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the responses to psychological and emotional challenges in our day - to - day life are involved in the development of cardiovascular diseases like hypertension. approximately 50 million people in the usa have hypertension, and the prevalence is increasing. it has become a major problem throughout the world, especially in developing countries like iran. its prevalence in iran is as high as in the usa, and because it is a major risk factor for coronary heart disease, stroke, and congestive heart failure, even small reductions in its prevalence could have a potentially large public health and financial impact. also, prevention and treatment strategies are urgently needed to prevent prehypertensive people from developing hypertension and cardiovascular disease. non - pharmacological interventions, such as complementary methods, for hypertension provide an effective means to lower the blood pressure, and have been emphasized increasingly as a useful method for both the prevention and treatment of hypertension. olney also reported that complementary medicine reduces stress and controls the blood pressure, and recent researches indicate that massage therapy is the most popular among the patients. so, nowadays, due to increase in people 's interest to use some of these techniques, particularly massage therapy, this medicine is under the coverage of insurance and is considered as a part of care plans in the hospitals. it was even included in a part of skill and educational curriculum of the physicians and nurses. some of the complementary medicine techniques such as massage are described as part of the nursing job, and today, nurses frequently use complementary methods like massage to provide comfort and to relieve their patients from the feeling of distress. on the other side, perceiving the experiences of patients that clarify diseases and health is a necessary event for nurses to propose effective solutions in the care plans for their patients, especially those with chronic illnesses. qualitative research is particularly suited to the study of complex topics or less - known issues, and is used to describe the world of human experience. on the other hand qualitative research has great potential to give information on massage therapy practice. outcomes, context, and process factors enable the development and provision of more effective and appropriate treatment plans. lastly, qualitative research makes a practitioner more aware of the role of massage therapist - how the practitioner 's own beliefs and values can affect or influence a client 's experience, perceptions, and outcomes. the insights, provided by an awareness of how others practice, can be used to reflect one 's own practice. qualitative research findings, therefore, will not only help massage therapists practice more effectively, but also differently, with greater awareness and mindfulness. the purpose of the present article is to assess and analyze women 's experiences of receiving massage. the aim of the present study was to explore the experiences of women who had prehypertension and received therapeutic massage. the descriptive phenomenological design used in the present study is that of colaizzi, who advocated a descriptive approach in order to gain a description of the meaning of an experience from the participant 's point of view. each person has an understanding of the world that is based upon their own experiences ; therefore, one way to ascertain the perceptions of subjects about their experiences of the massage therapy is to find what they have received by interviewing. the study population included adult non - pregnant women (18 - 60 years) referring to sedighe tahereh cardiovascular research center, isfahan, iran, chosen by purposive sampling. sampling continued until data saturation (nine women). in the present study, by massage therapy we meant swedish massage, which was conducted for the patient in the supine position with non - aromatic topical lotion on the face, neck, shoulders, and upper chest using superficial and deep stroking, three times a week (morning to noon, 8 - 12 am) for 3.5 weeks, in a total of 10 sessions, with each session lasting 10 - 15 min. next, a deep and semi - structured interview was conducted with nine prehypertensive women who received swedish massage. data analysis was done by colaizzi 's method. in a meeting with the participants, the nature, objective, method of study, and their role each interview took 15 - 60 min, and after audiotaping, it was transcribed verbatim and the recorded information was rechecked. the tape scripts were studied and revised meticulously, and the statements related to the phenomena were extracted in order to understand the feeling of participants. next, codes were organized in categories, which were confirmed by participants. in the next stage, the results were integrated using a complete description of the studied phenomenon and revised to have clear concepts. validity and reliability were obtained with respect to measures such as real value, applicability, continuity, and authenticity. this study focused on the patients experiences of massage, as it not only presents a physical therapy but also a personal encounter involving social contacts and a formalized way of providing touch. the participants in this study were nine prehypertensive women (18 - 60 years) who referred to the sedighe tahereh cardiovascular center. they included relaxation, sleeping better, reduction of anxiety and tension, reduction of fatigue, invigorating experience, and improve connecting. the relaxation was felt with an element of surprise ; some were surprised at the speed and ease with which the relaxation occurred. the patients let their daily concerns, worries, and thoughts of their grueling situation briefly dissolve as they received massage. the massage sessions created an atmosphere of peacefulness : the first time i was very apprehensive because i was nt sure and i always found it hard to relax and i did not think about it at all. (p1) it was very relaxing, i could forget about everything. (p7) massage was surprisingly relaxing ; it helped me relax, especially in such a short time. they emphasized that falling asleep resulted from the massage, even in cases of severe insomnia. the massage reduced the anxiety and, consequently, it induced relaxation and sleep : i could nt sleep at all, i mean, i was looking at the clock every hour, but after receiving my massage in the last few weeks, i ve had good night 's sleep. i looked at the clock every hour during the night, waiting for the time to pass. since i had the massage last week, i am able to sleep well, which i could not do for weeks. the massage helped me relax and enabled me to have a good sleep. it affected me positively for the rest of the day as i became much calmer and it also became easier for me to get to sleep at night. it really helped me. (p9) many women mentioned the calming effect of massage. as a result of massage, they experienced a decrease in anxiety and tension they had before. the massage was, therefore, seen as a mode of control ranging from physical to mental aspect of their selves, as no anxiety ruled during and immediately after the session : fantastic, i think it must be very helpful for people who are very stressed and anxious. i felt at ease somehow, calm and wonderfully at peace floating away on clouds. (p1) it was difficult to explain, i think that is probably relieving my anxiety, relieving some of the tension that i experienced ; i did nt notice anything else. massage allowed them to have a time of rest and to respite from weak and poor body and from bothering thoughts as it stimulated energy and eliminated fatigue from the body and mind in daily life : fantastic, i do not feel tired after the massage. i could follow my responsibility, and interestingly all my daily fatigue was eliminated with massage. (p7) massage was so interesting, i felt all my daily fatigue was lost, i could experience something i had never experienced before, it gave me energy and i felt my body worked in different way. now, i feel i can do whatever i want. (p2) several women felt invigorated or energized by the experience. even though they were in a frail condition, massage gave them a temporary subjective feeling of physical and mental strength : it was really fantastic, really relaxing. i can conquer the world now ; i m in better control of myself, physically and mentally, and i found it so enjoying. i feel invigorated now ; i felt ten to fifteen years younger when i received massage. with massage, i could do my duty in the best way and it (massage) helped me somehow to manage the days in a good way. i was cheerful the day that i received massage, i felt more composed somehow, it 's difficult to explain but i felt strengthened in some way. (p4) several women felt improved relationship by the experience. as a whole, massage gave rise to existential respite, mainly by counteracting loneliness, meaninglessness, and anxiety that interfere with family and social relationship. the patients had a positive sense of luxury and existential well - being, and they were in a space away and beyond their current situation that influenced their relationships and improved their connections with others. this feeling seems to be related to taking pleasure in the massage therapy sessions : i felt better than before i can communicate with others, it affected me positively for the rest of the day and i preferred to be with others than being alone. i think it was very good ; i was much better in family relationships by the experience and had no problem and argument. when i get a massage, i have a better relationship with my husband, children, parents, friends and everyone who was in contact with me during the day ; i can say that i m more sociable and friendly now. the relaxation was felt with an element of surprise ; some were surprised at the speed and ease with which the relaxation occurred. the patients let their daily concerns, worries, and thoughts of their grueling situation briefly dissolve as they received massage. the massage sessions created an atmosphere of peacefulness : the first time i was very apprehensive because i was nt sure and i always found it hard to relax and i did not think about it at all. (p1) it was very relaxing, i could forget about everything. (p7) massage was surprisingly relaxing ; it helped me relax, especially in such a short time. the subjects reported that they were sleeping better. they emphasized that falling asleep resulted from the massage, even in cases of severe insomnia. the massage reduced the anxiety and, consequently, it induced relaxation and sleep : i could nt sleep at all, i mean, i was looking at the clock every hour, but after receiving my massage in the last few weeks, i ve had good night 's sleep. i looked at the clock every hour during the night, waiting for the time to pass. since i had the massage last week, i am able to sleep well, which i could not do for weeks. the massage helped me relax and enabled me to have a good sleep. it affected me positively for the rest of the day as i became much calmer and it also became easier for me to get to sleep at night. the massage was, therefore, seen as a mode of control ranging from physical to mental aspect of their selves, as no anxiety ruled during and immediately after the session : fantastic, i think it must be very helpful for people who are very stressed and anxious. (p8) relaxing and interesting. very useful, my anxiety is greatly reduced. i felt at ease somehow, calm and wonderfully at peace floating away on clouds. (p1) it was difficult to explain, i think that is probably relieving my anxiety, relieving some of the tension that i experienced ; i did nt notice anything else. (p5) massage allowed them to have a time of rest and to respite from weak and poor body and from bothering thoughts as it stimulated energy and eliminated fatigue from the body and mind in daily life : fantastic, i do not feel tired after the massage. i could follow my responsibility, and interestingly all my daily fatigue was eliminated with massage. (p7) massage was so interesting, i felt all my daily fatigue was lost, i could experience something i had never experienced before, it gave me energy and i felt my body worked in different way. now, i feel i can do whatever i want. (p2) they experienced a pleasant feeling of inner force and power. even though they were in a frail condition, massage gave them a temporary subjective feeling of physical and mental strength : it was really fantastic, really relaxing. i can conquer the world now ; i m in better control of myself, physically and mentally, and i found it so enjoying. i feel invigorated now ; i felt ten to fifteen years younger when i received massage. with massage, i could do my duty in the best way and it (massage) helped me somehow to manage the days in a good way. i was cheerful the day that i received massage, i felt more composed somehow, it 's difficult to explain but i felt strengthened in some way. (p4) several women felt improved relationship by the experience. as a whole, massage gave rise to existential respite, mainly by counteracting loneliness, meaninglessness, and anxiety that interfere with family and social relationship. the patients had a positive sense of luxury and existential well - being, and they were in a space away and beyond their current situation that influenced their relationships and improved their connections with others. this feeling seems to be related to taking pleasure in the massage therapy sessions : i felt better than before i can communicate with others, it affected me positively for the rest of the day and i preferred to be with others than being alone. (p6) i thought it was actually beneficial and i think it was very good ; i was much better in family relationships by the experience and had no problem and argument. (p1) when i get a massage, i have a better relationship with my husband, children, parents, friends and everyone who was in contact with me during the day ; i can say that i m more sociable and friendly now. key benefits reported included relaxation, sleeping better, relief of anxiety and tension, reduction of fatigue, invigorating experience, and improve relationship and connection everywhere. the observations from this study using massage provided additional information about the experience of receiving a supportive intervention. for the participants, the treatments were valued as a means of reducing anxiety, facilitating relaxation, distraction, and by giving a sense of well - being. for many recipients, the psychological and emotional challenges that we face in our daily life are involved in the development of diseases like hypertension with an increasing prevalence. it has become a major problem throughout the world, especially in developing countries like iran. its prevalence in iran is as high as in the usa, and also, it is higher in women than in men. meanwhile, the results of some studies indicated that women expressed relaxation and reduction of anxiety on experiencing massage. for example, campeau., in their study on 100 patients with various cancers, concluded that patients who had received massage expressed reduction of anxiety directly after the intervention. the results of the present study are consistent with the results of olney, hernandez., and post - white., which reported reduction of anxiety and increase of relaxation caused by massage therapy. the effects of the aromatherapy - massage in reduction of anxiety and depression are consistent with those reported in other studies on massage in depressed mothers who received massage. the experiences of anxiety reduction are also reported in other studies on aromatherapy - massage in patients in palliative or postoperative icu care. the results of this research indicated that women were sleeping better as an effect of having massage. ferrell - torry and glick and meek found that relaxation, comfort, and sleep were promoted in people who received massage therapy. many researches have reported that massage could relieve fatigue. according to the results of jin and kim 's study, elderly women receiving foot reflexology massage experienced a good sleep and had their fatigue relieved after the massage therapy sessions. according to the cochrane database systematic review, the findings of mackereth. suggested that chair massage treatment led to a feeling of invigoration or energized several patients. in their research on 57 pregnant women and their partners who were recruited for a pregnancy massage study during their second trimester of pregnancy, concluded that subjects relationship improved in the massage group. reported an improvement in relationships following massage, which possibly resulted from diminished pain and the decrease in depression and anxiety. on the other hand, the improved relationships in the present study may be due to improved confidence of patients, as the massage therapy improves confidence. many scientists suggest massage as a way to improve self - confidence, social confidence, and communication, possibly due to improvement of communication and relationships. it is concluded that a body - centered intervention like massage could be valuable to increase relaxation, sleep quality, invigorating experience, and relationship quality, and to decrease fatigue, anxiety, and tension in prehypertensive women, which can influence the general well - being of the people and also have a potentially large public health and financial impact in the society. finally, nurses could focus on the experiences of prehypertensive women and use massage as a complementary and alternative medicine to plan appropriate interventions in order to help patients achieve general well - being. although the results obtained from nine patients can not be generalized, the findings can raise some important concerns and questions, which challenge the approaches currently adopted by health care practitioners. the study relied on immediate written feedback, so it can not be an indication of long - terms benefits. in future studies, using a control group and a different intervention such as relaxation techniques may help to collect data concerning assessment of the therapeutic outcomes of massage therapy. | background : hypertension has become a major problem throughout the world, especially in developing countries like iran. as it is a major risk factor for coronary heart disease, even small reductions in the prevalence can have potentially large public health benefits. among the complementary methods, massage provides an effective means to lower the blood pressure. if nurses perceive the experiences of hypertensive patients receiving massage, they can use massage more effectively in their care plan.materials and methods : this is a descriptive phenomenological study. deep interviews were conducted with nine prehypertensive women who received swedish massage three times a week in a total of 10 sessions, with each session lasting 10 - 15 min. then, the researcher conducted an interview using a grand tour question (open ended question) and the participants were then encouraged to speak freely explaining their thoughts and feelings about the experience of massage therapy. data analysis was done by colaizzi 's method. validity and reliability were obtained through measures such as real value, applicability, continuity, and authenticity.results:women evaluated the massage therapy positively. the findings yielded six themes, including relaxation, sleeping better, reduction of anxiety and tension, reduction of fatigue, invigorating experience, improve connecting.conclusions:this study demonstrates that a body - centered intervention like massage can be valuable in a multidisciplinary approach to women with prehypertension. this method is easy to learn and relatively short (10 - 15 min) to administer as a suitable complement in nursing care for this group of patients. |
seminal vesicle cysts can be acquired or congenital the latter is generally associated with other developmental anomalies of the genitourinary tract. the combination of a seminal vesicle cyst and ipsilateral renal agenesis is called zinner syndrome. when these are not detected in early childhood, the seminal vesicle cysts are discovered upon symptoms such as : inflammation ; voiding dysfunction ; abdominal, pelvic, or post - coital pain ; hematuria ; hematospermia ; and sub- or infertility. due to efficient diagnostic techniques, symptomatic cases are usually recognized and treated properly ; however, the surgical removal of the seminal vesicle cyst may become necessary in these patients. recent publications have presented efficient and minimally invasive surgical treatment of the disease in complicated situations [2, 3, 4 ]. according to the medline database, 23 authors presented 35 standard laparoscopic or robot - assisted seminal vesicle cyst removals as of april 2010. however, only a few articles presented data on fertility. in light of our successfully resolved cases we would like to stress the importance of preserving fertility in patients of fertile age, and why publishing data such as this in the future is worthwhile. a 31-year - old man with a history of cardiac arrhythmia, right renal agenesis with normal renal function, and recurrent urinary tract infections. ultrasonography (us) detected a 28x32 mm seminal vesicle cyst on the right side. after an unsuccessful transrectal ultrasound - guided puncture, a transurethral resection (tur) of the verumontanum temporarily resolved the symptoms for five years. pelvic magnetic resonance imaging (mri) and renal scintigraphy showed an enlarged right seminal vesicle, a dilated right ureter, and the absence of the right kidney. because of repeated failure of other treatment options, a transperitoneal laparoscopic nephroureterectomy with removal of the seminal vesicle was performed. during the procedure the patient was placed in the trendelenburg position. the same transperitoneal approach with five ports used for radical prostatectomy after insufflation of the peritoneum with co2 to 12 mm hg, the rectovesical peritoneum was transversely incised. the right seminal vesicle and the ampulla of the vas deferens were identified and carefully dissected sharply with limited use of bipolar coagulation. the seminal vesicle was deflated, sectioned, and its orifice over the ampulla was closed with two stitches. a hypoplastic kidney was found at the usual kidney location, which was removed en bloc. the operation time was 195 minutes, and the estimated blood loss was 300 ml. the patient was free of symptoms 24 months after the procedure, and his sperm count was normal (table 1). patients data one week before and 18 months after operation abbreviations : yrs years, ipss international prostate symptom score, ql quality of life, iief international index of erectile function, conc. semen analysis revealed asthenozoospermia. abdominal ultrasound and computed tomography (ct) showed a large (58 mm) right seminal vesicle cyst and absence of the right kidney. due to the persistent symptoms and the size of the cyst,, we removed the seminal vesicle cyst with the control of the right vas deferens using the aforementioned technique, but in this case the bladder was filled with methylene blue for better exposure. the control semen analysis presented the same motility and a recovery of sperm concentration (table 1). a 31-year - old man with a history of cardiac arrhythmia, right renal agenesis with normal renal function, and recurrent urinary tract infections. ultrasonography (us) detected a 28x32 mm seminal vesicle cyst on the right side. after an unsuccessful transrectal ultrasound - guided puncture, a transurethral resection (tur) of the verumontanum temporarily resolved the symptoms for five years. pelvic magnetic resonance imaging (mri) and renal scintigraphy showed an enlarged right seminal vesicle, a dilated right ureter, and the absence of the right kidney. because of repeated failure of other treatment options, a transperitoneal laparoscopic nephroureterectomy with removal of the seminal vesicle was performed. during the procedure the patient was placed in the trendelenburg position. the same transperitoneal approach with five ports used for radical prostatectomy after insufflation of the peritoneum with co2 to 12 mm hg, the rectovesical peritoneum was transversely incised. the right seminal vesicle and the ampulla of the vas deferens were identified and carefully dissected sharply with limited use of bipolar coagulation. the seminal vesicle was deflated, sectioned, and its orifice over the ampulla was closed with two stitches. a hypoplastic kidney was found at the usual kidney location, which was removed en bloc. the operation time was 195 minutes, and the estimated blood loss was 300 ml. the patient was free of symptoms 24 months after the procedure, and his sperm count was normal (table 1). patients data one week before and 18 months after operation abbreviations : yrs years, ipss international prostate symptom score, ql quality of life, iief international index of erectile function, conc. semen analysis revealed asthenozoospermia. abdominal ultrasound and computed tomography (ct) showed a large (58 mm) right seminal vesicle cyst and absence of the right kidney. due to the persistent symptoms and the size of the cyst, after the nephroureterectomy, we removed the seminal vesicle cyst with the control of the right vas deferens using the aforementioned technique, but in this case the bladder was filled with methylene blue for better exposure. the control semen analysis presented the same motility and a recovery of sperm concentration (table 1). after the successful treatment of two patients with zinner syndrome we were able to prove with internationally used questionnaires and semen analysis that laparoscopic removal of seminal vesicle cysts can resolve symptoms while preserving fertility and erectile function. after reviewing the published standard laparoscopic or robot - assisted cases we found that the symptoms disappeared in the majority of patients but only a few papers reported data on fertility. sperm analysis was performed in two cases before the operation, but the authors did not record sperm count or motility after cyst removal. one of mcdougall 's patients had hypospadiasis and left cryptorchidism in his medical history and semen analysis revealed azoospermia before the operation. among the three reported patients, none had erectile problems after the procedure but the methodology of the registration was not described. in the paper of selli, a patient with normal sperm count was presented, but he did not have any children yet. in another three articles the drained fluid was analyzed in one case spermatozoa and in two cases normal sperm cells were found [7, 8, 9 ]. other authors reported patients having children in three cases [4, 6, 10 ]. one patient fathered a child between successful laparoscopic removal and a robot - assisted removal of bilateral seminal vesicle cysts. in the majority of the reported cases the authors made some efforts during the procedure to preserve fertility, but this may not be successful in some complicated situations like serious congenital malformations or chronic inflammations. sixteen of the 35 reported patients were in the most active sexual period of their lives (16 - 39 years), and five more patients were in the fifth decade. only four of all these cases had at least data from cystic fluid regarding sperm morphology, the others did not. however, with a disease such as this, which changes the anatomy of the reproductive tract and can affect fertility, it is considerable to perform and report semen analysis and erectile function. also, limited use of bipolar coagulation, careful dissection, and the administration of methylene blue for better exposure might be key factors in the preservation of fertility during these procedures. large cohort studies are limited because of the low number of cases, thus gaining as much experience as possible is essential in order to set up guidelines for the proper treatment of these patients. andrologists and urologists alike must keep these rules in mind, because they are the ones who meet and treat these patients in the vast majority of the cases. in cases of symptomatic and complicated seminal vesicle cysts, a surgical procedure should be considered. the advantages of laparoscopic and robotic approaches include the possibility of preserving erectile function and fertility during seminal vesicle cyst removal. registration of fertility parameters is of utmost importance in young patients. due to the rarity of seminal vesicle cysts, preoperative examinations and treatment modalities should focus not only on the relief of symptoms but also on the preservation of fertility and erectile function, which have hardly been reported in the reviewed cases. such data would reveal the real advantages of laparoscopy over other treatment options and could serve as a guide for how and when to treat these patients. | seminal vesicle cysts can cause sub- or infertility. minimally invasive techniques have the advantage of preserving the vas deferens by the treatment of symptomatic cases. after reviewing the published articles, only a few of them presented data on fertility before and after surgery. the authors now report the successful treatment of two patients with seminal vesicle cysts, in which laparoscopic cyst removal resolved the symptoms, preserving fertility and erectile function. due to the rarity of seminal vesicle cysts, preoperative examinations and treatment modalities should focus not only on the relief of symptoms but also on the preservation of fertility and erectile function. |
the calcifying cystic odontogenic tumor (ccot) was described by gorlin. in 1962, as a distinct entity derived from odontogenic epithelial remnants, showing various clinical and histological features. it is an extremely rare lesion representing 2% of all odontogenic pathological changes in the jaw. the ccot usually arises intraosseously, but it may also occur extraosseously, with about equal frequency in the mandible and maxilla. the age of the patients may range from 5 to 92 years, with peak incidence in the second and sixth decade of life. the unique histopathological features include a fibrous wall and a lining of odontogenic epithelium with columnar or cuboidal basal cells resembling ameloblasts. ghost cells, which may occasionally become calcified, situated in the cyst lining or in the adjacent fibrous tissue. hyalinized eosinophilic material, suggestive of immature or dysplastic dentin, is present, and is closely associated with the epithelial lining. this report describes a case of ccot that occurred in the mandible of a 75-year - old male, with no reports of recurrence since its complete removal in september 2007. a 75-year - old male was referred to m m college of dental sciences and research for a swelling on left side of the mandible. the patient first noticed the swelling five years back when he underwent uneventful extraction of 33, 34. initially the swelling was minimal, but it had grown slowly with time to the present size. clinical examination disclosed a 4 2 cm, ovoid firm swelling on the residual ridge in the region of 32 to 34 [figure 1 ]. radiographically a poorly demarcated unilocular radiolucency with radiopaque foci in the region of 33, 34 was seen [figure 2 ]. swelling of the left anterior residual ridge diffuse unilocular radiolucency with radio - opaque foci in the region of 33, 34 the lesion was excised under local anesthesia. after fixation in buffered formalin the tissue was processed and stained with h and e stain. the h and e stained section showed a cystic cavity lined by odontogenic epithelium of varying thickness (13 cell layers). the basal cells were cuboidal to columnar in shape with their nuclei pushed away from the basement membrane resembling ameloblasts. the outer layers of the cells were composed of loosely arranged angular cells resembling stellate reticulum, which in some areas were eosinophilic, ballooned, and keratinized forming ghost cells [figure 3 ]. the nuclei of these cells were pushed to the periphery and eventually disappeared as the cells enlarged. individual cells became confluent with the adjacent ballooning cells and gradually lost their cell boundaries, making large sheets of amorphous, acellular eosinophilic material, filling the cystic lumen. some ghost cells were also seen in the underlying connective tissue, which was loose and vascular, with hemorrhagic areas along with some variable - sized islands of odontogenic epithelium. irregular masses of hyalinized acellular calcified material (dentinoid) were also observed in the connective tissue, in relation to both epithelial lining and masses of ghost cells [figure 4, figure 5 ]. photomicrograph showing odontogenic cyst lining with ghost cell proliferation (h and e, 10) photomicrograph showing dentinoid material and odontogenic islands (h and e, 20) photomicrograph showing sheets of ghost cells lined by epithelium adjacent to bone (h and e, 10) based on these features the lesion was diagnosed intraosseous calcifying cyst odontogenic tumor. healing was uneventful and in the six - month follow - up there were no signs of recurrence. in 1971, calcifying odontogenic cyst was described as a nonneoplastic cystic lesion, but in 1992 the who classified this lesion with odontogenic tumors because of its histological complexity and diversity. in 2005, the who renamed it as a calcifying cystic odontogenic tumor. clinically, ccot corresponds to 2% of the odontogenic tumors with the age range between 5 to 92 years. several articles have reported that a greater incidence takes place in the second decade, but other authors have noticed a bimodal distribution with a second peak of incidence in the sixth and seventh decade of life. in the present case the patient was in his eighth decade of life. both central and peripheral forms occur with equal frequency in the maxilla and mandible with a majority of lesions being located in the incisor - canine area. the reported lesion was of the central variety and was present in the lower left mandibular region. the central ccot occurs as an asymptomatic hard swelling in the jaws, which produces expansion of bone, whereas, the peripheral variant presents as a nonspecific well - circumscribed sessile or pedunculated mass, with a smooth surface. the presented case, similar to other reports in the literature, was an asymptomatic hard swelling present since the past five years. radiopaque structures within the lesion, either irregular calcification or tooth - like densities are present in about one - third to half of the cases. the roots of the adjacent teeth are displaced or resorbed from the expansion of the lesion. radiographically the present case shows a diffuse unilocular radiolucency with some radio - opaque foci in the region of 33 and 34. although histological criteria have been established for the diagnosis of the ccot, its pathogenesis is still speculative. freedman. proposed that the neoplastic cell originated from a well - differentiated ameloblast, and its neural crest origin confers to this cell a pluripotential capacity to undergo terminal differentiation. starting from the postulate that ameloblasts are stem cells, terminal differentiation is not necessarily required to originate the ccot neoplastic cells. believe coc cystic epithelium originates from the reduced enamel organ, from islands of odontogenic epithelium within the tooth follicle or from the remnants of odontogenic epithelium in the bone or gingival tissue. in the reported case, we believe the neoplastic epithelium arose from the remnant of odontogenic epithelium present in the bone. the presence of the so - called ghost cells, which are assumed to represent an abnormal form of keratinization, is the most conspicuous feature of the lesion. however their mere presence does not justify a diagnosis of ccot, as other lesions also show their formation. hence diagnosis of ccot should only be made for a lesion in which the formation of ghost cells takes place in a typical epithelial cyst lining, presenting a basal layer of cuboidal or short cylindrical cells and an overlying layer consisting of cells that bear resemblance to stellate reticulum - like cells. the formation of dentinoid or osteoid, which is frequently described as being present in connection with masses of ghost cells is another characteristic finding of the lesion. gorlin. considered the appearance of dentinoid material in ccot to represent an inflammatory response of the body tissue toward masses of ghost cells. abrams and howell further stated that masses of ghost cells induce granulation tissue to lay down juxtaepithelial osteoid which may calcify. contrary to these interceptions sauk stated that the juxtaepithelial osteoid and dentinoid are frequently found in areas free of either granulation tissue or ghost cells and postulated that it might be an inductive phenomenon. to date it remains to be clarified as to whether this material represents a true inductive effect or merely a metaplastic change in the connective tissue. nevertheless in the present case these dentinoid - like regions were observed both in association with epithelium and ghost cells. due to the clinical characteristics of this lesion, the differential diagnosis of ccot must be done with regard to the ameloblastoma, dentigerous cyst, and keratocysts. in more advanced stages it could be confused with calcifying epithelial odontogenic tumor, fibro odontoma, adenomatoid odontogenic tumor and partially mineralized odontoma. histopathologically the differential includes ameloblastic fibro odontoma, complex and compound odontoma, ameloblastoma, and carcinomas. in relation to the treatment recurrence of ccot is rare, only eight cases of recurrences have been reported in english literature. the lack of recurrence depends on the complete excision. in the reported case complete excision of the tumor | the calcifying odontogenic cyst was first reported by gorlin. in 1962. it had been classified as a neoplasm related to the odontogenic apparatus because of its histological complexity and morphological diversity until it was renamed as a calcifying cystic odontogenic tumor by the who, in 2005. here we describe a case of mandibular calcifying cystic odontogenic tumor in a 75-year - old male, which was present since five years, with a history of occurrence after the extraction of teeth in the involved region. the lesion was surgically removed and a histopathological examination revealed a cystic tumor with predominance of ghost cells and some amount of dentinoid tissue. |
the control of human posture partly depends on proper patterns of muscle activity1. after receiving efferent impulses from the central nervous system, muscles usually react to balance disturbances by extra - recruitment of specific motor units with appropriate temporal and spatial relations2, 3. depending on the range of the destabilizing stimulus, optimal strategies for balance recovery are activated4. the distal - proximal sequence of muscles actions, defined as the ankle strategy is realized when a person stands in an upright bipedal position on a wide support plane with attempts to reduce the influence of destabilizing forces5,6,7,8. muscles acting at the ankle are activated first in response to impulses destabilizing upright posture, inducing body shifts mainly in the sagittal plane9, 10. it can be assumed that ankle strategy can be changed in patients with a history of sciatica, and from a clinical point of view, balance training is required to prevent the recurrence of low back pain. previous studies revealed that other joints in the lower extremities are stabilized in these conditions by passive properties of muscles and surrounding tissues11. the activity of lower extremity muscles is coordinated by spinal centers receiving afferent input from many types of proprioceptors, which results in adaptation of muscle motor unit recruitment12, 13. in bipedal upright position in healthy subjects, the center of foot pressure (cop) is kept minimally forward from the ankle and oscillates in a limited area created by the contour of the foot support surface11. patients with chronic low back pain of different etiologies present the abnormal patterns in sustaining a stable upright position. the sway of cop in the upright position in these patients is pathologically increased, particularly in the sagittal plane. the mechanism of this disturbed coordination remains unclear12, 14, 15. from the report by chen., it can be assumed that patients with sciatica present changes in proprioception and a significant decrease in force and endurance in paravertebral and lower extremity muscles16. these muscles dysfunctions as well as diminished physical activity in patients with low back pain are likely to disturb maintenance of a stable posture14, 17. geisser.18 and jacobs.15 provided evidence for increased amplitudes on electromyography (emg) recordings from back and lower extremity muscles in patients with low back pain. this increase is a sign of abnormal extra - activity of all muscles engaged in posture control, which primarily limits cop sway14, 15, 19. this prolonged muscles abnormal activity leads to greater muscle fatigue, resulting in a further increase in cop sway1, 17. the question arises whether there are persistent alterations in activity in muscle motor units engaged in ankle strategy following the successful treatment of sciatica and pseudo - sciatica. such alterations should be detectable by surface - recorded polyelectromyography and by cop sway velocities and center of spectrum measurements. examinations revealing such alterations may enable the choice of optimal physiotherapeutic procedures to prevent subsequent postural disturbances. twenty patients (11 women and 9 men) aged 3555 years (45.5 years on average) with a history of low back pain radiating to one leg were studied. a control group of 9 healthy volunteers (5 women and 4 men) aged 26 to 51 years (46.4 years on average) with a good health status were also studied. each subject was informed about the aim of the study. written consent for participation in this project and data publication was obtained from every participant. ethical considerations conformed with the declaration of helsinki, and approval was also received from the bioethics committee. following sciatica in 11 patients, the cause of pain was found to be unilateral l5-s1 disc - root conflict confirmed by magnetic resonance imaging (mri) and clinical assessment20. these patients previously reported characteristic radiation of pain in the lower extremity in an l5-s1 dermatome distribution, with decreased sensory perception and weakness of muscles innervated by l5-s1 nerve roots. patrick s and gaenslen s tests were also positive in patients with pseudo - sciatica syndrome. pretreatment electroneurography (eng) showed a decrease in amplitude in m - wave evoked potentials and frequency of recorded f - waves following stimulation of l5-s1 motor fibers only in patients with a root conflict. a previous study described the function of muscles acting at the ankle 6 months following successful treatment21. no sciatica or pseudo - sciatica symptoms were reported or detected on the day of examination. no changes in motor fiber transmission of neural impulses during eng were found ; therefore no invasive needle emg recordings were necessary. the results of surface (semg) and postural examinations performed once were compared to results obtained in the control group (table 1table 1.results of examinations performed with balance platform and surface electromyographynormal standing (ns)examined parameterhealthy subjects n=9following sciatica n=11following pseudo - sciatica n=9cop sway velocity (mm / s)4.71.95.52.14.91.4cop center of spectrum (mm)0.330.030.410.07 0.370.1examined muscleaffected sideunaffected sideaffected sideunaffected side(amplitude in v) semg gastrocnemius238.3115.2240.9151.6245.4155.3248.997.5251.1146.3semg extensor dig.115.595.4120.9108.4127.7102.1105.577.3102.240.2(no of fluctuations) semg gastrocnemius10.55.21.61.51.20.93.42.5 3.01.1semg extensor dig.11.95.210.74.49.62.99.04.98.94.8tandem position (t)examined parameterhealthy subjects n=9 following sciatica n=11 following pseudo - sciatica n=9 cop sway velocity (mm / s)13.18.416.18.815.73.9cop center of spectrum (mm)0.490.130.50.170.490.11examined muscleaffected sideunaffected sideaffected sideunaffected side(amplitude in v) semg gastrocnemius383.3245.3748.2356.8 338.1211.9502.2349.5311.1207.3semg extensor dig. 319.4203.7681.8455.1 310263.6561.1231.5383.3246.2(no of fluctuations) semg gastrocnemius11.76.04.32.94.42.5 4.72.8 5.43.9semg extensor dig.13.55.56.54.4 9.44.97.73.2 8.13.7cop : center of foot pressure ; semg : surface recorded electromyogram amplitude. statistics are shown as mean values and standard deviations. differences at p<0.05 between results detected following sciatica and pseudo - sciatica syndromes versus healthy subjects ; differences at p<0.01 are marked ; differences at p<0.05 between results recorded in patients from both groups). statistics are shown as mean values and standard deviations. differences at p<0.05 between results detected following sciatica and pseudo - sciatica syndromes versus healthy subjects ; differences at p<0.01 are marked ; differences at p<0.05 between results recorded in patients from both groups a rehabilitation physician qualified each subject for the study and conducted a clinical examination. the neurophysiologists did not know which group a subject (healthy volunteer or patient) belonged to. physiotherapists performed cop sway velocity / center of spectrum measurements with the same rigour as neurophysiologists. the metitur good balance (m good balance system) platform was used for the evaluation of sway velocity and measurements of the center of spectrum for the tested posture (cop) in static conditions (fig. 1.photographs illustrating normal (aa) and tandem (ba) positions during standing on a balance platform. bilateral locations of surface electrodes over extensor digiti (ab, bb) and gastrocnemius (ac, bc) muscles during semg examinations performed in normal standing and tandem positions, respectively, are presented. examples of semg original recordings in healthy subjects (ca, da), following pseudo - sciatica syndrome (cb, db), and following sciatica (cc, dc) during normal standing (c) and tandem (d) positions are shown in the lower part of the figure. the two upper traces were recorded from gastrocnemius muscles on right and left sides while the lower two were from extensor digiti muscles in the same manner. calibration bars for sensitivity (200 v / d) and time base (4 s / d) used in semg recordings are shown in the left bottom corner. arrowheads mark greater amplitude and less frequent fluctuations in recordings from the extensor digiti muscles of affected legs in patients with sciatica.). first, measurements were performed for normal standing position (ns) with feet placed parallel 20 cm apart. a patient was asked to stand motionless in an upright position for 30 s with eyes open. photographs illustrating normal (aa) and tandem (ba) positions during standing on a balance platform. bilateral locations of surface electrodes over extensor digiti (ab, bb) and gastrocnemius (ac, bc) muscles during semg examinations performed in normal standing and tandem positions, respectively, are presented. examples of semg original recordings in healthy subjects (ca, da), following pseudo - sciatica syndrome (cb, db), and following sciatica (cc, dc) during normal standing (c) and tandem (d) positions are shown in the lower part of the figure. the two upper traces were recorded from gastrocnemius muscles on right and left sides while the lower two were from extensor digiti muscles in the same manner. calibration bars for sensitivity (200 v / d) and time base (4 s / d) used in semg recordings are shown in the left bottom corner. arrowheads mark greater amplitude and less frequent fluctuations in recordings from the extensor digiti muscles of affected legs in patients with sciatica. the second test position with eyes open (lasting 20 s) was standing posture with the feet in tandem position (t). a patient was instructed to stand motionless in an upright position for 20 s with eyes open and with the feet in tandem position. the average cop sway velocity in mm / s was calculated for both tested postures for changes in anterior - posterior direction. the velocity of cop sway was calculated by evaluating the total amount of displacement. a higher velocity indicated greater instability of posture related to disturbances in ankle strategy realization. the other parameter was the cop center of spectrum measured in mm and counted with fourier transform formulas. the center of spectrum was the median value of body oscillations in the anterior - posterior direction according to a description by the metitur good balance platform manufacturer. the resolution of precise cop center of spectrum measurement was estimated at 0.05 mm. the assessment of the function of muscles engaged in ankle strategy was performed with semg recorded from the gastrocnemius and extensor digiti muscles in both extremities. pairs of standard bipolar agcl - gelled electrodes with 5 mm recording surface were placed on the skin over the muscle belly and tendon. an eight - channel keypoint system (medtronic a / s, skovlunde, denmark) (fig. 1abc and 1bbc) muscle motor unit activity was tested in two positions (ns and t). the keypoint system enables automatic acquisition of mean frequency and amplitude parameters. because the activity of motor units in all emg recordings ranged from 70 to 90 hz, the parameter of mean amplitude fluctuation was mainly analyzed in semg recordings. sensitivities in semg recordings were adjusted at 200 v / d and time base at 4 s / d. parameters of mean amplitude and number of fluctuations recorded in muscles of both lower extremities were analyzed in healthy volunteers and patients. the amplitude of recruiting motor unit action potentials was measured as the maximal - minimal value of negative - positive inflections with reference to the isoelectric line. fluctuation was defined as a period of temporal amplitude change (increase or decrease) lasting more than 1 second during semg recording of muscle motor unit activity. ethical considerations conformed with the declaration of helsinki (1975, revised 1983). approval was also received from the bioethics committee of the university of medical sciences in pozna, poland. results of tests for both groups of patients were evaluated with analysis of variance (anova) procedures for repeated measurements. more important differences between analyzed parameters in all groups of patients were found at p<0.01. values for cop sway velocity and cop center of spectrum parameters were greater in t than in ns position in all subjects during postural studies (table 1). moreover, these studies revealed an increase in cop center of spectrum values during ns recordings only following sciatica in comparison to healthy volunteers (p<0.05). activity of examined muscles was statistically greater following sciatica during semg studies in t position at p<0.05, in comparison to healthy volunteers and patients following pseudo - sciatica syndromes. it is noteworthy that mean amplitude values recorded from both examined muscles were greater in t than in ns positions in all patients (fig. 1, the number of fluctuations in raw semg recordings from gastrocnemius muscles in both ns and t positions was significantly fewer in patients following sciatica (at p<0.01) than following pseudo - sciatica syndromes (at p<0.05) in comparison to healthy volunteers. moreover, the same parameter differentiated patients following sciatica from those following pseudo - sciatica syndromes at p<0.05. fluctuation changes in semg recordings from extensor digiti muscles were significant at p<0.05 during measurements on the affected sides only in t positions (table 1 ; fig. 1, the results of semg recordings indicated a persistent abnormal pattern in activity of selected ankle muscles following sciatica. this may also suggest abnormalities in ankle strategy confirmed by a significant increase in the cop center of spectrum values in patients in whom stability of upright posture was changed in normal standing. the findings of other authors indicate that patients with low back pain are characterized by disturbed body stability caused by impairments at the lumbosacral spine and reduced proprioception from the lower extremities12, 14. jo. described global postural imbalance with disturbances of trunk stability at lumbosacral levels in patients with low back pain24. results of postural examinations performed by della volpe. indicated that changes in lower extremity proprioception in patients with low back pain influenced the number of anteroposterior oscillations of cop in different tested positions12. in the present study, all examined subjects presented greater cop sway velocities in t than ns positions, which suggests that the t position is less stable. however, it should be noted that cop sway velocity measurement did not allow for differentiation between patients and healthy volunteers (table 1), in contrast to cop center of spectrum parameters, but only in ns position. stated that patients with chronic low back pain try to compensate for deficits of postural stability by increasing anteroposterior oscillations, which allows for their differentiation from healthy subjects25. the abnormal activity of ankle muscles presented by patients with a history of sciatica may result from disturbances of afferent proprioception, which influences sensory integration processing at spinal and supraspinal centers. during semg recordings in t position, an increase in gastrocnemius muscle motor unit activity was found on the previously affected side, but only following sciatica. such changes were not found in a group of patients with a history of pseudo - sciatica episodes, which differentiated patients in both examined groups. similarly, jacobs. observed that chronic low back pain is related primarily to wider - band muscle activity, which probably prevents excessive horizontal displacement of the trunk15. they also observed an emg amplitude increase during recording from the gastrocnemius muscle in response to an attempt to cause the patient to lose balance. in the present study, an additional characteristic property of semg recordings from the gastrocnemius muscle was the smaller number of amplitude fluctuations that were more significantly expressed following sciatica compared with pseudo - sciatica syndromes, but were again detected in less stable t position. tried to explain these fluctuations in healthy people as temporary and spatial activation of new motor units, and deactivation of motor units acting for a prolonged duration2. it seems that a smaller number of fluctuations found in a group of patients with a history of sciatica is a sign of persistently disturbed somatosensory integration, which is too small, however, to evoke a significant change in cop velocity values. the data presented in table 1 regarding the parameters of semg indicate that the number of detected fluctuations in recordings performed on patients was significantly lower in comparison with healthy volunteers. it is doubtful that changes that were significantly different in the three examined groups were caused by movement - related artifacts, but rather reflected different mechanisms of motor unit activation. confirmation of disturbances in motor unit activity in muscles on the previously affected side during tandem position recordings in patients following sciatica can be reflected in an increase in semg amplitude values. this is in all probability a sign of adaptation in muscle activity in realizing ankle strategy. according to horak and nasher, disturbances in realization of ankle strategy the clinical importance of this study consists in the necessity of introducing elements of balance training in rehabilitation of patients with sciatica. the aim is to diminish the probability of lumbosacral pain arising from abnormalities in ankle strategy realization. the strength of the study is in the application of two complementary functional methods for evaluation of abnormalities in postural control in patients with a history of sciatica ; a weakness is the limited number of patients in this study. in conclusion, the results of this study suggest that an abnormality in activity of gastrocnemius and extensor digiti muscle groups may appear following sciatica, resulting in disturbances of proper ankle strategy realization. | [purpose ] it is hypothesized that ankle strategy can be changed in patients with a history of sciatica. the aim of this study was to detect residual disturbances following successful treatment. [subjects and methods ] in patients with a history of sciatica (n=11) and pseudo - sciatica (n=9), differences in muscle activity were recorded with bilateral surface polyelectromyography and stability measurements (center of foot pressure sway and center of spectrum) in normal standing and tandem positions. results were compared with recordings in healthy people (n=9) to identify abnormalities in electromyographic and postural studies. [results ] increased amplitude of electromyographic recordings from the gastrocnemius and extensor digiti muscles on the affected side was detected more in patients with a history of sciatica than pseudo - sciatica syndromes in tandem position. fewer amplitude fluctuations were observed in both positions preferably in patients following sciatica. changes in center of foot pressure sway and center of spectrum during balance platform studies were detected in normal standing position in this group of patients. no similar abnormalities in electromyographic and postural studies were detected in healthy people. [conclusion ] sciatica and pseudo - sciatica evoke persistent disturbances in activity of muscles responsible for ankle strategy. electromyography differentiates the two groups of patients better than postural studies. |
the medical records of 12 culture - proven mdr - pa keratitis who presented from 2005 to 2013 were retrospectively reviewed for clinical features, ulcer progression, microbiology findings, treatment details, and outcome. each isolate was labeled resistant (resistant and intermediate) or susceptible to a particular antibiotic based on the zone of inhibition around the antibiotic impregnated filter paper disc. multi - drug resistance was considered if the bacterial isolate exhibited resistance pattern to three classes of antibiotics. initially following gram - stain report these patients had been treated with 0.3% ciprofloxacin or ofloxacin, 1% atropine sulfate and oral ibuprofen. two nonconventional antibiotics were used in four patients - 1.6% colistimethate sodium and 5% imipenem / cilastatin. the former was prepared by reconstituting 1 million iu/80 mg of colistimethate sodium (xylistin, cipla ltd., mumbai, india) powder with 5 ml distilled water, giving a concentration of 16 mg (200,000 iu)/ml (1.6%) and the latter by reconstituting 50 mg of imipenem / cilastin (cilaster, alkem ltd., mumbai, india) with 10 ml of distilled water to obtain a 5% concentration. patients were admitted, and the antibiotics were instilled half - hourly for 4872 h and then 1 hourly during daytime and 3 hourly during the night and thereafter the frequency was reduced according to the clinical response. before the use of these antibiotics, the patients were explained about the empirical nature of the treatment and informed consent was obtained. the ulcer was considered to be healed once there were corneal epithelialization and resolution of the infiltrate and inflammation. the demographic details, clinical features, treatment, and outcome of the 12 patients are given in table 1. there were 8 (67%) males and 4 (33%) females with age ranging from 6 to 64 (mean : 44.33 18.81) years. the mean time interval to the doubling of ulcer size or progression to perforation was 4.16 2.41 (range : 210) days. seven(58.33%) patients progressed to corneal perforation while 4(33%) patients had endophthalmitis like patient 3 [fig. 1 ]. the outcome in eight patients treated only with fluoroquinolones was evisceration in 4 (50%) patients, corneal graft in 1 (12.5%) patient, phthisis bulbi in 1 (12.5%) patient, and no clinical improvement in 2 (25%) patients. the only patient treated with imipenem / cilastatin did not improve clinically and underwent therapeutic corneal grafting. clinical features, treatment details and outcome clinical slit - lamp photograph of the left eye of a 35-year - old farmer (patient no. 3) who had presented on august 07, 2006 with a pigmented fungal corneal ulcer (a) caused by curvularia lunata and on the same day underwent superficial keratectomy to remove the pigmented plaque (b). repeat scraping isolated multi - drug resistant pseudomonas aeruginosa and 5 days later there was total corneal involvement with perforation and endophthalmitis (d) results of antimicrobial susceptibility test by kirby bauer disc diffusion clinical slit - lamp photographs of the right eye of a 55-year - old farmer (patient no. 10) who presented on january 10, 2010 with a corneal graft infiltrate 2 months after undergoing penetrating keratoplasty (a). two days later the infiltrate had doubled with corneal melting (b) necessitating the application of cyanoacrylate adhesive. three weeks later the infiltrate had reduced significantly (c) and (d) complete healing occurred by 3 months clinical slit - lamp photographs of the left eye of a 60-year - old retired gentleman (patient no. 11), who had been previously treated for presumed herpes stromal keratitis, presented on march 08, 2012 with a corneal ulcer (a) which 48-h later had doubled in size (b). at this point, four days later (c) the infection appeared to be under control and eventually healed (d) by 2 weeks clinical slit - lamp photographs of the left eye of an 18-year - old male (patient no. 12) who presented on may 06, 2013 with a corneal ulcer following trauma with a metallic foreign body (a). the ulcer had doubled 48-h later with corneal melting (b) when 1.6% colistimethate treatment was initiated. four days later (c) there was reduction in infiltrate density and corneal melting appeared to have ceased and subsequently the ulcer healed (d) by 30 days increasing resistance to topical ciprofloxacin, the drug of choice in pa bacterial keratitis has been reported since the late 1990s. alternative antibiotics suggested in different reports included amikacin, or combinations of tobramycin and ticarcillin, or ceftazidime and a fluoroquinolone. however, when the isolate is resistant to commonly available antibiotics the options are limited. therefore, on the basis of reports from systemic infections published in nonophthalmic literature, we selected two alternative antibiotics imipenem / cilastatin and colistimethate. our clinical experience with imipenem / cilastatin was not favorable, although the isolate was susceptible to the drug in vitro. this physical deterioration of the drug may have affected its potency and be the reason why treatment failed, and the patient eventually required therapeutic keratoplasty. however, in a report, where meropenem, a newer carbapenem, was used, no such physical change was reported by the authors and all of their four cases had healed. all the three patients in our series, of whom two had moderately severe ulcer [table 2 ], who were treated with 1.6% colistimethate healed. due to the limited treatment options in systemic infections with mdr gram - negative bacteria, colistimethate (or polymyxin e) a polypeptide antibiotic introduced initially in the 1960s is seeing a resurgence in use. after the submission of our paper for publication, a report by jain. was published, where the authors reported the use of 0.19% colistimethate in eight patients with keratitis due to mdr - pa. similar to our series, the clinical presentation of their patients was characterized by rapid progression of the ulcers and nonresponsiveness to initial broad - spectrum antibiotics. the concentration of colistimethate used in this study, 16 mg / ml, was higher than the study reported by jain. who used 0.19%. the choice of 0.19% concentration was explained by the authors to be based on a previous paper published by lund and plains however, none of these reports, provide any scientific evidence or explanation based on which a 0.19% concentration was selected. despite a higher concentration we did not observe any ocular features of toxicity. lund. did not observe any ocular or systemic toxicity in experimental rats and dogs, by using oral doses of 6.67, 20 mg and 60 mg base / kg daily for a period of 90 days. colistimethate sodium is hydrolyzed to colistin sulfate, the active form of the drug and other sulfomethylated derivatives. the use of a higher concentration of colistimethate sodium would theoretically provide a higher concentration of colistin base as cited by jain. the higher concentration would be advantageous as it would besides increasing efficacy, also prevent the development of bacterial resistance. the preparation of 1.6% concentration of colistimethate involves a single step of dilution in contrast to 0.19% concentration, which requires double dilution. thus, the former process is simpler and would make compounding the solution less cumbersome in eye wards and pharmacies. further studies are required to gain information on pharmacokinetics and pharmacodynamics of colistimethate in ocular use to come to an ideal concentration. the clinical progression in mdr - pa keratitis is fulminant and so rapid that the eye is often beyond salvage even before the susceptibility report is available. four of the eight patients in the series reported by jain. required additional surgical intervention such as application of cyanoacrylate adhesive and corneal patch graft. of the three patients that we treated with colistimethate sodium, one (patient no. 10) [fig. 2 ] required application of cyanoacrylate adhesive for impending perforation. in an earlier paper, the same authors had suggested performing early keratoplasty in larger ulcers as a globe - saving procedure. a lower performance of therapeutic corneal grafts that resulted in a higher evisceration rate in our patients who were treated with conventional antibiotics is due to lack of donor corneal tissue, a limitation which is common in almost all developing countries. furthermore, antibiotic susceptibility testing in an ophthalmic microbiology laboratory uses a limited number of antibiotics leading to delay in identifying alternative drugs. to avoid this delay we now routinely include colistimethate, piperacillin, and meropenem in antibiotic susceptibility test for pseudomonas isolates. although this series is small, it underlines the severity of infection with mdr - pa and the efficacy of 1.6% colistimethate. there is a need for further studies, but as observed by others the low incidence rate of this particular infection is likely to make larger clinical trials a difficult proposition. pooling of data from multiple centers may give more information on antibiotic resistance trends and provide evidence - based solutions. nevertheless, this study along with other reports provide an important evidence to support the use of these alternative antibiotics in an infection where treatment options are extremely limited. | the purpose was to evaluate the clinical outcome in multi - drug resistant pseudomonas aeruginosa (mdr - pa) bacterial keratitis and report the successful use of an alternative antibiotic, topical colistimethate in some of them. the medical records of 12 culture - proven mdr - pa keratitis patients, all exhibiting in vitro resistance by kirby bauer disc diffusion method to three classes of routinely used topical antibiotics were reviewed. eight patients were treated with 0.3% ciprofloxacin or ofloxacin, 1 patient with 5% imipenem / cilastatin and 3 patients with 1.6% colistimethate. the outcomes in 8 eyes treated with only fluoroquinolones were evisceration in 4 eyes, therapeutic corneal graft in 1 eye, phthisis bulbi in 1 eye, and no improvement in 2 eyes. the eye treated with imipenem / cilastin required a therapeutic corneal graft. all the three eyes treated with 1.6% colistimethate healed. colistimethate may prove to be an effective alternative antibiotic in the treatment of mdr - pa keratitis. |
one of the main goals of cell biology is to understand how a cell works as a machine : where and how key proteins interact and achieve a desired function. it is no surprise, then, that an experimental article very often ends with a final figure that distills insights from results into a cartoon - like qualitative model a mechanistic blueprint depicting protein machinery at work. more often than not, this is all that is needed, but there are cases in which there is a clear question that requires a mathematical or computational model. there could be various incentives for modeling for example, to make sure that the cartoon does not contradict the rules of physics and chemistry, or the cartoon could be so complex that mathematics rather than limited intuition is necessary to demonstrate that the protein machine works as hypothesized (mogilner., 2006). as experimental biology becomes more quantitative and complex, the number of cases in which modeling accompanies experimental studies grows. one of them is that modeling approaches have diversified so much recently that it is not easy to grasp how models work. if one looks at the greatest early successes of mathematical cell biology, it is hard to overlook two iconic examples : in the middle of the 20th century, turing (1952) demonstrated mathematically that two chemicals, a slowly diffusing activator and a rapidly diffusing inhibitor, can generate spatially periodic patterns, which created a fundamental paradigm for morphogenesis. at around the same time, hodgkin and huxley (1952) showed numerically that a complex combination of ion fluxes through highly nonlinear voltage - gated channels can account for excitable electric pulses in nerve cell membrane. these studies and hundreds of others used the powerful technique of differential equations (des), which goes back to isaac newton, who discovered that laws of mechanics can be expressed by relating rates of change of position and velocity (time derivatives) to position - dependent forces. solutions of resulting ordinary differential equations (odes) turned out to be spectacularly effective in astronomy and physics. later, fourier proposed a diffusion equation one of the most important partial differential equations (pdes)according to which the rate of change of a concentration at a given point is proportional to the spatial derivative of the spatial gradient of this concentration. indeed, newton said that the laws of nature are written in the form of des, but he mostly meant physics. the reason is very simple : a great number of biological problems deal with many molecules that move randomly and/or directionally and engage in chemical reactions (figure 1). very often, one can reasonably well describe the molecular ensemble by a continuous concentration / density function ; the local rate of change of density can be described by a sum of a diffusion term (second spatial derivative of density, accounting for the random movements), a drift term (first spatial derivative of density, accounting for the directional movements), and an algebraic reaction term accounting for chemical reactions (murray, 2002). a powerful analytical and numerical apparatus was developed over centuries to solve and understand des, and the comparison of the model s predictions with experiments is in principle straightforward : de solutions for the density as functions of time and the spatial coordinates could be compared with microscopy data. recently de models started to lose out to so - called agent - based (ab) models. these are computational models simulating actions and interactions of autonomous agents (either individual molecules or collective molecular ensembles). the key notion on which ab models are based is that multiple agents interact according to simple rules ; these rules are easy to encode in a computer program, and interactions are easy to simulate. however, despite their simplicity, these rules and interactions can generate complex emergent behavior. to give an example of an ab model, one can consider the reaction - drift - diffusion system (see figure 1 and the section comparison of de and ab modeling). whereas a de model would deal with solving pdes describing molecular density, an ab model would start with describing rules of behavior of individual molecules : at each small time interval, a molecule jumps randomly in space due to diffusion, shifts directionally due to drift, and, with a certain probability depending on the proximity of other molecules, disappears or appears or turns into another molecule. then a great number of molecules would be simulated in parallel, giving us very complex spatial - temporal patterns (figure 1 and comparison of de and ab modeling). the growing popularity of ab models is explained by many factors, one of which is that biological systems are, in fact, often characterized by complex emergent behavior, almost impossible to intuit, and based on simple interactions of a great number of molecular agents. turing, hodgkin, and huxley foresaw the paramount importance of computers for simulating complex sets of des that arise in biology. advances in computing technology since then have made numerical solutions of des relatively easy and more recently have made possible challenging simulations of ab systems, finally bringing modeling to the experimental lab. here we review the history of modeling of cleavage furrow positioning in cytokinesis to illustrate and compare de and ab models as clearly, simply, and concisely as possible at the expense of not discussing subtleties and not being comprehensive., 2007 ; an., 2009 ; holcombe., cell division at the end of mitosis starts with a cleavage furrow developing at the cell equator. physically, the furrow is due to the contraction of the actomyosin ring at the cell cortex. many serendipitous observations and ingenious micromanipulation experiments made over > 100 years (rappaport, 1961, 1971) have converged on four qualitative hypotheses - cartoons of the furrow positioning (mishima, 2016 ; figure 2a). three of these models posit that the mitotic spindle activates, in a spatially precise way, the onset of the actomyosin ring assembly and contraction. (rappaport, 1961) posits that astral microtubules (mts) deliver a molecular activator to the cell actomyosin cortex ; due to the overlap of the mt asters from the two sister centrosomes, the number of astral mts at the cortex might peak at the equator, causing the furrow to form there. the polar relaxation model (wolpert, 1960 ; white and borisy, 1983), in contrast, proposes that the spindle restricts furrowing to the cell equator by means of the mt asters inhibiting cortical contractility at the cell poles. the central spindle model suggests that a molecular activator is delivered to the equator from a special bundle of mts developing in the spindle midzone in anaphase. finally, the mitotic apparatus independent model hypothesizes that a mechanochemical pattern self - organizes in the cortex without the spindle as a central governor and positions the furrow to certain molecular cues in this pattern. (b) top, detailed astral stimulation model (devore., 1989) ; bottom, predicted distribution of the signaling molecule in the cell (modified from devore. density plots surrounding the spherical cell show various averaged densities of centralspindlin (taken from figure 1b in odell and foe, 2008). a very clear quantitative question asking for mathematical modeling is immediately apparent when one looks at the cartoons of the astral stimulation and polar relaxation models : given certain geometries of the spindle mt asters and cell shape, would more signal be delivered to the equatorial or polar regions of the cortex ? the former or latter could then be used as an argument in favor of the astral stimulation or polar relaxation model, respectively. white and borisy (1983) computed the surface density of the astral mt plus ends at the cortex from two spindle asters in a spherical cell. in their simulations, the spindle was symmetrically positioned in the cell, and the mts were stable and straight, stretching from the centrosome to the cortex. the strength of the signal from an individual mt was taken as proportional to an inverse power of the mt length (signal weakens with either centrosome cortex distance, with mt splaying apart, or both). this study found that the maximal signal is at the poles, and therefore the polar relaxation mechanism is more likely. however, the polar relaxation model has been shown to predict incorrectly the effects on cytokinesis of at least 15 experimental modifications of cellular shape (devore., 1989). for example, if the cell is an elongated cylinder with a small spindle in the middle, then clearly very little signal would be delivered to the cell poles. according to the polar relaxation model, this would imply furrows forming at the poles, in contradiction with experimental results. (1989) therefore proposed the following modification based on visual examinations of spindle micrographs : there is a conical region for each aster in the middle of the spindle where the mts do not grow, perhaps because they bump into the large structures of the opposite aster and destabilize (figure 2b). (1989) assumed that signaling molecules drift with a constant flux to the mt plus ends, detach from the plus ends near the cortex, diffuse, and are spontaneously degraded in the cytoplasm (figure 2b). then they translated these assumptions into a reaction - diffusion - drift pde and solved this pde numerically. the solutions of the pde in such geometry predicted that the signal maximum at the poles of the spherical cell is only local ; the global signal maxima are achieved at the equator (figure 2b), leading the authors to conclude that the astral stimulation model accounts for the experimental data, whereas the data falsify the polar relaxation model. this and another modeling study (harris and harris, 1989) scanned a number of cell geometries and key model parameters (i.e., the angle defining the mt - void sector in the spindle and the inverse power of the length dependence for the signal), lending additional support to the astral stimulation model. another, later model (yoshigaki, 2003) added the central spindle hypothesis and tested all three mitotic apparatus subsequent experimental studies, inspired in no small part by these modeling efforts, led to the discovery of molecular pathways causing the activation of cortex contractility. these pathways include, but are not limited to, centralspindlin, a complex consisting of a kinesin - type molecular motor delivering the signal to the mt plus ends, and a rho - family gtpase - activating protein triggering reactions resulting in actomyosin assembly and contraction (for a comprehensive review, see davino. it would seem that the discovery of centralspindlin supports only the cortex activation models, that is, the astral stimulation and central spindle models. (2003), indicated that mts have simultaneously opposite effects : stable mts boost, but dynamically unstable mts suppress, cortical activation. again, one of the ways to answer this question is by the use of modeling. this question was addressed by odell and foe (2008) by means of ab modeling. 1) there is an indirect cortical relaxation effect, because centralspindlin binds to and walks along all mts but rarely reaches the cortex on dynamically unstable mts because they continually depolymerize before centralspindlin arrives. dynamically unstable mts thus sequester centralspindlin away from the cortex, suppressing actomyosin activation globally. 2) some astral mts, aimed primarily toward the cell equator, stabilize during anaphase and thereby become effective rails along which centralspindlin reaches the equatorial cortex. to simulate this scenario, a few hundred thousand agents were used : hundreds of mts, each consisting of hundreds of polymer segments, thousands of centralspindlin complexes drifting on mts and diffusing in the cytoplasm, and so on even yolk particles to account for the excluded volume of the cytoplasm. tens of microscopic processes were simulated, including transport and diffusion of centralspindlin, repeated growth and shrinking of mts, nucleotide exchange on tubulin subunits governing mt dynamic instability, elastic bending of mts, and much more. massive simulations taking tens of hours on a powerful computer cluster resulted in stunning snapshots (figure 2c). of interest, the authors failed to find a reasonable combination of parameter values for which these two effects caused a significant buildup of centralspindlin at the equator. this is because diffusion scattered centralspindlin as soon as the motors walked off the mt tips. therefore the authors posited a third hypothesis : upon reaching the mt plus end, centralspindlin does not walk off but stalls at the tip. this model brings to mind a colorful analogy made by the grandfather of cytokinesis research, ray rappaport, who said that cytokinesis is not a beautifully made, finely adjusted swiss watch, but rather an old maine fishing boat engine : overbuilt, inefficient, yet never failing. before we start to compare de and ab modeling in general, let us mention briefly that after we understand better how the spindle positions the furrow, the next question is how the spindle itself is positioned. many studies, including modeling ones, have been devoted to this question, culminating recently in a combination of microscopy, micromanipulation, and ab modeling approaches that resulted in a model according to which dynein motors distributed both on the cell cortex and cytoplasmic scaffold pull on the astral mts, creating a force balance that positions the spindle (minc., 2011). in addition, the fourth model, cortex self - polarization, has recently attracted quantitative modelers. using reaction - drift - diffusion pde systems to describe the dynamic densities of cytoskeletal and par proteins, they have found combinations of nonlinearities in the reaction terms and key feedbacks that could support self - polarization (dawes and munro, 2011 ; goehring., 2011), finally, there are fundamental questions about the assembly and mechanics of the contractile actomyosin cortex ; in this case, too, modeling offers insight and possible answers. one of the earliest of these questions was asked by white and borisy (1983) : if there is an initial uniform distribution of elastic contractile elements around the cortex of the cell with high hydrostatic pressure inside and then the contractility is relaxed at the poles, would the model predict the observed sequence of cytokinetic cell shapes from one spherical to two daughter cells ? a de model gave a negative answer because the saddle - like curvature of the deepening furrow at the cell equator attenuates the effective force of scission. note that such a de model requires simulating mechanics equations, the physics of which is based on force balances (danuser., 2013). mathematical forms of both force balance equations and reaction - diffusion - drift equations that we discussed earlier are very similar. note also that other examples of ab modeling are the very useful (and very computationally expensive) molecular dynamics simulations based on solving mechanics equations describing molecules interacting with each other through realistic physical forces in aqueous media. white and borisy (1983) then realized that full cell division can be explained if contractile elements are pulled into the cell equator by the actin flow and align along the equator. later, much more sophisticated de models were able to mimic the mechanics of cell division with even greater precision (he and dembo, 1997 ; poirier., 2012). recently a detailed de model of the actin cortex that includes signaling dynamics of rho gtpases governing the cortical mechanics predicted highly nontrivial oscillatory excitable behavior at the onset of cytokinesis, which was confirmed experimentally (bement., 2015). not only de models proved to be useful to describe the mechanics of cytokinesis : an ab model (vavylonis., 2008) answered another fundamental question how does a contractile structure self - assemble from a disordered array of actin and myosin filaments ? this question asks for an ab modeling approach because the nature of this self - assembly is microscopic and random, involving a relatively small number of discrete agents characterized not only by positions but also by orientations and lengths. the emergent pattern formation, based on the coalescence of random search - and - capture driven actin myosin contractile units, is too complex to intuit and too multidimensional, heterogeneous, and noisy to be described by pdes.., 1989 ; odell and foe, 2008), both of them unquestionable but not absolute modeling successes, illustrates how to choose between two model types. (1989) wanted to explore a conceptual rather than detailed model because molecular pathways of stimulating the furrow were not known at the time. therefore the number of molecular species was minimal, explored geometries and dynamics were simple, and the resulting pde were easily solvable numerically even at the time when computer simulations were much less routine. perhaps the greatest advantage of the resulting model was the ease of scanning the parameter space. this said, their de model was based on the unexamined approximation that discreteness and randomness of the finite number of mts, which are significantly splayed apart near the cell cortex, do not affect the continuous deterministic result. in addition, another assumption, that the signal delivered by mts decreases as mt inverse length in some power, was relatively drastic and not supported by explicit modeling. if this study was done now, it would be a good idea to complement the de model with the ab model to see how good the approximations that stem from these two assumptions are. the most desirable practice is to combine a more conceptual de model with more detailed ab model, although this is not always possible, not to mention very laborious. odell and foe (2008) made the decision to use ab modeling mainly because mt dynamic instability was hypothesized to be of primary importance for the astral stimulation mechanism and they wanted to develop a detailed model with a great number of processes with known or hypothesized rates included and they wanted to exclude approximations of truly random processes with deterministic ones. however, assuming a great number of mts, one could easily investigate a de model with mt dynamic instability accounted for by analytical expression for average mt density. comparing predictions of such a simplified de model with the full ab model would be very informative. ultimately, the most important aspect one has to think about is what not to include into the model. for example, in the study of odell and foe (2008), one can not help but wonder whether simulating mt elasticity and steric interactions, which consumed most of the computational time, was really necessary. without those features, the model s parameter space probably could have been explored better. these two studies illustrate that the choice between the modeling types is nontrivial and not unambiguous. ab models are gaining popularity and for a number of reasons will likely become the dominant modeling tool in the future : one reason is that ab models are often considered to be in silico reconstitutions of biological systems because, if sophisticated enough, such models produce a life - like simulation of the cellular subsystem. the ab model allows computer experiments with in silico systems with an exquisite control of all parameters and ease of simulating biochemically or genetically perturbed systems. because biological systems do actually consist of a large number of agents whose simple interaction rules produce mind - bogglingly complex behavior, the ab philosophy is very close to biology, and thus, often fewer approximations have to be made to build an ab model. an ab model is often much simpler than a de model, especially in cases in which complex geometries, a high number of dimensions (including, besides spatial dimensions, distributions in angle and size), heterogeneity and anisotropy, and a great number of types of agents are involved. in addition, ab models capture stochastic effects more naturally than stochastic de models. when a biological system consists of few discrete objects, the continuous approximation required for a de model is not faithful. last but not least, computer - savvy and quantitative - minded biologists can do ab modeling without the need to study mathematics. however, of course, a number of catches ensure that de models will never cease to be useful. the main problem with ab models is that it is often much harder than with de models to get a qualitative insight : in silico systems tend to become too complex ; in a way, we get interesting results but can only guess how the assumptions led to them. some artifacts are inherent to some types of ab models ; for example, there are artificial oscillatory solutions in boolean models and difference equations that do not correspond to reality usually, multiple simulations and vast and nontrivial statistics are necessary to extract meaningful insight from an ab model. last but not least, ab modeling is hard to teach ; unlike des, to which many textbooks are devoted, descriptions of ab modeling tools are scattered and not systematized. many questions have to be considered before embarking on the hard modeling journey : is modeling needed for this study at all (what is the question to be answered by it) ? do we honestly list and examine all assumptions and simplifications ? do we really look for a prediction, even if it contradicts the data, or are we subconsciously trying to confirm our preconceptions ? only when these questions are pondered can we start deciding on the method and type of the model. i. biology : both ab and de modeling start with formulating a hypotheses about the key players involved in the process in question. each agent (e.g., protein, molecule, organelle, cell) interacts with other agents and/or the environment. typical examples are biochemical (signaling, binding / unbinding) and physical (e.g., elastic collisions or excluded - volume effects) interactions. these interactions can change the position (transport effects of diffusion and directional drift) or the nature of the agent (chemical reactions). the model more often than not includes stochastic effects, reflecting variabilities in size, speed, or other properties of the agents, and random movements. ii. model : a typical ab model consists of a large system of equations simultaneously updating positions and chemical states of each agent. here (figure 1, ab - model), we show one such equation (langevin equation in this case, but many other types of equations, or even equation - less particle rules, are often used), which updates the position of the ith agent in small time increments due to its stochastic brownian motion (first line) and due to deterministic directional shifts as the result of interactions with all other agents (second line). for simplicity, we do not show reaction terms here. to translate the biological problem into a de model, a continuous function describing the density of the agents has to be introduced, and reaction, drift, and diffusion terms containing partial derivatives of the density have to be derived. many explicit and hidden approximations go into such derivations. in the model shown here (figure 1, de - model), we exhibit the diffusion term and the term responsible for the drift and the reactions. results : ab models can be explored by performing many simulations using either available software or custom - made coding. typical approaches include monte carlo or gillespie algorithm simulation, in which many (often costly) simulation rounds are necessary to determine the mean behavior of the system. an advantage, however, is that one also gains knowledge about the variability of certain quantities (e.g., as depicted in figure 1, ab - model results, the mean density of an agent). each problem at hand comes with a set of parameters (such as particle speed, reaction rates), and the system s overall behavior might depend crucially on them. in figure 1, we show aggregation and uniform distribution as examples of possible behaviors. for ab models, it is usually impossible to explore the whole parameter space (not only because of the computational cost), and therefore there is always a risk that a set in parameter space and the corresponding behavior are missed (compare ab and de results in figure 1). in other words, ab models rarely allow a full understanding of the system. for de models, on the other hand, the existing toolboxes include both analytical and numerical methods. in rare cases, analytical solutions for mean sometime a complete phase diagram dependence of all possible patterns and behaviors of the system on the whole set of parameters can be deduced (figure 1, de - model results). all this, however, can be done only for relatively simple de models relying on assumptions that are sometimes hard to verify and on gross approximations. iv. note : even though many of the mathematical models used in biology are ab or de based, there of course exist combinations of those two approaches, as well as models employing other techniques. examples include models using recurrence relations, graph theory, or integral and integrodifferential equations. in connection with models for swarming and collective motion, the vicsek model (vicsek., 1995), a minimalistic ab model, has been used as a starting point for derivations of macroscopic, de models (e.g., degond and motsch, 2008). systematically deriving such continuous models from their microscopic counterparts | the number of studies in cell biology in which quantitative models accompany experiments has been growing steadily. roughly, mathematical and computational techniques of these models can be classified as differential equation based (de) or agent based (ab). recently ab models have started to outnumber de models, but understanding of ab philosophy and methodology is much less widespread than familiarity with de techniques. here we use the history of modeling a fundamental biological problem positioning of the cleavage furrow in dividing cells to explain how and why de and ab models are used. we discuss differences, advantages, and shortcomings of these two approaches. |
