancy itself is commonly regarded as a state in which what would normally be supererogatory becomes morally required.26 pregnant women often behave in ways that in any other context would be deemed clearly supererogatory perhaps even pathologically so. i have argued elsewhere that the demands placed on mothers and pregnant women are unreasonable and excessive.27 it is because of this that fetal surgery makes us uneasy, i would suggest. expectations of maternal sacrifice in conjunction with new technological means of intervention have led us to an uncomfortable place. the accepted view seems to be that we should erect a variety of barriers that constrain women 's options within parameters that others have deemed reasonable. in short, a reversion to paternalism : it can not be safely left to pregnant women to decide. this perpetuates and reinforces the idea that pregnant women are so extravagantly altruistic that their choices must be restricted in their own interests. in essence, because our ideas of maternal altruism do not fit into an autonomy - based ethics system, we are tempted to exclude pregnant women from their place in that system by a system of paternalistic restrictions. laura purdy28 has demonstrated the level to which paternalistic assumptions infiltrate arguments related to selective reduction another facet of fetal surgery. perhaps this encroachment of paternalism is a subversion of medical ethics on a greater scale than the problem that it is attempting to solve. a better approach might be to look to the social world in which women 's freedom to make choices is formed. it is a mistake here to suppose that non - pregnant women, and men, are perfectly free in their choices and decisions. it is always likely to be imperfect, but if we feel that social pressures are coming to bear too heavily on a particular area of decision - making, there may be an opportunity to reconsider and question that social pressure, rather than hastening towards the refuge of paternalism. it illustrates in the most graphic way the collision of two highly problematical assumptions : first, that ever - greater surveillance of pregnancy is beneficial, or at least not harmful, and second, that pregnant women should sacrifice their bodily integrity for the welfare of their child. | fetal surgery has been practised for some decades now. however, it remains a highly complex area, both medically and ethically. this paper shows how the routine use of ultrasound has been a catalyst for fetal surgery, in creating new needs and new incentives for intervention. some of the needs met by fetal surgery are those of parents and clinicians who experience stress while waiting for the birth of a fetus with known anomalies. the paper suggests that the role of technology and visualisation techniques in creating and meeting such new needs is ethically problematic. it then addresses the idea that fetal surgery should be restricted to interventions that are life - saving for the fetus, arguing that this restriction is unduly paternalistic. fetal surgery poses challenges for an autonomy - based system of ethics. however, it is risky to circumvent these challenges by restricting the choices open to pregnant women, even when these choices appear excessively altruistic. |
metagenomics is defined as the study of genomic content of microbial communities in their natural environments, bypassing the need for isolation and laboratory cultivation of individual species. its importance arises from the fact that over 99% of the species yet to be discovered are resistant to cultivation. this limitation imposed by cultivation of isolated clones has severely skewed our view of microbial diversity. metagenomics promises to enable scientists to study the full diversity of the microbial world, their functions and evolution, in their natural environments. next generation sequencing (ngs) technologies generate data more efficiently, economically, and with a greater depth than ever before. ngs has opened up an array of possibilities for many applications including whole - genome sequencing, epigenetics, and metagenomics. of these, the characterization of diversity of heterogeneous microbial environments, metagenomes, has recently gained significant interest. although a host of methods for whole - genome assembly have been developed, reconstruction of individual clones from metagenomes still remains a challenge. as compared to existing technologies, reads produced by ngs are typically shorter and more error - prone. the growth in the size of the datasets is fast outpacing the computational power needed to analyze it. the computational method we present here aims to quantify the microbial diversity within a metagenome based on a set of deep sequencing reads. in single genome sequencing, we can be certain that all extracted dna fragments belong to the same genome. however, in majority of metagenomic samples, it is not possible to isolate and culture individual clones. it is further complicated by the fact that the data comes from heterogeneous microbial communities, where the number of species as well as their relative abundance is unknown. sequence data is usually partial and fragmentary, as environmental sequence sampling rarely produces all the sequences required for assembly. moreover, metagenomic datasets are beset with increased amounts of polymorphism and horizontal gene transfer. sequences from closely related species will most likely have homologous sequences shared between them, hindering their separation. as a result, the reconstruction of a whole genome is generally not possible. in the light of new data, an additional step in metagenomics that is not required in single genome assembly is binning the reads belonging to different species that is the need to associate the reads with its source organism. clustering methods aim to identify the species present in the sample, classify the reads by their species of origin, and quantify the abundance of each of these species. most of the existing classification methods are supervised and depend on the availability of reference data for training [49 ]. a metagenomic dataset may, however, contain reads from unexplored phyla which can not be labeled into one of the existing classes. poor performance on the short fragments is mostly due to the high dimensionality and sparsity associated with the data. to overcome the limitation imposed by the length however, there is the danger of assembling reads from different species together, thereby creating interspecies chimeras. moreover, the abundances of different species can be potentially skewed such that the within - species variance overwhelms the between - species variance. in this paper, we develop a method for clustering the short metagenome reads that addresses the challenges posed by the nature of metagenomic data. we formulate an unsupervised two - way multispecies, multidimensional mixture model to represent reads from a metagenome. we model the distribution of word counts along a genome as a gaussian for shorter, frequent words and as a poisson for longer words that are rare. we employ either a mixture of gaussians or a mixture of poissons to model reads within each bin. the proposed model is used to cluster metagenomic reads by their species of origin and to characterize the abundance of each species. it is a composition - based method that seeks to distinguish between genomes based on their characteristic dna compositional pattern. our method handles the high - dimensionality and sparsity associated with the data by grouping the set of words comprising the reads, to regularize the parameters in the mixture model. this implies that, for every group, only one statistic of the words in this group is needed to cluster reads. we show that a high clustering accuracy can be obtained at a much lower dimension. we provide a framework that complements existing similarity - based methods. later in the paper, we evaluate the applicability of the multidimensional mixture of distributions and its ability to estimate the parameters of genome abundance accurately, for simulated and real metagenomes. we compare the performance of our method with likelybin and scimm, two other unsupervised composition - based method. also, we demonstrate the robustness of our method to changes in the relative abundance of different species. the last decade has seen an explosion in the number of computational methods developed to analyze metagenomic data. literature abounds in methods for classifying (as opposed to clustering) metagenome reads into taxon - specific bins [5, 7, 8 ]. current approaches to metagenomics clustering can be classified into two main categories : similarity - based and composition - based methods. the similarity - based approaches align the reads to close phylogenetic neighbors and hence depend on the availability of closely related genomes in existing databases [6, 11, 12 ]. megan, a metagenome analysis software system, is a representative example of this kind. it uses sequence homology to assign reads to common ancestors based on best match as given by blast (basic local alignment search tool). as most of the extant databases are highly biased in their representation of true diversity, such methods fail to find homologs for reads derived from novel species. these methods rely on the intrinsic features of the reads such as oligonucleotide distributions [7, 8, 14 ], codon usage preference, and gc composition to differentiate between reads belonging to different species. a significant limitation of most composition - based methods developed so far is that they do not perform well on reads shorter than 500 bp. phylopythia is a supervised composition - based classification method that trains a support vector machine to classify sequences of length greater than 1 kbp. phymm uses interpolated markov models to characterize variable length dna sequences by their phylogenetic group. its accuracy of assignment drops drastically (to just 7.1% at genus level) for short reads and reads from unknown species. nasser. demonstrated that a k - means based fuzzy classifier, trained using a maximal order markov chain, can separate fragments that are about 1 kbp long at the phylum level with a high accuracy. compostbin is a semisupervised algorithm for grouping fragments that uses a novel weighted pca (principal component analysis) and a normalized cut clustering algorithm to classify the sequences. they have demonstrated an error rate bounded by 10%, when guided by information from phylogenetic markers, on datasets of low complexity. however, the accuracy of this method on reads less than 1 kbp has not been shown. recently, chan. developed a semisupervised seeded growing self - organizing map (s - gsom) to cluster metagenomic sequences. it extracts 813 kbp of flanking sequences of highly conserved 16s rrna from the metagenome and uses them as seeds to assign the remaining reads using composition - based clustering. the caveat with soms is that it was shown to work well only on dna fragments that are longer than 8 kbp and lose much accuracy for reads with length below 1 kbp. all the above supervised methods a metagenomic dataset may, however, contain reads from unexplored phyla which can not be labeled into one of the existing classes. the accuracy of these methods on dataset containing reads from unknown species is yet to be demonstrated. likelybin is an unsupervised method that clusters metagenomic sequences via a monte carlo markov chain approach. scimm is a recently developed model - based approach to sequence clustering where interpolated markov models represent clusters and optimization is performed using a variant of the k - means algorithm. in the results section, we compare the accuracy of our proposed method with likelybin and scimm on datasets of different divergences. however, if it is known a priori that the reads differ widely in their abundances, then we recommend using abundance bin over other binning methods. one of the most common genome signatures is the frequency of occurrence of words (or oligomers) in a dna sequence. in our method, we model each cluster, containing reads from a species, as a function of probability distributions of words comprising them. the inherent basis of this method is that the set of reads sequenced from a species have a characteristic genome signature that distinguishes it from reads belonging to other species. the distribution of word counts along a genome can be approximated as a gaussian for shorter, frequent words and as a poisson for longer words that are rare. we propose an unsupervised multidimensional naive bayes poisson mixture model and derive an expectation maximization algorithm for the same. the corresponding algorithm for gaussian mixture model however, with the increase in the length of words, the dimensionality of the data increases exponentially, while the word counts become sparse. to tackle high dimensionality and sparsity of word counts in essence, the proposed method provides a general statistical framework for associating each read with its species of origin, based on its genome signatures. a genome signature is a compositional parameter reflecting the relative abundance of different words along the genome. in general some words that are deemed to be biologically significant are very common in a genome, while others may never be encountered. composition - based methods use genome signatures to ascertain the origin of the dna reads. the underlying basis is that the distribution of words in a dna is specific to each species and undergoes only slight variations along the genome. by establishing the dictionary of words used by a species and their frequency of occurrence literature abounds in methods that study the statistical distribution of the word locations along a sequence and word frequencies [21, 25 ]. the exact distribution of count of words is known under the hypothesis that the letters are independent (bernoulli) or under the markov model. however, in practice, it is extremely time consuming to compute the exact distribution for long sequences or for frequent words. distribution of word counts along a genome can be approximately modeled as a gaussian distribution for short words (that are more frequent), or a poisson distribution for longer words (that are rare). as different words occur with different frequencies along the genome, each word follows its own distribution. thus, reads belonging to a species can be modeled as a multidimensional distribution of words (one dimension for each word) comprising them. figure 1 illustrates the distribution of dimer and pentamer counts across reads sampled from the genome of haemophilus influenzae (for the purpose of clarity, only a few distributions are shown). we see that count of each dimer (a short word) across the reads tends to a gaussian distribution with a different mean and standard deviation and that of a pentamer tends to a poisson distribution. hence, the problem of clustering metagenomic reads can be cast as a multidimensional mixture of gaussians (or poissons for longer words) where distribution of each word is modeled as a gaussian (or poisson). in other words, this corresponds to the multidimensional naive bayes model, where each dimension is modeled as a unimodal gaussian (or poisson) distribution. such a general statistical model takes into account the compositional heterogeneity of words along the genome. in this paper, we formulate an unsupervised multidimensional poisson mixture model for clustering reads within a metagenome by their species of origin. we propose to model the reads from a species as a multidimensional distribution of the words comprising them. therefore, each cluster is represented by the distribution of word counts within the species. mixture models cover the data well, that is, dominant patterns in the data are captured by the component distributions. they allow better approximations of the true distributions, and their parameters are relatively easy to estimate. an additional advantage of using generative models is that they are flexible and can handle a large number of classes. for instance, a mixture of poissons can be multimodal, while a poisson distribution is always unimodal. we begin with a metagenome, x = { x1, x2,, xn }, containing n reads from m species. let m be the proportion of species m in the dataset, with m=1m = 1. we assume that x is observed and is governed by some density function p(x |) with parameter. our goal is to cluster the reads by their species of origin, based on the frequency of words that appear in the reads. for every species m, we want to determine m, its proportion in the dataset, and, the parameter governing the distribution of words within the reads. we assume that yi = m for m 1, m if the ith read belongs to the mth species. we call (x, y) the complete dataset. for a word of length l, we obtain p = 4 different words (combinations of a, c, t, g), denoted by w = { w1, w2,, wp}. each read xi is represented by a p - dimensional feature vector, xi = { xi1, xi2,, xip }, where xij is the count of word wj in read xi. we model the distribution of words within every species m by a multidimensional poisson distribution, say m = { m1, m2,, mp}. that is, given that read xi belongs to species m, the distribution of each word wj is poisson with parameter mj, where m = 1,2,, m and j = 1,2,, p, (1)p(wj mj)=(wj mj)=emjmjxijxij!. we assume independence between features of read vector. the probability of a read xi, given it belongs to species m, is, (2)p(xi yi = m,)=p(xi m)=j=1p(xij mj). at first glance, it might seem imprudent to represent a read as a collection of words comprising it, because it leads to the loss in information about the sequencing read. strictly speaking, even if the sequence of bases in a dna is independently and identically distributed, distribution of word occurrences are not independent, due to overlaps. bayesian networks or belief networks can be used to represent the conditional dependencies between the words comprising the reads. although, in practice, methods for exact inference in bayesian networks are often computationally expensive. an attractive alternative to bayesian networks is the naive bayes algorithm that assumes independence between the different features of the read. naive bayes is known to perform well on complex models and takes time that is linear in the number of components. in addition, lost information can be restored at later stages. in this paper, we have presented the formulation of mixture models with the assumption that the different features (word counts) of the read are independent of each other. to initialize the estimation algorithm, we randomly assign each read to a cluster m. the posterior probability qi, m is set to 1, if read i is assigned to cluster m and 0 otherwise. with the initial posterior probabilities, a maximization step (m - step) expectation stepwe estimate the posterior probability qi, m of read xi belonging to species m. by bayes theorem, we have (3)p(yi = mxi,)=mp(xim)k=1mkp(xik)=qi, m, qi, mmj=1p(xij mj) subject tom=1mqi, m=1. we estimate the posterior probability qi, m of read xi belonging to species m. by bayes theorem, we have (3)p(yi = mxi,)=mp(xim)k=1mkp(xik)=qi, m, qi, mmj=1p(xij mj) subject tom=1mqi, m=1. maximization stepthe m - step uses qi, m to compute the expectation of complete data log likelihood, (4)q((t+1),(t))=ep(y x,)[log p(x, y)]=m=1 m i=1np(yi = m xi,(t)) log(p(xi, yi = m (t+1)))=m=1 m i=1n(qi, mlog(mp(xi m))). we also take into account the constraint, which requires that m 's sum to 1 by adding a lagrange multiplier : (5)q((t+1),(t))=m=1 m i=1n(qi, mlog(mp(xi m))) + (m=1mm1). we maximize the above expression with respect to the parameters, = arg maxq(,), and update the parameters, (6)m(t+1)=i=1nqi, mn, mj(t+1)=i=1nqi, mxiji=1nqi, m. finally, these two steps are repeated as necessary. each iteration is guaranteed to increase the log - likelihood, and the algorithm is guaranteed to converge to a local maximum of the likelihood function. the m - step uses qi, m to compute the expectation of complete data log likelihood, (4)q((t+1),(t))=ep(y x,)[log p(x, y)]=m=1 m i=1np(yi = m xi,(t)) log(p(xi, yi = m (t+1)))=m=1 m i=1n(qi, mlog(mp(xi m))). we also take into account the constraint, which requires that m 's sum to 1 by adding a lagrange multiplier : (5)q((t+1),(t))=m=1 m i=1n(qi, mlog(mp(xi m))) + (m=1mm1). we maximize the above expression with respect to the parameters, = arg maxq(,), and update the parameters, (6)m(t+1)=i=1nqi, mn, mj(t+1)=i=1nqi, mxiji=1nqi, m. finally, these two steps are repeated as necessary. each iteration is guaranteed to increase the log - likelihood, and the algorithm is guaranteed to converge to a local maximum of the likelihood function. higher - order words are known to be more discriminative than shorter ones. with the increase in length of the word firstly, the distribution of words tends to poisson and not gaussian (by law of rare numbers), see figure 1. secondly, the length of the read vector grows exponentially (e.g., for l = 10, 4 10). with increase in dimensions, many words will tend to have similar distributions and hence can be clustered together into a word group. at the same time, the number of distinct words in any read is usually substantially smaller than the number of dimensions. hence, the model may fail to predict the true feature distribution of different components. however, reduction of the number of words using feature selection can not be too aggressive, otherwise the clustering accuracy will suffer. a supervised two - way poisson mixture model with word grouping was originally proposed by li and zha for simultaneous document classification. such a two - way clustering involves simultaneous clustering of reads as well as of words. the clusters means are regularized by dividing the words into groups and constraining the parameters for the words within the same group to be identical. the grouping of the words is not predetermined but optimized as part of the model estimation. this implies that, for every group, only one statistic for all the words in this group is needed to cluster reads. for instance, in figure 1, we observe the distribution of pentamers falls into three distinct group. therefore, words following similar distributions can be clustered together into a word group. we extend our formulation to an equivalent two - way unsupervised poisson mixture model in order to simultaneously cluster word features and classify reads and derive an expectation maximization algorithm to estimate its parameters. note that we make a distinction on the use of cluster to refer to binning of reads belonging to the same species and group to refer to binning of words within read in a cluster. recall that the genome signature is similar between closely related species and dissimilar between nonrelated species. therefore, we can assume that, within each cluster, words in different reads have equal poisson parameters, while, for reads in different clusters, words may follow different poisson distributions. for simplicity, we assume that all clusters have the same number of word groups. it is trivial to extend to the case where different clusters may have different number of word groups., l denote the word groups. we define a group assignment function c(m, j) 1,2,, l, which denotes the group to which word wj belongs in class m. words in the same word group will have the identical parameters, that is, mk = mj = m, l, if c(m, k) = c(m, j). the likelihood of xi is now (7)p(xi m)=j=1pp(xij mj)=j=1pp(xij m, c(m, j)). we can derive an em algorithm similar to the one outlined above to estimate the poisson parameters m, l where m 1,, m, l 1,, m, j 1,, p and the prior mixture components m, for m 1,, m. we initialize by setting each value of the group assignment function c(m, j) randomly to a number in 1,, l. we start with the same word group partition for all the clusters, that is, c(m, j)s are initially identical over m. we update the parameters as follows : (8)m(t+1)=i=1nqi, mn, m, l(t+1)=i=1nqi, mj1xijmli=1nqi, m, where c(m, j)=l. once ml is fixed, the word cluster index c(m, j) can be found by doing a linear search over all components : (9)c(t+1)(m, j)=argmaxli=1qi, m(xijlogm, l(t+1)m, l(t+1)). xp) are independent poisson variables with parameters 1, 2,, p, respectively, then the conditional distribution of (x1, x2,, xp) given that x1 + x2 + +xp = n is multinomial with parameters j/, where = j, that is, mult(n,), where = (1/, 2/,, p/). if (x1, x2,, xp) are independent poisson variables with parameters 1, 2,, p, respectively, then the conditional distribution of (x1, x2,, xp) given that x1 + x2 + +xp = n is multinomial with parameters j/, where = j, that is, mult(n,), where = (1/, 2/,, p/). the above theorem implies that the unconditional distribution (x1, x2,, xp) can be factored into a product of two distributions : a poisson for the overall total and a multinomial distribution of x, x ~ mult(n,). therefore, the likelihood - based inferences about are the same whether we regard x1, x2,, xp as sampled from p independent poissons or from a single multinomial. here, n refers to the length of the reads and our interest lies in the proportion of words in the reads. any estimates, tests, inferences about the proportions will be the same whether we regard n as random or fixed. we can now derive the naive bayes mixture of multinomials as standardized mixture of poissons. we assume that the distribution of words within the reads of a species is governed by the parameters of a multinomial distribution = (1, 2,, m), where each m is the parameter for species m and therefore, the likelihood of the data will be (10)p(xi yi = m)=p(xi m)=ni!j=1pxij!j=1pmjxij, the sum of the probabilities satisfies the constraint j=1mj = 1. the em algorithm for naive bayes mixture of multinomials can be derived similarly, and we only give the final set of equations : (11)m=i=1nqi, mn,mj=i=1nqi, mxiji=1nj=1pqi, mxij=i=1nqi, mxiji=1nqi, mni. if we assume the length of each read to be a constant n, we get the same results as that with poisson distribution ; hence, the two distributions are equivalent in modeling the distribution of words within reads of a species. also, since the multinomial distribution is single distribution, we do not perform a two - way dimension reduction on it. the algorithm has been implemented in matlab and c. the space and time complexity scale linearly with the number of reads and species. our method converged for all the cases we tested and was robust to the choice of initial conditions. metagenomics being a relatively new field lacks standard datasets for the purpose of testing clustering algorithms. as the true solution for sequence data generated from most metagenomic studies we also apply our method to the actual acid mine drainage dataset to identify the dominant species. in order to test the accuracy of our proposed method, we used metasim to simulate synthetic metagenomes. metasim takes as input the sequencing technology to be used (sanger, 454, exact), a set of known genomes, length of the reads, and an abundance / coverage profile which determines the relative abundance of each genome in the simulated dataset. the genomes used for generating the synthetic metagenomes were downloaded from national center of biotechnology information (ncbi). we generated datasets with reads of lengths between 50 and 1000 bp and various abundance ratios. in the first part of this section, we demonstrate the performance of the multidimensional gaussian mixture model on several datasets. as the number of dimensions is relatively small, we do not perform word grouping. next, we describe the results using the two - way poisson mixture model with a word length of 5. the method has been implemented for word lengths from 2 to 9. in order to calculate the clustering accuracy, we assign each cluster to the source species that is most frequent in the cluster. the number of species in each dataset is supplied as an input. determining the number of clusters from a statistical perspective is a difficult problem and has been addressed by. previously, 16 s/18 s rdna have been used for phylotyping and assessing species diversity using a rarefaction curve. tools such as metaphyler and treephyler can be used for making an educated guess of the number of species [34, 35 ]. estimating species diversity is still an active area of research, and we do not address it in this paper. experiments in the 1960s and 1970s have shown that the dinucleotide relative abundance in a genome is a remarkably stable property [36, 37 ]. closely related organisms display more similar dinucleotide composition than do distant organisms. in, the authors proposed a measure of intergenomic difference between two sequences f and g, called the average dinucleotide relative abundance, (12)(f, g)=116x, y|xy(f)xy(g)|, where xy(f) = fxy/fyfy and fx denotes the frequency of x in f. a measure of intergenomic difference was obtained by comparing different genome signatures. in order to assess the robustness of our method, we test it across datasets representative of values ranging from 34 to 340. in general, lower values correspond to closely related species and higher values correspond to distant species. in figure 3, we plot the performance of our proposed multidimensional poisson model over 450 datasets with values ranging from 34 to 340. we observed a positive correlation between the intergenomic difference and the accuracy of our method, as also noted in. the initial increase in the accuracy with word length is justified by the increased discriminative power of higher order words. however, any further increase in word length has to be accompanied by dimension reduction, otherwise owing to the high dimensional and sparse nature of feature matrix, the accuracy begins to drop. in figure 4, we compare the accuracy of our proposed multidimensional gaussian model with two other unsupervised composition - based methods likelybin and scimm on several datasets. we varied the read length between 200 to 500 bp, values from 60 to 300, and the abundance ratio up to 1 : 5. note that the distribution of dimers tends to a gaussian. as the number of dimensions is relatively small (4 = 16) our method clearly outperforms likelybin and performs as well or better than scimm on most instances. another point worth noting from the figure is that our method 's error rate is bounded by 10% for datasets with read length as short as 200 bp. we analyzed the accuracy and applicability of our method on binning reads from low complexity communities, containing 35 species (see table 1). with the increase in number of species, there was a slight degradation in performance, though the accuracy was consistently above 85%. this is in agreement with the results from the 2 species dataset, considering that the total coverage of each species is much lower in a multispecies dataset (reads from b. burgdorferi form only 6% of the 5th dataset). next, we evaluated the robustness of our method to changes in the abundance ratio between species as well as the length of the reads. we varied the abundance ratio from 10 : 1 to 1 : 10 in stages for the two species. from figure 5, we note that there was only a slight drop in performance for extreme abundance ratios. it is noteworthy to point out that estimates are good at all abundances. in order to test the usefulness of the method for analyzing data produced by the current ngs technologies (especially solexa and solid) that generate short reads, we tested three datasets of varying values for read lengths between 50 and 1000 bp. with the decrease in read length from 1000 to 50 bp, the drop in accuracy of our method is bounded by 15%. recall that with the increase in the length of the words and the simultaneous increase in the number of dimensions, the distribution of the words tends to a poisson and word grouping becomes necessary. in this section, we present the clustering results obtained by estimating the two - way poisson mixture model with different number of word groups l. we observed the variation in classification accuracy to be more prominent for lower values of l. therefore, in table 2, we report the results for values of l < 50 for a 2 species dataset. if word grouping is not performed, then clustering based on mixture model is essentially the naive bayes algorithm with each dimension modeled by a poisson distribution (last column of table 2). from the results, we can infer that word grouping resulted in considerable increase in accuracy compared to the naive bayes algorithm. that is, the characteristic vectors are of a much lower dimension with l p. also, a high clustering accuracy can be achieved using no more than ml dimensions, significantly smaller than the original dimension, 1024. note that it is difficult to know a priori, the exact value of l that yields the best clustering. however, among the values we tested, lower values of l provided a higher accuracy. in table 3, we compare the performance of our 2-way poisson mixture model with gaussian mixture model for datasets with low values. in real situations, it is difficult to know beforehand, the most discerning order of the word to use. however, from our experiments, we can infer that higher - order word - based models, in general, tend to be more discriminatory than those based on lower order words. if it is known a priori that lower - order words (of length 2 - 3) are more discriminatory in the dataset, then we recommend using a gaussian mixture model. for other datasets, our method 's accuracy in classifying reads from the datasets composed of species across various taxonomic ranks is reported in table 4 we used the poisson mixture model without word grouping. we can infer that the accuracy is mostly correlated to the phylogenetic distances between the species. for example, reads from datasets containing species with taxonomic differences at the level of class were classified with a very high accuracy. the ultimate goal of binning methods is to cluster reads in a real metagenome, by their species of origin. clustering in real situations is error - prone and affects our final estimates of species abundance. moreover, evaluating clustering methods on real metagenomes can be problematic as the true taxonomic composition of the data is mostly unknown. the accuracy of unsupervised clustering methods decreases with increase in the complexity of metagenomes and for species present at very low abundances. however, the composition of acid mine drainage metagenome has been substantially characterized, and we used this dataset to evaluate the performance of our proposed method. the amd microbial community is reported to consist of two dominant populations (ferroplasma sp. group ii) and three other less abundant ones (ferroplasma acidarmanus type i, leptospirillum sp. we downloaded the reads, as well as the scaffolds assembled from the reads for the 5 species of the actual amd dataset from ncbi. only 58% of the amd reads can be mapped back to the assembled scaffolds using blast. therefore, in order to compute the accuracy of our method, we simulated a metagenome with reads sampled from the downloaded scaffolds. the simulated amd dataset consisted of 110,000 reads of average length 732 bp (average read length in the actual amd dataset) from the 5 species, in the ratio 4 : 4 : 1 : 1 : 1. therefore, we can simplify the problem by first separating the reads into two bins based on their abundance. in the first stage, the reads were grouped into two bins, using abundance bin, with a resulting accuracy of 93.3%. the bins corresponding to the abundance levels of 4 and 1 had a cluster purity of 93.2% and 98.2%, respectively. in the next stage, we used the reads from each of the bins output by abundance bin, as an input to our proposed 2-way poisson mixture model, to further classify the reads by their species of origin. our method clustered the reads from the bin containing dominant species into two clusters corresponding to ferroplasma sp. the other bin consisted of very few reads from the remaining three species ferroplasma acidarmanus type i, leptospirillum sp. our method clustered the reads from this bin into three clusters, with an accuracy of 70.34% (with l = 10). in this paper, we formulated an unsupervised two - way multispecies, multidimensional mixture model to represent reads from a metagenome. we used the proposed model to cluster metagenomic reads by their species of origin and to characterize the abundance of each species. the distribution of word counts along a genome can be approximated as a gaussian for shorter, frequent words and as a poisson for longer words that are rare. therefore, we use a multidimensional mixture of gaussians or poissons to model the reads from each bin. an additional reason to use these distributions this is critical for success as most metagenomic datasets contain reads from unexplored phyla which can not be labeled into one of the existing classes. our probabilistic approach can be used to identify reads which belong to more than one species and occlude the cluster boundaries. note that our proposed method is primarily a composition - based method that seeks to distinguish between genomes based on their characteristic dna compositional pattern. therefore, it can not distinguish between genomes unless their dna compositions are sufficiently divergent (see figure 4, dataset with b. burgdorferi and c. jejuni). it is unlikely that our method will be able to accurately distinguish between strains of the same species. for such datasets, composition - based methods must be used in conjunction with other similarity - based methods and abundance - based methods to yield better performance. note that the two - way poisson mixture model was originally proposed for classification of documents. in this work, we demonstrate the relevance and applicability of such a general statistical framework for modeling metagenome reads. we have illustrated that the proposed method can accurately classify reads from low to medium complexity datasets into taxon - specific bins, based on genome signatures. our framework complements the existing similarity - based and abundance - based methods and hence can be combined with such methods to obtain a better performance. we intend to develop such hybrid methods in the future that can tackle the problem of classifying sequences in complex metagenomic communities. | a major challenge facing metagenomics is the development of tools for the characterization of functional and taxonomic content of vast amounts of short metagenome reads. the efficacy of clustering methods depends on the number of reads in the dataset, the read length and relative abundances of source genomes in the microbial community. in this paper, we formulate an unsupervised naive bayes multispecies, multidimensional mixture model for reads from a metagenome. we use the proposed model to cluster metagenomic reads by their species of origin and to characterize the abundance of each species. we model the distribution of word counts along a genome as a gaussian for shorter, frequent words and as a poisson for longer words that are rare. we employ either a mixture of gaussians or mixture of poissons to model reads within each bin. further, we handle the high - dimensionality and sparsity associated with the data, by grouping the set of words comprising the reads, resulting in a two - way mixture model. finally, we demonstrate the accuracy and applicability of this method on simulated and real metagenomes. our method can accurately cluster reads as short as 100 bps and is robust to varying abundances, divergences and read lengths. |
over the last few years use of short message services (sms) and smartphone - based communication applications in health care has shown its wide application and potential to improve access to health care ; enhance efficiency of service delivery ; improve diagnosis, facilitate timely treatment and support public health programmes. this effective means of communication is providing timely and accurate results to the correct recipient, safeguarding their privacy and confidentiality and avoids misunderstanding and misinterpretation of the results. there are various uses of mobile phone message services and smartphone - based communication applications such as : appointment remainders ; to monitor chronic medical conditions ; to improve treatment compliance ; to provide psychological support ; managing communicable diseases via contact tracing and partner notification for sexually transmitted illnesses and to promote health promotion programs like smoking cessation. tata memorial hospital uses many modes of health related e - communications such as web based electronic medical records (emr) where patients and families can access their medical records, blood investigations, scans and all patient - related information using an individual patient login key and password. this information can be accessed at home or any other remote setting using a computer or smartphone. the other modes of e - communications used are e - mail, short message service (sms), multimedia messaging service (mms) and whatsapp (text, pictures, and video). although these applications are not substitute for face - to - face interview and clinical examination, in most of the situations especially in palliative care setting it minimizes needless transport of the patient, cost and logistics involved. it also provides a means for quick information sharing, cuts the waiting time and facilitates initiation of the treatment at the earliest. it also forms a means of communication with local general practitioner and local health care provider such that continuity of the care is maintained. a 60-year - old pleasant lady, widow, high school educated, who is known to have type 2 diabetes mellitus, ischemic heart disease, and metastatic carcinoma breast with nodal and skeletal metastasis was referred to palliative medicine for symptom control and supportive care. during her initial outpatient visit to palliative medicine department she had 8/10 somatic nociceptive pain over spine and ribs, extreme fatigue, anorexia and had an eastern cooperative oncology group (ecog) of 4. her mobility was impaired due to severe pain and spine metastasis, which had a great impact on her activity of daily living. initial symptom control and supportive care measures were instituted and she was referred to palliative home care services where she was triaged as priority 1 (scheduled home visit within 3 days). the homecare services are coded as high (01), medium (02) and low (03) priority. in high priority, patients received home visit in 0 - 3 working days, medium priority, in 0 - 10 working days and low priority, in 0 - 15 working days. she was on step 1 non - steroidal anti - inflammatory drugs (nsaids) + step 3 (transdermal fentanyl) + adjuvant (pregabalin) analgesics for pain. the immediate caregivers were distant relatives staying close to her house and visiting her daily. her niece, who is a practicing dermatologist, was facilitating her ongoing medical care. patient was fully aware of the diagnosis and prognosis, and maintained a positive outlook toward illness and life. she was on a regular follow - up with palliative home care and over next few weeks on a weekend she developed diplopia and painful weakness of the right upper limb. information of her illness was shared with the palliative home care doctors by her niece via text messages, patient pictures and a video through whatsapp. the doctor reviewing these e - communications requested for an urgent mr brain and plain x - ray of right shoulder. mr brain was suggestive of base of brain disease with leptomeningeal and skull base involvement. plain x - ray of right shoulder was suggestive of pathological fracture. scanned copy of the mr and x - ray reports were sent to the local general practitioner and management plan for raised intracranial tension and pathological fracture was communicated. patient 's daughters who returned to see her mother was in constant touch with the palliative home care doctors through call and text. they frequently use to send patient 's pictures and reports and occasionally patient videos via whatsapp and on call palliative homecare doctor was able to provide after - hours consultation. the timely symptom management improved the quality of life of the patient and caregivers were very satisfied by the support they received. her analgesic doses and complications (seizures, aspiration pneumonitis) were managed at home with the help of local general practitioner support with continued input from palliative home care. with this process needless hospitalization we felt smartphone applications played a crucial role in providing timely and continued palliative care input. a 64-year - old gentleman, known hypertensive, having advanced stage of adenocarcinoma of stomach with peritoneal metastasis, bilateral pleural effusion, and ascites was referred to department of palliative medicine for symptom control and ongoing supportive care. ascites needed paracentesis to relieve dyspnea and patient was maintained on oral frusemide / spironolactone combination for both ascites and edema. the daughter was counseled and poor prognosis of the patient was discussed. as per patient 's wishes, patient was cared at home. the wife was elderly, not literate and had poor perspective of illness or care process. the son was a young adult who studied and worked simultaneously and had little involvement in the care process. the daughter was married and has an 18-month - old child and resided in a different place. she was the major support and was main portal for communication between palliative home care team and family. she used whatsapp to send text messages, patient images and patient videos to the team. the team worked in close liaison with the family physician, which helped in maintaining the continuity of care. patient had a sudden worsening of clinical condition due to malignant bowel obstruction and also developed acute renal failure secondary to obstructive uropathy. in accordance to patient 's wishes and preferences, patient was managed at home with iv hydration, nasogastric tube, anti - secretory drugs, transdermal fentanyl and iv steroids. progress of management was monitored through continuous exchange of images and videos through whatsapp and symptom control was assessed through direct patient interview via video chat. this process complemented the homecare provided and enabled the daughter to balance her time both at work and home. patient died peacefully at home and the total duration of home - based palliative care contact was 39 days. bereavement follow - up showed patient 's family was coping well and were pleased with the care provided. a 60-year - old pleasant lady, widow, high school educated, who is known to have type 2 diabetes mellitus, ischemic heart disease, and metastatic carcinoma breast with nodal and skeletal metastasis was referred to palliative medicine for symptom control and supportive care. during her initial outpatient visit to palliative medicine department she had 8/10 somatic nociceptive pain over spine and ribs, extreme fatigue, anorexia and had an eastern cooperative oncology group (ecog) of 4. her mobility was impaired due to severe pain and spine metastasis, which had a great impact on her activity of daily living. initial symptom control and supportive care measures were instituted and she was referred to palliative home care services where she was triaged as priority 1 (scheduled home visit within 3 days). the homecare services are coded as high (01), medium (02) and low (03) priority. in high priority, patients received home visit in 0 - 3 working days, medium priority, in 0 - 10 working days and low priority, in 0 - 15 working days. she was on step 1 non - steroidal anti - inflammatory drugs (nsaids) + step 3 (transdermal fentanyl) + adjuvant (pregabalin) analgesics for pain. the immediate caregivers were distant relatives staying close to her house and visiting her daily. her niece, who is a practicing dermatologist, was facilitating her ongoing medical care. patient was fully aware of the diagnosis and prognosis, and maintained a positive outlook toward illness and life. she was on a regular follow - up with palliative home care and over next few weeks on a weekend she developed diplopia and painful weakness of the right upper limb. information of her illness was shared with the palliative home care doctors by her niece via text messages, patient pictures and a video through whatsapp. the doctor reviewing these e - communications requested for an urgent mr brain and plain x - ray of right shoulder. mr brain was suggestive of base of brain disease with leptomeningeal and skull base involvement. plain x - ray of right shoulder was suggestive of pathological fracture. scanned copy of the mr and x - ray reports were sent to the local general practitioner and management plan for raised intracranial tension and pathological fracture was communicated. patient 's daughters who returned to see her mother was in constant touch with the palliative home care doctors through call and text. they frequently use to send patient 's pictures and reports and occasionally patient videos via whatsapp and on call palliative homecare doctor was able to provide after - hours consultation. the timely symptom management improved the quality of life of the patient and caregivers were very satisfied by the support they received. her analgesic doses and complications (seizures, aspiration pneumonitis) were managed at home with the help of local general practitioner support with continued input from palliative home care. with this process needless hospitalization we felt smartphone applications played a crucial role in providing timely and continued palliative care input. a 64-year - old gentleman, known hypertensive, having advanced stage of adenocarcinoma of stomach with peritoneal metastasis, bilateral pleural effusion, and ascites was referred to department of palliative medicine for symptom control and ongoing supportive care. ascites needed paracentesis to relieve dyspnea and patient was maintained on oral frusemide / spironolactone combination for both ascites and edema. the daughter was counseled and poor prognosis of the patient was discussed. as per patient 's wishes, patient was cared at home. the wife was elderly, not literate and had poor perspective of illness or care process. the son was a young adult who studied and worked simultaneously and had little involvement in the care process. the daughter was married and has an 18-month - old child and resided in a different place. she was the major support and was main portal for communication between palliative home care team and family. she used whatsapp to send text messages, patient images and patient videos to the team. the team worked in close liaison with the family physician, which helped in maintaining the continuity of care. patient had a sudden worsening of clinical condition due to malignant bowel obstruction and also developed acute renal failure secondary to obstructive uropathy. in accordance to patient 's wishes and preferences, patient was managed at home with iv hydration, nasogastric tube, anti - secretory drugs, transdermal fentanyl and iv steroids. progress of management was monitored through continuous exchange of images and videos through whatsapp and symptom control was assessed through direct patient interview via video chat. this process complemented the homecare provided and enabled the daughter to balance her time both at work and home. patient died peacefully at home and the total duration of home - based palliative care contact was 39 days. bereavement follow - up showed patient 's family was coping well and were pleased with the care provided. moribund patients with advanced life - limiting illness can receive continued care at home with the help of collaborative and ongoing support from the palliative home care team and local family physician. audio - visual communications aided through smart phone applications can improve this facilitation. using applications like whatsapp messenger, which allows the user to share clinical images, clinical video, photograph of reports, photograph of medications and text messages can bring palliative home care management to a new level by enhancing the channels of communication between patient, palliative care doctor, caregiver and local family physician. this technology is useful in palliative care for the patients lacking access to medical services due to both debilitating medical condition and geographic isolation. a study done by coyle. demonstrated certain benefits such as limited need for daily physical examination and assessment, screening for a need for a clinical visit or admission, communication assistance to patients who can not speak or hear and increased satisfaction by the patient and the caregivers. in a palliative care setting it also decreases monetary burden on caregiver as it saves the cost of travel and consultations and prevents unnecessary hospital admission and investigations. the current smartphone applications like whatsapp messenger is much cheaper and easily available when compared to the teleconferencing systems. the most teleconferencing solutions pertain to the transfer of electronic medical data from one institution to another, only a small percentage of such programs can be applied to home health care. it is a worldwide phenomenon to incorporate the newer technologies into the field of health and social care, even though there is lack of evidence to support this. the rapid development, adoption and use of various smartphone applications also present with crucial challenges for clinicians and users. the important challenges are to ensure patient confidentiality, safety, establish cost - effectiveness and engage patients and clinicians to optimize their use in health decision - making. although healthcare and academic institutions should support the improvements offered by technological advances, they must do it within a supervised framework, after thoroughly evaluating clinical outcomes and unintended consequences. challenges in using mobile phone applications in health care also include incomplete coverage of mobile networks across regions, lack of standards, and possible information overload. at present we have a mobile user society and the worldwide deployment of mobile and wireless networks and the wireless infrastructure in health care is able to provide healthcare to anyone, anytime, and anywhere without constraints of location or time. it 's application in palliative home care has demonstrated significant improvement in control of physical symptoms, address non - physical issues and improves patient satisfaction. special attention needs to be given to its potential benefits and challenges in palliative care and certainly its open a new area of research in palliative care. smartphone applications in palliative homecare are a novel cost - effective approach in providing timely and effective careimproves symptom control, helps in continued care at home and prevents unnecessary hospitalizationimproves patient and caregiver satisfaction. smartphone applications in palliative homecare are a novel cost - effective approach in providing timely and effective care improves symptom control, helps in continued care at home and prevents unnecessary hospitalization improves patient and caregiver satisfaction. | smartphone applications in healthcare delivery are a novel concept and is rapidly gaining ground in all fields of medicine. the modes of e - communications such as e - mail, short message service (sms), multimedia messaging service (mms) and whatsapp in palliative care provides a means for quick tele - consultation, information sharing, cuts the waiting time and facilitates initiation of the treatment at the earliest. it also forms a means of communication with local general practitioner and local health care provider such that continuity of the care is maintained. it also minimizes needless transport of the patient to hospital, prevents needless hospitalization and investigations and minimizes cost and logistics involved in the care process. the two case studies provided highlights the use of smartphone application like whatsapp in palliative care practice and demonstrates its utility. |