modern liver techniques allowed the development of segment - based anatomical liver resections [13 ]. nevertheless, there is still a place for nonanatomical liver resections. enucleation of small lesions located near the hepatic surface is achieved with low morbidity and mortality and is a current practice among surgeons. however, in some cases, there is a need for enucleation of deep located liver tumors. another option is to use radioablation of these tumors, but this option is not always available and may result in high recurrence in inexperienced hands. the main problem with enucleation of a liver tumor deeply located in the middle of the liver is the control of bleeding resulting from the rupture of small or medium vessels. the authors describe a simple way to control the bleeding without the use of any special instrument or material. this technique can also be used to control bleeding from stellate liver lesions after blunt or penetrating liver injury. after identification of the lesion by inspection and intraoperative ultrasonography, glisson 's capsule is marked with eletrocautery 2 cm away from the tumor margin. the marked area is checked by ultrasonography to ascertain surgical margin right before liver transection. hemihepatic ischemia or pringle maneuver can be applied, according to location of the tumor. after removal of the tumor, a large and deep defect is produced, often with minor or major bleeding. the technique consists of filling the defect with any absorbable hemostatic tissue available, such as surgicel (ethicon, cincinnati, oh, usa), and wait until this hemostatic tissue is imbibed with blood. the cautery is put at maximum power in coagulate mode and the blood is cooked until a crust is formed (fig. 1). figure 1:technique of bleeding control. (c) hemostatic tissue is imbibed with blood and liver borders are brought together with manual compression. (d) the cautery is put at maximum power in coagulate mode and the blood is cooked until a crust is formed. (e) the lateral compression is released and some residual bleeding is controlled with cautery. (f) final view after the use of the technique (larger arrow). note that another enucleated area was controlled with the same technique (smaller arrow). (c) hemostatic tissue is imbibed with blood and liver borders are brought together with manual compression. (d) the cautery is put at maximum power in coagulate mode and the blood is cooked until a crust is formed. (e) the lateral compression is released and some residual bleeding is controlled with cautery. (f) final view after the use of the technique (larger arrow). note that another enucleated area was controlled with the same technique (smaller arrow). although anatomical resection, which allows a better clearance between tumor deposits and the cut section of the liver, is recommended as a standard procedure for liver tumors, nonanatomical resection is quite feasible for small tumors with little risk of microscopic local invasion [57 ]. indeed, liver resection of peripheric small lesions is feasible and can be done with minimum morbidity and mortality rates. enucleation is also used to remove small liver tumors from the future liver remnant after major liver resections. enucleation of deep located liver tumors may result in rupture of small venous or portal branches that are not always easy to control. another common situation that may be a good indication for the present technique is penetrating abdominal trauma with liver injury. usually, multiple stellate liver injuries are not easy to control and liver packing may be necessary for damage control. this technique may help dealing with these injuries without the use of any special equipment. it did not require any fancy device or instrument, and it is an important adjunct for the treatment of multiple liver tumors. we have been used this technique for hemostasis control after nonanatomical liver resections since 2012, with excellent results. this technique is useful in combination with anatomical liver resections, such as right and left hemihepatectomies, or two - stage liver resections. it is also useful to control the bleeding from penetrating liver injury and therefore can be an important tool for the general surgeon. | modern liver techniques allowed the development of segment - based anatomical liver resections. nevertheless, there is still a place for nonanatomical liver resections. however, in some cases, there is a need for enucleation of deep located liver tumors. the main problem with enucleation of a liver tumor deeply located in the middle of the liver is the control of bleeding resulting from the rupture of small or medium vessels. the authors describe a simple way to control the bleeding without the use of any special instrument or material. this technique can also be used to control bleeding from penetrating liver injury. |
with advances in stabilization devices, surgical techniques, and anesthetic management strategies, multivessel off - pump coronary artery bypass graft surgery (opcab) has gained increased popularity worldwide (1, 2). during opcab, displacing the heart for exposure of the anastomotic field can elicit a significant hemodynamic deterioration (3, 4). in addition, interruption of the coronary flow for precise vessel anastomosis may result in various degrees of ischemic injury and myocardial dysfunction (3, 5 - 7). various anesthetic and surgical techniques to minimize the myocardial dysfunction and subsequent hemodynamic deterioration have been proposed (3, 5, 8, 9). among them, insertion of intracoronary shunt has been advocated by many authors with favorable results, which provides bloodless surgical field and some degree of distal flow at the same time (8, 10 - 12). however, additional technical complexity and intracoronary shunt - induced endothelial damage have raised concerns about the routine use of intracoronary shunt (13 - 15). stenotic lesion of right coronary artery (rca) in patients with right dominant coronary circulation has been known to be closely associated with right ventricular (rv) function (16 - 18), which has been suggested as an important prognostic factor of survival (19). although numerous studies have validated the efficacy of intracoronary shunt on reducing left ventricular dysfunction during opcab (8, 10, 11), there is lack of evidence supporting its role on rv function during rca revascularization. the myocardial dysfunction due to ischemic insult during grafting could be more profound in patients with poor collateral formation (3, 5, 7, 8). therefore, we studied the effect of intracoronary shunt on global rv function during off - pump rca grafting without angiographically visible collateral supply in patients with right dominant coronary circulation using fast - response thermodilution pulmonary artery catheter in a prospective, randomized and controlled trial. after approval of institutional review board and patients ' consent, 40 patients with either proximal or mid rca stenosis scheduled for multivessel opcab including mid or distal rca grafting were studied. all patients had right dominant coronary circulation without visible collateral supply to the rca territory confirmed by preoperative angiography. patients with preexisting pulmonary disease, left ventricular ejection fraction (lvef) of less than 40%, and systolic rv pressure of more than 40 mmhg as measured by preoperative transthoracic echocardiography were excluded. the patients were randomized to rca revascularization either with intracoronary shunt (shunt flo - thru, synovis surgical innovations, st. paul, mn, u.s.a., n=20) or with the proximal rca occluded by a soft snare (n=20) using a computer - generated randomization table. all patients received intramuscular premedication (0.05 - 0.1 mg / kg morphine) 1 hr before operation. upon arrival at the operating room, standard monitoring devices were applied including a fast - response thermodilution pulmonary artery catheter (swan - ganz ccombo cco / svo2, edwards lifesciences llc, irvine, ca, u.s.a.), which was inserted via the right internal jugular vein before induction of anesthesia. the pulmonary artery catheter was positioned in the pulmonary artery using pressure waveform analysis and carefully advanced until pulmonary capillary wedge pressure (pcwp) tracing was obtained. after that, the catheter was connected to an analysis system (vigilance, edwards lifesciences llc, irvine, ca, u.s.a.) and the position of the catheter tip was adjusted to obtain a signal quality index of 2 on the analysis system. anesthesia was induced with intravenous midazolam (2.0 - 3.0 mg) and sufentanil (1.5 - 3.0 g / kg) and maintained with isoflurane (0.5 - 1%) and continuous infusion of sufentanil (0.5 - 1.0 g / kg / hr). neuromuscular blockade was achieved by administering rocuronium (0.9 mg / kg) and maintained with continuous infusion of vecuronium (1 - 2 g / kg / min). after induction of anesthesia, transesophageal echocardiography probe was inserted in all patients to monitor newly developing segmental wall motion abnormalities. isosorbide dinitrate 0.5 g / kg / min was infused in all patients throughout the study period. diagnostic criteria for post grafting myocardial infarction were new q waves of greater than 0.04 mm or a reduction in r waves greater than 25% in at least 2 leads, or both, new st elevation in at least 2 contiguous leads measuring more than 0.2 mv in leads v1-v3 or more than 0.1 mv in all the other leads, or new left bundle branch block (20). all surgical procedures were performed through a median sternotomy, and the heart was displaced for optimal exposure of the anastomotic field using posterior pericardial stitch, large (1270 cm) gauze swabs and tissue stabilizer (octopus tissue stabilization system, medtronic inc. the sequence of grafting was always the left anterior descending (lad) coronary artery first, followed by the circumflex coronary artery (lcx) and the rca. intravascular volume replacement was managed with crystalloid and colloid solutions to maintain the pcwp between 8 - 14 mmhg before first grafting. during the period of heart displacement, mean systemic arterial pressure (map) was maintained above 70 mmhg either with 10 - 20 tredelenburg position and/or norepinephrine infusion. hemodynamic variables including heart rate (hr), map, central venous pressure (cvp), mean pulmonary arterial pressure (mpap), pcwp, cardiac index (ci), mixed venous oxygen saturation (svo2), rv end diastolic volume index (rvedvi), and rvef were recorded 15 min after returning the heart to its natural position following completion of grafting at the left coronary arteries before rca grafting (baseline, t1), 10 min after application of tissue stabilizer for distal anastomosis of the rca (t2), and 15 min after completion of rca grafting with the heart in its natural position (t3). the amount of infused norepinephrine at t2 and the total amount of infused fluid throughout the study period were also recorded. corresponding pulmonary vascular resistance index (pvri) and rv stroke work index (rvswi) were calculated. we determined that 17 patients would be required in each group with 80% power to detect a 5% difference in rvef between the groups with a standard deviation (sd) of 5% and an alpha level of 0.05 using independent t - test. data between the groups were compared using chi - square test, fisher 's exact test, or independent t - test as appropriate. changes between time points within the groups were compared using univariate analysis of variance with post hoc comparison using dunnette 's test. patients ' characteristics were similar between the groups including preoperative lvef and percentage of rca stenosis (table 1). opcab could be successfully performed in all patients without any occurrence of dangerous arrhythmia requiring prompt treatment or conversion to cardiopulmonary bypass during rca grafting. there were no newly developed segmental wall motion abnormalities or ecg changes indicative of myocardial infarction after grafting. none of the patients required intraoperative inotropic support and the amount of infused norepinephrine during rca grafting was similar between the groups (snare group, 7149 g ; shunt group, 6547 g). the time required for rca grafting was similar between the groups (snare group, 15.41.3 min ; shunt group, 16.11.2 min). the total amount of infused fluid during the surgery was also similar between the groups (snare group, 3,923876 ml ; shunt group, 4,063737 ml). in intergroup comparisons, there were no significant differences of the hemodynamic variables except the map, which was higher in the snare group during rca grafting (p=0.021, table 2). in intragroup comparisons, hr, mpap, cvp, pcwp, and pvri were significantly increased in both groups during rca grafting compared to baseline values of each group, but returned to baseline values at t3 except hr (table 2). svo2, rvef, and rvswi were significantly decreased in both groups during rca grafting compared to baseline values of each group, but returned to baseline values at t3 (table 2, 3). other variables did not show any significant changes compared to baseline values (table 2, 3). in this study, we evaluated the effect of intracoronary shunt during rca anastomosis on global rv function in patients theoretically vulnerable to ischemia - induced rv dysfunction during rca grafting with extraluminal coronary occlusion. insertion of intracoronary shunt was not associated with any positive effects on global rv function in terms of rvef and rvswi during rca grafting. in both groups, rvef and rvswi decreased compared to baseline values during rca grafting and returned to baseline values after reperfusion. favorable results have been reported supporting the use of intracoronary shunt during lad grafting regarding global as well as segmental lv function and reduced postoperative troponin i level (8, 10 - 12). however, brief periods of extraluminal coronary occlusion are generally well tolerated in most patients, and evidence of intracoronary shunt - induced endothelial damage that was not found with extraluminal coronary occlusion with soft snare, has been reported raising concerns about long - term graft patency (13 - 15). therefore, insertion of intracoronary shunt was recommended in selective cases with severe, multivessel coronary disease and poor cardiac function or poor collateral formation (8, 10, 12, 13). rv failure is being increasingly recognized as an important cause of perioperative morbidity, and global rv function is predominantly determined by the rv free wall, which is primarily supplied by the rv branches (16, 17, 19). we have enrolled patients undergoing mid or distal rca revascularization where coronary hemostasis with soft snare could impair flow to the rv branches as well as acute marginal and posterior descending branches. in addition, all of our patients had right dominant coronary circulation without angiographically visible collateral formation where the rv receives its blood supply almost exclusively from the rca. therefore, the effect of intracoronary shunt in minimizing global rv dysfunction was assumed to be significant in this study. in contrast to the above - mentioned favorable results regarding the lv function with insertion of intracoronary shunt during lad grafting (8, 10 - 12), we could not observe a significant difference in global rv function. during rca grafting, the heart is displaced more, resulting in greater alteration of the normal geometry than during lad grafting (3, 4). the subsequent increase in rv afterload, which could be observed as increase in pvri in both groups in this study, could have offset the potentially favorable effect of intracoronary shunt insertion on global rv function. under normal conditions, rv function is load - dependent, and even though rvedvi remained constant in this study, abnormally increased afterload becomes the major determinant of the rv function (21). also, prior grafting of the lad could have affected our result. since the lad supplies part of the rv anterior wall (16), this could have supported rv function during rca grafting, although global rv function is predominantly determined by the rv free wall (16, 17). in addition, prior lad and lcx revascularization might have resulted in improved recruitable collateral flow to rca territories. ischemia - related transient segment wall motion abnormalities of the rv during rca grafting could have developed in the snare group, which was unable to detect with the fast - response thermodilution pulmonary artery catheter. unlike the diagnosis of ischemia - related segmental wall motion abnormalities in the lv, measurement of rv contractility and segmental wall motion is difficult with tee because of the complex shape of the rv and is especially limited during opcab when the heart is displaced from its original position (3, 22, 23). the fast - response thermodilution pulmonary artery catheter continuously measures rvef by a thermodilution technique, which has a high reproducibility and a good correlation with other methods of measurement (22 - 24). the observations that rvef and rvswi returned to baseline values after reperfusion and that none of our patients showed newly developed segmental wall motion abnormalities or ecg changes indicative of myocardial infarction afterwards indicate no clinically beneficial effect by intracoronary shunt insertion with regard to ischemia - related rv function occurrence of serious arrhythmia and/or hemodynamic collapse requiring immediate cardiopulmonary bypass has been reported during rca grafting, especially when the atrioventricular (av) nodal artery flow is interrupted (3, 6). although av node is supplied by the rca in 80% of normal human hearts, the remaining 20% receives its blood supply either by the lcx or both lcx and rca (25). all of the patients in our study tolerated well with extraluminal coronary occlusion and the incidence of such critical event is very low (6). therefore, in order to validate the safety of the extraluminal coronary occlusion with regard to serious arrhythmia, further studies with a larger number of patients are warranted. another limitation of this study is that collaterals visualized on preoperative angiography may not reflect accurately the total collateral flow and recruitable collaterals might have existed (26). the result that map was significantly higher during rca grafting in the snare group might have resulted in better recruitable collateral flow. however, map was maintained above 70 mmhg in both groups during rca grafting and the effect of difference in map on recruitable collateral flow should be negligible. to avoid distorting the comparative study outcome, we have excluded patients with decreased lvef and systolic rv pressure 40 mmhg who could be more susceptible to ischemia - induced rv dysfunction, and the results of our study should not be extended to these patients. in conclusion, we could not observe beneficial effects with intracoronary shunt insertion compared to temporary extraluminal coronary occlusion with regard to global rv function during rca grafting even in ischemia - susceptible patients without angiographically visible collateral formation. regarding the possibility of long - term graft failure by intracoronary shunt - induced endothelial damage, routine use of intracoronary shunt during rca grafting in patients with preserved biventricular function is not recommended. | although numerous studies have validated the efficacy of intracoronary shunt on reducing left ventricular dysfunction during off - pump coronary artery bypass surgery (opcab), there is lack of evidence supporting its role on right ventricular (rv) function during right coronary artery (rca) revascularization. therefore, we studied the effect of intracoronary shunt during grafting of dominant rca without visible collateral supply on global rv function using thermodilution method. forty patients scheduled for multivessel opcab with right dominant coronary circulation without collateral supply confirmed by angiography were randomized to rca revascularization either with a shunt (n=20) or soft snare occlusion (n=20). rv ejection fraction (rvef) was recorded at baseline, during rca grafting, and 15 min after reperfusion. corresponding rv stroke work index (rvswi) was calculated. rvef and rvswi decreased significantly during rca grafting and returned to baseline values after reperfusion in both groups without any significant differences between the groups. intracoronary shunt did not exert any beneficial effect on global rv function during rca grafting, even in the absence of visible collateral supply. regarding the possibility of graft failure by intracoronary shunt - induced endothelial damage, routine use of intracoronary shunt during rca grafting is not recommended in patients with preserved biventricular function. |
our model is an extension of a previously published model (11), using as its platform the published model 's analytic algorithm but changing treatment regimens and inputs, in keeping with the newer medications being considered. adults enter the analysis at diabetes diagnosis and progress through the model until death or age 95. only patients between the ages of 25 and 64 with newly diagnosed diabetes patients have an annual risk of diabetes complications, modified by age, race, and sex, time since diabetes onset, time since diagnosis, treatment, a1c achieved, smoking, hypertension, and/or concomitant hypercholesterolemia. it is assumed that hypertensive patients develop complications more rapidly than nonhypertensive patients and that glycemic control has no impact on the progression of coronary heart disease. the summary metric used to estimate the value of exenatide and sitagliptin is the incremental cost - effectiveness ratio (icer). in this analysis, icer = (costs of treatment averted diabetes complication - related costs)/(quality - adjusted life years [qalys ] gained). costs were calculated from a health care system perspective, using a lifetime analytic horizon. side effect related quality - of - life assumptions a positive number (utility) indicates a gain in quality of life, and a negative number (disutility) indicates a loss in quality of life. the overall disutility was calculated as the weighted sum of the side effect utilities / disutilities, where the weights were 1) the probability a patient was on a given medication at a point in time and 2) the probability the side effect occurred. we assumed that intensive glycemic control is now the standard of care in the u.s. three intensive glycemic control strategies were modeled : 1) glyburide as second - line treatment strategy, 2) exenatide as second - line treatment strategy, and 3) sitagliptin as second - line treatment strategy. in each strategy, patients were treated with combinations of metformin, the second - line agent specific to the strategy, rosiglitazone, and nph insulin. in all three strategies, if glycemic control was not achieved with metformin alone, other medications were added, based on modeled rates of treatment failure (see supplementary table, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-1488/dc1). the methods used to estimate the probabilities of diabetes complications have been described elsewhere (15). in brief, probabilities depended on time since diagnosis, time between onset and diagnosis, age, sex, race, glycemic levels, smoking, cholesterol levels, and hypertension (table 1). age, sex, smoking, and cholesterol level affected transition probabilities associated with coronary heart disease (chd) and stroke. race affected glycemic levels and mortality. alternative treatment strategies affected transition probabilities by altering a patient 's modeled trajectory of a1c levels over time. all three strategies were assumed to provide the same degree of glycemic control and, hence, the same effects on risks of diabetes complications. this assumption was based on results from clinical trials of sitagliptin and exenatide (1621). side effect profiles were developed for each of the second - line medications (glyburide, exenatide, and sitagliptin) based on literature review. utilities (positive gains in quality of life) or disutilities (losses in quality of life) were applied to reflect these profiles. we grouped these effects into five categories : weight gain / loss, hypoglycemia, nausea / other gastrointestinal effects, upper respiratory infections, and the disutility associated with an injectable formulation. each side effect could be experienced by the proportion of the population receiving a given drug at a given time. the effects of weight gain / loss, nausea and upper respiratory infection were assumed to last for 2 years (13) ; all others were assumed to last for the duration that the medication was taken. glyburide was associated with a weight gain of 3% experienced by all, nausea experienced by 4.2%, and hypoglycemia experienced by 36.1%. exenatide was associated with a weight loss of 5% (experienced by all), hypoglycemia (experienced by 16%), nausea and other gastrointestinal effects (experienced by 57%), and a disutility because it was an injectable medication. sitagliptin was associated with weight neutrality, hypoglycemia (experienced by 6.2%), and an increased risk of minor upper respiratory infections (experienced by 3.5%). all three side effect profiles resulted in a net disutility for each year the respective medication was taken (table 2). it was assumed that patients with hypertension would receive antihypertensive medications and that patients with hypercholesterolemia would be given statins. costs of glycemic control included the costs of the drugs themselves, the costs of equipment needed for self - injection of insulin, the costs of glucose monitoring, and the costs of outpatient care associated with routine follow - up for diabetic patients. costs of diabetes complications were drawn from the same literature sources and used the same methods of calculation as in the model published previously (11). these costs included costs of procedures, inpatient and outpatient care, specialist visits, and medications required for the management of diabetic nephropathy, neuropathy, retinopathy, chd, and stroke. prevention of diabetes complications results in a reduced risk of mortality and improved quality of life. in the model, strategies associated with improved glycemic control reduced the transition probabilities leading to diabetes complications at all stages, thereby reducing the risks of death due to chd, stroke, nephropathy, or neuropathy. retinopathy was assumed to lead to blindness but not to alter the risk of death. quality of life was captured by incorporating health utilities (table 1) into the model, where a utility of 1 describes a period of time lived in perfect health and a utility of 0 is assigned to death. utility values between 0 and 1 describe life lived in less than perfect health and are used in the calculation of qalys (22). all analyses were performed with custom software built by the original study team (15). we assumed that intensive glycemic control is now the standard of care in the u.s. three intensive glycemic control strategies were modeled : 1) glyburide as second - line treatment strategy, 2) exenatide as second - line treatment strategy, and 3) sitagliptin as second - line treatment strategy. in each strategy, patients were treated with combinations of metformin, the second - line agent specific to the strategy, rosiglitazone, and nph insulin. in all three strategies, if glycemic control was not achieved with metformin alone, other medications were added, based on modeled rates of treatment failure (see supplementary table, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-1488/dc1). the methods used to estimate the probabilities of diabetes complications have been described elsewhere (15). in brief, probabilities depended on time since diagnosis, time between onset and diagnosis, age, sex, race, glycemic levels, smoking, cholesterol levels, and hypertension (table 1). age, sex, smoking, and cholesterol level affected transition probabilities associated with coronary heart disease (chd) and stroke. race affected glycemic levels and mortality. alternative treatment strategies affected transition probabilities by altering a patient 's modeled trajectory of a1c levels over time. all three strategies were assumed to provide the same degree of glycemic control and, hence, the same effects on risks of diabetes complications. this assumption was based on results from clinical trials of sitagliptin and exenatide (1621). side effect profiles were developed for each of the second - line medications (glyburide, exenatide, and sitagliptin) based on literature review. utilities (positive gains in quality of life) or disutilities (losses in quality of life) were applied to reflect these profiles. we grouped these effects into five categories : weight gain / loss, hypoglycemia, nausea / other gastrointestinal effects, upper respiratory infections, and the disutility associated with an injectable formulation. each side effect could be experienced by the proportion of the population receiving a given drug at a given time. the effects of weight gain / loss, nausea and upper respiratory infection were assumed to last for 2 years (13) ; all others were assumed to last for the duration that the medication was taken. glyburide was associated with a weight gain of 3% experienced by all, nausea experienced by 4.2%, and hypoglycemia experienced by 36.1%. exenatide was associated with a weight loss of 5% (experienced by all), hypoglycemia (experienced by 16%), nausea and other gastrointestinal effects (experienced by 57%), and a disutility because it was an injectable medication. sitagliptin was associated with weight neutrality, hypoglycemia (experienced by 6.2%), and an increased risk of minor upper respiratory infections (experienced by 3.5%). all three side effect profiles resulted in a net disutility for each year the respective medication was taken (table 2). it was assumed that patients with hypertension would receive antihypertensive medications and that patients with hypercholesterolemia would be given statins. dollars (table 1). both costs and health benefits were discounted at 3% annually and estimated from the health care system perspective. costs of glycemic control included the costs of the drugs themselves, the costs of equipment needed for self - injection of insulin, the costs of glucose monitoring, and the costs of outpatient care associated with routine follow - up for diabetic patients. costs of diabetes complications were drawn from the same literature sources and used the same methods of calculation as in the model published previously (11). these costs included costs of procedures, inpatient and outpatient care, specialist visits, and medications required for the management of diabetic nephropathy, neuropathy, retinopathy, chd, and stroke. prevention of diabetes complications results in a reduced risk of mortality and improved quality of life. in the model, strategies associated with improved glycemic control reduced the transition probabilities leading to diabetes complications at all stages, thereby reducing the risks of death due to chd, stroke, nephropathy, or neuropathy. retinopathy was assumed to lead to blindness but not to alter the risk of death. quality of life was captured by incorporating health utilities (table 1) into the model, where a utility of 1 describes a period of time lived in perfect health and a utility of 0 is assigned to death. utility values between 0 and 1 describe life lived in less than perfect health and are used in the calculation of qalys (22). all analyses were performed with custom software built by the original study team (15). all three strategies were assumed to confer the same benefits in terms of reductions in major health outcomes as a result of diabetes - related complications. they were assumed to differ in their side effect profiles only, and these side effects were not assumed to alter risks of complications. the impacts of these side effects were incorporated into the model as quality - of - life gains or losses. use of sitagliptin as a second - line treatment for type 2 diabetes in adults < 65 years of age is associated with additional intervention costs of $ 20,213 per person over a lifetime compared with a baseline strategy using glyburide as second - line therapy. use of exenatide as a second - line treatment is associated with an additional cost of $ 23,849 over a lifetime compared with glyburide as second - line therapy. the differences in intervention costs among the three strategies were due to differences in medication costs, summarized in fig. over 15 years, the average daily treatment costs for the glyburide, sitagliptin, and exenatide strategies were $ 2.98, $ 6.51, and $ 7.26, respectively. changes in costs and qalys were calculated using comparisons to the next most expensive strategy, as well as to the common baseline of the strategy incorporating glyburide as second - line therapy. the strategy incorporating sitagliptin as second - line therapy was associated with an incremental cost - effectiveness ratio of $ 169,572 per qaly saved, relative to glyburide as second - line therapy. because exenatide was 1) associated with an injectable formulation with an accompanying disutility and 2) had higher medication - associated costs, the strategy incorporating exenatide as second - line therapy was dominated by that incorporating sitagliptin, meaning that it was both more expensive and less effective in terms of qalys saved. results of cost - effectiveness analysis changes in costs in $ and qalys were calculated relative to the next most expensive treatment strategy. in one - way sensitivity analysis, when the disutility associated with an injectable medication was set to 0, exenatide ceased to be dominated and was associated with an incremental cost - effectiveness ratio of $ 932,308 per qaly saved. in two - way sensitivity analysis, when the disutility associated with an injectable medication was set to zero and the medication cost of exenatide was decreased by 25%, exenatide exerted weak, also termed extended, dominance over sitagliptin and was associated with a cost - effectiveness ratio of $ 167,002 per qaly saved. when utility gains and losses associated with weight changes were assumed to last a lifetime, the incremental cost - effectiveness ratio associated with sitagliptin was $ 141,833 per qaly saved. in this analysis, exenatide ceased to be dominated and was associated with an incremental cost - effectiveness ratio of $ 932,308 per qaly saved. in both the analysis for which the disutility associated with injectable medication was negated and in this lifetime weight change analysis, the net disutility associated with exenatide was 0, leading to similar results. when incremental cost - effectiveness analysis was performed using metformin and glyburide with insulin as third - line therapy (as opposed to rosiglitazone), exenatide remained dominated and the cost - effectiveness ratio associated with sitagliptin changed minimally to $ 173,300 per qaly saved. finally, when discount rates for costs and qalys were assumed to be 5%, the exenatide strategy remained dominated, and the incremental cost - effectiveness ratio associated with sitagliptin was $ 154,389 per qaly saved. our results suggest that widespread use of sitagliptin and exenatide as second - line agents in the glycemic control of patients with diabetes could be associated with $ 731 to $ 862 million additional direct health care costs in the u.s. additional quality - adjusted life would be gained because of improved side - effect profiles associated with these drugs. these gains would cost roughly $ 170 thousand per qaly for sitagliptin. in the base case analysis, exenatide is dominated, being both more costly and generating fewer qalys than sitagliptin. a prior analysis of the cost - effectiveness of glycemic control, based on the uk prospective diabetes study, was published before fda approval of many new medications used in diabetes management (11). our model represents an extension of this previously published model, which has now been used to address multiple diabetes - related policy questions (11,23,24). previous analyses compared sitagliptin and exenatide individually with generic drugs. because the treatment strategies used were different, countries under analysis were different, and comparator strategies were different, direct comparison of these analyses with the current analysis are difficult (1214). importantly, this is the first cost - effectiveness analysis to compare exenatide and sitagliptin to one another and to glyburide for second - line therapy in a single model. because intensive glycemic control has become the accepted standard of care for healthier individuals < 65 years of age, our analysis focuses on alternative strategies for achieving this goal (25). direct comparison of this analysis to the previous analyses is complicated by the fact that the current analysis is a comparison of intensive control strategies alone. in addition, unlike previous analyses, all three strategies made use of a metformin - first approach, with rosiglitazone as a third - line option and second - line therapy varying by strategy. finally, our analysis incorporated hypertensive control and statin therapy, as would be current standard practice. nonetheless, review of previous studies makes clear that the extent to which nonglycemic control effects are attributed to newer glycemic control agents influences cost - effectiveness (1214)., relative to that of insulin glargine, was found to be dominated by insulin glargine in one study, whereas its use was found to be cost - effective (with a cost - effectiveness ratio of 22,420) in another. these different results are explained by the former model not attributing to exenatide improvements in blood pressure and in lipid profile (leading to improved cardiovascular outcomes), whereas the latter study did make these attributions. because clinical trials of exenatide and sitagliptin have not shown significant differences in diabetes complication rates, including cardiovascular events, we chose not to ascribe such benefits to either medication. our findings suggest that the potential scale of health benefits gained by use of exenatide and/or sitagliptin, as a result of improved side - effect profiles, may be substantial. relative to glyburide as second - line therapy, we found the use of exenatide and sitagliptin to be associated with an additional 0.09 and 0.12 qalys per patient. this result is comparable to the scale of health benefits provided by a number of highly effective preventive and treatment strategies. for example, the use of aspirin for secondary prevention of myocardial infarction in 45-year - old men has been estimated to provide a qaly gain of 0.04 per patient (26). the use of statins in the secondary prevention of coronary artery disease has been associated with a qaly gain of 0.25 per patient (27). the use of 23-valent pneumococcal vaccine to prevent disease in elderly patients has been associated with a qaly gain of 0.003 per patient (28). we found results to be sensitive to assumptions regarding medication cost, incidence of medication effects, and disutilities due to medication effects. given the impact that such effects have on patients ' daily lives and the importance of these quality - of - life effects on cost - effectiveness, further empirical study is necessary to understand the preference - weighted quality of life impact of these effects, their costs, and their consequences. the american diabetes association and european diabetes have recently published consensus guidelines for sequencing existing and new classes of medications as initial therapies in diabetes (29). these recommendations are consistent with meta - analyses indicating that antiglycemic oral agents and insulin used to treat diabetes have comparable efficacy, (30,31), although they differ in other effects and significantly in costs. spending on antidiabetic agents nearly doubled from 6.3% of all prescription drug spending in 2004 to 12.3% in 2006, and costs of treatment increased sharply (9.5%) because of higher prices for nongeneric drugs and a shift in treatment mix toward newer, more expensive products (32). although side effects associated with older medications may justify the use of newer ones in individual cases, our study suggests that the additional costs of newer classes of drugs, when widely used in the large u.s. diabetic patient population, require that the value of these drugs be supported by substantial gains in health outcomes to be recommended on a population basis. better understanding of the quality - of - life impacts of these drugs is necessary to make such a case strongly. for example, understanding the duration of weight loss effects of some of these medications and the potential downstream effects on macrovascular events (coronary heart disease and stroke) could contribute substantially to the value of some of these medications. recently published long - term follow - up studies of intensive glucose control have demonstrated extended and improved treatment benefits, despite post - trial loss of between - group glycemic differences (4). our model did not integrate such legacy effects, relying instead on a direct relationship between glycemic control and diabetes complications. if such legacy effects differ by medication class, then significant adjustment of the economic model would be necessary. the model incorporates disutilities due to medication effects, but does not yet account for costs due to management of side effects or medication switches that may occur due to side effects. diabetes is an epidemic disease that imposes substantial morbidity, premature mortality, and costs on the u.s. appropriate treatment choices are necessary to minimize the economic burden associated with this prevalent disease. our study suggests that to provide good economic value, newer medications, such as sitagliptin and exenatide, need to confer health benefits in scale with the additional costs they bring to the health care system. | objectivenewer medications offer more options for glycemic control in type 2 diabetes. however, they come at considerable costs. we undertook a health economic analysis to better understand the value of adding two newer medications (exenatide and sitagliptin) as second - line therapy to glycemic control strategies for patients with new - onset diabetes.research design and methodswe performed a cost - effectiveness analysis for the u.s. population aged 2564. a lifetime analytic horizon and health care system perspective were used. costs and quality - adjusted life years (qalys) were discounted at 3% annually, and costs are presented in 2008 u.s. dollars. we compared three glycemic control strategies : 1) glyburide as a second - line agent, 2) exenatide as a second - line agent, and 3) sitagliptin as a second - line agent. outcome measures included qalys gained, incremental costs, and the incremental cost - effectiveness ratio associated with each strategy.resultsexenatide and sitagliptin conferred 0.09 and 0.12 additional qalys, respectively, relative to glyburide as second - line therapy. in base case analysis, exenatide was dominated (cost more and provided fewer qalys than the next most expensive option), and sitagliptin was associated with an incremental cost - effectiveness ratio of $ 169,572 per qaly saved. results were sensitive to assumptions regarding medication costs, side effect duration, and side effect associated disutilities.conclusionsexenatide and sitagliptin may confer substantial costs to health care systems. demonstrated gains in quality and/or quantity of life are necessary for these agents to provide economic value to patients and health care systems. |
this paper describes the anatomic features of the temporal lobe which are important in the most commonly performed surgical approaches for temporal lobe epilepsy (tle) : the anterior temporal lobectomy (atl), the transcortical selective amygdalohippocampectomy (tcah), and the transsylvian selective amygdalohippocampectomy (tsah). the differences between these approaches and the expected outcomes are best understood by knowing their microanatomical differences. two main objectives in epilepsy surgery are removal of the epileptogenic tissue and avoidance of surgical morbidity. three approaches will be reviewed from the perspective of (1) avoidance of visual pathways (optic tract, lateral geniculate body meyer 's loop, and optic radiations), (2) white matter pathways involved in the neurocognitive sequelae, (3) extent of the incision to the temporal stem, (4) extent of amygdalectomy, and (5) avoidance of vascular injury. the temporal lobe has four surfaces : lateral, medial, superior, and inferior (figure 1(a)). while its anterior and inferior limits are natural bone structures, the temporal lobe is separated posteriorly from the occipital lobe by the lateral parietotemporal line, an imaginary line connecting the preoccipital notch and the parietooccipital sulcus, and it is also separated from the parietal lobe by the occipitotemporal line, a line connecting the most posterior limit of sylvian fissure with the lateral parietotemporal line (figure 1(a)). the lateral surface of temporal lobe consists of three gyri : superior, middle, and inferior, which are separated by two parallel sulci : superior and inferior. while the superior temporal sulcus lies between the superior and middle temporal gyri, the inferior temporal sulcus courses between the middle and the inferior temporal gyri. the superior temporal gyrus is continuous with the transverse temporal gyri on the temporoopercular surface. the angular gyrus, a parietal lobe structure, caps the most posterior end of the superior temporal sulcus. the temporal gyri, especially the inferior temporal gyrus, are often separated into small parts by sulcal bridges (figure 1(a)). stated that the inferior temporal sulcus is separated into three or more parts in 92% of cases. this surface, from anterior to posterior, has three parts : the planum polare, the anterior transverse temporal gyrus or heschl 's gyrus, and the planum temporale (figure 1(e)). the planum polare is a relatively flat surface without any gyri on the anterior part of the superior surface. it is limited posteriorly by the heschl 's gyrus, the most anterior of the transverse temporal gyri, which blends around the margin of the sylvian fissure into the superior temporal gyrus. posterior to the heschl 's gyrus lies the planum temporale, which consists of middle and posterior transverse temporal gyri. the sylvian fissure is a very important landmark on the lateral surface of the cerebrum. it crosses deep between opercular surfaces of the frontal, parietal, and temporal lobes reaching the carotid cistern anteriorly and the insular surface posteriorly. the stem begins at the anterior clinoid process in the frontobasal area, extends lateral and adjacent to the sphenoid ridge, and divides at the surface into three rami : anterior horizontal, anterior ascending, and posterior. the deep part of the sylvian fissure, referred to as the sylvian cistern, has two compartments : the sphenoidal and the operculoinsular. the sphenoidal compartment is positioned between the carotid cistern medially, the posterior part of the frontoorbital area superiorly and the planum polare of the temporal lobe, and the amygdala inferiorly. the opercular cleft lies between the opercular surfaces of frontal and parietal lobes superiorly, and opercular surface of the temporal inferiorly. the insular cleft has an upper lip that lies between the frontal and the parietal lobes and the insular surface, and a lower lip that lies between the temporal lobe and the insular surface. the sulci and gyri, which constitute the inferior surface of the temporal lobe, are continuous with the basal surface of the occipital lobe (figure 1(b)). this surface is formed by three gyri and two longer and one shorter sulci [2, 5 ]. of these longer sulci, the occipitotemporal sulcus courses laterally and separates the lower surface of the inferior temporal gyrus and the fusiform gyrus. the other longer sulcus, the collateral sulcus, separates the fusiform gyrus from the medially placed parahippocampal gyrus. the rhinal sulcus, the shorter sulcus, courses at the lateral edge of the uncus between the uncus and the fusiform gyrus and may or may not be continuous with the collateral sulcus. the indentation of the collateral sulcus towards the temporal horn generates a prominence at the floor of the temporal horn, called the collateral eminence, and at the floor of the atrium, called the collateral trigone (figures 3(f), 5(a), 5(e), and 5(f)). the medial surface of the temporal lobe, or medial temporal region (mtr), is the most complicated of cortical surfaces. the anterior segment begins where the rhinal sulcus turns superiorly at the anterior edge of the uncus and ends at the posterior limit of the uncus ; the medial segment begins at the posterior edge of the uncus and ends at the level of the quadrigeminal plate ; the posterior segment begins at this point and ends at the calcarine point where the parietooccipital and calcarine sulci join (figure 1(d)). the uncus has an anterior and a posterior segments, which come together at a medially directed prominence, the apex of the uncus. the anterior segment of the uncus belongs to the parahippocampal gyrus and contains two gyri : the semilunar gyrus and the ambient gyrus. the semilunar gyrus is positioned on the upper part of the anterior segment, above the cortical nucleus of amygdala. the semilunar gyrus is separated from the anterior perforated substance by the entorhinal sulcus and optic tract superolaterally and from the ambient gyrus by the semiannular sulcus medially and anteriorly. the ambient gyrus, formed mainly by the entorhinal cortex, occupies the anterior and inferior parts of this segment (figures 2(a)2(c)). the posterior segment of uncus has two parts, superior and inferior, which are separated by the uncal sulcus. the inferior part, formed by the parahippocampal gyrus, is occupied by the entorhinal area. the entorhinal area also occupies the inferior surface of the anterior segment of the uncus and is limited on the lateral side by the rhinal sulcus anteriorly and the collateral sulcus posteriorly. the posterior limit of the entorhinal area is accepted as the posterior limit of the uncus. the entorhinal area has an important role in afferent and efferent connections of the hippocampus. the superior part of the uncus is formed by the hippocampal head and has the fimbria of fornix at its posterior limit. this part is the site of three small gyri, the uncinate gyrus, the band of giacomini, and the intralimbic gyrus. the anterior segment of the uncus faces the carotid cistern, the internal carotid artery (ica), and the proximal m1 segment of middle cerebral artery (mca) ; the apex of the uncus faces the oculomotor nerve ; the posterior segment of the uncus faces the crural cistern, the posterior cerebral artery (pca) below and the anterior choroidal artery (acha) above, and the crus cerebri. the middle mtr, when viewed medially, is formed from inferior to superior by the parahippocampal gyrus, the dentate gyrus, and the fimbria of fornix (figure 2(a)). the fimbriodentate sulcus separates the fimbria of fornix and the dentate gyrus, and the hippocampal sulcus separates the dentate gyrus and the parahippocampal gyrus. in this region, the presubiculum, a six - layered modified cortex between the subiculum and the cortex of the parahippocampal gyrus, occupies the medial surface of the parahippocampal gyrus. the posterior mtr is formed by the posterior end of the parahippocampal gyrus, which is divided by the anterior end of the calcarine sulcus into the isthmus of the cingulate gyrus superiorly and the lingual gyrus inferiorly. functionally, the parahippocampal gyrus shows a closer functional relationship with the isthmus of the cingulate gyrus, because the cingulum bundle passes from the isthmus into the parahippocampal gyrus. superiorly, the fimbria of the fornix courses posteriorly to become the crus of the fornix, which wraps around the posterior aspect of the pulvinar. the hippocampal tail passes posterior to blend into the fasciolar gyrus, just below the splenium of the corpus callosum (figure 3(b)). the main debate in selecting the best surgical approach for temporal epilepsy, other than the long - term seizure control, focuses on two topics : damage to the optic radiations and memory deficit after the surgery. however, there are clinical series that favor different types of surgery understanding the microanatomy of white matter bundles is important to achieving better results [711 ]. the optic radiations, the geniculocalcarine pathway, have a complex anatomy [1214 ]. the visual input is carried to the lateral geniculate body (lgb) of the thalamus via the optic nerve, the optic chiasm, and the optic tract. the lgb is located on the inferolateral side of the thalamus and just posterior to the cisternal side of the inferior choroidal point. the posterior bundle passes posteriorly to reach the superior lip of the calcarine sulcus without making any anterior curve. the middle bundle makes a partial anterior curve on its course to the calcarine cortex, and the anterior bundle of optic radiations makes a prominent anterior curve, referred to as the meyer 's loop, at the roof of the temporal horn on its way to the inferior lip of the calcarine sulcus. the optic radiations are separated from the temporal horn by a thin layer of tapetal fibers originating from the corpus callosum. the optic radiations completely cover the roof of the temporal horn and exceed the anterior wall of the temporal horn by a few millimeters. they also cover the lateral wall of the temporal horn except its anterior part [1214 ]. at the level of the atrium, optic radiations cover only the lateral wall ; the medial wall of the atrium is always free from the optic radiations. the uncinate fasciculus (uf) and inferior longitudinal fasciculus (ilf) also have important role in epilepsy surgery because of their importance in memory function. the uf connects the anterior temporal lobe with the orbitofrontal cortex by forming a curve deep to the limen insula and courses within the ventral extreme and external capsules. the uf occupies the anterior part of the temporal stem (figures 3(c)3(e)). its functions are not fully understood, but the orbitofrontal cortex and the anterior temporal lobe are reported to have an important role in recognizing faces, actions, and objects and also emotion [1820 ]. the ilf courses adjacent to the inferior part of the lateral wall of the temporal horn and is located lateral and inferior to the optic radiations (figures 3(c) and 3(f)). the ilf connects the anterior temporal lobe to the fusiform gyrus and dorsolateral parts of the occipital lobe. it is suggested that the ilf has a role in learning and remembering visual stimuli. stated that the process of learning to read occurs by remembering visual stimuli of words. they suggested that the posterior part of the occipitotemporal sulcus referred to as the visual word form area is involved in this process. it was debated, until recently, whether the ilf consists of only long horizontal fibers ; however, catani. have shown in their dti study that the ilf consists of both long horizontal fibers (direct part) and interconnecting u fibers (indirect part). the arcuate fasciculus (af) is considered to be a subsegment of the superior longitudinal fasciculus [24, 25 ]. the structure of this white matter bundle was further detailed in a dti study by catani.. in their study, they proposed that the af consists of two indirect parts and one direct part. the first indirect part connects the inferior frontal gyrus to the supramarginal gyrus, and the second indirect part connects the supramarginal gyrus to the posterior part of superior temporal gyrus. the direct part of the af is proposed to connect the inferior frontal gyrus to the posterior temporal areas by coursing dorsal to the insula by forming an arch (figure 3(g)). the af has a major role in language processing, which is thought to involve both a ventral pathway and a dorsal pathway. it is suggested that the af forms the dorsal pathway and plays a role in the phonological part of language. therefore, damage to the af causes a deficit in the production of speech or appropriate words. the ioff connects the inferior frontal cortex and the dorsolateral prefrontal cortex to the posterior part of the inferior surface of the temporal lobe and to the parts of the occipital lobe superior to the calcarine sulcus. from a functional point, the ventral pathway connecting areas known to have a role in picture naming and object recognition has a semantic role in the naming of sounds or recognition of speech [30, 31 ]. in a previous report from the laboratory of the senior author, it was shown that the ioff occupies the ventral part of the extreme and external capsules and the ventral claustrum (figure 3(c)). white matter tracts connecting the temporal lobe with other parts of the brain, such as the insula, basal ganglia, and frontal and parietal lobes, course through temporal stem. in the transsylvian route to the mtr ebeling and von cramon stated that the temporal stem is the area between the roof of the temporal horn and the inferior limiting sulcus. on the other hand, choi. described the temporal stem as the area between the inferior limiting sulcus, limen insula, entorhinal sulcus, and tail of caudate nucleus. according to this organization, the following fiber tracts are included in the temporal stem : the extreme capsule, the uf, ioff, the anterior commissure, the ansa peduncularis, and the inferior thalamic peduncle, which includes optic radiations. separation of these white matter tracts, located around posterior limit of the inferior limiting sulcus, is very difficult. this intermixed structure of fiber tracts, including the ioff, optic radiations, the anterior commissure, and the ilf, is referred to as the sagittal stratum. the anatomy of the temporal horn and atrium will be discussed with their relationship to the three parts of the mtr [6, 34 ]. the posterior limit of this part is the inferior choroidal point, which is the entry point of the acha into the temporal horn in most cases (figures 4(a), 4(f), 5(f), and 5(g)). the inferior choroidal point is located at the lower end of the attachment of the choroid plexus and is positioned just behind the head of the hippocampus. the uncal recess is situated anterior to the hippocampal head and separates it from the amygdala. it forms the anterior wall and the anterior part of the roof of the temporal horn. the bulge of the amygdala to the ventricle is the intraventricular representation of the semilunar gyrus, which occupies the anterior segment of the uncus. note that the amygdala in this region is the temporal amygdala and contains the basolateral, the corticomedial, and the central nuclei groups. the extended amygdala is in close relation with the ventral striatum and the anterior commissure and should be avoided during surgery for temporal lobe epilepsy. the collateral eminence located lateral to the head of the hippocampus occupies the floor of the temporal horn and is the indentation of the collateral sulcus into the temporal horn (figures 4(a), 4(d), and 4(f)). the posterior part of the temporal horn is related to the middle mtr and begins at the inferior choroidal point to open to the atrium. the floor of this part is formed by the collateral eminence similar to the anterior part. the collateral eminence is a reliable landmark to determine the medial limit of the neocortical removal. the medial surface of the temporal lobe and the perimesencephalic cisterns can be reached by opening the choroidal fissure. the choroidal fissure begins at the inferior choroidal point, which is located at the posterior limit of the uncus. the choroidal fissure is a natural cleft between the thalamus superiorly and the fimbria of the fornix inferiorly. the choroid plexus is attached on each side to the thalamus and the fimbria of the fornix, via the taenia choroidea and taenia fimbria, respectively. the choroid plexus is adjacent to the body of the hippocampus in this part of the temporal horn. the head of the hippocampus is located in the anterior mtr ; therefore, it is not related to the choroidal fissure and the choroid plexus. the ambient cistern can be reached by opening the choroidal fissure in the medial mtr. the roof and the lateral wall of the temporal horn are formed by a layer of tapetal fibers and then are covered by optic radiations. optic radiations do not cover the most anterior part of the lateral wall of the temporal horn. the floor of the atrium is formed by the collateral trigone that is the indentation formed by the posterior end of the collateral sulcus. the intraventricular part of the tail of the hippocampus is located at the most anterior part of the floor. the anterior wall of the atrium has two parts : a lateral part formed by the pulvinar and a medial part formed by the crus of fornix. the medial wall of the atrium is formed by two prominences : the inferior prominence, referred to as the calcar avis, is the indentation of the calcarine sulcus into the ventricular space, and the superior prominence, referred to as the bulb of corpus callosum, is formed by the callosal fibers of the forceps major. the pca, the mca, the acha, and also the ica are met in surgical interventions directed to the different parts of the temporal lobe. a clear understanding of microvascular anatomy of the temporal lobe is needed to perform epilepsy surgery with minimal morbidity. the pca has the main role of supplying the mtr and the inferior surface of the temporal lobe. the pca is divided into 4 segments : p1, p2, p3, and p4 (figures 4(e) and 4(f)). the p1 arises at the bifurcation of the basilar artery and is situated medial to the anterior segment of the uncus during its course in the interpeduncular cistern. the p1 ends after posterior communicating artery (pcoa) joining the pca and the p2 begins. the p2 terminates at the posterior margin of the midbrain and is divided into an anterior segment (p2a) and a posterior segment (p2p). the p2a courses in the crural cistern located between the posterior segment of the uncus and the cerebral peduncle. the p2p courses in the ambient cistern located between the parahippocampal gyrus and the midbrain. the p3 begins at the posterior part of the ambient cistern, posterolateral to the midbrain, and courses back into the quadrigeminal cistern to end at the anterior end of the calcarine sulcus. these branches, the anterior inferior temporal artery, the anterior hippocampal - parahippocampal artery, and the main trunk of p2a, have the main contribution to the arterial supply of the anterior mtr. the pca shows a bifurcation or trifurcation in most hemispheres (in 89% of hemispheres) at the middle part of this region at a distance of 6.7 mm (range 021 mm) to the posterior limit of the uncus. the most common bifurcation pattern is a division into a parietooccipital arterial trunk and a posterior inferior temporal artery. the next common bifurcation pattern is a division into a parietooccipital arterial trunk and an inferior common temporal artery, which then divides into anterior, middle, and posterior inferior temporal arteries (figures 4(c), 4(d), 6(c)6(e)). the posterior parahippocampal arteries originating from the posterior inferior temporal artery give the arterial supply to the middle mtr. the p3 and the p4 segments are related to the posterior mtr and do not give any branches to the inferior surface of the temporal lobe. the pca courses below the isthmus of the cingulate gyrus towards the calcarine sulcus to make its distal bifurcation into the parietooccipital artery and the calcarine artery (figure 4(c)). the posterior inferior temporal artery, the posterior hippocampal arteries, the splenial artery, the calcarine artery, and the parietooccipital artery are the branches of the pca emerging at this segment which contribute to the arterial supply of the posterior mtr. the c4 segment of the ica begins where the artery enters the dura and courses superior, posterior, and lateral to its bifurcation into the anterior and middle cerebral arteries. the c4 is divided into 3 subsegments : the ophthalmic, the communicating, and the choroidal. the first segment after the ica enters into the intradural space is the ophthalmic segment, which has the origin of the ophthalmic artery at its distal limit. the second segment is the communicating segment, which starts at the origin of the ophthalmic artery and ends at the origin of the pcoa. the third segment is the choroidal segment, which continues from the origin of acha to the bifurcation of the ica. the surgeon finds the acha before the pcoa because the acha is closer to the bifurcation of the ica, the acha originates closer to the lateral wall of the ica than the pcoa, and the acha follows a more lateral course than the pcoa after their origin. fernndez - miranda. stated that arterial branches of the ica, which supply the mtr, were present in 45% of hemispheres. if present, these arteries always arose from the choroidal segment of the ica. they suggested that their presence is in close relation to the absence of the branches of the acha and the mca. its origin is nearer to the origin of the pcoa than to the bifurcation of the ica in most cases. it can arise from the posterior wall of the ica and then immediately divide into two arteries, or the acha arises as two different trunks from the c4. the cisternal segment follows an initial posteromedial direction in the carotid cistern but then follows a posterior, lateral, and superior direction under the optic tract by staying lateral to it. it follows the superior surface of the posterior segment of the uncus and enters into the ventricle through the choroidal fissure. another entry point is also possible a few millimeters posterior to the inferior choroidal point. the length of this segment was measured as 23 mm and 24 mm in two different neuroanatomical studies [6, 34 ]. this arterial supply can be divided into three areas : anterosuperior, medial, and inferior. the anterosuperior area is the anterior segment of the uncus and is supplied by the anterior uncal arteries. the medial area depicts the posterior segment of the uncus and is supplied by the posterior uncal arteries. described that the acha gives only a minimal contribution to the arterial supply of this area (figure 6(b)). the arterial supply of the entorhinal area, which occupies the anterior portion of the parahippocampal gyrus, depends mainly on the pca and the mca. the pca gave the anterior hippocampal - parahippocampal arteries and its branches, called the anterior parahippocampal arteries, to the entorhinal area in all cases (figure 6(e)). the plexal segment of the acha begins at the entry point of the artery into the ventricle. this segment mainly gives the arterial supply of the choroid plexus in the temporal horn, but it may also supply the choroid plexus at more posterior levels. the mca is divided into four segments (figures 4(e) and 5(c)) : m1 or the sphenoidal segment, m2 or the insular segment, m3 or the opercular segment, and m4 or the cortical segment. the m3 is the segment within the sylvian cistern starting at the circular sulcus of the insula to end at the surface of the sylvian fissure. the bifurcation of the mca occurs just proximal to the end of the m1 ; thus, the m1 is divided into prebifurcation and postbifurcation parts. two other important vascular structures arise from the m1 other than the superior and inferior trunks after the bifurcation. the cortical branches arising from the m1 proximal to the bifurcation are referred to as early branches. early branches may reach to the frontal or the temporal lobes with the temporopolar area being the most common temporal area supplied by an early branch. the m1 supports the arterial supply of the mtr with 1 to 3 branches in 94% of the specimens. they described that these branches originate from the main m1 trunk in 42% of the cases, from a temporopolar artery in 41% of the cases, and from an early branch other than temporopolar artery in 14% of the cases. the anterior group, which supplies the anterior segment of the uncus, was named as the anterior uncal arteries. the anteroinferior group, which has the distribution to the anterior entorhinal area, was named as the unco - parahippocampal arteries. these two groups contain 92% of all branches directed to the mtr from the mca. the third group of arteries, which gives supply to the anterior part of the entorhinal area, was named as the anterior parahippocampal arteries. while anterior uncal arteries arise mostly from the main m1 trunk, the unco - parahippocampal arteries and anterior parahippocampal arteries arise mainly from the temporopolar artery. the arteries at the superior surface of the temporal lobe belong to the opercular segment (m3), which come into view during the splitting of the sylvian fissure ; the cortical branches at the lateral surface belong to the m4 which are grouped into distribution areas as, from anterior to posterior, the temporopolar, anterior, middle, and posterior temporal areas. the venous drainage of the cerebrum is divided into a superficial group and a deep group. the microvascular anatomy of the venous drainage of the cerebrum was described in other reports [39, 40 ], and the venous drainage of the sylvian fissure was further detailed by tanriover.. two most important venous structures at the lateral surface of the temporal lobe are the superficial sylvian vein (ssv) and the vein of labbe. the ssv usually arises as a single trunk at the most posterior end of the sylvian fissure but may also start as two trunks that join together before their drainage. tanriover. described that the ssv emptied into the sphenoparietal sinus in 35 of 43 hemispheres. in the remaining hemispheres, the ssv drained directly into the cavernous sinus or into a sphenopetrosal sinus. in their study, the mean distance between the limen insula and the junction of the ssv with the sphenoparietal sinus is measured 24.8 mm. the vein of labbe is the largest vein on the lateral surface of the temporal lobe that connects the ssv and the transverse sinus and is located in the drainage area of the middle temporal vein in more than half of the hemispheres. the most consistent dominant superficial anastomotic veins are the ssv and the vein of labbe followed by vein of labbe and the vein of trolard. oka. divided the superficial venous anatomy of the temporal lobe into a lateral group and an inferior group [4, 39 ]. the lateral group of veins is further divided into two additional groups : an ascending group consists of temporosylvian veins that drain towards the sylvian fissure and a descending group consists of anterior, middle, and posterior temporal veins that drain into the tentorial sinuses. tanriover. stated that there are a mean of four temporosylvian veins draining into ssv. these veins drain the temporopolar area and the superior temporal gyrus anterior to the posterior limit of the sylvian fissure. also, temporosylvian veins have a minor role at the venous drainage of the inferior limiting sulcus and the posterior long gyrus of the insula. the anterior temporal vein collects the venous drainage from the anterior one - third of the lateral surface except the superior temporal gyrus and drains mostly into a tentorial sinus. the middle temporal vein drains the midportion of the lateral surface and empties into the transverse sinus, a tentorial sinus, or the vein of labbe. the posterior temporal vein drains the posterior third of the lateral convexity of the temporal lobe and courses in a nearly vertical route to drain into a tentorial sinus or less commonly into the vein of labbe. the lateral part of the inferior surface of the temporal lobe is drained via the anterior, middle, and posterior temporobasal veins. the deep middle cerebral vein and the venous drainage of the mtr empty into the deep venous system. the deep venous system begins with the basal vein which has three segments (figures 7(a)7(c)). the deep middle cerebral vein is formed by the junction of the insular veins at the level of limen insula. the first (striate) segment is composed of the deep middle cerebral vein and inferior striate veins that unify to form the basal vein. the joining of the anterior cerebral, olfactory, and frontoorbital veins completes the striate segment, which courses posterior at the upper part of the anterior segment of the uncus to meet with the peduncular vein at the apex of the uncus. the second (peduncular) segment begins at this point to course posterior in the crural cistern. the anterior part of the peduncular segment is referred to as the anterior basal anastomotic vein, because it joins the striate segment and the posterior part of the peduncular segment. the posterior peduncular segment starts where the inferior ventricular vein joins the basal vein and finishes where the lateral mesencephalic vein joins the basal vein. the third (mesencephalic) segment is also known as the posterior anastomotic vein, since it unifies the peduncular segment with the vein of galen. this region shows mainly two types of drainage. if typical variant is present, the venous drainage empties into the posterior peduncular segment via the anterior basal anastomotic vein. the other variant is present if there is no anastomosis between the striate segment and the posterior peduncular segment. in this group, the venous drainage of the anterior mtr emptied into the cavernous sinus or into the sphenoparietal sinus via a large preuncal vein. the middle mtr is in close relation with the posterior peduncular segment and the proximal part of the mesencephalic segment. ventricular vein drains the roof and the lateral wall of the temporal horn as well as the anterior part of optic radiations. in the absence or hypoplasia of the peduncular segment, the inferior ventricular vein forms the second segment of the basal vein. other tributaries to the second segment of the basal vein are anterior hippocampal vein, which drains the extraventricular head of hippocampus, and anterior longitudinal hippocampal vein, which drains the body of the hippocampus (figures 7(a), 7(d)7(g)). the posterior mtr is in close relation with the distal part of the mesencephalic segment of the basal vein, which courses in the quadrigeminal cistern and drains into the vein of galen or the internal cerebral vein. the tributaries from this region into the basal vein are the posterior longitudinal hippocampal vein, medial temporal vein, lateral and medial atrial veins. the medial temporal vein drains the inferior surface of the posterior part of the parahippocampal gyrus. although thousands of patients with temporal lobe epilepsy were operated, there is still controversy regarding the best surgical approach [8, 10, 43, 44 ]. the main risks for functional decline are visual deficit or neurocognitive damage, which may be related to the choice of operative route to the medial temporal lobe. surgical methods for temporal lobe epilepsy can be divided in three groups from a neuroanatomical view : lateral approaches, inferior approaches, transsylvian approaches. the lateral approaches are the anterior temporal lobectomy (atl) and the transcortical selective amygdalohippocampectomy (tcah) ; the inferior approaches are the subtemporal approach and the transparahippocampal approach ; and the transsylvian approaches are the transsylvian selective amygdalohippocampectomy (selah) and the transcisternal approach. in this chapter, the atl, tcah, and the selah will be discussed. the subtemporal approach, the transparahippocampal approach, and the transcisternal approach will not be discussed since they are performed only in selected centers. after sufficient exposure of the lateral surface of the temporal lobe, the sulcal and vascular anatomy should be inspected carefully. the arterial structures in view are the m4 segment of the mca and divided into the temporopolar area and anterior, middle, and posterior temporal areas. this vein is located at the drainage area of the middle temporal vein in most cases, and care should be taken to preserve it, since it is commonly the dominant superficial anastomotic vein. after the neocortical removal, the temporal horn comes into view which has the collateral eminence at its floor. the collateral eminence is the indentation of the collateral sulcus towards the temporal horn and lies lateral to the hippocampus. the neural tissue lateral to the collateral eminence can be removed safely without any risk of damaging midbrain structures. another reliable landmark to achieve a safe neocortical resection is the tentorial edge (figure 5(a)). as long as the resection is aimed lateral to the free edge of the tentorium, the damage to the inferior limiting sulcus, the sylvian fissure, and midbrain structures will be avoided. yeni. stated that this risk is higher during the atl comparing to the tsah. the meyer 's loop was found to cover the roof of temporal horn and to exceed the anterior wall of the temporal horn a few millimeters, thus opening the ventricle at the superior wall may damage optic radiations. additionally, meyer 's loop was found an average of 31.4 mm posterior from the temporal pole. therefore, any resection posterior to this extent carries the risk of causing damage to optic radiations. the ilf connects anterior parts of the temporal lobe to the fusiform gyrus and the occipital lobe. the neocortical resection may include the ilf itself or the cortical areas it originates. and since the areas the ilf connects are thought to have role in learning and remembering of visual stimuli, the neocortical resection can cause memory problems. a cortical incision to the middle temporal gyrus or deep to the superior temporal sulcus is made at the tcah to reach to the ventricle through its lateral wall. olivier described that the cortical incision is made to the middle temporal gyrus rather than the superior temporal sulcus. although transsulcal approach has less distance to reach to the temporal horn, the need for the retraction of the sulcal lips and possible presence of vascular structures deep to sulcus makes this incision demanding. the difficulty of this technique is to find the temporal horn, because a blind dissection in the white matter can lead to significant deficits. the tcah carries the risk of damaging both optic radiations and the ilf. but the risk of damaging optic radiations is less than the risk with the atl, since optic radiations do not cover the anterior portion of the lateral wall of the temporal horn. the tsah, described by wieser and yasargil, is performed with a different route to the temporal horn. the incision is made near to the inferior limiting sulcus after the sylvian fissure is opened. this incision is placed at the temporal stem between the temporopolar artery and anterior temporal artery and carried back 1520 mm from the limen insula. this approach has its own risks to cause functional deficits by damaging optic radiations, the uf, and the ioff. the distance of the lgb to the limen insula is on average 25.1 mm, and there is no clear demarcation between the lgb and the thalamus. during the incision to the inferior limiting sulcus, choi. stated that it is impossible to avoid the optic radiations if the incision is carried 1520 mm posterior to the limen insula. optic radiations can be avoided at a level 10 mm posterior to the limen insula, if the dissection is made between 7 and 25 degrees medially from the sagittal plane, while damaging the mesencephalon and diencephalic structures is highly possible with such dissection. they described that an incision straight inferiorly to the limen insula and to the following 5 mm of the inferior limiting sulcus avoids meyer 's loop. although the anatomy is more complicated with the tsah, the risk of damaging optic radiations is lower than the risk atl carries. it is impossible to preserve the uf with an incision extending posterior from the limen insula. the destruction of this white matter bundle can be the cause of the memory deficits occurring after tsah, because it is stated that the uf functions at recognizing faces and objects by connecting the orbitofrontal and temporopolar areas [1820 ]. the incision to the inferior limiting sulcus can cause damage to the ioff and produce semantic paraphasias. the hippocampectomy begins with the opening of the choroidal fissure beginning at the inferior choroidal point anteriorly. the acha enters into the temporal horn through the inferior choroidal point or just a few millimeters posterior to it. the inferior choroidal point is also the intraventricular representation of the posterior limit of the uncus and determines the border between anterior and middle mtr. the head of the hippocampus is situated anterior to inferior choroidal point, thus located in the anterior mtr. the body of the hippocampus located posterior to this point is in the middle mtr. since the choroid plexus is attached to the choroidal fissure, the choroid plexus is related only to the body of the hippocampus. the choroid plexus originates from the tela choroidea, which is attached to fimbria of the fornix and to the thalamus at the edges of the fissure. the tela attached to the fimbria and thalamus is called the taenia fimbriae and taenia thalami. the choroidal fissure should be opened on the forniceal side to avoid damage to the diencephalic structures and to the vascular structures, such as the acha, lateral posterior choroidal arteries, and the inferior ventricular vein, by leaving them at the thalamic side (figures 4(a), 5(b), and 7(g)). once the anterior part of the choroidal fissure is opened, the pca comes into view under the arachnoidal membrane of the ambient cistern. one should note that the pca is related to the inferior part of the posterior segment of the uncus. the parahippocampal gyrus coursing medially throughout the ambient cistern can be seen once the opening of the choroidal fissure is extended posterior. at this point, the next step is the anterior and medial disconnection of the head of hippocampus via the uncal recess that leads the surgeon to the apex of the uncus. the oculomotor nerve located medial to the apex of the uncus and the p2a located inferomedial to the posterior segment of the uncus may come into the view under the arachnoid membrane. this view shows that the inferior part of the posterior segment of the uncus is also removed. after the choroidal fissure is opened, an arachnoid membrane adjacent to the hippocampus is met before reaching the arachnoid membrane of the crural and the ambient cisterns. the medial disconnection will be carried out by separating the structures of the mtr from these arteries by following a direction towards the inferior surface of the parahippocampal gyrus until reaching the collateral sulcus. the posterior limit of the tail of hippocampus is where he tail meets the calcar avis, which is the inferior prominence at the medial wall of the atrium. the removal of the anterior segment of the uncus exposes the ica and the proximal m1. the oculomotor nerve and the pca resection of the superior part of the uncus carries risk of damaging the globus pallidus unintentionally, since there is no clear border between the globus pallidus and the amygdala. the proximal part of the cisternal segment of the acha is related to the superior surface of the anterior segment of the uncus in 68% of the hemispheres and the distal part of the cisternal segment of the acha is related to the superior surface of the posterior segment of the uncus in 86% of the hemispheres. since this relationship is not present in every case, the acha itself is not a reliable landmark to determine the superior limit of resection of the uncus and the amygdala. wen. described an imaginary line starting at the ica bifurcation or the proximal m1 and ending at the inferior choroidal point (carotid - choroidal point) important in avoiding extension of the amygdala resection in the globus pallidus, but add that the optic tract may also be useful to determine the superior extent of the amygdala removal in tsah (figure 4(b)). while the main goal in epilepsy surgery is long - term seizure control, avoidance of motor, visual, and cognitive deficits helps to optimize an improved quality of life for these patients. despite advances in image guided neurosurgical navigation, a three - dimensional knowledge of the microsurgical anatomy is the best resource for precise tle surgery. a thorough knowledge of the microanatomy of this region leads epilepsy surgeon to a better understanding of the functional anatomy, and a better appreciation of predicted results from different surgical approaches. | objective. we review the neuroanatomical aspects of the temporal lobe related to the temporal lobe epilepsy. the neuronal, the ventricular, and the vascular structures are demonstrated. methods. the previous articles published from the laboratory of the senior author are reviewed. results. the temporal lobe has four surfaces. the medial surface has a complicated microanatomy showing close relation to the intraventricular structures, such as the amygdala or the hippocampus. there are many white matter bundles in the temporal lobe showing relation to the extra- and intraventricular structures. the surgical approaches commonly performed to treat temporal lobe epilepsy are discussed under the light of these data. conclusion. a thorough knowledge of the microanatomy is necessary in cortical, subcortical, and intraventricular structures of the temporal lobe to achieve better results. |
to determine if a threshold of a 1-hour glucose challenge test (gct) eliminates the need for a 3-hour glucose tolerance test (gtt). gdm was diagnosed using national diabetes data group (nddg) and carpenter coustan(cc) criteria. sensitivity, specificity, and predictive values were calculated for 1-hour gct values of 160 to 220 mg / dl. of 6218 patients, 988(15.9%) had an elevated gct and 753(12.1%) underwent a gtt. 165(2.7%) were diagnosed with gdm using nddg criteria, and 250(4.0%) by cc criteria. the positive predictive value of a 1-hour gct200 mg / dl for gdm was 68.6% by nddg and 80.0% for gdm by cc criteria. although the predictive value of an elevated 1-hour 200 mg / dl for gdm was high, 1 in 3 to 1 in 5 women would be overdiagnosed with gdm if the 3-hour gtt were omitted. gestational diabetes mellitus (gdm) is a common complication of pregnancy, affecting nearly 6% of all pregnancies. the american college of obstetricians and gynecologists (acog) recommends a 2 step screening process, using a 50 gram glucose challenge test (gct) for screening, followed by a diagnostic three hour glucose tolerance test (gtt) using 100 grams of glucose for those individuals with one hour glucose levels 130140 mg / dl. two main diagnostic criteria can be used for the diagnosis of gdm, the national diabetes data group (nddg) criteria, or the more stringent lower thresholds of the carpenter - coustan criteria (cc). although use of the cc criteria results in approximately 50% more diagnoses of gdm, neither criteria has been shown to more favorably improve pregnancy outcomes and both are acceptable in current clinical practice. some studies suggested that women with a very high 1-hour gct might not need a 3-hour gtt to diagnose gdm. as would be expected, higher 1-hour gct thresholds result in lower sensitivity but increased specificity and decreased false positive rates in diagnosing gdm. however, the positive predictive value of an extremely elevated 1-hour gct has varied widely across studies, ranging from 50%95% for a threshold of 180 mg / dl in some reports and from 79%100% for a threshold of 200 mg / dl or greater in others. these studies are limited by their small sample sizes, by the use of single - ethnicity populations, and by the lack of contemporary data evaluating this question. because current data are unclear, there are varied clinical practices regarding patients with extremely elevated 1-hour results, with some institutions managing those patients as diabetics without further testing and others proceeding with the 3-hour gtt for definitive diagnosis. although forgoing the 3-hour gtt in those with a very high 1-hour could allow for earlier treatment of gdm, eliminate the inconvenience and cost of the additional test, and avoid extremely elevated blood glucose levels induced by a 3-hour gtt, it could also lead to over diagnosis with unnecessary treatment of those who would not actually have gdm based on 3-hour testing. our aim was to estimate if a threshold of a 1-hour gct, alone or in combination with maternal risk factors, could achieve high enough specificity and positive predictive value to eliminate the need for a 3-hour gtt. this was a retrospective cohort study of all consecutive patients undergoing a 1-hour, 50 gram gct at barnes jewish hospital between 2004 and 2008. women were included in the study if they had a singleton gestation, did not have type i or type ii diabetes, and completed 1-hour gct testing followed by 3-hour gtt testing as appropriate after 20 weeks gestation. women were excluded if there were no 3-hour gtt values available in the medical record. the study was conducted after approval from the washington university school of medicine human research protection office. given the retrospective nature of the study, the need for informed consent was waived. screening was conducted between 2428 weeks unless risk factors suggested need for earlier testing, although only those with testing performed after 20 weeks were included for this analysis. risk factors leading to early testing included a history of previous gdm, obesity with body mass index (bmi) 30.0 kg / m, history of macrosomic infant in a prior pregnancy, first degree relative with diabetes mellitus, or glycosuria. for women with a normal early 1-hour gct, screening was repeated between 2428 weeks and only the second was included for analysis. for those with an elevated 1-hour gct 140 mg / dl, prompt diagnostic testing with a 3-hour gtt was completed, generally within 1 week of initial screening test. gdm was diagnosed by having 2 or more abnormal values using national diabetes data group (nddg) criteria (fasting 105 mg / dl, one hour 190 mg / dl, two hour 165 mg / dl, three hour 145 mg / dl). analysis was also performed for gdm as diagnosed using more stringent carpenter coustan (cc) criteria (fasting 95 mg / dl, one hour 180 mg / dl, two hour 155 mg / dl, three hour 140 mg / dl). information on maternal baseline characteristics, obstetric history, including prior gestational diabetes (based on patient report or available medical records from prior pregnancy), medical history of comorbid conditions that are associated with gdm, body mass index at time of presentation to prenatal care, and laboratory data were obtained from the prenatal record. baseline characteristics and outcomes were compared between women with and without an extremely elevated 1-hour. as originally suggested by carpenter and coustan, an extremely elevated 1-hour was defined as > = 180 mg / dl. student s t test or mann whitney - u test were used for continuous variables and chi - squared tests were used for dichotomous variables as appropriate with a two - sided alpha of 0.05 considered significant. for continuous variables, one hour gct results were then categorized by 20 mg / dl increments between 160 mg / dl and 220 mg / dl. receiver operating characteristic curve (roc) analysis was used to evaluate the test characteristic. the c - statistic, or area under the curve (auc), is used to evaluate the efficacy of screening tests, with auc approaching 1.0 and the far left corner of a roc graph for more effective tests, and auc paralleling the diagonal and nearing 0.5 for tests that are not better than chance. the auc was calculated for each of the thresholds between 160 mg / dl and 220 mg / dl for the 1-hour gct to diagnose gdm using both cc and nddg thresholds. the optimal cutpoint was identified using the youden index which maximizes the sum of sensitivity and specificity. sensitivity, specificity, positive and negative predictive values were also calculated for each of the thresholds. analysis was then repeated for each of the thresholds amongst women with individual and combinations of specific risk factors, including maternal bmi 30 kg / m, history of gdm, and maternal age. all statistical analyses were performed using stata 12, special edition (stata, college station, tx, usa). of 6218 women screened, 988 (15.9%) had an elevated 1-hour gct and 5230 (84.1%) did not (figure 1). of the 988 women with an elevated 1-hour, 235 (23.8%) were excluded, with 152 (15.3%) lost to follow up and 59 (6.0%) patients treated as gdm without a 3-hr gct based on provider preference (figure 1). the cohort was 51.3% african american, 33.6% caucasians, 8.0% hispanic, and 5.0% asian (figure 1). of the eligible 753 women with an elevated 1-hour, 165 women, or 2.7% of the total cohort were diagnosed with gdm by nddg criteria, and 250 (4.0%) were diagnosed with gdm by cc criteria. this increase of gdm in our cohort by 51.5% using the more inclusive cc criteria is similar to other published results. women with an extremely elevated 1-hour 180 mg / dl were similar with respect to race, bmi, history of chronic hypertension, and alcohol, tobacco, and drug use to those with a 1-hour between 140 mg / dl and 180 mg / dl, the groups (table 1). women with an extremely elevated 1-hour 180 mg / dl were more likely to be older than 30 (rr 1.30, 95% ci 1.05, 1.62) and more likely to have a history of gdm (rr 3.48, 95% ci 2.40, 5.02) than those with a 1-hour between 140 mg / dl and 180 mg / dl (table 1). roc analysis was used to evaluate the predictive ability of a 1-hour gct to detect gdm by both nddg and cc criteria in the cohort of women with elevated 1-hour results (figures 2 and 3). the area under the curve (auc) was for 0.730 for gdm as diagnosed by nddg criteria and 0.693 for cc criteria, with the youden maximal cutpoint 157.5 mg / dl for nddg criteria and 158.5 for cc criteria (figures 2 and 3). analysis was performed for each 20 mg / dl threshold between 160 mg / dl and 220 mg / dl with increasing specificity, decreasing sensitivity, at each threshold using both nddg and cc criteria (table 2). as evidenced by the non - overlapping 95% confidence intervals, these changes in sensitivity and specificity were statistically significant moving from 160 mg / dl to 180 mg / dl and to 200 mg / dl, but were not significantly different at 220 mg / dl, possibly due to smaller sample sizes (table 2). additionally, the auc decreased at each threshold, although the auc were not significantly different from one another based on the 95% confidence intervals. the specificity increased at higher thresholds, ranging from 92.2%99.6% for values from 180220 mg / dl using both diagnostic criteria (table 2). the positive predictive values also increased at each threshold, ranging from 52.1% at 180 mg / dl to 72.7% at 220 mg / dl using nddg criteria (table 3). positive predictive values were higher using cc criteria, ranging from 64.6% at 180 mg / dl to 81.8% at 200 mg / dl (table 3). the predictive characteristics of each threshold were evaluated in women with individual and combinations of specific risk factors. diagnostic performance of each threshold was evaluated in women with the addition of clinical risk factors for gdm, including age over 30, history of gdm, and obesity (table 3). for each threshold amongst women with high risk characteristics, sensitivities and specificities the positive predictive values increased for each threshold in the higher risk subgroups, with the most significant improvements in predictive value coming with the addition of history of gdm, and nominal increases in predictive value with the addition of other characteristics (table 3). when considering women who were obese (bmi 30.0 kg / m) in addition to being older than age 30 and having a history of gdm, the positive predictive value at 180 mg / dl increased to 82.6% for gdm as diagnosed by nddg criteria and 91.3% for gdm as diagnosed by cc criteria (table 3). the positive predictive value increased to 100% for those with a history of gdm and an elevated 1-hour 220 mg / dl, but this included only 7 women in our cohort, yielding a wide 95% confidence interval (table 3). in an institution that employs universal gdm screening, we found that even with extremely elevated 1-hour gct results from 180 mg / dl to 220 mg / dl, the positive predictive values ranged from 52.1%81.8%. the predictive value of an extremely elevated 1-hour gct was not substantially improved by applying cc criteria although the cc criteria did predictably increase the rates of diagnosis of gdm. the addition of maternal characteristics that place women at higher risk of gdm improved the positive predictive value to 100%, but only for those with a 1-hour result 220 mg / dl. these results suggest that a 3-hour gtt should be performed even in the setting of an extremely elevated 1-hour gct to avoid over diagnosis and treatment of gdm in those who might not need it. although carpenter and coustan described a greater than 95% positive predictive value for gdm in those patients with elevated 1-hour 182 mg / dl, subsequent studies have found mixed results. bobrowski, friedman, and landy found a 100% positive predictive value for gdm with elevated 1-hour results greater than 220 mg / dl, 200 mg / dl, and 220 mg / dl, respectively. our positive predictive values are more similar to the contrasting findings of shivvers, who found that only 81% of those with a 1-hour greater than 200 mg / dl were ultimately diagnosed with gdm. similarly, yogev found that only 34% of mexican - americans with 1-hour results 200 mg / dl had gdm. some of these studies have been limited by small sample size which has precluded the statistical approach used in the current study. most of the studies only evaluate the 1-hour with use of the higher thresholds of the nddg criteria. additionally, the studies of yogev and friedman were in single ethnic populations, limiting their generalizability to multi - ethnic populations such as ours. finally, other studies are largely based on databases from the 1990s, with the most recent including data from 2004. given the intervening rise in obesity as well as rates of gestational diabetes, our results add evidence on the predictive value of an extremely elevated 1-hour in a more modern cohort. additionally, the overall prevalence of gdm in our cohort, which was 2.7% using nddg criteria and 4.0% using cc criteria, shows our prevalence to be in the range of findings from other studies, where estimates for prevalence of gdm in the united states range from 2.014% depending on which criteria are used. this makes our data externally generalizable to populations with similar gdm prevalence and similar ethnic makeup. additionally, we attempted to use maternal clinical and demographic data to provide more robust and clinically relevant prediction of the diagnosis of gdm and compared these predictive criteria to both diagnostic approaches currently used in the united states first, it is based on an available retrospective cohort at our institution from 20042008. although this represents older data, guidelines for gdm diagnosis at our institution have not changed since that time. second, 235 people, or nearly 24% of those with elevated 1-hour gct were excluded. these included some patients lost to follow - up as well as 59 (6.0%) patients who were treated for gdm without further diagnostic testing. as expected, these included patients with extremely elevated 1-hour gct results ; 43 (4.6%) patients had a 1-hour gct 180 mg / dl. however, in a sensitivity analysis comparing those with an extremely elevated 1-hour who were treated as gdm without further testing to those who underwent traditional 3-hour testing and were not diagnosed with gdm, there were no significant differences in baseline characteristics between the two groups. nevertheless, the inclusion of these patients, had they undergone diagnostic testing and tested positive for gdm, likely would have increased the predictive values of an extremely elevated 1-hour result. including these patients as if they all tested positive for gdm raises the predictive value of a 1-hour gct 180 mg / dl to 66.9% using nddg criteria and 75.5% using cc criteria. additionally, even in this large cohort, having an extremely elevated 1-hour result was still a relatively rare event. the group with high risk characteristics for whom the 1-hour result was 100% predictive included only 7 women. these results may not be applicable to a population with a much higher prevalence of women with extremely elevated 1-hour gcts or with a much higher prevalence of gdm. it further should be noted that significant hyperglycemia 250 mg / dl occurred during diagnostic testing of 10 patients with a 1-hour gct 200 mg / dl who were subsequently diagnosed with gdm. this hyperglycemia would have been avoided if no further testing had been performed on patients with fasting blood glucose on the day of the 3-hr gtt test 120 mg / dl. finally, the current study does not address pregnancy outcomes in women with an extremely elevated 1-hour gct. previous studies have shown that gdm is associated with higher rates of pregnancy induced hypertension, cesarean deliveries, operative deliveries, shoulder dystocia, and macrosomia. additionally, the hyperglycemia and adverse pregnancy outcome study confirmed a continuous relationship between maternal glucose levels and adverse pregnancy outcomes including cesarean delivery and macrosomia. therefore, some have suggested that those with an extremely elevated 1-hour result may have some degree of glucose intolerance and may benefit from treatment for gdm. further study is needed to evaluate pregnancy outcomes in those with an extremely elevated 1-hour who do not have gdm on diagnostic testing. despite these limitations, our study adds to the literature by demonstrating that even with an extremely elevated 1-hour gct result 200 mg / dl, 20%33% of patients would be over diagnosed with gdm if the 3-hour gtt was omitted. although the addition of maternal risk factors marginally improves the specificity and positive predictive value of an extremely elevated 1-hour, it would only eliminate the need for a 3-hour gtt in a few select patients, making this less practical. these findings support the need for a diagnostic 3-hour gtt even in those patients with extremely elevated 1-hour results. | objectiveto determine if a threshold of a 1-hour glucose challenge test (gct) eliminates the need for a 3-hour glucose tolerance test (gtt).study designa retrospective cohort of patients undergoing gtt after gct was 140 mg / dl. gdm was diagnosed using national diabetes data group (nddg) and carpenter coustan(cc) criteria. sensitivity, specificity, and predictive values were calculated for 1-hour gct values of 160 to 220 mg / dl.resultof 6218 patients, 988(15.9%) had an elevated gct and 753(12.1%) underwent a gtt. 165(2.7%) were diagnosed with gdm using nddg criteria, and 250(4.0%) by cc criteria. the positive predictive value of a 1-hour gct200 mg / dl for gdm was 68.6% by nddg and 80.0% for gdm by cc criteria.conclusionalthough the predictive value of an elevated 1-hour 200 mg / dl for gdm was high, 1 in 3 to 1 in 5 women would be overdiagnosed with gdm if the 3-hour gtt were omitted. |