they are a congenital disturbance of the position of the eyelashes, which are usually on the lateral quadrant of the upper eyelid or conjunctival surface of the eyelid. the origin of these abnormally placed cilia is not clear, but a previous theory of meibomian gland substitution has been refuted and an embryologic origin suggested.1,2 in most cases, it causes no apparent medical morbidity and there is no positive family history. here we report a case of a patient with ectopic cilia and hypochromic patches on the right arm. a 6-year - old girl was referred for evaluation of a congenital lateral upper eyelid ectopic cilia cluster (figure 1). on physical examination, she had hypochromic lesions on the right arm, diagnosed by the dermatology service as hypochromic nevi (figure 1). the specimen comprised a circular 106 mm portion of skin and contained a centrally placed tuft of strong and pigmented hairs. longitudinal sections and histologic analysis showed dermal and subcutaneous pilosebaceous follicles (figure 1a and 1b), packed in a dense fibrous tissue core (figure 1b) extending through the eyelid from the subcutaneous area, scarcely represented in the specimen. well developed sebaceous lobules (figure 1c), bulbs of pilous follicles (figure 1d), and apocrine - lined epithelial ducts consistent with glands of moll (figure 1e) and associated with pilous follicles were observed. occasional striated orbicularis muscle fibers were present in the depths of the specimen (figure 1f), with the lowest hair bulbs (figure 1f) superficial to them. they have been observed to protrude from the anterior surface of the eyelid skin and the posterior aspect of the tarsal plate. there are 15 cases located on the anterior surface of the eyelids.1,2 histologic examination has demonstrated the presence of apocrine and pilosebaceous glands, and there is evidence that ectopic cilia in the palpebral conjunctiva are acquired aberrant cilia, in contrast with anterior ectopic cilia, which are congenital.3 in this case, histopathologic examination of the specimen revealed the typical characteristics of ectopic cilia described in the rare cases reported in the literature. we found strong and pigmented hairs in a dense fibrous stroma associated with sebaceous and apocrine glands located in the dermis and subcutis and superficial to the orbicular muscle fibers. importantly, ectopic cilia must be differentiated from abnormally placed hairs. as in our present case, the diagnosis of ectopic cilia is confirmed histologically by identification of sweat glands of the apocrine type attached to the follicles.4 recent reports also underscore the importance of morphologic observation of ectopic cilia in association with lobules of sebaceous glands and apocrine glands.5 in the present case, the cilia arose from the anterior surface of the tarsal plate, being classified as anterior - type ectopic cilia, which are congenital, in contrast with posterior ectopic cilia that arise in the palpebral conjunctiva and are acquired aberrant cilia.3 although previous reports have not correlated ectopic cilia with other skin hypopigmentation disorders, it is important to note that congenital hypochromic patches (hypochromic nevi) were observed in this case. | ectopic cilia are rare in humans. we report a 6-year - old girl with typical characteristics of ectopic cilia as described in the rare cases reported in the literature, in association with cutaneous lesions that appeared to be hypochromic nevi. this framework could be a different clinical presentation of ectopic cilia. |
arteriovenous malformation (avm) probably occurrs from loss of the regulatory sphincteric function of the arteriolarcapillary junction which results in overflow of the arterial blood into the capillaries and venules, producing an arteriovenous shunt.1 pancreatic avm is a rare disease which can be complicated with abdominal pain, gastrointestinal bleeding, portal hypertension, and duodenal ulcer.2 however, formation of the pancreaticoduodenal fistula by pancreatic avm is extremely rare. herein, we report a case of pancreaticoduodenal fistula associated with pancreatic avm which induced recurrent anemia and ascending infection. a 58-year - old man was admitted to chonnam national university hospital with recurrent fever and epigastric pain for 2 months. two years ago, the patient visited for exertional dyspnea and he was diagnosed as iron deficiency anemia with pancreatic arteriovenous malformation communicating gastroduodenal artery and pancreaticoduodenal artery with superior mesenteric vein (fig. 1). since then he experienced recurrent anemia and he took oral iron preparations occasionally. the body temperature was 38.2. laboratory investigation revealed leukocyte 12,900/mm (neutrophil 79.5%), hemoglobin 14.1 g / dl, platelet 239,000/mm. the blood chemistry were protein 6.1 g / dl, albumin 3.2 g / dl, ast 127 u / l, alt 96 u / l, alkaline phosphatase 145 u / l, total bilirubin 1.1 mg / dl, gamma glutamyl transpeptidase 190 u / l, amylase 85 u / l, lipase 34 u / l, crp 9.1 mg / dl, prothrombin time 11.8/92.1/1.05 sec/%/inr. despite he underwent thorough examinations including computerized tomography (ct) of chest and abdomen, esophagogastroduodenoscopy and colonoscopy, the body temperature was 36.5. laboratory investigation revealed leukocyte 9,900/mm (neutrophil 75.5%), hemoglobin 12.2 g / dl, platelet 337,000/mm. the blood chemistry were protein 6.8 g / dl, albumin 3.8 g / dl, ast 16 u / l, alt 22 u / l, alkaline phosphatase 156 u / l, total bilirubin 0.5 mg / dl, gamma glutamyl transpeptidase 94 u / l, amylase 159 u / l, lipase 34 u / l, crp 2.0 mg / dl, prothrombin time 11.5/96/1.03 sec/%/inr and the urinary analysis was within normal limit. abdominal ct showed abnormally dilated vessels which abutted with descending duodenum and communicated with gastroduodenal artery and superior mesenteric vein. the head of pancreas was focally enlarged, soft tissue was infiltrated in the peripancreatic fat plane, and air densities in the main pancreatic duct were detected (fig. when the radiocontrast dye was injected through the fistula, the dilated pancreatic duct was visualized (fig. review of the past abdominal ct films revealed air densities in the main pancreatic duct also. on hospital day 5, he complained abdominal pain and the body temperature was 39.5. the leukocyte was 12,900/mm (neutrophil 91.7%) and citrobacter freundii was cultured from peripheral blood. on hospital day 8, the fever and abdominal pain was relieved with antibiotics. but, he refused surgery and other conservative treatment including angiographic embolization or transjugular intrahepatic portosystemic shunt. and although avm can be occurred frequently in digestive organ such as cecum, ascending colon, jejunum, and ileum, pancreatic avm is rare3 and reported first by halpern.4 doppler ultrasonography and ct can be helpful, angiography has played the most important role in the diagnosis and planning the therapy of pancreatic avm. the angiographic findings of pancreatic avm include 1) dilated and tortuous feeding arteries, 2) racemose intrapancreatic vascular network followed by a transient dense pancreatic strain, 3) early venous filling into the portal vein, and 4) early disappearance of the pancreatic strain.5 the vessels most commonly affected by a pancreatic avm are the splenic artery (42%), gastroduodenal artery (22%), and small pancreatic arteries (25%).6 the clinical symptoms of pancreatic avm were variable including gastrointestinal bleeding, abdominal pain, jaundice, portal hypertension, pancreatitis, and duodenal ulcer.2,7 gastrointestinal bleeding can be occurred by following mechanisms, first, direct intestinal bleeding from intestinal mucosa, second, bleeding from pancreatic duct, and third, bleeding from esophagogastric varices associated with portal hypertension.8 in the present case, although there was no evidence of massive gastrointestinal bleeding, chronic occult blood loss might have occurred from telangiectasia associated with pancreatic avm. duodenal ulcer or duodenitis appears to be associated with regional ischemia caused by the diseased mucosa.9 recurrent inflammation of duodenum and pancreas might have induced the fistula tract between pancreatic duct and duodenum. however, choledochoduodenal or pancreaticoduodenal fistula associated with pancreatic avm is extremely rare, and according to the english literature, only 2 cases have been reported10,11 and so far, there was no report about recurrent ascending infection caused by pancreaticoduodenal fistula associated with pancreatic avm. citrobacter freundii is gram negative enteric bacilli which can be found in human intestine and infects urinary and biliary tract. in the present case invasion of citrobacter freundii via pancreaticoduodenal fistula might have induced pancreatitis and bacteremia. in the present case, chronic ischemia caused by pancreatic avm could induce chronic pancreatitis and chronic pancreatitis was acutely exacerbated at each time of ascending infection by pancreaticoduodenal fistula. it is known that pancreatitis associated with pancreatic avm can be occurred by bleeding from avm into the pancreatic duct and ischemia of the pancreas.7 ascending infection via pancreaticoduodenal fistula should be considered as a cause of pancreatitis. complete cure is achieved by total resection of the affected organ, or at least the involved portion.12 once after portal hypertension developed, it can not be corrected even after surgical resection, so early diagnosis and proper treatment is important.5 in case of inoperable conditions, transcatheter arterial embolization,12 radiation therapy,13 and transjugular intrahepatic portosystemic shunt14 seems to be the alternative treatment for complications of pancreatic avm. in summary, herein, we report a case of pancreaticoduodenal fistula associated with pancreatic avm which induced recurrent anemia and ascending infection. | although arteriovenous malformations (avm) occur frequently in digestive organs, pancreatic avm is rare. the clinical symptoms of pancreatic avm are variable and include gastrointestinal bleeding, abdominal pain, jaundice, portal hypertension, pancreatitis, and duodenal ulcer. however, choledochoduodenal or pancreaticoduodenal fistulas complicated with ascending infection and pancreatitis is extremely rare. herein, we report a case of pancreaticoduodenal fistula associated with a pancreatic avm that induced recurrent anemia and ascending infection. |
migrated tooth is defined as the movement of teeth into altered positions in relation to the basal bone of the alveolar process and the adjoining and opposing teeth due to loss of approximating or opposing teeth, occlusal interferences, habits, or inflammatory and dystrophic disease of the attaching and supporting structures of the teeth. pathologic tooth migration (ptm) is defined as tooth displacement that occurs when the balance among the factors that maintain the physiologic tooth position is disturbed by periodontal disease. ptm is a periodontal disease that has aesthetic effects. despite the prevalence of pathological migration among periodontal patients, there is limited data on the different aspects of this problem. according to some studies, ptm is the one of the most common complaint that leads periodontal patients to dental clinics. case studies and clinical observations on this subject have shown that etiology of pathologic migration is multifactorial and lack of research in this field may be as a result of this complexity. ptm may be an early sign of the disease or may be related to gingival inflammation and pocket formation during the progression of the disease. even during the early stages of the disease, some degree of bone destruction may occur. diagnosis in its early stages and prevention of severe complications by eliminating the causes has a major advantage. pathologic migration occurs most often in the anterior region but may also involve the posterior areas. moreover, the prevalence of ptm in moderate to severe periodontitis has been studied. severity of periodontitis can be characterized based on the amount of clinical attachment loss (cal) as follows : mild = 1 or 2 mm cal, moderate = 3 or 4 mm cal, and severe = 5 mm cal. in teeth with ptm, mean of cal greater than 3.33 mm these measures are related to the moderate and advanced stages of periodontitis and are not related to the early stages. nevertheless, the prevalence of ptm in mild periodontitis has not yet been studied. this study aims to determine the prevalence of ptm in terms of the patient 's periodontal condition and the severity of disease based on measures of cal. this cross - sectional epidemiological study was carried out in the shiraz dental school and included 370 recorded files of patients with periodontal disease. the number of studied cases belonged to periodontal patients who were referred to our clinic in a 5-year period. patients with background characteristic or factors of smoking, diabetes, oral breathing, bruxism, and patients who were simply suffering from gingivitis (without periodontitis) were excluded (n = 56). periodontitis was diagnosed in patients based on clinical examination according to the 1999 american academy of periodontology classification. severity of periodontitis (mild, moderate, severe) was classified based on the amount of cal as mentioned previously. cal was measured as the distance between the cementoenamel junction (cej) and the base of the pocket. cal at 6 points of each tooth was measured by the periodontal probe manually (mesiofacial, midfacial, distofacial, mesiolingual, midlingual, and distolingual). mean and standard deviations were calculated and subjected to t - test and chi - square test. record files of 314 patients with periodontitis (228 females, 86 males ; mean age = 37.7 years ; age range : 1770 years) were studied. the mean age of females and males were 36.53 10.93 and 41.02 12.05, respectively, and there were no significant differences. characteristics of patients with periodontitis regarding tooth migration the prevalence of pathologic migration in terms of periodontal status was as follows : pathologic migration was not observed in patients with mild chronic periodontitis ; it was seen in 5.2% of patients with moderate chronic periodontitis ; pathologic migration was seen 51% of patients with severe chronic periodontitis whereas it was seen in 50% of patients with aggressive periodontitis (localized and generalized) [figure 1 ]. the maximum number of migrated teeth in patients was 5 teeth [table 2 ]. frequency distribution of number of patients with pathologic tooth migration in different severity of periodontitis number of migrated teeth according to the severity of periodontitis in each patient. the diagnostic criteria for the diagnosis and classification of periodontitis are based on the 1999 american academy of periodontology classification among the 228 females with periodontitis, ptm was seen in 26 patients (11.40%), and among 86 males with periodontitis, 9 patients (10.46%) had ptm. nineteen patients (33%) in the five groups (more than 3 teeth loss) had ptm. distribution of patients with periodontitis and prevalence of pathologic tooth migration according to the number of lost teeth in this cross - sectional epidemiological study, the prevalence of ptm was studied with respect to the severity of periodontitis. the results of this study suggest that ptm is a common finding in periodontal patients (11.14%), and is also more prevalent in the more advanced stages of periodontal disease. the highest prevalence of ptm was seen in the severe form of chronic periodontitis and aggressive periodontitis. to the best of our knowledge, this is the first study on the prevalence of ptm in different classes of periodontitis based on the 1999 american academy of periodontology classification. the first study of prevalence of ptm was published in 1997 by towfighi. ; the study only included patients with moderate to severe periodontitis. in our study, the mean of cal in migrated teeth has been reported to be between 3.3 and 5.5 mm. according to these studies, it can be assumed that, in the initial stages of periodontal disease, ptm does not occur. according to some studies, ptm is one of the most common complaints that lead periodontal patients to dental clinics. pathological migration may be an early sign of the disease or may be related to gingival inflammation and pocket formation during the progression of the disease. normally, there is a balance between the forces that hold the tooth in normal position and occlusal and muscular forces the tooth should normally bear. the weakened supporting tooth is unable to maintain its normal position and moves away from the opposing force. all degrees of pathological migration have been observed and may involve one or more teeth. treatment of ptm in anterior teeth can be long and complex, and a multidisciplinary approach is often necessary. carried out a cross - sectional study to determine the prevalence of ptm and assess its relation to bone loss, tooth loss, gingival inflammation, age, lingual interposition, parafunctions, and oral habits. in their study, 852 patients were examined and the diagnosis of ptm was based on the presence of a developing diastema in the upper anterior sextant, which was not present in the past or already existing but was increased. they reported a significant relationship of ptm with bone loss, missing teeth, and gingival inflammation, whereas significant relationship between ptm and age and parafunction habits and tongue interactions have not been observed. one of the highlights of their study, despite the significant sample of radiographic and clinical records (e.g., assessment of gingival inflammation), was that the patients were examined by only one person. due to the exclusion of patients with tooth loss in the anterior region, the prevalence of ptm was expected to be under - reported, however, due to subjective criteria utilized in their study, the amount estimated for ptm may be more than normal. prevalence obtained from their study (55.8%) is significantly higher than the results of our research (11.14%), and this may be as a result of the subjective nature of diagnosis of ptm in their study, which can overestimate the results. the prevalence of ptm in the study by towfighi. was estimated as 30.03% 2.5 in a group of patients with moderate to severe periodontitis. in our study, prevalence of ptm in patients with periodontitis (including mild periodontitis) was 11.14%, however, when patients with mild periodontitis were excluded, the prevalence of ptm was 21.73%, which was less different from the results of our research. the use of archival records in this study can be considered as an advantage because the examiners were been informed about the research. in the present study, recorded files of patients referred to dental school were used ; hence, they are not representative of all segments of the society. hence, it is suggested that further studies should be carried out on large samples that are representative of all strata of the society. the results of this study suggest that ptm is more prevalent in more advanced stages of periodontal disease. we would like to emphasize that we did not find any tooth migration among patients with mild periodontitis ; nevertheless, the prevalence of ptm was seen in the severe form of chronic periodontitis and aggressive periodontitis, indicating that diagnosis and treatment of periodontitis in the early stage could reduce the incidence of pathologic tooth migration. accordingly, there will be no need for time consuming, complex interdisciplinary treatments. | aim : pathologic tooth migration (ptm) has been defined as tooth displacement that occurs when the balance among the factors that maintain physiologic tooth position is disturbed by periodontal disease. the aim of this cross - sectional epidemiological study was to determine the prevalence of pathologic tooth migration among patients with periodontitis.materials and methods : recorded documents of 370 patients (72.4% females, 27.6% males) within the age range of 17 to 70 years (mean 37.77 11.46) were studied. statistical analysis was carried out using t - test and chi - square test.results:pathologic migration prevalence was 11.4% (35/314 patients), however, there was no pathologic migration in patients with mild chronic periodontitis. the chi - square test showed that there was no statistically significant difference between males and females.conclusions:the results of this study confirm that pathologic tooth migration is relatively common among periodontal patients and its prevalence is increased by the severity of periodontal disease. |
percutaneous coronary intervention (pci) for chronic total occlusion (cto) has a lower success rate than pci for non - cto lesions. a cto lesion was defined as an obstruction of a coronary artery with thrombolysis in myocardial infarction or a flow grade of 0, with an estimated duration of at least 3 months prior to the event. in 1990, the first retrograde approach technique was applied for cto via a degenerated saphenous vein graft. over the next 2 decades, variable and new retrograde techniques were reported over the next few years, including the following : kissing wire technique, knuckle wire technique, the controlled antegrade and retrograde subintimal tracking (cart) technique, the reverse cart technique, the modified reverse cart technique, the wire trapped technique, and the reverse wire trapped technique. despite the development of these various approaches, cto lesion still remains a challenge. in this situation, information about the cto lesion from intravascular ultrasound study (ivus), cardiac ct angiography is a very useful approach, and is required for greater success in pci. in this case, we report our experience using the reverse cart technique under the aid of ivus, cardiac ct angiography for an ambiguous cto of proximal right coronary artery (p - rca) (c, 100%, 0) with grade iii collateral flow. a 68-year - old man with a history of hypertension presented with worsening exertional dyspnea. fifteen months prior, angiography performed at a different hospital showed total occlusion of the p - rca (100%, c, 0) and distal left circumflex artery (lcx) (100%, c, 0) with significant stenosis of the proximal left anterior descending artery (lad) (95%, b2, ii). at that time, pci using drug - eluting stent was performed in the proximal lad and distal lcx, only because multiple trials through the anterograde approach of cto of p - rca failed. bilateral coronary angiography demonstrated total occlusion of the p - rca and rentrop grade 3 collateral flow from lad, filling in a retrograde manner, the distal right coronary artery (rca). a small vessel of rca proximal portion was looked as conus branch in coronary angiography (fig. 1). but, ivus study via anterograde approach in p - rca revealed tapered - type cto lesions, which was looked as conus branch. and, this distinction was detected in previous cardiac ct angiography, too (fig. multiple guide wires were used to bypass the lesion through use of the collaterals, retrospectively. finally, the guide wire (fielder fc 300 cm asahi intecc, osaka, japan) passed the lesion via the septal branch to mid rca. then, a 2.515 mm size percutaneous transluminal coronary angioplasty balloon was inserted through the septal branch via retrograde guide wire and inflated to 12 atm. two cypher stents, 3.528 mm and 3.533 mm, were implanted in the mid- and proximal - rca. several studies show that successful recanalization of the cto improved angina pectoris, survival, and left ventricular systolic function.1 - 5) despite this necessity, the success rate of recanalization was still unsatisfied. the most common reason for pci failure in cto is passage failure of guidewire.6) ivus can give adjunctive information to detect the area of occlusion in selective cases from adjacent side branches or from the false lumen and the anatomical information about the distal segment of the totally occluded coronary artery, when dye could not penetrate.7) in addition, cardiac ct angiography can help identify features that most influence the current success rates of pci, such as marked calcifications at the stump, severe tortuosity of the proximal vessel, long length of the occluded segment, as well location of the vessel distal to the occlusion, which often can not be well visualized using conventional angiography.8) in this case, cardiac ct angiography and ivus in addition to angiography were performed to evaluate cto lesions because of the previous failure of intervention for p - rca. segmental calcified and soft plaques were detected in p - rca with cto by cardiac ct angiography. and, the conus branch was made out at the proximal portion of the plaques. thus, we identified the lesion that firstly considered as tapered stump was conus branch. and it looked as tapered stump. retrograde approach through the collateral channels has been proposed recently and has the potential to improve the success rate of pci towards the treatment of cto lesions.9) in addition, rathore.10) demonstrated a 100% success rate of cto recanalization using reverse cart. in general, this technique is performed after previous techniques have failed via the anterograde approach, with the presumption that the distal cap of the cto lesion may be softer than the proximal cap. several strategies using the retrograde approach may be proposed to cross the occlusion, according to the cto lesion characteristics, and sometimes, a very complex cto lesion may require a combination of several techniques. the reverse cart technique has been shown to be safe and feasible, with a high success rate when performed by highly experienced operators. in conclusion, if previous attempts to cross the totally occluded lesion from anterograde approach fail, the retrograde approach from collateral should be considered first, as in our case. with the help of diagnostic imaging, such as ivus and cardiac ct angiography,, the development of diagnostic methods will be reduced to reduce the failure to passage the cto lesion. | passage failure of guidewire is still remained most common reason for percutaneous coronary intervention (pci) failure in chronic total occlusion (cto). intravascular ultrasound study (ivus) and cardiac ct angiography can help identify features that most influence current success rates of pci. we report our experience using the reverse controlled antegrade and retrograde subintimal tracking technique under the aid of ivus, cardiac ct angiography for an ambiguous cto of proximal right coronary artery. |