odontogenic tumors (ots) are a group of heterogenous lesions derived from epithelial and/or mesenchymal elements that are part of the tooth forming apparatus. although, some of them represent hamartomas, there are many others that are true benign and malignant neoplasm with different degrees of aggressiveness. ots are rare lesions of the mandible and maxilla that must be considered as a differential diagnosis of lesions that occur in the jaws. in humans, ots comprises about 1% of all jaw tumors, and are located mainly in the maxilla and mandible, and occasionally in the gingiva. in 1971, the first edition to classify this group of lesions was published by the world health organization (who) and in 2005 a revised third edition was done. there are not enough reports, to our knowledge, on ots in iranian population, thus our analyses of ot in isfahan population could provide useful additional data to the literature. this study evaluates the relative frequency of odontogenic tumors recorded at the dental faculty of isfahan, iran over a period of 23 years (1988 - 2010) and compares these data with other parts of the world. we reviewed 6,860 samples of oral and biopsy from the patient records of the oral pathology service, dental faculty of isfahan, iran over a 23-year period (1988 - 2010). data were analyzed for age, gender, tumor site and histopathologic typing according to 2005 who classification. those cases lacking information about one of the above features were documented in a new column in their relative table. we divided ots in to 2 major groups based on who classification : benign and malignant. the jaw was divided in to 3 areas : anterior region (from canine to canine), posterior region (posterior to the canine) and anterior posterior region, posterior area of the mandible included the ascending ramus. data was analyzed by the spss for windows (version 13.0) statistical software package. we found 260 cases of oral cavity and jaw lesions referred to oral pathology laboratory of dental faculty of isfahan, iran during this 23-year period were ots, which constituted 3.79 % of these lesions. benign tumors presented a male : female ratio of 1:1.05 and that one malignant tumor was found in 26-year - old male. table 1 shows the frequency and gender distribution of odontogenic tumors listed based on who classification of odontogenic tumors. frequency and gender distribution of odontogenic tumors listed by diagnostic type, who classification of odontogenic tumors table 2 shows the mean age of this patient population is 27.8. most cases (n = 179) were found in the second, third and fourth decades, with the peak in the second decade (32.3%). the most prevalent ot arose in the second decade of life was odontoma (29.8%), following by ameloblastoma (27.4%), aot (16.7%), and myxoma (10.7%). age distribution of 260 odontogenic tumors table 3 shows the distribution of benign and malignant ot based on their location. the posterior region of the mandible and the anterior region of the maxilla were the most frequent affected area, that 33% and 10.4% of all tumors were appeared there, respectively. the anterior zone was the most common involved area for each ots that occurred in the maxilla, with the one exception of fibromyxoma. distribution of 260 benign and malignant tumors by location ameloblastic carcinoma, ameloblastic fibroma and squamous odontogenic tumor were only detected in the mandible. 91.6% of the ameloblastoma were affected the mandible and most frequently the posterior zone ; besides, odontoma and odontogenic myxoma were mostly occurred in the posterior area of mandible, 30.2% and 20.8%, respectively. cases of odontogenic tumors which affected both area of each jaw, anterior and posterior, were odontoma and ameloblastoma. table 4 gather prevalence of all histopathologic types of ots from different studies and compare them with our study. in the ochsenius series, fernandes series and saghravanian series we can notice the referrals from other places. comparison of the relative frequency of odontogenic tumors from selected references of different countries and this study there are a few numbers of published studies that reveal about odontogenic tumors, besides based on our information there is just one comprehensive related survey in our country, which was done in mashhad, so this made us to plan this survey in another state of our country in order to collect more reasonable data source about odontogenic tumors incidence in iran. in the present study we made the comparison the saghravanian, fernandes, ochsenius, ledeinde, adebayo, olgac and lu series. in this study, ots found as an infrequent lesions (3.79%) among jaw and oral biopsies, which is similar to many published series and different with some others which carried out in nigeria that ots represent 9.6% and 32% of all oral lesions, respectively. according to sex distribution, in this study the male to female ratio of 1:1.05 was found. in saghravanian series it is 1:1.17, in fernandes series it is 1:1.21, in ochsenius series it is 1:1.16, in olgac series it is 1:1.16, in lu series it is 1:1.3, but in ledeinde and adebayo series the ratio was found 1.03:1 and 1.3:1, respectively. by all these studies these tumors were mostly detected in the mandible (3.42:1). in nigeria and china this ratio was found 4.1:1, 4.4:1 and 3.2:1, respectively, which could be due to the greater prevalence of ameloblastoma. while another iranian, brazilian, chilean and turkish series showed a slight frequency for the mandible (2.4:1, 1.9:1, 1.1:1 and 1.9:1 respectively). ameloblastoma was the most common tumor in this study (36.5%) which is comparable with the reports in iran ; mashhad, brazil, nigeria, istanbul and china but, in contrast of studies in america, canada and chile. in latest series, odontoma was the most frequent tumor (73.8%, 45.8% and 44.7% respectively). in our study, we considered on incidence of ameloblastoma of 35.7% to occur in the posterior site of mandible, while in other studies it shows major frequency in this area for this tumor. its prevalence is less than the result from chile, usa and canada but more than the results from iran ; mashhad, brazil, nigeria, turkish and china. in the present study, male to female ratio for odontogenic myxoma was found 1:1.4 that is agree with saghravanian, lu, fernandes and olgac studies. with regard to myxoma, we did not found any gender predilection. other studies of ot reported a female predilection.[11131921 ] in our study, ameloblastic fibro odontoma was more common (6.5%) than other reports.[915 ] our findings show a mean age of 33.9 years for ameloblastoma, which is comparable with other reports[91013 ] which shows 38.5, 27.7, 37.4, 31.7, 29 years respectively for mean age. in the present study, this rarity was also confirmed by studies in iran ; mashhad, brazil, chile, istanbul and america while in african and chinese series we can see significantly higher frequency for malignant tumors (3.4% and 6.1% respectively). it should be mentioned that in 2005 who classification, odontogenic keratocyst has been renamed as keratocystic odontogenic tumor and coc classified as a tumor. however, some of the standard references in oral pathology still use the term okc and definitely classify coc as a cyst, so we have not termed okc and coc as tumor in this study. in conclusion, this study shows odontogenic tumors are not common among oromaxillofacial lesions and malignant types are very rare. these tumors mostly occurred in posterior site of mandible and there is a slight prevalency for females. these data are in correlation with those mentioned in saghravanian study in the other parts of iran. so, maybe we can extent these findings to other site of our country, but for better understanding and perfect conclusion it seems more studies in other areas are necessary. | background : the aim of this study was to record the relative frequency of odontogenic tumors (ots), evaluate and analyze the epidemiological features of the lesions in patients referring to dental faculty of isfahan and compare these data with previous studies.materials and methods : in this study, we reviewed the records of 6,860 lesions from 1988 to 2010 archived in the oral pathology department of dental faculty of isfahan retrospectively and using criteria for histological typification published by the who in 2005. age, sex, site and extent of tumors were analyzed.results:among recorded lesions 260 were ots (3.79%). of these, 259 were benign and just 1 was malignant. the most common lesions were ameloblastomas (n = 95) followed by odontomas (n = 86), odontogenic myxomas (n = 24) and others. there were a few more female patients (n = 133, 51.15%) than male and the mean age of patients was 27.8 years (range 1.5 - 80 years).the posterior of mandible was the most common site (n = 86, 33%).conclusion : according to accumulated data, odontogenic tumors are uncommon lesions and malignant tumors are very rare. |
destructive periodontal disease is defined as an inflammatory condition of infectious primary cause, resulting in marginal alveolar bone resorption and attachment loss. as the destruction of the periodontium progresses apically, the furcation of multirooted teeth is exposed, leading to irreversible bone loss in the interadicular area. the glossary of periodontal terms defines furcation as " the anatomic area of a multirooted tooth where the roots diverge " and furcation invasion refers to the " pathologic resorption of bone within a furcation ". effective instrumentation of furcation defects have always been a challenge for dentists due to the limited accessibility through the furcation entrances as well as the complex anatomy and morphology of molar teeth. in addition, the morphology of the furcation region provides an environment favorable to bacterial plaque retention, which hampers professional and personal dental plaque control and affects positively the pathogenesis of periodontal destruction. for those reasons, teeth with furcation involvement in periodontal disease therefore, the dentist must have a thorough understanding of furcation anatomy to accurately assess etiological factors, diagnose the furcation involvement and treat this condition appropriately. correct definition of the horizontal furcation involvement degree is important to choose the best treatment and determine the prognosis of furcated teeth. the main anatomical considerations are : root trunk and furcation entrance dimensions, root surface area, and root separation distances. according to previous studies, root trunk dimensions play an important role in the periodontal disease process due to its significant relation to both prognosis and treatment of the tooth. concerning the furcation entrance dimensions, a high percentage of mandibular first molars have width values equal to or less than 0.75 mm. these values are smaller than the width of common curettes, which means that such instruments do not clean appropriately the dental surface in the furcation entrance area. thus, the effectiveness to instrument the furcation entrance area is compromised because such curettes do not fit in this area. another feature of molar anatomy is root separation area in furcation region, which corresponds to the portion where the roots are separated by alveolar bone. the measurement of this area tends to increase apically demonstrating how divergent the roots can be. interadicular separation higher than or equal to 2 mm has been associated with the improvement of the furcation healing after regenerative therapies. the aim of the present study was to investigate the morphology of the furcation area of mandibular first molars comparing buccal and lingual sides, based on the limited information regarding this comparison. the experimental sample consisted of 233 mandibular first molars from the human tooth bank of the laboratory of anatomy, department of morphology, araraquara dental school, unesp, brazil. for sample selection, teeth should present good conditions, in other words, teeth with caries, fused roots, calculus or restorative treatment that could interfere in the area of interest were excluded. the reasons for tooth extraction and any information about possible periodontal treatment before extraction could not be identified. the present study was approved by the research ethics committee of unesp (protocol number 13/05). after selection of teeth, reference points were marked on the buccal and lingual surfaces of each tooth with a 0.3 mm graphite pencil, under a stereomicroscope leica mz6 (leica microsystems, heerbrugg, switzerland). initially, a line was drawn on the cementoenamel junction (cej), and a point mark indicated the fornix of the furcation entrance. in each root, two other dots were performed, at 1 mm and 2 mm apical to the fornix point. after, the teeth were fixed in a red wax plate, in order to standardize tooth position and facilitate points and lines visualization. a small piece of millimeter paper was fixed on the tooth crown to allow resolution adjustment and stereometric measurements conversion from pixels to millimeter after taking the photomicrographs (figure 1). buccal (a) and lingual (b) surfaces of a mandibular molar with line and reference points photomicrographs were taken at 15x magnification using a digital camera dxc-107 a/107 ap (sony electronics inc., tokyo, japan). the images were transferred to a microcomputer and image analysis software (jandel sigma scan pro, jandel corporation, san rafael, ca, usa) was used for stereometric analysis. in the digital images, the following lines were drawn : line 1) a horizontal line tangent to the highest points of the cej of each root ; line 2) a line parallel to the previous one, passing through the point of the fornix ; lines 3) and 4) horizontal lines binding the points of each root, corresponding to 1 mm and 2 mm apical to the fornix. lines 1 and 2 were used to obtain the measurement of the root trunk (rt). lines 3 and 4 determined the interadicular distance. after the lines were drawn, the following measurements were taken (figure 2) : reference lines 1, 2, 3 and 4 for stereometric analysis rt (root trunk)= distance between the fornix and the highest point of the cej ; d1= distance between the mesial and distal roots 1 mm apical to the fornix ; d2= distance between the mesial and distal roots 2 mm apical to the fornix ; ia (interadicular angle)= angle of separation formed by the buccal and lingual roots of the furcation. for the ia measurement, two lines were drawn along the inner wall of the mesial and distal root. the angle formed between the intersection of these two lines also, the average of three measurements corresponding to 1 mm in the grid paper on each digital image was obtained. all measurements obtained in this study had normal distribution according to gaussian curve, allowing parametric statistical analysis. hence, all data were expressed as the meanstandard deviation of buccal and lingual furcations and comparative analysis between both sides was performed. for the evaluation of the variables rt, interadicular distance at 1 mm (d1) and 2 mm (d2) and ia statistical analysis was performed using a paired t test, comparing buccal and lingual measures. correlations between buccal and lingual measurements were calculated using the pearson 's correlation coefficient. statistically significant differences were found between all measured parameters for buccal and lingual sides (p<0.05). the interadicular distance increased apically for both sides and the buccal side of the furcation presented greater d1 and d2 distances than the lingual side (p<0.001). the ia of the lingual side was significantly smaller than that of the buccal side (p<0.001). mean and standard deviation (sd) values of root trunk height (rt), d1 and d2 linear measurements (mm) and interadicular angle (ia) measured on the buccal and lingual surfaces of mandibular first molars (n=233) pearson s correlation analysis between buccal and lingual surfaces for root trunk height (rt) (a), d1 (b), d2 (c) and interadicular angle (ia) (d) measurements (n=233, p<0.001) thorough knowledge of root anatomy is mandatory in periodontal therapies as it is intimately associated with the establishment of an accurate diagnosis and the correct choice of the treatment modality to provide optimal long - term prognosis of the teeth. the main finding of the present study was that the lingual furcation is anatomically different from the buccal furcation for all measurements evaluated, which probably affects disease establishment and prognosis. the severity of furcation involvements is directly associated with the relationship between the amount of attachment loss and the rt length. in the present study, morphometric analysis of mandibular first molar furcation area revealed longer lingual root trunk in comparison with buccal root trunk, as shown in figure 3. short rt is more likely to develop early furcation involvement and attachment loss in the presence of periodontal disease because it has less surface area for periodontal attachment. even though, once the disease is installed, reduced rt lengths tend to lead to satisfactory periodontal treatment outcomes because of their easier access also, short rt has been associated with longer individual roots and, consequently, greater potential for corrective therapy. however, in the initial stages of the periodontal disease, long rt has a more favorable prognosis compared to the short one, because it protects the furcation from disease involvement. on the other hand, if the furcation is affected, the prognosis is poorer for longer rt, because the access for instrumentation is hampered and the roots are shorter indicating reduced chance of repair after periodontal therapy. in addition, it has been reported that there is no root trunk longer than 6 mm, which implies that if you have 6 mm of attachment loss in a multirooted tooth, you are probably dealing with a tooth with furcation involvement. the furcation entrance measure is extremely important in anticipating the success of periodontal therapy. in this study, the lingual furcation was statistically narrower than the buccal furcation based on the interadicular distances (d1 and d2) and on the interadicular angle differences. this is the first study to find this particular feature of the first mandibular molar in respect to its interadicular width and relationship between the buccal and lingual surfaces. narrow furcation implies an increased difficulty of access through furcation entrances for complete root debridement leading to a poor periodontal outcome. on the other hand, longer root trunk ends up compensating for this characteristic of the lingual side because it makes the furcation access more difficult, preventing early periodontal involvement. this is a positive relationship between root trunk dimension and interadicular distance / angle found in this study, since these features reduce the chance of dental plaque accumulation in the furcation entrance. curettes are the manual instruments commonly used during periodontal therapy to produce a smooth and biologically acceptable surface and to permit satisfactory healing. the blades of these instruments play an important role since they must present a width that allows correct and effective root debridement. however, narrow furcation entrance dimensions may complicate the periodontal treatment of furcation involvements as the active tip of most instruments (e.g. : gracey curette) present width of 0.95 - 1.2 mm and do not fit in the furcation region. considering the furcation entrance as well as the blade width of periodontal instruments, various studies have found this type of difficulty in periodontal therapy in molar furcations. it is important to emphasize that the interadicular distance of 1 mm from the furcation fornix (d1) is the most critical measurement obtained because it is narrower and more coronal, corresponding almost to the furcation roof of the teeth. this is the first area to be infected when the furcation is involved, and the most difficult region for the dentist to access and perform a correctly instrumentation during the periodontal treatment. a recent study evaluating the radiographic characteristics of furcation involvements showed that narrower root furcations may have better outcomes after nonsurgical periodontal therapy because they are less exposed to contaminants and have less root irregularities. (1991) have stated that interadicular separation of 2 mm or greater provides more favorable regenerative healing. on the other hand, pontoriero, (1988,1989) have found that furcation width - interadicular separation area - greater than 4 mm and entrance height of 3 mm or greater failed to heal with complete defect closure. this means that there may be limitation values for interadicular separation - size and height of the furcation defect - that promotes a favorable healing on regenerative therapies. the positive correlation obtained in this study between buccal and lingual sides suggests that teeth with difficult access to one surface will probably have the same difficulty on the opposite side. reports by other studies confirm that the morphology / anatomy of mandibular first molars is extremely complex and must be thoroughly understood to improve success rate of periodontal therapy the results of this study call attention to the importance of the furcation dimensions in these teeth to a better clinical practice, involving diagnosis, prevention and treatment of periodontal disease. this is the first study that compared buccal and lingual surfaces of first mandibular molar demonstrating significant differences based on the interadicular width. according to the present findings, mandibular first molars have anatomically narrower interadicular distance and greater root trunk height in the lingual surface than in the buccal surface. these observations have implications for clinical practice in the treatment planning and prognosis determination of furcation involvements in patients with periodontal disease. marcelo brito conti, laboratory technician from the department of morphology, for technical support. | furcation involvement in periodontal disease has been a challenge for the dentist.objectivethe aim of this study was to investigate root dimensions in the furcation area of 233 mandibular first molars. material and methodsdigital photomicrographs were used to obtain the following measurements on the buccal and lingual surfaces of each tooth : root trunk height (rt), horizontal interadicular distance obtained 1 mm (d1) and 2 mm (d2) below the fornix and interadicular angle (ia). resultsmean standard deviation of buccal and lingual furcation measurements were, respectively, 1.370.78 mm and 2.040.89 mm for rt ; 0.860.39 mm and 0.710.42 mm for d1 ; 1.500.48 mm and 1.380.48 mm for d2 ; 41.6813.20 and 37.7813.18 for ia. statistically significant differences were found between all measured parameters for buccal and lingual sides (p<0.05, paired t test). conclusionsin conclusion, the lingual furcation of mandibular first molars presented narrower entrance and longer root trunk than the buccal furcation, suggesting more limitation for instrumentation and worse prognosis to lingual furcation involvements in comparison to buccal lesions. |
human adenoviruses (ads) are nonenveloped viruses responsible for respiratory, ocular, and enteric infections. their icosahedral capsid, containing the 36-kpb double - stranded dna genome, is composed of three major proteins : the hexon, the penton base, and the fibre. at the 12 vertices of the capsid it has been reported that the fibre interacts with high affinity with a primary receptor, enabling a subsequent interaction of the penton base rgd motif to cellular integrins that trigger endocytosis [1, 2 ]. remarkably, had3 penton - base expressed in the baculovirus system led to the formation of symmetric complex of 12 pentameric penton bases called base - dodecahedron (bs - dd). we have previously reported that bs - dd interacts with cellular heparan sulfate proteoglycans (hspgs) and facilitates in turn the particle binding to integrins that is a prerequisite for entry [4, 5 ]. due to its great internalisation efficiency, dodecahedron has already been exploited as a versatile vector in dna or protein delivery [3, 6, 7 ]. here, we describe that a heparan sulphate (hs) oligosaccharide binds to the bs - dd rgd - loop and that a basic sequence located next to the rgd motif is critical for integrin recognition. hs octasaccharide (dp8) was obtained by partial heparinase i digestion of heparin and purification by size exclusion chromatography. this dp8 oligosaccharide is made of four sulfated dissacharide repeats of n - sulfated glucosamine and iduronic acid. for cryo - microscopy reconstruction, the structure of an octamer of n - acetyl glucosamine (pdb 1ehn) has been used due to its similarity to dp8. bs - dd (0,5 mg / ml) was incubated for 18 h at 4c with dp8 (0,5 mg / ml) in pbs, corresponding to a 20 : 1 ratio of oligosaccharide per penton base monomer. excess of dp8 was withdrawn by 8 cycles of centrifugation and pbs addition in a microconcentator (microcon 30,000 mwco ; millipore). 1,200 single bs - dd / dp8 particles out of a total of 2,000 coming from 16 micrographs imaged with a jeol 2010 feg electron microscope at a magnification of 50,000 times were used to generate the 3d structure of the complex. the resolution of the 3d structure of this virus was estimated to be 20 resolution (0.5 cutoff for the fourier shell correlation). mutations were achieved using the stratagene quick change mutation kit on the pacuw31 vector encoding the wild type had3 penton base. after dna sequencing to control mutation, baculovirus were built according to the clontech protocol, as previously described. home - made rabbit anti - ad3 penton base antibody and mutants were then purified by ultracentrifugation onto a 1540% sucrose gradient, as previously described. negative staining electron microscopy was performed using uranyl acetate staining and grids were observed on philips cm10 electron microscope. hela or cho cells were resuspended in pbs (5.10 cells in 200 l) supplemented or not with 1 mm cacl2. cells were incubated for 1h at 4c with equal amounts (15 ug / ml) (micrograms) of either bs - dd, mutaqas, or mutrge. home - made rabbit serum directed against the ad3 penton base, then with fitc labelled secondary antibody. cells were analysed by flow cytometry using the facs fluorescein channel (becton dickinson ; cellquest). for experiments using the integrin blocking drug, cells were preincubated with 3 nm of s36578 (kindly given by dr tucker, servier laboratory) for 30 minutes at 4c prior to dodecahedron addition. for experiments using oligosaccharide, dp8 (10 g / ml) was preincubated with dodecahedron for 30 minutes at 4c prior to the incubation with cells. in order to localize the hspg recognition surface on the bs - dd particle, cryo - electron microscopy (cryo - em) was performed on bs - dd alone or preincubated with an hs octasaccharide (dp8). reconstruction of bs - dd / dp8 particles was performed to generate the 3d structure of the complex at a resolution estimated to 20 (figure 1(a)). an extradensity located on spikes at the top of the particle corresponding to the rgd loop [810 ] was clearly visible in the reconstruction of bs - dd / dp8 complex when compared to the previously determined bs - dd structure filtered to 20 (figure 1(a)). as shown in figure 1(b), this trilobed density can easily accommodates an octamer of n - acetyl glucosamine (which should basically have the same length as our dp8 molecule and of a similar structure). the extra density has the shape of an isosceles triangle with one less defined edge (marked # on figure 1(b)) suggesting that only the right part of the oligosaccharide is attached to the bs - dd rgd loop ; the left part is floating around and occupying a different position on the # marked edge in figure 1(b). such location for an hspg binding site suggests a close relationship between hspg and integrin recognition that are both involved in bs - dd attachment and entry into the cell [4, 5 ]. this observation is reinforced by the presence of a consensus (+ x++) basic sequence known to interact with hs just downstream the rgd motif (figure 2(a)). in order to investigate the role played by this basic sequence, mutations were performed to change kqkr into aqas (mutaqas) and an rgd - to - rge mutation (mutrge) was also produced to investigate the role played by integrins (figure 2(a)). baculovirus - expressed mutants were checked by western - blotting using an anti - dodecahedron serum (figure 2(b)) and then purified by ultracentrifugation on sucrose gradient. negative staining electron microscopy showed that neither pentamerisation nor dodecamerisation of the proteins was affected by the mutations (figure 2(b)). we have previously demonstrated that hspgs are the main receptors involved in bs - dd attachment to hela cells. neutralisation of the putative hs binding site in the particle would then result in a decrease of its binding to this cell line. to investigate this point, hela cells were incubated at 4c with equal amounts of either bs - dd, mutaqas or mutrge. as expected, a strong binding of bs - dd was observed, contrasting with the low background raised by the control cells incubated with antibodies only (figure 3(a), green and black histograms). a weak but relevant shift of the mutrge signal towards the lower intensity was observed (purple histogram), showing that direct attachment to integrins was prevented, but that the main attachment to hspgs still occurred. interestingly, binding of mutaqas (light blue histogram) was dramatically reduced, suggesting a significant contribution of the kqkr sequence to cellular receptor recognition likely through reduction of hspg recognition. to better understand the role played by integrins in the attachment, similar experiments were performed on cells preincubated for 30 minutes at 4c with the integrin blocking drug s36578 as previously described. a similar profile was observed (figure 3(a), right panel). to look closer to the effect of s36578 addition on particles binding, each mutant was individually compared in respect of the presence or absence of the integrin blocking drug (figure 3(a), bottom panels). it was clear that integrin inhibition by the drug slightly decreased the wild type particle binding, reflecting the contribution of integrins direct attachment occurring beside the main attachment to hspgs. a similar trend was seen with mutrge in presence of the s36578 drug, showing that the rgd - to - rge mutation was not sufficient to totally abolish the direct integrin recognition on hela cells. on the contrary, this was unexpected as it was thought, in light of figure 1, that this sequence was only involved in hspg but not in integrin recognition. this nonresponse to the s36578 drug suggests that beside a reduction in hspg recognition on hela cells, integrin binding is abolished by this mutation despite the presence of an intact rgd sequence. to further investigate the role of the basic sequence on integrin binding bs - dd binding to this cell line was observed as seen on figure 3(b) (left panel, blue histogram). on these cells, integrins are the unique bs - dd receptor, as pretreatment with s36578 drug totally abolished particle binding (figure 3(b), right panel). interestingly, mutrge was not able to bind these cells (even in absence of s36578) indicating that mutation as discrete as conversion of an aspartate to glutamate (only one supplementary methyl in the lateral chain) was sufficient to totally prevent dodecahedron docking and thus that integrins are the unique receptor for bs - dd on this cell line. surprisingly, mutaqas supposed to be affected only in hspg recognition showed a reduced binding capacity on this hspg deficient cell line (80% of inhibition in absence of s3578, figure 3(b) left panel) compared to bs - dd. this experiment reinforces the previous observation on hela cell showing that despite the presence of an intact rgd sequence, mutation in the kqkr sequence affects integrin recognition. this observation is in agreement with the hypothesis stating that although eight different integrins have been shown to utilise the rgd sequence, their specificity might be principally governed by the local conformation adopted by this tripeptide in a particular ligands. as adenovirus penton base displaying a great variation in the rgd loop size (42 aminoacids for had3 but only 14 for had12 and up to 75 for had2), it could be conceivable that integrin recognition is influenced by the loop flexibility. beside the length of the loop, its aminoacids composition may also play an important role. indeed, it has been reported for the had2 penton base that replacement of the rgd flanking region (hairgdtfa to sfgrgdirn) did not impair v3 recognition, but nearly abolished v5 and 51 binding [12, 13 ]. by mimicking the fibronectin sequence (vtgrgdspa), ad2 derived penton base exhibited high binding to v3 and 51 but not to v5. in the light of our results, it is now clear that the putative basic hspg binding site next to the rgd sequence is implicated in the recognition of integrins. to test whether hs binding to bs - dd influences the integrin recognition efficiency, cho-2241 cells were incubated in pbs 1 mm cacl2 at 4c with different concentrations of bs - dd, with or without 10 g / ml of dp8 (figures 4(a) and 4(b)). histograms clearly showed that oligosaccharide had no effect on binding whatever the bs - dd concentration. on the contrary, a similar experiment performed in pbs without calcium showed a different feature (figure 4(a) and 4(b)). indeed, bs - dd binding to cho-2241 was completely abolished in the absence of calcium indicating that cations were required for interaction. this effect could be explained by a structural change induced by the oligosaccharide on the rgd loop resulting in a change of the affinity for integrins. indeed, it is known that the rgd loop in the adenoviruses is flexible and normally not resolved by cryo - em and image analysis neither in the had5 cryo - em structure nor in the had3 bs - dd cryo - em structure (fuschiotti. the fact that this loop is longer in complex with oligosaccharide compared to the bs - dd alone (figure 1(a) and brown part in figure 1(b) right panel) could be explained by its rigidification induced by a structural change (and enabled us to visualize the dp8 at the apical end of this protuberance). this rigidification has no effect on binding under favourable conditions (i.e., in presence of cations, figure 4) but seems crucial when interaction between the particle and the receptor is weakened by the absence of the stabilizing cation [15, 16 ]. it could be also hypothesized that bs - dd, integrins, and hs would be associated in a ternary complex in which the hs oligosaccharide could consolidate the interaction by forming a bridge between the pseudoviral particle and integrins. indeed, close relationship between integrins and hspgs has been described for different ligands such as extracellular matrix proteins like fibronectin, but also for viruses. among them, foot and mouth disease virus (fmdv) and adenovirus - associated virus (aav-2) behave differently upon hspg binding. indeed, despite the close proximity of hspg binding site located at the interface between vp1/vp2/vp3 proteins and the vp1 protein 's rgd loop, no structural changes occur upon oligosaccharide binding on fmdv. on the contrary, hspg binding to the aav-2 vp3 protein is thought to trigger structural changes on the adjacent integrin binding site and modulate its affinity according to a click to fit mechanism. our data supports the idea of a close relationship between hspg and integrin receptors that might be finely tuned by structural changes upon docking to the cell surface. our cryo - em reconstruction is an important step towards the understanding of this mechanism. even though the biological role of dodecahedron is not yet understood, a better knowledge of the mechanism used by this particle to enter cells is of interest in biotechnology. indeed, we have previously described the utility of dodecahedron in dna and protein delivery [3, 6, 7 ] and the comprehension of the entry mechanism used by this particle will be helpful in vectorology. | human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases) features the ability to enter cells through heparan sulphate proteoglycans (hspgs) and integrins interaction and is used as a versatile vector to deliver dna or proteins. cryo - em reconstruction of the pseudoviral particle with heparan sulphate (hs) oligosaccharide shows an extradensity on the rgd loop. a set of mutants was designed to study the respective roles of the rgd sequence (rge mutant) and of a basic sequence located just downstream. results showed that the rge mutant binding to the hs deficient cho-2241 cells was abolished and unexpectedly, mutation of the basic sequence (kqkr to aqas) dramatically decreased integrin recognition by the viral pseudoparticle. this basic sequence is thus involved in integrin docking, showing a close interplay between hspgs and integrin receptors. |
the prevalence of obesity continues to rise worldwide with alarming rates in most of the world countries. evidence shows that obesity is a major cause of death in both developed and developing countries. these increases over the last several decades as well as growing reports of co - morbidities and mortality, has led to considerable interest in estimating and evaluating obesity possible health effects. most of recent global, national, and sub - national studies approved the body - mass index (bmi), as a reasonably good measure of general adiposity. raised bmi is an established risk factor for several causes of death, including ischemic heart disease (ihd), stroke, hypertensive diseases (hypertension [htn ]), diabetes mellitus (dm), and cancers. it has been suggested that obesity is second only to smoking as a preventable cause of death accounting for as many as 300,000 globally deaths per year as a public health threat. in iran, studies emphasized on noticeable increase in overweight and obesity prevalence for all ages and both sexes. considering the higher population of young people, ignoring the priority of the problem and lack of effective policies, burden of obesity in the near future, become the irreparable concern of health. to designing the controlling programs and allocating resources, a determinant question is the role of risk factors on mortality. population attributed fraction (paf) is one of the important parameters of measuring health effect of risk factors and evaluating their potential impact of preventive programs in community level. evidence reveal that for obesity, pafs may be best - considered indicators of attributed outcomes effects. although some of them are conducted based on exact methodological approaches, there are limited to some national and socioeconomic - geographical regions estimations. the previous related study was based on data that were not nationally representative. moreover, it estimated the effects of bmi defined clinical thresholds, even though epidemiologic studies reveal that the association between bmi and its attributed outcomes continues below such thresholds. in this estimations, incompleteness of the vital registration system and misclassification of cause of death, and uncertainty of the estimated number of deaths were other problems. considering above, using the representative national and provincial data, benefiting from the most comprehensive of mortality data, and following the global burden of diseases (gbd) studies approach, the aim of this study was to estimate and compare the mortality attributable to excess bmi in iranian population. using the paf, we estimated mortality attributable to excess bmi in iranian adults of 2565 years old, at the national and sub - national levels. paf provides the fraction by which the occurrence of an interesting disease / risk factor would be reduced under a sustained alternative, more favorable, exposure distribution. to assess the full effects of bmi distribution as an exposure, following the comparative risk assessment (cra) project, we used the counterfactual of bmi distribution with a mean of 21 kg / m and a standard deviation (sd) of 1 kg / m. in summary, to calculate deaths attributable to excess bmi, the total number of deaths is multiplied by the paf which may be interpreted as the proportion of deaths attributable to excess bmi : these aggregates of population risk can be thought of as proportional to areas under the curve, made up, in a discrete approximation, of small strata of proportions exposed at a given level times the risk at that level, so that : where p1 refers to the factual (or predicted) and p2 to the counterfactual bmi distribution. in the cra method, the relative risks (rrs) for all positions on the counterfactual exposure distribution are set to 1 and given that the sum of the probability distribution is 1, the formula simplifies to : for bmi values, we used data from the noncommunicable disease surveillance survey (ncdss) which followed step wise approach of who guidelines. this is the only complete data source that contains the information of bmi, separately by age and sex at provincial and district levels for adults of 2565 years old. after refine processing, we had 3182 point of data that was representative of 169,347 populationthe effect size of excess bmi for outcomes specific mortality was included by rr. aim to that we used recent relevant meta - analyses of international cohorts evidencemortality data are from death registration system (drs) that is currently collected by ministry of health and medical education (mohme) and national organization for civil registration (nocr). because of delayed registration and inaccurate recording of cause of deaths in nocr data, only mohme data were employed in this study. the mohme drss collected by deputy of research and technology from 1996 to 2001 and by deputy of public health from 2001 to 2010. this system registers the death by age, sex, cause of death, date of death, place of residence, place of death, and other informationpopulation data are from the censuses which are systematically iterated every 10 years, except periods. accordingly, seven censuses have been conducted so far (19862011, province, district). for bmi values, we used data from the noncommunicable disease surveillance survey (ncdss) which followed step wise approach of who guidelines. this is the only complete data source that contains the information of bmi, separately by age and sex at provincial and district levels for adults of 2565 years old. after refine processing, we had 3182 point of data that was representative of 169,347 population the effect size of excess bmi for outcomes specific mortality was included by rr. aim to that we used recent relevant meta - analyses of international cohorts evidence mortality data are from death registration system (drs) that is currently collected by ministry of health and medical education (mohme) and national organization for civil registration (nocr). because of delayed registration and inaccurate recording of cause of deaths in nocr data, only mohme data were employed in this study. the mohme drss collected by deputy of research and technology from 1996 to 2001 and by deputy of public health from 2001 to 2010. this system registers the death by age, sex, cause of death, date of death, place of residence, place of death, and other information population data are from the censuses which are systematically iterated every 10 years, except periods. accordingly, seven censuses have been conducted so far (19862011, province, district). we considered 9 attributable outcomes of excess bmi for loss of life, based on deaths assigned codes of icd-10. these include : ihds, stroke, hypertensive heart disease htn, dm, colon cancer, cancer of the body of the uterus (only in females), breast cancer (only in 45-year - old females), kidney cancer, and pancreatic cancer. these losses are expressed in the metrics of deaths. using simulation methods, combining the uncertainties of bmi distributions, rrs, and outcome - specific mortality, we estimated the corresponding uncertainty intervals of deaths estimations. by assuming an sd of 20% of the estimated completeness, the uncertainty of incompleteness of death registration considered as the variance of the estimated level of completeness. computation were based on 1000 draws for each mentioned parameter in repeated calculations and reported 95% uncertainty intervals based on the distributions of 1000 estimated attributable deaths. the uncertainty of the basic assumptions of the extrapolation of age patterns and rrs across population is not considered in this analysis. these analyses were performed by stata software (version 11) and r software (version 3.0.2). for bmi values, we used data from the noncommunicable disease surveillance survey (ncdss) which followed step wise approach of who guidelines. this is the only complete data source that contains the information of bmi, separately by age and sex at provincial and district levels for adults of 2565 years old. after refine processing, we had 3182 point of data that was representative of 169,347 populationthe effect size of excess bmi for outcomes specific mortality was included by rr. aim to that we used recent relevant meta - analyses of international cohorts evidencemortality data are from death registration system (drs) that is currently collected by ministry of health and medical education (mohme) and national organization for civil registration (nocr). because of delayed registration and inaccurate recording of cause of deaths in nocr data, only mohme data were employed in this study. the mohme drss collected by deputy of research and technology from 1996 to 2001 and by deputy of public health from 2001 to 2010. this system registers the death by age, sex, cause of death, date of death, place of residence, place of death, and other informationpopulation data are from the censuses which are systematically iterated every 10 years, except periods. accordingly, seven censuses have been conducted so far (19862011, province, district). for bmi values, we used data from the noncommunicable disease surveillance survey (ncdss) which followed step wise approach of who guidelines. this is the only complete data source that contains the information of bmi, separately by age and sex at provincial and district levels for adults of 2565 years old. after refine processing, we had 3182 point of data that was representative of 169,347 population the effect size of excess bmi for outcomes specific mortality was included by rr. aim to that we used recent relevant meta - analyses of international cohorts evidence mortality data are from death registration system (drs) that is currently collected by ministry of health and medical education (mohme) and national organization for civil registration (nocr). because of delayed registration and inaccurate recording of cause of deaths in nocr data, only mohme data were employed in this study. the mohme drss collected by deputy of research and technology from 1996 to 2001 and by deputy of public health from 2001 to 2010. this system registers the death by age, sex, cause of death, date of death, place of residence, place of death, and other information population data are from the censuses which are systematically iterated every 10 years, except periods. accordingly, seven censuses have been conducted so far (19862011, province, district). we considered 9 attributable outcomes of excess bmi for loss of life, based on deaths assigned codes of icd-10. these include : ihds, stroke, hypertensive heart disease htn, dm, colon cancer, cancer of the body of the uterus (only in females), breast cancer (only in 45-year - old females), kidney cancer, and pancreatic cancer. these losses are expressed in the metrics of deaths. using simulation methods, combining the uncertainties of bmi distributions, rrs, and outcome - specific mortality, we estimated the corresponding uncertainty intervals of deaths estimations. by assuming an sd of 20% of the estimated completeness, the uncertainty of incompleteness of death registration considered as the variance of the estimated level of completeness. computation were based on 1000 draws for each mentioned parameter in repeated calculations and reported 95% uncertainty intervals based on the distributions of 1000 estimated attributable deaths. the uncertainty of the basic assumptions of the extrapolation of age patterns and rrs across population is not considered in this analysis. these analyses were performed by stata software (version 11) and r software (version 3.0.2). in 2011, in adults of 2565 years old, at the national level, excess bmi was responsible for 39.5% (10,210 deaths) of total 25,860 deaths that were attributed to 9 bmi paired outcomes. considering bmi outcomes, the highest mortality was attributed to ihd (55.7%) which was followed by stroke (19.3%), and dm (12.0%). except for ihd and colon cancer, attributed death to all of other common outcomes, considerably, were higher in females. figure 1 shows the national attributed deaths (and uncertainty intervals) trends of excess bmi, by outcomes, sex, and age groups. the national attributed deaths (and uncertainty intervals) trends of excess body mass index, by outcomes, sex, and age groups (20052011) in figure 2, the provincial patterns of excess bmi attributed death have been presented by outcomes and sex, in 2005 and 2011. based on the provincial patterns, the most incremental changes happened in central and western provinces. these increases are more impressive in females than males and in ihd, htn, and cancers than other outcomes. the provincial patterns of excess body mass index attributed deaths by outcomes and sex, in 2005 and 2011 figure 3 shows the provincial excess bmi attributed deaths by outcomes, sex, and age groups, in 2011. for all of the outcomes, the highest death was, mostly, happened in 5559 and 6064 age groups. isfahan, mazandaran, and khuzestan were the first leading province for ihd attributed death in males. for females, the highest deaths for stroke were aligned, respectively, to tehran, isfahan, and khuzestan in males and tehran, khuzestan, and east azerbaijan in females. khorasan razavi and isfahan were the first two provinces with the highest htn death, for both sexes, which were followed by tehran in males and east azerbaijan in females. at the same way, khuseatan and isfahan were two top ranked of highest dm death that were followed by razavi khorasan in males and mazandaran in females. for cancers mortality : tehran, isfahan, and khuzestan, unanimously, were three first leading provinces in both sexes. it is mentionable that in females, west azerbaijan was together with khuzestan stand at the third ranks. the provincial excess body mass index attributed deaths by outcomes, sex, and age groups, in 2011 given the comparative results at national levels, between 2005 and 2011, obtained pafs, were increased for all 9 excess bmi attributed outcomes. considering the age - specific changes, these increasing detected in most of adult age groups. except for colon cancer, the remaining 6 common outcomes of ihd, stroke, htn, dm, kidney cancer, and pancreatic cancer, were higher for women than men. it is noteworthy that colon cancer showed higher attributed risk in all of age groups of males, compared with females. figure 4 compares the national pafs of all 9 excess bmi attributed outcomes, by sex, by age groups, between 2005 and 2011. based on the categorical references, for breast cancer, data were estimated only for over 45 age groups. the national pafs of excess body mass index attributed outcomes, by sex, by age groups, in 2005 and 2011 in tables 1 and 2, respectively, for males and females, the pafs of three leading cause of excess bmi attributed death, ihd, stroke, htn, and dm compared between different age groups at national and provincial levels. in each province, the lowest and highest levels have been showed, respectively, in green and red. the national and provincial population attributable fractions of excess bmi on ihd, stroke, htn, and dm in iranian males by age groups in 2011 the national and provincial population attributable fractions of excess bmi on ihd, stroke, htn, and dm in iranian females by age groups in 2011 overlay at national level, in males, the highest paf for ihd, stroke, htn, and dm were, respectively, detected in 3539, 3539, and 4044, and 4549 age groups. at the same way, for all three outcomes, the highest pafs for ihd were in kohgiluyeh and boyer - ahmad and mazandaran (0.74), for stroke, in ardabil and kohgiluyeh and boyer - ahmad (0.69), for htn, in ardabil and kohgiluyeh and boyer - ahmad (0.65), and for dm, in mazandaran (0.87) provinces. in females, the highest national paf for ihd (0.58), stroke (0.54), and htn (0.60) was, respectively, detected in 4044 age groups. two age groups of 3539 and 5054 also had the highest value for paf of stroke (0.54). the highest national paf for dm (0.54) was detected in 5054 age groups. except for all other three outcomes, with lowest pafs of 2529 age groups, for dm the lowest value was found in 6064 age groups. at provincial levels, the highest pafs for ihd were in markazi and zanjan (0.77), for stroke, zanjan (0.74), for htn in markazi (0.79), and for dm ardebil (0.90) provinces. using paf, widely regarded as an estimate of the proportion of disease / risk factor(s) burden, we estimate the mortality attributable to excess bmi in iranian population. we followed the methodology which was used in previous researches. in 2011, in adults of 2565 years old, at national level, excess bmi was responsible for 39.5% (10,210 deaths) of total 25,860 deaths that were attributed to 9 bmi paired outcomes, of them 5619 (55.0%) were males. considering bmi outcomes, the highest mortality was attributed to ihd (55.7%) which was followed by stroke (19.3%), and dm (12.0%). based on the pafs estimations in 2011, except for colon cancer, the remaining 6 common outcomes of ihd, stroke, htn, dm, kidney cancer, and pancreatic cancer, were higher for women than men. there is a considerable variety among studies regarding the methods and results for paf calculation and the selection of appropriate counterfactuals. in iran, studies are limited, and there is no previous study on provincial estimation of pafs or excess bmi mortality (10, 20, 28). based on the results of previous study, in 2010, excess bmi was the third cause of cardiovascular disease (cvd) deaths in women, which was responsible for about 17,000 (14,00020,000) national deaths. for men, excess bmi as the fourth cause of cvd death led to 13,000 (11,00015,000) deaths. comparing four socioeconomic - geographical regions of the country, the lowest bmi attributed death was estimated for both males and females in south - east region 700 (600800). the highest estimations were in the western region, 5400 (46006200) and 7100 (59008300), respectively, for men and women. considering the differences, the highest attributable mortality rate was 1.22.2 times that of the lowest. our analysis extends the relevant evidence on national and sub - national cra analyses. considering the previous studies in iran, the present study has several achievements. this study is the first analysis of national and sub - national estimation of mortality attributable to excess bmi in iranian population. benefited from gbd studies approaches, using ncdss data, we estimate the trends of attributed deaths and their correspond uncertainties, at the national and provincial levels from 2005 to 2011, by sex and age. it is northworthy that for the first time, we had access to most comprehensive data of mortality, at the national and sub - national levels. considering the geographical changes in provincial divisions, dealing with misalignments problems, for a better comparison first of all, there is a potential source of bias in methods when estimations used with rrs adjusted for confounding. obesity is correlated with other exposures or unobserved factors in the population resulting in under- (when there is a positive correlation) or over- (negative correlation) estimation of the true paf when used with adjusted rrs. we did not have data on dietary factors, physical activity, alcohol and drug use, or other metabolic risk factors. moreover, causes other than those considered in our study may be positively associated with excess adiposity, which biases our estimates of attributable deaths downward. as another important limitations, our exposure data did not include people 65 years of age and older, which limited the scope of our analysis. it is mentionable that to estimate the number of deaths, we used recorded death data that did not consider incompleteness. in our future plan, our death estimations would be presented dealing with these limitations. considering all of strengths and limitations, under the national plan of comprehensive nasbod project, using most updated modern methodologies and regarding more wide age ranges from 25 up to more than 80 years old, we are going to estimate trends and burden of bmi, obesity, and overweight, at the national and sub - national levels, for adult iranian population from 1990 to 2014. the present results show a growing need to comprehensive implications for the national and sub - national health policies and interventional programs in iran. despite the priority of the problem, these considerations should be followed at the shortest possible time through the primary health care system or community - based lifestyle and dietary interventions. suggested strategies including family - oriented interventions on healthy eating and physical activity by a dietitian, integrating obesity prevention and control programs in the primary health care system, and identifying, implementing, and evaluating of applied interventions are particularly solutions emphasized for dealing with ascending trends of overweight and obesity in iran and worldwide. in these regards, evidence - based documents emphasize specifically on solutions and strategies that could be implemented at the level of the individual. these plans should be designed and followed through a comprehensive by collective societal actions to change the food and activity environments. we must also help to bridge the division between individual and collective responsibility for successful long - term weight loss maintenance. according to our knowledge, this is the first analysis of the national and sub - national estimation of mortality attributable to excess bmi in iranian population. excess bmi was responsible for 39.5% (10,210 deaths) of total attributed deaths to 9 bmi paired outcomes. considering the priority of the problem, presented estimations provide practical information for health policies and programs. | background : the prevalence of obesity continues to rise worldwide with alarming rates in most of the world countries. our aim was to compare the mortality of fatal disease attributable to excess body mass index (bmi) in iran in 2005 and 2011.methods:using standards implementation comparative risk assessment methodology, we estimated mortality attributable to excess bmi in iranian adults of 2565 years old, at the national and sub - national levels for 9 attributable outcomes including ; ischemic heart diseases (ihds), stroke, hypertensive heart diseases, diabetes mellitus (dm), colon cancer, cancer of the body of the uterus, breast cancer, kidney cancer, and pancreatic cancer.results:in 2011, in adults of 2565 years old, at the national level, excess bmi was responsible for 39.5% of total deaths that were attributed to 9 bmi paired outcomes. from them, 55.0% were males. the highest mortality was attributed to ihd (55.7%) which was followed by stroke (19.3%), and dm (12.0%). based on the population attributed fractions estimations of 2011, except for colon cancer, the remaining 6 common outcomes were higher for women than men.conclusions:despite the priority of the problem, there is currently no comprehensive program to prevention or control obesity in iran. the present results show a growing need to comprehensive implications for national and sub - national health policies and interventional programs in iran. |
urothelial carcinoma accounts for more than 90% of bladder tumors, and about 70% of urothelial carcinomas are non - muscle - invasive bladder cancers (nmibcs) at the time of the initial diagnosis. although nmibc is usually not life - threatening in the early stage, more than half of these tumors will relapse and approximately 10% to 20% of these tumors will develop into muscle - invasive bladder tumors. although several studies have been performed, a single prognostic factor has not yet been identified because nmibc is considered to be a heterogeneous disease. to predict the risk of recurrence and progression, the european organization for research and treatment of cancer (eortc) developed a simple scoring system that includes factors such as the number of tumors, tumor size, prior recurrence rate, cancer stage, presence of carcinoma in situ (cis), and world health organization (who) grade on the basis of data from 2,596 patients with nmibc. to correct the overestimated risks of recurrence and progression owing to the low rate of bacillus calmette - guerin (bcg) instillation, the spanish urological club for oncological treatment (cueto) proposed a modified model using gender, age, recurrent tumor, number of tumors, cancer stage, cis, and who grade on the basis of data from 1,062 patients who were treated by bcg instillation. these scoring systems can help in individualizing patients ' follow - up schedules and in the decision making process for performing an early cystectomy. the goal of this study was to confirm the utility of the eortc and cueto scoring systems in a korean population. between january 1985 and december 2011, 531 patients underwent transurethral resection of bladder tumor (tur - bt) at chung - ang university hospital owing to a histological diagnosis of nmibc. we retrospectively analyzed the patients ' medical records, which contained information on age, gender, prior recurrence rate, number of tumors, tumor size, cancer stage, presence of cis, who grade, intravesical treatment, recurrence, and progression of bladder tumor. the follow - up period of the patients was checked from the first operation to the last cystoscopy procedure. patients who were found to have advanced bladder tumors, ureteral tumors, or nonurothelial carcinoma at the first operation were excluded from the study. after pathologic confirmation, all patients underwent follow - up cystoscopies at the authors ' clinic and received intravesical treatment when indicated. patients who had a t1 tumor, grade 3, or no muscle lesion on pathology underwent repeat tur - bt within 6 weeks. we calculated the time to first recurrence (disease - free interval) as months to detect recurrence on cystoscopy after the diagnosis of bladder cancer. patients alive without recurrence were censored at the time of the last available follow - up cystoscopy. we calculated the time to progression as months to detect muscle - invasive disease on pathological examination or metastasis on radiologic imaging after the diagnosis of bladder cancer. patients alive without stage t2 or higher disease in the bladder were censored at the time of the last available follow - up cystoscopy. twenty - four patients had undergone cystectomy or had experienced recurrence at the ureter or prostatic urethra after the first diagnosis of nmibc. urine cytology and computer tomography were used according to the european association of urology (eau) guideline, and intravesical biopsy was performed for suspicious lesions. the scores for risk of progression and recurrence were estimated by using the eortc and cueto models. cumulative incidence probabilities of recurrence and progression at 1 and 5 years were analyzed with 95% confidence intervals (95% cis). the kaplan - meier survival analysis was used to assess recurrence and progression curves in both models. we assessed the predictive performance for recurrence and progression by using a receiver operating characteristic (roc) curve of the eortc score and cueto score and calculated the cutoff values. univariate and multivariate cox proportional hazards regression analyses were used to identify the prognostic factors for recurrence and progression. all statistical analyses were conducted by using ibm spss ver. 18.0 (ibm co., armonk, ny, usa), and r ver. 3.0.2 (r foundation for statistical computing, vienna, austria). the basic characteristics of the patients are shown in table 1 for comparison between the eortc and cueto models. the patients ' mean age was 63.7 years (range, 22 - 93 years) at the time of diagnosis of bladder tumor, and the median follow - up duration was 58 months (range, 3 - 321 months). one hundred seventy - five patients (33.0%) had a relapse of bladder tumor within a mean follow - up of 19.0 months (range, 3 - 321 months). forty - eight patients (9.0%) showed progression to muscle - invasive disease within a mean period of 33.6 months (range, 3 - 301 months). the 60- to 70-year - old age group was the largest (37.1%), and 88.7% of the patients were male. single tumors were observed most frequently (62.0%), and a tumor size less than 3 cm (71.4%) was observed more frequently than a tumor size greater than 3 cm. the presence of cis was observed in a small proportion of patients (6.6%). in our study, the cancer stages were divided almost equally. by use of the eortc model, 55.9% of patients had ta cancer, and by use of the cueto model, 19.4% of patients did. also in our study, patients with who g1 disease were few (4.0%). with the eortc model, 43.2% of patients had g1 disease, and with the cueto model, 15.2% of patients had g1 disease. a total of 282 patients (53.1%) received bcg instillation as the intravesical treatment. 1a and b show kaplan - meier survival curves of the 4 recurrence risk groups according to each model. by use of the eortc model, all groups had statistically significant differences except between the group with a score of 0 and the group with a score of 1 - 4 (p=0.301). by use of the cueto model, the harrell 's concordance index using the eortc and cueto models was 0.759 and 0.836 for recurrence, respectively. calibration of eortc and cueto models showed p - values of 0.856 and 0.688. in fig. 1c and d, the patients were divided into 4 groups according to model score, and the time to progression is presented for each group. by use of the eortc model, a significant difference was observed between all groups except between the group with a score of 2 - 6 and the group with a score of 7 - 13 (p=0.303). by use of the cueto model, a significant difference was observed only between the group with a score of 0 - 4 and the other groups ; the other groups did not differ significantly (score 5 - 6 vs. score 7 - 9, p=0.616 ; score 5 - 6 vs. score 10 - 14, p=0.121 ; score 7 - 9 vs. score 10 - 14, p=0.307). the harrell 's concordance index using the eortc and cueto models was 0.704 and 0.745 for progression, respectively. calibration of the eortc and cueto models showed p - values of 0.974 and 0.994. our probabilities of recurrence and progression at 1 and 5 years were compared by use of the eortc risk table and the cueto risk table as shown in tables 2, 3. our results for recurrence at 1 and 5 years showed larger differences between each score group than the reference probabilities provided by the eortc and cueto risk tables. however, our results on comparison with the eortc system were more distinguishable than our results on comparison with the cueto system for progression at 1 and 5 years. by use of the roc curve, the eortc and cueto scores were found to be useful predictors of recurrence with area under the curve (aucs) of 0.832 (95% ci, 0.794 - 0.868) and 0.894 (95% ci, 0.865 - 0.923), respectively. comparison of the aucs of the eortc and cueto models showed the auc of the cueto model to be significantly higher (p=0.0002). when we applied a cutoff score for recurrence of 5.5, the eortc system had a sensitivity of 74.3% and specificity of 75.3% in predicting recurrence, and the cueto system had a sensitivity of 83.4% and a specificity of 82.0% in predicting recurrence. the eortc and cueto scores were not good predictors of progression, with aucs of 0.722 (95% ci, 0.649 - 0.779) and 0.724 (95% ci, when we applied a cutoff score of 4.5 for progression, the eortc system showed a sensitivity of 93.8% and a specificity of 43.9%, and the cueto system had a sensitivity of 91.7% and a specificity of 46.8%. in the multivariate analysis of recurrence, prior recurrence rate, cis, and grade were significant prognostic factors by use of the eortc model, and recurrent tumor, number of tumors, cis, and grade were significant prognostic factors by use of the cueto model. in the multivariate analysis of progression, recurrent tumor, cancer stage, and cis were significant predictors by use of the eortc model, and recurrent tumor, cancer stage, cis, and grade were significant predictors by use of the cueto model. rates of smoking and chemical exposure vary, and both of these are known causes of bladder cancer. bladder cancer incidence is approximately 3 times higher in white men than in african american men. furthermore, the survival rate for african americans is worse than for asians and whites. in a study predicting the recurrence and progression of bladder cancer by use of the surveillance, epidemiology, and end results medicare data, stage t1 was associated with a higher rate of recurrence, and female gender, black race, grade, and cis with t1 were associated with a higher risk of progression. however, a definite method for predicting prognosis has not yet been found, and we should determine risk factors for the korean population. xu. investigated 363 chinese patients with nmibc and found that the eortc model was more accurate in predicting recurrence and progression than was the cueto model. they stated that the reason for this occurrence was that most of their patients received intravesical chemotherapy, similar to the patients in the eortc study. xylinas. explained that both models showed a poor discrimination ability because of overestimation of the risk of recurrence and progression in high - risk patients. the cueto model showed a higher probability in predicting tumor size, cancer stage, and cis than did the eortc model. in the eortc study, 78% of the patients received intravesical treatment, mostly with chemotherapy, and a few patients were treated with bcg instillation. in the cueto study, however, 100% of the patients received bcg instillation and 15% of the patients were additionally treated with mitomycin c. meta - analyses have shown that bcg instillation after tur - bt reduces the risk of recurrence and progression. in our study, 53% of patients received bcg instillation, and tumor size, cancer stage, and cis were better predictors than in the cueto study, which resulted in a similar recurrence rate (33%) to that in the cueto study (32.6%). in addition, in multivariate analyses of data from the cueto study for recurrence, although age and gender were not significant, other significant variables, especially recurrence, showed a high hazard ratio (4.875). hence, this may have affected the distinguishable and significant kaplan - meier curve of cueto for recurrence. however, compared with the eortc study, the variables that were significant in multivariate analyses of data from the cueto study varied, and the effect of a high hazard ratio was less than in the eortc study. the harrell 's concordance indexes for recurrence were more distinguishable in the cueto model than in the eortc model (p=0.01). also, the auc for the cutoff score for recurrence was more significant in the cueto model than in the eortc model. however, the eortc model was found to be a valid tool for assessing the probabilities of recurrence and progression at 1 and 5 years. the weakness of both of these models is the low positive predictive value for progression, especially in patients with high - grade disease. our results were more distinguishable using not only the eortc model but also the cueto model than were the original references provided by the eortc and cueto studies. although their variables were not optimal for our subjects for assessing the results of univariate and multivariate analyses, sylvester 's report showed that the variables in the eortc and cueto model were not significant in several studies. the eortc model was strong for predicting the probabilities of recurrence and progression at 1 and 5 years, and this could help clinicians in individualizing the follow - up schedule according to the patient 's risk score. before performing cystoscopy, assuming an eortc recurrence score of greater than 5 implies a recurrence rate of greater than 50% at 5 years could help clinicians avoid missing the probability of recurrence. in addition, patients treated with bcg instillation who had a cueto recurrence score of greater than 5.5 could indicate that there is a high probability of failure of intravesical therapy and the clinician should consider early cystectomy for such patients. our study had some limitations. because of the retrospective analysis and long follow - up period, not all of the patients were treated by the same regimen or the same clinician. also, we did not assess intravesical chemotherapy or dosage and discontinuation of bcg instillation owing to incomplete old records. there are recent studies on low - dose bcg and the use of a short period to reduce the side effects of bcg ; even reduced bcg instillation has been found to be clinically effective. in our hospital, we used the 1973 grading system of the who between 1985 and 2006 and the 2004 who grading system from 2007 onwards. g1 of the 1973 who grading system can correspond to papillary urothelial neoplasm of low malignant potential or low - grade tumor. g2 of the 1973 who grading system can correspond to low - grade tumor or high - grade tumor of the 2004 who grading system, and g3 of the 1973 who grading system can correspond to high - grade tumor of the 2004 who grading system. seventy - eight patients were graded by using the 2004 who grading system. since the eortc and cueto models used the 1973 who grading system, but the 2004 grading system is used currently in our hospital, our aim was to confirm the applicability of both models to our conditions. chen. found that both the 1973 and the 2004 who classifications were effective in predicting progression, whereas the 1973 who classification was more suitable for predicting recurrence. according to the european guidelines, the other limitation of our study that we could not identify new variables associated with a poor prognosis, such as prostatic urethral involvement or bladder neck involvement, molecular markers, and snps. however, research on these variables has only recently begun and they may not be recommended for routine examination. the eortc and cueto scoring systems showed value in predicting recurrence and progression in korean patients with nmibc. especially, the cueto model showed statistically significant results for recurrence in our study, because of the effect of bcg instillation and the heterogeneous patient characteristics. these models can help clinicians in individualizing the appropriate treatment and follow - up schedule. prospective, multicenter, and large - scale studies using modified eortc and cueto models are needed to predict the accurate recurrence and progression of bladder cancer in the korean population. | purposethis study aimed to confirm the utility of the european organization for research and treatment of cancer (eortc) and the spanish urological club for oncological treatment (cueto) scoring systems and to determine which model is preferred as a prognostic model in korean patients with non - muscle - invasive bladder cancer.materials and methodsbetween 1985 and 2011, 531 patients who were treated by transurethral resection of bladder cancer were retrospectively analyzed by use of the eortc and cueto models. statistically, we performed kaplan - meier survival analysis ; calculated harrell 's concordance index, receiver operating characteristic (roc) curve, and cutoff values ; and performed univariate and multivariate cox proportional hazards regression analyses.resultsfor risk of recurrence, with the use of the eortc model, all groups had statistically significant differences except between the group with a score of 0 and the group with a score of 1 - 4. with the use of the cueto model, all groups differed significantly. for risk of progression, with the use of the eortc model, significant differences were observed between all groups except between the group with a score of 2 - 6 and the group with a score of 7 - 13. with the use of the cueto model, a significant difference was observed between the group with a score of 0 and the other groups. the concordance index of the eortc and cueto models was 0.759 and 0.836 for recurrence and 0.704 and 0.745 for progression, respectively. the area under the roc curve for the eortc and cueto models was 0.832 and 0.894 for recurrence and 0.722 and 0.724 for progression, respectively.conclusionsboth scoring systems, especially the cueto model, showed value in predicting recurrence and progression in korean patients, which will help in individualizing treatment and follow - up schedules. |