the retina is a complex multilayered structure composed of two components, a photosensitive layer made of rods and cones and the neural connections, and the other part being the retinal pigment epithelium (rpe) and its basal lamina called bruch 's membrane, which helps to maintain the integrity of the barrier between the choroid and the retina. retinal degeneration occurs in different forms of retinal diseases including retinitis pigmentosa (rp), age - related macular degeneration (amd), glaucoma, and diabetic retinopathy. age - related macular degeneration (amd) is a complex disorder with multifactorial etiology affecting the macula of the eye and involves the retinal pigment epithelium (rpe), bruch 's membrane (bm), and choriocapillaris which results in progressive and irreversible loss of central vision. estimates from the world health organization (who) indicate that there are nearly 161 million visually impaired people worldwide, 37 million of which are blind, with a yearly increase of 1 to 2 million and that the number of people with amd will increase due to an increase in the ageing population. dry type of amd or nonexudative amd refers to the condition in which, due to advancing age, the retina accumulates waste material which leads to amorphous deposits termed as drusen and the retinal pigment epithelial cells degenerate leading to loss of central vision. wet type of amd or exudative amd refers to the condition in which new blood vessels from the choroid grow into the subpigment epithelial and subretinal spaces due to loss of integrity of the bruch 's membrane and these new blood vessels are leaky, leading to edema, which progressively disrupts visual function. in both types of amd, studies have shown that there is diffuse loss of photoreceptors in nonexudative amd and a severe loss of photoreceptors in exudative amd, both of which dominated by the loss of rods. with regard to the retinal neurons, it has been reported that loss of ganglion cells is profound (nearly 47% of the ganglion cells are lost in end - stage disease) in exudative amd while in nonexudative amd, the ganglion cell layer is preserved. though ganglion cells are preserved in nonexudative amd, it has been indicated that their functioning may not be normal. thus nearly all the layers of the retina are affected by this condition. from its first description in the medical literature in 1874, amd continues to be a disorder with no permanent solution [8, 9 ]. current therapeutic approaches to amd include thermal laser photocoagulation, surgical approaches like excision or displacement, photodynamic therapy and antivascular endothelial growth factor (anti vegf) therapies. the search for an ideal therapeutic approach that would help restore permanent vision in amd is continuing. in this regard, cell transplantation holds some of the greatest promise because it addresses the root cause of disease by replacing the dysfunctional cells with healthy ones. we will limit this review with regard to cell - based therapies for damage to the various retinal layers that occur mainly in the amd. development of regenerative strategies is usefully guided by studying the difference in the regenerative potential between organisms. retinal regeneration takes place in a variety of vertebrates including fish, amphibians, birds, and mammals, although it is restricted to certain stages of development in most animals. the urodelian amphibians have the ability to regenerate the whole retina even after complete removal of retina, the cellular source of regeneration being the rpe which transdifferentiates into retinal progenitor cells. the stem cell in the ciliary marginal zone contributes to the retinal regeneration in these animals. it has been shown that retinal regeneration occurs in the embryonic stages of anuran amphibians and avian embryos.. showed that xenopus laevis retains the ability to regenerate the retina and lens following the surgical removal of theses tissues even in adult animals. in adult animal species such as fish, amphibians, and birds, in addition to retinal stem cells or precursor cells in the ciliary marginal zone, the rod precursors or mller glial cells are also a source of regenerating retina. in teleost fish, retinal regeneration occurs even after surgical removal of a portion of the central part of the retina. intrinsic retinal regeneration, which occurs in fish and chick embryos by the formation of new retinal neurons from the progenitor / stem cells residing at the ciliary marginal zone, does not occur in mammals including humans. in mammals, however, in response to retinal damage, mller glia will proliferate and give rise to neuronal cells but the regenerative capacity of mller glia is limited in mammals than that in fish and birds. hence, there arises the search for alternative cell sources to regenerate the retinal cells when there is a cell loss. there are different cell sources for retinal cell transplantation and we hypothesize that the choice of cell source should depend on the level of damage in the retina that occurs due to amd. there are a range of cell sources ranging from adult retinal pigment epithelial cells, bone marrow stem cells (bmscs), fetal stem cells, embryonic stem cells (escs), induced pluripotent stem cells (ipscs), and so forth. in this short review, we herein describe the various cell sources, which can be considered for repair and regeneration of different grades of damage to the retina in amd, for optimal regeneration and efficient utilization of cell sources. when the damage is restricted to the rpe layer (figure 1), the choice we suggest would be rpe cells. in 1959, the first fetal retinal transplant into the anterior chamber of the eyes of rats was reported. cultured human rpe cells were transplanted into the eyes of monkeys, first with open techniques and methods and later with closed cavity vitrectomy techniques [1719 ]. the therapeutic potential of transplantation of the rpe was demonstrated at the royal college of surgeons in an animal model where a suspension of rpe cells labeled with carboxyfluorescein diacetate succinimidyl 5, 6-ester was injected in the subretinal space and the rpe cells were able to phagocytose the outer segments of photoreceptors. in 1991, peyman. transplanted rpe in humans but later, allogenic fetal rpe cell transplantation was tried in which immune rejection of the graft was a major problem. it has also been observed that the rejection rates were lower in dry amd than that in wet amd. autologous rpe transplantation is conventionally done employing two techniques, namely, rpe suspension and autologous full - thickness rpe - choroid transplantation [2226 ]. encouraging clinical outcomes has already been reported with the transplantation of the autologous rpe choroid from the periphery of the eye to a disease affected portion [25, 27, 28 ]. however, inability to transplant a uniform layer and formation of multilayered folds and contraction continue to be some of the challenges in rpe transplantation. rpe cell suspensions also might not survive in an aged or defective host basal lamina after transplantation. to overcome this difficulty, use of biologically derived basal lamina, amniotic membrane, descemet 's membrane, lens capsule and so forth the use of these biological scaffolds has the risk of biological contamination and disease transmission. hence, polymers both natural and synthetic have also been tried for growing rpe cells in the form of a layer and transplanting as a rpe scaffold layer construct for better in vivo survival characteristics and improving the efficacy. in this regard, an article by lee. explores the microprinting of retinal pigment epithelial cells and iris pigment epithelial cells onto lens capsules and coating inhibitory molecules on the lens surface to control the organization of the cells growing on them. in the case of autologous graft, the size of the full - thickness rpe graft that can be taken from the periphery of the same eye to patch the defect leads to insufficiency of the graft and for repeated rpe transplantation procedures, this approach is not feasible. recently, the concept of retinal pigment epithelial cell expansion has been reported, in which a synthetic polymer scaffold has been used to support proliferation of the retinal pigment epithelial cells in vitro. this approach could offer a potential solution for the quantity of cells required for transplantation wherein a small quantity of rpe cells can be expanded for patient use and also in allogenic transplantation, rpe cells from a single cadaver retina can be expanded for use in many patients. in a pilot study on 14 eyes (13 patients) with amd who underwent subretinal surgery for treatment of foveal choroidal neovascularization, transplantation of retinal pigment epithelium harvested from the nasal subretinal area of the same eye was performed and a best - corrected visual acuity and satisfactory reading vision between jaeger 1 and 4 were achieved in three eyes with no significant intraoperative or postoperative complications. in a prospective trial on 56 patients who had foveal choroidal neovascularization (fcnv), subretinal surgery combined with simultaneous transplantation of autologous rpe cells resulted in improved visual acuity in eyes which received rpe transplantation compared to eyes which had membrane excision alone. these results provide evidence that transplantation of rpe may be regarded as a reasonable treatment option for amd when the damage is limited to rpe layer. however, the rpe transplantation is not without limitations. first, the extensive surgical intervention apart from requiring technical skills followed by a learning curve has its adverse outcomes as well. the technique such as neural retinal bleb detachment technique has issues such as the surgeon 's view of the foveal structures may be hampered and forcing the surgeon to work using one hand through the neural retinal hole which might expand during repeated manipulations. moreover, the ability to separate the rpe layer alone without disturbing the choroid is another technical hurdle one has to keep in mind. the use of aged rpe cells whose function might not be optimal as that of young rpe cells is also of concern. also autologous rpe in amd may carry the same genetic information which might lead to recurrence of the disease. to overcome the limitations of the neural retinal bleb detachment approach, the use of a 180 degree retinotomy to create a pedicled graft is recently being followed with good outcomes [37, 38 ]. this technique, since it allows the neural retina to be folded nasally offers a better visual operative field for the surgeon. the use of ips cell technology to generate large numbers of rpe cells for transplantation along with the feasibility to correct the genetic disease in the rpe cells created using ips technology offers exciting arenas for future research. use of rpe transplantation as a prophylactic procedure in amd before neovascularization or geographic atrophy sets in can be considered as a step towards a permanent solution for amd. this is advantageous because rpe transplantation has shown to stabilize the retinal vessels and prevents neovascularization. this requires more refined surgical techniques, improved in vivo visualization, and research to develop rpe monolayers which will not be rejected after transplantation. as wet type of amd is more difficult to treat with rpe transplantation compared to dry amd, rpe cells themselves can be used as drug carriers for sustained release of anti - vegf agents to prevent neovascularization in amd. rpe transplantation can also be considered for patients who are unresponsive to anti - vegf therapies. these are some areas of research which are worthwhile looking into, as we progress further in the field of rpe transplantation. if the damage extends up to the rods and cones (figure 2), the choice of cell source would be bmscs as there have been studies which have demonstrated the differentiation of bmscs into photoreceptors [4345 ] and also due to the fact that bmscs are an established cell source for therapies. studies on animal models have shown that the injection of bmscs into the eye can potentially rescue injured retinal tissue. it has also been observed that intravitreally injected adult bone - marrow - derived hematopoietic stem cells stabilize and rescue retinal blood vessels that would ordinarily completely degenerate in these retinal degeneration animal models. in the retina, cellular differentiation in which the injected bmscs might transdifferentiate to cells of the retina or fuse with the host cells, the bone - marrow - derived microglial cell formation and activation, which might clear away the cellular debris produced by retinal damage, production of neurotrophic factors thereby repairing retinal damage, are the proposed mechanisms of retinal repair by bmscs. paracrine effects like increasing angiogenesis, decreasing inflammation, activating neighboring resident stem cells, antiapoptotic and chemotactic signaling, and beneficial remodeling of the extracellular matrix are also implicated in the mechanism of how bmscs contribute to retinal repair. clinical studies of intra - vitreal bmsc transplantation have been reported with the jonas. study in 2008 establishing safety of intra - vitreal injection of the autologous bmscs in a 43-year - old patient with diabetic retinopathy. in 2010, the same group reported a study of autologous bmsc intra - vitreal injection in three patients with diabetic retinopathy, age - related macular degeneration, and optic atrophy. however application of bmscs for wet type of amd is contraindicated, as the angiogenic potential of bmscs would further worsen the condition. in addition to bmsc, the photoreceptor transplantation is another potential option for grade ii damage particularly in wet amd where bmsc transplantation is not indicated. pearson. demonstrated that the transplanted photoreceptor precursors in rod deficient mice were able to form the classic triad synaptic connections and visual signals could be generated by these transplanted rod photoreceptors. were able to achieve integration of the transplanted photoreceptors across a range of inherited retinopathies. these recent reports on the transplantation of photoreceptor cells for improving the vision may also be considered as a choice for damage extending to the photoreceptors, as more evidences gather. though the above evidences are very much promising, when translating to human trials, one has to keep in mind the limitations of using bmsc derived photoreceptors or bmscs per se or precursors of photoreceptors for grade ii damage to the retina, as the reproducibility of the parameters such as synapses formation, posttransplantation viability of the cells, and their integration with other layers of retina might vary between animal models which are young having undergone a controlled damage versus a human patient of a chronic pathophysiology. when the damage extends to the retinal neurons (figure 3), a stem cell population, which has inherent capacity to give rise to neuronal lineage, is preferred. in this, we describe the various cell sources, which could give rise to the neurons of the retina. developmentally, the nonneural retina and the neural retina share common origin from the optic vesicle. experiments in chick embryos showed that the rpe cells in the eye, in explant, or in a dissociated cell culture can give rise to cells resembling retinal neurons when reprogrammed with appropriate regulatory genes involved in retinal neurogenesis. however, whether this can be applied to mammals can be answered only by future studies. since the rpe is located adjacent to the neural retina, rpe reprogramming, if successful in mammals, may offer an approach to repopulate the neural retina. several studies on reprogramming retinal pigment epithelium to differentiate into retinal neurons with sox2, neurogenin 1, ash 1, and so forth have been reported with success [5356 ]. recently it has been reported that a subpopulation of adult human rpe cells can be activated in vitro to a self - renewing cell, the retinal pigment epithelial stem cell (rpesc) that loses rpe markers, proliferates extensively, and can redifferentiate into stable cobblestone rpe monolayers. bmscs, especially hematopoietic stem cells (hscs), are another preferred source for generating neurons because mammalian bmscs have been observed to differentiate into neural cells in several in vitro and in vivo studies [5860 ]. it has been proven by studies that hscs retains the capacity to differentiate into cell types like oligodendrocytes progenitors, ependymal cells, neurons, and astrocytes. an animal study by sigurjonsson. proved that substantial proportions of adult human hscs differentiate into full - fledged neurons in lesions of the developing spinal cord in the chicken embryo. in a laser - induced bruch 's membrane rupture mice model of choroidal neovascularization, it was demonstrated that reconstitution of gfp+ hscs in lethally irradiated c57bl6/j mice resulted in gfp+ cells adopting the morphological and immunological characteristics of endothelial cells, pericytes, astrocytes, rpe, and macrophages, and the study also showed that these hscs participate in repair of the cnv lesion. in another study, intra - vitreal injection of mouse and human adult bone - marrow - derived lineage - negative hematopoietic stem cells resulted in neurotrophic rescue of retinal degeneration.. another choice would be human embryonic stem cells (hescs), as two prospective clinical studies have started recently to establish the safety and tolerability of subretinal transplantation of hesc - derived rpe in patients with stargardt 's amd and dry amd. the finding of both studies suggests that the there was no sign of hyper proliferation, abnormal growth, or immune mediated transplant rejection in the patients. the best corrected visual acuity improved from hand motions to 20/800 in the study on the patient with stargardt 's macular dystrophy, and vision improvement thus, human embryonic stem cells could also be considered for regeneration of the retinal layers when damage extends to the neurons. however, issues with hescs like immune rejection, risk of teratogenicities, and ethical barriers need to be overcome and safety has to be firmly established before they are brought into routine clinical procedures. there are several studies reported on generating retinal neural cells from induced pluripotent stem cells (ipscs). chen. demonstrated that ipscs derived from mouse fibroblast inherently express the retinal progenitor cells - related genes and overexpression of math5 with addition of noggin can help in generation of retinal ganglion - like cells from these ipscs. in another study by tucker., subretinal transplantation of retinal precursors derived from ipscs resulted in these cells taking up residence in the retinal outer nuclear layer leading to increased electroretinal function. thus, ipscs could also be a very good candidate for cell replacement therapy in amd when the damage extends to the neural cell layers. the study by zhao., which reported that in contrast to derivatives of escs, some cells differentiated from ipscs had an abnormal gene expression which can induce t - cell - dependent immune response in syngeneic recipients, and the studies, which showed that ips cells show rapid telomere shortening, dna chromosomal damage, and increased p21 expression that cause cell growth arrest, caution us from the use of ipscs clinically but future research will witness the generation of safe as well as viable human ips - derived somatic cells including rpe. it will also be worthwhile to consider the differentiation ability of neural stem / progenitor cells to retinal neurons which has been reported in studies. therefore, if the damage extends to the retinal neurons, any cell population capable of differentiating into neuronal lineage can be considered, provided the capability of the transplanted cells either pluripotent stem cells like the hescs, ipscs, or neurons generated from hscs or rpescs in forming neuronal connections with the lateral geniculate ganglion or higher areas in the visual cortex is proven first by appropriate in vitro experiments followed by in vivo studies which though might be a long way ahead according to the authors but is worth considering. in this short review, the authors place their thoughts on the choice of cell source for transplantation to treat retinal damage depending on the extent of damage. this is important in the wake of multitude studies that have been reported and are being reported on cell - based therapies for amd and other retinal diseases which creates a dilemma for the clinicians, and researchers to choose the right cell source. while a question may arise as to how the extent of damage can be assessed, it should be understood that the level of damage might not be uniform throughout the retina even in the same patient and even within the same eye. the tissue damaged most can be deciphered based on the following.pathogenesis of the disease, the fact that amd affects the macula and the rpe primarily, should be kept in mind and early disease versus late disease can help the clinician to hypothesize whether the damage would have involved the other layers too enabling further assessment. optical coherence tomography (octs) can give a picture of gross morphology of the macula. it gives an almost histopathological section of the retina and the area of tissue damage can be visualized using oct but the functional damage can not be assessed from this. newer octs are capable of providing high tissue definitions.auto fluorescence, red - free imaging, fluorescein, and indocyanine angiograms can also give information about the extent of damage to the rpe, the outer and inner blood retinal barrier, and a clue about accumulation of fluid within the retina and subretinal space which can be indirect indicators of tissue damage.functional assessment is possible to a reasonable extent in the early stages of the disease with electrodiagnostic tests such as the erg, multifocal erg, and the electrooculogram. pathogenesis of the disease, the fact that amd affects the macula and the rpe primarily, should be kept in mind and early disease versus late disease can help the clinician to hypothesize whether the damage would have involved the other layers too enabling further assessment. investigations that may aid to decide on the extent of damage are as follows. optical coherence tomography (octs) can give a picture of gross morphology of the macula. it gives an almost histopathological section of the retina and the area of tissue damage can be visualized using oct but the functional damage can not be assessed from this. newer octs are capable of providing high tissue definitions.auto fluorescence, red - free imaging, fluorescein, and indocyanine angiograms can also give information about the extent of damage to the rpe, the outer and inner blood retinal barrier, and a clue about accumulation of fluid within the retina and subretinal space which can be indirect indicators of tissue damage.functional assessment is possible to a reasonable extent in the early stages of the disease with electrodiagnostic tests such as the erg, multifocal erg, and the electrooculogram. optical coherence tomography (octs) can give a picture of gross morphology of the macula. it gives an almost histopathological section of the retina and the area of tissue damage can be visualized using oct but the functional damage can not be assessed from this. auto fluorescence, red - free imaging, fluorescein, and indocyanine angiograms can also give information about the extent of damage to the rpe, the outer and inner blood retinal barrier, and a clue about accumulation of fluid within the retina and subretinal space which can be indirect indicators of tissue damage. functional assessment is possible to a reasonable extent in the early stages of the disease with electrodiagnostic tests such as the erg, multifocal erg, and the electrooculogram. the caveats are that damage to one tissue does result in damage to the other. for instance, damage to rpe will cause retinal degeneration over time and damage to the photoreceptors in turn can cause ganglion cell loss. hence, it would be difficult to quantify the degree of damage to each of the tissue layers of the retina with certainty. however, importance is to be attached to the macula while assessing the extent of damage. thus the etiopathogenesis of the disease, the severity, and clinical evaluation will be able to provide an idea of the damage to the retinal layers. once the level of the damage has been assessed, the cell source most appropriate for regeneration of that particular layer must be chosen. the reason why rpe transplantation is suggested over other cell sources in case of damage mostly limited to rpe (grade i damage) is because of the fact that a cell source for transplantation is available from the same tissue of origin. it is logical to conceive that use of cell source from same the tissue of origin, that is, rpe itself, is safe and is expected to provide better outcomes compared to cells from distant tissue or origin. also rpe cells are a relatively mature population compared to other cell sources like bone marrow, embryonic stem cells, and so forth, which increase the safety and autologous transplantation of rpe from the same eye is also possible. choice of bmscs for damage extending to the photoreceptors (grade ii damage) would be based on the advantage of easy availability and accessibility in obtaining bone marrow and the fact that bmscs have been established as a promising source of stem cells in regenerative therapies for a variety of diseases and disorders including cardiac diseases, liver cirrhosis, neurodegenerative disease, and bone and cartilage diseases [6872 ]. transplantation of photoreceptors or photoreceptors from human embryonic stem cells or induced pluripotent stem cells presents a possibility but the difficulties associated with the isolation and culture of these cell types along with the ethical issues surrounding the use of embryonic stem cells should be considered. in the case of damage extending to the neurons, we have to consider using embryonic stem cells, hscs, ipscs, or cells with neuronal lineage differentiation capability as neuronal differentiation presents a complex phenomenon compared to retinal pigment epithelial or photoreceptor differentiation. this differential choice of cells for each grade of damage would help clinicians make the right choice in the appropriate situations as otherwise they may end up choosing inaccessible cell sources and cell sources with complex culture methodologies thereby leading to a waste of resources, time, and personnel involved in establishing that particular cell source for clinical application. though, at this point, rpe transplantation for grade i damage to the retina according to the authors is very much promising, the specific cell sources for grade ii and iii damages need extensive research, while keeping in mind that the varying grades of damage in the clinical settings of a human patient might not be reproducible in in vitro models or animal experiments. thus, this paper provides valuable suggestions for researchers and clinicians on the appropriate cell sources for different grades of damages of the retina because identifying the right source would be the first step for a truly successful cell therapy. we understand that though several cell sources have been described in this paper, only some of the cell sources have entered clinical trials. also among all approaches, the rpe transplantation approach has provided encouraging results and all other cell sources for repair, rejuvenation, or restoration of the degenerated retina need significant validation before they can be considered for clinical translation, which is worth an effort considering the light at the end of the tunnel. | background. age - related macular degeneration (amd) is a complex disorder that affects primarily the macula involving the retinal pigment epithelium (rpe) but also to a certain extent the photoreceptor layer and the retinal neurons. cell transplantation is a promising option for amd and clinical trials are underway using different cell types. methods. we hypothesize that instead of focusing on a particular cell source for concurrent regeneration of all the retinal layers and also to prevent exhaustive research on an array of cell sources for regeneration of each layer, the choice should depend on, precisely, which layer is damaged. results. thus, for a damage limited to the retinal pigment epithelial (rpe) layer, the choice we suggest would be rpe cells. when the damage extends to rods and cones, the choice would be bone marrow stem cells and when retinal neurons are involved, relatively immature stem cell populations with an inherent capacity to yield neuronal lineage such as hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells can be tried. conclusion. this short review will prove to be a valuable guideline for those working on cell therapy for amd to plan their future directions of research and therapy for this condition. |