uranium, an actinide element that has been present since earth s formation, is used as fuel for nuclear power plants due to its reactivity. in the case of internal exposure, accidental intake of uranium induces acute renal toxicity in humans and animals through accumulation in the kidneys. in parenteral ingestion of soluble - form uranium, the uranium rapidly enters the systemic circulation as uranyl ions, and it is also immediately deposited in the kidneys.uranium accumulates mainly in the renal proximal tubules of the outer stripe of the outer medulla, and a high accumulation of uranium causes tubular damage such as renal tubular necrosis. uranium is also one of the bone - seeking elements, and it is deposited on the bone surface, where it remains for a long period. as one of the alpha - particle - emitting radionuclides, uranium is thus thought to possibly increase the risk of a stochastic effect, such as bone malignancies. following an accidental intake of toxic levels of uranium, decontamination therapy should therefore be performed to prevent uranium toxicity including acute renal toxicity and the risk of bone cancer development. in decontamination therapy, uranium excretion - enhancing drugs such as chelating agents can be used to induce the excretion of as much uranium as possible in the early period. the treatment regimen described in a 2010 national council on radiation protection & measurements (ncrp) report is a slow intravenous infusion of sodium bicarbonate solution, or an oral administration of sodium bicarbonate tablets until the urine reaches a ph of 8.0 to 9.0. although increasing the blood level of bicarbonate ions and alkalinizing the urine were thought to effective for amelioration of acute uranium contamination in affected humans, the effectiveness of uranium decontamination by sodium bicarbonate has not been supported by controlled studies with laboratory animals under realistic conditions. indeed, two research groups reported that treatment with sodium bicarbonate produced almost no decontamination effects in uranium - contaminated rats. 0.1 g / kg, which is almost equal to the clinical human dose, and the urine ph of the treated animals was not monitored in these studies. in addition, sodium bicarbonate showed urinary alkalinization at a dose level higher than the clinical human dose. chiu. reported that cortical uptake of gentamicin was inhibited by urinary alkalinization due to a 1-h infusion of a solution of sodium bicarbonate (0.3 mol / l) at a dose of 6.3 ml / h (calculated as approx. 0.5 g / kg) preceding the administration of gentamicin, and by a continuous infusion of 0.15 or 0.30 mol / l sodium bicarbonate for 3 h (calculated as approx. 0.75 or 1.5 g / kg) after the administration of gentamicin. hattori. reported that sodium bicarbonate at a dose of 1 g / kg showed a significant urine alkalinization effects in rats. we thus hypothesized that the lack of a uranium decorporation effect in the laboratory animals in the henge - napoli. and fukuda. studies described above may have been due to the dosage of sodium bicarbonate, which produced insufficient urine alkalinization. in the present study, to determine whether sodium bicarbonate at a dose producing urine alkalinization has uranium decontamination effects, we used a uranium - contaminated rat model to examine the effectiveness of sodium bicarbonate for removing uranium and protecting against uranium - induced acute renal toxicity. the uranium was dissolved in distilled water, and the administration volume was 1 ml / kg body weight. the sb was dissolved in distilled water, and the administration volume was 10 ml / kg body weight. we used 8-wk - old male crl : cd (sd) rats (n=24 ; charles river laboratories japan, kanagawa, japan). all animals were administered 1 mg / kg of uranyl nitrate intramuscularly into the right femoral muscle at 10:40 a.m. twenty minutes after the uranium injection, the animals in the four groups were given a single oral administration of sb at the dose levels of 0, 0.1, 0.3 and 1 g / kg, respectively. the dosages used were based on the report by hattori. for the uranium control group, distilled water (dw) only was administered in the same manner as that used for sb. the urine of each animal was collected just before and at 1, 2, 4, 9 and 23 h after the uranium injection. each animal was placed in a plastic animal cage, and the naturally excreted urine was collected. the ph of the urine was measured with a ph meter (model d-51, horiba, kyoto, japan). at 24 h after the uranium injection, the animals were euthanized under ketamine / xylazine anesthesia, the left kidney was removed, and the concentration of uranium in the kidney was measured with an inductively coupled plasma - mass spectrometer (icp - ms, sii spq9700-ii, sii nanotechnology, chiba, japan) after separating the uranium from matrix components using a closed - vessel microwave digestion system (discover sp - d, cem corp., matthews, nc, usa). based on the concentration of uranium in the kidney, the uranium amount per left kidney was calculated and used for the evaluation. for this experiment, we used 8-wk - old male crj : cd rats (n=60 ; charles river laboratories japan inc. all animals were administered 1 mg / kg of uranyl nitrate intramuscularly into the right femoral muscle at 10:40 a.m. at 20 min after the uranium injection, the animals in the three groups were given a single oral administration of sb at the dose levels of 0 (uranium control), 0.1 and 1 g / kg, respectively. six animals in each group were autopsied at 1, 3 and 7 days after the uranium injection, respectively. two dose groups of sb were selected based on the results of experiment i. the high dose was expected to show significant urine alkalinization, and the low dose was expected to show no significant urine alkalinization. before autopsy, 24-h urine collection was performed using metabolic cages. from this 24-h urine, 05-h the urinary volume was measured, and the urine samples were subjected to a uranium amount analysis and urinary biochemistry. the animals were euthanized under ketamine / xylazine anesthesia, and blood samples obtained from each animal were used for plasma biochemical analyses. plasma urea nitrogen (un), plasma creatinine (cre), urinary total protein (utp) and urinary glucose (uglu) were analyzed using an automatic biochemistry analyzer (ca 400, furuno, nishinomiya, japan). beta-2-microblobulin (2-mg) in urine was determined by a commercial enzyme - linked immunosorbent assay (elisa) kit (lsi medience corp., we evaluated the total urinary excretions of utp, uglu, and 2-mg in the 24-h urine samples. in addition, each rat s right kidney was removed, and a part of the kidney was fixed in 10% neutral buffered formalin. paraffin sections cut at 5 m were stained with hematoxylin and eosin and subjected to histopathological examinations. the left kidney and the left femur were weighed and stored in a freezer until analyses. the concentrations of uranium in the left kidney, left femur and urine were measured by the method described above. based on the concentrations of uranium in the left kidney, left femur and urine, the uranium amount per left kidney or left femur and 24-h urinary uranium excretion were calculated and used for the evaluation. these animals were administered dw intramuscularly into the right femoral muscle, and 20 min after administration of the dw, the animals were given a single oral administration of dw. the rats 24-h urine was collected and used for determination of the normal level of urinary 2-mg excretion. one day after the dw administration, the animals were euthanized under ketamine / xylazine anesthesia, blood samples were obtained, and the separated plasma was used for plasma biochemical analyses. the statistical analysis was performed using dunnett s test after an analysis of variance (anova). all animal experiments were carried out with permission and under regulation of the institutional committee for animal safety and welfare at the national institute of radiological sciences. the uranium was dissolved in distilled water, and the administration volume was 1 ml / kg body weight. the sb was dissolved in distilled water, and the administration volume was 10 ml / kg body weight. we used 8-wk - old male crl : cd (sd) rats (n=24 ; charles river laboratories japan, kanagawa, japan). all animals were administered 1 mg / kg of uranyl nitrate intramuscularly into the right femoral muscle at 10:40 a.m. twenty minutes after the uranium injection, the animals in the four groups were given a single oral administration of sb at the dose levels of 0, 0.1, 0.3 and 1 g / kg, respectively. the dosages used were based on the report by hattori. for the uranium control group, distilled water (dw) only was administered in the same manner as that used for sb. the urine of each animal was collected just before and at 1, 2, 4, 9 and 23 h after the uranium injection. each animal was placed in a plastic animal cage, and the naturally excreted urine was collected. the ph of the urine was measured with a ph meter (model d-51, horiba, kyoto, japan). at 24 h after the uranium injection, the animals were euthanized under ketamine / xylazine anesthesia, the left kidney was removed, and the concentration of uranium in the kidney was measured with an inductively coupled plasma - mass spectrometer (icp - ms, sii spq9700-ii, sii nanotechnology, chiba, japan) after separating the uranium from matrix components using a closed - vessel microwave digestion system (discover sp - d, cem corp., matthews, nc, usa). based on the concentration of uranium in the kidney, the uranium amount per left kidney was calculated and used for the evaluation. for this experiment, we used 8-wk - old male crj : cd rats (n=60 ; charles river laboratories japan inc., kanagawa, japan). all animals were administered 1 mg / kg of uranyl nitrate intramuscularly into the right femoral muscle at 10:40 a.m. at 20 min after the uranium injection, the animals in the three groups were given a single oral administration of sb at the dose levels of 0 (uranium control), 0.1 and 1 g / kg, respectively. six animals in each group were autopsied at 1, 3 and 7 days after the uranium injection, respectively. two dose groups of sb were selected based on the results of experiment i. the high dose was expected to show significant urine alkalinization, and the low dose was expected to show no significant urine alkalinization. before autopsy, 24-h urine collection was performed using metabolic cages. from this 24-h urine, 05-h the urinary volume was measured, and the urine samples were subjected to a uranium amount analysis and urinary biochemistry. the animals were euthanized under ketamine / xylazine anesthesia, and blood samples obtained from each animal were used for plasma biochemical analyses. plasma urea nitrogen (un), plasma creatinine (cre), urinary total protein (utp) and urinary glucose (uglu) were analyzed using an automatic biochemistry analyzer (ca 400, furuno, nishinomiya, japan). beta-2-microblobulin (2-mg) in urine was determined by a commercial enzyme - linked immunosorbent assay (elisa) kit (lsi medience corp., we evaluated the total urinary excretions of utp, uglu, and 2-mg in the 24-h urine samples. in addition, each rat s right kidney was removed, and a part of the kidney was fixed in 10% neutral buffered formalin. paraffin sections cut at 5 m were stained with hematoxylin and eosin and subjected to histopathological examinations. the left kidney and the left femur were weighed and stored in a freezer until analyses. the concentrations of uranium in the left kidney, left femur and urine were measured by the method described above. based on the concentrations of uranium in the left kidney, left femur and urine, the uranium amount per left kidney or left femur and 24-h urinary uranium excretion were calculated and used for the evaluation. these animals were administered dw intramuscularly into the right femoral muscle, and 20 min after administration of the dw, the animals were given a single oral administration of dw. the rats 24-h urine was collected and used for determination of the normal level of urinary 2-mg excretion. one day after the dw administration, the animals were euthanized under ketamine / xylazine anesthesia, blood samples were obtained, and the separated plasma was used for plasma biochemical analyses. the statistical analysis was performed using dunnett s test after an analysis of variance (anova). all animal experiments were carried out with permission and under regulation of the institutional committee for animal safety and welfare at the national institute of radiological sciences. in experiment i, the urinary ph of the uranium - treated rats fell immediately after the uranium injection and returned to near neutral more than 4 h post injection (fig. changes in urinary ph of rats treated with uranyl nitrate alone or in combination with sodium bicarbonate (sb). p<0.05 vs. uranium control group at each examination time point (dunnett s test) ; p<0.01 vs. uranium control group at each examination time point (dunnett s test).). sodium bicarbonate significantly suppressed the decrease in urinary ph of the uranium - treated rats at a dosage of 0.3 g / kg or more. the highest dose of sb raised the urinary ph from 2 h until more than 4 h after the treatment, and the urine alkalinization effect was maintained until 9 h after treatment. the ph of the urine of the middle - dose group was significantly higher than that of the uranium control group from 2 until 4 h after treatment. although the lowest dose of sb did not alkalinize the urinary ph, the acidity of the urea in the uranium - treated rats was significantly improved in this group at 4 h after treatment. the sb treatment dose - dependently reduced the uranium amounts in the kidney (fig. uranium amount of the left kidney of rats treated with uranyl nitrate alone or in combination with sb. p<0.01 vs. uranium control group (dunnett s test).). changes in urinary ph of rats treated with uranyl nitrate alone or in combination with sodium bicarbonate (sb). p<0.05 vs. uranium control group at each examination time point (dunnett s test) ; p<0.01 vs. uranium control group at each examination time point (dunnett s test). uranium amount of the left kidney of rats treated with uranyl nitrate alone or in combination with sb. p<0.01 vs. uranium control group (dunnett s test). in experiment ii, one animal in the uranium control group died on day 7 due to acute renal failure induced by uranium. the urine volume increased in the uranium control group at 3 and 7 days after uranium treatment. however, the urine volume of the sb low - dose group increased in a manner similar to that in the uranium control group (fig. 3fig. changes in the urine volume of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point ; p<0.01 vs. uranium control group at each examination time point. # p<0.05 vs. uranium control group before administration (dunnett s test) ; # # p<0.01 vs. uranium control group before administration (dunnett s test).). a significant increase in urinary uranium excretion was noted in the first 24-h urine of the sb high - dose group (fig. urinary uranium excretions in 24-h urine of rats treated with uranyl nitrate alone or in combination with sb. the bar with a grid pattern represents the collected urine during 05 h post administration of uranyl nitrate. p<0.01 vs. uranium control group at each examination time point (dunnett s test).). this increase was due to the increase in the uranium excretion in 05-h urine of the sb high - dose group, and the uranium excretion in the 524-h urine of the sb high - dose group was similar to that of the uranium control group (fig. 4). the urinary uranium excretion at 3 or 7 days after the uranium treatment decreased in the sb high - dose group (fig. the uranium amounts in the kidney and in the femur were significantly decreased in this group on day 1 and remained at a low level throughout the experiment period. the uranium amounts in the kidney and in the femur of the sb low - dose group were lower than those in the uranium control group on day 1. however, the uranium amounts in the kidney and in the femur were similar to those in the uranium control group on days 3 and 7 (fig. 5fig. uranium amounts in the left kidney (a) and left femur (b) of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point (dunnett s test) ; p<0.01 vs. uranium control group at each examination time point (dunnett s test).). changes in the urine volume of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point ; p<0.01 vs. uranium control group at each examination time point. # p<0.05 vs. uranium control group before administration (dunnett s test) ; # # p<0.01 vs. uranium control group before administration (dunnett s test). urinary uranium excretions in 24-h urine of rats treated with uranyl nitrate alone or in combination with sb. the bar with a grid pattern represents the collected urine during 05 h post administration of uranyl nitrate. p<0.01 vs. uranium control group at each examination time point (dunnett s test). uranium amounts in the left kidney (a) and left femur (b) of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point (dunnett s test) ; p<0.01 vs. uranium control group at each examination time point (dunnett s test). the clinical chemistry results showed that the high dose of sb had remarkable protective effects against uranium - induced acute renal toxicity. plasma un and cre were increased in the uranium control group after day 3, whereas the levels of these clinical markers in the sb high - dose group remained almost normal (fig. blood biochemical analyses. concentrations of urea nitrogen (a) and creatinine (b) in the plasma of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point ; p<0.01 vs. uranium control group at each examination time point. # # p<0.01 vs. normal control group (dunnett s test).). the levels of utp, uglu and 2-mg, which indicate uranium - induced renal tubular damage, were increased in the uranium control group on day 3, and then began to recover (fig. total urinary excretions of total protein (a), glucose (b) and 2-microglobulin (c) in 24-h urine of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point ; p<0.01 vs. uranium control group at each examination time point. # p<0.05 vs. uranium control group before administration (a, b) or normal control (c) (dunnett s test) ; # # p<0.01 vs. uranium control group before administration (a, b) or normal control (c) (dunnett s test).). these markers were significantly lower in the sb high - dose group than in the uranium control group. the low - dose sb group did not show marked improvement of these renal markers ; only a mild but significant suppression of 2-mg on day 3 was noted. blood biochemical analyses. concentrations of urea nitrogen (a) and creatinine (b) in the plasma of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point ; p<0.01 vs. uranium control group at each examination time point. # # p<0.01 vs. normal control group (dunnett s test). urinary biochemical analyses. total urinary excretions of total protein (a), glucose (b) and 2-microglobulin (c) in 24-h urine of rats treated with uranyl nitrate alone or in combination with sb. p<0.05 vs. uranium control group at each examination time point ; p<0.01 vs. uranium control group at each examination time point. # p<0.05 vs. uranium control group before administration (a, b) or normal control (c) (dunnett s test) ; # # p<0.01 vs. uranium control group before administration (a, b) or normal control (c) (dunnett s test)., mild degeneration and single - cell necrosis of the tubular epithelium in the outer stripe of the outer medulla were sporadically seen in the uranium control group (fig. light micrographs of the kidneys from the uranium control group (a c) and the group that received uranium combined with high - dose sb (d f). the outer stripe of the outer medulla of the kidney from rats treated with uranyl nitrate on day 1 (a, d), day 3 (b, e) and day 7 (c, f).). on day 3, severe tubular necrosis was observed mainly in the outer stripe of the outer medulla in the uranium control group (fig. 8b). in these lesions, in addition to the necrosis and/or degeneration of proximal tubular epithelial cells (which were usually detached from the basement membrane), basophilic epithelial cells were also observed in the affected tubules. on day 7, although marked regeneration of damaged renal tubules was observed, tubular dilatation with casts consisting of cellular debris was observed in the uranium control group (fig. the cellular casts were commonly seen in the outer stripe of the outer medulla, and proteinaceous casts and mild congestion were observed in the inner stripe of the outer medulla of the rats in the uranium control group (fig. light micrographs of the kidneys from the uranium control group (a c) and the group that received uranium combined with high - dose sb (d f). the outer stripe of the outer medulla of the kidney from rats treated with uranyl nitrate on day 1 (a, d), day 3 (b, e) and day 7 (c, f). glomerular abnormalities were not seen in the uranium control group. in the sb high - dose group, 8d) on day 1, and only mild degeneration and necrosis of the tubular epithelium were observed (fig. mild and focally regenerated tubules were seen as basophilic tubules in the sb high - dose group (fig. the renal lesions in the sb low - dose group observed on days 1, 3 and 7 were comparable to those in the uranium control group at each time point (data not shown). the results of the present study demonstrated that sodium bicarbonate had a decorporating effect for uranium contamination at the dosage showing urine alkalinization. from the results of experiment i, we found that oral treatment with sodium bicarbonate decreased renal uranium deposition at the dosage showing urinary alkalinization, and we suspect that the degree of the reduction of renal uranium deposition was related to the dose level of sodium bicarbonate. the results of experiment ii clearly demonstrated that sodium bicarbonate protected the rats against uranium - induced renal toxicity at the dosage showing urinary alkalinization. in light of the results of experiments i and ii, we speculate that 0.1 g / kg of sodium bicarbonate which slightly improved the urinary ph of uranium - contaminated rats but did not alkalinize their urine ph might have a weak or limited decorporating effect, resulting in the apparent renal - protection effects not being seen in the rats administered 0.1 g / kg of sodium bicarbonate. in contrast, the urine alkalinization caused by 1 g / kg of sodium bicarbonate immediately after the uranium challenge enhanced urinary uranium excretion and showed subsequent renal protective effects. these results indicate that urinary alkalinization immediately after uranium ingestion is important for uranium decorporation. uranyl tricarbonate is a dominant species at about ph 8.0 or more, and it is considered to be stable. sodium bicarbonate may increase the uranyl tricarbonate levels in blood and urine by increasing the blood level of bicarbonate ions, and the increased stable uranyl ion complex may lead to a decrease in both the interaction between uranyl ion and renal tubular cells and the deposition of uranium in the tubular epithelial cells of the kidney. ethane-1-hydroxy-1,1-bisphosphonate (ehbp), a bisphosphonate used for the treatment of paget s disease and the prevention of osteoporosis, has been reported to chelate uranium and show a uranium decorporation effect in rats, and it is listed as one of the possible agents for uranium decontamination therapy in humans. in a comparison of the degree of effectiveness of sodium bicarbonate and that of ehbp, the effectiveness of sodium bicarbonate was thought to be larger than that of the decorporating effect of ehbp in uranium - contaminated animals. in that report, ehbp was administered 5 or 30 min after an intramuscular injection of uranyl nitrate in rats, and the deposition in the kidney was decreased on the first day by a factor of approx. 5 or 2, respectively. in our experiment, the uranium deposition in the kidney was decreased by a factor of approx. 5 with 1 g / kg of sodium bicarbonate treatment 30 min after the uranyl nitrate treatment. in conclusion, our present findings clearly demonstrate that the urine alkalinization agent sodium bicarbonate had a significant decorporation effect in the uranium - contaminated rat model. regarding optimization of the decontamination treatment, further studies using sodium bicarbonate in rats could be conducted to examine parameters such as a delay between exposure and treatment of 30 min or more. in addition, urine alkalinization medicine may be useful as a decorporation agent for uranium - decontamination therapy. | to evaluate the effectiveness of sodium bicarbonate (sb) in removing uranium and protecting animals from uranium toxicity, we intramuscularly administered 1 mg / kg of uranyl nitrate to 8-wk - old male sd rats, and 20 min after administration of uranyl nitrate, the animals were given a single oral administration of sb at 0.1, 0.3 or 1 g / kg. the sb treatment at a dose of 0.3 g / kg or more raised the ph of the rats urine until 4 h after treatment, and it significantly reduced the uranium amounts in the kidneys at 1 day after treatment. in another experiment, rats were intramuscularly administered 1 mg / kg of uranyl nitrate, and 20 min later, the animals were treated with sodium bicarbonate (0.1 or 1 g / kg). the rats were autopsied at 1, 3 and 7 days after uranium treatment. high - dose sb resulted in a significant increase in urinary uranium excretion in the first 24 h and a reduction of uranium deposition in the kidneys and femurs, and it also significantly suppressed uranium - induced renal toxicity, as shown by both histopathology and clinical chemistry at 3 days after uranium treatment. low - dose sb did not show such marked effects. our findings demonstrated that the uranium decorporation effect of sodium bicarbonate was observed at the dosage showing urine alkalinization in rats and that decorporation effect of sodium bicarbonate might be beneficial if it is administered immediately after incorporation of soluble uranium. |
strain cbs10496 was isolated from a 31-year - old aids patient from montreal, quebec, canada, who had traveled to mexico 15 months before cryptococcosis was diagnosed. the patient died despite extensive antifungal treatment with ketoconazole and amphotericin b (11). cbs10496 has been identified as c. gattii serotype b (cited as c. neoformans var. aflp, amplified fragment length polymorphism ; cbs, fungal biodiversity centre ; amc, netherlands reference laboratory for bacterial meningitis, academic medical center, amsterdam, the netherlands ; na, not applicable ; csf, cerebrospinal fluid. origin and genetic composition of the strains are indicated. aflp fingerprint genotype (7), followed by corresponding m13 pcr fingerprint genotype (8). the ploidy of cbs10496 was determined by using flow cytometry (10) with the sequenced haploid strains cbs8710 and cbs10510 as references. coloration of colonies grown on canavanine - glycine - bromthymol blue (cgb) medium (12) was determined after incubation at 24c for 6 and 15 days. the serotype of cbs10496 was determined by using the cryptocheck serotyping kit (iatron laboratories, tokyo, japan). dna of these colonies was used for amplified fragment length polymorphism (aflp) analysis (7). the partial sequence of 6 nuclear regions was determined for reference isolates cbs10488cbs10490, cbs1622, cbs6992, and the putative hybrid isolate cbs10496. selected nuclear regions were those for internal transcribed spacer (its) region, intergenic spacer region, laccase (cnlac1), 2 rna polymerase ii subunits (rpb1 and rpb2), and translation elongation factor 1 (tef1) (9,10). the g1 peak of reference strains was located at positions 31.6 (cbs8710) and 31.1 (cbs10510), and the g2 peak was located at positions 65.8 (cbs8710) and 56.4 (cbs10510). the g1 peak of cbs10496 was located at position 57.5, and the g2 peak was located at position 115.7. thus, the g1 peak of cbs10496 coincided with the g2 peak of the haploid strains (figure 1, panel a), which indicates that cbs10496 has 2 more dna than haploid strains. staining with 4,6-diamidino-2-phenylindole showed that cells of cbs10496 were monokaryotic (figure 1, panel b). a) determination of ploidy of the novel cryptococcus neoformans c. gattii serotype ab hybrid isolate cbs10496 by flow cytometry. the first peak corresponds to the g1 phase ; the second peak corresponds to the g2 phase. haploid reference strain cbs10510 is shown by the red line ; cbs10496 is shown by the black line. the g1 peak of cbs10496 coincided with the g2 peak of strain cbs10510, which indicated that strain cbs10496 has approximately twice the amount of dna than cbs10510. b) nuclear staining of isolate cbs10496 with 4,6-diamidino-2-phenylindole, showing that cells are monokaryotic. reaction of cbs10496 on cgb medium was negative, which corresponds to c. neoformans (12). the cryptocheck serotyping kit serum factors 5 (corresponding to serotype b) and 7 (corresponding to serotype a) agglutinated, which indicated that cbs10496 is a serotype ab strain. the aflp fingerprint obtained by analysis of colonies of cbs10496 did not match any of the previously defined aflp genotypes. the fingerprint of cbs10496 was compared with aflp fingerprints of reference strains cbs8710 and cbs9172, which are aflp1/vni, and e566 and cbs10510, which are aflp4/vgi. the aflp fingerprint of cbs10496 contained fragments characteristic of aflp1/vni and aflp4/vgi (figure 2), which indicated that genetic material from these 2 genotypes was present in this isolate. amplified fragment length polymorphism (aflp) fingerprint of 3 colonies of the novel cryptococcus neoformans c. gattii hybrid serotype ab isolate cbs10496 and 4 reference strains. cbs9172 and cbs8710 are c. neoformans var. grubii (aflp1/vni) strains ; e566 and cbs10510 are c. gattii (aflp4/vgi) strains. two alleles representing aflp1/vni and aflp4/vgi were found when fragments of rpb1, rpb2, cnlac1, and intergenic spacer region of cbs10496 were cloned and sequenced. however, after 30 clones were sequenced, only 1 allele was obtained for tef1, i.e., aflp4/vgi, and its, i.e., aflp1/vni. our results indicate that genetic material from aflp1/vni and aflp4/vgi was present in cbs10496, although only 1 allele was obtained for tef1 and its. specific primer pair resulted in an amplicon. when mata and the mata serotype a in addition, cbs10510, a mat serotype b strain, was amplified with the mat specific primer pair, and e566, a mata serotype b strain, yielded an amplicon with the mata - specific pcr. these results indicate that a c. gattii and a mat serotype a background are present in cbs10496. because the mating type of the c. gattii background within cbs10496 was unknown, 30 mat clones of cbs10496 were sequenced to determine whether a mat serotype b allele could be identified. however, all clones were mat serotype a ; no mat serotype b clones were found. our results indicated that cbs10496 is a monokaryotic, diploid, or aneuploid strain with the novel ab serotype. grubii (serotype a, aflp1/vni) and c. gattii (serotype b, aflp4/vgi). cbs10496 had been identified as c. gattii on the basis of a weak positive reaction on cgb medium (11). although a negative response on cgb medium has been shown for other c. neoformans c. gattii hybrids (10,14), weak and delayed positive reactions on cgb medium may occur in c. neoformans c. gattii hybrid isolates (10,14). inconsistent serotyping results have been reported for other hybrids (10,15) and may result from differences in specificity and potency among different batches of factor serum. all c. neoformans c. gattii hybrids discovered have originated from clinical sources (13 ; f. hagen and t. boekhout, unpub. although an amplicon was obtained with c. gattii specific mating - type primers, the c. gattii background could not be linked to a mating type. we hypothesize that the serotype ab c. neoformans c. gattii hybrid cbs10496 was formed by mating of a mata serotype b strain with a mat serotype a strain and subsequent loss of the mata serotype b allele. detection of single its and tef1 alleles in cbs10496 further supports our findings because it indicates that other alleles were also lost. loss of genetic material has been observed in other hybrids, such as serotype ad and bd hybrids (14), and seems to be a normal process in cryptococcal hybrids. our results show that the c. gattii parent of the serotype ab hybrid belongs to the aflp4/vgi genotype, as was the case for serotype bd hybrids (10). the c. gattii parental sequence of all known serotype bd c. neoformans c. gattii hybrid isolates was identical to sequences of aflp4/vgi strains cbs1622 and cbs6992 in all regions studied (9). aflp4/vgi subgroup in all isolated c. neoformans c. gattii hybrids may indicate that this subgroup preferentially forms interspecies hybrids. | interspecies hybrids of cryptococcus neoformans and c. gattii have only recently been reported. we describe a novel c. neoformans c. gattii hybrid strain (serotype ab) that was previously described as c. gattii and that caused a lethal infection in an aids patient from canada. |
endometrial and placentomal tissues were collected at various stages during the estrous cycle and gestation from japanese black and holstein cows in a local abattoir and the national institute of agrobiological sciences (nias), japan. the stage of the estrous cycle was estimated from the gross appearance of the ovaries and uterus, and the gestation date was estimated from the crown - rump length. the stages of the cycle were designated as the early luteal (stage i ; n=3), middle luteal (stage ii ; n=3), middle - to - late - luteal (stage iii ; n=3) and follicular (stage iv ; n=3) phases. in pregnant cows, the stages of gestation were designated as the peri - implantation period (stage pi ; n=3), early gestation (stage eg ; days 50 to 90, n=3), mid - gestation (stage mg ; days 100 to 180, n=3) and late gestation (stage lg ; days 210 to 270, n=3). the stage pi samples were collected from a designated pregnant cow that had been subjected to artificial insemination at the nias. the day of insemination was designated as day 0 of gestation, and the pi samples were collected on days 19, 20 and 25 of gestation. endometrial and placentomal tissues were collected and fixed in 10% formaldehyde (ph 7.4). the postfixed tissues were dehydrated in alcohol and xylene and embedded in paraffin wax. the embedded samples were cut into 5 m sections with a rotary microtome and placed on slides (matsunami, kishiwada, japan) overnight at 40 c. all animal care and experimental procedures were carried out in accordance with the guidelines of the animal care and use committee of the iwate university and nias. anti - cd3 (mm1a, diluted 1:100 ; vmrd, pullman, wa, usa) monoclonal antibody, which reacts specifically with an antigen molecule on the surface of the bovine tcr / cd3 complex, was used to detect t cells. tissue sections were permeabilized for 20 min with 0.3% hydrogen peroxide in methanol and washed three times in phosphate - buffered saline (pbs, ph 7.4). they were then incubated with pbs containing 1.5% normal donkey serum and 0.1% triton x-100 for 30 min and incubated with the primary antibody in 0.1% triton x-100/pbs overnight at 4 c ; 0.1% triton x-100/pbs was used for the controls. after the tissue samples were washed 6 times in pbs, they were incubated with histofine simple stain maxpo (m) (nichirei, tokyo, japan) as a secondary antibody for 30 min at room temperature. subsequently, they were washed 3 times in pbs and incubated in 3,3-diaminobenzidine tetrahydrochloride (0.2 mg / ml, ph 7.5) for 5 min at room temperature. the slides were photographed using an eclipse e600 microscope with an attached digital sight camera (nikon, tokyo, japan). the number of cells in the digital images (200-fold magnification) was counted using the imagej software. the numbers of cd3 cells from the digital images were expressed per 0.1 m tissue area. the cells were counted in at least three different intercaruncular areas in each slide, and the numbers of cd3 cells per 0.1 m were averaged. all the scores are presented as mean standard error (se) values. data were analyzed using the jmp software (sas institute, cary, nc, usa) with one - way anova followed by a tukey - kramer multiple comparison test. endometrial and placentomal tissues were collected at various stages during the estrous cycle and gestation from japanese black and holstein cows in a local abattoir and the national institute of agrobiological sciences (nias), japan. the stage of the estrous cycle was estimated from the gross appearance of the ovaries and uterus, and the gestation date was estimated from the crown - rump length. the stages of the cycle were designated as the early luteal (stage i ; n=3), middle luteal (stage ii ; n=3), middle - to - late - luteal (stage iii ; n=3) and follicular (stage iv ; n=3) phases. in pregnant cows, the stages of gestation were designated as the peri - implantation period (stage pi ; n=3), early gestation (stage eg ; days 50 to 90, n=3), mid - gestation (stage mg ; days 100 to 180, n=3) and late gestation (stage lg ; days 210 to 270, n=3). the stage pi samples were collected from a designated pregnant cow that had been subjected to artificial insemination at the nias. the day of insemination was designated as day 0 of gestation, and the pi samples were collected on days 19, 20 and 25 of gestation. endometrial and placentomal tissues were collected and fixed in 10% formaldehyde (ph 7.4). the postfixed tissues were dehydrated in alcohol and xylene and embedded in paraffin wax. the embedded samples were cut into 5 m sections with a rotary microtome and placed on slides (matsunami, kishiwada, japan) overnight at 40 c. all animal care and experimental procedures were carried out in accordance with the guidelines of the animal care and use committee of the iwate university and nias. anti - cd3 (mm1a, diluted 1:100 ; vmrd, pullman, wa, usa) monoclonal antibody, which reacts specifically with an antigen molecule on the surface of the bovine tcr / cd3 complex, was used to detect t cells. tissue sections were permeabilized for 20 min with 0.3% hydrogen peroxide in methanol and washed three times in phosphate - buffered saline (pbs, ph 7.4). they were then incubated with pbs containing 1.5% normal donkey serum and 0.1% triton x-100 for 30 min and incubated with the primary antibody in 0.1% triton x-100/pbs overnight at 4 c ; 0.1% triton x-100/pbs was used for the controls. after the tissue samples were washed 6 times in pbs, they were incubated with histofine simple stain maxpo (m) (nichirei, tokyo, japan) as a secondary antibody for 30 min at room temperature. subsequently, they were washed 3 times in pbs and incubated in 3,3-diaminobenzidine tetrahydrochloride (0.2 mg / ml, ph 7.5) for 5 min at room temperature. the slides were photographed using an eclipse e600 microscope with an attached digital sight camera (nikon, tokyo, japan). the number of cells in the digital images (200-fold magnification) was counted using the imagej software. the numbers of cd3 cells from the digital images were expressed per 0.1 m tissue area. the cells were counted in at least three different intercaruncular areas in each slide, and the numbers of cd3 cells per 0.1 m were averaged. all the scores are presented as mean standard error (se) values. data were analyzed using the jmp software (sas institute, cary, nc, usa) with one - way anova followed by a tukey - kramer multiple comparison test. 1. localization of cd3 cells in the endometrium during the estrous cycle and gestation. immunohistochemistry was used to detect cd3 cells in the endometrium during the estrous cycle (a d and a) and gestation (e h). (a) stage iv, (b) stage i, (c) stage ii, (d) stage iii, (a) stage iv in an enlarged portion of the frame shown in a, (e) peri - implantation period (pi), (f) early gestation (eg), (g) mid - gestation (mg), and (h) late gestation (lg). le, luminal epithelium ; c, conceptus.), and significant numbers of these cells were observed throughout the estrous cycle in the stratum compactum, that is, the sublayer of the stratum functionalis facing the interior of the uterus (fig. the endometrial distribution was similar to that seen during the estrous cycle, but the number of cells decreased with the progression of gestation. furthermore, in the placentome, specifically in the tissues near the fetus, including the trophoblastic area, no t cells were found. in addition, a certain number of positive cells were detected in the lower part of the caruncle close to the area with the highest density of blood vessels, which was near the myometrium (fig. immunohistochemistry was used to detect cd3 cells in the placentome during mid - gestation. the top and bottom of (a) show the fetal side and maternal side, respectively. (b)(d) these are 200-fold magnified images of the frames shown in (a). immunohistochemistry was used to detect cd3 cells in the endometrium during the estrous cycle (a d and a) and gestation (e h). (a) stage iv, (b) stage i, (c) stage ii, (d) stage iii, (a) stage iv in an enlarged portion of the frame shown in a, (e) peri - implantation period (pi), (f) early gestation (eg), (g) mid - gestation (mg), and (h) late gestation (lg). immunohistochemistry was used to detect cd3 cells in the placentome during mid - gestation. the top and bottom of (a) show the fetal side and maternal side, respectively. (b)(d) these are 200-fold magnified images of the frames shown in (a). scale bars=50 m. the notable changes in the number of cd3 cells in the intercaruncular endometrium during the estrous cycle and gestation are shown in fig. number of cd3 cells in the intercaruncular endometrium during the estrous cycle and gestation. the values indicate the number of cd3 cells per 0.1 m area and are shown as mean se values. pi, peri - implantation period ; eg, early gestation ; mg, mid - gestation ; lg, late gestation.. the number of cd3 cells was higher during stages i iii of the estrous cycle than during stage iv, but the differences were not statistically significant. a similar number of cells was detected until early gestation. however, during mid to late gestation, the numbers of cd3 cells were significantly lower than those seen during stages i iii, and this reduction in the number was seen until late gestation. number of cd3 cells in the intercaruncular endometrium during the estrous cycle and gestation. the values indicate the number of cd3 cells per 0.1 m area and are shown as mean se values. pi, peri - implantation period ; eg, early gestation ; mg, mid - gestation ; lg, late gestation. dynamic changes in t - cell distribution were detected immunohistochemically in the bovine endometrium during the estrous cycle and gestation. this finding suggests a new aspect of the t cell role : a regulatory function for endometrial coordination with embryonic information, which is limited around the time of implantation in cows. in the present study, we examined the endometrial t - cell distribution throughout the reproductive cycle, namely, during the different stages of the estrous cycle and gestation ; this aspect has not been studied previously. an additional refinement was the use of a t cell - specific marker antibody rather than a pan - leukocyte marker antibody or morphological features, as used in previous studies [9, 13,14,15 ]. immunological endometrial and immune cell functions are crucial factors in the establishment of implantation and placentation, and therefore, various studies have been carried out in humans and rodents ; less work has been done on t cells in cows throughout the estrous cycle and gestation. in previous studies, bovine endometrial lymphocytes were detected, although their numbers decreased from early to mid gestation, and no lymphocytes were detected in the caruncular endometrium [9, 14 ]. generally, the detection of cd3 t cells in the current study was in agreement with previous reports : cell numbers decreased as gestation progressed, and no t cells were found in the caruncle. however, specifically in cows, the placentome develops in the caruncular area, and no t cells were found there, while a small number of t cells were recognized in the intercaruncular area, and this number decreased with the progress of gestation. considering this and the endometrial expression of t cells during the estrous cycle, the localized disappearance of t cells may be necessary for the maintenance of gestation in bovines. 1, t cells were equally numerous throughout the endometrium, with no difference between the caruncular and intercaruncular epithelium in this regard. during the estrous cycle, however, there were some discrepancies between our findings and those of previous studies. although the current study showed that the number of t cells was higher in stages i and ii than in stage iv, cobb and watson, using anti - cd5 antibody immunohistochemistry, reported that the number of t cells increased during stage iv. cd5 may be expressed in t cells, but it is also expressed by b cells and endothelial cells in the endometrium [13, 22 ], whereas cd3 antibody recognizes a specific t - cell marker. therefore, this discrepancy may depend on the specificity of the antibodies used ; the cd3 antibody may be more specific. cd3-positive cells may be excellent markers for investigating the immune tolerance of the endometrium. when endometrial aberration occurs, cd3 cells appear and may therefore be related to preventing placental disorder. changes in the endometrial t - cell distribution are believed to be regulated by adhesion molecules and humoral factors. in the ovine endometrium during early gestation, vascular cell adhesion molecule 1 (vcam-1) is expressed, and differences are seen between the vcam-1 expression in the caruncular and intercaruncular endometrium. furthermore, it is considered that humoral factors, including hormones, cytokines and chemokines are involved in the infiltration and migration of lymphocytes. there is a close association between endometrial leukocytes and decidualization, and our results indicate that the t - cell distribution in the bovine endometrium differs from that in the human endometrium. these differences are possibly related to the structural variations in the placenta, specifically the epitheliochorial and hemochorial placenta. in the former, the placental barrier includes an intact uterine epithelium, but the latter is the most invasive form of placentation, as there is loss of all the maternal tissue including the endothelium of the maternal blood vessels at the site of placentation, and maternal blood irrigates the fetal membrane directly. therefore, although endometrial t cells play a coordinating role at the feto - maternal interface, different functions may be involved in cattle and humans through changes in t - cell distribution and humoral regulation. although little is known about the in vivo functions of endometrial t cells, the appearance of t cells may be related to feto - maternal interactions. in somatic cloned cows, t - cell expression seems to indicate placental function abnormalities involving major histocompatibility antigen complex (mhc) class i expression, and the number of t cells has been reported to be significantly higher than that in control. t cells contribute to the placental functions in many species because the emergence of t cells is closely related to abortion and placental retention. furthermore, maternal lymphocytes promote tissue remodeling in stimulating endometrial angiogenesis. immunological regulation during gestation is still a disputed issue, but t cells play a crucial role in coordination between the mother and fetus, especially during the implantation period when endometrial tissue remodeling through lymphocyte functions is necessary for fecundity. further research is required to investigate endometrial t - cell subsets and to determine the role of each subset in the bovine endometrium. in conclusion, the dynamics of the bovine endometrial t - cell distribution were determined throughout the estrous cycle and gestation. the number of t cells was higher in the early - mid luteal phase, but after implantation, t cell numbers decreased as gestation progressed. no t cells were found in the placentome. the t - cell distribution might contribute to implantation and endometrial tissue remodeling. thus, bovine endometrial t cells might be closely related to the success and maintenance of bovine gestation in a spatiotemporal manner. | t cells are the dominant lymphocytes in the endometrium and are considered to play a crucial role in implantation and in the maintenance of gestation through cytokine production and immune regulation. the mechanisms underlying immunoregulation at the feto - maternal interface are still obscure for this complex system. understanding the role of t cells is a key factor in understanding the endometrial immune system. in this study, the distribution of endometrial cd3 + t cells in bovines was examined by immunohistochemical analysis. the estrous cycle and gestation was divided into 4 stages, and the number of cd3 + -positive t cells was counted in each stage. cd3 + cells were found in the endometrium in significant numbers throughout the estrous cycle and were mostly located in the subepithelial area. the number of cd3 + cells significantly increased in the early and mid - luteal phases but decreased after implantation with the progression of gestation. no t cells were found in the placentome or specifically in the tissues near the fetus, including the trophoblastic area. in addition, very few t cells were found in stromal regions close to the myometrium of the endometrium. these findings suggest that downregulation of bovine endometrial cd3 + t - cell functions is closely related to the successful maintenance of gestation in a spatiotemporal manner. |
the proprotein convertases (pcs) are members of a mammalian family of endoproteases related to the bacterial subtilisin and the yeast kexin. there are seven pcs that cleave proteins at paired basic amino acid residues, namely furin, pc2, pc1/3, pc4, pace4, pc5/6, and pc7. the optimal pc recognition sequence is r - x - k / r - r, while the minimal consensus sequence is r - x - x - r. a variety of substrates have been described including precursors of hormones, enzymes, growth factors, receptors, cell membrane proteins, and plasma proteins but also a number of pathogenic proteins such as viral glycoproteins and bacterial toxins. our previous work showed that pace4 has a role in prostate cancer cellular proliferation. pace4 has a wide expression pattern and is constitutively secreted into the extracellular media. it has been suggested from immunohistochemical observations that in addition to its localization within the secretory pathway, pace4 is also localized at the cell surface through interactions between its cysteine - rich domain (crd) and heparan sulfate proteoglycan (hspg) or tissue inhibitors of metalloproteinases (timps). recently, two independent studies (including one from our group) showed a specific overexpression of pace4 mrna in prostate cancer tissues. this overexpression is correlated with higher circulating protein levels in some patients. using a molecular inhibition approach, the relevance of pace4 in a prostate cancer model has been demonstrated. as the expression levels of other pcs remains unchanged, it was suggested that a selective pace4 inhibitor, with limited inhibition toward furin, might provide a useful tool against prostate cancer. to our knowledge, no such inhibitor has been yet reported (for complete review see ref.). designing specific pc inhibitors represent an important challenge. the high homology level deep within the catalytic cleft suggests that small - molecule inhibitors acting as competitive inhibitors will be unlikely to produce any specificity. indeed, structural evidence indicates that the pc active sites are nearly identical in their s1s4 subsites.a however, there are notable differences found at the s5 subsite and beyond. this suggests that peptide - based inhibitors could be designed to achieve the desired specificity, although they would require a minimum of six residues. there is some proof for this concept based on discovered endogenous peptide inhibitors, such as the 7b2 ct - peptide, which is a highly potent (nm range) and specific pc2 inhibitor. of course, each pc also has an endogenous inhibitor within its structure, namely their prodomains, of which the c - terminal region provides the critical positions for inhibition at the catalytic sites. pc prodomains first act in the er as intramolecular chaperones and then as activity delayers through interactions in cis with the active site of their cognate pc. the derived prodomains have been shown to be potent pc inhibitors in trans but display minimal levels of specificity. last, the screening of peptide combinatorial libraries has led to the identification of polyarginine peptides as furin inhibitors, however, these are also not highly specific. the present study reports the development of a new pace4 inhibitor, named the multi - leucine (ml)-peptide. our focus remains primarily on discriminating between our target pace4 and furin, which is the only ubiquitously pc enzyme in normal tissues. since it is known that furin inhibition can be lethal (i.e., as demonstrated in furin knockout mice), it appears logical to design an inhibitor that primarily avoids furin as a target. we also present some of the characteristics of the ml - peptide as a potent inhibitor of proliferation in prostate cancer cell lines. pc prodomains act as cis regulatory inhibitors during the maturation process and have been considered as lead compounds for pc inhibition. our previous work assessed the inhibitory potency of the prodomains as trans competitive inhibitors. however, their inhibitory potency has never been carried out for pace4 until now (figure 1a). we observed that most pc prodomains displayed similar inhibition potencies for pace4 and furin, with ki values ranging from low nanomolar (12.4 nm) to subnanomolar levels (0.34 nm). the inhibition ratios (furin / pace4) were always in the range of 15 for all prodomains, with the exception of the pc7 prodomain, which displayed a 36-fold inhibition preference for pace4 over furin. the ki obtained with the pc7 prodomain on pace4 (0.34 nm) is lower than the ki previously reported for inhibition of pc7 by its own prodomain. (a) pcs prodomains were produced and purified to perform inhibition assays toward pace4 and furin. the ratio between ki for furin and pace4, namely the specificity ratio, point out the selectivity of pc7 prodomain toward pace4. kis in the table are means and standard deviations of three independent experiments. (b) pcs prodomain sequence alignment was performed for the region p7p1 downstream primary cleavage site. dark background indicates conserved residues, while light - gray background indicates residues of same type than consensus. uniprotkb accession numbers are the following : hfurin (p09958), mpc1/3 (p63239), hpc2 (p16519), mpc4 (p2921), hpc5/6 (q92824), hpc7 (q16549), and hpace4 (p29122). to further investigate the selectivity of pc7 prodomain inhibition toward pace4, a sequence alignment was performed, focusing on the c - terminal regions (figure 1b). although prodomains are approximately 100 amino acids in length, our analysis focused on the dissimilarities in the p1p7 region of the primary cleavage site. because those residues are directly implicated in molecular recognition by pcs, we hypothesized that the 36-fold difference might be related to this c - terminal region. the sequence alignment (figure 1b) reveals that the pc7 prodomain differs from the consensus in position p6, as it does not exhibit a basic amino acid at this position. apart from the pc2 prodomain, a poor inhibitor of both furin and pace4, only pc7 is dissimilar from consensus in this position. it is well - known that the presence of a basic residue in position p6 promotes increased inhibition potency for furin. however, the observation of leu residues at p6 and p7 positions of the pc7 prodomain suggests that pace4 may have a preference for hydrophobic residues. the use of combinatorial libraries has proven its relevance for the rational design of inhibitors. this method has also been used for pc recognition pattern studies. an 8-mer positional scanning - synthetic peptide combinatorial libraries (ps - spcl) approach was used to further study differences in furin and pace4 inhibition. because it is hypothesized that specific pc inhibition will be reached by varying the p5p8 positions, this library was synthesized by fixing the p1p4 positions with the core consensus motif rvkr while using the combinatorial method for the p5p8 positions. this pattern ensures that each peptide in the library has the ability to be recognized and bind to the pc s active site. the inhibition profiles for pace4 and furin at the p6 position are shown in (figure 2). as expected, peptides with arg and lys at the p6 position were the most potent pace4 inhibitors (ki = 40 and 37 nm, respectively). it is interesting to note that peptides with hydrophobic leu residue displayed equally strong pace4 inhibitory potency (ki = 49 nm) (figure 2). in contrast, when testing furin inhibition, peptides containing leu in p6 had a much higher inhibition constant (900 nm) whereas peptides with lys and arg at this position maintained mid - nm range inhibition constants (230 and 300 nm, respectively). for this purpose, the specificity ratios were most important and led to the observation that peptides containing a leu residue showed a 18-fold ratio (furin / pace4). the second highest ratio was obtained with peptide containing his residue with a 11-fold specificity ratio. it is relevant to mention that ph differences in the enzymatic assays might influence this result (ph 6.5 for pace4 and ph 7.5 for furin) given that the histidine side - chain pka is 6.08. use of sp - spcl to profile pc - inhibitor recognition. to better understand the recognition patterns of pace4 and furin, sp - spcl was used toward both enzymes. for each sample in this table, the general recognition pattern rvkr is present in positions p1p4 and p6 position are occupied by a unique amino acid. the other positions of those peptides are occupied by an equimolar mixture (x) of the 19 natural amino acids, cysteine excluded. ki was calculated from ic50 using the cheng and prussof equation for competitive inhibitors. thus, this partial combinatorial library approach confirmed that pace4 tolerates the presence of a hydrophobic amino acid in the p6 position whereas furin did not and presents a clear preference for basic amino acids in this position. on the basis of these results, peptides containing leu at the p6 position as a leu containing peptide could offer a selective inhibition toward pace4, the effects of leu n - terminal extensions of the core sequence ac - rvkr - nh2, a poor micromolar inhibitor of pcs, was investigated in vitro (figure 3). the peptide ac - llrvkr - nh2 was a midnanomolar inhibitor of pace4, but the progressive addition of n - terminal leu decreased the inhibition constant to the low nanomolar range (1822 nm) for both ac - lllrvkr - nh2 and ac - llllrvkr - nh2. however, the subsequent addition of leucine residues increased ki values, reaching higher nanomolar values (300 nm) for the decapeptide ac - llllllrvkr - nh2. peptides containing three to four leu residues were the most potent inhibitors of pace4 evaluated in this study and were significantly more effective on pace4 than furin (2022-fold). the peptide ac - llllrvkr - nh2 and now designated as the ml - peptide was chosen as lead inhibitor for further characterization on pace4 inhibition. the inhibitory potency of the ml - peptide was also assayed with other members of the pc family and also showed high levels of specificity (supporting information table s1). multi - leucine peptides. to stabilize pc inhibitor interaction, n - terminal leucine extensions were added to the core rvkr sequence. (a, b) each peptide was assayed with pace4 and furin in an inhibition assay. (c) kis in this table are means and standard deviations of three independent experiments. peptides ac - lllrvkr - nh2 and ac - llllrvkr - nh2 were the most potent and the most selective inhibitors of pace4 of this library. the peptide ac - llllrvkr - nh2, named multi - leu peptide (ml), was selected as lead compound for further studies. in a previous study, pace4 was proposed as a new therapeutic target in prostate cancer based on a molecular inhibition approach using the prostate cancer cell line du145. because the molecular inhibition of pace4 in du145 cells had dramatic effects on cell proliferation in vitro and in vivo, we decided to test the ml - peptide as a pharmacological inhibitor to achieve identical results. in the present study, two additional prostate cancer cell lines thus, pace4 expression level was first evaluated in lncap and pc3 cell lines in comparison to du145 cells using a rt - qpcr approach (figure 4a). pace4 was most highly expressed in lncap cells, with nearly 6-fold higher levels than du145 cells, but was almost absent in pc3 cells. du145 and lncap cells also exhibited higher levels of furin mrna than pc3 cells. similar expression levels were observed for pc5/6 and pc7 within all cell lines investigated and pc1/3 and pc2 were undetectable. the effect of the multi - leu peptide on cellular proliferation of each cell line was evaluated using mtt assays (figure 4b). the ml - peptide showed a very poor inhibition of pc3 cells, whereas the half - maximal inhibitory concentrations (ic50) were in the micromolar range for du145 and lncap cells (100 10 and 180 60 m, respectively). thus, the ml - peptide inhibited the proliferation of du145 and lncap cells, but not pc3 cells, showing a strong correlation with cellular pace4 expression. in previous work, our research team proposed pace4 as a therapeutic target against prostate cancer progression. (a) du145, lncap, and pc3 prostate - cancer derived cell lines were first screened to compare their pcs expression levels using rt - qpcr. (b) to assess the efficiency of our new pace4 inhibitor in such context, mtt assays were performed on those cell lines. ml is efficient to inhibit proliferation and metabolic activity in du145 and lncap, two pace4-expressing cell lines, indicating a possible role for ml as a prostate cancer therapeutic. (c) to prove that the inhibition observed is a pcs dependent mechanism, the peptide ac - llllrvk[darg]-nh2 was used as negative control. because the p1 position is a key residue of the recognition pattern, the replacement of p1 arg by darg significantly affected the ki for this peptide. (d, e) as expected from inhibition constant values, the peptide ac - llllrvk[darg]-nh2 is a poor proliferation inhibitor in a mtt assays with du145 and lncap. an additional control experiment was performed to test the pc - specific interaction of the ml - peptide resulting in cell proliferation inhibition by designing a ml - peptide substituted at the p1 position with a darg. as the p1 arg position is critical for pc recognition, this modification should strongly abrogate the observed effects unless they are not pc - mediated. as expected, the peptide ac - llllrvk-[darg]-nh2 showed a substantial loss of affinity in vitro going from a nm to a m inhibitor (figure 4c) (kis = 1380 and 2600 nm for pace4 and furin, respectively). consistent with this affinity loss, this peptide also showed a significant loss of potency in both du145 (figure 4d) and lncap (figure 4e) cell - based assays (ic50 440 80 and 390 10 m, respectively). these data make a strong case that decreased cell proliferation is mediated by pc inhibition. pace4 is localized both at the cell surface and within the intracellular secretory pathway. thus, it was relevant to determine whether the inhibitory effect of the ml - peptide is mediated by cell surface and/or intracellular pace4. to elucidate this question, ml - peptide analogues bearing n - terminal modifications with strikingly different cell penetration properties was coupled with an n - terminal amino - poly(ethylene glycol)-8 (-amine--propionic acid octaethylene glycol, known as peg8) group that should render the peptide much less hydrophobic, whereas a n - terminal hydrophobic 8-amino - octanoyl (c8) chain was used in order to increase its hydrophobicity. in vitro enzymatic activity assays demonstrated that pegylation and alkylation of the ml - peptide did not alter its potency or selectivity for pace4 inhibition (figure 5a). the uptake of fitc - labeled versions of these ml - peptide analogues were tested on du145 cells and analyzed using a fluorescence - activated cell sorter (facs) (figure 5b). the fitc--ala - ml - peptide had excellent cell penetration properties, considering it is relatively unmodified, suggesting that the hydrophobic content of this peptide is sufficient to penetrate cell membranes (see also confocal microscopy in supporting information figure s1). however, cells treated with fitc-[peg8]ml - peptide showed a geometric mean of the distribution (geomean) of about 1 log unit lower than the one measured for the fitc--ala - ml - peptide (5.437.84 vs 48.9052.82). these results indicate that the fitc-[peg8]-ml - peptide penetrates the cell membrane very poorly. as expected, the fitc - c8-ml - peptide displayed greater cell penetration properties with 5-fold greater geomean compared to the control fitc--ala - ml - peptide (260.08261.95 vs 48.9052.82, respectively). when all three peptides were tested on du145 cells, the [c8]-ml - peptide gave very similar results to the ml - peptide by maintaining antiproliferative effects (ic50s 80100 m) while the [peg8]-ml - peptide could no longer reduce cell proliferation (ic50 was estimated to be > 500 m) (figure 5c, d). this result provides strong evidence that the antiproliferative actions of the ml - peptide were largely mediated by intracellular pace4. a second conclusion is that the cell penetration properties of the unmodified ml - peptide are sufficient to achieve a near maximal effect. ml - peptide targets intracellular pace4 to inhibit prostate cancer cell lines proliferation. (a) to determine whether the cell proliferation effects are mediated by cell surface or intracellular pace4, ml n - terminal extensions were design to modify cell penetration properties of our inhibitor. neither pegylation (peg8) nor alkylation (c8) modified the affinity of multi - leucine inhibitor toward pace4 and furin, as determined in a kinetic assay. (b) the uptake of fitc - labeled peptides was tested on du145 cells and analyzed by facs. comparison of geomeans (numbers adjacent the peaks) obtained with these three peptides indicates that pegylation prevents cell entry, whereas alkylation increases the uptake of the inhibitor. (c) using a mtt metabolic assay, it was clearly demonstrated that alkylation increases inhibitory potency of ml peptide, whereas pegylation leads to a poor proliferation inhibitor in du145 cell line. (d) ic50 in the table are means of five independent experiments. n.c. means the curve did not converged to 50% with doses up to 500 m. this assay demonstrates that ml peptide must enter the cell in order to inhibit du145 proliferation. to obtain further support for the antiproliferation effects observed, a dna content analysis on lncap treated cells was performed with 100 or 200 m of ml - peptide (figure 6). as these assays are performed over a 96 h period, which could result in peptide degradation, a modified analogue ac-[dleu]lllrvkr - nh2 was also used. the substitution of the n - terminal leu by a dleu provided improved stability of the peptide to amino - peptidases (unpublished data). a dose - dependent g0/g1 accumulation and s phase decrease were observed following exposure to ml - peptide and ml - peptide analogue. following a 200 m treatment with the ac-[dleu]lllrvkr - nh2, a 10% increase in the g0/g1 population was observed along with an increase in cells with hypodiploid dna content (sub - g1) proportions, which represent apoptotic cells. the effect of pace4 inhibition on cell cycle distribution was observed by flow cytometry. lncap cells were treated for 96 h with 100 or 200 m of peptide. (a) ac - llllrvkr - nh2 or (b) ac-[dleu]lllrvkr - nh2. cell cycle distribution was assessed from cellular dna content analysis of cells treated with propidium iodine. the significance of the results were established from an unpaired two - tailed t test. (p < 0.05 ; p < 0.01 ; p < 0.001). (c) data in the table are mean and standard deviation of a representative experiment. furin was the first discovered pc, and ever since the discovery of additional pcs, issues of distinct and redundant functions have been debated for this family of enzymes. while this remains a fundamental interrogation, it can also be envisaged that an answer to this question could have repercussions on the druggability of pcs in various pathologies. structural data have been very clear that within the deep subsites of the catalytic cleft (s1s4), the pcs appear to be virtually identical. this has an immediate consequence from a medicinal chemistry point of view, that small - molecule inhibitors (i.e, small organic compounds) will not be specific if they are competitive inhibitors. it can not be excluded that small molecules could specifically inhibit pcs if they have an allosteric mechanism of action, but to date no such inhibitors have been reported nor have druggable allosteric pc sites been established. therefore, the most probable option presently is to use peptides as lead compounds to design specific pc inhibitors. this is plausible based on the observed structural differences within the s4s7 subsites, as deduced by homology modeling of the furin structure. peptide - based inhibitors afford a number of advantages, the principal one being a great variety of structures that could provide an optimal fit, providing the sought specificity. the disadvantages of peptide - based inhibitors are in their further use as in vivo drug compounds because they can be readily degraded, may be excreted rapidly, and are said not to be very cell penetrable. there is mounting evidence of the potential benefits of inhibiting pace4 in prostate cancer cells. should a therapeutic application be possible, it would have to be through the development of a relatively specific pace4 inhibitor. thus, we have focused our studies on a peptide - based approach to target pace4, with the full knowledge of the obstacles that may arise. as the starting point to design a specific pace4 inhibitor, the potency and the selectivity of purified pc prodomains toward pace4 and furin were evaluated (figure 1a). as furin is the only basic amino acids cleaving pc ubiquitously expressed (i.e., defined as expressed in every single normal cell) and furin full knockouts in mice have proven to be lethal, it seemed appropriate to establish an inhibitor that favors pace4 as much as possible over furin. this objective may appear almost insurmountable due to the stated structural similarities of the two enzymes and multiple data suggesting very similar substrates. nonetheless, the peptide designs focus on obtaining the best specificity ratio of furin / pace4 inhibitory potency (furin ki /pace4 ki). as previously reported in various studies, including our own previous work, pcs prodomains are highly potent inhibitors of pcs (nm and sub - nm range), except for pc2 prodomain, which displays no inhibition at concentrations up to 1 m. however, one observation stands out, namely the specificity ratio of the pc7 prodomain, which was 36-fold (furin ki / pace4 ki). the alignment of pc prodomains was performed for region p7 to p1, as this region contains key residues for substrate - pcs recognition (figure 1b) and highlights the higher density of hydrophobic residues in this region. the pc7 prodomain clearly stands out as it differs from the consensus in position p6. this suggests that basic residue in p6 position could be crucial for potent furin inhibition, whereas pace4 may tolerate a broad range of residues for this position. the combinatorial peptide library provided additional support for this hypothesis (figure 2). this library was anchored at the c - terminal with the consensus motif rvkr to allow the study of p5p8 positions. it was first observed that kis were higher for furin than pace4 for every peptide mixture. as positions p8, p7, and p5 of those samples are occupied by an equimolar mix of the 19 natural amino acids (cysteine excluded), this could indicate that pace4 can accommodate a more diversified selection of amino acids. thus, when looking at kis for position p6 for furin, it can be concluded that furin has a marked preference for basic residues, as lys and arg peptides are the only midnanomolar inhibitors of this enzyme in this library. on the other hand, although p6 lys and p6 arg peptides are mid - nm inhibitors of pace4, similar levels of inhibition can be reached with p6 leu peptides. the highest specificity ratios for p6 position were obtained from leu, his, val, ile, and ala containing peptides. on the basis of the hydrophobic nature of leu, val, ile, and ala, this suggests that pace4 tolerates the presence of hydrophobic residues in position p6 and this opens possibilities to design specific inhibitors. the synthesized series of ml - peptides validated the differences between furin and pace4 in the p5p8 region. to date, the ml - peptide is the best pace4 specific inhibitor described. although furin and pace4 share very identical catalytic domains and an almost identical catalytic cleft, our design of the ml - peptide selective pace4 inhibitor shows that even with these similarities, both enzymes have different binding affinities. the results highlight the fact that furin does not provide nonpolar peptide stabilization in its catalytic cleft while pace4 has good binding affinity with both cationic and nonpolar peptides. to hypothesize on the origin of partial selectivity between differently charged substrates, a homology model of pace4 containing peptide ac - rvkr - cmk in the binding cleft was built using modeler 9v8 from crystalline furin 1p8j(31) (figure 7). stereoscopic views of (a) mouse furin crystal catalytic cleft (1p8j) and (b) pace4 homology model. asp and glu negative charges are shown in red, whereas arg and lys positive charges are shown in blue. green spheres represent dicationic calcium ions and decanoyl - rvkr - cmk inhibitor has been modified to ac - rvkr - cmk for clarity. a close examination of the whole catalytic domain of pcs shows various degrees of charge excess, as previously reported for other subtilisins. by looking at the number of positively charged arg and lys (i.e., his being mostly neutral at cytoplasmic ph is left out) and negatively charged asp and glu, the overall total charge calculated for both convertase catalytic domains are 7 for human furin and 0 for human pace4 (i.e., the catalytic domain of furin goes from n - terminal prodomain primary cleavage site asp108 to val444 and from gln150 to ala496 in pace4). the active site of both furin and pace4 also reveals that the s1, s2, and s4 sites are formed by residues with similar properties, e.g., glu257 and glu230 in furin replace asp309 and asp282 in pace4, respectively (figure7). on the other hand, the differences in affinity observed with multiple leu extension in the ml - peptide propose the existence of notable differences in subsite s5 and beyond. to gain a deeper insight of the phenomenon, it is necessary to dissect the binding cleft of both pcs. one common feature among all the subtilases is the presence of a groove running from the catalytic site to helixes 3 and 4 that stabilizes a peptidic substrate in an antiparallel -sheet conformation through an induced fit process. this interaction can also be seen in the noncatalytic pcsk9 structure (2pmw) between the prodomain and the region homologous to the catalytic cleft of the other pcs (ser147 to gln152). despite the fact that other residues might be involved, the same trend remains for furin and pace4 : a way of hosting a peptidic substrate or inhibitor adopting an antiparallel -sheet. on the basis of these data, we propose that the linear conformation of the peptide brings the p6p8 residues of the inhibitor in the vicinity of the residues located at the tip of helix 4, and this region of the enzyme would represent the subsites s6, s7, and s8. we also focused our analysis on the disparities in this region to better understand the specificity observed for ml - peptide because the solvent accessible residues of helix 4 differ in pace4 and furin. from the homology model of pace4, it appears that the most critical dissimilarity in the helix 4 comes from the replacement of acidic residues in furin by either basic or neutral residues in pace4 (glu271 and glu272 in furin exchanged for lys323 and gln324 in pace4). consequently, the n - terminal end of helix 4 has a charge of 1 in furin and + 2 in pace4. finally, an intramolecular quench of charges in pace4 may explain its potential to complex neutral ligands. such self - quenching is unlikely for furin in the n - terminal region of the helix 4. in sum, the fact that furin catalytic clef is more negatively charged than pace4 might very well account for much of the selectivity showed by the two pcs. the cellular effects of the ml - peptide are remarkable, as they phenocopy the antiproliferation effects observed in our previous molecular studies knocking down pace4 in du145 cells. this study also provided additional evidence for the pace4 targeted antiproliferation using lncap cells. however, the effects of the ml - peptide are largely ineffective in the pc3 cell lines, as it expresses very little if any pace4. as a control, the polybasic peptide ac - rarrrkkrt - nh2, which is a potent furin inhibitor of similar length, was tested in the mtt cell - based assays (with lncap and du145 cells) and no antiproliferative effects could be observed with this peptide (supporting information figure s2). we are confident that the furin target was reached by the peptide, as in a previous study we showed that this potent furin inhibitor has cell penetration properties. additional control peptides provide evidence that the ml - peptide indeed targets pace4 because the ac - llllrvk[darg]-nh2 shows a substantial loss of affinity in vitro and is also a poor inhibitor of du145 and lncap cell proliferation (figures 4d, e). pace4 is localized at the cell surface and in the extracellular matrix due to interactions between its cycteine - rich domain (crd), heparin sulfate proteoglycans (hspgs), and tissue inhibitors of metalloproteinases (timps). to assess the importance of cell surface pace4 in tumor progression, the ml - peptide was modified with n - terminal pegylation, significantly reducing its cell penetration property while not affecting its inhibitory potency (i.e, pegylated ml - peptide has a similar ki to the ml - peptide). because the pegylated ml - peptide significantly losses its ability to inhibit du145 proliferation, it leads to the conclusion that intracellular pace4 is required for this function. a reduction of cellular proliferation could occur by a number of mechanisms downstream of pace4 inhibition, but most likely a number of important growth factors which are substrates of pace4 have lost their activity due to lack of processing. this general lack of growth factor activity would eventually have effects on the cell cycle. therefore, a cell cycle analysis was performed to evaluate if cell cycle arrest or slow - down was possible. a dose - dependent accumulation of cells in g0/g1 phase was observed, thus preventing cells entry into s phase. the transition between g1 and s phase is a finely regulated mechanism controlled by a combination of environmental considerations mostly influenced by the presence of growth signals and the discontinuation of extracellular inhibitory signals. this could suggest that the ml - peptide inhibits the processing of growth factors required to go beyond the restriction point and therefore triggers a cell cycle arrest at this point. however, while outside of the scope of the present manuscript, further studies identifying these substrates and signaling pathways involved will be required. nonetheless, various potential substrates have been suggested in various other studies. of interest was the observation of apoptosis following an exposure to 200 m ac-[dleu]-lllrvkr - nh2 in lncap cells. this phenomenon may be easily explained by the fact that cell cycle arrest is usually poorly tolerated and prolonged cytostasis must be escaped by cell death. if this is true, then more potent and stable versions of the ml - peptide inhibitor could result in exceptional drugs that reduce prostate cancer cell proliferation as well as inducing specific cell death. in the context of the metastasis phase of prostate cancer synthesis of sp - spcl peptide library was performed as described previously (torrey pines institute for molecular studies). all other peptides were obtained by solid - phase peptide synthesis (50 mol scale) on a polystyrene resin, tentagel s ram (rapp polymere, capacity 0.23 mmol / g), with a pioneer peptide synthesizer (applied biosystems), according to standard coupling procedures and fmoc strategy. the protected amino acids were coupled at 3-fold excess using hatu as coupling agent in the presence of dipea in dmf. after the final fmoc deprotection, with the exception of fitc - labeled peptides, n - terminal acetylation was carried out in dmf with acetic anhydride (0.5%) and 2.6-lutidine (0.6%). the fitc - peptides were labeled with fluorescein isothiocyanate isomer i (fitc) through their n - terminus (-ala was used as a spacer for the peptide : fitc-[-ala]-llllrvkr - nh2). dcm (1:4) was added to the resin and allowed to couple overnight. after completion of the synthesis, the protected peptidyl resins were treated with tfa h2o tis (95:2.5:2.5) and stirred for 3 h. the solutions with the released peptides were filtered and evaporated in vacuo to a volume of about 1 ml. then the peptides were precipitated with diethyl ether to afford crude products. the crude compounds were purified by semipreparative hplc (agilent technologies, 1100 series hplc equipped with a diode array detector (dad)) on reversed - phase support agilent c18 column (15 m, 100, 7.8 mm 300 mm). the purity of the peptides was controlled using analytical hplc. a seldi - tof mass spectrometer (bio - rad laboratories) was used to confirm the identity of the pure products (molecular ion). according to both hplc and mass spectrometry, the peptide content analyses were performed in vacuo with a beckman 120c autoanalyzer following 24 h hydrolysis in 5.7 n hcl at 110 c and analyzed using a varian ds604 system integrator. recombinant soluble human pcs were produced from s2 insect cells and purified as previously described. briefly, s2 conditioned medium was purified using ultrafiltration, anion exchange chromatography, hydrophobic interaction chromatography, and gel filtration. the prodomains of pcs were produced in one shot top 10 cells (invitrogen) transformed with cdna construct of full - length prosegment. the recombinant prodomains were purified from bacterial lysate by nickel chromatography and reverse - phase hplc as previously described. enzyme inhibition assays for furin were performed in 100 mm hepes ph 7.5, 1 mm cacl2, 1 mm -mercaptoethanol, and 1.8 mg / ml bsa, while assays for pace4 were performed in 20 mm bis - tis ph 6.5, 1 mm cacl2, and 1.8 mg / ml bsa. all assays were performed with the substrate pyroglu - arg - val - lys - arg - methyl - coumaryl-7-amide (bachem, ca) at 100 m. assays were carried out at 37 c for 60 min, and real - time fluorescence was measured using a gemini em 96-well spectrofluorometer (molecular devices, ca) (em, 370 nm ; ex. inhibitory peptides and prodomains were added to the assays at decreasing concentrations to perform a competitive inhibition assays. kinetics assays were analyzed using softmaxpro5, and ki values were determined from ic50 using cheng and prusoff s equation with km values of 4.61 m for furin and 3.5 m for pace4. the quality of the total rna sample was assessed using an agilent bioanalyzer with the rna nano chip (agilent technologies). briefly, 1 g rna was reverse transcribed and the obtained cdna was used to carry out qpcr analysis. relative expression were calculated using -actin as a reference gene and the formula (1 + amplification efficiency) (ct) for each cell line, as described previously. to perform mtt assays, both du145 and pc3 cell lines were seeded at a density of 1500 cells per well in 96-wells plates. lncap cells were seeded at a density of 2500 cells per well in a poly lysine coated 96-well plate. after 24 h, media was changed and inhibitory peptides were added to the cells. the peptides were incubated with the cells for 72 h prior to addition of mtt reagent at a final concentration of 1 mg / ml. mtt reagent was incubated 4 h with du145 and lncap and 6 h with pc3 cells, and then media was removed and formazan was solubilized with 100 l of 2-propanol hcl (24:1n). the total metabolic activity was normalized relatively to vehicle treated cells (sterile bidistilled water 0.1% dmso). each step of mtt, as well as maintenance of du145, lncap, and pc3 cells, were carried in rpmi 1640 5% fbs for du145 and 10% fbs for lncap and pc3. the uptake of fitc peptides was tested on du145 cells and analyzed with a facscan cytometer (becton dickinson, mountain view, ca). first, 4 10 cells were incubated 1 h in serum - free rpmi media with 10 m of fitc--ala - ml, fitc-[peg8]-ml, or fitc-[c8]-ml and collected by centrifugation. then the cell pellets were washed twice with trypsin (0.05% v / v) during 5 min at 37 c to remove nonspecific interactions with the membrane. cells were incubated 2 min with propidium iodine (10 g / ml) in order to exclude cells with altered membrane. a minimum of 10000 events per sample was acquired, excluding cell clumps and debris. geomeans were determined using cellquest software (becton dickinson, mountain view, ca). to perform cell cycle analyses on lncap, 4 10 cells were seeded in 10 cm culture dishes and grown for 24 h without treatment. cells were then treated with vehicle (0.1% dmso) or with 100 m or 200 m of peptides ac - llllrvkr - nh2 or ac-[dleu]lllrvkr - nh2. treatments were carried out in complete medium (10% fbs) for a period of 96 h, and cell media were changed every 24 h to offset peptide degradation. cells were harvested using trypsin, washed once with pbs, resuspended in 0.5 ml of pbs, and fixed by dropwise addition of 1.5 ml of ice - cold ethanol. after a 30 min incubation at room temperature, cells were washed with pbs and dna staining was performed in 20 mm hepes ph 7.2, 0.16 m nacl, and 1 mm egta buffer containing 10 g / ml of rnasea and 10 g / ml of propidium iodine. flow cytometry was performed using a facscan cytometer (becton dickinson, mountain view, ca) equipped with a 15 mw argon ion laser tuned at 488 nm. forward and side scatter signals were used to establish the live gate to exclude debris and cell clumps and a second live gate was set using the fl3-a and fl3-w parameters of the doublet discrimination module (ddm), allowing single cell measurements. the percentages of cells in different phases of cell cycle were calculated by modfit software (verity software house, topsham, me). | the proprotein convertases (pcs) play an important role in protein precursor activation through processing at paired basic residues. however, significant substrate cleavage redundancy has been reported between pcs. the question remains whether specific pc inhibitors can be designed. this study describes the identification of the sequence llllrvkr, named multi - leu (ml)-peptide, that displayed a 20-fold selectivity on pace4 over furin, two enzymes with similar structural characteristics. we have previously demonstrated that pace4 plays an important role in prostate cancer and could be a druggable target. the present study demonstrates that the ml - peptide significantly reduced the proliferation of du145 and lncap prostate cancer - derived cell lines and induced g0/g1 cell cycle arrest. however, the ml - peptide must enter the cell to inhibit proliferation. it is concluded that peptide - based inhibitors can yield specific pc inhibitors and that the ml - peptide is an important lead compound that could potentially have applications in prostate cancer. |
uprighting springs or mesiodistal root uprighting springs are an indispensable component of light wire differential force technique, introduced by begg in 1956. besides being used for mesiodistal root uprighting during stage iii of begg 's technique, they are also used as braking auxiliaries to increase the anchorage to carry out various tooth movements. however, with the advent of the preadjusted edgewise appliance, the use of light wire technique has drastically reduced resulting in a lack of familiarization of the begg uprighting spring among contemporary orthodontists. of late, the various possibilities of this spring as an adjunctive in contemporary preadjusted edgewise appliance systems containing vertical slot is being explored. the additional anchorage potential provided by uprighting springs can be used to treat different situations successfully (e.g. space closure by posterior teeth protraction). these springs are made from 0.009 " to 0.018 " australian archwire depending upon the type of tooth (i.e. incisors, canines, premolars and molars) and the purpose (e.g. mesiodistal uprighting or as brakes to provide additional anchorage) for which we need to use these auxiliaries. although these uprighting springs are easily available in prefabricated form, here we present a simple and quick chair side method to fabricate and customize uprighting springs according to the required crown / root movement for correction. determine the type of moment (clockwise or counterclockwise), required for mesiodistal root uprighting either clinically or with the help of opg. this will vary depending upon tooth type and quadrant of the arch under consideration in the maxilla or mandible.figure 1, represents a situation, where upper right 2 premolar needs clockwise moment for root uprighting.hold approximately 6 cm of australian arch wire, as shown in figure 1a, and bend the right plank of the wire in a clockwise direction. then, the wire is bent around beak of 139 pliers keeping this end under the left plank [figure 1b ]. figure 1d completes the residual wire bending as per requirement of the degree of activation. determine the type of moment (clockwise or counterclockwise), required for mesiodistal root uprighting either clinically or with the help of opg. this will vary depending upon tooth type and quadrant of the arch under consideration in the maxilla or mandible. figure 1, represents a situation, where upper right 2 premolar needs clockwise moment for root uprighting. hold approximately 6 cm of australian arch wire, as shown in figure 1a, and bend the right plank of the wire in a clockwise direction. then, the wire is bent around beak of 139 pliers keeping this end under the left plank [figure 1b ]. figure 1d completes the residual wire bending as per requirement of the degree of activation. (a) hold the australian wire in 139 pliers showing required direction of the bend ; (b) bend the right plank of wire in a clockwise direction, keeping this end under left plank ; (c) make two and half turns and (d) complete the residual wire bending and final clockwise type uprighting spring optimum angulation is 135 between arm and stem of uprighting spring. for anchorage reinforcement, 4.figure 2, represents a situation, where lower right 2 premolar needs anticlockwise moment for root uprighting [figure 2a ]. the wire is bent around beak of 139 pliers keeping this end under the right plank. give two and a half turns around the beak [figure 2c ]. complete the residual wire bending required at activation arm and stem of uprighting spring that is anticlockwise type [figure 2d ]. figure 2, represents a situation, where lower right 2 premolar needs anticlockwise moment for root uprighting [figure 2a ]. the wire is bent around beak of 139 pliers keeping this end under the right plank. complete the residual wire bending required at activation arm and stem of uprighting spring that is anticlockwise type [figure 2d ]. step by step procedure to fabricate anticlockwise type uprighting spring (a) hold the australian wire in 139 pliers showing required direction of the bend ; (b) bend the left plank of wire in anticlockwise direction, keeping this end under right plank, (c) make two and half turns and (d) complete the residual wire bending and final anticlockwise type uprighting spring alternatively, bending directly from the spool is advisable to prevent wastage. contemporary world of orthodontics utilizes different kinds of preadjusted appliances, where all the features to correct 1, 2 and 3 order discrepancies, are incorporated therein. however, certain clinical situations exist where we need to increase or reinforce the anchorage value at a particular location, to carry out various types of orthodontic tooth movements. some of these clinical situations are shown in figure 3, where begg 's uprighting spring has been used as an adjunct to facilitate tooth movements. barbieri and barbieri reported various clinical situations for the use of uprighting spring to reinforce anchorage. various uses of begg 's uprighting spring in different clinical situations (a) stage iii of begg 's technique as uprighting of canine and premolars (b) shows uprighting spring acting as braking auxiliary to protract lower posterior segment ; (c) and (ssd) uprighting spring on upper left and right canine respectively with preadjusted edgewise appliance bracket these auxiliaries can also be useful in diverging the roots of canines and or first premolars in presurgical orthodontics for anterior maxillary osteotomy by wassmund 's or wunderer 's technique. physical factors like gauge and resiliency of the wire, size of the helix, number of the coils, direction of activation are discussed besides their classical use as mesiodistal uprighting auxiliary. the relationship between diameter of the wire and diameter of the loop referred to as spring coil index should be at least six as described by thurow. this means that loop diameter should be at least 6 times the diameter of the wire (e.g. for wire diameter of 0.012 ", loop diameter should be 0.072 ") for its optimal performance. lesser than this will lead to the creation of internal strain and increases the risk of failure. so also, while using such kind of auxiliaries, the utmost care must be exercised to prevent or minimize their side effects like labial crown movements, extrusion of anteriors, intrusion of posteriors and buccal crown movements. it is, therefore, absolutely essential that the base archwire be rigid enough, preferably 0.020 " premium or 0.018 " premium plus, pulse straightened australian archwires to overcome the side effects. this can find use to enhance uprighting (quality and quantity), reinforce anchorage, and presurgical root positioning along with contemporary preadjusted edgewise (or tip - edge type) appliances. merely determining the direction (clockwise or anticlockwise) of moments, will help to customize these uprighting springs as per individual needs as shown in figure 3. additional advantages are that there is no need for stocking / storing, or any extra investment over the purchase of prefabricated ones. | uprighting springs, an integral part of the begg ligsht wire differential force technique is gaining more and more popularity, as a useful adjunct in contemporary preadjusted edgewise appliance systems as well. it can be used with brackets containing vertical slots for mesiodistal crown uprighting, or as braking auxiliaries providing additional anchorage while protracting posteriors. here, we present a simple and quick chair side method of fabricating and customizing uprighting springs according to the required crown / root movement for correction. this communication would serve as a ready reckoner during fabrication of the springs, thus dispelling the confusion that usually arises regarding direction and position of the coil and active arm. |
endometrial cancer (ec) is a common gynecological cancer worldwide, accounting for about half of all gynecological cancers. risk factors for ec include age 40 years, obesity, diabetes, hypertension, estrogen using, tamoxifen treatment, and family history of malignant tumor. an effective screening strategy for women with high - risk factors may contribute to early detection and management of ec. direct endometrial sampling procedures, including dilatation and curettage (d&c) and biopsy, are traditional and efficacious diagnostic methods in ec since they can obtain endometrial specimens for histopathological analysis. therefore, d&c is a less practical screening tool and usually performed in hospital settings. cytology - based papanicolaou smears have steadily decreased the incidence and mortality of cervical cancer in countries with successful national screening programs. liquid - based cytology (lbc) is a method approved by the food and drug administration for cervical cancer screening in america in 1996. lbc prepares samples for cytology examination by depositing the collected sample into a bottle of preservative liquid. through removing obscuring factors such as mucus or blood, lbc can reduce obscuring factors and provide thin - layer specimens for cytology examination. cell block (cb) is prepared from the residual cytological specimens. both the morphology of endometrial cells and glandular architectures are critical to ec diagnosis. lbc is helpful for studying cell details, whereas the evaluation of glandular architecture relies indirectly on the morphology of cell clumps. cb can maintain cell morphology and tissue architecture and is thus a useful complement to liquid - based smears for definitive diagnosis. cb preparation is used as a complementary diagnostic tool in gynecocytology, fine - needle aspiration, and effusion cytology. in the current study, we compared the specimen adequacy of lbc and cb preparation. in addition, we investigated the diagnostic accuracy of lbc and cb in detecting atypical endometrial hyperplasia and carcinoma. the cytological specimens were obtained from may 2014 to april 2015 at department of obstetrics and gynecology, xuanwu hospital, capital medical university, beijing. the inclusion criteria included : women aged 40 years, abnormal uterine bleeding (aub), or thickened endometrium (postmenopausal women 4 mm or menopausal women 20 mm). the exclusion criteria included malformation of the genital tract, adhesion of endometrial cavity or cervical canal, and intrauterine contraceptive device. after signing informed consent, all women were submitted first to endometrial cytological test (ect), then to d&c or biopsy guided by hysteroscopy. cytological sampling of endometrium was performed using a sap-1 sampler (saipujiuzhou co., beijing, china) without cervical dilatation. the device was made of soft plastic, which measured 3 mm diameter and 25 cm length, consisting a scalable latex ring with some fine teeth inside a 16-cm outer protective sheath to prevent contamination from endocervical and vaginal cells. after collection of the endometrial sample, the ring was then immersed in surepath cell preservative container (surepath preservative fluid ; bd diagnostic, burlington, nc, usa), where it was shaken to allow cells to release. the 5 ml specimens were transferred into centrifuge tubes with some density reagent (bd diagnostic, burlington, nc, usa) to remove blood and mucus. the centrifuge tube was inserted into the surepath semi - automated slide processor and stained using papanicolaou stain [figure 1a and 1b ]. (a) secretory phase endometrium in liquid - based cytology preparation (papanicolaou, original magnification 40). (b) endometrial carcinoma in liquid - based cytology preparation (papanicolaou, original magnification 100). (c) secretory phase endometrium in cell block preparation (he, original magnification 40). (d) endometrial carcinoma in cell block preparation (he, original magnification 40). the residual fluid was collected in a 10-ml disposable centrifuge tube and then centrifuged at 1000 round / min for 2 min and 15 s and 2000 round / min for 10 min and 15 s (rotina 46s, hettich co., germany). the cell pellets with the endometrial cells were wrapped up in a cassette and fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (he) [figure 1c and 1d ]. histological samples were routinely fixed in neutral buffered formalin, embedded in paraffin, and stained with he. histological sections from cb and histology were executed blindly by two clinical pathologists according to the who classification scheme. the findings of lbc and cb were correlated with the histological results. in cases of discordant diagnosis we classified the endometrial lesions into four categories : normal endometrium, nonatypical hyperplasia, atypical hyperplasia, and carcinoma. normal endometrium and nonatypical hyperplasia were considered as negative while atypical hyperplasia and carcinoma as positive. statistical analyses were performed using the statistical program / spss for windows (version 10.0, spss inc., a double access table was created to evaluate the accuracy, sensitivity, specificity, the positive predictive value (ppv), and negative predictive value (npv) of lbc smears and cbs. the accuracy was defined as dividing the true positive and true negative cases by all the cases studied. the sensitivity was defined as dividing the number of true positive cases by all the positive cases confirmed by histology. the specificity was defined as dividing the number of true negative cases by all the negative cases. the ppv was defined as dividing the true positive cases by the overall true positive and false positive cases. the npv was defined as dividing the true negative cases by the overall true negative and false negative cases. the cytological specimens were obtained from may 2014 to april 2015 at department of obstetrics and gynecology, xuanwu hospital, capital medical university, beijing. the inclusion criteria included : women aged 40 years, abnormal uterine bleeding (aub), or thickened endometrium (postmenopausal women 4 mm or menopausal women 20 mm). the exclusion criteria included malformation of the genital tract, adhesion of endometrial cavity or cervical canal, and intrauterine contraceptive device. after signing informed consent, all women were submitted first to endometrial cytological test (ect), then to d&c or biopsy guided by hysteroscopy. cytological sampling of endometrium was performed using a sap-1 sampler (saipujiuzhou co., beijing, china) without cervical dilatation. the device was made of soft plastic, which measured 3 mm diameter and 25 cm length, consisting a scalable latex ring with some fine teeth inside a 16-cm outer protective sheath to prevent contamination from endocervical and vaginal cells. after collection of the endometrial sample, the ring was then immersed in surepath cell preservative container (surepath preservative fluid ; bd diagnostic, burlington, nc, usa), where it was shaken to allow cells to release. the 5 ml specimens were transferred into centrifuge tubes with some density reagent (bd diagnostic, burlington, nc, usa) to remove blood and mucus. the centrifuge tube was inserted into the surepath semi - automated slide processor and stained using papanicolaou stain [figure 1a and 1b ]. (a) secretory phase endometrium in liquid - based cytology preparation (papanicolaou, original magnification 40). (b) endometrial carcinoma in liquid - based cytology preparation (papanicolaou, original magnification 100). (c) secretory phase endometrium in cell block preparation (he, original magnification 40). (d) endometrial carcinoma in cell block preparation (he, original magnification 40). the residual fluid was collected in a 10-ml disposable centrifuge tube and then centrifuged at 1000 round / min for 2 min and 15 s and 2000 round / min for 10 min and 15 s (rotina 46s, hettich co., germany). the cell pellets with the endometrial cells were wrapped up in a cassette and fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (he) [figure 1c and 1d ]. histological samples were routinely fixed in neutral buffered formalin, embedded in paraffin, and stained with he. histological sections from cb and histology were executed blindly by two clinical pathologists according to the who classification scheme. the findings of lbc and cb were correlated with the histological results. in cases of discordant diagnosis we classified the endometrial lesions into four categories : normal endometrium, nonatypical hyperplasia, atypical hyperplasia, and carcinoma. normal endometrium and nonatypical hyperplasia were considered as negative while atypical hyperplasia and carcinoma as positive. statistical analyses were performed using the statistical program / spss for windows (version 10.0, spss inc., chicago, il, usa). the adequacy of specimens was compared using chi - squared test. a p < 0.05 was considered statistically significant. a double access table was created to evaluate the accuracy, sensitivity, specificity, the positive predictive value (ppv), and negative predictive value (npv) of lbc smears and cbs. the accuracy was defined as dividing the true positive and true negative cases by all the cases studied. the sensitivity was defined as dividing the number of true positive cases by all the positive cases confirmed by histology. the specificity was defined as dividing the number of true negative cases by all the negative cases. the ppv was defined as dividing the true positive cases by the overall true positive and false positive cases. the npv was defined as dividing the true negative cases by the overall true negative and false negative cases. direct endometrial specimens were collected from 198 perimenopausal and postmenopausal women. among them, 89 (44.9%) were postmenopausal. the 198 cases consisted of one hundred cases with thickened endometrium, 13 cases with aub, and 85 cases with both thickened endometrium and aub. of the 198 cases, 44 (22.2%) cb specimens were inadequate, whereas 14 (7.1%) lbc specimens were inadequate. the specimen inadequate rate of cb was significantly higher than lbc (= 18.18, p < 0.01). among 184 adequate lbc smears, 144 (78.3%) residual specimens were processed into cbs. however, among 14 inadequate lbc smears, 10 residual specimens were successfully processed into cbs. in addition, postmenopausal women accounted for 63.6% in 44 inadequate cb cases and 35.7% in 14 inadequate lbc specimens. a total of 144 specimens were adequate for both cb and lbc preparation [figure 2 ]. lbc : liquid - based cytology ; cb : cell block. among 144 specimens adequate for both cb and lbc, histological results demonstrated that there were 115 negative cases (37 normal endometria and 78 nonatypical hyperplasia endometria) and 29 positive cases (11 atypical hyperplasia endometria and 18 endometrial carcinomas). lbc correctly recognized 23 positive cases and 112 negative cases, whereas six positive cases were underrated and three negative cases were misdiagnosed as positive. cb correctly diagnosed 24 positive and 113 negative cases, whereas five positive cases were underrated and two negative cases were misdiagnosed. a combination of lbc and cb correctly diagnosed 26 positive cases and 112 negative cases. there were three positive cases (atypical hyperplasia) underrated as negative and three negative cases (nonatypical hyperplasia endometria) misdiagnosed as positive (atypical hyperplasia). the diagnostic accuracy of lbc was 93.8%, with a sensitivity of 79.3%, specificity of 97.4%, ppv of 88.5%, and npv of 94.9%. the diagnostic accuracy of cb was 95.1%, with a sensitivity of 82.8%, specificity of 98.3%, ppv of 92.3%, and npv of 95.8%. when combined lbc with cb, the diagnostic accuracy was improved to 95.8%, with a sensitivity of 89.7%, specificity of 97.4%, ppv of 89.7%, and npv of 97.4% [table 1 ]. n / n) lbc : liquid - based cytology ; cb : cell block ; ppv : positive predictive value ; npv : negative predictive value. direct endometrial specimens were collected from 198 perimenopausal and postmenopausal women. among them, 89 (44.9%) were postmenopausal. the 198 cases consisted of one hundred cases with thickened endometrium, 13 cases with aub, and 85 cases with both thickened endometrium and aub. of the 198 cases, 44 (22.2%) cb specimens were inadequate, whereas 14 (7.1%) lbc specimens were inadequate. the specimen inadequate rate of cb was significantly higher than lbc (= 18.18, p < 0.01). among 184 adequate lbc smears, 144 (78.3%) residual specimens were processed into cbs. however, among 14 inadequate lbc smears, 10 residual specimens were successfully processed into cbs. in addition, postmenopausal women accounted for 63.6% in 44 inadequate cb cases and 35.7% in 14 inadequate lbc specimens. a total of 144 specimens were adequate for both cb and lbc preparation [figure 2 ]. among 144 specimens adequate for both cb and lbc, histological results demonstrated that there were 115 negative cases (37 normal endometria and 78 nonatypical hyperplasia endometria) and 29 positive cases (11 atypical hyperplasia endometria and 18 endometrial carcinomas). lbc correctly recognized 23 positive cases and 112 negative cases, whereas six positive cases were underrated and three negative cases were misdiagnosed as positive. cb correctly diagnosed 24 positive and 113 negative cases, whereas five positive cases were underrated and two negative cases were misdiagnosed. a combination of lbc and cb correctly diagnosed 26 positive cases and 112 negative cases. there were three positive cases (atypical hyperplasia) underrated as negative and three negative cases (nonatypical hyperplasia endometria) misdiagnosed as positive (atypical hyperplasia). the diagnostic accuracy of lbc was 93.8%, with a sensitivity of 79.3%, specificity of 97.4%, ppv of 88.5%, and npv of 94.9%. the diagnostic accuracy of cb was 95.1%, with a sensitivity of 82.8%, specificity of 98.3%, ppv of 92.3%, and npv of 95.8%. when combined lbc with cb, the diagnostic accuracy was improved to 95.8%, with a sensitivity of 89.7%, specificity of 97.4%, ppv of 89.7%, and npv of 97.4% [table 1 ]. n / n) lbc : liquid - based cytology ; cb : cell block ; ppv : positive predictive value ; npv : negative predictive value. endometrial cell morphology may be influenced by certain hormonal situations which makes the diagnosis more confusing. therefore, it is challenging to detect ec by cytological screening and the incidence and mortality increase annually in most developed countries. however, the overall death rate in japan was reduced from 20/100,000 in 1950 to 8/100,000 in 1999. this reduction was mainly attributed to the endometrial cytological screening, a widely accepted method in japan. cb slides can preserve both cellular (nuclear and cytoplasm) characteristics and architectural patterns of endometrial glands and stroma, thus increasing the diagnosis accuracy. moreover, cb preparation also allows for long - term preservation and provides more materials for immune stains in later studies. in the current study, we compared the specimen adequacy of lbc and cb preparation. our result demonstrated that the inadequate rate of cb (22.2%) preparation was significantly higher than that of lbc (7.1%) (p < 0.01). garcia. showed that the inadequate rate of lbc was 15%, lower than that in endometrial biopsies (26%). a validation study of 1514 ect showed that the inadequate rate of cytology was lower than that of histology collected from d&c or biopsy. in particular, the inadequate rate was markedly lower in lbc than in endometrial biopsies in postmenopausal women. the high inadequate rate of cb in our study might be due to several reasons. cb was collected from the residual specimens of lbc and cb preparation processes result in cell losses. garcia. performed a prospective study of 103 symptomatic women and reported a sensitivity of 78% and specificity of 96% with ppv of 78% and npv of 96% in the detection of endometrial abnormalities. a review by fambrini. showed that the overall sensitivity of lbc in diagnosing ec was from 78% to 100% in different literatures, with a specificity of 95100%. our results showed that the diagnostic accuracy of lbc was 93.8%, with a sensitivity of 79.3% and specificity of 97.4%. in addition, the diagnostic accuracy of cb in our study was 95.1%, with a sensitivity of 82.8% and specificity of 98.3%. in the present study, the combination of cb and lbc improved the diagnostic accuracy of ec to 95.8%, with a sensitivity of 89.7% and specificity of 97.4%. importantly, no endometrial carcinoma was missed or misdiagnosed when cb and lbc were combined. they showed that the diagnostic accuracy was improved significantly in hyperplasia with atypia, from 55% to 95.3% and in adenocarcinoma, from 98.6% to 100%. they concluded that cb preparation was an excellent adjunctive tool in the evaluation of endometrial lesions because of its advantages to preserve the quality for immunohistochemistry. however, few studies investigated the combination of cb and lbc in the diagnosis of endometrial lesions. in conclusion, cb is a feasible and reproducible adjuvant method in diagnosing endometrial lesions. however, cb demonstrates a higher specimen inadequate rate than lbc smears. in addition, not all cytological specimens are cost - effective for cb preparation and it may be used in confusing cases as an auxiliary diagnosis tool. used together, lbc and cb can improve the diagnostic accuracy of ec. | background : liquid - based cytology (lbc) offers an alternative method to biopsy in screening endometrial cancer. cell block (cb), prepared by collecting residual cytological specimen, represents a novel method to supplement the diagnosis of endometrial cytology. this study aimed to compare the specimen adequacy and diagnostic accuracy of lbc and cb in the diagnosis of endometrial lesions.methods:a total of 198 women with high risks of endometrial carcinoma (ec) from may 2014 to april 2015 were enrolled in this study. the cytological specimens were collected by the endometrial sampler (sap-1) followed by histopathologic evaluation of dilatation and curettage or biopsy guided by hysteroscopy. the residual cytological specimens were processed into paraffin - embedded cb after lbc preparation. diagnostic accuracies of lbc and cb for detecting endometrial lesions were correlated with histological diagnoses. chi - square test was used to compare the specimen adequacies of lbc and cb.results:the specimen inadequate rate of cb was significantly higher than that of lbc (22.2% versus 7.1%, p < 0.01). there were 144 cases with adequate specimens for lbc and cb preparation. among them, 29 cases were atypical endometrial hyperplasia (11 cases) or carcinoma (18 cases) confirmed by histology evaluation. taking atypical hyperplasia and carcinoma as positive, the diagnostic accuracy of cb was 95.1% while it was 93.8% in lbc. when combined lbc with cb, the diagnostic accuracy was improved to 95.8%, with a sensitivity of 89.7% and specificity of 97.4%.conclusions : cb is a feasible and reproducible adjuvant method for screening endometrial lesions. a combination of cb and lbc can improve the diagnostic accuracy of endometrial lesions. |
clinicaltrials.gov identifier, nct00121641, nct00316082, nct00698932, nct00918879, nct00121667, nct00661362, nct00313313, nct00295633, nct00757588. worldwide, diabetes is the leading cause of chronic kidney disease (ckd), which is defined by an estimated glomerular filtration rate (egfr) of 50 ml / min/1.73 m, n = 13,916), moderate renal impairment (egfr 3050 ml / min/1.73 m, n = 2240), or severe renal impairment (egfr < 30 ml / min/1.73 m, n = 336). compared with placebo, saxagliptin (5 or 2.5 mg / day in patients with egfr 50 m) significantly reduced a1c in all egfr subgroups at 1 year, which persisted through a median follow - up of 2 years. saxagliptin also significantly reduced progressive microalbuminuria in patients with normal or mildly reduced renal function (p < 0.0001) and in those with moderate - to - severe renal impairment (p = 0.04) based on improvement or less worsening of urinary albumin : creatinine ratio compared with placebo. in conclusion, saxagliptin at doses of 2.5 or 5 mg / day was equally well tolerated and improved glycemic control in patients with moderate renal impairment who have limited treatment options. w. cook and b. hirshberg are employees of medimmune, llc, a wholly owned subsidiary of astrazeneca. all procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1964, as revised in 2013. this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors. this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. | introductiontype 2 diabetes (t2d) is the leading cause of chronic kidney disease (ckd). the recommended dose of the dipeptidyl peptidase-4 inhibitor saxagliptin is 2.5 mg in patients with moderate or severe renal impairment (creatinine clearance 50 ml / min). in this post hoc analysis, we assessed the effect of saxagliptin 2.5 and 5 mg / day versus placebo on glycemic measures in patients with t2d and estimated glomerular filtration rate 4560 ml / min/1.73 m2.methodsefficacy and safety data were pooled from nine 24-week, randomized, placebo - controlled clinical trials.resultsthe majority (5661%) of patients were women aged < 65 years with glycated hemoglobin (a1c) 8.18.2% ; half of the patients had a t2d duration 5 years. mean change from baseline in a1c was significantly greater with saxagliptin 2.5 (0.6%, p = 0.036 vs placebo) and 5 mg / day (0.9%, p < 0.001 vs placebo) compared with placebo (0.2%). there were numerically greater reductions in fasting plasma glucose and 2-h postprandial glucose, and a significantly greater proportion of patients achieved a1c < 7% with saxagliptin 5 mg / day (44.8%) compared with placebo (20.0%, p = 0.004 vs placebo). the incidence of hypoglycemia was not significantly different across groups (16.2% in the saxagliptin 5-mg / day, 12.2% in the saxagliptin 2.5-mg / day, and 11.3% in the placebo groups).conclusionthese results suggest that saxagliptin 2.5 and 5 mg / day improve glycemic control and are generally well tolerated in patients with t2d and moderate ckd.trial registrationclinicaltrials.gov identifier, nct00121641, nct00316082, nct00698932, nct00918879, nct00121667, nct00661362, nct00313313, nct00295633, nct00757588.fundingastrazeneca, gaithersburg, md, usa. |
the weqaya screening program commenced in april 2008 for uae nationals (aged 18 years) residing in abu dhabi linked to the provision of free comprehensive health insurance (called thiqa) (3). individuals consented in line with the principles of the abu dhabi medical research council (6). further details about the screening program are described elsewhere (3), but in summary, it was conducted at a series of dedicated primary health care facilities with a systematic screening methodology (available at http://www.haad.ae/haad/linkclick.aspx?fileticket=sj-gi8-biv4%3d&tabid=820). screening recorded demographics and self - reported indicators ; anthropometric measures included waist - to - hip ratio, bmi, and a single - arterial blood pressure reading ; and blood testing included nonfasting samples for glucose, ldl and hdl cholesterol, and hba1c. mmol / l) or missing hba1c and glucose data from the first round of screening were invited back for further investigation. fasting glucose levels (12 h fasting), oral glucose tolerance test (ogtt) using a 75-g glucose load in line with world health organization guidelines (7), and hba1c levels were recorded at follow - up. only individuals attending public facilities hba1c was measured on whole blood using the cobas integra instrument in line with the national glycohemoglobin standardization program, standardized to the diabetes control and complications trial reference assay (8). all statistical analyses were conducted using stata version 10.0 (statacorp lp, college station, tx). the screening test was hba1c (ranging from 6.1 to 7%) and random glucose (11.1 diagnostic testing determined sensitivity, specificity, positive predictive value, and negative predictive value. the receiver operating characteristic (roc) curve areas were determined to compare area under the curve (auc) for sensitivity versus 1 specificity. all statistical analyses were conducted using stata version 10.0 (statacorp lp, college station, tx). the screening test was hba1c (ranging from 6.1 to 7%) and random glucose (11.1 diagnostic testing determined sensitivity, specificity, positive predictive value, and negative predictive value. the receiver operating characteristic (roc) curve areas were determined to compare area under the curve (auc) for sensitivity versus 1 specificity. mean (95% ci) for bmi and waist circumference were 30.4 kg / m (29.930.9) and 97 cm (95.898.1). means for systolic blood pressure and diastolic blood pressure, ldl and hdl cholesterol, triglyceride, and fasting and 2-h postload glucose levels were within normal ranges. hba1c was diagnostic of diabetes using the american diabetes association criteria in 216 (21.0%) of the study sample. table 1 shows the sensitivity, specificity, positive predictive value, negative predictive value, and auc for various thresholds of hba1c against ogtt as the reference test. the results show that the hba1c threshold of 6.4% had the highest auc of 0.78 (95% ci 0.750.82) with sensitivity of 72% (6578%) and specificity of 84% (8287%). using an hba1c threshold of 6.4% to diagnose diabetes would have resulted in 72% of patients with diabetes being correctly diagnosed and 16% being incorrectly diagnosed. sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and area under the receiver operating characteristics curve comparing various thresholds of hba1c with the who criteria for diabetes data in parentheses are 95% cis. the following criteria were used : fasting glucose 7.0 mmol / l or 2-h glucose (post75-g glucose load) 11.1 mmol / l, plus random glucose level 11.1 mmol / l. a comparison of the study cohort with the full weqaya cohort found higher mean age in this study at 42 years compared with 35 years (p < 0.001). nondiabetic patients in this cohort had higher mean age, bmi, waist circumference, systolic blood pressure, diastolic blood pressure, and ldl cholesterol than nondiabetic patients from the weqaya cohort. for diabetic patients, mean age and systolic blood pressure were lower in this cohort than in diabetic patients in the weqaya cohort. this is one of the first studies to demonstrate the utility of hba1c for diagnosing diabetes and has shown that 6.4% is an optimum hba1c threshold for the abu dhabi national population. studies repeatedly demonstrate that within populations, over one - third of all patients with diabetes remain undiagnosed, with resulting complications due to late diagnosis (9). the availability of a single nonfasting blood test could facilitate successful population - level screening programs. hba1c is an independent predictor of cardiovascular events in patients with diabetes and nondiabetic subjects (10), which makes hba1c data from screening programs a valuable resource for public health planning. in this study, an hba1c threshold of 6.4% yielded the highest auc (0.78). a recent study in china comparing hba1c screening with ogtt demonstrated sensitivity, specificity, and auc of 63%, 96%, and 0.86, respectively (11). sensitivities and specificities reported using fasting plasma glucose as the reference in the chinese study were 56 and 97% for their optimum hba1c threshold of 6.4% (11) and in national health and nutrition examination survey data were 59 and 97% for their optimum hba1c threshold of 5.8% (12). the higher sensitivity and lower specificity in the weqaya study may be due to population factors affecting glycation of hemoglobin, such as hemoglobinopathies and other abnormal hemoglobin types, anemias (13), vitamins c and e, iron deficiency, and some medication use (14). different ethnic groups have also been shown to have hba1c levels up to 0.40.7% higher than caucasian populations despite similar glucose levels (15). the choice of hba1c threshold for clinical practice will depend on whether the tool is being used for screening or diagnosis. because hba1c has now been recommended for use as a diagnostic test, specificity is increasingly important, and the threshold of 6.4% in this study provides a high specificity without too much compromise of sensitivity. this study is the first in a middle eastern population to screen at population level using hba1c and has been shown to be representative (3) ; however, some differences were shown in nondiabetic subjects between this study sample and the overall weqaya population. hba1c has been validated as a suitable test for the definitive diagnosis of diabetes in the weqaya population screening program in a high - risk middle - eastern population. this is a major step in the fight to tackle the increasing burden of diabetes in the uae. studies repeatedly demonstrate that within populations, over one - third of all patients with diabetes remain undiagnosed, with resulting complications due to late diagnosis (9). the availability of a single nonfasting blood test could facilitate successful population - level screening programs. hba1c is an independent predictor of cardiovascular events in patients with diabetes and nondiabetic subjects (10), which makes hba1c data from screening programs a valuable resource for public health planning. in this study, an hba1c threshold of 6.4% yielded the highest auc (0.78). a recent study in china comparing hba1c screening with ogtt demonstrated sensitivity, specificity, and auc of 63%, 96%, and 0.86, respectively (11). sensitivities and specificities reported using fasting plasma glucose as the reference in the chinese study were 56 and 97% for their optimum hba1c threshold of 6.4% (11) and in national health and nutrition examination survey data were 59 and 97% for their optimum hba1c threshold of 5.8% (12). the higher sensitivity and lower specificity in the weqaya study may be due to population factors affecting glycation of hemoglobin, such as hemoglobinopathies and other abnormal hemoglobin types, anemias (13), vitamins c and e, iron deficiency, and some medication use (14). different ethnic groups have also been shown to have hba1c levels up to 0.40.7% higher than caucasian populations despite similar glucose levels (15). the choice of hba1c threshold for clinical practice will depend on whether the tool is being used for screening or diagnosis. because hba1c has now been recommended for use as a diagnostic test, specificity is increasingly important, and the threshold of 6.4% in this study provides a high specificity without too much compromise of sensitivity. this study is the first in a middle eastern population to screen at population level using hba1c and has been shown to be representative (3) ; however, some differences were shown in nondiabetic subjects between this study sample and the overall weqaya population. hba1c has been validated as a suitable test for the definitive diagnosis of diabetes in the weqaya population screening program in a high - risk middle - eastern population. this is a major step in the fight to tackle the increasing burden of diabetes in the uae. | objectivethe validity of hba1c as a population diagnostic tool was tested against oral glucose tolerance testing in abu dhabi nationals.research design and methodsthe screening tool of hba1c and random glucose was validated against the gold standard oral glucose tolerance test according to world health organization criteria.resultsthe hba1c threshold of 6.4% provided the optimum balance between sensitivity (72.0%) and specificity (84.3%) with positive and negative predictive values of 47.9 and 93.7% and area under the receiver operating characteristics curve of 0.78. this threshold compares with a threshold of 6.5% recommended by the international scientific committee and american diabetes association and of 6.3% in a recent study in china.conclusionsthis study successfully validates the feasibility and threshold of hba1c for diagnosis of diabetes at the population level in a middle - eastern population. this result is a major step in the fight to tackle the increasing burden of diabetes in the united arab emirates. |
the american demographer and sociologist valerie oppenheimer wrote a series of influential articles in which she emphasized the role of men s socioeconomic position in demographic change, in particular in the declining rates of marriage and the underlying tendency to increasingly postpone and perhaps even forego marriage (oppenheimer 1988, 2000, 2003 ; oppenheimer. i review oppenheimer s original theoretical study, i discuss how her study was held up in empirical research in europe, and i provide a new test of the theory for the european setting. in doing so, i try to resolve some remaining gaps in the empirical literature, and i evaluate whether the theory is equally valid in different countries that make up the european context. given the recent economic crisis in the united states and in europe, and the growing concerns about economic inequality, the influence of men s economic position on marriage and family formation remains a vital concern. at the time oppenheimer began writing her articles on how men s economic position influenced marriage formation in the late 1980s and early 1990s this was generally not a popular idea. the declining rates of marriage and increasing rates of divorce were typically conceptualized in terms of an " erosion of marriage. " one theory looked for the culprit in the growing economic role of women in society. this theory was voiced by demographers and economists working from a micro - economic perspective (becker 1981 ; espenshade 1985 ; farley 1988), although, as oppenheimer noted (1988, p. 575), it bore a strong resemblance to classic sociological theories formulated by functionalists like talcot parsons (parsons 1949). the explanation basically argued that more symmetrical economic roles of men and women would lead to a decline in the gains to marriage, or to put it in parsonian terms, would undermine marital solidarity. the second explanation argued that the decline of marriage was related to value change, and in particular to the increasing need for individual autonomy on the one hand, and the ideological condemnation of traditional institutions like marriage on the other. this second perspective was expressed more strongly by european demographers like lesthaeghe and van de kaa although it was also used by the influential american demographers at the time (bumpass 1990 ; rindfuss and van den heuvel 1990). in their second demographic transition theory, lesthaeghe and van de kaa argued that ideological change in combination with secularization was driving not only the postponement of marriage, but also the increase in cohabitation, the rise in divorce, and the decline of fertility (lesthaeghe 1983 ; lesthaeghe and meekers 1986 ; lesthaeghe and surkuyn 1988 ; van de kaa 1987). while the first explanation saw the engine of the demographic transition in economic change, the second emphasized the primacy of cultural change. both theories, however, were pessimistic about the future of marriage : the economic perspective saw marriage as incompatible with symmetrical gender roles, the second saw it as incompatible with individualistic values. while there was a considerable debate between the proponents of economic and cultural explanations, oppenheimer criticized both perspectives. for example, she noted that there were no signs of a so - called independence effect. women with attractive economic resources were not less likely to enter marriage, as would be predicted from the micro - economic perspective (oppenheimer and lew 1995). although women s employment and education had an effect on fertility and divorce, this did not appear to be the case for marriage timing (oppenheimer 1997). oppenheimer also had empirical critique on the cultural perspective. when looking at simple descriptive statistics on what people want for themselves on people s hopes and desires she noted that the majority of both single men and women still wanted to be married (oppenheimer 1994). the anti - marriage ideology may have existed in feminist circles or in the pop culture of the sixties, but it had not spread to a larger audience in the way that, for example, egalitarian gender norms had done. oppenheimer also had theoretical criticisms of the two explanations (oppenheimer 1994, 1997). first, she believed that the theories were basically about nonmarriage and not about delays in marriage. as other demographers also had observed, the declining marriage rate was primarily driven by increases in the age at marriage, and not so much by a decline in the proportion of persons who marry eventually, although the latter could of course not yet be observed in the late 1980s. this seems by and large correct now, although the proportion of the marrying persons among the lower educated in the united states did appear to decline (goldstein and kenney 2001). a second part of her theoretical critique was against the micro - economic model of specialization. quoting historical demographic work, oppenheimer noted that wives in the past had always worked for pay when circumstances required this. wives worked to make ends meet when the husband was not making enough money, when he was unemployed, or when household costs were temporarily pressing (oppenheimer 1982). oppenheimer argued that specialization in marriage is an inflexible and risky strategy in many different societal contexts. if marriage was not based on a model of full specialization in the more distant past, oppenheimer argued, why would it then cease to exist in the modern era in which wives began to work ? oppenheimer not only criticized the then dominant perspectives on demographic change, she also presented an alternative. her explanation can be placed in the economic rather than the cultural camp, but it was different in that it focused on men rather than women. during the 1980s and 1990s, young men s economic position in the united states had deteriorated quickly, especially for those with little schooling. in the poor and uncertain economic prospects of young men, oppenheimer saw an important potential for understanding the decline of marriage. because the earlier explanation had focused more on women especially through arguments about women s economic independence one could say that oppenheimer was in fact " bringing men back into the debate. " first, she reinstated older malthusian ideas about the economic costs of marriage (hajnal 1965 ; easterlin 1980). as setting up and running a household costs money, men who are unable to fulfill oppenheimer recognized that this traditional male - breadwinner hypothesis may have lost some of its force when gender roles become more symmetrical. nonetheless, she argued that it would also be naive to expect men s economic resources to become unimportant in influencing marriage prospects : this would be " throwing out the baby with the bathwater. " the second way in which she brought men back in the debate was through her uncertainty hypothesis (oppenheimer 1988). the argument is that unstable careers, as indicated by low - status jobs, nonemployment, and irregular and temporary employment, signal uncertainty. this uncertainty applies not only to whether the husband will be able to provide in the future, but also to the type of life he will lead. work structures the lifestyle a person will develop, and when men have not yet settled in their career it is difficult to predict what married life will be like. in this way. an important difference between the breadwinner and the uncertainty hypotheses is that the former focuses primarily on the financial aspects of employment whereas the latter is also concerned with its social consequences. an implication is that the neo - malthusian argument would be fully covered by effects of income, whereas the uncertainty argument would also be reflected in indicators like irregular attachment to the labor market, the amount of work experience, career trajectories, and temporary employment. in a separate article, oppenheimer also developed and operationalized the concept of a stopgap job, i.e., a job that is not a reflection of an employee s educational credentials and that is meant as temporary by both employer and employee (oppenheimer and kalmijn 1995). men in such stopgap jobs would postpone marriage because they are not settled in their career and therefore can not yet make a suitable match in the marriage market. compared to the other two perspectives, oppenheimer s theory has a more optimistic implication for the future of marriage. after all, female labor force participation was unlikely to decline in the future and individualism did not appear to be receding. in oppenheimer s theory, the economic position of young men largely depends on macro - economic conditions. because unemployment rates tend to have cyclical rather than linear trend patterns, the economic position of young men could improve and this would then have positive repercussions for marriage. moreover, the theory only implies the postponement of marriage until men accumulate more work experience and become settled in their career, and not an erosion of the institution of marriage, as the other theories seem to imply. oppenheimer 's explanation had a second attractive feature : it could also explain another important demographic trend, namely the rise in cohabitation (oppenheimer 2003). oppenheimer argued that a man s failure to provide economically would be less of a problem for cohabitation than for marriage. for many couples, cohabitation is a trial stage before marriage, and it may be that uncertainty about a young man s position is more tolerable during the cohabiting stage than it would be for a long - term commitment to marriage. assuming that the costs of breaking up a cohabiting union are lower, cohabitation can therefore provide a way for couples to reduce uncertainty about future career prospects. in a sense, oppenheimer argued that a cultural innovation like cohabitation before marriage (on a massive scale) was the outcome of economic needs rather than the result of ideological change. in line with this, other authors even argued that cohabitation is a rational response to uncertainty : a flexible partnership well - suited for a flexible labor market (mills. american research generally supports the view that poor economic prospects for men are associated with a delay in marriage. this has been demonstrated for a range of indicators, including employment per se, unstable employment, low earnings, and other indicators of career " immaturity " (clarkberg 1999 ; lichter. 1992 ; lloyd and south 1996 ; mare and winship 1991 ; oppenheimer 2003 ; oppenheimer. 1997 ; sassler and schoen 1999 ; smock and manning 1997 ; sweeney 2002 ; xie. there is also evidence in the united states that cohabitation is less strongly influenced by men s economic position than marriage, although there is no clear reverse income effect, i.e., that the poor are being selected into cohabiting unions. furthermore, in the united states, the income effect on marriage timing appears to be stable over time. sweeney (2002) compared two cohorts in the united states and found that in the cohort marrying during the 1980s and 1990s, men s income had an equally strong positive effect on the entry into marriage as in the cohort marrying during the 1960s and 1970s (sweeney 2002). in this article, i develop a new test of oppenheimer s theory for the european context. first, the demographic trends that occurred in europe were similar, although sometimes less dramatic and sometimes occurring later. the age at marriage has risen, the rate of marriage has declined, and cohabitation has increased as well (kiernan 2002 ; lesthaeghe 1983 ; van de kaa 1987). second, many european countries experienced economic problems that were similar to those in the united states. they were especially salient for outsiders on the labor market, such as young adults, ethnic minorities, and women. several authors argued that partly in response to economic globalization, young european men (and women) faced increasing levels of economic uncertainty in their transition from school to work (blossfeld. 2005). in many european countries, especially in southern europe, levels of youth unemployment were even higher than in the united states, a phenomenon which has often been linked to the higher degrees of employment protection in europe (mller and gangl 2003 ; nickell 1997). there are also reasons to believe that the theory would be less applicable to europe. one counter argument lies in the role of the welfare state. in several european societies, and particularly in social - democratic welfare states like sweden, denmark, and the netherlands, social security is more generous and more universally provided than it is in the united states (arts and gelissen 2002 ; esping - andersen 1993). this means that in many european countries, young men receive unemployment benefits when they are out of work. moreover, for those who have never worked, basic welfare is provided, albeit at a minimum level. as a result following the neo - malthusian argument, it could thus be argued that employment problems do not per se lead to marriage postponement in europe. a rebuttal of this point is that oppenheimer s argument about uncertainty and assortative mating, which is not only about money, but also about stability and predictability, could still apply to europe. a young man who is on unemployment benefits remains an uncertain candidate on the marriage market even if he has the financial means to support a household at that point in time. another important difference between the american and the european case lies in the degree of heterogeneity. although the united states is certainly not a homogeneous country there are important ethnic, racial, and regional differences it is fair to say that europe is more heterogeneous (at least regionally) than the united states. in comparative studies, it has often been argued that european countries can be rated on a continuum from more traditional societies such as spain, greece, and italy on the one hand, to more modern and more economically developed societies such as sweden and the netherlands on the other (hagenaars. these differences are expressed in a number of social and cultural domains, including differences in marriage and family living. for example, in more traditional european societies, cohabitation and divorce are less common and less accepted, marriage has a higher social status, gender roles in marriage are more unequal, female labor force participation is lower, and extended family ties are stronger (hans - peter blossfeld and hakim 1997 ; gelissen 2003 ; kalmijn 2003 ; knudsen and waerness 2008 ; reher 1998). these indicators are strongly correlated, both with each other and with the level of economic development in a country (gdp). this degree of heterogeneity suggests that oppenheimer s theory may not apply equally to all european countries. for example, in contexts where gender roles are more egalitarian, men s economic situation could be less important for the entry into marriage and cohabitation. in these settings, men are not the only breadwinners and women s economic resources should be of growing importance, making men s economic resources less important. what has the evidence in europe shown so far ? perhaps the most important source of evidence comes from a large multi - nation project initiated by the german sociologist blossfeld and his colleagues (blossfeld. in this project, blossfeld brought together a number of demographers and sociologists from different parts of the world (with an overrepresentation of european countries), with the aim of examining the effect of men and women s individual economic resources on the timing of marriage and parenthood. while the authors used their own country - specific longitudinal data, they used similar methods and variables, leading to a reasonably uniform and comparable set of outcomes. the project s goal was to test the exact same set of hypotheses in each country. the hypotheses were borrowed in part from oppenheimer s work but they were translated by mills and blossfeld to make them fit for a broader societal setting (mills and blossfeld 2005). the articles were combined in a volume for which oppenheimer wrote the foreword (blossfeld. the articles in blossfeld s volume provide generally positive evidence for the theory in the european countries studied (germany, the netherlands, france, sweden, hungary, great britain, italy, and spain). in virtually all countries, school enrollment one of the indicators of uncertainty negatively affected the entry into marriage. more importantly, men s unemployment appeared to lower the chances of entering marriage in most countries (bernardi and nazio 2005 ; kieffer. 2005 ; liefbroer 2005 ; noguera. 2005 ; robert and bukodi 2005). in britain, an effect of unemployment was observed only on the transition from cohabitation to marriage, and not on the transition from being single to living together (francesconi and golsch 2005). in sweden, only unemployment after leaving school appeared to delay marriage formation, not unemployment after a period of employment (bygren. 2005). some evidence was also found for the effect of temporary contracts. in italy, spain, france, and the netherlands, it was shown that men who were employed temporarily were less likely to enter marriage than men who had permanent employment. in germany and hungary, there was no effect of temporary work, however, and in several other countries, the effect could not be studied. a recent analyses of fertility in europe has also pointed to the delaying effect of temporary contracts (adsera 2011). outside the blossfeld project, there were a number of important individual articles in which aspects of oppenheimer s theory were tested. for example, in sweden, it was found that men s employment increased the chances of union formation while it did not affect the chances of marriage after cohabitation (bracher and santow 1998). in norway, men s employment increased the chances of marrying after being single and the chance of marrying after living together (kravdal 1999). in the netherlands, men s employment had a stronger effect on direct marriage than on cohabitation but there was no effect of employment on marriage after cohabitation (kalmijn and luijkx 2005 ; cf. the evidence outside europe (i.e., israel) has been supportive as well (raz - yurovich 2010), as has been the evidence in central and eastern europe, a region not included in the present article (gerber and berman 2010). while the role of employment has often been studied in europe, less is known about how men s income and earnings affect union formation. many of the studies discussed above were based on retrospective rather than prospective longitudinal data. the panel data that exist have been collected by economists and labor market researchers and do not always have the desirable demographic indicators. because income can not be measured well in a retrospective fashion, this has also meant that we know little about the income effects on marriage and divorce in europe. this is unfortunate because employment and income need to be examined simultaneously, especially if one wants to make a distinction between the neo - malthusian breadwinner hypothesis on the one hand, and oppenheimer s uncertainty hypothesis on the other. because in many european welfare states nonemployment does not, per se, another drawback of the prevailing evidence is that most studies are based on single countries. blossfeld s multi - nation project is clearly a major step forward in trying to summarize the evidence for europe as a whole, but the analyses are not pooled so the results can only be summarized verbally. moreover, possible differences that exist between countries can be described but they can not be compared or tested in a more rigorous fashion. for these reasons, in answering this question, i not only look at employment, but also i look at men s income, work experience, and type of labor contract. by looking at income and employment patterns simultaneously, i obtain more direct evidence on the underlying mechanisms. the period for which i answer unemployment rates increased substantially in the early 1990s before declining again in the mid to late 1990s (oecd 2009). in italy, greece, and belgium, unemployment remained high in the late 1990s but began to decline later, in the early 2000s. in other words, for most countries, the period that i examine covers a recovery stage of the economy, a stage which should have been positive for marriage and family formation. second, are the effects of men s economic position similar or different for the chances of entering marriage and the chances of entering a cohabiting union ? with this part of the study, i replicate the last influential study of oppenheimer (2003), in which she studied this issue for the united states. we would expect effects to be weaker for cohabitation than for marriage : marriage would require a stronger economic underpinning than cohabitation (kravdal 1999 ; oppenheimer 2003). in addition, i examine the chances that cohabiting unions turn into marriage. here too, oppenheimer expects men s economic position to have an effect, but because those who cohabit already have an independent household, the effects of income will probably be weaker. third, to what extent are the effects of men s economic position on union formation different across societal contexts ? in this part, i focus on differences between traditional and egalitarian societies. the expectation is that men s economic characteristics remain important in traditional societies but are less important in more modern, egalitarian ones. by looking at differences among societal contexts, i try to generalize the cross - cohort comparison that sweeney (2002) made for the united states. answering this question is also of more general importance because if we find conditions under which the theory is (not) true, this could in principle lead to theoretical progress in the field. i use panel data that are collected in the same format for a number of european countries, i.e., the european community household panel (echp). the echp was an annual panel survey held between 1994 and 2001 (behr. samples are large and representative, and (almost) the same questionnaire was used each year. for the analyses in this article, i use data from 13 countries : denmark, finland, germany, austria, the netherlands, belgium, france, the united kingdom, ireland, portugal, spain, italy, and greece. my sample is limited to men who were never married in the first wave of the panel. although i am able to exclude previously married men, i can not exclude men who ended a cohabiting union before the panel began. never married men who were cohabiting in the first wave are also included because these men can make the transition from cohabitation to marriage. the first wave of data from the netherlands is excluded because no information is available on cohabitation status there have been previous demographic analyses of the echp most notably by adsera (2011) who analyzes the effect of individual and aggregate labor market characteristics on fertility. although adsera focused more on women than on men, her general conclusion is that labor market uncertainty is very influential in delaying fertility, in line with the perspective suggested above. in the current article, we go back one step by analyzing how labor market uncertainty affects union formation, a transition which probably remains the most important necessary condition for family formation. i use discrete time event - history analysis by estimating logistic regression models on a person - year file. the person - year file begins at the first wave and ends in the last wave or when a transition is made. as is the case with all event - history analyses of panel data, such left truncation problems can be solved in principle, but not without losing our time - varying independent variables (guo 1993). the first logit model is estimated for person - years in which men are single and never married. the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t + 1 (union formation). in the second logit model, i estimated which choice was made, cohabitation or marriage, using only the person - years in which the event occurred. this sequential approach is slightly different from the approach taken by oppenheimer, who estimated multinomial logit (i.e., competing risk) models (2003). the sequential approach does not need the assumption implicit in a competing risk model that the two choices this assumption is problematic because it is plausible that unmeasured factors like personality, wealth, attractiveness, and the like, influence marriage and cohabitation to the same extent (hill. if a person is single in year t, missing in year t + 1, and married or cohabiting in year t + 2, i also regarded this as an event. duration dependency is modeled with two age effects, according to the approach developed by blossfeld and huinink (1991). blossfeld used log (age a1) and log (a2age) to capture the nonlinear age - dependency of union formation, where a1 is the lowest and a2 the highest age in the sample. there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage. the second logit model is estimated for person - years in which men were living with a partner without being married. the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t + 1 (marriage after cohabitation). respondents who were living alone in wave t + 1 are truncated. hence, separation is treated as a competing risk. if a person is cohabiting in year t, not in the panel in year t + 1, and married in year t + 2, i also regarded this as an event. duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel, no data on the start of the union is available. as an alternative age is obviously less ideal than duration since persons enter a cohabiting union at different ages. it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave. hence, i could not check if a man changed (cohabiting) partners between the subsequent waves, nor could i check if the married partner was the same person as the cohabiting partner in the previous wave. all independent variables, except where noted, refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t + 1, the independent variables precede the dependent variable in time. the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15 h a week. the second variable indicates other less common forms of employment, such as military service, apprenticeships, unpaid family work, and working less than 15 h. given that a man is employed, i also considered the type of contract he has. temporary contracts include fixed - term or short - term contracts, casual work with no contract, and " some other working arrangement " that is not a permanent contract (all included within the 15 + hours category). the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract. i also include whether a person is enrolled in full - time schooling in the interview week. data on work history are obtained from a monthly calendar that all respondents had to fill out. for the year t 1, i counted the number of months that a man was employed, self - employed, or in school (hereafter called " active "). i checked whether the effects of this variable were different when excluding the netherlands but this was not the case. i consider personal income and not only income from work since social security income may also provide a stable source of income. ideally, we would like to measure the income a man had in the interview month or in the 12 months before the interview. unfortunately, incomes are measured for (full) calendar years only. to solve this, i took a weighted average of the income in calendar year t 1 and the income in calendar year t, using information on the month of interview. for example, if the interview was in september 1994, income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993. it is noted that some of the income in year t may be earned after a man marries. marrying in the year of the interview is more likely when the interview took place early in the year, but then, this income receives a lower weight. i corrected all incomes for changes in the consumer price index and converted them to pounds sterling. to estimate effects of income in a comparable way across countries i first calculated income quintiles in each country using data from all men in the first wave of the echp. next, i used these quintiles to categorize the men in the person - year file. i also use a linear income variable which is coded 15 for the five quintiles. three control variables are used which may affect union formation : general health (time varying), the highest level of educational attainment, and the year of the interview (using dummies). health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities (waldron. descriptive findings for the independent variables for each country are presented in table 1.table 1means of independent variables in person - year file by countryyear (18)agegeneral health (15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active (012)relative income (15)income in poundsuk (n = 7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland (n = 7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany (n = 9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria (n = 5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france (n = 11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium (n = 3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands (n = 5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy (n = 18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece (n = 7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain (n = 12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal (n = 10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland (n = 5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark (n = 5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total (n = 111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp (own calculations) means of independent variables in person - year file by country source : echp (own calculations) to explore differences across societies, i constructed two measures at the macro level. first, i considered a measure of the division of household labor in marriage in a society. this measure was obtained from a article by knudsen and waerness (2008) and measures the extent to which husbands and wives share four household tasks (i.e., laundry, grocery shopping, meal preparation, caring for sick family members). the second measure is the female labor force participation rate in the 1990s, which is defined as the percentage of women aged 2049 who are active in the labor force (obtained from ilo, geneva).. countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally (r =.74). the most traditional societies are southern european countries, the most egalitarian are northern european countries. i construct a single macro - level indicator, which is the means of the two standardized items. i use discrete time event - history analysis by estimating logistic regression models on a person - year file. the person - year file begins at the first wave and ends in the last wave or when a transition is made. as is the case with all event - history analyses of panel data, such left truncation problems can be solved in principle, but not without losing our time - varying independent variables (guo 1993). the first logit model is estimated for person - years in which men are single and never married. the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t + 1 (union formation). in the second logit model, i estimated which choice was made, cohabitation or marriage, using only the person - years in which the event occurred. this sequential approach is slightly different from the approach taken by oppenheimer, who estimated multinomial logit (i.e., competing risk) models (2003). the sequential approach does not need the assumption implicit in a competing risk model that the two choices this assumption is problematic because it is plausible that unmeasured factors like personality, wealth, attractiveness, and the like, influence marriage and cohabitation to the same extent (hill. if a person is single in year t, missing in year t + 1, and married or cohabiting in year t + 2, i also regarded this as an event. duration dependency is modeled with two age effects, according to the approach developed by blossfeld and huinink (1991). blossfeld used log (age a1) and log (a2age) to capture the nonlinear age - dependency of union formation, where a1 is the lowest and a2 the highest age in the sample. there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage. the second logit model is estimated for person - years in which men were living with a partner without being married. the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t + 1 (marriage after cohabitation). respondents who were living alone in wave t + 1 are truncated. hence, separation is treated as a competing risk. if a person is cohabiting in year t, not in the panel in year t + 1, and married in year t + 2, i also regarded this as an event. duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel, no data on the start of the union is available. as an alternative, age is obviously less ideal than duration since persons enter a cohabiting union at different ages. it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave. hence, i could not check if a man changed (cohabiting) partners between the subsequent waves, nor could i check if the married partner was the same person as the cohabiting partner in the previous wave. all independent variables, except where noted, refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t + 1, the independent variables precede the dependent variable in time. the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15 h a week. the second variable indicates other less common forms of employment, such as military service, apprenticeships, unpaid family work, and working less than 15 h. given that a man is employed, i also considered the type of contract he has. temporary contracts include fixed - term or short - term contracts, casual work with no contract, and " some other working arrangement " that is not a permanent contract (all included within the 15 + hours category). the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract. i also include whether a person is enrolled in full - time schooling in the interview week. data on work history are obtained from a monthly calendar that all respondents had to fill out. for the year t 1, i counted the number of months that a man was employed, self - employed, or in school (hereafter called " active "). i checked whether the effects of this variable were different when excluding the netherlands but this was not the case. i consider personal income and not only income from work since social security income may also provide a stable source of income. ideally, we would like to measure the income a man had in the interview month or in the 12 months before the interview. unfortunately, incomes are measured for (full) calendar years only. to solve this, i took a weighted average of the income in calendar year t 1 and the income in calendar year t, using information on the month of interview. for example, if the interview was in september 1994, income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993. it is noted that some of the income in year t may be earned after a man marries. marrying in the year of the interview is more likely when the interview took place early in the year, but then, this income receives a lower weight. i corrected all incomes for changes in the consumer price index and converted them to pounds sterling. to estimate effects of income in a comparable way across countries, i first calculated income quintiles in each country using data from all men in the first wave of the echp. next, i used these quintiles to categorize the men in the person - year file. i also use a linear income variable which is coded 15 for the five quintiles. three control variables are used which may affect union formation : general health (time varying), the highest level of educational attainment, and the year of the interview (using dummies). health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities (waldron. descriptive findings for the independent variables for each country are presented in table 1.table 1means of independent variables in person - year file by countryyear (18)agegeneral health (15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active (012)relative income (15)income in poundsuk (n = 7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland (n = 7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany (n = 9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria (n = 5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france (n = 11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium (n = 3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands (n = 5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy (n = 18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece (n = 7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain (n = 12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal (n = 10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland (n = 5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark (n = 5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total (n = 111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp (own calculations) means of independent variables in person - year file by country source : echp (own calculations) to explore differences across societies, i constructed two measures at the macro level. first, i considered a measure of the division of household labor in marriage in a society. this measure was obtained from a article by knudsen and waerness (2008) and measures the extent to which husbands and wives share four household tasks (i.e., laundry, grocery shopping, meal preparation, caring for sick family members). the second measure is the female labor force participation rate in the 1990s, which is defined as the percentage of women aged 2049 who are active in the labor force (obtained from ilo, geneva).. countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally (r =.74). the most traditional societies are southern european countries, the most egalitarian are northern european countries. i construct a single macro - level indicator, which is the means of the two standardized items figure 1 shows that there are small differences within europe in the age at first union entry. in most countries, male age at entry is low in portugal and austria, and high in the netherlands and italy. for example, the age at union formation is quite high in italy and greece where the family is a strong institution. europe, youth unemployment is high. when we look at the type of first union chosen by men, presented in the left side of fig. 1 even more in northern europe, direct marriage is a minority experience. in many countries, more than 80% of men cohabit first. in southern europe, the proportions are almost reversed : 6080% of the men in these countries marry directly. the united kingdom resembles western europe but ireland resembles the south, with most men choosing marriage as their first way to enter a union. 1men s first union formation in 13 countries men s first union formation in 13 countries the effects of men s economic characteristics on union entry are presented in table 2. the odds of entering a union are 58% higher for employed men than for men who are not employed and not in school (e). the type of labor contract also matters for the choice between marriage and cohabitation. compared to men with a permanent job, men with a temporary job who enter a union are 23% less likely to choose marriage than cohabitation. we also note that school enrollment has a negative effect on union entry while it does not affect the type of union.table 2discrete time event history models of union formation : logit regression coefficients and p - values in parentheses union formationmarriage vs. cohabitationmarriage after cohabitationmodel amodel bmodel cmodel dmodel emodel fmodel gmodel hmodel iln (age15)0.841 (0.000)0.661 (0.000)0.676 (0.000)0.746 (0.000)0.745 (0.000)0.741 (0.000)0.230 (0.040)0.317 (0.006)0.316 (0.006)ln (65age)1.872 (0.000)1.816 (0.000)1.847 (0.000)0.810 (0.000)0.813 (0.000)0.809 (0.000)0.296 (0.038)0.264 (0.061)0.268 (0.058)general health0.106 (0.000)0.100 (0.000)0.098 (0.000)0.135 (0.012)0.135 (0.013)0.135 (0.012)0.107 (0.008)0.104 (0.010)0.103 (0.011)education secondary versus primary0.029 (0.491)0.002 (0.955)0.007 (0.867)0.046 (0.655)0.035 (0.731)0.047 (0.648)0.005 (0.951)0.012 (0.890)0.014 (0.873)education tertiary versus primary0.225 (0.000)0.141 (0.003)0.122 (0.010)0.113 (0.311)0.149 (0.194)0.111 (0.326)0.408 (0.000)0.361 (0.000)0.349 (0.000)working 15 + hours versus no work / school0.457 (0.000)0.193 (0.003)0.220 (0.001)0.450 (0.003)0.419 (0.007)0.445 (0.004)0.178 (0.166)0.090 (0.495)0.102 (0.434)other employed versus no work / school0.144 (0.088)0.204 (0.016)0.188 (0.026)0.279 (0.176)0.350 (0.096)0.282 (0.173)0.237 (0.277)0.250 (0.254)0.245 (0.263)temporary versus fixed contract0.067 (0.182)0.026 (0.609)0.031 (0.544)0.253 (0.033)0.262 (0.028)0.251 (0.035)0.120 (0.249)0.086 (0.410)0.090 (0.386)in school versus no versus no work / school0.623 (0.000)0.431(0.000)0.468 (0.000)0.180 (0.388)0.089 (0.679)0.173 (0.412)0.515 (0.015)0.410 (0.059)0.424 (0.046)months worked last 2 years0.022 (0.000)0.006 (0.243)0.007 (0.178)0.008 (0.518)0.011 (0.405)0.009 (0.504)0.031 (0.010)0.021 (0.089)0.023 (0.066)first income quintile versus third quintile0.604 (0.000)0.272 (0.054)0.256 (0.044)second income quintile versus third quintile0.253 (0.000)0.039 (0.735)0.182 (0.037)fourth income quintile versus third quintile0.232 (0.000)0.136 (0.210)0.094 (0.224)fifth income quintile versus third quintile0.366 (0.000)0.127 (0.328)0.109 (0.233)linear income (15)0.239 (0.000)0.007 (0.861)0.098 (0.001)constant11.814 (0.000)10.677 (0.000)11.595 (0.000)7.903 (0.000)7.789 (0.000)7.895 (0.000)3.346 (0.000)2.771 (0.000)3.138 (0.000)n776217762177621447244724472116781167811678224024602451162516301625335348346note : controlled for country and year dummiesp <.05, p <.01, p <.001 discrete time event history models of union formation : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies p <.05, p <.01, p <.001 next to the effects of current employment characteristics, we see effects of men s work history. the more months men were employed or otherwise active in the past calendar year, the more likely they are to enter a union in the next two calendar years. men who were active for an entire year were e = 30% more likely to enter a union in the next 2 years than men who were inactive the entire year. this effect comes on top of the effect of a man s current employment situation. there is no effect of work history on the choice between marriage and cohabitation. in sum, the findings from model a and model d confirm that men who are not yet settled in their career postpone union formation. the findings also confirm that cohabitation is less sensitive to economic insecurity than marriage, although this applies more to employment per se and the type of employment than to a man s work history. in model the poorest quintile of men are 45% less likely to enter a union than the middle quintile, while the richest quintile are 44% more likely to do so. the income effects increase monotonically but the differences between the quintiles become smaller at higher income levels. a formal test shows that the effect is not strictly linear : the nonlinear model for union formation (model b) has a better fit than the linear model (model c, = 9.3, p =.03). when we look at the income effect on choice between the marriage and cohabitation, we only see one marginally significant effect. men in the lowest quintile who enter a union are 31% more likely than the middle quintile to choose cohabitation rather than marriage (p =.054). this result seems to confirm the notion of cohabitation as the poor man s marriage, although the poor are still more likely not to enter a union in the first place. when we compare the two models for union entry (model a and b), the employment effects that we observed in model a are reduced by more than half once income is added to the model. in other words, the effects of employment and work history run in part via income. nevertheless, the employment effect remains statistically significant even when income is included, which means that the influence of employment on union formation also has a non - financial element. it is also interesting to observe that the effects of employment and temporary jobs on the choice between the marriage and cohabitation are not affected by whether or not income is added (compare model d and e). hence, the non - financial aspects of work are more important for the type of union than for the chance of entering a union in the first place. in this sense, oppenheimer s uncertainty theory seems to work better for the type of union than for union formation per se. this confirms theories about selection into marriage and suggests that screening or selection may be less strong for cohabitation. education, finally, has a positive effect on union entry but these effects are reduced when income is controlled for. in the last columns of table 2, we focus on marriage formation after cohabitation (model g, h, and i). we see that it is not affected by employment, but being enrolled in school does reduce the chance of marrying. we also find that men who worked more months in the past are more likely to marry. the higher the men s income during cohabitation, the more likely they are to change from cohabitation to marriage. when comparing the linear income effect on marriage after cohabitation with the linear effect on initial union formation, it appears that income is less important for marriage after cohabitation (b =.10) than it is for union formation after being single (b =.21). this is in line with the neo - malthusian hypothesis, which suggests that it is primarily for initial union entry that the costs of setting up a household play a role (although the wedding is a cost factor which applies specifically to marriage). men in good health are more likely to experience a transition from cohabitation to marriage, suggesting that the selection effects also play a role in the decision to marry, not only in the decision to live together. we also see that higher educated cohabiting men are more likely to marry, hence, for the higher educated, cohabitation is less often seen as a long - term option. do the effects we found vary across societal contexts ? to assess this, i present interaction effects of key independent variables with the macro - level indicator of traditional versus egalitarian societies. the p - values are based on standard errors that are corrected for the clustering of cases within countries. this yields a more conservative test for macro - level effects and is a good alternative to multilevel models when the number of units at the macro level is limited. to check for outliers at the country level, i calculated dfbetas for the interaction effects of modernization and employment, and of modernization and income, and re - estimated the models while leaving out countries for which dfbetas exceeded the critical value (kalmijn 2010 ; ruiter and de graaf 2006). dfbeta is calculated as the difference in an effect with and without the outlier divided by the standard error of the effect. these outlier analyses are presented in the third and fourth columns of table 3.table 3discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parenthesesmodel a all casesmodel b all casesmodel a w / o outliersmodel b w / o outliersln (age15)0.953 (0.000)0.781 (0.002)0.943 (0.001)0.686 (0.036)ln (65age)1.970 (0.000)1.956 (0.000)1.934 (0.001)2.040 (0.010)general health0.121 (0.001)0.107 (0.002)0.102 (0.006)0.119 (0.016)education secondary versus primary0.071 (0.287)0.023 (0.732)0.031 (0.638)0.115 (0.079)education tertiary versus primary0.271 (0.000)0.152 (0.014)0.248 (0.000)0.224 (0.001)working 15 + hours versus no work / school0.501 (0.000)0.194 (0.000)0.549 (0.000)0.175 (0.000)in school versus no versus no work / school0.672 (0.000)0.604 (0.000)0.614 (0.000)0.673 (0.000)income linear0.242 (0.000)0.243 (0.000)index (traditional egalitarian)1.916 (0.177)2.075 (0.144)2.092 (0.262)2.039 (0.327)index ln (age15)0.278 (0.058)0.314 (0.037)0.311 (0.110)0.217 (0.272)index ln (65age)0.233 (0.442)0.262 (0.386)0.288 (0.471)0.309 (0.485)index health0.003 (0.914)0.004 (0.872)0.007 (0.718)0.005 (0.897)index secondary education0.079 (0.333)0.087 (0.285)0.086 (0.368)0.121 (0.041)index tertiary education0.119 (0.106)0.130 (0.079)0.139 (0.127)0.205 (0.000)index working0.158 (0.001)0.122 (0.001)0.152 (0.000)0.098 (0.043)index enrolled0.236 (0.001)0.210 (0.002)0.215 (0.004)0.288 (0.000)index income0.002 (0.935)0.037 (0.008)constant13.107 (0.000)12.953 (0.000)12.853(0.000)13.112 (0.000)n77621776216883758354note : controlled for country and year dummies. outliers for model a are denmark and greece, for model b these are italy and the united kingdom p <.05, p <.01, p <.001 discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies. outliers for model a are denmark and greece, for model b these are italy and the united kingdom p <.05, p <.01, p <.001 in table 3, we see a negative interaction of the societal index with men s employment (b =.16, p <.01). hence, the average effect of employment (b =.50) is reduced by 32% for each standard deviation increase in the egalitarian context (.16/.50). this shows that the effect of men s employment on the entry into a union is considerably weaker in more egalitarian countries than in more traditional countries. i also present the effects for each country separately in a graph (fig., the effect of selected independent variables is plotted against the macro - level index. although the graph does not provide a test, like the interaction effect, it does give a good intuitive feel of the importance of the interaction effect. in line with expectations, the graph shows that in more traditional societies like spain, italy, and ireland, the effect of men s employment is quite strong. in more egalitarian countries like the netherlands, denmark, and finland, men s enrollment in school deters union formation but this effect is weaker in more egalitarian societies. s economic characteristics on union entry effects of men s economic characteristics on union entry table 3 further shows no negative interaction of the societal index with men s income effect on union formation. after deleting two outliers (italy and the united kingdom), the interaction becomes negative and significant (b =.037, p =.01). hence, for these 11 countries, the effect of men s income on union formation is weaker in more egalitarian countries than in more traditional countries. the magnitude of the effect is a 15% reduction in the income effect per standard deviation increase in the egalitarian context (.037/.234). when we look at the graph, the pattern appears to be weaker than it was for employment. spain and portugal reveal strong effects of men s income and denmark and the netherlands have weak effects. however, there are also outliers like the united kingdom (stronger effect than expected) and italy and belgium (weaker effect than expected). so far, these results apply to union formation regardless of the type of union. traditional and egalitarian societies also differ in the extent to which cohabitation occurs. the more egalitarian countries like denmark, finland, and the netherlands also have the highest levels of cohabitation (soons and kalmijn 2009). because the effects of men s economic position differ depending on whether marriage or cohabitation is the outcome, the results could in part be due to such compositional differences. for this reason, i also look at the entry into marriage only (either after being single or after cohabitation). the interaction with employment is.066 (p =.11), the interaction with enrollment is.326 (p <.01), and the interaction with income is.073 (p <.01). hence, the employment interaction effect is weaker in this model, although still negative, while the other interaction effects are not affected. we therefore conclude that part of the reason why the employment effects depend on the context lies in the fact that unions are more often unmarried unions in more egalitarian societies. even apart from that, however, there is evidence that men s economic status matters more for marriage in more traditional societies. this article has re - examined the importance of oppenheimer s theory on marriage timing in the european context. by and large unemployment, little work experience, low income, and temporary employment on the part of men deter union formation. by analyzing income and employment effects simultaneously in a panel perspective, it was possible to obtain more direct evidence for the two contrasting hypotheses suggested by oppenheimer, i.e., the neo - malthusian male breadwinner hypothesis and the career uncertainty hypothesis (sometimes also called the career instability or immaturity hypothesis). many previous european analyses have not been able to take income into account and have therefore not been able to separate these two mechanisms empirically. my analyses first show that income effects are strong and significant, which supports the male breadwinner hypothesis. second, the income effects explain about half of the effects of employment and work experience, suggesting that employment effects by themselves are not sufficient evidence for the uncertainty hypothesis. this suggests that employment effects on union formation are more than just a matter of financial resources, in line with the uncertainty hypothesis. by analyzing the choice between cohabitation and marriage, marriage in europe appears to be more sensitive to men s economic position than cohabitation. men who are not employed or who have temporary jobs are more likely to choose cohabitation rather than marriage. this finding provides additional evidence for the uncertainty hypothesis since cohabitation is more like a trial marriage and hence, more compatible with uncertainty in other life domains (i.e., employment). we do not, however, see a clear income effect on the choice between marriage and cohabitation. hence, the choice between marriage and cohabitation has more to do with employment uncertainty than with income. there is a small effect of the lowest income group, however. among men who enter a union, hence, there is some evidence that cohabitation is the " poor man s marriage. " following oppenheimer s last study, i also examined the transition from cohabitation to marriage. we would expect economic uncertainty to also reduce the chances of a transition from cohabitation to marriage, but in general, the effects we find are weaker for this transition. the weaker income effect could be due to the fact that the cost of setting up a household plays no role for this transition. while this is in line with the neo - malthusian argument, the finding that the employment effects on the transition from cohabitation to marriage are also weaker is unexpected. other authors have found this too (e.g., bracher and santow 1998 ; liefbroer 2005). if couples buy a house or have a child, they may decide to marry. although such transitions may be partly governed by economic uncertainty, they may also be exogenous and hence reduce the effects of other determinants like employment. another speculation has to do with the lack of information on the (female) partner. the partner may have become economically more secure during the cohabitation stage, thereby promoting the transition to marriage, but we do not observe such changes. european countries differ considerably in terms of their economic, social, and cultural characteristics, and so, it is important to also examine country variation in the degree to which the theory applies. we hypothesized that men s economic position would be less influential in more developed countries where gender roles are more symmetrical. the effects of men s employment and school enrollment on union formation are stronger in more traditional societies than in more egalitarian ones. income effects are also weaker in egalitarian societies but the evidence for this pattern is weaker. our interpretation is that in egalitarian settings, the costs of setting up a household are more often shared between men and women. hence, men can afford to experiment with their career if they have a partner who has a (more) stable career. similarly, women may attach less weight to the career options of men than they did in more traditional circumstances and for instance, pay more attention to other traits, such as men s willingness to participate in child rearing. my finding is in contrast to a previous cohort comparison for the united states which suggested that men s economic effects on marriage timing did not change over time (sweeney 2002). perhaps this difference is due to the limited time period that was examined in sweeney s trend study, a design which may have reduced the variance in contextual gender roles. at the same time, however, it needs to be investigated whether the patterns that i found still hold when a larger number of countries is considered. the income interaction effects, for example, are sensitive to outliers and therefore less convincing. more units at the macro - level will be needed to confirm this finding. in closing, it is important to re - emphasize on the role of women. although oppenheimer was justifiably " bringing men back " into the debate at a time when there was too little attention on the changing economic fate of young (american) men, the growing egalitarian model that is now supported by many couples in europe and the united states, suggests that men and women should be examined simultaneously for a better understanding of trends and differentials in marriage and cohabitation. traditionally, studies of women were largely done from a beckerian perspective in which it was argued that women s status would lead to a decline of marriage (and fertility). currently, we can speculate that a strong economic position and career stability on the part of women might foster marriage. it could be that certainity on just one side either the man or the woman alternatively, it could be that both the man s and the woman s career need to have been settled before couples enter a more committed union. in this respect, it is somewhat unfortunate that analyses of marriage (timing or formation) have often been one - sided, focusing either on men or on women. this design is unavoidable given that the partner of a respondent is typically observed too late, i.e., when the respondent is married or living with the partner. empirically, this problem does not exist when we observe cohabiting couples chances of marrying. some authors in the past have regarded the transition from cohabitation to marriage as a two - sided problem and have analyzed economic characteristics of both partners in one model (lichter. these studies have not been able to examine the more important entry into a first union, however. for this transition, the problem can, in principle, be solved by examining dating couples in a more systematic fashion. for example, prospective surveys could collect data on the school and work careers of both members of a dating couple and subsequently analyze whether or not (and when) they begin to live together, while using the dissolution of the dating relationship as a competing risk. in this way, the analysis of marriage and cohabitation can become truly two - sided and the economic characteristics of men and women can be included in one model. there are some innovative sociological studies of transitions from dating to cohabitation and marriage, but so far, they have not focused on the partners economic characteristics (joyner and kao 2005). | this article discusses oppenheimer s theory on marriage timing, reviews the way this theory was received in european demography and family sociology, and develops a new test of the theory using annual panel data from 13 european countries for the period 19942001. several indicators of men s economic status are used, including school enrollment, employment, type of labor contract, work experience, income, and education. effects of these indicators are estimated for the transition to marriage and cohabitation, as well as for the transition from cohabitation to marriage. country differences in these effects are examined as well. the evidence provides strong support for the male breadwinner hypothesis on the one hand, and for oppenheimer s career uncertainty hypothesis on the other. however, the relevance of these hypotheses also depends on the national context, and especially on the way gender roles are divided in a society. |