rnrs of the cauda equina were first described by verbiest14) in 1954, and subsequently named by cressman and paul1) in 1968. rnrs is characterized by a tortuous, serpentine, large and elongated nerve root of the cauda equina10). it has been reported that rnrs develop as a response to lumbar spinal stenosis3,7,10). to our knowledge, most rnrs are associated with lumbar spinal stenosis, and the rnrs associated with lumbar disc herniation has not been reported until now. therefore, we present a rare case of unusual rnrs associated with lumbar disc herniation mimicking intradural disc herniation on magnetic resonance imaging study. a 50-year - old man who worked at a rice - cake shop has suffered from increasing low back pain and radiating pain on his left leg for five years. he had a nerve root block on l5 level five years ago, and the pain subsided temporarily. he was having conservative treatment but mild chronic pain still lasted and gradually aggravated and abruptly extended to the right leg a few days before his admission. a straight leg raise test was positive on the left side and the bilateral knee and ankle jerk was normal. sagittal and axial t2-weighted magnetic resonance image reveals broad base central disc protrusion at l4 - 5 level, and about 0.70.52.5 cm sized ovoid nonenhancing lesion in dorsal aspect of l5-s1, which seemed to be intradural disc herniation rather than intradural mass (fig. 1). although the image clearly demonstrated neither the classical ring enhancement of the intradural fragments on gadolinium contrast mri or discontinuity of the pll disrupted at the disc level, this finding was thought as an intradural disc herniation rather than an intradural extramedullary (idem) tumor such as meningioma. ct myelography showed central disc herniation at l4 - 5 and intradural lesion looked like intradural disc or intradural mass within the thecal sac below l4/5 (fig. a decompressive laminectomy was carried out at l4 - 5 levels and a herniated disc at l4 - 5 was removed. and we found the fragments of ruptured disc and adhesion with the dura. to determine whether there was a coexisting intradural disc rupture, we carried out an intradural exploration. after durotomy, a long, edematous and tortuous nerve root had forced out through a dural incision (fig. we explored the thecal sac, but there was no other intradural abnormality such as intradural disc herniation or intradural mass. after rearrangement of rnrs of the cauda equina to be released from the tangled position, the dura was carefully closed without tension. the rnrs of the cauda equina has been discussed sporadically in both the neurological and radiological literature. characteristically, the patients are men in their 4 to 6 decade, and the l3 to s1 nerve roots are most commonly involved4). rnrs were found in 42.3% of the patients with severe dural constriction and were seen on 8.2% of cadavers of aged people in autopsy10). low back pain, lumbar radiculopathy, and intermittent claudication are common presenting features3). associated bladder and sexual dysfunction can also occur3,6). the disorder is usually found in older patients and the onset of symptoms exhibits a long period10). some authors suggested that congenitally long nerve roots in a narrow canal create a block1,8). there are many reports on the relationship between rnrs and constriction of the spinal canal and the most substantial mechanism seems to be an acquired elongation of nerve roots due to mechanical trapping at the site of spinal stenosis2,3,4,10,13,14). the inhibition of normal sliding and mobility causes repeated stretching of the nerves, leading to elongation above the level of constriction. when positional changes (e.g., flexion) cause a cephalic pull on the nerve roots, they are caught at the constriction and are unable to slide through. this repeated stretch causes elongation of the roots above the block. when the nerve roots are relieved, the elongated segments relax and pile up in redundant loops above the block2). different from many cases of rnrs presenting above the stenosis, redundant nerve roots were below l4/5 level that had a broad base central disc protrusion in our case. we suppose that disc protrusion had an effect on the thecal sac and caused the rnrs. but, it is still a question why the rnrs developed below the block, and not above, and there is lack of sufficient analysis about the rnrs developed below the block in the previous study., the evaluations were mainly based on myelographic findings. though the appearance of serpiginous filling defects can be helpful in differentiating from other causes, it can also cause confusion occasionally, so it must be differentiated from vascular lesions such as arteriovenous malformation or dilated tortuous veins due to other causes2,3). arteriovenous malformations can also produce serpiginous filling defects, but usually are not associated with extradural blocks. they, usually thoracic in location, show no variability with positional change and often have large feeding vessels2,9). venous engorgement resulting from impaired venous drainage secondary to a block produces filling defects below the block. rnrs could also be diagnosed on sagittal t2-weighted mr images. in general, the positive rate of rnrs examined by the mr imaging was as high as 97.6% when the nerve roots appeared as loop - shaped lesion on myelograms, whereas the positive rate was only 23.5% when the nerve roots appeared as serpentine - shaped lesions. according to the previous study, magnetic resonance imaging appeared to demonstrate more advanced rnrs, and therefore mr imaging features of the rnrs are thought to represent important clinical findings7). unfortunately, mr imaging of the present case did n't show definite features of usual rnrs in spinal stenosis and it was not enough to exclude intradural herniation or intradural mass. untypically, the rnrs looked like intradural mass such as idem tumor, but the lesion was not enhanced unlike intradural mass. the rnrs had similar signal intensity to the cauda equina on both t1 and t2-weighted images but on enhanced images, it is observed as a non - enhancing lesion protruding on intradural space of l4 - 5. in addition, there was disc protrusion on l4/5 accompanied by non - enhanced intradural lesion just below l4/5. also, ct myelography showed central disc herniation at l4/5 and intradural lesion looking like intradural disc or intradural mass within the thecal sac below l4/5. it is possible to mistake the intradural lesion as the downward migrating intradural ruptured disc, so we performed the intradural exploration to confirm. to avoid misdiagnosis and unnecessary procedure, careful analysis and wide experiences are needed. early and sufficient decompressive laminectomy with duroplasty is recommended to relieve the nerve root compression before neurological deficits appear3,6). patients with severe dural constriction caused by lumbar spinal stenosis should be treated surgically before the nerve roots undergo irreversible damage10) and intradural exploration should be considered where there still exists a question of tumor or avm. we experienced untypical rnrs and the rnrs with lumbar disc herniation could have been misdiagnosed as the intradural disc herniation before operation. to avoid misdiagnosis, it needs to be notice that lumbar disc herniation could be causative of rnrs. so we report a rare case of rnrs associated with lumbar disc herniation mimicking intradural disc herniation. | redundant nerve roots (rnrs) is an uncommon clinical condition characterized by a tortuous, serpentine, large and elongated nerve root of the cauda equina. to our knowledge, most cases of rnrs are associated with lumbar stenosis, and rnrs associated with lumbar disc herniation has not been reported until now.here we present a rare case of unusual rnrs associated with lumbar disc herniation mimicking intradural disc herniation. |
whereas varicocele before puberty is rare, the prevalence of this disease in adolescence is equivalent to that in the general male population : 12.4 to 16.2%, with an average of 15%. in addition, the incidence of varicocele in male patients investigated for infertility is approximately 40%. varicocele can negatively and progressively affect the testicular growth, histology and function resulting in progressive decline in fertility. fifty percent to 60% of men who have had their varicocele treated show improvement in semen quality. in the last few years the present study compares the laparoscopic and conventional open methods for high ligation of varicocele. sixty - five consecutive patients who were referred to the urology department (group i) had open varicocelectomy by palomo 's technique. one hundred and twenty - eight patients (group ii), referred to the general and pediatric surgery departments, had laparoscopic varicocelectomy. the ages of the patients in group i ranged between 8 and 42 years, with an average of 24.4 years. those in group ii were between 8 and 39 years, with an average of 21.3 years. the majority of school age patients were asymptomatic, and the disease was discovered during routine medical check - up. while testicular pain and/or swelling were the main complaint among patients between 15 and 25 years of age, subfertility was the major presentation among those above 25 years of age. the diagnosis of varicocele was established mainly by clinical examination with the patient in upright position. the disease was graded according to the criteria published by lion. in the majority of cases, nine patients in group i (13.8%) and 21 patients in group ii (16.4%) had recurrent disease. sixty - two patients in group i (95.4%) and 109 patients in group ii (85.2%) had left - sided disease. bilateral varicocele was present in 3 patients in group i (4.6%) and in 19 patients in group ii (14.8%) (table 1). doppler ultrasound scan was done in all cases to confirm the diagnosis and to evaluate the testicular size pre- and postoperatively. for patients with subfertility, seminal analysis was done at least twice preoperatively, and then checked every 6 months for 18 to 24 months postoperatively. the examination included sperm count, the percentage of sperm motility each hour for four hours, and the presence of abnormal forms. results of semen analysis were evaluated according to the criteria of the world health organization laboratory manual. all patients in group i had high ligation of the dilated testicular veins by the technique described by palomo in 1969. laparoscopic varicocelectomy was performed with the patient in the supine or trendelenburg position (20 - 30 degrees head down). a urinary catheter was routinely inserted after the induction of anesthesia to evacuate the bladder. a veress needle for the creation of pneumoperitoneum was introduced into the abdomen through a supra - umbilical transverse skin incision. carbon dioxide insufflation was maintained through the veress needle at a rate of 1 - 2 liters per minute, and the intra - abdominal pressure was kept between 11 - 14 mm hg, depending on the patient 's age. after the withdrawal of the veress needle, a 5-mm trocar was inserted through the umbilical incision, connected to the carbon dioxide insufflator, and a 0 or 30 degree laparoscope was introduced into the peritoneal cavity. in all cases, the abdominal and pelvic viscera were examined in addition to the spermatic vessels and internal inguinal rings on both sides. for unilateral varicoceles, the working ports consisted of two 5-mm trocars introduced via a stab in the ipsilateral iliac fossa and in the contralateral iliac fossa in the midclavicular lines. the operator was standing on the contralateral side of the operating table using the working ports, while the assistant surgeon was standing on the ipsilateral side and controlling the laparoscope. after identifying the spermatic vessels, the overlying peritoneum was caught with a blunt grasper in the ipsilateral port. using a blunt endo - scissors in the contralateral port, a t - shaped incision was made parallel to and lateral to the spermatic vessels. the vascular bundle was then carefully grasped and dissected approximately 3 - 5 cm from the parietal peritoneum and underlying structures using a blunt endo - scissors or a diathermy hook. the testicular artery was not always searched for ; this was relatively more difficult in the pediatric age group. the testicular veins were clipped approximately 3 - 5 cm above the internal inguinal ring and then divided between the clips. at the conclusion of the procedure, in group ii, other procedures were concomitantly performed, including right orchidopexy in 14 patients (11%), repair of right inguinal hernia on 5 patients (4%) and removal of a chronically inflamed appendix in one child (0.8%). in group i, the average operative time was 38.5 minutes for unilateral cases and 69.5 minutes for bilateral cases. for laparoscopic varicocelectomy, the overall average of the operative time for the unilateral cases was 58.3 minutes and 74.2 minutes for bilateral cases. the average operative time for the first 20 operations was 69.8 minutes for unilateral operations and 92.5 minutes for bilateral cases. with increasing familiarity with the technique, the timings were gradually reduced in the remaining cases to 42.3 and 71.8 minutes, respectively (table 2). while no collateral veins were reported at operation in group i, retropubic collateral channels were identified in 8 patients (6.3%), and lateral collaterals were found in 22 patients (17%) during laparoscopic varicocelectomy. all the collateral veins were interrupted laparoscopically using either clipping or diathermy coagulation, according to their sizes. the testicular artery was detected in 49 patients (75.4%) in group i and in 120 patients (93.8%) in group ii. three patients (2.3%) had pneumoscrotum, which has spontaneously resolved within 24 - 48 hours. the postoperative hospital stay for patients in group i was 2 - 8 days, with a mean of 3.5 days. in group ii, the postoperative hospital stay was shorter, 1 to 3 days, with a mean of 1.3 days. one patient in each group developed wound infection that responded to systemic antibiotics. almost all wound complications in group ii occurred at the site of the umbilical port. scrotal edema occurred in 11 patients in group i (17%) compared to only 3 patients in group ii (2.3%). postoperative wound pain was subjectively evaluated among patients above the age of 12 years, and its intensity was estimated on the basis of the number of narcotic injections administered during the first 24 hours after surgery. while all patients in group i required one or more narcotic injections, 87% of those in group ii did not require any narcotic injection (table 3). five children (3.9%) in group ii had mild postoperative shoulder pain, which was gradually resolved in 24 hours. patients who had open surgery returned to their normal activities after 7 - 14 days, with a mean of 8.9 days compared to 3 - 7 days for those who had laparoscopic varicocelectomy, with a mean of 4.5 days. ipsilateral hydrocele developed in three patients in each group (4.6% and 2.3% respectively). seven patients in group i (10.8%) and five patients in group ii (3.9%) had recurrence of the disease within 6 to 18 months postoperatively. no testicular atrophy was reported in any case in the study, regardless of whether the testicular artery was clipped or not. this was based on the postoperative seminal parameters and the measurement of testicular size. seminal analysis was done for 21 patients in group i and 45 patients in group ii. according to the results of the preoperative semen analyses, the postoperative seminal parameters were classified into : improved, no change, or improvement of seminal parameters after varicocele ligation was observed in 43% of cases in group i and in 51% of cases in group ii (table 4). varicocele has generally been attributed to the absence or incompetence of valves in the internal spermatic veins. however, with the help of spermatic venography, bypassing collateral channels have been found in about 20% of patients with varicocele despite competent venous valves. in the present study, the sole indication for surgery in the present study was the presence of varicocele, even when asymptomatic. this was based on the concept that early correction of varicocele will alter not only the progressive decline in fertility but will also prevent future infertility in younger male patients. the relatively higher rate of reversal of the seminal parameters (51% vs 43%) and the fewer incidences of recurrent varicocele (3.9% vs 10.8%) after laparoscopic varicocelectomy can be attributed to better visualization and access provided by the laparoscopic approach. the mean operative time of laparoscopic varicocelectomy reported in the present series was similar to that reported by donovan and winfield and tan. technical failures and problems with the laparoscopic instruments constituted the major causes of prolonged operative time for laparoscopic varicocelectomy. furthermore, adhesions of the bowel to the parietal peritoneum of the groin were found in 19 out of 21 patients with recurrent varicocele after open surgical varicocelectomy. neither obesity nor the presence of bilateral or recurrent disease was a problem for performing varicocelectomy via laparoscopy. although laparoscopic varicocelectomy has been performed by many surgeons on a day - surgery basis, the mean hospital stay after laparoscopic varicocelectomy in our study was relatively longer than was anticipated. almost all our patients prefer to remain in the hospital and do not wish to resume activities until complete pain relief. additional factors that contributed to prolonged hospital stay were postoperative wound complications and the performance of additional operative procedures, particularly inguinal hernia repair. the hospital stay after laparoscopic varicocelectomy was not affected by whether the disease was unilateral or bilateral. similar to other studies, we did not find any significant difference between testicular artery ligation and preservation during varicocelectomy. laparoscopic varicocelectomy is a minimally invasive procedure that is easy to perform with simple instruments. not only can the bilateral varicocele be easily dealt with through the same ports, but other procedures, such as hernia repair and orchidopexy, can also be simultaneously performed. the clear visualization and magnification it provides facilitate control of the affected vessels and enable detection of abnormal collateral channels, one of the major reasons for postoperative recurrence. compared to the open technique, laparoscopic varicocelectomy has minimal postoperative morbidity, shorter convalescence and a faster return to normal activities. therefore, we recommend that the laparoscopic technique for varicocele ligation replace the conventional open method. | the purpose of this study is to determine the relative advantages of laparoscopic varicocelectomy compared to the conventional open high ligation of palomo. we studied 193 patients who presented with varicocele. while 65 patients were treated by open high ligation of the testicular veins, 128 patients had laparoscopic varicocelectomy. in addition to varicocele ligation, 14 patients (11%) had laparoscopy - assisted right orchidopexy, and 5 patients (4%) had laparoscopic repair of concomitant right inguinal hernia. the mean hospital stay was 3.5 days and 1.3 days, respectively, and the recurrence rates were 10.8% and 3.9%, respectively. return to normal activity was significantly earlier in group ii (mean 4.5 days) compared to group i (mean 8.9 days). there was no incidence of testicular atrophy in any case in the study, regardless of whether the testicular artery was ligated or preserved during surgery. we conclude that laparoscopic varicocelectomy is safe, effective and minimally invasive. in addition to its better cosmetic results and advantage in case of bilateral disease, it allows excellent exposure and control of the affected vessels. furthermore, the shorter hospital stay and the earlier return to normal activities are very important advantages in recommending this technique as an efficient alternative to the open surgical method. |
the bethesda system for reporting thyroid cytopathology (tbsrtc) was introduced in 2007 in an attempt to standardize international terminology and to categorize morphological criteria in fine - needle aspirations (fnas) from patients with thyroid nodules. fna is currently the preferred screening test for guiding the diagnosis and treatment of thyroid nodules. tbsrtc establishes six diagnostic categories for fna results and assigns a malignancy risk and recommendations for patient management for each category. global studies of the incorporation of tbsrtc in diagnostic algorithms for patients with thyroid nodules have concluded that tbsrtc reduces unnecessary thyroidectomies while also ensuring the quality of thyroid malignancy detection. in colombia, the health system provides the existence of general care hospitals and hospitals specializing in the care of patients with cancer. in both types of hospitals, there are trained cytologists and pathologists for the diagnosis of the most common diseases, and it is supposed that an oncologic pathologist outperforms the general pathologist in the cytological diagnosis of thyroid samples. few studies have been published on the implementation of the tbsrtc in colombian health institutions and its diagnostic correlation with final histopathological results. the objective of this study was to determine the correlation between diagnoses made based on fna results interpreted using tbsrtc and the final histopathological diagnosis for patients at oncology hospitals (ohs) and non - oncology hospitals (nohs). a cross - sectional study was designed using fna results of patients with thyroid nodules classified based on the tbsrtc for whom histopathological reports on the resected surgical lesion were available (n = 196) and who were treated between 2010 and 2012. the study included 104 (53%) patients at an oh, in bogot, and 92 (47%) patients at nohs in bucaramanga and bogot, colombia. the histopathological reports on partial or total thyroidectomies of patients who participated in the study were diagnosed based on the world health organization (who) 2004 classification for tumors of the thyroid gland. frequency measures are reported for the categorical variables and central tendency and dispersion measures are reported for continuous variables. in addition, frequency tables were constructed for each category in tbsrtc for comparison with the final histopathological report. frequency measures are reported for the categorical variables and central tendency and dispersion measures are reported for continuous variables. in addition, frequency tables were constructed for each category in tbsrtc for comparison with the final histopathological report. a total of 196 patients were included in the study, 174 of whom were women (88.78%), with a mean age of 50.8 years (range : 14 - 88 years). characteristics of the study population the following fna results by tbsrtc category were obtained for the total population : non - diagnostic or unsatisfactory (nd / uns), 4.1%;benign, 23.5%;atypia of undetermined significance or follicular lesion of undetermined significance (aus / flus), 2%;follicular neoplasm or suspicious for a follicular neoplasm, 16.8%;suspicious for malignancy (sm), 37.2% ; andmalignant, 16.3%. when grouped by oh and nohs, more categories v, sm and vi, malignant (68.3%) and less category ii, benign (6.7%) were diagnosed at the oh, whereas more benign fna results (42.4%) were obtained at the nohs, followed by category v, sm (37%) and category iv, follicular neoplasm or suspicious for a follicular neoplasm (18.5% ; table 2). non - diagnostic or unsatisfactory (nd / uns), 4.1% ; atypia of undetermined significance or follicular lesion of undetermined significance (aus / flus), 2% ; follicular neoplasm or suspicious for a follicular neoplasm, 16.8% ; suspicious for malignancy (sm), 37.2% ; and malignant, 16.3%. when grouped by oh and nohs, more categories v, sm and vi, malignant (68.3%) and less category ii, benign (6.7%) were diagnosed at the oh, whereas more benign fna results (42.4%) were obtained at the nohs, followed by category v, sm (37%) and category iv, follicular neoplasm or suspicious for a follicular neoplasm (18.5% ; table 2). fna results and histopathology based on type of hospital about the histopathological diagnoses, 64.3% were malignant and 35.7% were benign. the most frequent benign diagnosis was goiter (20.4%) and the most frequent malignant diagnosis was papillary carcinoma (59.7%). when the results were compared based on type of hospital, the oh made a final malignant histopathological diagnosis in 83.6% of cases, whereas nohs made a final malignant diagnosis in 42.4% of the cases [table 2 ]. when the results of fna and histopathology were compared, fna categories v and vi were associated with a histopathological malignancy frequency of 90.4% and 100%, respectively (this last category was not used for any fna at the nohs), while category ii, benign, was associated with a benign histopathological diagnosis in 86.9% of all cases. with respect to category iv, follicular neoplasm or suspicious for a follicular neoplasm, 39.4% of cases were associated with a final malignant histopathological result [table 3 ]. fna frequency and histopathological final diagnosis based on institution a comparison of fna with histopathology results based on institution type revealed that fna category v was associated with a higher frequency of final malignant histopathological results at the oh (oh 97.4% vs. nohs 82.3% ; p = 0.03). for benign category ii fna, no significant difference between type of institution was observed when correlations between final benign and malignant histopathological results were compared (oh : 85.7% benign and 14.3% malignant vs. nohs : 87.2% benign and 12.8% malignant ; p = 0.6). for a fna follicular neoplasm or suspicious for a follicular neoplasm category iv, a greater proportion of benign results were obtained at the nohs (76.5% nohs vs. 43.8% oh), and a greater proportion of malignant histopathological results were obtained at the oh (56.3% oh vs. 23.5% nohs ; p = 0.05) [table 3 ]. thyroid carcinoma is the most frequent endocrine neoplasm ; it presents at all ages and predominantly affects women. based on the globocan 2012 results, the estimated incidence per 100,000 persons per year of thyroid cancer varies from country to country : the incidence is 13.2 in the united states, 8.4 in france and 5.1 in colombia, clearly indicating regional differences in the incidence of thyroid cancer and the need to establish diagnostic correlations between fna and histopathology for each country. according to the national institute of cancer, colombia, thyroid carcinoma is the fifth most frequent malignant neoplasm in colombia. when used, analyzed and interpreted correctly, fna reduces the number of unnecessary thyroidectomies in patients with benign nodules and is highly sensitive for diagnosing patients with malignant thyroid tumors. tbsrtc is a unified system that complements cytological findings from thyroid fna and represents a consensus among multiple experts in different areas of medicine. in colombia, implementation of tbsrtc at a single institution that specializes in the treatment of cancer patients revealed an 89% correlation with the final histopathological report for multiple malignant lesions ; however, the sample size and the population selection bias limits the external validity of these results. no data are available on the diagnostic correlation between tbsrtc and the final histopathology report in nohs in colombia and whether there are differences in the implementation of tbsrtc and diagnostic accuracy among pathologists and cytologists with more training in the diagnosis of cancer at oh and those trained in diagnosing general diseases at nohs. in this study, histopathology and cytopathology results were compared for the same thyroid lesions in patients at three different health institutions : one oh and two nohs. all institutions participating in the study used tbsrtc for the interpretation of fna ; however, at the nohs, cytopathologists did not use category malignant in their fna reports regarding the characteristics of the study population in these hospitals, which have a relatively low frequency of carcinomas, producing distrust of the use of the category. we do not take into account the percentage of incidental papillary carcinoma in the resections for analysis. histopathological and cytopathological diagnoses of benign lesions were frequent in the nohs, whereas diagnoses of malignant neoplasms were more frequent in the oh. comparison of the distribution of the samples in each tbsrtc category with previous reports reveals similarities in the proportion of fna category iii aus / flus (2% vs. 3 - 3.2%). these results reflect a strict adherence to the established criteria for this category and contradict the general belief that this category represents a catchall for those cases that can not be classified as any other category. however, the same comparison for category iv, follicular neoplasm or suspicious for a follicular neoplasm, reveals that the proportion varies from 5.5% and 11.6% in the literature, while the value in the present study was greater, 16.8% (oh 15.4% vs. nohs 18.5%). of these cases, 39.4% were associated with a final malignant histopathology report, the majority of which were the follicular variant of papillary thyroid carcinoma, and a statistically significant difference was noted between the oh and nohs for the final malignant