carotid atherosclerotic disease is a major risk factor for stroke and a major of systemic plaque burden2,6,8,13). also, the progression of carotid atherosclerosis can be contributed to variable factors as followed ; systemic factors such as age, sex, and statin therapy and local factors such as intraplaque hemorrhage (iph), ulcer, and thin fibrous cap6,17,19,20,21). in multiple trials using 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors or statins, reduction in plaque burden as measured by intima - media thickness complex carotid plaques included iph, ulcer, or thin - fibrous cap were associated with the occurrence of subsequent cerebrovascular events10,22,23). especially, iph into the carotid atherosclerotic plaque is significantly associated with more rapid progression in wall and lipid - rich necrotic core (lrnc) size, as well as more rapid progression in luminal stenosis20). high - resolution mri, as a noninvasive imaging tool, has proved to be a modality with excellent capability for discriminating tissues of the carotid plaque, including the status of the fibrous cap, lrnc, calcium, and hemorrhage10,19,20,21,22,23,27). t1-weighted mr sequences and three - dimensional (3d) time - of - flight (tof) angiography are commonly used to detect iph24,25,26). far more accurate detection of iph is now accomplished using advanced, heavily t1-weighted techniques including the magnetization - prepared rapid acquisition with gradient - echo (mprage) sequence. the mprage sequences for detection of iph using histological confirmation has demonstrated significantly higher sensitivity and specificity compared with the conventional t1 or tof sequences9). mcnally.7) reported that in the workup of acute stroke, carotid mprage - positive signal was associated with an increased risk of territorial cerebral ischemic events as detected objectively by brain diffusion tensor imaging. the relative risk of stroke was increased in all carotid stenosis categories but was most elevated in the mild stenosis category. however, this study has only documented the relationship between mprage - positive signals and cerebral ischemic events in western populations. the purpose of our study was to assess prevalence of iph and associations between territorial acute infarction and iph on mprage in patients with acute neurologic symptoms., 136 patients who underwent stroke mr protocol because of acute neurologic symptoms were imaged with the additional carotid mprage sequences. of these patients, 22 were excluded due to poor image quality and 31 were excluded from the analysis : 1) co - existent > 50% ipsilateral intracranial artery stenosis or luminal irregularity (n=13) ; 2) non - atherosclerotic vasculopathy, such as dissection or moyamoya disease (n=3) ; 3) evidence of cardioembolism such as atrial fibrillation or previous stroke history of cardiac origin (n=15). 166 arteries of 83 patients (61 males, 22 females ; age range 59 - 89, median age 69 years - old) are eligible for data analysis. main neurological symptoms of these patients were as fallows ; 21 dizziness, 14 dysarthria, 37 unilateral weakness, 9 mental change, and 2 hyperesthesia. all patients with acute neurological symptoms underwent the baseline brain ct scans for evaluating intracranial or subarachnoid hemorrhage in the emergency department. mr examination was obtained with an achieva 3.0-t scanner (philips medical systems, best, the netherlands) with a 16-channel head coil. stroke mr imaging was performed immediately following ct scanning with the following techniques : 1) diffusion - weighted imaging (dwi) ; 2) 3d tof mr angiography (mra) of the intracranial arteries ; 3) susceptibility - weighted imaging (swi) ; 4) perfusion - weighted imaging (pwi) ; and contrast - enhanced mra for evaluation of carotid artery. dwi was conducted with a spin - echo - type echo - planar imaging sequence, with 3 b - values of 0, 500, and 1000 sec / mm along all 3 orthogonal axes : repetition time (tr)/echo time (te)=3000/80 ms ; flap angle (fa)=90 ; sensitivity encoding=3 ; field of view (fov)=220220 mm ; matrix=128128 ; section thickness / gap= 5 mm/30% ; scanning time=35 - 38 seconds. axial dynamic gradient - echo echo - planar pwi was performed after tracking a bolus of 0.03 mmol / kg of gadofosvesettrisodium (vasovist, schering, berlin, germany). acquisition parameters were as follows : tr / te=1850/35 ms ; fa=40 ; fov=230230 mm ; matrix= 132132 ; and section thickness / gap=5 mm/30%. after image reconstruction and processing, pwi data were transferred to a workstation (adw 4.2 ; ge healthcare, milwaukee, wi, usa). for mra, 3d multislab tof - mra from the petrous portion of the internal carotid artery (ica) was generated with the following parameters : tr / te=23/3.45 ms ; fa=20 ; fov=200200 mm ; matrix=488249 ; sensitivity encoding factor=2.5 ; slice thickness=1.2 mm ; echo train length (etl)=1 ; and number of average=1. we also performed contrast - enhanced carotid mra from the aortic arch to whole brain by using coronal planes with a 3d spoiled gradient echo sequence optimized for high spatial resolution and an intravenous bolus injection of 0.03 mmol / kg of gadofosvesettrisodium (vasovist, schering, berlin, germany), which was administered with an automatic injector at a rate of 1 ml / s through an 18-gauge cannula placed in the antecubital vein of the right arm, followed by 15 ml of saline solution, and the following imaging parameters ; tr / te=4.9/1.7 ms, flip angle=27, slice thickness=1.0 mm, matrix size=384384, voxel size=110.7 mm. for 3d mrprage sequence, segmental acquisition was performed with the sequence tr, the inversion preparation time (ti), and the phase encoding order of the mprage sequences adjusted to optimally identify iph as hyperintense. image parameters were as follows : tr / te / ti=8.7/5.3/304 ms, fa=15, etl=32, fov=140140 mm, matrix=216192. images were obtained 20 mm below the carotid bifurcation to 20 mm above the carotid bifurcation at 1.0-mm slice thickness. the ti was chosen relative to the phase encoding acquisition to maximize contrast between hemorrhage and inflowing blood. was defined by the location in the plaque and at least 2 image slices with at least 2-fold higher signal intensity compared to adjacent muscle (fig. two experienced neuroradiologists determined mprage status in this objective manner independent of brain mr findings. also, the presence of carotid plaque and stenosis was calculated by one neuroradiologist using the north american symptomatic carotid endarterectomy trial criteria. dwi positive was defined as hyperintense signal on dwi trace with associated decreased signal on the apparent diffusion coefficient map, corresponding to an acute ischemic event at the time of the scan (fig. acute territorial ischemic events were first classification based on distribution (ipsilateral ica territory, ipsilateral basal ganglia, and posterior circulation). only dwi positive events in the ipsilateral ica territory were placed in the dwi - positive category. statistical analysis of carotid mprage signal and brain dwi signal was performed using fisher exact tests. likelihood ratios from the 22 table were used to determine relative risk of a cerebrovascular ischemic event in mprage - positive lesion with a 95% confidence interval., 136 patients who underwent stroke mr protocol because of acute neurologic symptoms were imaged with the additional carotid mprage sequences. of these patients, 22 were excluded due to poor image quality and 31 were excluded from the analysis : 1) co - existent > 50% ipsilateral intracranial artery stenosis or luminal irregularity (n=13) ; 2) non - atherosclerotic vasculopathy, such as dissection or moyamoya disease (n=3) ; 3) evidence of cardioembolism such as atrial fibrillation or previous stroke history of cardiac origin (n=15). 166 arteries of 83 patients (61 males, 22 females ; age range 59 - 89, median age 69 years - old) are eligible for data analysis. main neurological symptoms of these patients were as fallows ; 21 dizziness, 14 dysarthria, 37 unilateral weakness, 9 mental change, and 2 hyperesthesia. all patients with acute neurological symptoms underwent the baseline brain ct scans for evaluating intracranial or subarachnoid hemorrhage in the emergency department. mr examination was obtained with an achieva 3.0-t scanner (philips medical systems, best, the netherlands) with a 16-channel head coil. stroke mr imaging was performed immediately following ct scanning with the following techniques : 1) diffusion - weighted imaging (dwi) ; 2) 3d tof mr angiography (mra) of the intracranial arteries ; 3) susceptibility - weighted imaging (swi) ; 4) perfusion - weighted imaging (pwi) ; and contrast - enhanced mra for evaluation of carotid artery. dwi was conducted with a spin - echo - type echo - planar imaging sequence, with 3 b - values of 0, 500, and 1000 sec / mm along all 3 orthogonal axes : repetition time (tr)/echo time (te)=3000/80 ms ; flap angle (fa)=90 ; sensitivity encoding=3 ; field of view (fov)=220220 mm ; matrix=128128 ; section thickness / gap= 5 mm/30% ; scanning time=35 - 38 seconds. axial dynamic gradient - echo echo - planar pwi was performed after tracking a bolus of 0.03 mmol / kg of gadofosvesettrisodium (vasovist, schering, berlin, germany). acquisition parameters were as follows : tr / te=1850/35 ms ; fa=40 ; fov=230230 mm ; matrix= 132132 ; and section thickness / gap=5 mm/30%. after image reconstruction and processing, pwi data were transferred to a workstation (adw 4.2 ; ge healthcare, milwaukee, wi, usa). for mra, 3d multislab tof - mra from the petrous portion of the internal carotid artery (ica) was generated with the following parameters : tr / te=23/3.45 ms ; fa=20 ; fov=200200 mm ; matrix=488249 ; sensitivity encoding factor=2.5 ; slice thickness=1.2 mm ; echo train length (etl)=1 ; and number of average=1. we also performed contrast - enhanced carotid mra from the aortic arch to whole brain by using coronal planes with a 3d spoiled gradient echo sequence optimized for high spatial resolution and an intravenous bolus injection of 0.03 mmol / kg of gadofosvesettrisodium (vasovist, schering, berlin, germany), which was administered with an automatic injector at a rate of 1 ml / s through an 18-gauge cannula placed in the antecubital vein of the right arm, followed by 15 ml of saline solution, and the following imaging parameters ; tr / te=4.9/1.7 ms, flip angle=27, slice thickness=1.0 mm, matrix size=384384, voxel size=110.7 mm. for 3d mrprage sequence, segmental acquisition was performed with the sequence tr, the inversion preparation time (ti), and the phase encoding order of the mprage sequences adjusted to optimally identify iph as hyperintense. image parameters were as follows : tr / te / ti=8.7/5.3/304 ms, fa=15, etl=32, fov=140140 mm, matrix=216192. images were obtained 20 mm below the carotid bifurcation to 20 mm above the carotid bifurcation at 1.0-mm slice thickness. the ti was chosen relative to the phase encoding acquisition to maximize contrast between hemorrhage and inflowing blood. a positive signal on mprage sequence was defined by the location in the plaque and at least 2 image slices with at least 2-fold higher signal intensity compared to adjacent muscle (fig. two experienced neuroradiologists determined mprage status in this objective manner independent of brain mr findings. also, the presence of carotid plaque and stenosis was calculated by one neuroradiologist using the north american symptomatic carotid endarterectomy trial criteria. dwi positive was defined as hyperintense signal on dwi trace with associated decreased signal on the apparent diffusion coefficient map, corresponding to an acute ischemic event at the time of the scan (fig. acute territorial ischemic events were first classification based on distribution (ipsilateral ica territory, ipsilateral basal ganglia, and posterior circulation). only dwi positive events in the ipsilateral ica territory were placed in the dwi - positive category. statistical analysis of carotid mprage signal and brain dwi signal was performed using fisher exact tests. likelihood ratios from the 22 table were used to determine relative risk of a cerebrovascular ischemic event in mprage - positive lesion with a 95% confidence interval. of 166 arteries, 39 (23.5%) on contrast - enhanced mra and mprage sequence had a carotid artery plaque. mean percentage of carotid artery stenosis is 30% (range, 5 - 99%). of 39 arteries with carotid atherosclerotic plaque, 26 (66.7%) had carotid artery stenosis less than 50% and 13 (33.3%) had stenosis above 50%. in all carotid arteries, high - signal intensity on dwi was found in 17.5% (29/166). combined lesion with ipsilateral high - signal intensity on dwi and mprage - positive iph data of the mprage - positive iph and positive dwi in patients with carotid atherosclerotic plaque show in table 1. of 39 arteries with carotid artery plaque, mprage - positive iph was found in 30.8% (12/39) and match lesions with high - signal intensity on dwi and mprage - positive iph was found in 15.4% (6/39). mprage - positive iph was significantly higher prevalence in high - grade stenosis group (p=0.010). also, matched lesions between ipsilateral positive dwi and mprage - positive iph was significantly higher prevalence in high - grade stenosis (p=0.018). data of the relationship of mprage - positive iph with territorial dwi - positive acute stroke events shows in table 2. mprage - positive carotids were associated with increased risk of a dwi - positive acute territorial stroke event (fig. the relative risk of an acute territorial stroke event with an mprage - positive carotid compared with an mprage - negative carotid was 3.3 (p=0.005). the relative risk of an acute territorial stroke event with an mprage - positive carotid in patients with carotid atherosclerotic plaque compared with an mprage - negative carotid was 6.8 (p=0.01). this study used mri at 3-t to immediately undertake combined carotid mprage sequence and stroke mr protocol after admission at emergency room in patients with acute neurological symptoms. in this study, matched lesions between ipsilateral positive dwi and mprage - positive iph were significantly higher prevalence in high - grade stenosis. the relative risk of an acute territorial stroke event with an mprage - positive carotid compared with an mprage - negative carotid was 3.3-fold. the relative risk of an acute territorial stroke event with an mprage - positive carotid in patients with carotid atherosclerotic plaque compared with an mprage - negative carotid was 6.8-fold. early determination of the etiologic factors of acute ischemic stroke is essential for secondary prevention because the risk of recurrence is highly dependent on the underlying cause6). major identified causes of acute ischemic stroke are extra- or intracranial atheroma, cardioembolic sources, microvascular disease, aortic arch atheroma, and cryptogenic factors. most hospitals about patients with acute stroke performed stroke mri after initial brain ct at admission of emergency room. stroke mri had been demonstrated to be more sensitive for the detection of acute ischemia and more specific for delineation of infarction core volume when compared to ct3). recently, some studies reported brain mr examination combined sequences of carotid plaque characterization such as mprage, or conventional plaque mr and the relationship between vulnerable carotid plaque and territorial stroke event5,7). this study was performed the relationship between territorial acute ischemic stroke and carotid iph using acute stroke mr examination with mprage sequence. although iph in the carotid atherosclerotic plaque is significantly associated with more rapid progression in luminal stenosis20), the association of hemorrhage and symptoms remains a controversial subjects4,5,14,16,19). sitzer.16) demonstrated that ulceration and luminal thrombus were the main sources of downstream cerebral microemboli in patients with high - grade internal carotid stenosis. kolodgie.4) found evidence that showed a hemorrhage might represent a potential atherogenic stimulus. symptomatic groups had higher prevalence of complex (american heart association type iv) plaques on carotid plaque mr5,14). especially, fibrous cap rupture was associated with increases in dwi lesion in the brain5). takaya.21) reported that the presence of iph or fibrous cap rupture was significantly associated with subsequent carotid cerebrovascular event. some from carotid endarterectomy (cea) studies have supported the relationship of carotid iph with acute stroke. spagnoli.18) found that iph in cea specimens corresponded with a recent clinical stroke or transient ischemic attack based on the presence of neurological symptoms. early identification of patients with iph is very important for decrease of future sequela and optimizing management. the development of iph posed an immediate and long - term promoting effect on plaque progression. high - resolution mr imaging is recognized as a reliable method for the comprehensive characterization of atherosclerotic plaque tissue composition and the identification of iph in particular10,21,22,23,27). t1-weighted mr sequences are commonly used to detect iph owing to the degradation of hemorrhage into methemoglobin, which results in t1 shortening and correspondingly causes high signal intensity on t1-weighted mr images. among the t1-weighted mr sequences, black blood t1-weighted fast spin - echo and 3d tof angiography our study used mprage because recent research has shown this sequence has higher sensitivity and specificity in detecting hemorrhage compared with black blood t1-weighted fast spin - echo and tof angiography9). the mprage sequence combined with contrast - enhanced t1-weighted and tof angiography is more practical for many institutions for evaluation of carotid plaque components included iph. in the current study, the heavily t1-wighted signal of the mprage sequence was accomplished by a magnetization preparation inversion pulse1,28). from signal simulation that incorporated t1 values of muscle, blood, and hemorrhage, the segment tr and t1 in the mprage sequence were selected to suppress the flowing blood signal and enhance tissues with short t1 relative to muscle. current studies reported that carotid plaque mr with multiple sequences has used in the workup of acute stroke in a one week after symptom onset5,10). in situ type vi plaque identified by carotid mr imaging the prevalence of iph at baseline in western population was 49.0% and the presence of iph was associated with a 6-fold higher risk for events15). also, the annualized event rate in subjects with detectable iph was 17.71% compared with 2.43% in patients without iph. mcnally.7) performed that carotid mprage in western patients has been used in the workup of stroke in an emergent setting as our study. carotid mprage - positive signal was associated with an acute cerebral territorial ischemic event with a relative risk of 6.4. in our study, the prevalence of iph at baseline was 30.8% and mprage - positive carotid in our patients with acute neurologic symptoms had a further 3.3-fold increased risk of acute stroke on dwi compared with mprage - negative carotid. mcnally.7) reported that a high grade carotid stenosis had a relatively increased risk of acute stroke if the lesion was mprage - positive. our study shows that mprage - positive carotid in patients with carotid atherosclerotic plaque had a higher risk of acute stroke compared with mprage - negative carotid. first, scan time in acute stroke mr examination has important implications for the time onset of treatment and prognosis. therefore, acute stroke mr examination was generally used the limited sequences such as dwi, swi, pwi, tof - mra, and contrast - enhanced mra. scan time of mprage imaging was approximately 4 minutes, which was longer than required by other imaging methods. however, we believe that the benefits of early and accurate detection of carotid iph awarded by this imaging technique outweigh the con of longer scan times. second, our study was focused the atherosclerotic lesion of intracranial and extracranial vessels, although patients with intracranial atherosclerotic plaque were excluded in this study. major causes of acute ischemic stroke are major arterial atheroma, cardioembolic sources, microvascular disease, and cryptogenic factors. acute ischemic stroke of vulnerable carotid plaque such as iph, fibrous cap rupture, or ulcer was relatively low prevalence compared with that of cardiac sources. ota.9) performed the diagnostic performances of three t1-weighted 3.0-t mr sequences at carotid iph imaging, with histologic analysis as the reference standard. mprage sequence, as compared with t1-weighted fast spin echo and tof sequences, demonstrated higher diagnostic capability for the detection and quantification of iph. mprage - positive carotid plaque in population was associated with increased risk of acute territorial ischemic strokes. especially, mprage - positive in patients with carotid atherosclerotic plaque was relatively higher risk of acute ischemic stroke. this study found that iph in carotid atherosclerotic plaque was present in a high proportion of acute ischemic strokes. therefore, routine use of the mprage sequence in stroke mr examination may lead to a more accurate stroke risk estimate and more accurate determination of a carotid source of ischemic events. | objectivethe purpose of our study was to assess prevalence of carotid intraplaque hemorrhage (iph) and associations between territorial acute infarction and iph on magnetization - prepared rapid acquisition with gradient - echo (mprage) in patients with acute neurologic symptoms.methods83 patients with suspected acute neurologic symptoms were evaluated with both brain diffusion weighted imaging (dwi) and carotid mprage sequences. carotid plaque with high signal intensity on mprage of > 200% that of adjacent muscle was categorized as iph. we analyzed the prevalence of iph and its correlation with territorial acute infarction.resultsof 166 arteries, 39 had a carotid artery plaque. of these arteries, 26 had carotid artery stenosis less than 50%. in all carotid arteries, mr - depicted iph was found in 7.2% (12/166). high - signal intensity on dwi was found in 17.5% (29/166). combined lesion with ipsilateral high - signal intensity on dwi and iph on carotid mprage sequence was found in 6 lesions (6/166, 3.6%). of patients with carotid artery plaque, mr - predicted iph was found in 30.8% (12/39) and match lesions with high - signal intensity on dwi and mprage was found in 15.4% (6/39). mr - predicted iph was significantly higher prevalence in high - grade stenosis group (p=0.010). relative risk between carotid mprage - positive signal and ipsilateral high - signal intensity on dwi in arteries with carotid artery plaques was 6.8 (p=0.010).conclusioncarotid mprage - positive signal in patients was associated with an increased risk of territorial acute infarction as detected objectively by brain dwi. the relative risk of stroke was increased in high - grade stenosis categories. |
pulmonary alveolar proteinosis (pap) is a rare disease characterized by the excessive accumulation of lipoproteins containing periodic acid / schiff reagent (pas)-positive surfactant in the lower respiratory tracts. it can be classified as autoimmune, secondary, or genetic. regarding the secondary type, secondary pap is believed to be caused by a relative deficiency of granulocyte macrophage colony - stimulating factor (gm - csf) and related macrophage dysfunction. in contrast, autoimmune pap is thought to be primarily caused by anti - gm - csf autoantibodies (1 - 3). in the present report, we describe a patient with pap who was positive for gm - csf autoantibodies and simultaneously exhibited a myeloproliferative neoplasm. he underwent an examination at a local clinic after becoming aware of swelling of the dorsum of the right foot for 2 weeks. regarding his lifestyle, he had smoked 30 cigarettes per day for 50 years and was never exposed to a dust explosion. on a physical examination, a fine crackle was audible on both sides of the back, and edema of the right dorsum of the foot was evident. the blood test results were as follows : white blood cells (wbcs) : 30,402 /l (differential count = myeloblasts : 2%, promyelocytes : 1%, myelocytes : 3%, metamyelocytes : 2%, band cells : 9%, granulocytes : 68%, lymphocytes : 7%, basophils : 1%, and monocytes : 7%), hb : 15.6 g / dl, reticulocytes (ret) : 2.2%, platelets (plt) : 195,000 /l, total protein : 7.4 g / dl, albumin (alb) : 4.1 g / dl, blood urea nitrogen : 18.2 mg / dl, creatinine (cr) : 1.18 mg / dl, aspartate amino transferase : 22 iu / l, alanine amino transferase : 19 u / l, lactate dehydrogenase (ldh) : 430 iu / l, total bilirubin : 0.5 mg / dl, uric acid : 6.6 mg / dl, sodium : 138 meq / l, potassium : 4.0 meq / l, chloride : 105 meq / l, calcium : 9.3 mg / dl, c - reactive protein : 0.71 mg / dl, antinuclear antibody : (-), cytoplasmic antineutrophil cytoplasmic antibodies : (-), perinuclear antineutrophil cytoplasmic antibodies : (-), cardioembryonic antigen (cea) : 15.8 ng / ml, krebs von den lungen-6 (kl-6) : 38,090 u / ml, surfactant protein d : 185.9 ng / ml, and -d - glucan : 9.5 pg / ml. plain chest x - ray and computed tomography (ct) showed ground glass opacity (ggo) accompanied by significant, diffuse hypertrophy of the interlobular septa of both lungs (fig. 1). no lymph node enlargement was noted, but the spleen was mildly enlarged. (a, b) on plain chest radiography, bilateral alveolar opacities located centrally in mid and lower lung zones are found. (c, d) on high resolution ct reveals ground glass opacity accompanied by significant, diffuse hypertrophy of the interlobular septa of both lungs referred to as respiratory function testing indicated that, on room air, the blood oxygen saturation level (spo2) was 93%, blood gas was ph 7.437, partial pressure of carbon dioxide was 33.6 mmhg, partial pressure of oxygen was 72.2 mmhg, base excess was -0.7 mmol / l, bicarbonate was 22.3 meq / l, vital capacity was 94.1%, forced expiratory volume in 1 second (fev1%) was 80.2%, and diffusing capacity for carbon monoxide was 11.39 ml / min / mmhg (80.8%). regarding leukocytosis, bone marrow testing indicated that the numbers of all types of granulocytes were elevated in the hyperplasic bone marrow without any abnormalities in differentiation of any lineages or dysplastic features. a small increase in the numbers of reticular fibers was detected using the silver impregnation method. chronic myelogenous leukemia (cml) was excluded because the g - banding chromosome analysis was 46,xy [20/20 ] and a fluorescence in situ hybridization analysis of the bcr - abl fusion gene was 0%. it was difficult to make an accurate diagnosis according to the who classification 2008 because mutations in genes such as jak2, v617f, and csf3r were not evaluated. on clinical presentation, there was no splenomegaly. on a peripheral blood analysis these findings excluded primary myelofibrosis, polycythemia vera, essential thrombocythemia, and chronic eosinophilic leukemia. there was no bone marrow dysplasia, so we excluded myelodysplastic syndrome (mds) and atypical cml. chronic neutrophilic leukemia was also excluded because a peripheral blood analysis revealed that the band cell and granulocyte levels were less than 80% and myeloblast levels over 1%. therefore, we diagnosed the patient with myeloproliferative neoplasm, unclassifiable (mpn u) by exclusion diagnosis. lung shadows on x - ray imaging suggested pap ; therefore, bronchoscopy was performed. iu / l, leukocytes were 1.110/ml, quantitative method for alb was 246.8 mg / cr, quantitative method for urinary protein was 70 mg / dl, and cea was 18.4 mg / ml. on transbronchial lung biopsy, the alveolar space was filled with pas - positive eosinophilic granule - like substances, consistent with pap (fig. we noted substances that tended to stain light green in the bal fluid and suctioned the sputum. macrophages were also present in the bal and sputum (fig. 2c and d). taken together, these findings were consistent with pap, and a diagnosis of pap was made. (a, b) on trans bronchial lung biopsy, the terminal bronchioles and alveoli are filled with a pas - positive eosinophilic material with a granular pattern. (c, d) on bronchoalveolar lavage fluid and suctioned sputum, we can find granule - like substances that tended to be stained light green and the presence of macrophages. our treatment strategy involved a conservative approach with regular follow - up observations for mpn. regarding pap, following discussion with respiratory specialists, given that the dyspnea on exertion was mild and did not interfere with the patient 's daily activities, we decided not to perform alveolar lavage or gm - csf inhalation therapy until more severe symptoms manifested. two years after the diagnosis, the wbc count reached over 70,000 /l, and we began treatment with hydroxyurea (500 mg / day). at that time, the respiratory symptoms and chest x - ray showed no signs of progression of pap. three years after the diagnosis, the levels of blast cells in the peripheral blood increased suddenly to 18%. at that time the spo2 was 94% on room air, and plain chest x - ray and ct showed no marked changes from the initial diagnosis of pap. the blood test results were as follows : wbcs : 13,710 /l (differential count = myeloblasts : 18%, myelocytes : 1%, band cells : 2%, granulocytes : 42%, lymphocytes : 19%, basophils : 1%, eosinophils : 1%, and monocytes : 16%), hb : 11.5 g / dl, ret : 1.5%, and plt : 43,000 /l. bone marrow testing showed that the blast cell levels were at 37%, and peroxidase staining results were positive. blast cells were positive for cd13, cd33, and hla - dr and negative for cd34 and cd117. the levels of monocytes also reached 10%, and a diagnosis of acute myeloid leukemia (aml) was made. the g - banding chromosome analysis was 46,xy[7/20 ], 46,xy, idic(17)[11/20 ], 47,xy,+21[2/20 ]. soon after admission, remission induction therapy (30 mg / m daunorubicin for 3 days and 200 mg / m enocitabine for 8 days) was introduced in accordance with the japan adult leukemia study group gml200 protocol (4). no severe adverse events occurred during the course, although on day 27 of the recovery phase of remission induction therapy, bone marrow aspirate showed residual myeloid blast cells (about 5 - 10%), and the patient failed to achieve remission. a large volume of hemoptysis began to continuously appear, and his oxygenation simultaneously worsened rapidly. plain chest x - ray and ct showed progression of bilateral ggo and pleural effusion. a respiratory specialist determined the patient to have alveolar bleeding due to thrombocytopenia and infection. despite intensive care, the patient died on day 16 of the second induction. throughout the course from diagnosis to death, we deemed the state of pap to be unchanged, despite the progression to leukemia. this meant that we did not provide therapy for pap, such as alveolar lavage. the gm - csf autoantibody levels could not be evaluated periodically because this was not covered by his medical insurance. the abnormal gm - csf function caused by autoimmune mechanisms is the main cause of adult pap (5 - 7). of the 248 reported cases of pap in japan, 223 (89.9%) twenty - four patients (9.7%) were diagnosed with secondary pap that was negative for gm - csf autoantibodies or an underlying illness. the remaining patient (0.4%) had disease of unknown cause (7). this report shows that autoimmune pap is the most common form. in the present case, tests for gm - csf autoantibodies were positive, and the disease was thus considered to be autoimmune pap. the causes of secondary pap are likely related to relative decreases in the levels of gm - csf and corresponding macrophage dysfunction. only one case of secondary pap related to indium - tin oxide exposure with gm - csf autoantibodies was observed (8). other reports on secondary pap have shown that most cases were negative for gm - csf autoantibodies and did not involve autoimmune mechanisms (1,3,9,10). one report showed that 40 cases of pap secondary to hematologic malignancy were negative for gm - csf autoantibodies (3). in contrast, the present case of pap was positive for gm - csf autoantibodies and had mpn. although the median antibody titer for autoimmune pap was reported to be 15.29 g / ml (7), the antibody titer in our case was 56.45 g / ml, which was relatively high. we can not exclude the possibility that the onset of pap was a result of mpn, but we regarded this case as one of autoimmune pap with independent mpn. secondary pap with hematological disease and without autoimmune mechanisms is common, but autoimmune pap with hematological disease, as in the present case, has rarely been reported. previous case reports of pap with mpn are listed in table (11 - 14). as noted in other reports (9), cml and mds are the most common cause of pap, and there are no reports of pap with mpn u such as ours. in one case of pap, gm - csf autoantibodies were detected, and it was speculated that abl tyrosine kinase inhibitor - induced mechanisms were involved (14). interestingly, three out of four cases listed in table were diagnosed with pap when mpn progressed or disseminated infection occurred. in the remaining case, however, in our case, the relationship between the clinical course of mpn and pap was not apparent. one report compared the ct findings between autoimmune pap and secondary pap and showed that the typical high - resolution ct (hrct) findings for autoimmune pap were ggo with a patchy geographic pattern, subpleural sparing, crazy - paving appearance, and predominance in the lower lung field. these findings are uncommon for secondary pap (15). in the present case, additionally, subpleural sparing and the crazy - paving appearance were seen, patterns which are more similar to autoimmune pap. however, ggo was not predominant in the lower lung field, and we could not confirm the typical appearance of an autoimmune pap pattern. when reviewing the therapeutic strategies for pap, it is important to note that the various forms of pap may require different treatments, even though whole - lung lavage is the current therapeutic standard (16,17). gm - csf inhalation therapy (18) and rituximab (19) may also be effective treatments for autoimmune pap. several reports have shown that alveolar lavage and serial measurements of serum gm - csf autoantibodies may prove useful for monitoring disease activity and response to treatment (20). however, it has been reported that pap may also improve if hematological malignancy can be controlled (2). pap did not progress after mpn transformed to aml in our case, and this observation was consistent with autoimmune pap. this report shows that evaluating the levels of gm - csf autoantibodies is critical for distinguishing secondary and autoimmune pap, even if the patient has pap with hematological disease. | pulmonary alveolar proteinosis (pap) is classified as autoimmune, secondary, or genetic. we herein describe a 69-year - old man with autoimmune pap, simultaneously diagnosed with myeloproliferative neoplasm (mpn). two years after the diagnosis, the mpn progressed to acute myeloid leukemia, and the patient died from an alveolar hemorrhage during remission induction chemotherapy. throughout the clinical course, no progression of pap was observed, despite the progression to leukemia. there are few reports of autoimmune pap with hematological malignancy, and this case demonstrated that an evaluation for gm - csf autoantibodies is important for distinguishing the autoimmune and secondary forms of pap, even if the patient has hematological malignancy. |
leptin, a 167 amino acid peptide, is the prototypical adipose tissue - derived hormone related to the regulation of energy homeostasis. in the 1970s, coleman postulated that the relentless hyperphagia and morbid obesity observed in obese ob / ob mice was due to the absence of a circulating satiety hormone from adipose tissue. circulating leptin levels increase in proportion to an increase in fat mass in the body. leptin exerts pleiotropic effects by binding to and activating its receptors in the hypothalamus and other tissues. it is well documented that hyperleptinemia, caused by increased fat mass, disturbs various physiological functions, such as blood pressure, renal function, angiogenesis, wound healing, immune function, bone formation, and glucose leptin, a regulatory signal in glucose homeostasis, can directly suppress insulin release from -cells and acts independently of peripheral and central targets that express the biologically active long form of the leptin receptor ob - rb. a peripheral adipo - insular feedback loop was hypothesized to strictly regulate leptin and insulin secretion because leptin can suppress insulin release from -cells. however, in vivo evidence has revealed an alternative route through which leptin can act on central targets, not involving a peripheral mechanism, to suppress insulin secretion from -cells. it was later shown that the intraventricular infusion of leptin suppressed blood insulin levels and increased receptor sensitivity. hyperleptinemia, which is caused by increased adipose tissue, inhibits leptin transport across the blood - brain barrier (bbb), thereby inducing leptin insufficiency in the hypothalamus [figure 1 ]. because of sustained leptin insufficiency, the hypothalamic restraint on pancreatic insulin secretion is lost, and both glucose metabolism and energy expenditure are diminished. by elucidating the mechanism of leptin insufficiency syndrome, a role for leptin in the hypothalamus has been revealed ; leptin restrains rhythmic pancreatic insulin secretion while enhancing glucose metabolism and non - shivering thermogenic energy expenditure. a flowchart of sequential events initiated by the consumption of energy - enriched diets leading to leptin insufficiency in the brain and derangements in the hypothalamic regulation of insulin secretion, glucose metabolism, and energy expenditure that together promote metabolic disorders (modified with permission from central leptin insufficiency syndrome : an interactive etiology for obesity, metabolic and neural diseases and for designing new therapeutic interventions. peptides 2008;29:127 - 38) two mechanisms are hypothesized to reduce leptin transport across the bbb, where this is associated with hyperleptinemia. the first mechanism is that target receptors become less responsive due to abnormal increases in rhythmic hormonal signaling. it is postulated that leptin receptors in the cerebral microvasculature are downregulated by increased pulsatile leptin secretion. the second line of defense is modulating leptin before it is transported across the bbb. both the increased binding of leptin to c - reactive protein (crp) and the modulation of blood - to - brain transport by metabolic variables in the peripheral circulation are included. leptin replacement significantly improves hyperinsulinemia and type 2 diabetes without affecting food intake and weight. because leptin replacement centrally abolished hyperinsulinemia in obese rodents, insulin hypersecretion by relieving the restraint on -cells was confirmed to be a necessary response for enhancing the conversion of excess energy into fat. leptin is a hormone that is related to the regulation of energy homeostasis, including glucose metabolism. in ob / ob mice, leptin - deficient mice, relentless hyperphagia, and morbid obesity neuropeptide y (npy), the most potent appetite transducer, regulates appetite in the arcuate nucleus npy, agouti - related peptide (agrp), gamma - amino butyric acid (gaba), and alpha-1 receptors simultaneously repress anorexigenic melanocortin signaling in the arc pvn axis. primarily, anorexigenic leptin from adipocytes inhibits npy expression, and orexigenic ghrelin from the stomach stimulates npy expression. leptin directly decreases ghrelin release from oxyntic cells of the stomach and antagonizes the ghrelin - induced stimulation of npy release in the arc pvn axis. in the hypothalamus, leptin increases the expression and release of two anorectic neuropeptides, pro - opiomelanocortin (pomc) and cocaine- and amphetamine - regulated transcript (cart), while suppressing the synthesis and release of two orexigenic neuropeptides, npy and agrp. through protease cleavage, pomc produces a number of products, including alpha - melanocyte stimulating hormone (a - msh). agrp and a - msh are the antagonist and agonist, respectively, of the melanocortin 4 receptor (mc4r). however, a - msh activates mc4r, reduces food intake, and increases energy expenditure. a decrease in npy or an increase in pomc by leptin in the hypothalamus not only suppresses food intake but also enhances sympathetic outflow to brown adipose tissue (bat). leptin - induced stimulation in energy expenditure is hypothesized to be due to increased uncoupled respiration (thermogenesis) in bat, which is indicated by markedly elevated uncoupling protein-1 (ucp-1) mrna and protein levels. in general, thus, the decrease in hypothalamic npy induced by leptin may increase energy expenditure via enhanced sympathetic outflow to bat. to understand the molecular basis of leptin resistance, it is important to define the signaling cascades that mediate its effects in pomc and agrp neurons. one of the hypothalamic signaling pathways mediated by the leptin receptor is the janus tyrosine kinase 2 (jak2)/signal transducer and activator of transcription protein 3 (stat3) pathway. briefly, when leptin binds to ob - rb, tyrosine phosphorylation of the receptor is catalyzed by jak2. when the leptin receptor is phosphorylated, cytosolic stat3 is activated through subsequent phosphorylation by jak2 and recruited. stat3 binding to the pomc promoter increases pomc mrna expression through the recruitment of histone acetylases. however, the expression of stat3 in agrp neurons decreases agrp (and possibly npy) expression by recruiting histone deacetylases. leptin resistance in obese rodents is hypothesized to be due to impaired intracellular signaling downstream of leptin - rb activation in the interactive appetite - regulating network (arn) targets. however, further experiments showed that leptin inhibited food intake independent of stat3 signaling and that varying degrees of hyperleptinemia were not associated with altered stat3 signaling. thus, it remains uncertain whether suppressed intracellular signaling downstream of leptin - rb activation in arn targets is a necessary component of leptin resistance in obese rodents. in addition to the jak2/stat3 pathway, there are other leptin - sensitive signaling pathways, such as phosphatidylinositol 3-kinase (pi3k) in pomc cells. pi3k, when activated, phosphorylates the membrane lipid phosphatidylinositol-4,5-bisphosphate (pip2) to phosphatidylinositol-3,4,5-trisphosphate (pip3). the accumulation of pip3 recruits several kinases to the plasma membrane that induce the signaling cascade. pi3k - mediated phosphorylation of foxo1 and the exclusion of foxo1 in the nucleus cooperatively increase pomc mrna expression. leptin also induces pi3k activation in npy / agrp neurons, and stimulated pi3k signaling appears to decrease npy and agrp mrna expression, although leptin can activate pi3k signaling not in agrp / npy neurons themselves, but through synaptic transmission. new therapies that would enhance leptin transport across the bbb or reinstate leptin restraint on npy signaling are expected for the treatment of type 1 and 2 diabetes. central leptin gene therapy or pharmacological leptin mimetics are likely to improve the pathophysiological sequelae of diabetes and related ailments of metabolic syndrome. replication - deficient recombinant adeno - associated virus (ravv) is non - immunogenic and non - pathogenic. it can infect both dividing and non - dividing cells, and after integration into the host genome, it evokes transgenic expression for the lifetime of the cell. as a newer therapy for obesity and metabolic disorders, the ravv vector engineered to encode the leptin gene (ravv - lep) in contrast to the intrahypothalamic injection of leptin, the systemic injection of ravv - lep may not be an appropriate strategy because ectopic leptin production is enhanced in peripheral tissues (skeletal muscle or liver) and the pleiotropic effects of leptin are exerted. for example, leptin influences immune functions, bone formation, and angiogenesis, and exerts effects on non - adipose tissues such as the kidney and pancreas. furthermore, lipotoxicity is induced by the sustained effects of hyperleptinemia in non - adipocyte tissues. however, when a leptin gene was transferred into the hypothalamus or into selected hypothalamic sites, such as the medial preoptic area (mpoa), paraventricular hypothalamus (pvn), ventromedial hypothalamus (vmh), or arc, ultradian insulin secretion from the pancreas was suppressed. it has also been suggested that after the intrahypothalamic injection of leptin, glucose uptake by some peripheral tissues is increased through the activation of the vmh - sympathetic nervous system. enhanced npy induces hyperphagia and decreases energy expenditure, obesity, dyslipidemia, glucose intolerance, insulin resistance, and hyperinsulinemia, which are all risk factors for type 2 diabetes. repressing the npy system with a stable increase in leptin availability in the hypothalamus inhibits age - related and high - fat - diet (hfd)-induced obesity, hyperinsulinemia, and diabetes. body fat depletion drastically decreases adipocyte hormones, including leptin and adiponectin, as well as tumor necrosis factor- (tnf-), free fatty acids (ffas), and pancreatic insulin. after either intracerebroventricular injection or microinjection of ravv - lep into the arc, vmn, or pvn of rats, the expression of the gene encoding npy is attenuated, whereas the expression of the gene encoding pomc is enhanced in the arc ; however, expression of the gene encoding agrp in the arc is unaffected. because npy and agrp are coexpressed in arc neurons, it is hypothesized that leptin injected in the hypothalamus acts selectively. it has also been shown that central pomc gene therapy overcomes leptin resistance, decreases weight, and improves insulin sensitivity in obese zucker and aged rats. thus, the central melanocortin system may be a useful drug target for combating obesity. a low dose of ravv - lep moderately suppressed weight gain in rodents without decreasing daily food consumption, but increased energy expenditure, which was reflected by enhanced ucp-1 mrna in bat. furthermore, whereas microinjection of raav - lep in the arc, vmn, and pvn of rats decreased food intake, weight, and adiposity, and enhanced thermogenic energy expenditure, a similar injection in the mpoa did not diminish daily consumption, but increased thermogenic energy expenditure. this result may be explained by leptin expression in the mpoa being ineffective for decreasing npy signaling, which affects appetite behavior. leptin insufficiency in the hypothalamus produced by either leptinopenia or hyperleptinemia is related to the pathogenesis of diabetes. increasing leptin levels in the hypothalamus by either leptin injection or leptin gene therapy normalizes glucose levels in the presence of insulin in wild - type rodents. increasing leptin levels in the hypothalamus also induces euglycemia in the absence of circulating insulin, as shown in akita mice and wild - type rodents pretreated with streptozotocin (stz) to produce insulitis. centrally infused leptin does not control serum glucose by regulating the food intake or peripheral insulin levels in stz - induced diabetic rats ; it does so by regulating hepatic glucose production, peripheral glucose uptake, and energy expenditure in these rats. through innervations from the hypothalamus, leptin - induced outflow is transmitted to upregulate glucose metabolism in the pancreas, liver, skeletal muscle, white adipose tissue (wat), and bat. for glucose homeostasis, optimal leptin signaling in the hypothalamus, which is independent of insulin secretion, is necessary. it has been demonstrated that injection of ravv - lep increases leptin levels in the hypothalamus and prevents early mortality in the stz - pretreated mice. increased central leptin levels also gradually ameliorated hyperphagia to normalize intake by week 20 and maintained body weight at significantly lower values than the control range in the stz - pretreated mice. the blood glucose levels in these mice began to decrease significantly by weeks 23 and were normalized by week 8, and euglycemia persisted during the remaining course of the experiment. these results show that central leptin gene therapy is effective for normalizing glucose levels for extended periods in the absence of insulin. leptin gene transfer by ravv is effective for hyperinsulinemia and hyperglycemia. from the various paradigms, it has been demonstrated that leptin in the hypothalamus suppresses episodic insulin secretion and inhibits hyperinsulinemia in diet - induced obese rodents and leptin - deficient obese ob / ob mice. increased hypothalamic leptin expression by ravv - lep injection in ob / ob mice suppressed body weight and adiposity and led to voluntarily decreased dark - phase food intake. increasing hypothalamic leptin expression through ravv - lep also suppressed plasma levels of adiponectin, tnf - a, free fatty acids, and insulin, normalizing glucose levels in ob / ob mice. there is also a report that leptin transgene expression in the hypothalamus induced the apoptosis of adipocytes in rats. in wild - type mice, after injection with ravv - lep, the body weight and plasma levels of leptin and metabolic variables were suppressed to a lesser extent without decreasing food intake. additionally, intracerebroventricular injection of ravv - lep in adult obese ob / ob mice displayed more than double the life span compared with control cohorts. these life - extending benefits were related to a drastic reduction in visceral fat, blood glucose, insulin levels, and anti - aging biomarkers. it was also reported that in wild - type and ob / ob mice, a single intracerebroventricular ravv - lep injection sustained the effects of suppressing food intake and body weight for the lifetime of the mice. furthermore, the persistent efficacy of central leptin gene therapy in suppressing weight gain was also shown through all phases of reproduction, lactation, and post - lactation in dams. in addition to ob / ob mice, an increase in hypothalamic leptin in extremely hyperglycemic hfd - consuming rodents reduced blood glucose levels and maintained euglycemia. increased central leptin levels induce euglycemia by accelerating glucose metabolism in bat, liver, skeletal muscles, and adipose tissue, independent of insulin involvement. central leptin gene therapy in rats consuming an hfd also affected food intake, body weight, and energy expenditure. ravv - lep treatment in rats consuming an hfd led to reduced food intake and prevented the hfd - induced increase in weight and adiposity. ucp-1 mrna expression in bat was augmented by treatment with ravv - lep in rats consuming an hfd, indicating increased thermogenic energy expenditure. by decreasing energy intake and increasing thermogenic energy expenditure, central leptin gene therapy efficiently blocked weight gain and increased adiposity and hyperinsulinemia in rats consuming an hfd. it has been reported that leptin transgene expression in the pvn normalizes weight and rapidly depletes adipose tissue in obese hyperleptinemic rats that continuously consumed an hfd. thus, the leptin supply in the pvn alone can reverse dietary obesity. in koletsky rats with a leptin receptor (ob - rb) gene mutation, after adenovirally inducing the expression of leptin receptors in the arc, peripheral insulin sensitivity was improved via suppression of hepatic glucose production without changing insulin - stimulated glucose uptake or disposal. furthermore, ob - rb expression in the arc through ravv reduced food intake, and ob - rb expression in all sites except for pvn increased energy expenditure, which was assessed through enhanced ucp-1 mrna expression. these studies show that a sustained increase in leptin availability in the hypothalamus alone can induce euglycemia and suppress body weight and adiposity. the fact that ravv vectors are nonpathogenic and nonimmunogenic and can be used for the long - term expression of therapeutic genes suggests that central leptin gene therapy is a viable therapeutic strategy to control weight and prevent adiposity - related metabolic disorders. ravv is expected to treat a wide spectrum of diseases, and several clinical trials have been conducted. for example, in patients with hemophilia b, after peripheral vein infusion of the codon - optimized human factor ix transgene, fix transgene expression levels became sufficient to improve the bleeding phenotype. currently, a clinical trial is being conducted to evaluate the safety and tolerability of gene therapy with an adeno - associated virus (aav) bearing the glutamic acid decarboxylase (gad) gene for parkinson 's disease. sustaining optimal sufficiency in leptin signaling solely in the hypothalamus is considered to be a novel strategy to treat obesity and metabolic syndrome. to decrease worldwide epidemic of obesity and diabetes, clinical tests should be conducted on central leptin gene therapy or novel long - acting leptin mimetics to facilitate their widespread use. | adipocyte - derived leptin is a hormone associated with the regulation of energy homeostasis, including glucose metabolism. hyperleptinemia, induced by the consumption of energy - enriched diets, inhibits leptin transport across the blood brain barrier, and thereby produces leptin insufficiency in the hypothalamus. as a result of sustained leptin insufficiency, the hypothalamic restraint on pancreatic insulin secretion is lost. additionally, both glucose metabolism and energy expenditure are also diminished, and both type 1 and type 2 diabetes are induced. a replication - deficient recombinant adeno - associated virus vector engineered to encode the leptin gene (ravv - lep) has been used in models of diabetes as a novel therapeutic approach. after ravv - lep injection in ob / ob mice, hypothalamic leptin expression was increased, body weight was suppressed, and hyperinsulinemia was ameliorated. additionally injection of ravv - lep into the hypothalamus suppressed the expression of orexigenic neuropeptide y (npy) and enhanced anorexigenic pro - opiomelanocortin (pomc) in the arcuate nucleus (arc) in rats. it is proposed that central leptin gene therapy should be tested clinically to reduce the worldwide epidemic of obesity, diabetes, and shortened life span. in this article, the information has been assembled from published review articles on this topic. |
l929 cells were maintained in minimum essential medium eagle (sigma) with 5% fetal bovine serum and penicillin / streptomycin. mefs and 293 t cells were maintained in dulbecco 's modified eagle 's medium with 10% fetal bovine serum and penicillin / streptomycin. mefs and 293 t cells were transiently transfected by fugene 6 (roche applied science). for the preparation of cell extracts, cells were lysed with lysis buffer (50 mm tris - hcl, ph 7.5, 150 mm nacl, 1 mm edta, 1% nonidet p-40, 0.1 mg / ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm sodium orthovanadate) and centrifuged at 245,000 g for 10 min. the supernatant was used for sds - page and electrophoretic mobility shift assays (emsas). oligonucleotides oligonucleotides (800 bp) corresponding to the coding sequence of green fluorescent protein were amplified by pcr using t7-primer (5-cgtaatacgactcactataggggatatcagcaaaggagaagaactttt-3) and t3-primer (5-gcaattaaccctcactaaagggaggcctagggagaagacagtgagctc-3). long dsrna (ds800) was prepared by annealing complementary strands separately synthesized by in vitro transcription using the ampliscribe t7 flash transcription kit (epicentre biotechnologies) and the ampliscribe sp6 high yield transcription kit (epicentre biotechnologies). the annealed dsrna was treated with s1 nuclease (takara bio) to generate a blunt end and alkaline phosphatase (takara bio) to remove 5-phosphate. p - ds800 was prepared by labeling the 800-bp dsrna using t4 polynucleotide kinase and [-p]atp. reporter assay mefs were transfected with p55-c1bluc, prltk, and expression plasmids for rig - i mutants or mda5 mutants. cells were split into two aliquots, stimulated with rna (5-pppgg25) (6) or poly(i - c) transfection or sendai virus (sev) infection for 12 h, and harvested at 48 h after dna transfection. l929 cells were transfected similar to mefs but were stimulated by newcastle disease virus infection. the rna transfection and poly(i - c) transfection were performed using lipofectamine rnaimax (invitrogen). the luciferase assay was performed with a dual - luciferase reporter assay system (promega). luciferase activity was normalized using renilla luciferase activity (prltk) as a reference. quantitative pcr assay plasmid constructs p-55c1bluc and prltk, pef - flag - rig - i, pef - flag - rig - icard, pef - flag - mda5, and pef - flag - mda5card were described previously (14). data on snps for rig - i and mda5 were obtained from the ncbi (www.ncbi.nlm.nih.gov) and hapmap databases. the expression plasmids for rig - i mutants (pef - flag - rig - ir7c, pef - flag - rig - is144f, pef - flag - rig - is183i, pef - flag - rig - it260p, pef - flag - rig - ii406 t, pef - flag - rig - id580e, pef - flag - rig - if789l, and pef - flag - rig - icards183i) and mda5 mutants (pef - flag - mda5t260s, pef - flag - mda5l274i, pef - flag - mda5k351e, pef - flag - mda5i442v, pef - flag - mda5h460r, pef - flag - mda5e627, pef - flag - mda5h843r, pef - flag - mda5i923v, pef - flag - mda5a946 t, and pef - flag - mda5d1014e) were generated using a geneeditor in vitro site - directed mutagenesis system (promega). antibodies and immunoblotting the anti - flag (m2 ; sigma) antibody is a commercial product. sds - page and immunoblotting were performed as described previously (14). emsa293 t cells (1 10/6-cm dish) were transfected with 1 g of expression vector. at 24 h after transfection, cell extract was prepared and mixed with anti - flag beads (sigma) to adsorb flag - tagged proteins. the beads were washed, and bound protein was eluted with flag peptide (sigma). construction of rig - i mutants and their biological activities in mefs derived from rig - i knock - out mice nssnps were selected from nucleotide sequence polymorphisms of human rig - i reported in databases and introduced into the rig - i expression vector by site - directed mutagenesis. mefs transiently transfected with these vectors, the wild type and seven human rig - i mutants were expressed at comparable levels (fig. these results indicate that none of the amino acid substitutions significantly affect the synthesis and/or stability of rig - i. the signaling function of the mutants was analyzed by temporarily complementing the function of rig - i in rig - i mefs using a virus - responsive luciferase reporter gene (fig. although the transfection of 5-ppprna did not activate the reporter gene significantly in rig - i mefs (fig. 2a, vector), the ectopic expression of wt rig - i restored the responsiveness to the ligand. all the mutant constructs exhibited functional complementation except the r7c, s144f, and s183i mutants, which exhibited a reduced response to 5-ppprna, particularly the s183i mutant, which exhibited a severe defect. because the activity of s144f was not reproducible, we did not investigate this mutant further. it has been shown that rig - i senses sev and activates the ifn promoter. therefore, the ability of the mutants to respond to this viral inducer was tested (fig. sev efficiently activated the reporter gene when wt rig - i was expressed ; however, s183i was virtually inactive and r7c exhibited partial activity. we further confirmed the effect of the r7c and s183i mutations by monitoring the expression of endogenous mouse ifn- mrna (fig. 2c). the results clearly demonstrate that s183i is barely active and that r7c is partially active. the above results strongly suggest that ser-183 is critical for rig - i to sense transfected 5-ppprna as well as sev - derived pamps. ser-183 resides within the second card, and this prompted us to explore the impact of the s183i substitution on the signaling function of the isolated rig - i card. unlike that of the full - length rig - i, overexpression of the truncated rig - i (1229), which encompasses the two repeats of card, constitutively activated the reporter p-55c1b without a viral stimulus (fig. rig - i (1229) with s183i failed to activate the reporter gene. in these cells, levels of rig - i (1229) with or without the s183i mutation were comparable, suggesting that ser-183 is critical for the signaling function but does not affect protein levels of rig - i card (fig. it was reported that human rig - i undergoes ubiquitination at lys-172 in the second card, and this process is essential for rig - i signaling (15). to compare the mutation at ser-183, we generated a k172r mutant and tested its activity (fig. 3, c and d). surprisingly, the mutant exhibited virus - induced signaling activity comparable with the wt, suggesting that the ubiquitination of lys-172 plays a minor role in the regulation of rig - i and that the phenotype of the s183i mutant is unlikely due to a failure of ubiquitination. a and b, rig - i mef cells were transiently transfected with p-55cibluc together with empty vector (vector) or the indicated constructs. the cells were subjected to a dual - luciferase assay after stimulation with 5-ppp - ssrna (12 h) (a) or sev (12 h) (b). the relative firefly luciferase activity, normalized to the renilla luciferase activity, is shown. error bars show the sds for triplicate transfections. mock, mock - treated. c, rig - i mefs were transfected with empty vector (vector) or expression vectors for wt rig - i or mutants as indicated (the total amount of plasmid was kept at 6gby adding empty vector). to observe the dose response cells were mock - treated or transfected with 5-ppp - ssrna for 12 h, and ifn- mrna was quantified by quantitative pcr by using the applied biosystems primer set for mouse interferon-1 : mm00439546_s1. a and b, rig - i mef cells were transiently transfected with p-55cibluc together with empty vector (vector) or the indicated constructs. the cells were subjected to a dual - luciferase assay after stimulation with 5-ppp - ssrna (12 h) (a) or sev (12 h) (b). the relative firefly luciferase activity, normalized to the renilla luciferase activity, is shown. error bars show the sds for triplicate transfections. mock, mock - treated. c, rig - i mefs were transfected with empty vector (vector) or expression vectors for wt rig - i or mutants as indicated (the total amount of plasmid was kept at 6gby adding empty vector). to observe the dose response, cells were mock - treated or transfected with 5-ppp - ssrna for 12 h, and ifn- mrna was quantified by quantitative pcr by using the applied biosystems primer set for mouse interferon-1 : mm00439546_s1. a, rig - i mef cells were transfected with reporter genes together with empty vector (vector) or plasmid expressing flag - tagged wt rig - i, rig - i card (the n - terminal region, amino acid 1229), or rig - i card s183i. after transfection (24 h), the cells were subjected to a dual - luciferase assay. b, each protein expressed in rig - i mef cells was detected by immunoblotting using an anti - flag antibody. c, reporter assay of the k172r mutant was performed as in fig. rig - i k172r and wt rig - i were expressed at comparable levels. e, empty vector or expression vectors for full - length rig - i with the t55i or s183i mutation were introduced into l929 cells (the total amount of plasmid was kept at 9 g by adding empty vector) and infected with newcastle disease virus, and reporter activity was analyzed as in panel a. to observe the dose response, cells received 1, 5, or 9 g of the expression plasmid for t55i and s183i as indicated. a, rig - i mef cells were transfected with reporter genes together with empty vector (vector) or plasmid expressing flag - tagged wt rig - i, rig - i card (the n - terminal region, amino acid 1229), or rig - i card s183i. after transfection (24 h), the cells were subjected to a dual - luciferase assay. b, each protein expressed in rig - i mef cells was detected by immunoblotting using an anti - flag antibody. c, reporter assay of the k172r mutant was performed as in fig. rig - i k172r and wt rig - i were expressed at comparable levels. e, empty vector or expression vectors for full - length rig - i with the t55i or s183i mutation were introduced into l929 cells (the total amount of plasmid was kept at 9 g by adding empty vector) and infected with newcastle disease virus, and reporter activity was analyzed as in panel a. to observe the dose response, cells received 1, 5, or 9 g of the expression plasmid for t55i and s183i as indicated. it is known that human rig - i with the amino acid substitution t55i acts as a dominant inhibitor (7). this mutation within the first card inactivates the signaling function of the isolated tandem cards. l929 cells were transfected with control vector or rig - i mutants and then activated by infection of newcastle disease virus. cells transfected with the vector exhibited ifn promoter activity due to endogenous rig - i (fig. expression of t55i as well as s183i significantly reduced the promoter activity in a dose - dependent manner, suggesting that s183i causes a dominant negative phenotype similar to t55i. construction of mda5 mutants and their biological activities in mefs derived from mda5 knock - out mouse next, the biological activity of human mda5 mutants was analyzed similarly using mda5-deficient mefs. ten nssnps including a946 t identified in familial t1d (16) were introduced into the mda5 expression vector (fig. the wild type and mutants were expressed in mda5 mefs at comparable levels (fig. 4b), showing that the mutations, including the e627 truncation, did not affect mda5 protein levels. the biological activity of the mutants was assayed similarly to that of the rig - i mutants. as a mda5 agonist, a commercial poly(i - c) with an average length of 2 kbp wild - type mda5 clearly conferred responsiveness to the poly(i - c) (fig. eight mda5 mutants, including a946 t, which was implicated in human t1d (16), exhibited complementing activity comparable with the wild type. the phenotypes of these mutations were further tested by transient expression in mda5 mefs and monitoring endogenous ifn- mrna (fig. 5b). although mda5 is absent, intracellular poly(i - c) induced ifn- gene expression in the control cells (vector). this is presumably due to the activation of rig - i by a short poly(i - c) present in the preparation we used. unlike the reporter assay, which monitors only transfected cells, this quantitative pcr assay detects ifn- transcripts from both transfected and non - transfected cells, increasing the background signal. irrespective of the background, overexpression of wt mda5 resulted in an enhancement of ifn- expression by poly(i - c), and this induction was not observed with e627 and i923v. the e627 mutant lacks a part of the helicase domain and the entire ctd in which ile-923 resides. figure 4.mda5 nssnp mutants and their expression in mefs. a, schematic representation of wt mda5 and its nssnps. b, flag - tagged mda5 snps were produced in mda5 mefs and detected by immunoblotting using an anti - flag antibody. mda5 nssnp mutants and their expression in mefs. a, schematic representation of wt mda5 and its nssnps. b, flag - tagged mda5 snps were produced in mda5 mefs and detected by immunoblotting using an anti - flag antibody. vector, empty vector ; mda5 full, full - length mda5. because in the case of rig - i, the ctd determines rna recognition specificity, we investigated the rna binding activity of these mutants by emsa using p - labeled poly(i - c). wild - type and recombinant mda5 proteins were expressed in 293 t cells and purified. the recombinant proteins were virtually free of other cellular proteins as analyzed by coomassie brilliant blue staining (fig. wild - type mda5 clearly formed a complex with poly(i - c) (fig. i923v and a946 t exhibited activity to bind poly(i - c) as strongly as the wild type under these conditions. 5e) and confirmed that mda5 wt and the i923v mutant bind to dsrna in a comparable fashion. these results suggest that the e627 mutant is biologically inactive due to its failure to recognize its agonist. human rig - i polymorphism we identified s183i as a loss of function mutation of rig - i. this serine residue is conserved in human, monkey, cow, and pig rig - i. the mutation apparently inactivates the tandem card, which relays signals downstream. furthermore, s183i exhibits a dominant inhibitory phenotype, suggesting that individuals retaining this mutation as a heterozygote would exhibit hypersensitivity to viral infections. it has been shown that rig - i mutants with a card deletion (rig - ic), card point mutation (t55i), and atp - binding site mutation (rig - i k270a) all function as a dominant inhibitor (3, 7). it has been reported that the repression domain present in the c - terminal region of rig - i and lgp2 dominantly suppresses the activation of rig - i in trans through interaction with card and the helicase loop region (7). because the repression domain encompasses the rna - binding domain of rig - i (6), one mechanism is likely competition of rna binding with wt rna. however, the rna binding - deficient rig - i mutants, k888a / k907a and k858a / k861a (6), functioned as a dominant negative inhibitor.3 interestingly, lgp2 mutants, k643e and k651e, which correspond to lys-888 and lys-907 of rig - i, lost rna binding activity but retain repression function (17). a, mda5 mefs were transfected with reporter genes together with the indicated constructs as in fig. 2a. after stimulation with poly(i - c) (12 h), the cells were subjected to a dual - luciferase assay. b, mda5 mefs were transfected with expression vectors for wt mda5 or e627 or ile-923 mutants (the total amount of plasmid was kept at 6 g by adding empty vector). to observe the dose response, cells were mock - treated or transfected with poly(i - c) for 12 h, and ifn- mrna was quantified by quantitative pcr as in fig. 2c. c, 293 t cells were transfected with empty vector, wt mda5, e627, i923v, or a946 t, and the produced proteins were purified using anti - flag (experimental procedures). the purified proteins were separated by sds - page and stained by coomassie brilliant blue. d, emsa of the purified mda5 proteins (500 ng) using p - labeled poly(i - c) as a probe. emsa was performed using 500, 300, and 100 ng of mda5 and ile-923 protein. a, mda5 mefs were transfected with reporter genes together with the indicated constructs as in fig. 2a. after stimulation with poly(i - c) (12 h), the cells were subjected to a dual - luciferase assay. b, mda5 mefs were transfected with expression vectors for wt mda5 or e627 or ile-923 mutants (the total amount of plasmid was kept at 6 g by adding empty vector). to observe the dose response cells were mock - treated or transfected with poly(i - c) for 12 h, and ifn- mrna was quantified by quantitative pcr as in fig. 2c. c, 293 t cells were transfected with empty vector, wt mda5, e627, i923v, or a946 t, and the produced proteins were purified using anti - flag (experimental procedures). the purified proteins were separated by sds - page and stained by coomassie brilliant blue. d, emsa of the purified mda5 proteins (500 ng) using p - labeled poly(i - c) as a probe. emsa was performed using 500, 300, and 100 ng of mda5 and ile-923 protein. one proposed function of the second card is to conjugate to ubiquitin (at lys-172), which may be essential for signaling activity. however, human rig - i with k172r, which is resistant to ubiquitination, did not affect phenotype (fig. moreover, the corresponding amino acid in mouse rig - i is glutamine, and the corresponding position of mda5 is glutamic acid (human, monkey, mouse, cow, and pig), suggesting that the ubiquitination of lys-172 has a minor impact on signaling activity. the precise function of the second card is not clear, but a comparative study of ser-183 and wt rig - i will elucidate the molecular function of this residue. human mda5 polymorphism we identified two loss of function mutations, e627 and i923v, in human mda5. each of these mutations is actually present in the human population (13). it is worth investigating the phenotype of the homozygote because mda5 knock - out mice clearly exhibit hypersusceptibility to the family picornaviridae, genus cardiovirus. e627, which lacks ctd and a part of the helicase domain, lost its dsrna binding activity ; hence there was no signaling activity. although the i923v mutation occurs in the ctd, this mutant exhibited intact dsrna binding, suggesting a novel function of ile-923 other than the recognition of rna. it is worth noting that this isoleucine is conserved in human, monkey, mouse, cow, and pig mda5. it is tempting to speculate that ile-923 participates in an interaction with some other domain of mda5 or other unknown regulatory protein(s). a new report by nejentsev. (13) describing the relationship between susceptibility to t1d and mda5 polymorphism was published. the report describes that four rare mutations (two mutations in the exon and two mutations in the intron) in the human mda5 gene are associated with protection against t1d. our analysis included the two exon mutations (e627 and i923v), which exhibited a loss of function phenotype. the genetic analysis included another rare nssnp, h460r, shown to be independent of t1d resistance. on the other hand, a946 t, which was suggested to associate with t1d in previous reports (13, 16), did not exhibit a loss of function phenotype. (13) suggested that the association of a946r with t1d was due to the effect of another nssnp, r843h, a combination of these mutations might confer loss of function on mda5. in summary, our analysis strongly suggests that loss of function mutations of mda5 have a causative role in resistance to t1d. although t1d has a complex pathology, these findings may provide a new strategy for establishing an animal model for t1d. the human genome encodes multiple sensors, including tlrs and rlrs for viral pamps. some of these may act redundantly to secure defense against viral infections. in the case of rlrs, rig - i and mda5 detect a distinct spectrum of viruses, as suggested from the phenotype of respective knock - out mice. there is variation within human populations in susceptibility to a particular viral infection. we argue for a possible contribution of the genetic diversity of rlrs, including those identified in the current investigation, to susceptibility. in addition to the impact of the infection itself, secondary effects such as autoimmunity, which is remotely triggered by certain viral infections, may be influenced at least in part through the functional diversity of rlrs. in summary, our results suggest a critical relationship between rlr polymorphisms and diseases including viral infections and autoimmunity. | retinoic acid - inducible gene i (rig - i) and melanoma differentiation - associated gene 5 (mda5) are essential for detecting viral rna and triggering antiviral responses, including production of type i interferon. we analyzed the phenotype of non - synonymous mutants of human rig - i and mda5 reported in databases by functional complementation in cell cultures. of seven missense mutations of rig - i, s183i, which occurs within the second caspase recruitment domain repeat, inactivated this domain and conferred a dominant inhibitory function. of 10 mutants of mda5, two exhibited loss of function. a nonsense mutation, e627, resulted in deletion of the c - terminal region and double - stranded rna (dsrna) binding activity. another loss of function mutation, i923v, which occurs within the c - terminal domain, did not affect dsrna binding activity, suggesting a novel and essential role for this residue in the signaling. remarkably, these mutations are implicated in resistance to type i diabetes. however, the a946 t mutation of mda5, which has been implicated in type i diabetes by previous genetic analyses, affected neither dsrna binding nor ifn gene activation. these results provide new insights into the structure - function relationship of rig - i - like receptors as well as into human rig - i - like receptor polymorphisms, antiviral innate immunity, and autoimmune diseases. |
why do neurons in the central nervous system (cns) fail to regenerate their axons after injury ? this fundamental question has obvious implications for human neurodegenerative disease and injury (moore and goldberg, 2010). in the cns, embryonic or neonatal neurons can regenerate their axons after injury, whereas postnatal or adult neurons can not (bregman and goldberger, 1982 ; kunkel - bagden., 1992). this is at least partially attributable to the development of an inhibitory cns environment, in which glial cells such as mature astrocytes and oligodendrocytes express molecules that suppress axon regeneration for example after injury in the adult mammalian cns (waxman, 1980 ; foran and peterson, 1992). after injury, damaged axons are exposed to myelin- and astrocyte - associated lipids and proteins that actively inhibit axon growth and regeneration (yiu and he, 2006). for example, astrocytes secrete chondroitin sulfate proteoglycans (cspgs) (snow., 1990 ; mckeon., 1999 ; becker and becker, 2002 ; jones., 2002, 2003 ; tang., 2003) and oligodendrocytes express myelin - derived axon growth inhibitors including nogo, myelin associated glycoprotein (mag), and oligodendrocyte myelin glycoprotein (omgp). when such molecules are genetically knocked out (bartsch., 1995 ; kim., 2003 ; simonen., 2003 ; zheng., 2003 ; su., 2008), neutralized through antibody treatments (caroni and schwab, 1988 ; bregman., 1995 ; tang., 2001), or enzymatically digested (crespo., 2007) in animal models, modest regeneration and improvement in behavioral assays is observed in animal models (schmandke., 2007). incomplete regeneration in all of these studies suggested the existence of other pathways involved in regenerative failure and motivated the search for signaling pathways intrinsic to the neurons themselves that may limit their regenerative ability. a number of signaling molecules have been identified to act in neurons to limit regenerative ability. some of these are trivial extensions of the extrinsic, inhibitory environment, such as receptors for inhibitory glial - associated proteins (cafferty and strittmatter, 2006) or signaling proteins downstream of these, such as rho and rho kinases (bertrand., 2007). others are signaling pathways involved more generally in cell growth regulation, such as anaphase promoting complex (apc) (konishi., 2004 ; lasorella., 2006), and phosphatase and tensin homolog (pten ; (park., 2008)) or neurotrophic factor responsiveness such as suppressor of cytokine signaling-3 (socs3 ; (smith., 2009)), cyclic adenosine 3,5-monophosphate (camp ; (cai., 2001)), and camp response element - binding protein (creb ; (gao., 2004)). over the past few decades, multiple studies have demonstrated a developmental decrease in the intrinsic ability of cns axons to rapidly elongate their axons (saunders., 1992 ;, 1997 ; dusart., 1997 ; blackmore and letourneau, 2006), which correlates with the developmental loss of regenerative capacity in vivo. for example, embryonic retinal ganglion cells (rgcs) grow their axons ten - fold faster than postnatal rgcs, and this rapid axon growth ability is lost around the time of birth (goldberg., 2002). what is the molecular basis for this loss ? when we analyzed microarray - derived transcriptomes from different ages of rgcs to identify developmentally regulated genes (wang., 2007), we discovered that expression of the transcription factor krppel - like factor 4 (klf4) significantly decreased neurite outgrowth in hippocampal and cortical neurons, and rgcs (moore., 2009). furthermore, klf4 knockout during early development increased neurite growth from rgcs in vitro, and increased axon regeneration in vivo after optic nerve injury (moore., 2009). interestingly, klf4 expression increases postnatally in rgcs, specifically during the period around birth, which is when rgcs lose their intrinsic axon growth ability (moore., 2009). moreover, another klf family member, klf9, also demonstrated a dramatic 250-fold increase in expression after birth. overexpression of klf9 was also shown to result in a significant decrease in neurite outgrowth in vitro (moore., 2009). in general, these data support a model whereby the increase in klf expression around birth, long after all rgcs have become post - mitotic, leads to a loss of regenerative ability of rgcs. given klf9 's higher expression levels after birth relative to klf4, and because it was closely related to subfamily members klf13 and klf16 that also suppressed axon growth, klf9 became an intriguing target for studying molecular mechanisms governing its activity in rgcs, but little was known about its regulation or gene targets. we have recently found that knocking down expression of klf9 with shrna constructs strongly promotes rgc axon regeneration after optic nerve injury (unpublished data), and can help to identify both ptms and downstream target genes required for klf9 to suppress axon growth in vitro and regeneration in vivo. many proteins have been shown to interact with and regulate transcription factors in the cns. participation in these interactions often involves post - translational modifications (ptms) of transcription factors such as acetylation, phosphorylation, ubiquitination, and sumoylation (savare., 2005 ; ceballos - chavez., 2012 ; goldberg and trakhtenberg, 2012 ; brochier such ptms are critical in functional outcomes such as neuronal differentiation, survival and neurite growth (cai., 2001 ; hirata., 2003 ; savare., 2005 ; raivich and makwana, 2007 ; lai and johnson, 2008 ; raivich, 2008 ; juliandi., 2010 ; ceballos - chavez., 2012 ; hasegawa., 2012 ; xie., 2012 ; brochier., 2013 ; watkins., 2013 ; welsbie., 2013, klf11 has been shown to interact with both histone acetyl and methyltransferases and these interactions have proven to be crucial in its functional role in regulating the dopamine d2 receptor in dopaminergic neurons (seo., 2012). klf5 has been shown to be activated by p300 and suppressed by set in its ability to bind dna and transactivate downstream target genes (miyamoto., 2003). phosphorylation is perhaps the best studied ptm of transcription factors, and numerous kinase families affect neurite growth and regeneration. the mapks (erks, jnks, and p38) and dlk families have been well studied in this regard. mapk activation upon neuronal injury leads to changes in gene expression patterns mediated by phosphorylated tfs such as c - jun, sox11, atf2, p311, and stat3 (raivich and makwana, 2007). these changes have varied effects including induction of heat shock proteins (hsp25/27) which spur the repair of the actin microfilament network (stokoe., 1992 ; cohen, 1996), promotion of survival by mitochondrial cytochrome c interference preventing caspase activation (benn., 2002 ; hirata., 2003 ; dodge., 2006), and induction of programmed cell death (pcd) upon ngf withdrawal via release of inflammatory cytokines such as il-1 and tnf- (xia., 1995). within the klf family, we have shown that a mapk family member regulates klf9 at two critical residues and that this regulation is crucial to its functional role as a neurite outgrowth suppressor in rgcs (unpublished data). this form of regulation via phosphorylation is not unique to klf9 among the klf family. klf6 has been shown to be regulated by phosphorylation in cos-7 cell lines metabolically labeled with radioactive phosphate, where it acts as a tumor suppressor (slavin., 2004), but the kinase responsible remains unidentified. using protein kinase inhibitors, it was demonstrated that klf5 is activated by phosphorylation in its role as an oncogene and interacts with protein kinase c (pkc) and p38 but not mapk in both human pancreatic and breast cancer cell lines (zhang and teng, 2003 ; mori., 2009). klf11 has been shown to be phosphorylated in 2 linker regions by erk in cho cell lines using in vitro phosphorylation assays (ellenrieder., 2002 ; ellenrieder, 2008). klf4 is phosphorylated by pkc at t401, an important regulatory residue, in vascular smooth muscle cell differentiation via smads in a tgf- and p38 dependent cascade (zhang., 2012). klf9, however, is thus far unique both in its capacity to bind the mapk family of kinases and known to be regulated by them within neurons. besides klf9, only klf10 and -11 possess the same structural motif necessary for klf9-mapk interaction. the involvement of these kinases in the regulation of klfs outside the cns makes them potential players in the intrinsic control of neurite growth both during normal neuronal development and after injury within the cns. characterizing the phosphorylation and phospho - regulation of other klf family members within the cns is thus an important goal for future study. the finding that kinases such as those in the mapk family, which are activated by extracellular signals such as neurotrophic factors or other signaling ligands, act on developmentally regulated transcription factors such as klfs, has important implications for understanding regenerative failure. such interactions may link the extrinsic regulators of axon growth including growth promoters and growth inhibitors to intrinsic, cell - autonomous signaling pathways. | molecular mechanisms of the krppel - like family of transcription factors (klfs) have been studied more in proliferating cells than in post - mitotic cells such as neurons. we recently found that klfs regulate intrinsic axon growth ability in central nervous system (cns) neurons including retinal ganglion cells, and hippocampal and cortical neurons. with at least 15 of 17 klf family members expressed in neurons and at least 5 structurally unique subfamilies, it is important to determine how this complex family functions in neurons to regulate the intricate genetic programs of axon growth and regeneration. by characterizing the molecular mechanisms of the klf family in the nervous system, including binding partners and gene targets, and comparing them to defined mechanisms defined outside the nervous system, we may better understand how klfs regulate neurite growth and axon regeneration. |
oncology includes a variety of different diseases and indications and can hardly be seen as one disease on its own. on the basis of the therapy advances in most recent years and the severity of the malignancy, the overall survival ranges from a few months to many years in the metastatic setting. in adjuvant indications where tumors are being detected at an early stage, cure could also be achieved.1 the goals of cancer care are to optimize both the length and quality of life,2,3 which could be achieved with different treatment options. in recent years innovative treatments have been introduced in oncology and hematology. oncology medications as monotherapy and combinations have an acceptable risk benefit profile ; however, they are also linked with more or less patient - relevant side effects.4 the aifa (agenzia italiana del farmaco, italian medicine agency) definition of side effects is as follows : harmful and unwanted event resulting from the use of a medicinal product. in fact with this definition, regardless of the use of the medicine, adverse reactions will be reported, including those arising from medication error, abuse, against misuse, off - label, overdose, and professional exposure.5 the adverse reactions are mainly related to the mode of action of these therapies. another important parameter of safety is the potential treatment error, which could occur in hospitals and might have an even bigger impact on adverse events due to a noncompliant and nonlabeled dosing.6 such errors could be assumed to occur more often with difficult dosing regimens and generally with therapies for which therapy dosing per patient (eg, per kg or per m) is administered. furthermore, errors could occur with oral medications in case these therapies are not taken at all or are administered to the wrong persons. such treatment errors can impact patients quality of life and sometimes survival.4,6 additionally, hospitals need to cover their liability with adequate insurances. the higher the risk for such treatment errors, the higher the insurance premium a hospital needs to pay annually. if a hospital would choose not to pay premiums, they would need to accrue higher amounts in their accounting for the potential financial implications of such an error. subcutaneous versions of rituximab and trastuzumab have been available since 2014.1,7 subcutaneous therapy should benefit all stakeholders in the health care system, especially if these are delivered as fixed - dose regimens. the analyzed hypothesis is a superior safety benefit of subcutaneous over intravenous therapies through fixed - dose administrations exemplary analyzed with trastuzumab and rituximab. as intravenous and subcutaneous were the only injection routes in medical administration available for trastuzumab and rituximab in oncology at the time of the study, these two therapies were chosen. the purpose of the underlying study was to analyze the risk impact of a subcutaneous therapy in comparison to an intravenous therapy in an italian health care setting focusing on breast cancer and nhl with trastuzumab and rituximab, respectively. in order to analyze the impact of a subcutaneous administration of an existing therapy in comparison to the intravenous mode, a primary research study in italy was executed. the primary objectives of the study were to quantify the risk and cost implications from different perspectives (patients, hospital administration, and medical staff) using the failure mode and effect analysis (fmea) approach.8 fmea was developed outside of health care and is now being used in health care to assess risk of failure and harm in processes as well as to identify the most important areas for process improvements. fmea has been used by hospitals in a variety of institute for healthcare improvement programs in the us, including idealized design of medication systems, patient safety collaboratives, and the patient safety summit.9 it involves reviewing as many components, assemblies, and subsystems as possible to identify failure modes and their causes and effects. for each component, the failure modes and their resulting effects on the rest of the system are recorded in a specific fmea worksheet. fmea includes the review of the following : steps in the processfailure modes (what could go wrong?)failure causes (why would the failure happen?)failure effects (what would be the consequences of each failure ?) failure modes (what could go wrong ?) failure causes (why would the failure happen ?) failure effects (what would be the consequences of each failure ?) within this approach, the critical treatment path is followed and risk classification for each individual step is estimated (figure 1). the method is explained in detail in ponzetti.10 in the first instance, four centers in two regions (emilia - romagna and lombardia) were identified in order to run a pilot study phase analyzing the feasibility. the pilot study was successful and demonstrated trends toward a benefit of the subcutaneous therapy.11 after the successful execution of the pilot study, 19 centers in six italian regions were recruited to participate in the study. the two largest regions participating in the study were emilia - romagna and lazio (table 1). information were collected for five patients per participating center using a validated questionnaire. within that survey the current information on the administration of trastuzumab in breast cancer and rituximab in nhl were collected and compared against the expected results for the subcutaneous therapy (figure 2). the rationale for the sample size per center was based on the average patient records per week. base assumptions were as follows : the health care processes are consistent and well - defined between the centers, and the sample did not have the purpose of being statistically representative but rather to provide an overview of operating modes consolidated. the route of administration was discussed and aligned with the participating centers in order to capture all relevant parts of the therapy. besides the location, the annual number of treated patients could also have an impact on the interpretability of the study. as it can been seen in table 2, 50% of participating hematology centers treat > 100 patients annually and are defined as being large centers. the proportion of medium and small - sized centers is quite evenly distributed with 24% and 29%, respectively. for the oncology centers with a focus on breast cancer patients, there are approximately 41% small study centers compared to 29% medium - sized and 24% large centers. for the rituximab and trastuzumab administration, 35 different risk steps were identified and assessed. a detailed description of all the 35 areas are available in table s1. after the identification of the risk levels for intravenous and subcutaneous administration, the monetary quantification of the insurance premium reduction was calculated as follows : it was assumed that risk classes of three or lower would not require separate insurance or would not have an impact on the insurance premiums for a hospital. for risk level 4, the following scenario was assumed : a female patient, 50 years of age, who receives a biologic therapy against her breast cancer would need to visit the hospital once a week. it is assumed that a treatment or administration error with a risk level 4 would impact the patient by a permanent invalidity of 40%. based on a decision by the milan court,12 a so - called table 2013 was published that shows that such a permanent disability would have a cost impact of minimum 234,371 such a risk would need to be insured additionally by a special insurance for each hospital. the exact premiums could not be calculated ; however, it could be assumed that a reduction in the likelihood of such a monetary impact would also have a proportional impact on the insurance premiums. when the risk classes are followed and calculated, eight areas were identified to be high risk for the administration of an intravenous therapy in hematology or oncology, 13 areas would be defined as having a median risk, and 14 areas would be classified as having a low risk classification (figure 4). the eight high - risk areas identified are as follows (numbers in brackets correspond to the respective fmea rankings in table s1) : overdosing of treatment due to dosage calculation error (2)overdosing of treatment due to wrong / missing prescription check (6)wrong patient treated due to wrong / missing prescription check (7)overdosing due to wrong therapy preparation (11)treatment to another (wrong) patient due to wrong drug arrival (15)treatment to another (wrong) patient due to wrong patient identification (17)treatment to another (wrong) patient due to missing check of patient and drug bag (label) (18)treatment to another (wrong) patient due to wrong check of patient and drug bag (label) (19) overdosing of treatment due to dosage calculation error (2) overdosing of treatment due to wrong / missing prescription check (6) wrong patient treated due to wrong / missing prescription check (7) overdosing due to wrong therapy preparation (11) treatment to another (wrong) patient due to wrong drug arrival (15) treatment to another (wrong) patient due to wrong patient identification (17) treatment to another (wrong) patient due to missing check of patient and drug bag (label) (18) treatment to another (wrong) patient due to wrong check of patient and drug bag (label) (19) when the new subcutaneous formulation would be applied, 23 different risk levels could be completely eliminated, which is a 65% reduction in risk levels (figure 3b). including those eliminations are the following important and high - risk classes : dose calculationpreparation and package labelingpreparation of the access to the veinpump infusion preparation and infusion monitoring preparation and package labeling preparation of the access to the vein pump infusion preparation and infusion monitoring including the infusion preparation and application of the infusion might also lead to the reduction of patient - relevant events such as infections and other infusion - related issues. furthermore, the following risk classes might possibly be moved from high risk or medium risk to the lower risk and lower likelihood of occurrence by changing the administration route to subcutaneous : wrong preparationmissing labelingwrong labelingwrong check of corresponding patient bag wrong check of corresponding patient bag the overall risk level for the intravenous administration was estimated to be 756 (ex - ante) and could be reduced by 70% to a risk score of 225 (ex - post). such an impact is patient relevant and also has a major impact on the insurance premiums being paid for such risks or the accruals a hospital needs to take for the potential financial implications. as the actual premiums could not be taken into account, an approximation based on the potential compensation for the harm was utilized. the likelihood for such a potential reduction is based on the lower risk index and lower risk potentials with the subcutaneous administration. based on the authors knowledge, this analysis is one of the first published articles for the italian health care setting evaluating the potential impact of a new subcutaneous formulation in hematology and oncology on the risk quantification. the results show a relevant decrease in the risk index and also a potential relevant financial impact with respect to insurance premiums being charged for each hospital or accruals to be taken to compensate the potential financial risks. in the current economic situation, hospitals in europe and especially in italy are under financial and health care quality pressure. these results have a large relevance in terms of therapy quality assurance from a patient s perspective linked with potential cost savings in terms of insurance premiums. furthermore, the underlying analysis shows potential for cost and resource savings in hospitals due to subcutaneous administration of trastuzumab and rituximab. the underlying analysis might be criticized based on the applied method. the analysis was done with a comparison of the actual situation with the intravenous therapy and was compared to the theoretical savings of a subcutaneous therapy. the theoretical risk reduction needs to be taken into account with the already existing risk mitigation strategies by hospitals and could hence be overestimated. furthermore, the real - life impact would need to be captured in a direct clinical study. finally, the impact on the insurance premiums for hospitals due to treatment errors would need further research. additionally, the number of centers were acceptable for such a research ; however, two out of 17 centers contributed > 50% of patients observed for the analysis in nhl and four out of 16 centers in breast cancer contributed 50% of participating patients. the availability and use of subcutaneous administration for oncologic or hematologic therapies might lower the risk of administration and treatment errors for patients and hence could indirectly have a positive financial impact for hospitals through lower insurance premiums against such risks. the availability of a subcutaneous version of rituximab and trastuzumab in the approved indications offers the availability of the current standard of care with a reduced risk of treatment errors. overview of all fmea activities included in the analysis and its respective rn abbreviations : fmea, failure mode and effect analysis ; rn, rank. | backgroundin oncology, an important parameter of safety is the potential treatment error in hospitals. the analyzed hypothesis is that of subcutaneous therapies would provide a superior safety benefit over intravenous therapies through fixed - dose administrations, when analyzed with trastuzumab and rituximab.methodsfor the calculation of risk levels, the failure mode and effect analysis approach was applied. within this approach, the critical treatment path is followed and risk classification for each individual step is estimated. for oncology and hematology administration, 35 different risk steps were assessed. the study was executed in 17 hematology and 16 breast cancer centers in italy. as intravenous and subcutaneous were the only injection routes in medical available for trastuzumab and rituximab in oncology at the time of the study, these two therapies were chosen.resultswhen the risk classes were calculated, eight high - risk areas were identified for the administration of an intravenous therapy in hematology or oncology ; 13 areas would be defined as having a median - risk classification and 14 areas as having a low - risk classification (total risk areas : n=35). when the new subcutaneous formulation would be applied, 23 different risk levels could be completely eliminated (65% reduction). important high - risk classes such as dose calculation, preparation and package labeling, preparation of the access to the vein, pump infusion preparation, and infusion monitoring were included in the eliminations. the overall risk level for the intravenous administration was estimated to be 756 (ex - ante) and could be reduced by 70% (ex - post). the potential harm compensation for errors related to pharmacy would be decreased from eight risk classes to only three risk classes.conclusionthe subcutaneous administration of trastuzumab (breast cancer) and rituximab (hematology) might lower the risk of administration and treatment errors for patients and could hence indirectly have a positive financial impact for hospitals. |