histology diagnosis in this category (56.3% oh vs. 23.5% nohs ; p = 0.05). differences in these categories are likely due to a cognitive bias of pathologists, i.e., the percentage of benign tumors is greater in nohs when using this category and the percentage of malignancies is higher in oh. in a meta - analysis that specifically evaluated category iv before and after the implementation of tbsrtc, an increase in the utilization of this category (6.1% to 7.4%, p = 0.0002) and an increase in the final frequency of histopathological malignancy (22% to 28%, p = 0.03) was observed, indicating the difficulty of differentiating neoplasms with follicular morphology from benign samples and confirming the necessity of histopathological examination for a final classification. our data differ from those in the literature for the relative frequencies of categories ii, benign (23.5 vs. 64.6 - 73.8%), v, sm (37.2% vs. 1.3 - 2.6%) and vi, malignant (16.3% vs. 5.2 - 7.5%). the distribution of cases in this study indicates a greater proportion of malignancies and a reduced proportion of benign results. as the incidence of thyroid carcinoma is 5.1 per 100,000 persons per year in colombia, this high rate of malignancy may be attributable to the population selection bias. the proportion of category i, nd / uns cases in this study is lower (4.1 vs. 10.4 - 11.1%) than that reported in similar studies ; however, the proportion of cases treated in the oh was greater than in the nohs (6.7% vs. 1.1%). tbsrtc assigns a risk of malignancy to each category in an ascending manner. in our series, this tendency is preserved, but the frequency of malignancy was greater for all categories when compared with tbsrtc, particularly for category iii, aus / flus (75% vs. 15% of tbsrtc ; table 4). notably, only four of 196 fnas were included in this category and, of these cases, three patients were judged to have carcinoma as the final histopathological report. the results of recently published studies demonstrate that the malignancy frequency for nodules classified as category iii aus / flus is greater than that estimated by tbsrtc, and a review of the guidelines and clinical protocols for tbsrtc is advised. comparison between the bethesda system overall categories and the colombian series : frequency of nodules included in each category and absolute risk of malignancy in conclusion, for the interpretation of aspiration cytology of thyroid lesions in colombia, tbsrtc is a useful tool that enhances the diagnostic accuracy of fna, exhibits an adequate diagnostic correlation with the final histopathological examination and enables a comparison of results between different institutions. significant differences in the diagnostic correlation of fna between the oh and the nohs in the study were observed, specifically for the percentage of malignancy in categories iv and v. it can be caused by selection bias of the population that consults each type of hospital and also by a cognitive bias of the pathologists who favor malignancy or benignity based on their own workload. | aim : to determine the correlation between the results of thyroid fine - needle aspirations interpreted using the bethesda system and final histopathological reports for patients at an oncology hospital (oh) and non - oncology hospitals (nohs).materials and methods : a retrospective, cross - sectional, descriptive study was performed to compare the cytology and histopathology results for patients with thyroid nodules in three colombian hospitals. the final correlation of diagnoses between the two methods is reported. in colombia, the health system provides the existence of general care hospitals and hospitals specializing in care of patients with cancer.results:a total of 196 reports were reviewed, of which 53% were from oh and 47% were from nohs. a greater proportion of category v (37.5%) was diagnosed at the oh, whereas nohs diagnosed a greater proportion of category ii (42.3%). the global correlation between diagnoses made using cytology and histopathology was 93.3% for categories v and vi (based on the final malignant diagnosis) and 86.9% for benign category ii. significant differences between institution types were observed when category iv and v and malignant histopathology were compared (56.3% oh vs. 23.5% noh ; p = 0.05 for category iv, 97.4% oh vs. 82.3% noh ; p = 0.03 for category v), while no significant difference between institution types was observed when category ii and final benign diagnosis were compared (p = 0.6).conclusions : the bethesda system for thyroid cytology correlates adequately with final histopathological diagnosis in colombia. significant differences were identified in the diagnostic correlation for malignant lesions between the oh and nohs in categories iv and v caused by selection bias of the population. |
since many years, a great number of studies have been discussing the role of keratinized gingiva (kg) in marginal periodontal behavior, either accepting that a minimum of 2.0 mm of kg width is required to maintain marginal periodontal health [16 ] or suggesting that kg width is negligible if excellent oral hygiene is performed [715 ]. areas with narrow width of kg seem to be prone to periodontal attachment loss, which could result in recession of the gingival margin in the presence of risk factors. lang and le have demonstrated that such areas show clinical signs of inflammation even in the absence of dental plaque, as evidenced by an increase in gingival crevicular fluid (gcf) flow rate, which suggests that tissue behavior is mainly associated to pathological more than to physiological processes under these conditions. an increased gcf flow rate from gingival sulcus is an early sign of clinical inflammation [1620 ]. in the absence of dental plaque, the increase of gcf flow rate in areas presenting < 2.0 mm of kg width could be influenced by a close proximity of the dentogingival unit to the alveolar mucosa, which is more permeable and mobile to allow primary defense against microorganisms and its products [5, 17 ]. some factors can explain the increase in gcf flow rate, including (i) increase in tissue hydrostatic pressure close to junctional epithelium induced by bacterial plaque accumulation [18, 19, 21 ] ; (ii) mobility of gingival margin and increase in marginal blood flow caused by mechanical stimulus from tooth mobility, frenum pull, toothbrushing, and chewing ; (iii) histamine intravenous injection or development of inflammation. these findings suggest that chemical or mechanical irritation is necessary to the production of gcf [21, 24 ]. gcf is considered to develop an important protective role on the defense mechanisms of gingival sulcus through the presence of defensive substance, such as pmn - neutrophils, and through mechanical properties, such as the flushing action, capable of removing carbon particles and bacteria from the gingival sulcus [20, 2428 ]. taken together, these findings suggest that the absence of gcf in the absence of mechanical stimulation would represent gingival health, while its presence in the absence of mechanical stimulation would represent gingival inflammation. to the best of our knowledge, no study has investigated if there are differences in gcf flow rate in areas with a narrow or wide width of keratinized gingiva, aiming at determining if a wider width of keratinized gingiva would allow a better dissipation of gcf. considering so, the aim of this study was to investigate the role of kg gingiva in the homeostasis of gingival margin by evaluating gcf flow rate in healthy sites before and after a mechanical stimulus. this study was approved by the committee of ethics in research of school of dentistry at bauru - usp (no. the sample was selected according to the following inclusion criteria : presence of a buccal healthy site showing 2.0 mm width of kg at bicuspids or molars and a homologous site presenting < 2.0 mm of kg ; good systemic health ; optimal oral hygiene status. it was excluded from the study pregnant or lactating women, patients prescribed with restricted drugs or antibiotics in the 6 and 3 months previous to data collection, those taking medicines capable of inducing gingival hyperplasia (e.g., calcium channel blockers, cyclosporine, and anticonvulsants), smokers, drugs, or alcohol abusers. a total of 60 patients were initially examined, but only 16 were found in accordance to inclusion and exclusion criteria (figure 1). group 1 was composed of 16 strictly healthy premolar and molar buccal sites showing 2 mm of kg width, and group 2 was composed of 16 strictly healthy premolar or molar buccal sites showing < 2 mm of kg width in the same patients, in a split - mouth design. in order to assure the absence of gingival inflammation, all patients were submitted to scaling and root planning and oral hygiene instruction before data collection. the reliability of these conditions was assessed by the evaluation of plaque index, sulcular bleeding index, and probing depth, measured with a millimeter periodontal probe (hufriedy, chicago, usa). kg width was measured by a digital caliper (727 me, wit, brazil) as the distance from gingival margin to mucogingival junction at buccal sites of the selected teeth, after staining of the tissues with a schiller solution (figure 2). all clinical examinations and procedures were under the responsibility of a single - trained examiner. clinical measurements were performed 2472 hours before gcf collection to avoid any interference on the gingival sulcus physiology. the collection of gcf was accomplished by imbibing of periopaper strips (oralflow, ny, usa) as proposed by lee and holm - pedersen. after preventing the contact of the tooth by the tongue the paper strips were closely fitted over the tooth crown and the buccal soft tissue surface, extending up to the alveolar mucosa (figure 3(a)), allowing the imbibing of the strips by fluid exuding through the bordering edge of alveolar mucosa (p1a) and through the gingival margin (p1b). gcf was also collected at superficial position (p2), at the entrance of gingival crevice (figure 3(b)), and deep intracrevicular position (p3), in which the strip was introduced to the base of gingival crevice (figure 3(c)), as determined by minimal tactile resistance upon its introduction [22, 25 ]. paper strips were kept in position for 60 seconds, allowing the imbibing of paper strips in a standardized period, as reported before [31, 32 ] and supported by the results obtained in a pilot study involving 3 patients (data not shown). gcf was collected in two different moments : before and after chewing a fresh fibrous food meal (cooked bovine steak) for 10 minutes. collection of gcf before chewing was performed 2472 hs after clinical measurements, in a rested position. after 24 hours, patients were recalled and eat the cooked fibrous meal during 10 minutes. the strips were then heat air dried and imbibed in a 2% alcoholic solution of ninhydrine, which provides comparable results to electronic devices and again heat air dried. comparisons between groups before and after chewing stimuli were analyzed by unpaired t - test. intragroup comparison of gcf collection before and after chewing stimuli was analyzed by paired t - test. a 95% confidence level was established for all statistical analysis (= 0.05). periodontal status of test and control sites before collection of gcf is shown at table 1. no differences were found between groups in probing depth, plaque index, and bleeding index. significant differences were observed between groups only in kg width (p < 0.05). the amount of gcf collected from groups 1 and 2 sites before and after chewing stimuli is described in table 2. significant differences between groups were found at p1a before mastication stimulus and at p2 and p3 after mastication stimulus. intragroup analysis by paired t - test showed significant increases in gcf at p1b and p3 in group 1 and at p1a in group 2. the results obtained in this study have shown that tissue fluid transudates rather through gingival sulcus than through alveolar mucosa in areas presenting at least a 2 mm kg width, while areas presenting lesser than 2 mm of kg width show transudation of tissue fluid by alveolar mucosa. these findings suggest that the protective role [16, 21, 2428 ] exerted by gcf is compromised in areas with a narrow width of kg. this study is important because no previous reports have evaluated the role of kg in the homeostatic response of marginal periodontal under strictly healthy and physiological conditions. miyasato, crigger, and egelberg also evaluated the homeostatic response of 16 dental students or members of the dentistry faculty showing appreciable (2 mm) or minimal amount of kg (< 1 mm) in plaque - free contralateral or unilateral sites and showed that both areas showed minimal amounts of gcf in a resting state, as observed in the present study. when plaque was allowed to accumulate, a significant increase in gcf flow rate was observed for both groups, with no differences between groups, as gingival inflammation developed, but no mechanical stimulus was performed to induce gcf flow rate. gcf flow rate is usually a measure of gingival inflammation, since its exudation increases in the presence of inflammation and either is not present or present in small quantities in healthy situations [16, 1921, 28, 29, 34 ]. indeed, the volume or resting gf increases as periodontal pockets develop [20, 34, 35 ]. while in healthy sites gcf represents a transudate of interstitial tissues, in the course of gingivitis and periodontitis, it is transformed into a true inflammatory exudate [19, 20 ]. in this context, the relevance of gcf in the control of a subgingival microbiota compatible with periodontal health should be emphasized [17, 20, 25 ]. no differences between groups were observed at gingival margin (p1b) and superficial (p2) and deep (p3) intracrevicular positions before chewing stimuli. this can be possibly explained by the fact that in clinically healthy conditions the amount of gcf transudation through gingival sulcus is minimal [16, 1921, 28, 29, 34 ], which might account for differences between the present study and others published in literature [1, 16 ]. however, a significant difference between groups was found at p1a (alveolar mucosa edge), indicating that, in a resting position, dissipation of tissue fluid occurs mainly by alveolar mucosa, which is more permeable and mobile to allow metabolic interchange. this result also seems to indicate that some tissue fluid from alveolar mucosa can dissipate toward the gingival sulcus in narrow kg, suggesting that the greater the distance from gingival sulcus to alveolar mucosa, the lesser the influence of alveolar mucosa on the physiological behavior of gingival sulcus. according to siegel, the identification of an increased flushing of tissue fluid through the epithelial barrier of the alveolar mucosa is highly suggestive of the permeability of this tissue. after mechanical stimuli provided by chewing a fibrous meal, a significant increase in transudation of gcf through gingival sulcus was observed at p1b and p3 positions for sites with 2 mm width of kg, with a trend to increase also at p2, although not statistically significant. the same stimulus resulted in increase in transudation of tissue fluid trough alveolar mucosa (p1a), while the amount of gcf collected from gingival sulcus (p1b, p2 and p3) suffered minor not significant variations in sites showing < 2 mm of kg width. these results are in agreement with other studies [1618, 20, 22, 25 ] showing that mechanical stimulus is capable of increasing gcf flow rate even in healthy areas. these findings also indicate a more pronounced permeability of alveolar mucosa than kg, which allows the crossover of substances from the inner to the outer environment and vice versa. this might be relevant in the control of periodontal homeostatic response through an early recognition of potentially aggressive bacterial antigens. the increased fluid rate observed at p1a position implies in increased alveolar mucosa transudation influx, probably related to the tissue blood supply necessary to accomplish the metabolic demands for functional tissue mobility. besides, the increase in gcf flow rate at gingival sulcus in group 1 reflects the role of gcf in flushing the gingival sulcus, corroborating the results obtained by previous studies [16, 17, 20, 25, 26 ]. this protective role was not observed in group 2, since gcf flow rate remained the same after chewing, suggesting that a wider area of kg is more compatible with marginal periodontium homeostasis, playing a regulatory role on the control of concentration and distribution of gcf flow rate [16, 17, 22, 25, 37, 38 ]. these results might explain why areas showing 2 mm of kg width are more compatible with marginal periodontal health than areas < 2 mm wide, which requires a strict maintenance program in order to prevent plaque accumulation [18, 11, 14 ]. kennedy. found that gingival health could be maintained in patients under professional plaque control but not in those without professional care in areas presenting an inadequate kg width. in their study, patients previously submitted to free gingival autografts procedures that did not participate in the supportive periodontal treatment for a 5-year period have not shown significant gingival inflammation and/or recession overtime, contrariwise to those portraying inadequate kg. from these results, it can be assumed, as in the present study, that the existence of an adequate kg width is a natural requirement to allow a reliable homeostatic response of the marginal periodontium when the patient performs personal routine dental plaque control. in the absence of dental plaque, the increase in flow rate of gcf in areas < 2 mm in width could be influenced by a close contact between the dentogingival unit and the alveolar mucosa, which is more permeable and mobile, thus allowing primary defense mechanisms to be activated against bacterial challenge. additionally, the increase in tissue hydrostatic pressure close to the junctional epithelium may contribute to a more pronounced flow rate of gcf in narrower areas [1821, 39, 40 ]. since the attached gingiva is a fraction of keratinized gingiva and extends up to the boundaries of the mucogingival junction, these areas must be capable of neutralizing the tension transmitted by the alveolar mucosa under muscle action in such a way that marginal gingival mobility is prevented. this study also showed that the mastication of natural fibrous food produces alterations in gcf flow rate which can vary according to the width of kg. this behavior could be engaged to the dental mobility induced by chewing, that would transmit a stimulus to gingival blood vessel walls through oxytalan fibers [4145 ], giving rise to an increased blood vessel transudation, which in turn would be related to the changes in gcf flow rate [18, 19 ]. additionally, the mobilization of gingiva can also be attributed to food excursion and/or functional demands of the alveolar mucosa, since both inadequate and adequate kg groups experienced significant variation in gcf flow rate, more pronounced in areas presenting an adequate width of kg. the main limitation of this study is the small number of subjects included in the study, due to difficulties in finding patients who presented homologous sites with or < 2 mm of kg width and did not present any of the exclusion criteria. this might explain why no significant differences were found at p2 position before and after chewing in group 1, since an increase in gcf was noticed. by the other side, this was a split - mouth design study, which implies that test and control sites were in the same patients. all patients were submitted to professional plaque control by scaling and root planning and prophylaxis, and oral hygiene instruction in order to assure that all patients were clinically healthy at the moment of gcf collection. these results suggest that kg plays a definite role in controlling the physiology of gingival sulcus, allowing the transudation of gcf through the gingival sulcus, resulting in an adequate protective role essential to the maintenance of gingival health and periodontal homeostasis. the results obtained in this study suggest that a wider area of keratinized gingiva favors physiological behavior of the gingival sulcus by a better dissipation of gcf ; a closer proximity of gingival margin and alveolar mucosa influences the dissipation of tissue fluid through alveolar mucosa, which is more permeable and mobile, impairing primary defense of gingival sulcus by the concentration of gcf. | objective. to shed light on the role of kg, its influence on periodontal behavior was investigated. methods. tissue fluid transudation was assessed in alveolar mucosa (p1a), outer gingival margin (p1b), at entrance of (p2) and within gingival sulcus (p3), before and after chewing of fibrous food in 16 patients portraying 2 mm kg at one tooth (group 1), and < 2 mm at another homologous tooth (group 2). results. there was a significant increase in gcf after chewing at p1b and p3 in group 1 and at p1a in group 2 (t - test, p < 0.05). conclusions. the results suggest that kg plays a role in marginal periodontal homeostasis. |
resorption is defined as a condition associated with either a physiologic or a pathologic process resulting in loss of dentin, cementum or bone. andreasen has classified tooth resorption as internal (inflammatory, replacement) and external (surface, inflammatory and replacement). internal root resorption is the progressive destruction of intraradicular dentin and dentinal tubules along the middle and apical thirds of the canal walls as a result of clastic activities. it is seen as a radiolucent area around the pulpal cavity, usually of incisors and mandibular molars. the various etiological factors suggested for internal root resorption include traumatic injury ; infection and orthodontic treatment. resorption occurs in two stages : degradation of the inorganic mineral structure followed by disintegration of the organic matrix. internal inflammatory resorption involves progressive loss of dentin, whereas root canal replacement resorption involves subsequent deposition of hard tissue that resembles bone or cementum but not dentin. clinically, the condition is usually asymptomatic, however, it may include the presence of a reddish area radiographs are mandatory for diagnosing internal resorption, which reveals a round - to - oval radiolucent enlargement of the pulp space. the margins are smooth and clearly defined with distortion of the original root canal outline. internal resorption can be detected by : visual examination based on changed color in tooth crown, radiographic diagnosis, conventional and cone beam computed tomography, light microscopy and electron microscopy. various materials available for the treatment of internal root resorption include mta, glass ionomer cement, super eba, hydrophilic plastic polymer (2-hydroxyethyl methacrylate with barium salts), zinc oxide eugenol and zinc acetate cement, amalgam alloy, composite resin and thermoplasticized gutta - percha administered either by injection or condensation techniques. perforating internal resorption may complicate the prognosis of endodontic treatment due to weakening of the remaining dental structure and possible periodontal involvement. however, prognosis of the tooth can be influenced by the biomaterial employed for the treatment. mta is most commonly used because of its biocompatibility, sealing ability and potential induction of osteogenesis and cementogenesis followed by thermoplasticized gutta - percha obturation techniques. this paper insights case series involving non - perforating and perforating internal resorption cases, which were successfully managed and showed successful healing after 8 months to 1 year follow - up period. a 45-year - old female patient came to the department with a chief complaint of dull pain in upper right front tooth region since one month. her medical history showed that she had stevens johnsons syndrome since the past five years and was under medication for the same. radiographic examination showed radiolucency in middle third of root surface of the lateral incisor indicating a case of internal resorption and an associated radiolucency in the periapical area of same tooth [figure 1a ]. also there was widening of periodontal ligament wrt 13. the diagnosis of necrotic pulp with chronic periapical abscess wrt 12 and asymptomtic apical periodontitis wrt 13 was made. (a) preoperative radiograph (b) postobturation radiograph (c) 1 year follow up radiograph access opening was initiated without local anaesthesia since the tooth was non - vital. biomechanical preparation of the tooth was done in the first visit. in the next visit, thick consistency of the sealer (endomethasone) was mixed and properly applied with lentulospiral into the canal and the resorptive defect followed by obturation of the canal using warm vertical condensation method [figure 1b ]. the patient was recalled on a regular basis for upto one year [figure 1c ]. the follow - up radiograph after one year showed peri - apical healing of the lateral incisor. a 38-year - old male reported to the department of conservative dentistry and endodontics with complaint of grossly decayed teeth in the mandibular right second molar region since one year. periapical radiograph revealed a radiolucent lesion in the apical third of the distal root continuous with the distal radicular surface, suggestive of a perforating resorptive defect [figure 2a ]. an oval shaped radiolucency was also seen in the coronal third of the distal root canal. (a) preoperative radiograph (b) 2b - mta plug (c) postoperative radiograph (d) 8 months follow up radiograph after rubber dam application, access opening was made. biomechanical preparation was done in the mesial root using m two - rotary system # 25 while the distal canal was prepared with hand k files till 50. the resorptive defect in the distal canal was the repaired with mta plug in the apical 3 mm of the canal [figure 2b ]. after the setting of the mta plug the tooth was obturated using obtura ii thermoplasticized gutta - percha [figure 2c ]. after post obturation an eight month follow - up radiograph showed uneventful healing of the lesion [figure 2d ]. a 25-year - old male reported to the department of conservative dentistry and endodontics with complaint of pain in the mandibular left second molar region since 15 days. radiographic examination showed inadequate obturation and persistent apical periodontitis with perforating internal root resorption with respect to tooth 37 [figure 3a ]. (a) preoperative radiograph (b) postoperative radiograph (c) 1 year follow up radiograph after rubber dam application access opening was modified. distal canal was prepared till # 50 and mesiobuccal and mesiolingual canals were prepared till # 25. the resorptive defect in the distal canal was the repaired with mta plug in the apical 3 mm of the canal. after the setting of the mta plug the canal was obturated with thermoplasticized gutta - percha. the mesial root canals were obturated with gutta - percha and endomethasone sealer using lateral condensation technique [figure 3b ]. a one year follow - up radiograph showed successful healing of the lesion [figure 3c ]. a 45-year - old female patient came to the department with a chief complaint of dull pain in upper right front tooth region since one month. her medical history showed that she had stevens johnsons syndrome since the past five years and was under medication for the same. radiographic examination showed radiolucency in middle third of root surface of the lateral incisor indicating a case of internal resorption and an associated radiolucency in the periapical area of same tooth [figure 1a ]. also there was widening of periodontal ligament wrt 13. the diagnosis of necrotic pulp with chronic periapical abscess wrt 12 and asymptomtic apical periodontitis wrt 13 was made. (a) preoperative radiograph (b) postobturation radiograph (c) 1 year follow up radiograph access opening was initiated without local anaesthesia since the tooth was non - vital. biomechanical preparation of the tooth was done in the first visit. in the next visit, thick consistency of the sealer (endomethasone) was mixed and properly applied with lentulospiral into the canal and the resorptive defect followed