flybase is the leading database for genomic and genetic information on the model organism drosophila melanogaster and other members of its clade (1). the website contains over 2.5 million pages covering 19 different data classes and 20 drosophila - sequenced genomes. flybase displays data on over 144 000 alleles, themselves associated with over 30 000 gene records. allele and gene reports accommodate phenotypic, genetic interaction and molecular data, much of which are extracted from the published literature by highly trained curators. flybase curators capture information from the published literature and integrate this with genome annotation, high - throughput datasets, stock information and other bulk datasets to provide a one - stop site for drosophila - related information. research trends and methods are constantly changing, and flybase curation has evolved over the last 20 years to accommodate these changes. during this period, the number of drosophila - related primary research articles published each year has steadily increased, from roughly 1000 in 1980, to over 2000 a year since 2001 (2). flybase genetic literature curation is performed on an article - by - article basis (as opposed to gene - by - gene or topic - by - topic) and until 2008, we used a variety of tier systems to list and prioritize journals for curation. the advantage of curating in an article - by - article manner is that each article is seen by one curator, rather than by a triaging curator and then a number of different curators, each curating a different aspect of the article (e.g. one for genetic interactions, one for phenotypes). for example, gene ontology (go) annotations are often taken from many parts of an article and can sometimes be formed only by taking into account information from different sections and figures. between 2005 and 2008, we had a three - tier system whereby journals (and the articles therein) were ranked based on a combination of their impact factor and an experienced - based estimation of the quantity of flybase - curatable drosophila - related information they contained. we had a watch list of 35 journals and each curator would select a journal from the list and inspect its latest issue in order to identify relevant articles. this method was not perfect however, as many data - rich articles from non - priority journals were only curated when we were alerted to them by users or from citations in other articles. novel strategies were needed to identify relevant data from a wider spectrum of drosophila literature and to rank articles by criteria more specific than parent journal. in 2008, we introduced our first - pass skim curation triaging system, where we extract a minimal level of information from every article and reserve full genetic curation for the more challenging data types. we brought authors into the process in 2011 with the introduction of an email - author system, whereby we contact authors of recently published articles and ask them to fill in an online fast - track your paper (ftyp) form that recapitulates our skim curation (2). in the first 9 months of e - mailing, corresponding authors skim curated 44% of newly published articles. we then began sending reminders to authors who have not responded and author - led skim curation has increased to 57%. another improvement we are exploring is the addition of text mining to the flybase curation pipeline. the continued increase in article number, data types and data density (within each article) is an on - going challenge to curation management. if software can shoulder some of the burden of paper triage and data extraction we can devote more curation time to the specialized task of full curation. this overview of flybase genetic literature curation practices highlights promising targets for effective text mining of drosophila data. it can take between 2 and 4 months on average for an article to be fully curated, from deposition in ncbi s pubmed to being available on our public web server (figure 1, curation pipeline). we perform a semi - automated search of ncbi - pubmed for drosophila - related publications every week. the title and abstract for each publication are checked by eye to confirm that the publication contains drosophila - related data, after which we semi - automatically add the basic citation details to the flybase database. the corresponding authors of these recently published articles are then sent an email inviting them to use our ftyp form to skim curate their article. after a suitable delay to allow the authors to respond, we then generate a list of all the publications that havent yet been either fully or skim curated. we do this by not only using those citations that we have just added, but also those citations that we havent yet curated from previous citation loads. untouched articles, i.e. all those that havent been curated in any form, either by us or by authors through our ftyp tool. each article on this list undergoes a first - pass skim curation by flybase curators. as the name suggests, skim curation is a shallow, quick curation, where the main genes mentioned within an article are associated with that publication. we also flag the article for the data types present, such as whether the authors generate a new allele or whether there is expression data in the article (we call this second step triaging) (table 1). these first - pass curation records are loaded into the database and made available to the public in the next release of flybase so that users can find all the references associated with their gene of interest. the citation details for these articles are added to our bibliography, and where possible, the associated pdf is downloaded. the correspondence email for each new article is extracted from the pdf and used to invite the author to use our ftyp tool. through use of this tool, or a flybase curator data types found in the article, flagged either by the authors or curators, are then used to generate a priority list for full curation, where we extract detailed genetic and molecular information. table 1data - type flags used in literature curation (taken from an article by bunt., 2012 2)data - type flagsdata presented in an articledrosophila reagents new allele or aberrationgeneration of a new classical allele or chromosomal aberration in a drosophilid genome new transgenegeneration of a new transgenic constructgene characterization initial characterizationinitial characterization of a drosophilid gene merge of gene reportsevidence suggesting the merge of two or more flybase gene reports gene renamenew gene symbol or name for an existing gene in flybaseexpression expression in a wild - type backgroundnew temporal or spatial expression data of any d. melanogaster gene in a wild - type background expression in a mutant backgroundexpression data of any d. melanogaster gene in a mutant background, or after environmental perturbationphenotypes and interactions phenotypic analysisnovel phenotypic data physical interactionphysical interactions involving d. melanogaster proteins or nucleic acidsgenome annotation data changes to d. melanogaster gene modelnew experimental data relevant to d. melanogaster gene model structure changes to non - d. melanogaster gene modelnew experimental data relevant to the gene model structure of non - d. melanogaster molecular mapping data cis - regulatory elements definedexperimental definition of cis - regulatory elements of d. melanogaster genes literature curation pipeline. the citation details for these articles are added to our bibliography, and where possible, the associated pdf is downloaded. the correspondence email for each new article is extracted from the pdf and used to invite the author to use our ftyp tool. through use of this tool, or a flybase curator data types found in the article, flagged either by the authors or curators, are then used to generate a priority list for full curation, where we extract detailed genetic and molecular information. data - type flags used in literature curation (taken from an article by bunt., 2012 ; see ref. 2) the data - type flags that were added during skim curation are stored in an internal database and used to generate a priority list for full curation. our current practice is to target articles with the broadest range of curatable data (i.e. the highest number of flags) first. essentially, the more flags an article has, the more relevant data types it contains and the higher its position in our priority list. the system is flexible so that we can change how the flags are used to prioritize articles as circumstances change (such as an increase in a particular type of data or funding changes). using this approach, each individual article is prioritized based solely on its relevance to flybase users. in full curation, literature curators extract genetic entities and related molecular and phenotypic data from the results (text, tables and figures) and methods sections of an article. only primary data are extracted, so no data are curated from the introduction, discussion or when they are referenced to another article. additional types of data include go annotations, genetic interactions (e.g. alleles of different genes), allelic interactions (e.g. alleles of the same gene), aberrations (e.g. deficiencies), allele classes (e.g. hypomorphs) and construct data (e.g. transgenic lines). curators interact with an article in differing ways. depending on the length of the article, some curators read the article from the computer screen, while others print the article out and highlight phrases with a marker. often, the find function of a pdf viewer is used to search for multiple occurrences of the same term during on screen curation. some curators skim through a whole article first in order to get a good overview of the work and then re - read it more thoroughly to extract the detail. others start curation from a specific section (not necessarily the abstract or the results section) and then move to another section in search of additional information about a specific concept, for example, a particular transgenic construct or gene function. each curator may have a different method by which they curate an article, but all curators follow strict guidelines as to what type and depth of data are curated and any inconsistencies in curation are identified in regular curator meetings. data from an article are added to structured template files using a range of valid symbols (and recorded synonyms), controlled vocabularies (cvs) (table 2) and free text. these proformae roughly correspond to the report pages found on the flybase website. for genetic curation, the proformae are arranged in a hierarchy, starting with a single publication proforma, followed by gene proformae and nested allele proformae as necessary, then by proformae for other genetic entities as required (figure 2a, proformae organization). (a) the proformae are ordered such that each curation record has to start with a publication proforma, so all objects mentioned subsequently can be attributed to the relevant publication. allele proformae are added underneath the parent gene proforma, so all allele information can be related back to the parent gene. the fields start with an exclamation mark (for processing) and each field has a field code, e.g. ga1a is the allele symbol field (all fields in the allele proforma are coded gax). table 2the main cvs used in literature curationontologyexample search term (cv id)fly_anatomydmp2 neuron (fbbt:00001602)fly_developmentpupal stage p6 (fbdv:00005353)term qualifiernutrition conditional (fbcv:0000714)phenotypic classsmell perception defective (fbcv:0000404)sequence ontologyengineered_foreign_gene (so:0000281)origin of mutationp - element activity (fbcv:0000486)allele classamorphic allele (fbcv:0000688)cellular componentgerm cell nucleus (go:0043073)molecular functionsatellite dna binding (go:0003696)biological processmrna processing (go:0006397) literature curation into proformae. (a) the proformae are ordered such that each curation record has to start with a publication proforma, so all objects mentioned subsequently can be attributed to the relevant publication. allele proformae are added underneath the parent gene proforma, so all allele information can be related back to the parent gene. the fields start with an exclamation mark (for processing) and each field has a field code, e.g. ga1a is the allele symbol field (all fields in the allele proforma are coded gax). the main cvs used in literature curation each proforma is composed of a number of fields, which are split into four main types (figure 2b, proforma fields). these contain the unique i d and official flybase symbol for the genetic entity of interest. these fields also include the symbols and names used to identify the object in the literature, so users can identify an object even if flybase labels the object with a different symbol / name to that used in a particular article. the second type of field houses cv lines constructed from one or more of the 16 ontologies (structured hierarchies) used in flybase (table 2). cv terms are used to annotate fields for many objects in flybase. for example, we use sequence ontology (so) (3) terms to denote the type of gene and go terms (4) to annotate these genes for molecular function, cellular component and biological process. we also use cvs to record both the class of a phenotype and the anatomical part that is affected. phenotypes are attached to alleles and genotypes (combinations of alleles, or alleles and chromosomal aberrations). an example piece of text from which we have extracted phenotype cv terms and free text is shown in figure 3. example data entries for a section of text [taken from an article by baines (2003), see ref. 5 ]. first, we identify the object we are ascribing the phenotype to, then we concisely curate the phenotype as free text, relating it to the object (which is placed between at sign symbols as these symbols are hyperlinked). we then annotate the phenotype to cv terms, in this case, to terms from our phenotypic class and fly_anatomy ontologies. example data entries for a section of text [taken from an article by baines (2003), see ref. 5 ]. first, we identify the object we are ascribing the phenotype to, then we concisely curate the phenotype as free text, relating it to the object (which is placed between at sign symbols as these symbols are hyperlinked). we then annotate the phenotype to cv terms, in this case, to terms from our phenotypic class and fly_anatomy ontologies. the third type of field is the so - called softcv field, which uses a limited set of terms to describe a feature in a semi - controlled manner. molecular lesions field in the allele proforma, which captures amino acid changes with the prefix amino acid replacement. the fourth type of field houses free text descriptions. these fields provide a level of detail that can not be captured by cv terms and describe phenotypes and molecular lesions in a more human readable form. an example of this field is the phenotype free text field, where we record the detail of a phenotype (by paraphrasing rather than cutting and pasting from the article) that is associated with an allele or genotype (figure 3). once genetic curation is complete, each curation record is checked using our in - house software, where each line is assessed for the correct structure and cv. the record is also checked for coherency between the fields, so for example, if a gene is renamed, we confirm that the new gene symbol has not been used before, and that a line is added in another field to attribute the rename to that particular article. once a curation record has been fully checked for clashes both within the record and with the existing database, it is integrated with existing data in the flybase chado database (6). currently, we update the public website with a new release of the data six times a year, at roughly 2-month intervals. it s a time - consuming task that can take an experienced curator a number of hours for a standard article. even then, curation is a social process, with curators seeking advice among the group. in this section, we will outline some of the problems we encounter in curation and describe how they could impact on the use of text mining in flybase. the most common problem encountered during curation is an ambiguous genetic entity (gene, mutant allele, transgene, etc.) this situation can arise when no unique identifier (such as a flybase gene identifier (fbgn) or a computed gene (cg) number for genes), or an accurate and explicit reference for a mutant or transgenic line is given. ambiguity is a particular problem when a generic symbol / name is used (e.g. actin or uas - notch), or when a symbol / name is used that is a synonym for a different entity (e.g. ras is the current flybase symbol for the raspberry gene, fbgn0003204, but is often used in the literature to refer to the ras85d gene, fbgn0003205). a further issue is that some symbols only differ in case - sensitivity for the first character, for example, the genes symbols these ambiguities can usually be resolved by searching for associated details about the entity in the article (e.g. the use of a specific mutant allele can identify the gene being discussed) or by consulting the supplemental information for additional details. sometimes we have to do some analysis ourselves, such as performing a blast search using any sequence data present in the article or supplementary files or executing an in - house script to report those entities used by a specified author in previously curated articles. as a final step, if we can not resolve a problem, we email the corresponding author for clarification. if the ambiguity still can not be resolved, then a curator will either associate a generic / unspecified entry for that entity with the article, or else omit the entity and add a (non - public) note to the curation record explaining the situation, with the hope that future publications will resolve the issue. one of the more esoteric problems found in curation is the fact that multiple relationships exist between the curated data types. p{ep}dpp insertion and disrupts the dpp gene. this can cause problems for text - mining assisted curation, as the data can be attributed to the wrong object due to sentence structure or the requirement of background or contextual knowledge found in other parts of the article. in cases like this, detailed knowledge of the flybase proforma and curation rules, as well as a good knowledge of drosophila biology, is necessary to ensure the correct proforma field is filled in. this is one of the reasons why we believe text - mining methods will assist manual curation rather than replace it in the near term. however, when broken down into discrete components, we believe, from experience, that the use of text - mining tools can be beneficial. we hope that, over the coming years, we will be able to integrate text mining into multiple areas within our curation pipeline and practice, to help streamline the process. in this section, we will outline our current progress and define our aims for the near future. flybase began interacting with the text - mining community in 2002 when we were involved in the kdd cup challenge (7), a forerunner to the biocreative initiative (8). in 2008, we developed a natural language processing (nlp) system that marked - up html versions of an article for gene / allele mentions and associated phenotypes (9). paperbrowser tool, written in java and on top of a web browser, is equipped with two navigation mechanisms called paperview and entitiesview. these are organized in terms of the document sectioning and possible relations between groups of words (noun phrases). vang) in the order in which they appear in each section of the article, while entitiesview lists noun phrases related to the gene symbols such as the wg pathway. clicking on a node in either paperview or entitiesview redirected the paperbrowser tool to the sentence that contains the corresponding gene symbol or noun phrase, so allowing the curator to navigate around the article to those sections involving the entity of interest, for example, a particular mutant allele or transgene. used in conjunction with a simple curation interface, the paperbrowser tool improved article navigational efficiency (the number of navigation events needed before the data are extracted) by 58% and provided curators with enhanced utility (the number of non - navigated events needed outside of the highlighted areas) by over 74% compared to using the the paperbrowser system is a good proof of concept, demonstrating that text mining can be successfully integrated with the flybase article - by - article curation system, which is something we would like to explore further in the future. flybase has collaborated with wormbase (10) and textpresso (11) since 2004 when we were consulted in the development of textpresso for fly, including during the creation of fly - specific vocabularies to use in the, we are exploring the use of support vector machine (svm) methods to triage primary research articles into categories based on our skim / author curation flags (12). we have trained the svm to triage articles for new alleles, new transgenes and gene renames. we have done this through the generation of positive and negative training sets, composed of articles that we have already curated (where we know whether they contain a particular data type or not). the positive training set contains between 500 and 1000 articles, while the negative training set contains over 2000 articles. we plan to re - train the svm for these flags periodically, to account for changes in the literature and to ensure continued low false - positive / negative rates. we are in the process of training the svm for some of the other data triage flags, in the hope of using the svm to triage those articles that havent been curated by the authors through our ftyp tool. we can envisage text mining being used at multiple points within our curation workflow. further to our current svm work, text mining could be a useful adjunct to our manual go annotation process. there are still many genes that lack functional information and, minimally, automated text mining could be used to identify articles with functional data for these genes. following the example set by wormbase (13), we hope to use text mining to generate suggested annotations for specific genes (subject to curator review) for at least go cellular component. we are about to embark on curating disease associations and anticipate that the textpresso disease category could also be helpful for this aspect of curation. while svm methods may replicate the triage aspect of skim curation, in order to fully automate the process, we need to also identify the genes mentioned in each publication. textpresso and the genetics society of america (gsa), in collaboration with wormbase, sgd (14) and flybase, have developed a journal article mark - up pipeline that links gsa journal articles and flybase gene symbols and ids (15). false positives are discovered (at a rate of 12%, with false negatives at 27%) and resolved by a curator through a manual quality control step, with the final marked - up document assessed by the authors as part of the proofing process. building on our collaboration with wormbase and the gsa, we hope to use text mining to extract genetic entity symbols from all drosophila - related literature. this, in combination with a document triage system, would form a text - mining equivalent of our skim curation, combining symbol extraction with document triaging. we are also interested in using text mining to suggest anatomy cv terms for specific genes from particular regions of text, along with gene and allele symbols and data triage flags. this would not extract the data and populate proforma, but simply highlight the text for the curator to assess and extract if appropriate. this would build on the success of the paperbrowser system and accelerate manual curation of an article. over the last 20 years, flybase has had to adapt and change to keep abreast of changes in biology and database design. a major challenge over the years is to deal with the massive increase in data that biologists are generating, both in genomic studies and also in the published literature. while it may be too ambitious to anticipate text mining replacing human curators for full curation, we can envisage a time when text mining will be regularly used in flybase curation. our aim when writing this article was to inform the text - mining community about our curation pipeline and practice. we hope that this will encourage collaboration with flybase so that we can put technologies in place that will aid curators both in flybase and in other databases to deal with this challenge. the national human genome research institute at the national institutes of health [p41 hg00739 ] and the medical research council, uk [g1000968 ]. funding for open access charge : the national human genome research institute, the national institutes of health [p41 hg00739 ]. | flybase is the model organism database for drosophila genetic and genomic information. over the last 20 years, flybase has had to adapt and change to keep abreast of advances in biology and database design. we are continually looking for ways to improve curation efficiency and efficacy. genetic literature curation focuses on the extraction of genetic entities (e.g. genes, mutant alleles, transgenic constructs) and their associated phenotypes and gene ontology terms from the published literature. over 2000 drosophila research articles are now published every year. these articles are becoming ever more data - rich and there is a growing need for text mining to shoulder some of the burden of paper triage and data extraction. in this article, we describe our curation workflow, along with some of the problems and bottlenecks therein, and highlight the opportunities for text mining. we do so in the hope of encouraging the biocreative community to help us to develop effective methods to mine this torrent of information.database url : http://flybase.org |
gallstone ileus is an infrequent cause of intestinal obstruction, responsible for 1 - 4% of all mechanical intestinal obstructions (1). it is due to a gallstone larger than 2.5 cm in diameter that usually obstructs terminal jejunum. the gallstone usually enters the bowel via a cholecystoenteric fistula (2), resulting from recurrent attacks of cholecystitis. gallstone ileus is usually a disease of the elderly and occurs more frequently in females (3). the present case is interesting because, 1) the patient was a young male ; 2) the ileus occurred 3 yr after cholecystectomy and 3) the localization of the obstruction was an old side - to - side ileoileic anastomosis due to a diverticulectomy following intussusception of meckels ' diverticulum at the age of 3. a 44-yr - old man presented to the emergency department with lower abdominal pain of 3 days ' duration, associated with nausea, diarrhea and failure to pass flatus and feces. the last year he presented again with an episode of severe abdominal pain, but the abdominal examination was unremarkable. his medical anamnesis reports gilbert syndrome, appendicectomy and meckel 's diverticulectomy due to intussusception at the age of three, and laparoscopic cholecystectomy three years before admission. physical examination showed mild abdominal distension, tenderness in the epigastrium, and dehydration with breaking of the skin and glue tongue. laboratory studies revealed a haematocrit of 35.4%, hemoglobin of 11.7 g / dl and white blood cell count 8.700/l with a predominance of polymorph nuclear cells (66.4%). his liver function tests were all within normal limits with total bilirubin of 7.6 mg / dl. plain films of the abdomen showed multiple air - fluid interfaces in the small intestine and colon full of gas. there was no evidence of pneumobilia, but there were surgical clips at the anatomic area of the right upper quadrant. two calcified opacities were detected in the left iliac fossa at the supine radiograph (rx), which changed location at the erect rx (fig. plain rx of the abdomen the two intraluminal abnormalities appeared in the dilated small bowel of terminal ileum (fig. 1), compatible with gallstones. ultrasonography (us) was performed revealing edema and thickening of the bowel wall and dilatation of the loops of small bowel, which showed active peristalsis diagnostic of mechanical small bowel obstruction. gallstones were identified within the dilated bowel as floating hyper - echoic foci producing distal acoustic shadowing reflections, which moved when the patient 's position was, changed (fig. the computed tomography (ct) showed multiple air - fluid interfaces, distended small bowel loops and two big ectopic peripheral calcified stones with radiolucent centers in the dilated small bowel of terminal ileum. one of them had a thick calcified surface and hypo - dense center which the form is like mercedes - benz figure. a new laterolateral ileoileic anastomosis was performed. in the excised intestine two calculi sized approximately 3 cm furthermore, in order to have chemical analysis the stones were powdered in a pestle and mortar and dissolved in different solvents depending upon the type of chemical constituent to be analyzed. the pathogenesis of gallstone ileus necessitates the presence of a gallstone in the intestinal lumen, the most common route of entry being a cholecystenteric fistula resulting from recurrent attacks of cholecystitis. a cholecystoduodenal fistula was demonstrated in 68% of patients with gallstone ileus by clavien. stones passing spontaneously through the ampulla of vater have also been reported to cause gallstone ileus (5), besides the fact that more than 80% of gallstones entering the gut are excreted uneventfully (6)., we ca n't identify the way through which the stones arrived to the intestine, but we are pretty sure that they enlarged in the gut, at the site of the old anastomosis. the stones had probably remained in the anastomosis and under dyskinesis and stasis the bacterial overgrowth caused large gallstone formation. the localization of the stones further explained the symptoms of the patient - notably intermittent partial obstruction - during the last two year. probably the bilirubin stones impacted and disimpacted within the old anastomosis that resulted in presentation and relief of the symptoms. rigler 's criteria (7) : 1) air or contrast medium in the biliary tree, 2) visualization of the stone in the intestine, 3) change in position of a previously observed stone, and 4) radiologic evidence of intestinal obstruction are pathognomonic in the diagnosis of gallstone ileus. in the present case three out of these four criteria were fulfilled and, even if the images were typical, the diagnosis of gallstone ileus was many times challenged due to the prior cholecystectomy. at this point (8) that ct shows important details such as : the evidence of endoluminal stones, their size and their number. ct may also detect ectopic stones and allow the diagnosis of gallstone ileus before severe intestinal obstruction from stone impaction occurs. although enterolithotomy alone remains the popular operative method in most reports, the one - stage procedure composed of enterolithotomy, cholecystectomy and repair of fistula is necessary, if indicated (9). tan. (10) compared the two surgical strategies of enterolithotomy alone and enterolithotomy with cholecystectomy for the emergent treatment of gallstone ileus, and concluded that both procedures are safe with no mortality, but the better surgical option is enterolithotomy. recently, chou. (11) proposed endoscopic approach to remove gallstones, but their size constitute a technical difficultly in order to apply. bowel resection is only indicated when there is intestinal perforation or ischemia (12). in the present case, furthermore, bowel resection was necessary due to the stenosis of the old anastomosis and the multiple adhesions. in conclusion, gallstone obstructive ileus has to be considered in a differential diagnosis of mechanical ileus, even in the absence of gallbladder. furthermore, even when not all of the ringler 's criteria are fulfilled then the biliary stone has to be considered as etiologic factor. finally, when suspicion for gallstone ileus appears then ct has to be the diagnostic modality to be employed. | the present case is one of gallstone obstructive ileus due to gallstones 3 yr after laparoscopic cholecystectomy. it is interesting because of the sex of the patient, the fact that ileus occurred 3 yr after cholecystectomy and that the localization of the obstruction was an old side - to - side ileoileal anastomosis due to a diverticulectomy following intussusception of meckels ' diverticulum at the age of 3. |
the 1983 amendments to the social security act (public law 98 - 21) enacted the medicare prospective payment system (pps) for the payment of hospital inpatient operating costs, but pending further study, continued cost - based payment of capital costs until october 1, 1986. department of health and human services (dhhs) issued proposed regulations for incorporating capital payments into pps. in july 1986, legislation (public law 99 - 349) was passed that extended the exclusion of capital from pps until october 1, 1987. in may and september 1987, dhhs issued proposed and final regulations to fold capital payments into pps. however, in october 1987, enactment of public law 100 - 119 prevented the final regulation from taking effect. the omnibus budget reconciliation act of 1987 (public law 100 - 203) subsequently required dhhs to implement prospective payment for capital starting october 1, 1992. in february and august 1991, dhhs once again issued proposed and final regulations for a third attempt to incorporate capital payments into pps. the august 1991 capital regulation took effect october 1, 1991, thus initiating a 10-year transition to full prospective payment of medicare 's share of inpatient capital - related costs. congressional persistence attests to the widespread belief that change was needed in medicare capital payment policy. in particular, there was recognition that to encourage efficient resource use, medicare payment policy should not influence hospitals ' choices of how to combine capital and operating inputs. there was also the understanding that with capital paid on a cost basis and operating costs on a prospective basis, medicare provided hospitals the incentive to substitute capital for operating cost. under these circumstances both the special nature of capital and the tradition of cost - based payment inhibited change. the durability and lumpiness of capital capital tends to be purchased in larger, less divisible quantities at less frequent intervals than operating inputs. the lumpiness of capital creates the need for long - term financing and the contractual commitments that accompany debt financing. debt obligations make it more difficult to change medicare capital - payment policy without imposing major adjustments on hospitals with very large debt service costs. another consequence of the lumpiness of capital is that, relative to its size, capital cost per discharge varies more among hospitals than does operating cost per discharge. as a result, a prospective payment based on the average capital cost will tend to have a greater relative impact in redistributing payments than was the case for operating cost. finally, the tradition of cost - based payment for hospitals has created a tendency to accentuate the uniqueness of capital in people 's thinking about medicare payment policy. only the tradition of cost - based payment is unique to hospitals. certainly, in the private sector, it is almost impossible to think of a good or service for which the unit of payment separately identifies capital from the other inputs used in producing the good or service. even in highly capital - intensive publicly regulated industries such as electric power, the return on capital is incorporated into a rate structure based on the unit of service (e.g., dollars per kilowatt hour). the experience of other industries clearly demonstrates that hospital capital can be paid on a per discharge basis and need not be paid separately from other input costs. however, a shift to prospective payment for hospital capital requires consideration of how to minimize the negative effects of transitional payment redistributions and how to structure the long run prospective payment for capital. the next section of this article addresses the issue of payment redistributions by presenting descriptive data on the variation of capital and operating costs. this information quantifies the extent to which capital costs are more variable than operating costs. the effect of capital - cost variability on total - cost variability is also assessed. the specifics of transitional payment mechanisms are beyond the scope of this article, but the details of the policy being implemented are described in the august capital regulation (federal register, 1991). the remainder of this article addresses the issue of the structure of the long - run prospective payment for capital by analyzing and comparing sources of variation in capital cost versus operating and total costs : the empirical models of capital- and total - cost variations that were estimated are described, and estimation results are presented. the regression results reported in this article differ from those found in the august capital regulation in two ways. first, given the focus of this article on comparative analyses, it was necessary to limit the data set to hospitals for which all variables were available. this meant that fewer hospitals were included in the regressions reported here than in the regression actually used to determine the capital payment adjustments in the august capital regulation. second, because the emphasis here is on understanding sources of cost variation rather than on determining specific payment adjustments, the variables included in the regressions are not identical to those reported in the august capital regression. the article identifies several problems in accounting for capital - cost variation and concludes that it is preferable to use the analysis of total cost (the sum of capital and operating cost) to develop a common set of adjustments for the capital and operating prospective payments rather than to use the analysis of capital cost to develop separate capital payment adjustments. it should be noted that this conclusion is not inconsistent with the august 1991 capital regulation, which establishes adjustment factors for the capital prospective payment based on analysis of total - cost variation. in the long run, it only makes sense to base the capital payment on total - cost variation if the operating payment is also based on total - cost variation. however, the health care financing administration (hcfa) currently lacks the statutory authority to modify the payment adjustments for the operating payment to conform with the capital adjustments. the logical conclusion is for legislative action to permit the use of the same payment adjustments for the operating and capital payments based on analysis of the combined capital and operating costs. data from medicare cost reports on the distributions of medicare inpatient capital and operating costs per discharge during the period 1985 - 89 are presented in table 1. medicare capital costs consist of medicare 's share of a hospital 's depreciation and interest expenses, plus capital - related insurance costs, property taxes, leases, and rent. the main components of medicare operating costs are routine and ancillary costs. excluded are capital costs and the costs of approved graduate medical education programs. table 1 contains only the portions of capital and operating costs that have been allocated to medicare inpatient services. although capital and operating costs per discharge have risen during this period, there was little change in the shape of their distributions (table 1). for example, the ratio of the interquartile range (the difference between the 75th and 25th percentile values) to the median has remained relatively constant for both capital and operating costs. the relative interquartile range for operating cost only varied from.53 to.54 during the 1985 - 89 period. for capital cost, the same measure was higher and somewhat more variable, ranging from.88 to.94. further, comparing the ratio of the mean to the median, it is clear that the capital - cost distribution is much more highly skewed to the right than the operating - cost distribution. for capital cost, for operating cost, the mean was only 6 - 8 percent higher than the median. this greater relative variation and skew ness illustrates that a capital payment system based on mean capital costs would tend to redistribute capital payments among hospitals to a greater extent than a comparable system for operating costs. however, because capital cost is on average much smaller than operating cost, the actual number of dollars redistributed would be expected to be smaller than in the case of operating cost. as shown in table 1, the mean capital cost in 1989 was $ 476, or about 11.7 percent of the mean operating cost ($ 4,067). the top 5 percent of hospitals had capital costs greater than $ 1,102, which is 27 percent of the mean operating cost but only 16 percent of the 95th percentile operating cost ($ 6,890). although smaller in absolute dollars than in the case of operating cost, the redistribution of capital payments could be large, especially for hospitals in the tails of the distribution. another way to look at the impact of capital - cost variation is to consider its impact on total cost variation. on the one hand, the fact that capital cost represents only about 10.5 percent of total cost will limit its impact. capital and operating inputs may sometimes substitute for one another, which would tend to make them negatively correlated, and at other times may complement each other, which would tend to make them positively correlated. the pearson correlation coefficients shown in table 2 indicate that on balance capital and operating inputs are positively correlated. during 1985 - 89, this fact will tend to increase the impact of capital cost on total cost variation. it also suggests that payment policy needs to treat both cost elements jointly, because hospitals ' decisions about capital and other inputs are related to one another. to estimate the effect on the variation in capital cost of capital 's relatively small share of total cost and the positive correlation between capital and operating cost, the increase in the standard deviation of total cost per discharge as a result of capital cost was calculated as follows : the variance (var) of a sum of two variables equals the sum of the two variances plus twice the covariance of the variables. noting that, by definition, the covariance is the product of the pearson correlation coefficient (rho) and the standard deviations (sd) of each variable yields : where tot, cap, and op represent total, capital, and operating costs per discharge. because the standard deviation (the square root of the variance) is measured in dollars per discharge, it provides a more interpretable measure of the increased variation than does the variance. if capital and operating costs were not correlated, the standard deviation of total cost would only be about 2 percent greater than the standard deviation of operating costs despite the much greater relative variation in capital cost. two percent of the 1989 standard deviation of total cost ($ 1,928) is about $ 38. however, taking into account the positive correlation between capital and operating costs makes the standard deviation of total cost about 11.5 percent (or $ 200) greater than the standard deviation of operating cost. medicare cost report data confirm that capital cost exhibits much greater variability among hospitals than does operating cost or total cost. the positive correlation between capital and operating costs is responsible for greater variation in total cost than would be expected based on capital 's relatively small share of total cost. the positive correlation between capital and operating costs also supports the view that medicare hospital payment policy should not attempt to deal with capital cost independently. for example, the variables used to explain operating - cost differences and/or to adjust the pps operating payment (case mix, wage index, teaching, disproportionate share, etc.) explain more than 70 percent of the variation in medicare operating cost (pettengill and vertrees, 1982 ; anderson and lave, 1986 ; sheingold, 1990.) however, the operating characteristics appeared to account for far less of the variation in medicare capital cost per discharge (anderson and ginsburg, 1983.) often observers lacked relevant data and merely assumed that the greater unexplained capital - cost variation was largely the result of capital 's special age and financing characteristics, which was attributed earlier to its lumpiness. two analyses (u.s. congressional budget office, 1988 ; prospective payment assessment commission, 1987) used examples for individual hypothetical hospitals to demonstrate the effect of age or position in the capital cycle on capital cost. direct empirical evidence on the impact of capital 's special characteristics has more recently begun to accumulate (american hospital association, 1990 ; cleverly, 1986). this article and the aha paper are among the first analyses to incorporate age and financing factors with the variables typically associated with operating - cost variation. this article differs from other work in its comparative analysis of the variation in capital and total costs. the approach taken in this article was to first apply a typical operating - cost model to capital cost. a log - linear cost function was estimated that included the following variables : the medicare case - mix index (cmi). the level of teaching activity (measured by the ratio of interns and residents to the average daily census : resday.) the proportions of poor medicare and medicaid patients (measured by the disproportionate - share percent with separate slopes for urban and rural hospitals with more and less than 100 beds : disproportionate share for urban hospitals with more than 100 beds (dsul), with less than 100 beds (dsus) ; disproportionate share for rural hospitals with more than 100 beds (dsrl), with less than 100 beds (dsrs). in addition, dichotomous variables were included for hospitals with at least 100 beds (bedsge100), hospitals in metropolitan areas with populations of at least 1 million (large), hospitals in other metropolitan areas (other), and type of ownership (proprietary : prop or government : govt.) rural hospitals and voluntary hospitals constitute the omitted categories. second, a capital - cost model was specified that added variables to the operating cost model that attempt to capture the distinctive features of capital. there were two steps in this process : variables that reflect differences in the timing of capital spending were added first. the second set of capital variables reflects differences in financing, occupancy rates, and construction costs. third, the operating - cost model was applied to total cost, and finally, the full - capital model, consisting of all operating variables and both sets of capital variables, was estimated for total cost. before proceeding to the estimation results, because capital has a useful life measured in years, it tends to be purchased in relatively large amounts with less frequency than operating inputs. when new capital is put into service, capital costs rise sharply and then, in the absence of further major capital spending, gradually decline until the next major expenditure. when major projects come on line, a hospital can be expected to move up dramatically in the cross - sectional distribution of capital costs. thus, two otherwise identical hospitals would have different levels of capital cost if the timing of their capital spending is not coincident. the term capital cycle was not used because there need not be regularity or uniformity among hospitals in the pattern of capital spending to produce this type of cost variation (federal register, 1991). to control for this effect requires a measure of differences among hospitals in the length of time between major capital expenditures. hospitals with recent expenditures will have relatively high capital costs, and hospitals that have not made a major capital purchase for a long time will have relatively low capital costs. however, estimates of the age of a hospital 's capital can serve as proxies. three variables were used in this analysis : the ratio of accumulated depreciation to current depreciation (age), remaining depreciable asset life (the ratio of net asset value to current depreciation : rlif), and the ratio of total fixed assets to total assets (tfata.) if a hospital had a single asset with a fixed useful life, rlif would be highly negatively correlated with age because net asset value is the difference between gross asset value and accumulated depreciation. of course, hospitals have many assets of varying age and useful lives, and rlif and age are in fact positively correlated. an old, mostly depreciated plant would tend to increase age, whereas rlif might be more affected by recent equipment purchases. first, because a hospital balance sheet typically includes a variety of capital purchases made at different times, age and rlif at best represent average values. second, they are distorted by the fact that book value and depreciation reflect historical cost. inflation in capital - goods prices means that assets purchased more recently will be more heavily weighted than older assets. as a result however, because depreciation is the largest single component of our capital - cost variable, the use of historical cost also affects our dependent variable. although there may be some impact on the values of the coefficients for age and rlif, it is not clear what effect this measurement problem has on the explanatory power of the equation. third, the ability to separate current depreciation from interest and other capital expenses in medicare cost reports is subject to measurement error. finally, tfata also suffers from the use of historical cost accounting, but it is not affected by limitations in separating depreciation from the other components of capital cost. it should be noted that including these variables will tend to control for differences in the relative frequency of capital spending, but they will not explain why differences in the frequency of capital spending occur. it is beyond the scope of this article to attempt to explain interhospital differences in me frequency of capital spending. the age variables are important because of their impact on the overall explanatory power of the model and the effects they may have on the coefficients of the other variables. consider the following cases : two hospitals are identical except for the fact that one renovated its facility 2 years ago, and the other carried out a comparable renovation 10 years ago. in this case, controlling for age of capital would correctly treat the hospitals as having the same capital cost. a hospital that has been financially successful recently made a large capital expenditure last year because it needed to put its surplus to work. a financially strapped hospital has been unable to make badly needed capital repairs. in this case, controlling for age is also appropriate. the financially successful hospital 's capital cost is in effect lowered, and the financially strapped hospital 's capital cost is raised. the two hospitals are not treated as having different capital costs just because of differences in their financial circumstances. further, if successful and strapped hospitals tend to consistently have certain characteristics found in the operating model, failing to control for age would indirectly attribute differences in financial status to those operating characteristics. a hospital located in a rapidly growing area has made large capital expenditures every year for the last 5 years in order to meet the needs of its population. another hospital located in an area with three other hospitals has experienced declining occupancy for several years and has not made a major capital expenditure for many years. in this case, controlling for age differences will treat the hospitals as being alike, when it would be desirable to recognize the appropriateness of the higher cost of the first hospital. it will not be possible to do so unless other variables that distinguish these hospitals ' circumstances can be identified. in this particular example, several possibilities may be available (occupancy rates, the rate of area population growth, degree of market competition, etc.). however, in general, it most likely will not be possible. the second factor specific to capital - cost variation pertains to debt financing. because medicare capital cost includes interest expense, hospitals that borrow relatively heavily will have higher medicare capital costs than hospitals that use more equity to finance their capital purchases. the ratio of total liabilities to total assets (tlta) was included in the cost equation to control for this source of variation. this variable was included instead of the ratio of long - term liabilities to total fixed assets because tlta was more highly correlated with capital cost. this result may occur because, on medicare cost report balance sheets, total categories appear to be more accurately reported than their components. it should be noted that tlta is included in the cost equation to account for variation in medicare capital cost ; no attempt is made to explain the variation in the relative use of debt financing. finally, two additional variables of special interest for capital were included : occupancy rate (occup) and a capital construction cost index (cci). the occupancy rate could have been included in the set of operating characteristics but given the more fixed nature of capital cost, occup is expected to have a stronger effect on capital cost. the first three columns contain the capital models, and columns 4 and 5 show the total cost results. column 1 includes only the variables commonly found in operating - cost models. in column 2 the three age - related capital variables intended to account for differences in the timing of capital spending have been added. column 3 presents the full capital model including the effects of relative debt, occupancy, and construction costs. column 4 shows the result of applying the operating model to total cost, and column 5 adds the full set of capital variables to the total - cost model. the data set comprises 2,859 hospitals whose capital variables passed a series of consistency edits. although very similar in many ways, the greatest differences appear to be in urban - rural location and teaching status. only 23 percent of the 2,859 hospitals are in metropolitan areas with a population of more than 1 million, compared with about 26 percent of all hospitals. almost 50 percent of the sample hospitals are in rural areas, compared with less than 47 percent of all hospitals. there may be a systematic relationship between complexity or special features of corporate structure and the absence of usable balance sheets. the effects of this limitation on the cost models reported in this article could not be determined. two years of medicare cost report data (pps-5 and pps-6) were pooled, and a dummy variable was included for the pps-6 year. all variables are derived from medicare cost reports with the exception of the cmi, which is based on fiscal year (fy) 1988 and fy 1989 medpar ; wi, which is constructed from the 1988 hcfa wage survey (excluding the effects of the 1990 - 91 decisions of the medicare geographical classification review board) ; and the cci, which was developed for hcfa by the center for health economics research (pope, 1991.) the functional form of all models is a hybrid log - linear - exponential function. the dependent - cost variables and all independent variables are in logarithms with the exception of the teaching variable (resday), the disproportionate share variables (dsul, dsus, dsrl, and dsrs), and other dichotomous variables that are exponential in form. table 3, column 1 shows that the operating - cost model explains 43 percent of the variation in capital cost. case mix shows the strongest effect, followed by the urban dummy variables and the beds variable. for example, the cmi coefficient is much greater than 1, which implies that capital cost increases disproportionately with case mix. pps operating payments are designed to increase proportionately with case mix, and operating - cost models have supported that relationship. in addition, the urban variables typically are thought of as picking up residual unexplained locational effects not captured by the other key variables. therefore, the large magnitude of the urban variables, combined with the statistically insignificant or marginally significant performance of the wage index, teaching, and disproportionate - share variables, is particularly troublesome. the coefficient of prop indicates that proprietary hospitals ' capital costs are 38 percent higher and (govt) hospitals ' costs are 9 percent lower than those of voluntary hospitals. table 3, column 2 indicates that adding the age of capital variables to the model improves the explanatory power noticeably (the r increases to.55). also, the behavior of the operating variables resembles somewhat more closely their behavior in operating - cost regressions (sheingold, 1990.) the wage index is now positive and significant, and, as might be expected, its coefficient is much smaller than in operating - cost regressions. the disproportionate - share variables are qualitatively similar to the typical operating - cost model. though not significant, the large urban disproportionate - share variable is now positive, and the small urban and rural variables are negative and generally significant. however, the cmi coefficient still greatly exceeds 1, and the teaching variable is very close to zero. interestingly, the urban and proprietary hospital dummy variables are reduced dramatically, and only the large urban variable remains statistically significant at the.05 level. this result implies that to a large extent, urban and proprietary hospitals ' higher capital costs result from their greater recent spending. in contrast, the coefficient of the bed - size variable is hardly affected by the addition of the age variables. the results for the full - capital model appear in column 3 of table 3. there is a small improvement in explanatory power compared with the age - only capital model (the r is.59). with the exception of the construction - cost index, all the capital variables are strongly statistically significant. the only operating variable that changes much is the wage index, which becomes statistically insignificant. the small, statistically insignificant coefficients of the wage- and construction - cost indexes suggest effects of multicollinearity. indeed, the two indexes are highly positively correlated (.80). however, regressions not reported in table 3 indicate that merely dropping one of the indexes does not strengthen the remaining index. the interrelationships appear to be more complicated and involve the combination of age and financing variables. to further explore the issue of multicollinearity, the klein test was performed for the full - capital model (maddala, 1977.) the r of each independent variable regressions was then compared with the r of the full - capital model. under the klein test, multicollinearity is likely to create problems when the r of an independent variable regression exceeds that of the main regression. only the wage- and construction - cost indexes fail the strict application of the klein test for all three capital models estimated. however, the cmi also fails the test for the model with only operating variables, and the occupancy rate would fail for similar models. unfortunately, the klein test does not provide a prescription for curing the ills of multicollinearity. a major econometric advantage of the total - cost models is that their higher overall explanatory power reduces the impact of the collinearities. table 3, column 4 shows the results of applying the operating - cost model to total cost. the r is.72, and all but two variables are statistically significant at the.05 level. the wage index behaves as expected ; there is a positive, highly significant teaching effect. the disproportionate - share variables for small urban and rural hospitals are negative and statistically significant.. the higher costs of proprietary hospitals compared with voluntary hospitals are statistically significant, but government hospitals ' costs do not differ significantly from those of voluntary hospitals. column 5 of table 3 presents the total - cost results using the full - capital model. although the addition of the capital variables increases the overall explanatory power only slightly (the r is.73), all of the capital variables are statistically significant with the exception of the variable rlif. however, the elasticities of the capital variables are no larger than.05, except for the construction - cost index, whose elasticity is.16. few of the operating - cost variables are affected by the addition of the capital variables, and their coefficients are very similar to those in column 4. the largest change is the wage index, whose coefficient declines from.71 in column 4 to.60 in column 5. the addition of the capital variables reduces the coefficient of prop, which is no longer significantly different from 0 at the.05 level. the results previously described demonstrate that the special characteristics of capital have an important effect on the cross - section variation in hospitals ' capital costs. the capital variables perform as expected and add substantial explanatory power to the capital - cost models. the chief implication of this finding is that hospitals ' past capital spending and financing decisions deserve serious attention in working out the transition from cost - based payment to prospective payment. in the long run, a hospital 's medicare prospective payment should, like market prices in general, be independent of the hospital 's capital timing and financing decisions. in this context, the age and financing variables are appropriately used as control variables, serving to prevent their effects from being attributed to other factors that may be used to adjust the long - run payment. however, other problems encountered in estimating the capital - cost models limit their usefulness for prospective payment. first, the medicare balance sheet data required for the construction of the key age and financing variables are unusable for a large number of hospitals. of special concern is the apparent systematic under - representation of certain types of hospitals, such as hospitals from large urban areas, major teaching hospitals, and proprietary hospitals. these types of hospitals may be more likely to have unusual capital items that complicate their balance sheets and result in their failing simple reasonableness screens. second, although the capital variables performed well in the capital - cost models, the key payment variables performed poorly compared with the total - cost model. the coefficient of the case - mix index was greater than 1, which implies that payments and costs would not be consistent with one another across the range of case - mix values (as previously discussed). also, multicollinearity made it impossible to obtain reasonable estimates of the effects of the wage- and construction - cost indexes. in addition to these limitations of the capital - cost models, there is another reason for preferring to base the long - run capital prospective payment on the total - cost model. in the total - cost model, the capital variables have little impact on the coefficients of the key operating variables. as a result, the capital variables could be ignored in designing payment adjustments based on total cost. assuming that this result holds more generally, the ability to estimate the total - cost model using all hospitals also offers a solution to the problem of sample unrepresentativeness encountered with the capital - cost model. as noted at the beginning of this article, capital 's special characteristics are not unique to hospitals. a single price covering both capital and operating inputs is the norm for most enterprises. a single price or, equivalently, a common set of adjustments for capital and operating payments is also the simplest way to make hospitals indifferent to medicare payment policy in choosing how to combine capital and operating inputs. the empirical findings of this article support using the total - cost models to develop a common set of adjustment factors for capital and operating prospective payment amounts. | the special characteristics of capital have an important effect on the cross - section variation in hospitals ' capital costs. variables reflecting capital age and financing differences perform as expected and add substantial explanatory power to capital cost models. however, even with the inclusion of these variables, the capital - cost models perform poorly compared with total - cost models. the empirical findings of this article support using the total - cost models to develop a common set of adjustment factors for capital and operating payment amounts in the medicare prospective payment system. |
endodontic treatment can be one of the most difficult dental procedures a practitioner encounters during clinical practice. due to the increase life expectancy in the population and the desire of individuals to preserve their natural teeth, there is an increasing demand for endodontic treatment and this will presumably increase in the years ahead. this reality necessitates dental students to be satisfactorily equipped with knowledge as well as experience in endodontic procedures prior to working independently. a dental student, upon graduation should have acquired the skills to make a sound diagnosis regarding endodontic cases, implement a reasonable treatment plan and carry out a qualified and safe endodontic treatment. it is a fact that different dental schools have varying prerequisites for graduation in each dental discipline and endodontics is no exception. the number of endodontic treatments a student is obliged to complete to be eligible for graduation differs from school to school and various factors such as the proportion of patient frequency to the number of enrolled clinical students of the related dental school may have impacts on this difference. on the other hand, there are some requirements and established competencies advocated by dental authorities and organizations that describe the minimum number of cases required to be completed prior to being licensed as a dental practitioner. an example to this is the statement in the undergraduate curriculum guidelines of the european society of endodontology advising the completion of root canal treatments of 20 teeth including extracted teeth prior to graduation. meanwhile, in the same report, it is regarded essential that students should have adequate experience of the treatment of endodontic emergencies. although quality of the completed work is a very significant parameter in deciding whether a student has gained enough proficiency, it is generally accepted that the more cases a dental student encounters during educational years, the more prepared he or she will be in terms of endodontic practice in the years of working independently. there are numerous references in the dental literature regarding the quality and outcome of endodontic treatments carried out by dental students ; however, there is scarce information regarding the way students perceive the branch of endodontology and their level of self - confidence about various aspects of endodontic treatment with respect to their future practice. the aim of this survey was to gather information about the general opinion of senior dental students enrolled in yeditepe university, faculty of dentistry, istanbul, turkey regarding endodontic treatment, to analyze their perception of this significant branch of dentistry and how they self - evaluate their confidence level in endodontic treatment a few months prior to graduation. following the approval of the institutional review board, anonymous survey forms, were handed to out to 48 senior year dental students enrolled in yeditepe university, faculty of dentistry. prior to the study, students were informed that they were not held obliged to complete and return the forms and completion of the survey would have no influence on their overall academic grading or performance. following some demographic information such as age and gender, the students were asked to score some endodontic procedures with different diagnosis, different steps of endodontic treatment as well as types of teeth according to their self - confidence levels. the students used the lickert 's scoring system from 1 - 5 to indicate their level of confidence as follows : 1 = very little confidence, 2 = little confidence 3 = neutral 4 = confident 5 = very confident. the survey continued with questions regarding students opinion about future endodontic practice while working independently, whether they wish to carry out all endodontic procedures by themselves or whether they would seek for the assistance of a specialist in case they felt necessary. students were also asked to share the most adverse experience they encountered during endodontic practice so far, if any. they were also asked to pick among some choices regarding the most significant innovation introduced into the science of endodontology that would increase practitioners performance in recent years. in the dental school where the study was conducted, students are held obliged to complete approximately 30 root canal treatments during their clinical years in order to be eligible for graduation. this number is dispersed between the clinical years, starting with a lower number of root canal treatments in the 3 year, gradually increasing until the senior year. during the survey, if case students felt that it was n't, they were asked to indicate the minimum number that they thought would be satisfactory to gain adequate proficiency. the survey was completed with a question which asked whether students wished to specialize in the branch of endodontology and additional comments. among the 48 students who were handed out the survey, 42 (88%) returned the forms. twenty - five students (59.5%) were females whereas 17 (40.5%) were males. the majority (42.9%) of the students rated endodontics as 3 in terms of difficulty [table 1 ] among other branches. the scorings regarding self - confidence levels of various aspects of endodontic treatment revealed that bleaching of endodontically treated teeth was the area where students felt the lowest confidence followed by rubber - dam application and management of interappointment flare - ups [table 2 ]. while scoring different types of teeth in terms of difficulty, maxillary and mandibular molars were the types of teeth that posed the most difficulty in terms of endodontic management [table 3 ]. root resorptions, endo - perio combined lesions and trauma cases were ranked as the situations in which students reported the lowest confidence levels [table 4 ]. the majority of the students (90.5%) reported that they would perform endodontic treatment of cases within their limit of expertise and skills in the future ; however planned to refer to a specialist when confronted with challenging situations beyond their experience level. only 4 students (10%) indicated that they are not planning to use any rotary instruments in the future. thirty - one students (73.8%) found the number of teeth to be treated satisfactory. one student commented that there should not be a limitation or prerequisite in terms of number of teeth to be completed. the students ranked the top 3 innovations brought into the science of endodontology in recent years as rotary instruments, mta and apex locators. different comments were made regarding the most negative experience during educational practices in terms of endodontic treatment. perforations, broken instruments and difficult retreatment cases that required prolonged visits were the predominant answers among students who wished to comment on this question. the number assigned to endodontics in case a sequence was made among dental disciplines in terms of difficulty average scorings of students regarding self - confidence of students about various endodontic perocedures average scorings of students regarding their self - confidence levels about the endodontic treatment of different types of teeth average scorings of students regarding their self - confidence levels during the management of different endodontic indications competency - based approach has recently replaced the traditional dental education methodology in most dental education programs and the aim of this modality is described as the understanding, skills, and professional values required of a student that are essential for beginning the unsupervised practice of dentistry. in the profile and competences for the graduating dentist released by the association for dental education in europe (adee), the competences, at the graduation, have been defined as the basic level of professional behavior, knowledge, and skills necessary for a graduating dentist to respond to the full range of circumstances encountered in general professional practice. consequently, the contemporary educational philosophy shows a competence fulfillment approach encompassing a wide spectrum of professional skills which is not limited to manipulative skills only. endodontics is a very significant branch in that respect, as it is frequently directly related with patient anxiety and pain. a dentist who has acquired the necessary competences in the field of endodontics is obliged to be equipped with multiple qualifications including appropriate patient approach and pain and anxiety management. student self - assessments of their own proficiency serve as helpful means to make a realistic evaluation of dental curricula and the assessment of the effectiveness of specific courses. meanwhile, scholarship in teaching and learning has started to be frequently pronounced recently and it has been indicated that this aspect of education should not be disputed in dentistry as well as other kind of higher learning. also, from the standpoint of training dentists as legitimate members of a learned profession, scholarship has been indicated to play a very important role. student surveys are significant in that respect as well and assist to unfold many issues that need to be resolved and reconsidered for better assimilation of knowledge and development of practical skills. though there are various studies that aim to evaluate the preparedness of the new graduate for clinical practice in general, to our knowledge there is no study that specifically focuses on endodontics and its clinical content. therefore, it is anticipated that the present study will be contibutory in drawing a general picture regarding students self - evaluation of themselves in a branch they will very frequently be involved in when they start working for the community. comments have been made by some authors regarding factors that may influence students self - confidence levels in clinical dental practice. defined one of the limits to developing confidence in performing clinical practices as insufficient clinical exposure within the undergraduate curriculum. lynch,. on the other hand, suggested that insufficient number of patients, lack of adequate physical space within the dental school, limitations posed by the busy curriculum and lack of well - trained staff are major obstacles, which may hamper high clinical self - confidence levels. in the dental school where the present study was conducted, it is not anticipated that the abovementioned parameters may be causative of lower confidence levels regarding various aspects of endodontic treatment. a significant proportion of the curriculum is dedicated to clinical practices, beginning from 23 out of 40 h / week in the 3 year, reaching 32 h / week in the second semester of the 5 year. there are sufficient number of cubicles that serve students needs where a daily rotation is established, when clinical students share their practical hours. an integrated clinical system is instilled where a student is responsible of all the dental treatment of a patient assigned to him / her, executing a holistic approach, which is not limited to only one single discipline. the student has the opportunity to disperse the allotted clinical period to any type of treatment necessary for the patient. moreover ; the clinical instructive staffs are all full - time employees specialized in the field of endodontics, with high level of clinical experience. there is also a high circulation of patients who are referred for their dental needs. when the graphs summarizing the results of the study are evaluated, it is observed that it is not generally the regular steps of endodontic treatment but rather more sophisticated aspects and indications related with endodontic treatment that lead to the reporting of relatively lower confidence levels. this is not quite unexpected as more challenging cases such as root resorptions, apexification procedures, retreatment and emergency cases and bleaching of endodontically treated teeth were those that were associated with relatively lower self - confidence levels. in case these types of cases are encountered at the student clinic, they are generally referred to the post - graduate clinic to be managed rather than undergraduate clinics. it is debatable whether students should be introduced to challenging cases during their educational years. it is quite likely that they will somehow encounter these situations in the future when they start to work independently. on the other hand, the profile and competences described by the association for dental education in europe indicates the acquisition of adequate competence by the undergraduate to perform endodontic treatment on uncomplicated single and uncomplicated multi - rooted teeth. recognizing indications for surgical and complicated non - surgical root canal therapy and taking appropriate action this implies that the student should at least adopt the skills to differentiate between cases within his / her level of expertise and refer to a specialist in case necessary. therefore, the relatively lower ratios reported in this study for more challenging cases should not create concern from an educational perspective, but should rather be regarded as a reflection of the current limitation of expertise expected from an undergraduate. when the types of teeth were scored in terms of self - confidence levels regarding endodontic treatment, molar teeth yielded relatively lower values consistent with the results of some other authors. bartlett,. indicated that dental schools might have the opinion that students can develop their skills in challenging cases better in general practice rather than the clinical environment offered by dental schools ; therefore, they might prefer to provide students with the knowledge of basic principles of these cases only. this comment may not be valid for the school in which this survey was conducted as students are expected to dedicate a significant proportion of their endodontic practice to molar endodontics. the lower result obtained may be rather the manifestation of inherent problems related with the management of molar teeth which may pose difficulty both in terms of their location and morphological characteristics. bleaching of endodontically treated teeth, rubber dam application and management of flare - ups were endodontic situations where students reported the lowest confidences. in the faculty where the study is conducted, bleaching is not a procedure that is required from students and it is generally a procedure undertaken by post - graduate students, so it is understandable that students may not feel themselves very confident over this type of practice about which they are generally theoretically instilled. however, rubber dam application is a prerequisite and students are not allowed to complete their treatments without the use of this significant adjunctive tool. a survey of the literature reveals a general underuse and some sort of resistance by dental practitioners as well as students regarding the use of the rubber dam. various factors have been proposed for the reluctance in the usage of this tool, including the difficulty of application and patients dislike. on the other hand, rubber dam is an indispensable element of contemporary endodontic practice and is not only a valuable tool but an ethical and medico - legal prerequisite for the dental practitioner. development of skills in terms of rubber dam application including management of difficult clinical cases with extensive tissue loss should be given priority by faculty and instructive staff in order for students to report higher levels of confidence in the future. flare - ups are undesirable situations that may arise during the course of endodontic treatment, requiring an unscheduled visit in some cases. it is also true that, flare - ups do not directly influence the outcome of the endodontic procedure, but are rather distressful situations resulting from the disruption of the balance between the host defense mechanism and irritating agents. one of the reasons for the occurrence of inter - appointment flare - ups may be procedural errors during the execution of endodontic treatment, such as extrusion of intracanal content inadvertently into the periradicular tissues. it can be speculated that flare - ups may be encountered more frequently in the students clinic, possibly due to inexperience of students allowing them to make some procedural errors such as overinstrumentation or extrusion of irrigants and intracanal debris. one possible explanation of this is that the students tactile skills have not developed as adequately as an experienced dentist. moreover, the patients need to be informed beforehand about the possibility of interappointment pain by the doctor and if they are done so, it may be easier for them to tolerate this complication. the students might have missed this detail during their communication with the patient, which may end up with negative reactions from the patient, making the treatment procedure more troublesome for the managing student. in terms of the most adverse occurrence students broken instruments and prolonged visits for retreatment cases were among the next most common statements by those who preferred to make a comment. in a study by balto,., root perforations in the 5-year students treatments were higher (3%) compared to the 4 year (0.3%). the authors commented that the relatively higher self - confidence and less clinical supervision of senior students might contribute to the high - risk of procedural errors during clinical practice. they also attributed the low percentage of adequate root canals assessed in their study to the fact that some of the supervision for undergraduate students was undertaken by non - specialists and not totally by endodontists. the clinical circumstance in the faculty where the study was conducted requires the complete monitorization of students by specialist endodontists. in spite of that, mishaps are always likely to occur probably due to relatively higher confidence of 5 year students enabling them to be more risk taking during difficult cases. it is generally traditional among dental schools to complete a threshold of clinical cases before they can be admitted to final examinations. it is also a widely accepted concept that repetition of clinical procedures is necessary to achieve clinical competence. chambers indicated that it is sometimes held that practice per se without regard for quality of outcomes, is a necessary if not sufficient condition for learning. the author also commented that the rationale for choosing the correct number of procedures and cases to ensure clinical competence is a traditional mystery. another point chambers drew attention was the different conceptual approaches between competency - based and the traditional systems towards achieving the adequate skills and competence. in the competency - based approach to dental education, individual student learning curves were allowed to vary based on practicality and the competence is fixed whilst in the traditional requirements system, a suggested or mandatory number of procedures were fixed but competence was permitted or expected to vary. the majority of the students who participated in the study stated that the number required for eligibility to graduate was satisfactory. on the other hand, based on the above - mentioned presumptions regarding the ambiguity of the number of necessary treatments before competence can be reached ; the reliability of the students comments is somewhat debatable. while it is true that students can make a better judgement of their clinical adequacy, there seems to be the necessity of very close and careful monitorization of each individual student and the development of an assessment strategy which is not dependent on numerary basis only. the question regarding students intention of using rotary instruments in clinical practice was presented in an attempt to acquire a general idea regarding their attitude towards contemporary aspects of endodontic care, same as the question which asked them to select in their opinion the best innovation brought into the science of endodontology recently. it is promising that almost all students expressed their wish in utilizing rotary instrumentation in their future practices. since rotary instrumentation techniques have gained widespread usage in dentistry, students willingness to incorporate these useful and time - saving tools in their routine care is an indication of their tendency towards using contemporary methodologies. this is also reflected in their ranking rotary instrumentation systems as the top in terms of beneficial innovations introduced in the branch of endodontology recently. the students also stated mineral trioxide aggregate and apex locators as the next 2 beneficial innovations brought recently. this result is rather pleasing from an educational perspective as these are noteworthy innovations and developments that have gained widespread attention and students seem to have gained adequate judgement abilities from what they have learned so far to appreciate contemporary methodologies developed to ease their performances. in summary, it can be stated that this study is conducted on a group of students and definitely reflects the opinions of only a limited group. on the other hand, it provides a general picture regarding students assessment of their abilities and limitations in the field of endodontics on the verge of graduation. there seems to be a tendency for students few months away from graduation to refer challenging cases to a specialist in future, however, this does not deny the fact that authorities should give priority to enhance the way information and experience is conveyed regarding various aspects of endodontic treatment. studies comprising other dental schools will be helpful in precisely determining the extent of instillation of adequate skills in endodontology and major missing areas that need further improvement. | objectives : the aim of this study was to obtain information about senior dental students perceptions and self - confidence levels regarding endodontic practice.materials and methods : anonymous survey forms were handed out to senior students at yeditepe university, faculty of dentistry. the students were asked to score their level of confidence using a 5-point scale and comment about future practices.results:the response rate of the survey was 88%. 11.9% expressed endodontics as the first branch in terms of difficulty. the majority (90.5%) indicated they would perform root canal treatments within their expertise limit in the future but refer difficult cases to an endodontist. bleaching of endodontically treated teeth, managing flare - ups, placement of a rubber dam were procedures in which students reported the lowest confidence (2.55 1.17, 3.24 0.96, 3.24 1.19, respectively). on the other hand, students felt the lowest confidence in the treatment of maxillary molars followed by mandibular molars (3.43 1.02 and 3.93 0.97, respectively). students also reported the lowest confidence in root resorptions, endo - perio lesions, traumas, retreatments and apexifications (2.93 1.16, 3.07 0.89, 3.24 0.85, 3.33 1.7 and 3.36 1.1, respectively).conclusions : the results showing students lower confidence in more challenging aspects of dentistry may be related with the attitude of dental schools to refer these cases to post graduate students and instilling information about these cases on a theoretical basis only. though there seems to be a tendency for students to refer challenging cases to a specialist in future, authorities should give priority to enhance the way information and experience is conveyed regarding various aspects of endodontic treatment. |