by obturation of the canal using warm vertical condensation method [figure 1b ]. the patient was recalled on a regular basis for upto one year [figure 1c ]. the follow - up radiograph after one year showed peri - apical healing of the lateral incisor. a 38-year - old male reported to the department of conservative dentistry and endodontics with complaint of grossly decayed teeth in the mandibular right second molar region since one year periapical radiograph revealed a radiolucent lesion in the apical third of the distal root continuous with the distal radicular surface, suggestive of a perforating resorptive defect [figure 2a ]. an oval shaped radiolucency was also seen in the coronal third of the distal root canal. (a) preoperative radiograph (b) 2b - mta plug (c) postoperative radiograph (d) 8 months follow up radiograph after rubber dam application, access opening was made. biomechanical preparation was done in the mesial root using m two - rotary system # 25 while the distal canal was prepared with hand k files till 50. the resorptive defect in the distal canal was the repaired with mta plug in the apical 3 mm of the canal [figure 2b ]. after the setting of the mta plug the tooth was obturated using obtura ii thermoplasticized gutta - percha [figure 2c ]. after post obturation an eight month follow - up radiograph showed uneventful healing of the lesion [figure 2d ]. a 25-year - old male reported to the department of conservative dentistry and endodontics with complaint of pain in the mandibular left second molar region since 15 days. radiographic examination showed inadequate obturation and persistent apical periodontitis with perforating internal root resorption with respect to tooth 37 [figure 3a ]. (a) preoperative radiograph (b) postoperative radiograph (c) 1 year follow up radiograph after rubber dam application access opening was modified. distal canal was prepared till # 50 and mesiobuccal and mesiolingual canals were prepared till # 25. the resorptive defect in the distal canal was the repaired with mta plug in the apical 3 mm of the canal. after the setting of the mta plug the canal was obturated with thermoplasticized gutta - percha. the mesial root canals were obturated with gutta - percha and endomethasone sealer using lateral condensation technique [figure 3b ]. a one year follow - up radiograph showed successful healing of the lesion [figure 3c ]. there is always a dilemma of whether to treat a tooth with a questionable prognosis endodontically or extract it and subsequently place an implant. it is a multifactorial process associated with various factors, which may be categorized in to physiological resorption, local factors, systemic condition and idiopathic resorption. internal resorption is the result of an inflamed pulp and the clastic precursor cells recruiting through the blood vessels. treatment of internal resorption is quite predictable as it is easy to control the process of internal root resorption via severing the blood supply to the resorbing tissues with conventional root canal therapy. in teeth with perforating defects, remineralization of the defect may occur, formation of hard tissue matrix against which permanent root canal filling is condensed or surgical approach and some cases may require extraction. in the present case series, different examples with available treatment approaches are included. in the case report 1, the systemic condition of the patient (steven johnson syndrome) could be the reason for the internal resorption. the defect was non - perforating which was filled with warm vertical condensation after cleaning and shaping of the canals. in case mta was used to make the apical plug of 3 mm because of its biocompatibility, sealing ability and potential induction of osteogenesis and cementogenesis. it was followed by thermoplasticized gutta - percha obturation to form a three - dimensional seal. hence, all the perforating resorptive defects were treated by sealing the perforation by mta and then filling the canal with thermoplasticized gutta - percha. in non - perforating internal resorption, early diagnosis, removal of the cause, proper treatment of the resorbed root is mandatory for successful treatment outcome. internal resorption is an uncommon resorption of the tooth, which starts from the root canal and destroys the surrounding tooth structure. it is easy to control the process of internal root resorption via severing the blood supply to the resorbing tissues with conventional root canal therapy. regular recall is important to check the status of healing and for the overall prognosis of the tooth. | management of internal root resorption is a challenge to the endodontists. it may occur in cases with chronic pulpal inflammation, following caries or due to trauma in the form of an accidental blow. most cases of internal root resorption are seen in anterior teeth, due to their susceptibility to trauma. however, it may be seen in posterior teeth, most likely because of carious involvement of the pulp. early diagnosis, removal of the cause, proper treatment of the resorbed root is mandatory for successful treatment outcome. this paper is an attempt to summarize the knowledge on internal root resorption and present various cases, which were successfully managed with different treatment modalities. |
human ether - a - go - go - related gene (herg) encodes the herg ion channel, which is a member of the voltage - dependent potassium channel (kv) family. herg mutations reduce the outward flow of potassium during repolarization and elongate the qt interval leading to polymorphic ventricular tachycardia, cardiac syncope, and sudden death. herg ion channels are expressed in the human pancreas where it has been shown to negatively regulate insulin secretion and positively regulate glucagon secretion. fluoroquinolones are antibiotics that are effective against gram - negative bacilli, but as drug development continues, their antibacterial spectra expand. fourth - generation fluoroquinolones, such as moxifloxacin and levofloxacin, are also effective against streptococcus pneumoniae, a gram - positive cocci, and anaerobic bacteria. with the widespread clinical use of these drugs, dysglycemia has been reported as an adverse effect. gatifloxacin, ofloxacin, and lomefloxacin have been reported to lead to hypoglycemia, whereas ofloxacin and gatifloxacin have been reported to result in hyperglycemia. although these side effects are rare, they require hospitalization and can endanger the life of a patient ; thus, patients taking fluoroquinolones should be monitored for dysglycemia. some fluoroquinolones cause hypoglycemia by blocking atp - sensitive potassium channels (katp) in pancreatic -cells leading to stimulation of insulin secretion. by stimulating histamine secretion gatifloxacin can also cause vacuolization of islet -cells leading to a decrease in insulin production and hyperglycemia. katp channels and other ion channels have been found to synergistically regulate insulin secretion ; thus, herg potassium channels and katp channels together may regulate insulin secretion by pancreatic -cells. because the ability of moxifloxacin to inhibit herg channels is unknown, this study explored the effect of moxifloxacin on herg channel currents and glucose metabolism in mice. c57bl/6 herg knockout mice were created using transcription activator - like effector nucleases (talens). this study was approved by the ethics committee of beijing tongren hospital, capital medical university, and was performed according to the principles of the declaration of helsinki ii. animal experiments followed the national ethical guidelines implemented by our institutional animal care and use committee and were approved by the ethical review committee at the institute of zoology, capital medical university, china. human pancreatic tissues were fixed by immersion in 4% paraformaldehyde for 2 h at 4c and were then embedded in paraffin. paraffin sections (5 m) were pretreated for antigen retrieval using a microwave. paraffin sections were incubated with primary antibody, goat anti - insulin antibody (diluted 1 : 100, santa cruz biotechnology inc., ca, usa) or rabbit anti - herg antibody (diluted 1 : 200, santa cruz biotechnology inc., the sections were washed three times with phosphate - buffered saline (pbs) and then were incubated with alexa fluor 555 donkey anti - goat - cy3 (diluted 1 : 200, santa cruz biotechnology inc., ca, usa) or alexa fluor 488 goat anti - rabbit - cy2 (diluted 1 : 200, santa cruz biotechnology inc., images were taken with a laser scanning confocal microscope (leica tcssp5 ; leica, wetzlar, germany). human embryonic kidney (hek) 293 cells were provided by the cell research center at basic medical peking union medical college. hek293 cells were cultured in a 37c incubator with 5% co2 and humidified air. the target gene, herg cdna, and the expression vector, gv314 (genechem co., shanghai, china), were transfected into cells using lipofectamine 2000 (invitrogen, carlsbad, ca, usa) according to the manufacturer 's protocol. vector gv314 was cotransfected because it contains the marker gene gfp, sv40, cmv promoter, and ampicillin resistance gene. cells were cultured in dulbecco 's modified eagle 's medium (dmem ; gibco, md, usa), 10% fetal bovine serum (gibco, md, usa), 1% penicillin - streptomycin (gibco, md, usa), and 1% sodium pyruvate (gibco, md, usa) for 4872 h after transfection. cells exhibiting strong fluorescence microscopically (te2000-u, niko, japan) were selected for patch clamp experiments. a patch clamp amplifier (epc-10, heka, germany) hek293 cells were bathed in 137 mmol / l of nacl, 4 mmol / l of kcl, 2 mmol / l of cacl2, 1 mmol / l of mgcl2, 10 mmol / l of hepes, and 10 mmol / l of glucose, ph 7.4. when the patch pipette was filled with the internal pipette solution, the pipette had a resistance of 2.54.0 m. the internal pipette solution contained 130 mmol / l of kcl, 5 mmol / l of mgatp, 1 mmol / l of mgcl2, 5 mmol / l of egta, and 10 mmol / l of hepes, ph 7.3. after membranes were ruptured using negative pressure suction, resistance ranged from 500 to 600 m. experiments were performed from 25 to 28c. louis, mo, usa) was dissolved to a concentration of 10 mmol / l with double - distilled water. this study contained four groups : wild - type mice that received physiological saline, wild - type mice that received moxifloxacin, herg knockout mice that received saline, and herg knockout mice that received moxifloxacin. the control groups received physiological saline at the same volume via the same route as the test groups. mice were fasted overnight for 16 h but were allowed free access to water. they were given 200 mg / kg of moxifloxacin or the same volume of physiological saline via gavage, and then they were given 2 mg / kg of glucose by intraperitoneal injection. blood samples were taken from the angular vein at 0, 15, 30, 60, and 120 min after glucose injection. blood glucose concentrations were assessed using an automatic glucometer (one touch, lifescan, usa). insulin concentrations were measured using a highly sensitive mouse insulin immunoassay kit (antibody and immunoassay services, hku, china) according to the manufacturer 's protocol. we used a dose - response curve fitted to a hill equation, y = [(a1 a2)/(1 + (x / c)nh) ] + a2, in which a1 represents 0 tail current inhibition, a2 represents 100% inhibition, c represents the ic50 concentration, and nh represents the hill slope. an independent sample t - test was used to compare two sets of data. a one - way anova followed by tukey 's or dunnett 's test pancreatic -cells, which are located in pancreatic islets, were visualized by insulin immunoreactivity (figure 1(b)). herg protein - positive cells were located in these islets (figure 1(c)). colocalization of insulin and herg by immunostaining indicated that herg is expressed in pancreatic -cells (figure 1(d)). hek293 cells were held at 80 mv for 1 s and then were depolarized for 3 s using 10 mv intervals from 60 mv to + 50 mv. the current initially increased and then decreased, in a time - dependent manner, as the voltage increased. a tail current was elicited for 3 s at 40 mv and the peak tail current at each voltage represented the degree to which the herg channel was open at that voltage. after perfusion of the transfected hek293 cells with 100 mol / l of moxifloxacin for 5 min, the current was reduced (figure 2(a)). curves i - v indicated that 100 mol / l of moxifloxacin significantly reduced the time - dependent and peak tail currents when the voltage was greater than 0 and 10 mv, respectively (figure 2(b)). cells were depolarized to + 20 mv for 3 s and peak tail currents were recorded at different moxifloxacin concentrations (10, 100, and 1000 mol / l). tail currents gradually decreased with the increasing concentrations of moxifloxacin indicating that inhibition by moxifloxacin was concentration - dependent. herg channel currents were completely inhibited at 1000 mol / l of moxifloxacin (figure 2(c)). by plotting moxifloxacin concentration (100 mmol / l) on the x - axis and tail current inhibition (%) on the y - axis, a dose - response curve was generated and the data were fitted to a hill equation, y = [(a1 a2)/(1 + (x / c)nh) ] + a2. mol / l and the hill slope, nh, was 0.96 (figure 2(d)). blood glucose and insulin concentrations of mice that underwent the glucose tolerance test 200 mg / kg of moxifloxacin led to a reduction in the blood glucose concentration at 60 min (p = 0.0346) (figure 3(a)). insulin levels in the moxifloxacin group were higher than those in the saline group at 60 min (p = 0.0373) (figure 3(b)). the areas under the blood glucose concentration versus time curve and the insulin concentration versus time curve differed (p = 0.0383 and 0.0383, resp.) there were no significant differences in blood glucose or insulin concentrations in herg knockout mice treated with moxifloxacin or saline (figures 3(c) and 3(d)). increased glucose levels trigger a series of reactions that ultimately lead to insulin secretion by islet -cells. the transformation and utilization of glucose increase the intracellular ratio of atp / adp causing katp channels to close the cell membrane is depolarized causing voltage - dependent calcium channels to open, which leads to an increase in the concentration of free intracellular calcium. in addition to katp channels, other voltage - gated potassium channels are expressed in pancreatic -cells and regulate insulin secretion by repolarizing membranes. were the first to report that herg potassium channels are expressed in human islets -cells, and they studied the effect of herg channels on insulin secretion using electrophysiological and molecular techniques.. also found herg in human islets using reverse transcription - polymerase chain reaction (rt - pcr), but they did not determine its specific distribution. the herg inhibitors e-4031, dofetilide, and verapamil impact channel gating to various degrees and can cause currents to be reduced or completely inhibited. basal insulin secretion in ins-1 -cells was shown to increase upon blockage of erg channels by e-4031. we demonstrated that the time - dependent and tail currents for herg decreased in response to 100 mol / l of moxifloxacin indicating that moxifloxacin inhibits the herg channel in hek293 cells. as moxifloxacin concentrations increased, tail currents were further reduced and completely inhibited at 1000 mol / l of moxifloxacin. we found that moxifloxacin inhibition was concentration - dependent and that the ic50 was 36.65 fluoroquinolones can induce life - threatening dysglycemia, but, in the clinical concentration range, fluoroquinolones do not initiate dysglycemia ; they rather enhance insulin secretion, which is induced by high glucose concentrations. fluoroquinolones can block herg channels and pancreatic -cell katp channels ; however, the structures of the fluoroquinolones that inhibit herg channels and katp channels differ. the amino group at position c5 of fluoroquinolones plays a major role in blocking herg channels, whereas the substituents at positions n1, c7, and c8 of fluoroquinolones are responsible for the inhibition of katp channels. inhibition of katp channels is required for fluoroquinolone - induced hypoglycemia, but channel inhibition alone does not sufficiently explain this side effect. when herg channels were selectively blocked by perfusion of islets with antiarrhythmic drugs, insulin secretion increased in the presence of glucose and arginine. a k current that is pharmacologically and biophysically similar to the herg current has been reported. these data suggest that herg channels contribute to the regulation of insulin secretion by human pancreatic -cells. patients receiving insulin concomitant with moxifloxacin have a significantly higher risk of hypoglycemia in taiwan. the fluoroquinolone moxifloxacin has been associated with the highest risk of hypoglycemia followed by levofloxacin and ciprofloxacin.. found that hypoglycemia occurred most often with moxifloxacin compared to levofloxacin and ciprofloxacin. ishiwata. reported that intravenous injection of 100 mg / kg of moxifloxacin increased the serum glucose concentration and temporarily elevated serum histamine and epinephrine concentrations in fasted rats. serum glucose, epinephrine, and histamine concentrations remained unchanged when moxifloxacin was administered at 75 mg / kg. serum insulin concentrations were not altered by moxifloxacin at both 75 and 100 mg / kg. this suggests that moxifloxacin can cause hyperglycemia by inducing histamine and epinephrine release. in our study, 200 mg / kg of moxifloxacin reduced blood glucose levels in wild - type mice 60 min after the start of the glucose tolerance test ; this reduction may be a result of a slight increase in insulin secretion. in contrast, moxifloxacin did not significantly alter blood glucose or insulin levels in herg knockout mice. serum glucose levels increased and insulin concentrations decreased in herg knockout mice. in a word, this is suggesting that moxifloxacin reduces blood glucose levels and increases insulin secretion via the herg protein. it is also not known whether other channels participate in the moxifloxacin - induced hypoglycemic effect. | the purpose of this study was to investigate the effect of moxifloxacin on herg channel protein and glucose metabolism. herg expression was investigated using immunohistochemistry. the whole - cell patch clamp method was used to examine the effect of moxifloxacin on herg channel currents. a glucose tolerance test was used to analyze the effects of moxifloxacin on blood glucose and insulin concentrations in mice. results show that herg protein was expressed in human pancreatic -cells. moxifloxacin inhibited herg time - dependent and tail currents in hek293 cells in a concentration - dependent manner. the ic50 of moxifloxacin inhibition was 36.65 mol / l. moxifloxacin (200 mg / kg) reduced blood glucose levels and increased insulin secretion in wild - type mice at 60 min after the start of the glucose tolerance test. in contrast, moxifloxacin did not significantly alter blood glucose and insulin levels in herg knockout mice. serum glucose levels increased and insulin concentrations decreased in herg knockout mice when compared to wild - type mice. the moxifloxacin - induced decrease in blood glucose and increase in insulin secretion occurred via the herg protein ; thus, herg protein plays a role in insulin secretion. |
prevalence estimates range from 4% to 29% for hazardous drinking and from < 1% to 10% for harmful drinking globally. similar rates of hazardous or harmful alcohol use in health care settings have been reported from different african countries. recent estimates suggest that about 2.5 million deaths each year are directly attributable to alcohol, with the highest percentage of alcohol - related deaths occurring among people between 15 and 29 years old ; with nearly 1 out of 10 deaths in this age group worldwide attributable to alcohol. alcohol use contributes to traumatic outcomes ; killing or disabling many at a relatively young age, and resulting in the loss of many years of life to death or disability. researchers have established a strong association between alcohol use and road traffic accidents in nigeria. in fact, welcome and pereverzev reported that approximately 50% of accidents on nigerian roads are related to alcohol use. there has been an increasing emphasis on the role of primary health care and family physician in the prevention and management of alcohol - related harm. the health professionals at these primary care settings are readily accessible to the general population and have a significant role in the delivery of advice about alcohol consumption. in addition, there is good evidence that brief interventions delivered by general practitioners have a positive impact on patients alcohol consumption. this study is aimed at determining the pattern and the predictors of alcohol use disorders (auds) among patients attending the family practice unit of our teaching hospital which provides essentially primary care services. the study population consists of patients attending family practice units of the ekiti state university teaching hospital, ado - ekiti, in south - western nigeria. the family physician clinic provides essentially primary care services and makes referrals to other specialist clinics. sample was selected through a random sampling from a population of patients, aged 18 - 65 years, attending the general medical unit of the hospital from january to april, 2013. patients with debilitating physical illnesses and those that came for routine medical check - up were not included in the study. the ethics and research committee of the university teaching hospital approved the study protocol and informed consents were obtained from the participants after the aims and objectives of the study had been explained to them. previously trained research assistants who are of psychology students and clinical psychologists explained the size of a standard drink to those agreeing to participate. a standard drink was defined as equivalent volumes containing an average of 13.5 g ethanol. data were collected using a pretested, semi - structured questionnaire incorporating sociodemographics, and the diagnoses made by the attending physicians. the participants also completed the alcohol use disorders identification test (audit) questionnaire and the patient health questionnaire (phq-9). the audit is a self - administered questionnaire including three items on the amount and frequency of drinking, three on alcohol dependence, and four on common problems caused by alcohol. several studies have shown its validity and reliability in the detection of hazardous drinking, alcohol misuse, and alcohol dependence. while this study, a score of 8 and above for men ; and score 7 and above for female indicated auds. subjects occupation was classified according to the system of boroffka and olatawura which is well - operationalized and is comparable to other systems like the international labor organization classificatory system. it has also been widely used in previous studies in this environment. the categories are as follows : group i consists of professionals with university degrees, e.g., doctors, lawyers, scientists, etc. ; group ii consists of professionals without university degrees and include teachers, administrators, large scale farmers, and armed forces officers ; group iii consists of clerks, motor vehicle drivers, mechanics, etc. ; group iv consists of cooks, small - scale farmers, barbers, etc. ; group v includes laborers and petty traders ; group vi includes full - time housewives, students, and unemployed educated youths. the phq-9 consists of nine items, each of which is scored 0 - 3, providing a 0 - 27 severity score. phq-9 severity is calculated by assigning scores of 0, 1, 2, and 3, to the response categories of - not at all, several days, more than half the days, and nearly every day, respectively. it consists of the nine criteria for depression from the diagnostic and statistical manual of mental disorders, fourth edition. the statistical package for the social sciences version 16 (spss.16) program was used for statistical analysis. results were calculated as frequencies (%), means, and standard deviations (sd). t - test for continuous data and chi - square for categorical data were used to examine differences between groups. the study population consists of patients attending family practice units of the ekiti state university teaching hospital, ado - ekiti, in south - western nigeria. the family physician clinic provides essentially primary care services and makes referrals to other specialist clinics. sample was selected through a random sampling from a population of patients, aged 18 - 65 years, attending the general medical unit of the hospital from january to april, 2013. patients with debilitating physical illnesses and those that came for routine medical check - up were not included in the study. the ethics and research committee of the university teaching hospital approved the study protocol and informed consents were obtained from the participants after the aims and objectives of the study had been explained to them. previously trained research assistants who are of psychology students and clinical psychologists explained the size of a standard drink to those agreeing to participate. a standard drink was defined as equivalent volumes containing an average of 13.5 g ethanol. data were collected using a pretested, semi - structured questionnaire incorporating sociodemographics, and the diagnoses made by the attending physicians. the participants also completed the alcohol use disorders identification test (audit) questionnaire and the patient health questionnaire (phq-9). the audit is a self - administered questionnaire including three items on the amount and frequency of drinking, three on alcohol dependence, and four on common problems caused by alcohol. several studies have shown its validity and reliability in the detection of hazardous drinking, alcohol misuse, and alcohol dependence. while this study, a score of 8 and above for men ; and score 7 and above for female indicated auds. subjects occupation was classified according to the system of boroffka and olatawura which is well - operationalized and is comparable to other systems like the international labor organization classificatory system. the categories are as follows : group i consists of professionals with university degrees, e.g., doctors, lawyers, scientists, etc. ; group ii consists of professionals without university degrees and include teachers, administrators, large scale farmers, and armed forces officers ; group iii consists of clerks, motor vehicle drivers, mechanics, etc. ; group iv consists of cooks, small - scale farmers, barbers, etc. ; group v includes laborers and petty traders ; group vi includes full - time housewives, students, and unemployed educated youths. the phq-9 consists of nine items, each of which is scored 0 - 3, providing a 0 - 27 severity score. phq-9 severity is calculated by assigning scores of 0, 1, 2, and 3, to the response categories of - not at all, several days, more than half the days, and nearly every day, respectively. it consists of the nine criteria for depression from the diagnostic and statistical manual of mental disorders, fourth edition. the statistical package for the social sciences version 16 (spss.16) program was used for statistical analysis. results were calculated as frequencies (%), means, and standard deviations (sd). t - test for continuous data and chi - square for categorical data were used to examine differences between groups. the age of the participants ranges from 18 to 65 years with a mean of 42.07 (sd = 13.93) 23 (9.4%) had audit score indicating auds. the audit scores of the participants ranges from 0 to 29 with a mean of 1.3 (sd = 4.08). the mean audit score for male is 2.68 sd = 5.875 and for female is 0.48 (sd = 1.889, p = 0.000). the phq score ranges from 0 to 23 with a mean of 5.99 (sd = 5.17). most (69.8%) were married to only 13 (5.3%) constituting either separated or divorced or widowed. sociodemographic characteristics of the respondents sociodemographic characteristics and auds of the 23 who had audit score indicating auds, 18 (18.9%) of males and the remaining 5 (4.0%) of women had auds. the association between gender and auds was statistically significant (= 10.136, p = 0.003). higher proportion (11.2%) of those aged 45 and above had auds compared to 7.8% of those aged below 45 years, however, this was not statistically significant (= 0.857, p = 0.239). a greater proportion of those with primary education (22.0%) ; compared to secondary (5.9%) or tertiary education (6.3%) had auds. the association between level of education and auds was statistically significant (= 8.198, p = 0.042). no statistically significant association was found between marital status (p = 0.170), average income per month (p = 0.463), religion (p = 0.714), and employment status (p = 0.629). as shown in table 3, analysis of occupational group in relation to auds shows that higher proportion of participants in occupational group iv had auds. categorizing occupational groups into upper, lower, and others shown that majority of those in the lower occupational group compared to other groups had aud, and this was statistically significant (p = 0.008). occupational group and auds table 4 shows that 18 (16.5%) of those who had significant depressive symptoms compared with 5 (3.7%) of those without significant depressive symptoms had auds. significant depressive symptoms was significantly associated with auds (= 11.722, df = 1, p = 0.001). the age of the participants ranges from 18 to 65 years with a mean of 42.07 (sd = 13.93) 23 (9.4%) had audit score indicating auds. the audit scores of the participants ranges from 0 to 29 with a mean of 1.3 (sd = 4.08). the mean audit score for male is 2.68 sd = 5.875 and for female is 0.48 (sd = 1.889, p = 0.000). the phq score ranges from 0 to 23 with a mean of 5.99 (sd = 5.17). most (69.8%) were married to only 13 (5.3%) constituting either separated or divorced or widowed. sociodemographic characteristics and auds of the 23 who had audit score indicating auds, 18 (18.9%) of males and the remaining 5 (4.0%) of women had auds. the association between gender and auds was statistically significant (= 10.136, p = 0.003). higher proportion (11.2%) of those aged 45 and above had auds compared to 7.8% of those aged below 45 years, however, this was not statistically significant (= 0.857, p = 0.239). a greater proportion of those with primary education (22.0%) ; compared to secondary (5.9%) or tertiary education (6.3%) had auds. the association between level of education and auds was statistically significant (= 8.198, p = 0.042). no statistically significant association was found between marital status (p = 0.170), average income per month (p = 0.463), religion (p = 0.714), and employment status (p = 0.629). as shown in table 3, analysis of occupational group in relation to auds shows that higher proportion of participants in occupational group iv had auds. categorizing occupational groups into upper, lower, and others shown that majority of those in the lower occupational group compared to other groups had aud, and this was statistically significant (p = 0.008). table 4 shows that 18 (16.5%) of those who had significant depressive symptoms compared with 5 (3.7%) of those without significant depressive symptoms had auds. significant depressive symptoms was significantly associated with auds (= 11.722, df = 1, p = 0.001). this study looked at the pattern and the predictors of harmful alcohol drinking among patients visiting the family physician at the general out - patient clinic. both in nigeria and likewise lower when compared with other studies in africa ; by kullgren. in uganda and peltzer in south africa. the difference observed in the prevalence rates may not be too surprising as difference sociocultural practices and cultural view of auds are influenced by prevalent norm in the society. it has been suggested, even though some similarities exist with respect to the definition of problematic alcohol use in ethnically diverse societies, very substantial differences also exist. the variations in the study methods used, including how alcohol use and various disorders are defined and the sensitivity of the instruments used, is another major factor. the variability in the response of the respondents to alcohol use may also be a factor. like in most studies, our study found a significant association between auds with some sociodemographic variables. the cultural practices and perception which is permissive to men drinking alcohol and the occupational engagement of men are possible reasons for this observation. besides, men also take alcohol to enhance sexual performance, for social acceptance and to overcome societal stresses. a higher prevalence of auds is seen among those age 45 years and above compared to those below 45 years. though our study did not find a significant association between age group and auds, the study by abiodun. reported a significant association between older age group and auds. nonetheless, there is a considerable variation in the pattern and prevalence of auds in different age groups. some authors have reported higher prevalence among younger age group while some have reported higher prevalence among older age group. the possible explanation for the pattern observed in this study may due to the fact that the study is based in the hospital ; and many young people with alcohol problems may not patronize the hospital but rather engage self - medication. studies have shown that young people tend to find it difficult to ask for help especially when it comes to health issues. thus, whenever young people face health concerns, they often seek health care informally ; in other words, they do not refer to health professionals or to formal health services first. there is a significant association between educational level and auds. even though, majority of patients in our study had tertiary education, however, a larger proportion of those with primary education compared with other groups had auds. this observation may also explain the higher prevalence of auds among those in lower occupational group. the fact that those with lower education engaged more in manual works such as driving, bricklaying, laborers and other occupation that expose them to the risk, and encourage use of drugs as an enhancer may possibly explain this observation. besides, this group is more likely to take more of local gin that contains a higher concentration of alcohol. in this study, auds is significantly associated with the presence of significant depressive symptoms. about a quarter of those with significant depressive symptoms compared to barely 4% of those without significant symptoms had auds, and most of these are likely to present with mild to moderate form of depression. previous studies have found that anxiety and depression were associated with auds and identifying alcohol use in these groups of people may be particularly important. people with auds often have co - occurring psychiatric disorders, most importantly depression ; furthermore, they experienced an adverse health outcome, and repeated hospital admissions. the association between auds and depression also increases the possibility of suicide because alcohol abuse can exaggerate depression and increases impulsiveness and impairs judgment. alcohol use disorders are relative lower in this population compared with other studies, but however significant. auds are associated with gender, low educational, low occupational group, and the presence of significant depressive symptoms mostly in the mild or moderate severity. identifying the group at risk in clinical setting may go a long way in reducing the adverse effect of auds in our society. | background : primary care has been identified as a key setting for the reduction of alcohol - related harm, while general practitioners are expected to play a significant role. the study aimed at identifying pattern of, and factors that are associated with alcohol use disorders (auds) among patients attending family medicine unit of state university teaching hospital.materials and methods : sample was selected through a random sampling from a population of patients, aged 18 - 65 years, attending the general medical out - patients unit of the hospital from january to april 2013. a pretested, semi - structured questionnaire was administered, incorporating sociodemographics and the diagnoses made by the attending physician. the participants also completed the alcohol use disorders identification test (audit) questionnaire and the patient health questionnaire-9.results:the prevalence of auds among the population of general out - patients was 9.7%. the audit scores of the participants range from 0 to 29 with a mean of 1.3 (standard deviation = 4.08). auds were significantly associated with gender, level of education, occupational class, and the presence of significant depressive symptoms (p < 0.05). there was no statistically significant association found for age, employment status, marital status, and religion.conclusion:the prevalence of auds among population studied was lower compared with a similar study in similar setting, but however, significant. auds were predicted by gender, lower education level, occupational group, and the presence of significant depressive symptoms mostly in the mild to moderate form. identifying the group at risk in clinical setting may go a long way in reducing the adverse effect of auds in our society. |
the endoscopic retrograde cholangiopancreatography (ercp)-related perforation are rare but sometimes carries 16% to 18% of death rate.1 the incidence of perforation has been reported from 0.3% to 2.2%.2 - 5 ercp - related perforation is classified into three or four types based on anatomical location and the mechanism of injury.2,3 the management of ercp - related perforation has been differentiated by the types of perforation. guidewire perforation are benign and in general do not require surgery.2,3 periampullary perforations with the diagnosis of retroperitoneal air may be managed nonsurgically such as by aggressive endoscopic drainage and medical treatment with broad spectrum antibiotics.3,5 but patients with retroperitoneal fluid collection have worse prognosis and require surgical intervention.2 here, we present our experience in a case of ercp - related severe ampullary perforation with retroperitoneal fluid collection that was treated nonsurgically with a covered metal stent. a 61-year - old korean woman was referred to our hospital for right upper quadrant pain. initial abdominal computed tomography (ct) showed biliary tree dilatation but, although periampullary swelling was suspected, no definite occluding lesion was evident. ercp was performed to obtain a biopsy specimen from the ampulla, which showed nonspecific dilatation of the common bile duct (cbd) without a definite mass around the ampulla. the day after the ercp, she developed a severe right flank pain and fever of 39, shortness of breath (26/min), and rapid pulse rate (102/min). abdominal ct revealed retroperitoneal air and fluid collection suggesting post - ercp ampullary perforation (fig. although her symptoms were severe, her condition looked stable and there was no sign of sepsis, and conservative treatment was considered. subsequent ercp revealed ampullary edema only, but the cholangiography showed continuous leakage of the radiocontrast media from the ampullary level. although the perforating point was not visualized, a 5-cm long, 10-mm in diameter, full - covered metal stent (niti - s ; taewoong medical, seoul, korea) was inserted to seal the perforation defect and to provide a lumen for biliary drainage (fig. 2).the fever and the abdominal pain after the ercp subsided at day 4 after the stenting. retroperitoneal air and fluid collection gradually resolved by follow - up abdominal ct and thus the stent was extracted at day 10 (fig. the patient was discharged on day 23 after ercp and consequent abdominal ct revealed complete resolution of the retroperitoneal fluid collection (fig. the recent paradigm of management, however, has shifted to a more selective approach that requires consideration of perforation type and surgical indications.2,3,6 the majority of patients without free wall duodenal perforation or peritoneal signs are being treated nonsurgically.2,4,7 the retroperitoneal air is a common finding indicating nonsurgical management and the amount of retroperitoneal air is not correlated with clinical course. for those treated conservatively, biliary drainage is the mainstay of treatment to reduce morbidity and mortality3 and is usually consisted of endoscopic nasobiliary drainage (enbd) and endoscopic retrograde biliary drainage (erbd). the finding of retroperitoneal fluid collection, however, suggests continued bile leak from the site of perforation. it is suggested that patients with retroperitoneal fluid collection have worse prognosis and require surgical intervention.2,6 however, the traditional drainage methods such as enbd or erbd may be limited for preventing bile and pancreatic fluid leakage, especially when severe cbd dilatation or a large perforation hole is present. surgery should be undertaken, therefore, if pain and abdominal signs are prominent, if suppuration is suspected, or if symptoms do not improve after a brief period of nonoperative management.8 biliary self - expanding metal stents have the advantage of being inserted with small sizes and provide large diameters for biliary drainage ; however, their use in benign conditions has been limited, mainly because of difficulty in extracting them. on the other hand, a covered stent has been occasionally used to treat an esophagorespiratory fistula or an esophageal rupture.9 in cases of ampullary perforation, a covered stent can provide complete sealing of the perforation defect and the stent lumen may allow physiologic drainage of bile and thus prevent additional fluid leakage. a small sized report suggested that fully covered metal stents were removed without any complication after being placed in the cbd for a mean time of over 4 months and that it could be used in the management of benign biliary conditions.10 recently, a case of persistent duodenal fistula caused by sphincterotomy - related duodenal perforation was reported. the patient underwent an ercp and sphincterotomy, after which a retroperitoneal duodenal perforation occurred. however, the fistula healed completely after the transient use of stents.11 based on this concept, we applied a covered stent to the perforation site immediately after the perforation developed, and achieved complete resolution of the retroperitoneal fluid collection and rapid clinical improvement. however, despite this treatment success, the merits of covered stents for ampullary perforation with retroperitoneal fluid collection have not been fully established because nonsurgical treatment failures have a high complication rate, with a potentially fatal outcome. therefore, management using a covered stent and serial follow - up by abdominal ct may be useful treatment option for ercp - related ampullary perforation with retroperitoneal fluid collection in selected patients. | endoscopic retrograde cholangiopancreatography (ercp)-related perforation is classified into three or four types based on anatomical location and the mechanism of injury. although ampullary injury, among them, may be managed nonsurgically, surgical management is required in cases of perforation with retroperitoneal fluid collection and severe condition. here, a patient with ercp - related severe ampullary perforation with retroperitoneal fluid collection that was treated nonsurgically with a covered stent is presented. |
the mayer- rokitansky - kuster - hauser (mrkh) syndrome is a rare anomaly characterized by congenital aplasia of the uterus and vagina in women showing normal development of secondary sexual characters and normal 44 xx karyotype. the uterine and vaginal agenesis in these karyotypic females may be accompanied by urogenital, skeletal or dental defects and other dysmorphias.[26 ] it is a rare disorder occurring in about 1 in 4000 to 5000 female births and is a common cause of primary amenorrhea. the diagnosis is often made during adolescence in the context of primary amenorrhea with normal puberty. the cause of this syndrome is still not completely understood, but the frequent association of other malformations involving the kidneys, skeleton, and ears suggests the involvement of major developmental genes, such as those of the homeobox (hox) family. these genes are known to play a crucial role during embryogenesis, particularly in the axial skeleton, the hindbrain, as well as, during urogenital differentiation. treatment of the mrkh syndrome consists of creating a neovagina, which can be offered to patients when they are emotionally mature and ready to commence sexual activity. treatment may either be surgical or non - surgical, but the chosen method needs to be tailored to the individual needs, motivation of the patients, and the options available. we report our experience in the management of two patients with congenital absence of the vagina due to the mrkh syndrome. faa was a 24-year - old student, referred to our unit with complaints of primary amenorrhea. she was said to have had delayed development of secondary sexual characters compared to her siblings, but no history of sexual exposure. the patient had no significant past medical history and no history of a similar problem in the family. examination showed a young female patient with normal secondary sex characters and well - developed breasts (tanner stage 4). vaginal examination showed normal vulva and urethral meatus, but a short (3 cm) blind ending vagina [figure 1 ]. preoperative picture of case no.1 showing blind ended vagina an abdominal ultrasound scan showed an enlarged right kidney, with a moderate hydrocalicosis and hydroureter. intravenous urography showed a low lying right hydronephrotic kidney and pelvi - ureteric junction (puj) obstruction. computerized tomography (ct) confirmed the absence of a uterus, vagina, and left kidney, as well as, the presence of a right kidney and puj obstruction [figures 2 and 3 ]. computerized tomography scan of the first patient showing absence of left kidney, and puj obstruction of right kidney computerized tomography scan of the first patient showing absence of uterus the patient had initial anderson - hynes dismembered pyeloplasty, with an uneventful postoperative recovery. the patient was counseled on the nature of the anomaly and the options available as well as the prospects of child bearing. postoperative follow up visit at one year and subsequent telephone communication indicated normal and satisfactory sexual intercourse with her spouse. intraoperative picture showing the prepared channel and distal vaginal segment ready for anastomosis with the colonic segment, to create a neovagina colonic segment prepared for vaginal reconstruction postoperative speculum examination of the first patient showing adequacy of the neovagina mm was a 24-year - old housewife who presented with complaints of primary amenorrhea, and inability to achieve penetrative sexual intercourse. there was no family history of a similar illness ; there was no significant past medical history. physical examination revealed a young female patient with normal secondary sex characters ; well - developed breasts (tanner stage 4), normal axillary hair and female pubic hair distribution ; there were no palpable groin or intra - abdominal masses. vaginal examination showed normal vulva and urethral meatus, but a blind ending vagina (about 3 cm). the patient received three courses of estrogen and progesterone, to assess the functional state of the uterus, but there was no evidence of increase in the uterine size. she was counseled on the nature of the anomaly and the treatment options available, as well as, the prospect of child bearing. she had excision of the grossly hypoplastic uterus and successful construction of a colovagina [figure 7 ]. the lady was discharged on the fifteenth postoperative day, and counseled to resume coitus three months after surgery. faa was a 24-year - old student, referred to our unit with complaints of primary amenorrhea. she was said to have had delayed development of secondary sexual characters compared to her siblings, but no history of sexual exposure. the patient had no significant past medical history and no history of a similar problem in the family. examination showed a young female patient with normal secondary sex characters and well - developed breasts (tanner stage 4). vaginal examination showed normal vulva and urethral meatus, but a short (3 cm) blind ending vagina [figure 1 ]. preoperative picture of case no.1 showing blind ended vagina an abdominal ultrasound scan showed an enlarged right kidney, with a moderate hydrocalicosis and hydroureter. intravenous urography showed a low lying right hydronephrotic kidney and pelvi - ureteric junction (puj) obstruction. computerized tomography (ct) confirmed the absence of a uterus, vagina, and left kidney, as well as, the presence of a right kidney and puj obstruction [figures 2 and 3 ]. computerized tomography scan of the first patient showing absence of left kidney, and puj obstruction of right kidney computerized tomography scan of the first patient showing absence of uterus the patient had initial anderson - hynes dismembered pyeloplasty, with an uneventful postoperative recovery. the patient was counseled on the nature of the anomaly and the options available as well as the prospects of child bearing. postoperative follow up visit at one year and subsequent telephone communication indicated normal and satisfactory sexual intercourse with her spouse. intraoperative picture showing the prepared channel and distal vaginal segment ready for anastomosis with the colonic segment, to create a neovagina colonic segment prepared for vaginal reconstruction postoperative speculum examination of the first patient showing adequacy of the neovagina mm was a 24-year - old housewife who presented with complaints of primary amenorrhea, and inability to achieve penetrative sexual intercourse. there was no family history of a similar illness ; there was no significant past medical history. physical examination revealed a young female patient with normal secondary sex characters ; well - developed breasts (tanner stage 4), normal axillary hair and female pubic hair distribution ; there were no palpable groin or intra - abdominal masses. vaginal examination showed normal vulva and urethral meatus, but a blind ending vagina (about 3 cm). the patient received three courses of estrogen and progesterone, to assess the functional state of the uterus, but there was no evidence of increase in the uterine size. she was counseled on the nature of the anomaly and the treatment options available, as well as, the prospect of child bearing. she had excision of the grossly hypoplastic uterus and successful construction of a colovagina [figure 7 ]. the lady was discharged on the fifteenth postoperative day, and counseled to resume coitus three months after surgery. the mrkh syndrome is a rare, but widely discussed developmental failure of a part of, or the whole mullerian duct. patients with this syndrome have a 46xx karyotype, normal female external genitalia, normal ovarian function, partial or incomplete absence of the vagina, and an absent or hypoplastic uterus with bilateral non - canalized tubes. while most patients present with the typical absence of the uterus and vagina, few present with the atypical form, where, in addition they have an asymmetric uterine remnant and abnormalities of the fallopian tubes. these patients suffer severe distortion of the body image, anxiety, depression, interpersonal sensitivity, and face a lot of psychological distress at diagnosis. in our environment, where gender role and identity are very crucial, these patients and families face difficult times. diagnosis is frequently made clinically, but often confirmed either radiologically or laparoscopically, in patients whose hormonal and karyotypic investigations for primary amenorrhea are normal. the use of ultrsonography, intravenous urography, computerized tomography, and magnetic resonance imaging provide information about the degree of abnormality and associated renal anomalies.[21113 ] management of vaginal agenesis in the mrkh syndrome remains controversial. the choice of procedure and patient age at reconstruction depend upon individual anatomy, fertility potential, and psychological and social factors. the ideal timing for intervention is at or after adolescence, when the woman has reached physical and psychological maturity. in the past, vaginal reconstruction procedures were performed on infants and pre - pubertal girls and this required inevitable surgical revision in adolescence before sexual activity. deferring treatment allows the woman herself to be involved in the decision making and also increases compliance with the adjuvant dilatation therapy that may be required. the goals of long - term treatments are to create a functional neo - vaginal canal with an adequate diameter and length, appropriate axial direction, and normal secretion / lubrication to accommodate sexual intercourse and to address the issue of fertility. there are two main types of procedures ; the first consists of the creation of a new cavity and can be done surgically or non - surgically. the second is vaginal replacement with the pre - existing canal lined with a mucous membrane (a segment of bowel). the most commonly used non - surgical procedure is the frank 's dilatation method, which involves the application first by the clinician and then by the patient of vaginal dilators, of progressively increasing length and diameter, and also ingram 's technique and its modifications.[1417 ] vaginal dilators have few complications as there are no anesthetic or surgical risks, but it is time consuming, causes the patient discomfort, and requires good patient motivation. surgical treatment of the mrkh syndrome is achieved by vaginal reconstruction, which includes ; williams vaginoplasty, which involves suturing the labia majora into a perineal pouch, but the vagina created is external, short, and unsatisfactory for penetrative intercourse ; this procedure is no longer practiced. the vecchietti procedure consists of increasing the vaginal size by gradually applying traction to the vaginal wall. finally, the neo - vagina can be created within the rectovesical space and lined by different tissues such as skin (mcindo - reed), peritoneum (davydov), and intestine. reconstructing the vagina using intestinal segments creates an aesthetically pleasing vagina, does not require moulds, dilatation or lubrication, and in children, the neo - vagina grows with the child with less risk of stenosis. the sigmoid colon has certain advantages, such as, a thick wall, large diameter, does not traumatize easily, has adequate mucosal secretion, which although adequate for lubrication is not excessive or irritating, and does not require regular dilatation after the postoperative period. both had an uneventful postoperative recovery and were satisfied with the immediate outcome. in conclusion vaginal reconstruction using the sigmoid colon has given a good, immediate outcome in this report. long - term follow - up will be needed to assess the long - term outcome, with regard to complications, sexual satisfaction of patients and spouses, as well as, their adaptation and resolution of fertility issues. | the mayer - rokitansky - kuster - hauser (mrkh) syndrome is a rare anomaly characterized by congenital aplasia of the uterus and vagina in women showing normal development of secondary sexual characters and normal 44 xx karyotype. we report our experience in the management of two patients with congenital absence of the vagina due to the mrkh syndrome. the first case was a 24-year - old student, who presented with primary amenorrhea, uterovaginal agenesis, right pelvi - ureteric junction obstruction, and left renal agenesis. the second patient was a 24-year - old housewife, who presented with primary amenorrhea and inability to achieve penetrative sexual intercourse. she had vaginal atresia and a grossly hypoplastic uterus. both had successful sigmoid colovaginoplasty and are sexually active. vaginal reconstruction using the sigmoid colon saw an immediate and satisfactory outcome in both patients |
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