since cancer stem cells (cscs) in solid cancers [1, 2 ] were first reported in the early half of the 2000s, the establishment of a treatment targeting cscs for radical cure of cancer has become an important goal. therefore, the search for markers to isolate cscs and characterize cells isolated with these markers has been active throughout the world. cd133 is a 120-kda glycoprotein with five transmembrane domains and is a csc marker. despite various theories, originally, cd133 was known as a surface marker of hematopoietic stem cells and progenitor cells, but cd133 has also recently been reported as a marker of cscs in solid cancers such as brain tumors, lung cancer, liver cancer, colon cancer [5, 6 ], pancreatic cancer, and prostate cancer. in addition, in lung cancer, breast cancer, hepatocellular carcinoma, colon cancer, and pancreatic cancer, cd133 expression has been reported to be strongly related not only to tumor progression, but also to treatment resistance. however, despite the large number of patients with gastric cancer in japan, cscs in gastric cancer have not been definitively reported, and few studies evaluating cd133 expression have been reported. in highly advanced gastric cancer and recurrent gastric cancer, compared to colon cancer, there is still no effective treatment, and survival rates remain low. therefore, identification of gastric cscs and establishment of treatment will be highly important in future gastric cancer therapy. regarding cscs, a hypoxic environment has recently been shown to be necessary to maintain cscs. hypoxia - inducing factor-1alpha (hif-1) is a downstream molecule in the mammalian target of rapamycin signaling pathway, is induced by hypoxemia, and acts as a transcription factor. hif-1 has attracted attention as a factor that regulates cd133 expression, and the relationship between cd133 expression and hif-1 expression has been investigated in various solid cancers. most studies have shown a correlation between cd133 expression and hif-1 expression [15, 16 ], but interestingly, downregulation of cd133 expression by hif-1 expression in a gastric cancer cell line has also been reported. in this study, we investigated the clinicopathological role of cd133 expression in gastric cancer by immunostaining clinical specimens from gastric cancer patients. in addition, we examined the relationship between cd133 expression and hif-1 expression using immunohistological staining of gastric cancer tissue specimens. paraffin specimens from 189 gastric cancer patients who underwent gastrectomy between january 2004 and august 2006 at kurume university hospital were selected. histopathological characteristics and each classification are defined in the japan classification of gastric carcinoma (14th edition). regarding histological type, we divided the patients into a differentiated group (tub1, tub2, pap), and an undifferentiated group (por1, por2, sig, muc). the residual tumor (r) was defined as r0, no residual tumor ; r1, microscopic residual tumor (positive resection margin or cy1) ; or r2, macroscopic residual tumor. none of the patients had received preoperative adjuvant therapy, but 82 patients had received postoperative adjuvant therapy.table 1patient informationcharacteristicnumber of patients (n = 189)age (mean sd), years66 11gender (male / female)133/56tumor size (mean sd), mm66 38histological type (differentiated / undifferentiated)81/108stage (i / ii / iii / iv)62/41/52/36surgery total gastrectomy (include remnant gastrectomy)72 distal gastrectomy99 proximal gastrectomy11 segmental gastrectomy7r (residual tumor), 0/1/2144/11/34adjuvant chemotherapy (/+)107/82regimen ts-1 37 oral anticancer drug except ts-125 ts-1 + continuous infusion anticancer drug15 details unknown5 sd standard deviation differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) r0, nonresidual tumor ; r1, microscopic residual tumor (positive resection margin or cy1) ; r2, macroscopic residual tumor ts-1 is an oral anticancer drug containing a 5-fluorouracil derivative (tegafur) sd standard deviation differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) r0, nonresidual tumor ; r1, microscopic residual tumor (positive resection margin or cy1) ; r2, macroscopic residual tumor ts-1 is an oral anticancer drug containing a 5-fluorouracil derivative (tegafur) this study was authorized in advance by the ethics committee of kurume university (study number 11040). surgical specimens were fixed in 10 % formaldehyde, embedded in paraffin, and cut into 4-m - thick sections. slides were heated at 120 c in an autoclave in 10 mm sodium citrate (ph 6.0) for 10 min and then cooled to room temperature. after blocking with 10 % horse serum, the sections were incubated overnight at 4 c with mouse monoclonal anti - cd133 antibody [milteny biotec, auburn, ca, usa ; diluted 1:100 in phosphate - buffered saline (pbs) ]. after washing, sections were overlaid with secondary antibody (vectastain elite abc kit universal ; vector laboratories, burlingame, ca, usa) for 30 min at room temperature. sections were incubated in 3.0 % hydrogen peroxide in pbs for 30 min to block endogenous peroxidase activity. negative control sections (isotype control) were incubated with normal mouse serum instead of the primary antibody. isobe. have reported detailed methods for immunohistochemical staining of hif-1. briefly, 4-m - thick sections were cut from archival formalin - fixed paraffin - embedded tissue blocks. slides were irradiated at 99 c in a microwave oven for 30 min in 10 mm citrate buffer (ph 9.0) and cooled to room temperature. the sections were incubated overnight at 4 c with rabbit polyclonal anti - hif-1 antibody h206 (santa cruz biotechnology, santa cruz, ca, usa ; diluted 1:100 in pbs), and reactions were developed using the same method as cd133 staining. to clarify the localization of cd133 in gastric cancer cells, double immunohistochemical staining with anti - cd133 the reaction was developed using anti - mouse poly - alkaline phosphatase (ap) and anti - rabbit poly - horseradish peroxidase (hrp) polymerization technology. we used a cocktail of primary antibodies containing the same anti - cd133 antibody (diluted 1:100 in pbs) and rabbit monoclonal anti - cytokeratin 8 antibody (abcam, cambridge, uk ; diluted 1:50 in pbs). after incubating overnight at 4 c, sections were overlaid with secondary antibody (mach2 double stain 1 mouse - ap rabbit - hrp ; biocare medical, concord, ca, usa). the alkaline phosphatase reaction for the anti - cd133 antibody was developed with vulcan fast red chromogen (biocare medical ; vulcan fast red chromogen kit2), and the peroxidase reaction for the cytokeratin 8 antibody was developed with 3,3-diaminobenzidine (dab). in addition, to confirm both cd133 and hif-1 expression in gastric cancer, double immunohistochemical staining was performed in some cases. we used a cocktail of primary antibodies containing the same anti - cd133 antibody (diluted 1:100 in pbs) and the same anti - hif-1 antibody (diluted 1:100 in pbs). the secondary antibody and the alkaline phosphatase reaction were developed using the same methods as described for the cd133 + cytokeratin 8 staining. for comparison of categorical data, the chi - square test and fisher s exact test were used. meier analysis, and differences between the groups were compared using the log - rank test. for multivariate analysis all statistical analysis was performed using statistical software (jmp 9.0 ; sas, cary, nc, usa). paraffin specimens from 189 gastric cancer patients who underwent gastrectomy between january 2004 and august 2006 at kurume university hospital were selected. histopathological characteristics and each classification are defined in the japan classification of gastric carcinoma (14th edition). regarding histological type, we divided the patients into a differentiated group (tub1, tub2, pap), and an undifferentiated group (por1, por2, sig, muc). the residual tumor (r) was defined as r0, no residual tumor ; r1, microscopic residual tumor (positive resection margin or cy1) ; or r2, macroscopic residual tumor. none of the patients had received preoperative adjuvant therapy, but 82 patients had received postoperative adjuvant therapy.table 1patient informationcharacteristicnumber of patients (n = 189)age (mean sd), years66 11gender (male / female)133/56tumor size (mean sd), mm66 38histological type (differentiated / undifferentiated)81/108stage (i / ii / iii / iv)62/41/52/36surgery total gastrectomy (include remnant gastrectomy)72 distal gastrectomy99 proximal gastrectomy11 segmental gastrectomy7r (residual tumor), 0/1/2144/11/34adjuvant chemotherapy (/+)107/82regimen ts-1 37 oral anticancer drug except ts-125 ts-1 + continuous infusion anticancer drug15 details unknown5 sd standard deviation differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) r0, nonresidual tumor ; r1, microscopic residual tumor (positive resection margin or cy1) ; r2, macroscopic residual tumor ts-1 is an oral anticancer drug containing a 5-fluorouracil derivative (tegafur) sd standard deviation differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) r0, nonresidual tumor ; r1, microscopic residual tumor (positive resection margin or cy1) ; r2, macroscopic residual tumor ts-1 is an oral anticancer drug containing a 5-fluorouracil derivative (tegafur) this study was authorized in advance by the ethics committee of kurume university (study number 11040). surgical specimens were fixed in 10 % formaldehyde, embedded in paraffin, and cut into 4-m - thick sections. slides were heated at 120 c in an autoclave in 10 mm sodium citrate (ph 6.0) for 10 min and then cooled to room temperature. after blocking with 10 % horse serum, the sections were incubated overnight at 4 c with mouse monoclonal anti - cd133 antibody [milteny biotec, auburn, ca, usa ; diluted 1:100 in phosphate - buffered saline (pbs) ]. after washing, sections were overlaid with secondary antibody (vectastain elite abc kit universal ; vector laboratories, burlingame, ca, usa) for 30 min at room temperature. sections were incubated in 3.0 % hydrogen peroxide in pbs for 30 min to block endogenous peroxidase activity. negative control sections (isotype control) were incubated with normal mouse serum instead of the primary antibody. isobe. have reported detailed methods for immunohistochemical staining of hif-1. briefly, 4-m - thick sections were cut from archival formalin - fixed paraffin - embedded tissue blocks. slides were irradiated at 99 c in a microwave oven for 30 min in 10 mm citrate buffer (ph 9.0) and cooled to room temperature. the sections were incubated overnight at 4 c with rabbit polyclonal anti - hif-1 antibody h206 (santa cruz biotechnology, santa cruz, ca, usa ; diluted 1:100 in pbs), and reactions were developed using the same method as cd133 staining. to clarify the localization of cd133 in gastric cancer cells, double immunohistochemical staining with anti - cd133 antibody and anti - cytokeratin 8 antibody was performed in some cases. the reaction was developed using anti - mouse poly - alkaline phosphatase (ap) and anti - rabbit poly - horseradish peroxidase (hrp) polymerization technology. we used a cocktail of primary antibodies containing the same anti - cd133 antibody (diluted 1:100 in pbs) and rabbit monoclonal anti - cytokeratin 8 antibody (abcam, cambridge, uk ; diluted 1:50 in pbs). after incubating overnight at 4 c, sections were overlaid with secondary antibody (mach2 double stain 1 mouse - ap rabbit - hrp ; biocare medical, concord, ca, usa). the alkaline phosphatase reaction for the anti - cd133 antibody was developed with vulcan fast red chromogen (biocare medical ; vulcan fast red chromogen kit2), and the peroxidase reaction for the cytokeratin 8 antibody was developed with 3,3-diaminobenzidine (dab). in addition, to confirm both cd133 and hif-1 expression in gastric cancer, double immunohistochemical staining was performed in some cases. we used a cocktail of primary antibodies containing the same anti - cd133 antibody (diluted 1:100 in pbs) and the same anti - hif-1 antibody (diluted 1:100 in pbs). the secondary antibody and the alkaline phosphatase reaction were developed using the same methods as described for the cd133 + cytokeratin 8 staining. for comparison of categorical data, the chi - square test and fisher s exact test were used. the overall survival rate was calculated using kaplan meier analysis, and differences between the groups were compared using the log - rank test. for multivariate analysis, prognostic factors were analyzed using cox s proportional hazard model. all statistical analysis was performed using statistical software (jmp 9.0 ; sas, cary, nc, usa). 1a, b). in differentiated gastric cancer in particular, we observed luminal expression of the gland (defined as l - type) as in colon cancer, and in undifferentiated gastric cancer in particular, we saw expression in the cytoplasm (defined as c - type) as in pancreatic cancer. in some cases, we saw both l - type and c - type on the same section. the frequency of cd133 expression cells in 1,000 cancer cells was 018.3 %. in 6 cases, we stained some sections from the gastric cancer tumor, but there were no differences in the cd133 expression frequency and expression type (data not shown). of the 189 total cases, we observed 56 cd133-positive cases (29.6 %), 33 l - type cases (17.4 %), and 23 c - type cases (12.1 %).fig. 1two general cd133 expression types were observed in gastric cancer. a luminal expression in the gland (l - type). a, b 200 two general cd133 expression types were observed in gastric cancer. a luminal expression in the gland (l - type). a, b 200 hif-1 expression was observed in the nucleus of cancer cells (fig. 2). we defined hif-1 as positive when more than 5 % of cancer cells showed positive nuclear expression. of the 189 total cases, 107 (56.6 %) were hif-1 positive.fig. 400 expression of hypoxia - inducible factor (hif)-1 was seen in the nucleus of cancer cells. 400 cytokeratin 8 is a cytoplasmic marker of adenocarcinoma including gastric cancer. double immunohistochemical staining with anti - cd133 antibody and anti - cytokeratin 8 antibody revealed that cytoplasmic expression of cd133 was present mainly in the intracytoplasmic lumen (icl) (fig. the occurrence of icl, which is well known in breast cancer, has been reported in gastric cancer.fig. 3double immunohistochemical staining with anti - cd133 antibody (red) and anti - cytokeratin 8 antibody (brown). in the c - type double immunohistochemical staining with anti - cd133 antibody (red) and anti - cytokeratin 8 antibody (brown). in the c - type, 400 gastric cancer specimens were confirmed to express both cd133 and hif-1. moreover, hif-1 expression in the nucleus tended to be present more often in c - type than l - type cases (fig. 4).fig. 4gastric cancer was confirmed to express both cd133 (red) and hif-1 (brown). 400 gastric cancer was confirmed to express both cd133 (red) and hif-1 (brown). we divided our cases into three groups : cd133 negative, l - type positive, and c - type positive. the rates of lymph node metastasis, peritoneal dissemination, vascular invasion, and advanced stage tended to be higher in the c - type - positive group than in the other two groups, and significant differences among these three groups were observed. thus, we hypothesized that cd133 expression in the cytoplasm was related to cancer progression.table 2comparison of clinical variables between cd133-positive and cd133-negative casesvariablecd133 expressionnegative (n = 133)positive p valuel - type positive (n = 33)c - type positive (n = 23)age (years) < 7074 (56 %) 15 (45 %) 11 (48 %) 0.503 7059 (44 %) 18 (54 %) 12 (52 %) gender male88 (66 %) 31 (94 %) 14 (61 %) 0.004 female45 (33 %) 2 (6 %) 9 (39 %) macroscopic type 043 (32 %) 9 (27 %) 5 (21 %) 0.176 1, 232 (24 %) 13 (39 %) 4 (17 %) 3, 458 (43 %) 11 (33 %) 14 (60 %) diameter (mm) < 7073 (55 %) 22 (67 %) 10 (43 %) 0.219 7060 (45 %) 11 (33 %) 13 (56 %) region u33 (25 %) 12 (36 %) 7 (30 %) 0.230 m37 (27 %) 3 (9 %) 6 (26 %) l63 (47 %) 18 (54 %) 10 (43 %) t classification 1, 259 (44 %) 12 (36 %) 7 (30 %) 0.374 3, 474 (55 %) 21 (63 %) 16 (69 %) n classification 071 (53 %) 16 (48 %) 4 (17 %) 0.006 1, 2, 362 (46 %) 17 (51 %) 19 (82 %) lymph node metastasis number 071 (53 %) 16 (48 %) 4 (17 %) 0.011 1623 (17 %) 10 (30 %) 7 (30 %) 739 (29 %) 7 (21 %) 12 (52 %) m classification 0114 (86 %) 27 (82 %) 13 (57 %) 0.003 119 (14 %) 6 (18 %) 10 (43 %) h classification 0132 (99 %) 29 (87 %) 20 (87 %) 0.001 11 (1 %) 4 (12 %) 3 (13 %) p classification 0121 (91 %) 32 (97 %) 16 (70 %) 0.024 112 (9 %) 1 (3 %) 7 (30 %) cy classification 0124 (93 %) 32 (97 %) 20 (87 %) 0.521 19 (7 %) 1 (3 %) 3 (13 %) stage i, ii80 (60 %) 17 (52 %) 6 (26 %) 0.009 iii, iv53 (39 %) 16 (48 %) 17 (73 %) histological type differentiated51 (38 %) 25 (75 %) 5 (22 %) < 0.001 undifferentiated82 (61 %) 8 (24 %) 18 (78 %) stroma med, int104 (78 %) 31 (94 %) 14 (61 %) 0.011 sci29 (21 %) 2 (6 %) 9 (39 %) inf a, b83 (62 %) 29 (87 %) 11 (48 %) 0.004 c50 (37 %) 4 (12 %) 12 (52 %) ly 0, 158 (44 %) 13 (39 %) 4 (17 %) 0.059 2, 375 (56 %) 20 (60 %) 19 (82 %) v 0, 1125 (94 %) 30 (91 %) 17 (74 %) 0.004 2, 38 (6 %) 3 (9 %) 6 (26 %) histopathological characteristics and each classification are defined according to the japan classification of gastric carcinoma (14th edition) l - type luminal expression of the gland type, c - type expression in the cytoplasm type differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) calculated with fisher s exact test comparison of clinical variables between cd133-positive and cd133-negative cases histopathological characteristics and each classification are defined according to the japan classification of gastric carcinoma (14th edition) l - type luminal expression of the gland type, c - type expression in the cytoplasm type differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) calculated with fisher s exact test figure 5 shows the relationship between prognosis and cd133 expression. the overall survival rate for the cd133-positive group was significantly worse than that in the cd133-negative group (fig. when the cd133-positive cases were divided into two expression types, the c - type - positive group showed significantly worse survival (fig., we controlled for t, n, and p factors, which were strong prognostic factors selected with backward stepwise regression, and for pathological type and hif-1 expression, which may be confounding factors. multivariate analysis revealed that cd133 c - type positive was an independent prognostic factor in gastric cancer (table 3).fig. 5kaplan meier survival curves of 189 patients with gastric cancer, stratified by cd133 expression. a the 5-year overall survival (os) rate of the cd133-negative () group was 66.6 % ; for the positive (+) group, os was 46.3 % (p = 0.004 with the log - rank test). b divided into two expression types, the 5-year os rate of the l - type (+) was 62.5 % ; for the c - type (+), the os was 22.7 %. p = 0.603 between cd133 () and l - type (+), and p < 0.001 between cd133 () and c - type (+). c survival curves of stage the 5-year os rate of c - type (+) was 60.0 %. there was no significant difference in the survival curves between cd133 () and c - type (+) (p = 0.191). d in stage iii / iv, the 5-year survival rate of c - type (+) was 11.7 %. there was a significant difference in the survival curves between cd133 () and c - type (+) (p = 0.017)table 3multivariate analysis of the relationship between cd133 expression type and overall survivalanalysiscd133 expressionnegativel - type positivec - type positiveunivariate hr11.183.62 95 % ci0.592.181.996.29 p value0.604<0.001multivariate model 1 hr11.163.59 95 % ci0.572.191.976.24 p value0.654<0.001 model 2 hr11.021.92 95 % ci0.501.941.023.45 p value0.9450.041 model 3 hr11.061.87 95 % ci0.522.051.003.39 p value0.8460.049we confined the variables that were incorporated into the analysis to nine from the number of events hr hazard ratio, ci confidence interval model 1 was analyzed using the cox proportional hazard model while controlling for age (< 70 years, 70 years) and gender model 2 includes model 1 variables plus t (1, 2/3, 4), n (0/1, 2, 3), and p (0/1) classifications model 3 includes model 2 variables + hif-1 expression and histological type (differentiated, undifferentiated) kaplan meier survival curves of 189 patients with gastric cancer, stratified by cd133 expression. a the 5-year overall survival (os) rate of the cd133-negative () group was 66.6 % ; for the positive (+) group, os was 46.3 % (p = 0.004 with the log - rank test). b divided into two expression types, the 5-year os rate of the l - type (+) was 62.5 % ; for the c - type (+), the os was 22.7 %. p = 0.603 between cd133 () and l - type (+), and p < 0.001 between cd133 () and c - type (+). c survival curves of stage i / ii. the 5-year os rate of c - type (+) was 60.0 %. there was no significant difference in the survival curves between cd133 () and c - type (+) (p = 0.191). d in stage iii / iv, the 5-year survival rate of c - type (+) was 11.7 %. there was a significant difference in the survival curves between cd133 () and c - type (+) (p = 0.017) multivariate analysis of the relationship between cd133 expression type and overall survival we confined the variables that were incorporated into the analysis to nine from the number of events hr hazard ratio, ci confidence interval model 1 was analyzed using the cox proportional hazard model while controlling for age (< 70 years, 70 years) and gender model 2 includes model 1 variables plus t (1, 2/3, 4), n (0/1, 2, 3), and p (0/1) classifications model 3 includes model 2 variables + hif-1 expression and histological type (differentiated, undifferentiated) furthermore, we investigated the relationship between cd133 expression and chemotherapy resistance and recurrence. we selected patients who underwent adjuvant chemotherapy and a curative resection, and disease - specific survival rate curves were compared among the cd133 expression types. in both the adjuvant chemotherapy group and the curative resection group, the survival rate curve of the c - type - positive group was significantly worse than that in the other groups (figs. 6kaplan meier survival curves of 82 patients who underwent adjuvant chemotherapy (adjuvant chemotherapy included all chemotherapeutic regimens and durations of administration). p = 0.341 between cd133 () and l - type (+) ; p < 0.001 between cd133 () and c - type (+) fig. 7kaplan meier survival curves of 144 patients with r0 status (r0 is a curative resection with negative resection margins). p = 0.563 between cd133 () and l - type (+), and p = 0.013 between cd133 () and c - type (+) kaplan meier survival curves of 82 patients who underwent adjuvant chemotherapy (adjuvant chemotherapy included all chemotherapeutic regimens and durations of administration). p = 0.341 between cd133 () and l - type (+) ; p < 0.001 between cd133 () and c - type (+) kaplan meier survival curves of 144 patients with r0 status (r0 is a curative resection with negative resection margins). p = 0.563 between cd133 () and l - type (+), and p = 0.013 between cd133 () and c - type (+) finally, we examined the correlation between cd133 expression and hif-1 expression. however, when we analyzed the groups according to expression type, the hif-1-positive rate was lower in l - type and higher in c - type cases. we found a significant difference in the hif-1 expression rate among these three groups (fig. 8a, b).fig. a hif-1 expression rate was 59.4 % in the cd133-negative group and 40.6 % in the cd133-positive group. there was no significant difference between the two groups (p = 0.234). the hif-1 expression rate was 33.3 % in the l - type - positive group and 73.9 % in the c - type - positive group. p = 0.041 among the three groups correlation between cd133 expression and hif-1 expression. a hif-1 expression rate was 59.4 % in the cd133-negative group and 40.6 % in the cd133-positive group. there was no significant difference between the two groups (p = 0.234). the hif-1 expression rate was 33.3 % in the l - type - positive group and 73.9 % in the c - type - positive group. p = 0.041 among the three groups we have previously reported that hif-1 expression is a poor prognostic factor in gastric cancer. in the present study, the overall survival rate curve of the hif-1-positive group was significantly worse than that in the hif-1-negative group (fig. however, if we divided the hif-1-positive group according to cd133 expression, both the cd133-positive and the hif-1-positive groups showed poor prognosis. interestingly, a similar result was seen without regard to the cd133 expression type (fig. 9kaplan meier survival curves of 189 patients with gastric cancer, stratified by hif-1 expression and cd133 expression. a the 5-year os rate was 70.2 % for the hif-1-negative () group and 53.2 % for the positive (+) group. b stratified by cd133 expression type, the 5-year os rate of the hif-1 (+) /cd133 () group was 63.0 %. for the hif-1 (+) /l - type (+) group, os was 27.2 %. for the hif-1 (+) /c - type (+) group, p = 0.332 between the hif-1 () and hif-1 (+) /cd133 () groups, p = 0.001 between the hif-1 () and hif-1 (+) /l - type groups, and p < 0.001 between the hif-1 () and hif-1 (+) /c - type (+) groups kaplan meier survival curves of 189 patients with gastric cancer, stratified by hif-1 expression and cd133 expression. a the 5-year os rate was 70.2 % for the hif-1-negative () group and 53.2 % for the positive (+) group. b stratified by cd133 expression type, the 5-year os rate of the hif-1 (+) /cd133 () group was 63.0 %. for the hif-1 (+) for the hif-1 (+) /c - type (+) group, os was 25.1 %. p = 0.332 between the hif-1 () and hif-1 (+) /cd133 () groups, p = 0.001 between the hif-1 () and hif-1 (+) /l - type groups, and p < 0.001 between the hif-1 () and hif-1 (+) /c - type (+) groups 1a, b). in differentiated gastric cancer in particular, we observed luminal expression of the gland (defined as l - type) as in colon cancer, and in undifferentiated gastric cancer in particular, we saw expression in the cytoplasm (defined as c - type) as in pancreatic cancer. in some cases, we saw both l - type and c - type on the same section. the frequency of cd133 expression cells in 1,000 cancer cells was 018.3 %. in 6 cases, we stained some sections from the gastric cancer tumor, but there were no differences in the cd133 expression frequency and expression type (data not shown). of the 189 total cases, we observed 56 cd133-positive cases (29.6 %), 33 l - type cases (17.4 %), and 23 c - type cases (12.1 %).fig. 1two general cd133 expression types were observed in gastric cancer. a luminal expression in the gland (l - type). a, b 200 two general cd133 expression types were observed in gastric cancer. a luminal expression in the gland (l - type). a, b 200 hif-1 expression was observed in the nucleus of cancer cells (fig. 2). we defined hif-1 as positive when more than 5 % of cancer cells showed positive nuclear expression. of the 189 total cases, 107 (56.6 %) were hif-1 positive.fig. 400 expression of hypoxia - inducible factor (hif)-1 was seen in the nucleus of cancer cells. 400 cytokeratin 8 is a cytoplasmic marker of adenocarcinoma including gastric cancer. double immunohistochemical staining with anti - cd133 antibody and anti - cytokeratin 8 antibody revealed that cytoplasmic expression of cd133 was present mainly in the intracytoplasmic lumen (icl) (fig. the occurrence of icl, which is well known in breast cancer, has been reported in gastric cancer.fig. 3double immunohistochemical staining with anti - cd133 antibody (red) and anti - cytokeratin 8 antibody (brown). in the c - type double immunohistochemical staining with anti - cd133 antibody (red) and anti - cytokeratin 8 antibody (brown). in the c - type, 400 gastric cancer specimens were confirmed to express both cd133 and hif-1. moreover, hif-1 expression in the nucleus tended to be present more often in c - type than l - type cases (fig. 4).fig. 4gastric cancer was confirmed to express both cd133 (red) and hif-1 (brown). 400 gastric cancer was confirmed to express both cd133 (red) and hif-1 (brown). we divided our cases into three groups : cd133 negative, l - type positive, and c - type positive. the rates of lymph node metastasis, peritoneal dissemination, vascular invasion, and advanced stage tended to be higher in the c - type - positive group than in the other two groups, and significant differences among these three groups were observed. thus, we hypothesized that cd133 expression in the cytoplasm was related to cancer progression.table 2comparison of clinical variables between cd133-positive and cd133-negative casesvariablecd133 expressionnegative (n = 133)positive p valuel - type positive (n = 33)c - type positive (n = 23)age (years) < 7074 (56 %) 15 (45 %) 11 (48 %) 0.503 7059 (44 %) 18 (54 %) 12 (52 %) gender male88 (66 %) 31 (94 %) 14 (61 %) 0.004 female45 (33 %) 2 (6 %) 9 (39 %) macroscopic type 043 (32 %) 9 (27 %) 5 (21 %) 0.176 1, 232 (24 %) 13 (39 %) 4 (17 %) 3, 458 (43 %) 11 (33 %) 14 (60 %) diameter (mm) < 7073 (55 %) 22 (67 %) 10 (43 %) 0.219 7060 (45 %) 11 (33 %) 13 (56 %) region u33 (25 %) 12 (36 %) 7 (30 %) 0.230 m37 (27 %) 3 (9 %) 6 (26 %) l63 (47 %) 18 (54 %) 10 (43 %) t classification 1, 259 (44 %) 12 (36 %) 7 (30 %) 0.374 3, 474 (55 %) 21 (63 %) 16 (69 %) n classification 071 (53 %) 16 (48 %) 4 (17 %) 0.006 1, 2, 362 (46 %) 17 (51 %) 19 (82 %) lymph node metastasis number 071 (53 %) 16 (48 %) 4 (17 %) 0.011 1623 (17 %) 10 (30 %) 7 (30 %) 739 (29 %) 7 (21 %) 12 (52 %) m classification 0114 (86 %) 27 (82 %) 13 (57 %) 0.003 119 (14 %) 6 (18 %) 10 (43 %) h classification 0132 (99 %) 29 (87 %) 20 (87 %) 0.001 11 (1 %) 4 (12 %) 3 (13 %) p classification 0121 (91 %) 32 (97 %) 16 (70 %) 0.024 112 (9 %) 1 (3 %) 7 (30 %) cy classification 0124 (93 %) 32 (97 %) 20 (87 %) 0.521 19 (7 %) 1 (3 %) 3 (13 %) stage i, ii80 (60 %) 17 (52 %) 6 (26 %) 0.009 iii, iv53 (39 %) 16 (48 %) 17 (73 %) histological type differentiated51 (38 %) 25 (75 %) 5 (22 %) < 0.001 undifferentiated82 (61 %) 8 (24 %) 18 (78 %) stroma med, int104 (78 %) 31 (94 %) 14 (61 %) 0.011 sci29 (21 %) 2 (6 %) 9 (39 %) inf a, b83 (62 %) 29 (87 %) 11 (48 %) 0.004 c50 (37 %) 4 (12 %) 12 (52 %) ly 0, 158 (44 %) 13 (39 %) 4 (17 %) 0.059 2, 375 (56 %) 20 (60 %) 19 (82 %) v 0, 1125 (94 %) 30 (91 %) 17 (74 %) 0.004 2, 38 (6 %) 3 (9 %) 6 (26 %) histopathological characteristics and each classification are defined according to the japan classification of gastric carcinoma (14th edition) l - type luminal expression of the gland type, c - type expression in the cytoplasm type differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) calculated with fisher s exact test comparison of clinical variables between cd133-positive and cd133-negative cases histopathological characteristics and each classification are defined according to the japan classification of gastric carcinoma (14th edition) l - type luminal expression of the gland type, c - type expression in the cytoplasm type differentiated (tub1, tub2, pap) ; undifferentiated (por1, por2, sig, muc) calculated with fisher s exact test the overall survival rate for the cd133-positive group was significantly worse than that in the cd133-negative group (fig. when the cd133-positive cases were divided into two expression types, the c - type - positive group showed significantly worse survival (fig., we controlled for t, n, and p factors, which were strong prognostic factors selected with backward stepwise regression, and for pathological type and hif-1 expression, which may be confounding factors. multivariate analysis revealed that cd133 c - type positive was an independent prognostic factor in gastric cancer (table 3).fig. 5kaplan meier survival curves of 189 patients with gastric cancer, stratified by cd133 expression. a the 5-year overall survival (os) rate of the cd133-negative () group was 66.6 % ; for the positive (+) group, os was 46.3 % (p = 0.004 with the log - rank test). b divided into two expression types, the 5-year os rate of the l - type (+) was 62.5 % ; for the c - type (+), the os was 22.7 %. p = 0.603 between cd133 () and l - type (+), and p < 0.001 between cd133 () and c - type (+). c survival curves of stage i / ii. the 5-year os rate of c - type (+) was 60.0 %. there was no significant difference in the survival curves between cd133 () and c - type (+) (p = 0.191). d in stage iii / iv, the 5-year survival rate of c - type (+) was 11.7 %. there was a significant difference in the survival curves between cd133 () and c - type (+) (p = 0.017)table 3multivariate analysis of the relationship between cd133 expression type and overall survivalanalysiscd133 expressionnegativel - type positivec - type positiveunivariate hr11.183.62 95 % ci0.592.181.996.29 p value0.604<0.001multivariate model 1 hr11.163.59 95 % ci0.572.191.976.24 p value0.654<0.001 model 2 hr11.021.92 95 % ci0.501.941.023.45 p value0.9450.041 model 3 hr11.061.87 95 % ci0.522.051.003.39 p value0.8460.049we confined the variables that were incorporated into the analysis to nine from the number of events hr hazard ratio, ci confidence interval model 1 was analyzed using the cox proportional hazard model while controlling for age (< 70 years, 70 years) and gender model 2 includes model 1 variables plus t (1, 2/3, 4), n (0/1, 2, 3), and p (0/1) classifications model 3 includes model 2 variables + hif-1 expression and histological type (differentiated, undifferentiated) kaplan meier survival curves of 189 patients with gastric cancer, stratified by cd133 expression. a the 5-year overall survival (os) rate of the cd133-negative () group was 66.6 % ; for the positive (+) group, os was 46.3 % (p = 0.004 with the log - rank test). b divided into two expression types, the 5-year os rate of the l - type (+) was 62.5 % ; for the c - type (+), the os was 22.7 %. p = 0.603 between cd133 () and l - type (+), and p < 0.001 between cd133 () and c - type (+).. the 5-year os rate of c - type (+) was 60.0 %. there was no significant difference in the survival curves between cd133 () and c - type (+) (p = 0.191). d in stage iii / iv, the 5-year survival rate of c - type (+) was 11.7 %. there was a significant difference in the survival curves between cd133 () and c - type (+) (p = 0.017) multivariate analysis of the relationship between cd133 expression type and overall survival we confined the variables that were incorporated into the analysis to nine from the number of events hr hazard ratio, ci confidence interval model 1 was analyzed using the cox proportional hazard model while controlling for age (< 70 years, 70 years) and gender model 2 includes model 1 variables plus t (1, 2/3, 4), n (0/1, 2, 3), and p (0/1) classifications model 3 includes model 2 variables + hif-1 expression and histological type (differentiated, undifferentiated) we selected patients who underwent adjuvant chemotherapy and a curative resection, and disease - specific survival rate curves were compared among the cd133 expression types. in both the adjuvant chemotherapy group and the curative resection group, the survival rate curve of the c - type - positive group was significantly worse than that in the other groups (figs. 6kaplan meier survival curves of 82 patients who underwent adjuvant chemotherapy (adjuvant chemotherapy included all chemotherapeutic regimens and durations of administration). p = 0.341 between cd133 () and l - type (+) ; p < 0.001 between cd133 () and c - type (+) fig. 7kaplan meier survival curves of 144 patients with r0 status (r0 is a curative resection with negative resection margins). p = 0.563 between cd133 () and l - type (+), and p = 0.013 between cd133 () and c - type (+) kaplan meier survival curves of 82 patients who underwent adjuvant chemotherapy (adjuvant chemotherapy included all chemotherapeutic regimens and durations of administration). p = 0.341 between cd133 () and l - type (+) ; p < 0.001 between cd133 () and c - type (+) kaplan meier survival curves of 144 patients with r0 status (r0 is a curative resection with negative resection margins). p = 0.563 between cd133 () and l - type (+), and p = 0.013 between cd133 () and c - type (+) there was no significant difference in hif-1 expression between the cd133-positive and cd133-negative groups. however, when we analyzed the groups according to expression type, the hif-1-positive rate was lower in l - type and higher in c - type cases. we found a significant difference in the hif-1 expression rate among these three groups (fig. a hif-1 expression rate was 59.4 % in the cd133-negative group and 40.6 % in the cd133-positive group. there was no significant difference between the two groups (p = 0.234). the hif-1 expression rate was 33.3 % in the l - type - positive group and 73.9 % in the c - type - positive group. p = 0.041 among the three groups correlation between cd133 expression and hif-1 expression. a hif-1 expression rate was 59.4 % in the cd133-negative group and 40.6 % in the cd133-positive group. there was no significant difference between the two groups (p = 0.234). the hif-1 expression rate was 33.3 % in the l - type - positive group and 73.9 % in the c - type - positive group. p = 0.041 among the three groups we have previously reported that hif-1 expression is a poor prognostic factor in gastric cancer. in the present study, the overall survival rate curve of the hif-1-positive group was significantly worse than that in the hif-1-negative group (fig. however, if we divided the hif-1-positive group according to cd133 expression, both the cd133-positive and the hif-1-positive groups showed poor prognosis. interestingly, a similar result was seen without regard to the cd133 expression type (fig 9kaplan meier survival curves of 189 patients with gastric cancer, stratified by hif-1 expression and cd133 expression. a the 5-year os rate was 70.2 % for the hif-1-negative () group and 53.2 % for the positive (+) group. b stratified by cd133 expression type, the 5-year os rate of the hif-1 (+) /cd133 () group was 63.0 %. for the hif-1 (+) /l - type (+) group, os was 27.2 %. for the hif-1 (+) /c - type (+) group, os was 25.1 %. p = 0.332 between the hif-1 () and hif-1 (+) /cd133 () groups, p = 0.001 between the hif-1 () and hif-1 (+) /l - type groups, and p < 0.001 between the hif-1 () and hif-1 (+) /c - type (+) groups kaplan meier survival curves of 189 patients with gastric cancer, stratified by hif-1 expression and cd133 expression. a the 5-year os rate was 70.2 % for the hif-1-negative () group and 53.2 % for the positive (+) group. b stratified by cd133 expression type, the 5-year os rate of the hif-1 (+) /cd133 () group was 63.0 %. for the hif-1 (+) /l - type (+) group, os was 27.2 %. for the hif-1 (+) /c - type (+) group, os was 25.1 %. p = 0.332 between the hif-1 () and hif-1 (+) /cd133 () groups, p = 0.001 between the hif-1 () and hif-1 (+) /l - type groups, and p < 0.001 between the hif-1 () and hif-1 (+) /c - type (+) groups we found that cd133 protein expression in gastric cancer clinical specimens was the same as that in other solid cancers. this expression could be broadly divided into two types : glandular - luminal cell membrane surface expression (luminal expression, l - type) and cytoplasmic expression (c - type). luminal expression was more common in differentiated gastric cancer, and cytoplasmic expression was more common in undifferentiated gastric cancer, and both expression types were seen in some tissue sections. also evaluated cd133 expression in gastric cancer using clinical specimens, and both reported two types of staining results, similar to our findings. originally, luminal expression of cd133 was reported in colorectal cancer, and cytoplasmic expression of cd133 was reported in pancreatic cancer [20, 21 ]. however, both expression types have recently been reported at the same time in colon cancer and in hepatocellular carcinoma [25, 26 ]. these reports also support the possibility of two types of cd133 expression in gastric cancer. a definitive difference was reported in another article about the expression of cd133 in gastric cancer. ishigami. evaluated the overall cd133 expression in gastric cancer, without dividing the cases into expression types. on the other hand, in our study, we focused on these two expression types, with the analysis divided for each expression type. ishigami. reported that cd133 expression in gastric cancer is a risk factor for tumor progression, prognosis, depth of invasion, and lymph node metastases. in our study, the cd133 cytoplasmic expression was certainly related to tumor progression, primarily metastasis such as lymph node metastasis, peritoneal dissemination, and vascular invasion. with multivariate analysis, however, we did not find a definitive relationship between luminal cd133 expression and the degree of malignancy. sasaki. reported that in hepatocellular carcinoma, cytoplasmic cd133 expression, rather than membranous expression, is related to the degree of malignancy and prognosis. in rectal cancer as well, cytoplasmic cd133 expression is related to local recurrence and prognosis in a group that underwent preoperative chemotherapy and radiotherapy. these reports suggest that cytoplasmic cd133 expression alone may also be involved in the degree of malignancy of gastric cancer. it is widely known that some proteins gain biological function based on their site of expression. what is the significance of cd133 luminal expression ? in an interesting report, fukamachi. used fluorescence - activated cell sorting (facs) analysis of gastric cancer tissue and reported that loss of cd133 expression on the glandular luminal surface may be related to gastric tumor progression. however, our study results suggest that release of cd133 from the cytoplasm of undifferentiated gastric cancer cells into the glandular lumen may promote gland duct formation. in any case, cd133 expression likely plays some role in differentiation, as shown by yang.. ? characteristics of cscs include tumorigenicity, treatment resistance, and tumor recurrence. among these the survival rate in the postoperative adjuvant chemotherapy group and the curative resection group was regarded as an indicator of treatment resistance and recurrence. among these patients, the survival rate in the cytoplasmic cd133 expression group was significantly lower than in the other groups. this finding showed that the cytoplasmic cd133 expression group acquired treatment resistance and was more likely related to tumor recurrence. in summary, cancer cells with cytoplasmic cd133 expression were related to tumor progression (primarily metastases) and prognosis, and were associated with more undifferentiated tumors, treatment resistance, and more likely recurrence. finally, regarding the relationship between cd133 expression and hif-1 expression, in the luminal cd133 expression group in our study, the hif-1 expression rate was lower, similar to that which was reported by matumoto. however, in the cytoplasmic cd133 expression group, the hif-1 expression rate was higher, similar to that reported for other organs. this finding suggests that for cytoplasmic cd133 expression with a high degree of malignancy, hif-1 may upregulate its expression. in fact, in our study as well, the survival rate in gastric cancer in the hif-1 expression group was lower. even among cases in the hif-1 expression group, in the group that was also cd133 positive, the survival rate was even lower. however, in our study, even when the hif-1 (+) cd133 (+) poor prognosis group was further classified based on cd133 expression type, no differences in survival rate based on expression type were observed. one possibility is that among the luminal cd133 expression group, those with hif-1 expression also showed cytoplasmic cd133 expression. considering the relationship between cd133 and hif-1 based on our study results, hif-1 expression is increased with hypoxia, cd133 is expressed or retained in cancer cell cytoplasm, and tumor progression occurs as a result of this csc - like function. in addition, release from this hypoxic state, namely via decreased hif-1, promotes the release of cd133 from the cytoplasm, which may then function in gland duct formation. if this is the case, then inhibiting hif-1 expression may lead to improved prognosis in gastric cancer. however, before reaching this conclusion, it will be necessary to establish an accurate method for isolating the cd133 expression types and to conduct studies in gastric cancer cell lines using these isolated expression types. in our present study, gastric cancer with cytoplasmic cd133 expression was associated with lymph node metastases, peritoneal dissemination, chemotherapy resistance, recurrence, and poor prognosis. evaluation of cytoplasmic cd133 expression in gastric cancer tissue sections may be useful in the future as a novel prognostic factor. moreover, a significant correlation between hif-1 expression and the cd133 immunohistochemical staining pattern was found. | backgroundcd133 is one of the most important stem cell markers in solid cancers. some recent reports have described a possible relationship between cd133 and hypoxia - inducing factor-1-alpha (hif-1). the aim of this study was to clarify the clinical role of cd133 expression in gastric cancer and to investigate the correlation between cd133 expression and hif-1 expression.methodswe studied 189 gastric cancer patients who underwent gastrectomy at kurume university hospital. cd133 and hif-1 expression was examined using immunohistochemical staining. fifty - six cases were cd133 positive, and they were divided into two expression types : luminal expression of the gland and cytoplasmic expression. we investigated the relationship among cd133 expression types, clinicopathological variables, prognosis, and hif-1 expression.resultswhen comparing clinicopathological variables, expression of cd133 in the cytoplasm was related to metastasis and tumor progression. however, this relationship was not observed with luminal expression of the gland type. the survival rate in patients with cytoplasmic cd133 expression was significantly worse than that in the cd133-negative group. this relationship was observed in the survival rate of the adjuvant chemotherapy group and the curative resection group. multivariate analysis revealed that the expression of cd133 in the cytoplasm was an independent prognostic factor in gastric cancer. regarding the correlation between cd133 expression and hif-1 expression, the hif-1 positive rate was lower in patients with cd133 luminal expression of the gland type and higher in patients with cytoplasmic expression of cd133.conclusiongastric cancer cells with cd133 expression in the cytoplasm were cells with high potential for malignancy, and this phenotype was associated with cancer progression, chemotherapy resistance, recurrence, and poor prognosis. cytoplasmic expression of cd133 may be a useful prognostic marker in gastric cancer. significant correlation was observed between hif-1 expression and the immunohistochemical staining pattern of cd133. |
mucopolysaccharidosis (mps) is a group of autosomal recessive metabolic disorders caused by the absence or malfunctioning of the lysosomal enzymes needed to break down molecules called glycosaminoglycans (gags). these are long chains of sugar carbohydrates in each cell that help build bone, cartilage, tendons, corneas, skin, and connective tissues. gags (formerly called mucopolysaccharides) are also found in the fluid that lubricates joints. people with mps either do not produce enough of one of the 11 enzymes required to break down these sugar chains into proteins and simpler molecules, or they produce enzymes that do not work properly. over time, these gags collect in the cells, blood, and connective tissues. this results in permanent, progressive cellular damage which affects the appearance, physical abilities, organ, and system functioning and, in most cases, mental development. common clinical presentation includes facial dysmorphism, hepatosplenomegaly, joint stiffness and contractures, pulmonary dysfunction, myocardial enlargement, valvular dysfunction, and neurological involvement.[15 ] we report this case of mps type ii because of its rarity and the atypical features. the purpose of presenting report is to highlight the distinctive manifestation of the hunter syndrome. an 11-year - old boy was referred to the outpatient department, college of dental surgery, aljabal - algharby - zawia university, zawia, libya, for routine dental examination. the boy had been followed regularly by the hospital pediatrics unit with the diagnosis of mps. there was no family history of similar symptoms. clinically, on physical examination the patient had retarded growth with a short stature for his chronologic age. bony deformities, including kyphosis, rotated legs, and short stubby hands with a clumsy and stiff gait were evident. the following facial features were observed : oblique palpebral fissures, along with skin eruptions which appeared as red colored rashes on the face, hands, legs, and abdomen [figure 1 ]. the patient also had multiple, whitish to red skin - coloured papules and nodules symmetrically distributed on the scapular region, the extensor aspects of the upper arms [figure 2 ] and thighs. there was mild mental retardation. on examination, the lips were incompetent and dry. intraorally, the enamel of permanent teeth was slightly hypoplastic with a clinical appearance of pitted enamel [figure 3 ]. there was a carious lower left deciduous first molar and root stumps in relation to lower right second deciduous molar. there was high arched palate, macroglossia, increased salivation, and anterior open bite. extra oral picture showing the facial features and skin eruptions on the forehead picture showing the skin lesions on the forehand intraoral picture revealing hypoplastic enamel and carious teeth iopa radiograph revealed cyst - like radiolucent lesion in between the mesial root of lower right first permanent molar and root stumps of deciduous second molar [figure 4 ]. the involved roots were pushed apart, and there was a well - defined radiolucency with a well - defined radio - opaque border. aspiration from the cystic lesion revealed a radicular cyst in relation to the root stump of second deciduous molar. iopa radiograph revealing a cystic lesion with an associated carious root stump of deciduous second molar opg showing the radiolucency in relation to right mandibular deciduous second molar analysis of the urine revealed a marked increase in dermatan sulfate and heparan sulfate. an enzyme assay for iduronate sulfatase could not be carried out due to limited availability of such tests. a definitive diagnosis was previously made upon detection of a significant increase of dermatan sulfate in urine and a marked deficiency of l - iduronidase activity in his leukocytes. our diagnosis of hunter syndrome mps ii was confirmed from his history, clinical examination and biochemical reports. mucopolysaccharidosis was first described by charles hunter, a canadian physician, who in 1917 described a rare disease found in two brothers. mucopolysaccharidosis is a group of inherited diseases characterized by defective lysosomal enzymes responsible for the degradation of mucopolysaccharides, which are major components of intercellular connective tissue. this leads to an accumulation of incompletely degraded mucopolysaccharides in the lysosomes which affect various body systems through enzymatic activity. the accumulation of gag within the lysosomes is responsible for the clinical manifestations of this disorder. mucopolysaccharidosis type ii or hunter syndrome is rare and is caused by a deficiency of iduronate-2-sulfatase. hunter syndrome is one of the most common mps with a prevalence of one in 170,000 male live births. mps type ii is classified into mild (type ii, hb) and severe (type ii, a) and this classification is based on the length of survival and the presence or absence of central nervous system (cns) disease., the clinical features appear between two and four years of age while in the mild form, the clinical features appear in the second decade of life. in the severe form death usually occurs in the first or second decade of life and the main cause of death is obstructive airway disease or cardiac failure. in the milder form, there is mild mental retardation but intelligence is normal, stature is near normal, and clinical features are less obvious and progress very slowly. we feel our patient may have a mild form of the disease. in patients with neurologic involvement, intelligence is impaired, and death usually occurs in the second decade of life, whereas those patients with minimal or no neurologic involvement may survive into adulthood with normal intellectual development.[68 ] in our case there was mild mental retardation with normal intellectual development. young established that patients with hunter 's syndrome did clearly fall into one of two groups according to the presence or absence of intellectual deterioration. yatziv suggested that the presence or absence of severe mental retardation and the longevity of the affected individuals be distinguishing factors in order to help clinically determine the difference between these two forms. patients with mps ii show dental abnormalities including enamel defects, carious teeth, dentigerous cysts, and abscesses. diagnosis often can be made through clinical examination and urine tests as excess mucopolysaccharides such as heparin and dermatan sufates are excreted in the urine. enzymes are also used to provide definitive diagnosis of one of the mucopolysaccharidoses. prenatal diagnosis using amniocentesis and chorionic villus sampling can verify if a fetus either carries a copy of the defective gene or is affected with the disorder. genetic counselling can help parents who have a family history of the mucopolysaccharidoses to determine if they are carrying the mutated gene that causes the disorders. enzyme replacement therapy has emerged as a new treatment for mucopolysaccharidosis disorders, including hunter syndrome. enzyme replacement therapy using idursulfase (elaprase), a recombinant human 12s produced in the human cell line, has been recently approved in the united states and the european union for the management of mps type ii. weekly intravenous infusion is given over 3 h at a dose of 0.5 mg / kg diluted in saline. bone marrow transplantation and umbilical cord blood transplantation are definitive treatments for mps. apart from these blood transfusion, infection, and nutritional management are also important in the management of mps type ii. a mild form is compatible with survival into adulthood, and reproduction is known to have occurred. six cases of this mild form of hunter syndrome have been described, and the patients survived to the ages of 65 and 87 in two cases. the enzyme deficient in this disorder is iduronate sulfatase, as described by neufeld. with the advent of hematopoietic stem cell transplantation and more recently, there exists a need for early diagnosis, better disease recognition, and management. the clinical features are limited to short stature, a large head, a short neck, coarse facial features, skin eruptions, and mild mental retardation with normal intelligence, anterior open bite due to enlarged tongue suggested of mucopolysaccharidosis. frequent upper and lower respiratory tract infections are common and occur secondary to the enlargement of tonsils and adenoids and enlarged tongue,[5781420 ] this presentation is evident in our patient. although the syndrome is uncommon worldwide, a higher incidence has been reported among jews in israel. cutaneous features are peculiar to this syndrome and may be the initial manifestation in the mild form of the disease although patients in both groups may have skin involvement. firm, skin - colored papules, 210 cm in diameter coalesce to form ridges on the scapular and posterior axillary lines. these pebbles are mainly found only in the hunter syndrome[2225 ] and helps in differentiating it from other mps. we emphasize that the skin eruption can be the earliest sign of hunter syndrome, particularly in the mild form presenting with normal development and growth as seen in our case. based on the clinical representation, it is possible to diagnose a case of mps. early detection of the disease and appropriate management through a multidisciplinary approach is recommended to improve the quality of life. a careful and systemic approach is needed to accurately diagnose the exact type as enzymatic studies are not available in most centres. | we report a rare case of hunter syndrome mucopolysaccharidosis type ii (mps ii) with atypical presentation of mild mental retardation, acrocephalic head without corneal clouding, and multiple skin eruptions along with oral, dental, and radiographic findings. it is a rare syndrome with a very low prevalence of 1:100,000 births and as such the clinician should be aware of this syndrome. |
an estimated 226,870 new cases of breast cancer are expected to be diagnosed in 2012 and account for about 29% of all new cancer cases among us women. adjuvant chemotherapy is one treatment option for breast cancer patients that is used because of its potential to reduce the risk of breast cancer recurrence and mortality ; however, not all patients benefit from adjuvant chemotherapy. moreover, chemotherapy may be detrimental to quality of life given its potential to produce toxicities, including myelosuppression. the quality of breast cancer care can be improved by informing treatment selection based on individual patient genomic risk profiles ; however, to realize the greatest benefits, advances in predictive models that inform treatment decisions must be accepted and used by healthcare providers and patients. one example of predictive modeling that is rapidly moving into the breast oncology care setting is oncotypedx testing [4, 6 ]. based on the expression of 21 genes obtained from tumor tissue, oncotypedx testing calculates the risk of breast cancer distant recurrence (i.e., the chance of breast cancer returning as metastatic disease) in patients with estrogen receptor (er) positive early breast cancer treated with adjuvant endocrine therapy and predicts the clinical benefit with additional adjuvant chemotherapy. given that approximately 75% of breast cancers are er positive and 61% of those cases are lymph node (ln) negative, many women with breast cancer may be qualified and benefit from oncotypedx testing. although there are clinical guidelines to identify patients who would derive the greatest benefit from oncotypedx testing, relatively few studies have been conducted to examine the impact of test results on treatment decisions. some research has indicated that oncotypedx recurrence score (rs) results have impacted receipt of chemotherapy, including a study of 276 patients who were newly diagnosed with breast cancer between 2005 and 2009. after controlling for nottingham prognostic index, adjuvant online mortality risk, progesterone receptor (pr) status, and medical oncologists ' blinded recommendation for adjuvant chemotherapy, only rs and patient age at diagnosis were significantly associated with receipt of adjuvant chemotherapy. other studies have linked rs results to changes in adjuvant chemotherapy plans [812 ]. for instance, one study conducted from 2004 to 2006 examined the impact of rs on 29 patients with er positive and ln negative breast cancer. results showed that rs changed chemotherapy plans for 9 patients such that 7 of 13 patients for whom chemotherapy was recommended did not receive it, and 2 of 16 patients received chemotherapy after initial recommendations against it. though some research has pointed to an association between rs and receipt of adjuvant chemotherapy, currently only one published study collected data beyond 2008. the current study serves to replicate these findings in another population, with the primary purpose of evaluating whether oncotypedx test results predict receipt of adjuvant chemotherapy in a cohort of consecutive patients with breast cancer who received an oncotypedx rs. the study population was comprised of patients treated at moffitt cancer center, a large national cancer institute - designated comprehensive cancer center in the southeastern us. approximately 60% of all patients seen at moffitt come from the surrounding seven county catchment area, with the remainder of patients coming from other florida counties, states, and countries. upon institutional review board approval, moffitt cancer center surgical pathology records were used to identify patients meeting the following criteria (1) diagnosed with breast cancer, and (2) had oncotypedx testing between december 2004 (the year in which the test was approved by the u.s. food and drug administration) and january 2009 (the year in which chart reviews were completed). based on this information, medical records were reviewed retrospectively to collect patient sociodemographic information, tumor characteristics, oncotypedx rs, and treatment - specific data. chart abstractions were performed by a study team member after training from the principal investigator. the senior study coordinator reviewed 10% of data files to assess the accuracy of collected data. finally, a medical oncologist reviewed the summarized data and identified a subset (~10% of charts) for additional review to ensure accuracy. sociodemographic data included age ; marital, parental, and menopause status ; race / ethnicity ; and family history of breast cancer (present / absent). clinical characteristics included breast cancer stage ; tumor size ; ln status ; histology ; modified combined histologic (nottingham) grade ; nuclear grade ; human epidermal growth factor receptor-2 (her2), er, pr, and angiolymphatic invasion status ; and rs. regarding rs, patients were classified into one of three groups based on cut points : low (rs 17), intermediate (rs = 1830), or high (rs 31). the primary outcome variable, receipt of adjuvant chemotherapy, was based on grouping women into those who had chemotherapy versus those who did not. this comparison was selected as it most closely reflects the primary treatment decision influenced by oncotypedx results [6, 13 ]. bivariate analyses were used to investigate the relationship between the primary outcome variable, receipt of adjuvant chemotherapy, and the sample sociodemographic and clinical characteristics. pearson chi - square or fisher 's exact tests were used to study relationships with each categorical variable of interest ; a t - test was conducted to examine differences in age and rs by chemotherapy uptake group. analyses used two - tailed tests of significance with the significance level set at p 1.0 cm (78.8%), and were ln negative (95.8%). the majority (84.8%) had invasive ductal carcinoma (idc), and over half (56.8%) of the tumors showed intermediate histologic grade. most patients ' tumors were her2 negative (94.9%), er positive (99.2%), pr positive (89.0%), and did not show angiolymphatic invasion (72.9%). the mean rs was 19.0 and the largest proportion of patients had a low rs (57.6%). in bivariate analyses, tumor size, histologic grade, nuclear grade, and rs were significantly associated with uptake of adjuvant chemotherapy (table 1). compared to patients who did not receive chemotherapy, those who received chemotherapy had a greater proportion of tumor size > 1.0 cm (91.4% versus 73.5%), high histologic grade (28.6% versus 8.4%), nuclear grade of 3 (40.0% versus 12.1%), intermediate (68.6% versus 15.7%) and high (20.0% versus 7.2%) rs category, and higher mean rs (26.4 versus 15.9). in multivariable analysis controlling for tumor size, histologic grade, and nuclear grade, only rs remained statistically significantly associated with chemotherapy receipt (table 2). relative to those with a low rs, those with an intermediate (adjusted odds ratio [aor ], 21.24 ; 95% ci, 3.62237.52) or high (aor, 15.07 ; 95% ci, 1.28288.21) rs had a greater odds of chemotherapy uptake. study results indicate that rs was significantly associated with adjuvant chemotherapy uptake, suggesting that in our sample of female breast cancer patients who underwent oncotypedx testing, the results of this gene assay were likely being used to inform treatment decision making, although it is unclear if and how the information was conveyed to patients. rs score was a significant predictor of chemotherapy uptake after controlling for more standard clinicopathological markers used to guide treatment selection, specifically tumor size, ln status, histologic grade, and nuclear grade. these results are consistent with earlier research that has shown that rs was associated with whether patients received adjuvant chemotherapy or not [812 ]. adjuvant chemotherapy is recommended for individuals with rs scores in the high - risk category, whereas it is unlikely to benefit individuals in the low - risk category [13, 14 ]. in our study, patients with a low rs were least likely to receive adjuvant chemotherapy (94% of low - risk patients did not receive chemotherapy). this finding is consistent with research showing that rs affects treatment decision - making for low - risk patients and is similar to the ademuyiwa study, which reported that 91% of patients with a low rs did not receive chemotherapy. although national comprehensive cancer network guidelines recommend chemotherapy for individuals with high rs, in our patient population, only 54% of individuals with high rs received chemotherapy. this percentage is lower than that of the ademuyiwa study, which reported that 96% of patients with high rs received chemotherapy. for our sample of patients in these higher risk categories, the receipt of chemotherapy is likely multifactorial (based on coexisting clinicopathological features). due to the small sample sizes in this study, we were unable to more fully investigate the patterns of treatment choice based on these multiple factors. of interest was the finding that one er negative patient and four ln positive patients received an rs, even though the test has been validated for er positive and ln negative breast cancer patients at the time oncotypedx testing was performed. all ln positive patients were her2 negative with primary tumor categories ranging from one to four. additionally, there were six her2 positive patients who received an rs, one of whom was t1an0, four were t1bn0, and one was t1cn0. it is possible that the rs was felt to be of utility for these patients, and perceived as an additional measure of breast cancer recurrence risk to support a decision about adjuvant chemotherapy, as was noted in one patient chart. most of the published literature regarding the use of rs to guide treatment selection focuses on physicians ' use of this information in guiding treatment selection [8, 10 ], with limited evaluation of the patient 's role in the decision to pursue a therapy. factors that patients may consider in their treatment decisions include side effects that can adversely affect quality of life, patient preference for participation in treatment decision - making, and understanding of the results ; the latter may be particularly critical where greatest uncertainty exists, that is, for patients with an intermediate rs. a preference for an active role in decision - making among those presented with oncotypedx results is related to health literacy, such that women with high - health literacy prefer a more active role in decision - making, whereas low - health literacy is related to preference for more shared and passive decision - making. our group recently completed a retrospective cross - sectional survey of a subset of the patients (n = 64) included in the present study and found that women incorrectly answered approximately half of a series of 14 items evaluating knowledge about rs. another similarly designed study of 77 breast cancer patients found that one - third of participants did not fully understand discussions related to rs. although not a focus of this study, it would be of interest to investigate how these variables interact to guide treatment planning for patients in the higher - risk rs categories. to date, much of the research on oncotypedx results ' association with treatment decision - making has occurred in an academic setting. some research conducted with an inner - city population suggests that oncotypedx results may influence chemotherapy treatment decisions in this setting. in this sample of 47 women who underwent oncotypedx testing, 5% of women with a low rs and 100% of women with a high rs received chemotherapy. the results for women with a low rs are aligned with the current study and ademuyiwa study, and results for women with a high rs are similar to the ademuyiwa study. more research needs to be conducted with larger samples of women from populations outside of the academic setting. first, the confidence intervals for the rs categories were wide, thus estimates may be imprecise. second, although several clinicopathological and demographic variables were included in the study, the small sample sizes limited our ability to further elucidate trends among individuals in the higher - risk rs categories who did not receive adjuvant chemotherapy. third, family history was recorded in the patient 's chart based on patient self - report and, like other self - report data, may be inaccurate ; however, some research on the concordance between self - report of family breast cancer history and cancer confirmation sources (e.g., state tumor registry) suggests a sensitivity ranging from 61% to 95% [20, 21 ]. fourth, as the study was retrospective, we were unable to assess definitively whether treatment planning was changed as a result of the availability of the rs score and whether patients themselves were able to use the rs to aid their treatment decision. also, we did not account for the possible role that comorbidity may have played in the uptake of chemotherapy. given that higher comorbidity has been linked to a decreased likelihood of receiving chemotherapy among women aged 55 and older, it is possible that comorbid conditions may also have been associated with chemotherapy among older women in the current study. although reflective of the patient population at this institution, findings may not be generalizable to other races and ethnicities. finally, some patients may have received chemotherapy after the data collection period ended and this change in chemotherapy receipt status was not reflected in the analyses. in summary, the current study offers relatively recent data to support results of previous research documenting a change in treatment selection based on the use of the rs [812 ]. the literature remains in need of studies investigating the variables associated with a patient 's use of this valuable health information, including the potential role of comorbidities, as well as studies conducted with populations outside of the academic setting. this paper has important implications for prevention, such that women with a lower risk of breast cancer recurrence as determined by oncotypedx testing may be able to avoid the potential toxicity associated with chemotherapy. | introduction. this study aimed to evaluate whether oncotypedx test results predict receipt of adjuvant chemotherapy in breast cancer patients who received an oncotypedx recurrence score (rs). materials and methods. pathology records were used to identify breast cancer patients who had oncotypedx testing between december 2004 and january 2009 (n = 118). patient sociodemographic information, tumor characteristics, rs, and treatment - specific data were collected via chart review. rs was classified as follows : low (rs 17), intermediate (rs = 1830), or high (rs 31). bivariate analyses were conducted to investigate the relationship between adjuvant chemotherapy receipt and each sociodemographic and clinical characteristic ; significant sociodemographic and clinical variables were included in a multivariable logistic regression model. results. in multivariable analysis controlling for tumor size, histologic grade, and nuclear grade, only rs remained significantly associated with chemotherapy uptake. relative to low rs, an intermediate (adjusted odds ratio [aor ], 21.24 ; 95% confidence interval [ci ], 3.62237.52) or high (aor, 15.07 ; 95% ci, 1.28288.21) rs was associated with a greater odds of chemotherapy uptake. discussion. results indicate that rs was significantly associated with adjuvant chemotherapy uptake, suggesting that oncotypedx results were used to inform treatment decision making, although it is unclear if and how the information was conveyed to patients. |
the danish head and neck cancer (dahanca) group was established in 1976 as a working group by the danish society for head and neck oncology with the primary aim to develop national guidelines for the treatment of head and neck cancer in denmark. dahanca group s activities have included creating and running a national database, development of treatment guidelines, performing numerous national clinical protocol studies (> 30), and providing quality assurance programs. the group is multidisciplinary and consists of representatives from all the oncology centers in denmark treating head and neck cancer, including oncologists, otolaryngologists, pathologists, imaging diagnosticians, experimental researchers, epidemiologists, and other expert support, eg, medical physicists. the dahanca model was behind the establishment of the structure of the danish multi disciplinary cancer groups (dmcg), and the dahanca group is thus the oldest of the still active dmcg. traditionally, these include cancer of the larynx, pharynx, oral cavity, sinuses, salivary glands, thyroid glands, and neck nodes from unknown primary type of cancer (corresponding to the international classification of diseases, tenth revision, classifications c.01c.11, c.30c.32, c.73, and c.80), but did not include cancer of the lip. the most significant types are cancer of the pharynx, larynx, and oral cavity. almost all of these are squamous cell carcinomas, which etiologically are strongly linked to tobacco and alcohol use and increasingly also to infections with especially human papilloma virus, since human papilloma virus - related oropharyngeal cancer has become the most rapidly increasing cancer type in denmark. the therapeutic strategy is therefore aimed toward eradicating the disease at the site of the primary tumor and the regional lymph nodes. such a treatment is typically surgery or radiotherapy, and over the past years in denmark, focus has deliberately been on radiotherapy as the primary modality, as this ensures the best possibility of organ conservation. dahanca group has been an active and well - established structure for 40 years and has provided a solid foundation for the treatment of head and neck cancer in denmark and has become recognized internationally as one of the major head and neck cancer groups. in this aspect, it should be noted that the treatment of head and neck cancer in denmark has rather a good outcome seen with international eyes. the registration of patients with head and neck cancer is the essential backbone of the dahanca structure, and the associated database includes currently > 33,000 patients, with an annual increase of 1,400 patients (table 1). historically, the database started as a local home - designed database at the department of oncology, aarhus university hospital, aarhus, denmark, where the medical physicist mogens hjelm hansen in the early 1970s created an elaborate program for the registration of head and neck cancer (the radiation physics department was among the first places where computers were introduced in health care). this program later formed the basis for equivalent databases in copenhagen and odense and a subsequent retrospectively registration of, especially, laryngeal cancer back to the early 1960s. the registration of cases later expanded to include the new oncology centers in aalborg and herlev, and a full national registration of larynx cancer has taken place since 1971. in the same time period, there have also been regional and more sporadic registrations of pharynx and oral cavity cancers. at a national level, the three initial databases included all patients with larynx cancer between 1971 and 1991. in connection with a large clinical trial,1 the national database was reconstructed and expanded to include all cancers of the larynx, pharynx, and oral cavity. each of the five oncology centers had their own databases (named da 15), which were all copies of a central master. thus, data were gathered locally and were immediately available for daily use and research purposes at the individual institutions. the da 15 data files were regularly merged into the joint database at the secretariat in aarhus and thus formed the national dahanca clinical database. also, the initial three databases named (akh, hlm, and hl3) were merged and restructured using the same program. the registration in da 15 covered the period 19921999. in 1995, a special dathyrca database for registering thyroid cancer2 and more lately databases for unknown primary neck tumor,3 salivary gland tumor,4 and sinonasal cancer were established.5 in 2000, the dahanca database was changed into a central web - based database (ocx), which allowed interactive registration directly on a central server with the immediate possibility for quality checks of the data and with instant correction of errors. this increased the quality assurance and gave direct access to information about the current status of the patients. the ocx database is based on the development tool clarion and uses hypertext transfer protocol secure certificate with an hypertext transfer protocol secure connection ; all communications are securely encrypted. the operating system is on a windows server 2012 platform, and the server is situated at aarhus university. the system is flexible and can be modified in connection with specific protocols and projects. all projects are carried out in accordance with the applicable legislation and regulations, including the european union s clinical trials directive, and corresponding regulations for the use of good clinical practice and quality assurance, and the database fulfills the danish data protection agency s description of standard specification and requirements for security. the ocx database has been active since 2000, and the structure has formed the basis for several other clinical quality cancer databases (eg, melanoma, sarcoma, ocular cancer, childhood cancer databases). moreover, in 2011, the dahanca database was transformed from being a private research database to a clinical quality database in public auspices under the danish clinical registries (rkkp), while still allowing the continued use for research purposes. this simplified the correlation with other public databases, such as the danish cancer registry (dcr), and allowed the database to be used as an indicator of quality. approximately 33,000 patients are included in the database, which has ~300 basic variables.6,7 additionally, there is the possibility of specially registered parameters in connection with specific projects. the aim is that established variables remain in the database, and to have, to a large extent, continuous variables that remain unchanged. international standardized classifications are used in this context where such classifications exist (eg, union for international cancer control tumor node metastasis classification). with regard to side effects, international principles are also applied, but the principles that we initially developed have now been adopted by other organizations, and thus, the dahanca database has contributed to the international standard with regard to registration of side effects from radiotherapy. as not all variables are in use at any time, the daily registration probably involves 100150 different parameters. the basic registration includes the following (refer forms at www.dahanca.dk) : on study : symptoms and the duration of the symptoms, etiological factors, pretreatment and diagnostic evaluation, including tumor metastasis classification, imaging, histopathology, and laboratory tests, primary treatment : semi - detailed information of radiotherapy, surgery, and medical treatment.follow-up : registration of vital and tumor status and acute and late side effects.recurrence : registration of relapse and treatment thereof.death form : registration of death and cause of death. on study : symptoms and the duration of the symptoms, etiological factors, pretreatment and diagnostic evaluation, including tumor node metastasis classification, imaging, histopathology, and laboratory tests, primary treatment : semi - detailed information of radiotherapy, surgery, and medical treatment. follow - up : registration of vital and tumor status and acute and late side effects. besides comparison with the dcr, vital status is checked regularly through updates from the civil registration number register. in certain situations, there have been correlations with specific extracts from the national patient discharge registry and the cause of death register. the principle of the dahanca database has throughout the years been a uniform structure of data description and a local registration. for the first many years, no actual clinical record forms existed, but the patient s journal was considered to be the paper - related data documentation and was used as a basis for data entry. for practical reasons, paper registration forms have been used since 1991 (refer forms at www.dahanca.dk). the gathering and recording of data has always been done by a few knowledgeable people, mainly senior specialist doctors. owing to the limited amount of such people, who have been involved over many years and who know the problems in detail, both communication and collection of data are done in a very uncomplicated and pragmatic fashion. this dependency on a few highly specialized persons is both the strength and weakness of the database, because it not only gives it extremely high quality but also makes it vulnerable to changes. the database has a number of error indicators built in, which safeguards against logical errors and at the same time has the option of reminders if information is missing. furthermore, lists of irregularities are instantly produced in a continuous attempt to collect missing data or correct any errors. the database is verified through the numerous related research projects.6,7 simultaneously, a specific comparison to the dcr is performed at least annually, where all cases in the dcr have been sought to be verified through the dahanca database, and in case of errors (in the dcr), these are explored when possible. thus, there is currently a full national registration of dahanca database ranging back to 1971 (table 1). this has shown that the dahanca database contains 99.7% of the originally known cases in the dcr, and an additional number of patients, currently 2%, have been identified, who were not in the dcr. furthermore, a number of erroneous registrations in the dcr have been corrected (in the dahanca database). the complete interaction between the two databases clearly shows that, in principle, there exists a total nationwide coverage in the dahanca database and further ascertains that the dahanca database has the potential of increasing the quality of the dcr s data. this investigative high - quality work has been performed in great detail, and in cases of doubt, relevant additional hospital records and/or other material have been obtained. thus, all patients in the database have had their hospital records scrutinized at least once, and all updates are verified by a specialist doctor. with the dcr s well - known almost complete coverage of cancer cases in denmark as a starting point, it is our impression that with the dahanca database we have a near - perfect clinical register, which probably is the world s most accurate recording of head and neck cancer treatment. the initial aarhus - derived database was approved with register regulations at vejle county. later, the national database was registered and approved as a research database by the danish data protection agency (record nos 1995 - 1200 - 282 and 2004 - 41 - 4802). since 2011, it has been a public clinical quality database (record no 1 - 16 - 02 - 249 - 14). jens overgaard is the overall data responsible head, and regionally, the responsibility lies with the consultant responsible for the head and neck cancer treatment at the respective oncological centers. as with other research - based databases, the project has initially been based on con amore gradually, the need for organized help has increased and the database has throughout the years been partly financed in connection with research projects, where among others, phd students were assigned to the activities mainly sponsored through the danish cancer society and the medical research council (we have constantly tried to avoid any commercial influence and sponsoring). however, the activities have throughout benefited from being part of the infrastructure at the department of experimental clinical oncology at aarhus university hospital and from the numerous dedicated persons who have used their spare time to strengthen the database. the value of any database is linked with its use, and the quality data are accessible through access to the rkkp (http://www.rkkp.dk/forskningsadgang/). throughout the years, the dahanca database has been extensively explored and has served as an active research database that has formed the basis for numerous scientific projects and publications, including 15 phd and doctoral theses (for full list, refer the dahanca homepage [www.dahanca.dk ]). it has been the supporting database for > 30 prospective clinical trials and has acted as a baseline for large retrospective studies describing the natural history and treatment outcome in head and neck cancer. examples of studies6,811 are given in figure 1, and more can be found on the dahanca web site. this means that the database is constantly revised and updated and, consequently, is extremely well verified. since many of the dahanca protocols have focused on biological - based improvement of head and neck cancer treatment, these translational studies have been based on a large amount of clinical material.1013 this dahanca - related biobank contains biological material (tumor, normal tissue cell lines, blood samples, dna, rna, etc) from > 4,000 patients with detailed clinical data and follow - up. currently, we are systematically collecting material from new patients in the national danish cancer biobank. the database has not only been strengthened by linkage to other registries6,7 but has also shown its value by being a supportive basis for studies of comorbidity,6 value of follow - up,14 or socioeconomical relationship in patients with head and neck cancer.15,16 in the foreseen future, it will be part of international comparisons of treatment strategies, and for such purpose, a clinical database is far superior than using cancer registry comparisons (such as the nordic nordcan study), since such databases are incomplete and inaccurate regarding clinical information. thus, it is important to use the right tools / databases for the right purposes, and when conclusions about therapeutic strategies are the aim, the data must be obtained from a well - verified clinical quality database. | aim of the databasethe danish head and neck cancer database is a nationwide clinical quality database that contains prospective data collected since the early 1960s. the overall aim of this study was to describe the outcome of the national strategy for multidisciplinary treatment of head and neck cancer in denmark and to create a basis for clinical trials.study populationthe study population consisted of all danish patients referred for treatment of squamous cell carcinoma of the larynx, pharynx, oral cavity, or neck nodes from unknown primary or any histopathological type (except lymphoma) of cancer in the nasal sinuses, salivary glands, or thyroid gland (corresponding to the international classification of diseases, tenth revision, classifications c.01c.11, c.30c.32, c.73, and c.80).main variablesthe main variables used in the study were symptoms and the duration of the symptoms ; etiological factors ; pretreatment and diagnostic evaluation, including tumor node metastasis classification, imaging, histopathology, and laboratory tests ; primary treatment with semidetailed information of radiotherapy, surgery, and medical treatment ; follow - up registration of tumor status and side effects ; registration of relapse and treatment thereof ; and registration of death and cause of death.main resultsdata from > 33,000 patients have been recorded during a period of > 45 years. in this period, the outcome of treatment improved substantially, partly due to better treatment as a result of a series of continuous clinical trials and subsequent implementation in national guidelines. the database has furthermore been used to describe the effect of reduced waiting time, changed epidemiology, and influence of comorbidity and socioeconomic parameters.conclusionhalf a century of registration of head and neck cancer treatment and outcome has created the basis for understanding and has substantially contributed to improve the treatment of head and neck cancer at both national and international levels. |
dry socket or localised alveolar osteitis is the most common complication of tooth extraction, with associated pain due to inflammatory changes in the exposed socket wall following breakdown of blood clot in extraction socket.1 the incidence of dry socket ranges from 0.6% to 5.6% in intra - alveolar extraction and 24.7% in trans - alveolar extraction.234 previous studies show predilection of females, mandibular teeth, patients in the 3 decade of life and smokers for alveolar osteitis.24 a reduced incidence of alveolar osteitis was reported in patients with good oral hygiene, avoidance of iatrogenic trauma to teeth and avoidance of surgery in days 1 and 22 of the menstrual cycle in non - menopausal females.5 recent report showed significantly better compliance among patients placed on verbal instruction than those placed on written instruction on the use of warm saline mouthwash after oral surgical procedures.6 however, the use of verbal and written post - surgical instruction was reported to enhance compliance.6 some studies on the use of post - extraction mouthwash (warm saline, hydrogen peroxide, chlorhexidine) and antibiotics (tetracycline, amoxicillin / clavulanic acid, clindamycin, metronidazole) have reported reduction in the incidence of post - extraction alveolar osteitis.1678910 there are previous nigerian reports that focussed mainly on incidence and pattern of alveolar osteitis,411 and the risk factors influencing the development alveolar osteitis following tooth extraction.35 apart from adebayo and dairo6 report on compliance to warm saline mouth wash after oral surgical procedure, no other study to our knowledge has evaluated compliance to various post - extraction regimen with the incidence of alveolar osteitis in our environment. this study aims to evaluate the effect of various combination of post - extraction regimen administered to patients who had intra - alveolar molar tooth extraction. this is a prospective study performed within a year (june 2001may 2002) involving patients who were placed on warm saline mouth rinse six to eight times daily for 1 week (verbal instruction) alone, warm saline mouth rinse with or without antibiotic (amoxicillin 500 mg and metronidazole 400 mg 8 hourly for 5 days) and or analgesic (paracetamol 1000 mg 8 hourly for 5 days) therapy (written prescription), or the antibiotic and analgesic therapy alone, after intra - alveolar molar tooth extraction in the department of oral and maxillofacial surgery and oral pathology, obafemi awolowo university teaching hospital, ile - ife (for the patients on warm saline mouth rinse alone, because this centre usually prescribes this post - extraction regimen) ; and department of oral and maxillofacial surgery and pathology, university of benin teaching hospital, benin city, nigeria (for patients on warm saline mouth rinse with or without antibiotics and patients on antibiotics and analgesic, because this centre usually prescribes these post - extraction regimen). consenting patients who had intra - alveolar molar tooth extraction and were placed on various post - extraction regimen based on the chosen hospital centres, and those who came for 1-week post - extraction review were selected for this study. the patients with history of smoking, frequent intake of alcohol, those suffering from diabetes mellitus, those on oral contraceptive and poor oral hygiene were excluded from this study. the study was carefully explained to the patients and they were assured of strict confidentiality of information obtained through questionnaire and clinical examination. the patients age, gender, site of the molar tooth, post - extraction regimen and alveolar osteitis were analysed. pearson 's chi square correlation was performed for the variables, with confidence level set at 95% and p value of < 0.05 was considered significant. of the 76 patients studied there were 49 (64.5%) females and 27 (35.5%) males, giving a male to female ratio of 1:1.8. the peak age group of the patients was the 3 decade of life (n = 29, 38.2%), with a mean age of 35 20 years [table 1 ]. forty - two (55.2%) of the patients had lower molar tooth extraction, 22 (29.0%) patients had upper molar tooth extraction, while 12 (15.8%) patients had upper molar and lower molar extraction. a total of 63 (82.9%) patients complied with the post - extraction regimen, giving a significantly high compliance to the post - extraction instructions (p = 0.001). twenty - two (29.0%) patients were compliant among the 29 (38.2%) cases placed on warm saline mouth rinse. of the 31 (40.8%) patients placed on warm saline mouth rinse, antibiotics (amoxicillin and metronidazole) and analgesic (paracetamol), 27 (35.5%) cases were compliant. twelve (15.8%) patients were on analgesic and warm saline rinse, among which 11 (14.5%) cases were compliant. of the 4 (14.5%) patients placed on antibiotics (age distribution of the patients placed on post - extraction regimen there were 10 (13.2%) cases of post - extraction localised alveolar osteitis, with predilection for the lower molar teeth (n = 6, 7.9%), followed by upper molar teeth (n = 3, 4.0%) and one (1.3%) case involving both upper and lower molar teeth. there was a significant female predilection for alveolar osteitis (n = 8, 10.5%) (p = 0.005) in this study [table 2 ]. there were five (6.6%) cases of localised alveolar osteitis in the compliant patients (n = 63, 29.0%) giving a ratio of 1:12.6. five (6.6%) cases of localised alveolar osteitis were found among non - compliant patients (n = 13, 17.1%), giving a ratio of 1:2.6. there were two (2.6%) cases each of localised alveolar osteitis in compliant patients (n = 22, 29.0%, ratio 1:11) and non - compliant patients (n = 7, 9.2%, ratio 1:3.5) on warm saline mouth rinse only. among the patients placed on warm saline mouth rinse, antibiotics and analgesic, there were three (4.0%) cases of alveolar osteitis in compliant (n = 27, 35.5%, ratio 1:9) and one (1.3%) case of alveolar osteitis in non - compliant patients (n = 4, 5.3%, ratio 1:4). among the patients placed on analgesic and warm saline mouth rinse, there was one (1.3%) case of alveolar osteitis in the non - compliant patient (ratio of 1:1). among the patients placed on analgesic and antibiotics, there was one (1.3%) case of alveolar osteitis in the non - compliant patient (n = 1, 1.3%, ratio of 1:1). there was significant association of non - compliance with post - extraction regimen and the incidence of localised alveolar osteitis (p = 0.015) [table 2 ]. age, gender and tooth type distribution of localised alveolar osteitis in the patients placed on post - extraction regimen although compliance to post - extraction regimen using mouth rinses and prophylactic antibiotics are reported to significantly reduce the incidence of localised alveolar osteitis,1678910 dental surgeons often administered post - extraction regimen without considering patients preference for any combination of the regimen based on predictable outcome of the regimen. this study showed significant compliance to the post - extraction regimen, which agrees with previous report by adebayo and dairo.6 a corresponding significant reduction in the incidence of localised alveolar osteitis was also observed among the compliant patients in this study following molar teeth extraction. conversely, a relatively higher incidence of localised alveolar osteitis was found among patients who were non - compliant with the post - extraction regimen. overall, a relatively higher incidence of localised alveolar osteitis was observed in this study, with significant predilection of the lesion for females and mandibular molar teeth. similarly, female predilection for dry socket has been previously reported in nigeria1112 and united kingdom.13 furthermore, female and mandibular molar teeth predilection for dry socket was also reported by oginni.5 however, equal incidence of dry socket in the maxilla and mandible, occurring exclusively in molar and premolar teeth was reported by ogunlewe.,11 the higher incidence of localised alveolar osteitis in this study, may be due to the selection of patients who had molar tooth extraction, a group known to be highly susceptible to alveolar osteitis.231113 although the mechanism of action of the post - extraction regimen in the prevention of dry socket is not very clear, previous report by cardoso.,14 states that irrigation of extraction socket with increasing amount of physiologic saline progressively decreases the incidence of dry socket,1516 while antibiotics prevent dry socket because of the antimicrobial effect against bacteria involved in pathogenesis of dry socket.17 when the use of various combination of the post - extraction regimen were compared with the incidence of developing alveolar osteitis in this study, compliance to post - extraction warm saline mouth rinse alone, as prescribed in the ile - ife centre was the most effective in the prevention of localised alveolar osteitis. this was followed by compliance to a combination of warm saline mouth rinse, antibiotic and analgesic as prescribed in the benin centre. this finding indicated that the warm saline mouth rinse alone has advantage of better compliance and consequently, it was more effective in reducing alveolar osteitis compared with the combination post - extraction regimen. but there was a higher risk of patients placed on warm saline mouth alone and warm saline mouth and analgesic without antibiotics developing alveolar osteitis. however, localised alveolar osteitis was observed mostly in non - compliant patients placed on warm saline mouth rinse and analgesic and patients placed on antibiotics and analgesic. there was reduced number of patients in this study because some of the consenting patients did not present in clinic for the 1-week post - extraction review. also, this study was not specifically focussed on patients preference for various combination of the post - extraction regimen. however, the findings of this study from the two centres, suggest that it may be needful to properly educate patients on the effect of compliance to various combination of post - extraction regimen in reducing the incidence of localised alveolar osteitis. thereafter, the patients preferred regimen is identified and accordingly prescribed to the patients after tooth extraction. furthermore, large randomised controlled studies is recommended to determine the rationale for individualising post - extraction regimen to each patient and to assess the effectiveness of each regimen, while taking into account the other known factors that could contribute to the effectiveness of the regimen. this study showed a significant patients compliance with post - extraction warm saline mouth rinse, prophylactic antibiotics and analgesic and a corresponding significant reduction in the incidence of localised alveolar osteitis following intra - alveolar molar tooth extraction. a relatively higher incidence of localised alveolar osteitis was observed in this study, with significant predilection of the lesion for females, mandibular molar teeth and non - compliance to post - extraction regimen. compliance to post - extraction warm saline mouth rinse, followed by compliance to a combination of warm saline mouth rinse, antibiotic and analgesic, may be the most effective measures of reducing the incidence of localised alveolar osteitis. this study emphasises the need to properly educate patients on the effect of compliance to various combination of post - extraction regimen in reducing the incidence of localised alveolar osteitis. | background : to evaluate the effect of various combination of post - extraction regimen administered to patients who had intra - alveolar molar tooth extraction.patients and methods : one year prospective study involving 76 consenting patients who came for 1-week post - extraction review. the patients were placed on warm saline mouth rinse with (verbal instruction) or without antibiotic and or analgesic therapy (written prescription), after intra - alveolar molar tooth extraction. information was obtained from the patients through questionnaire and clinical examination.results:the patients were placed on warm saline mouth rinse (n = 29, 38.2%) only, warm saline rinse, antibiotics (amoxicillin and metronidazole) and paracetamol (n = 31, 40.8%), paracetamol and warm saline rinse (n = 12, 15.8%) and antibiotics (amoxicillin and metronidazole) and paracetamol (n = 4, 5.3%). a total of 63 (82.9%) patients complied with the post - extraction regimen, giving a significant high compliance to the post - extraction instructions (p = 0.001). there were 10 (13.2%) cases of post - extraction localised alveolar osteitis, with predilection for the lower molar teeth (n = 6, 7.9%) and a significant predilection for females (n = 8, 10.5%) [p = 0.005 ]. overall, there were five (6.6%) cases each of localised alveolar osteitis in the compliant patients (n = 63, 82.9%) and non - compliant patients (n = 13, 17.1%), giving a ratio of 1:13 and 1:3, respectively. there was significant association of compliance with post - extraction instruction and the reduced incidence of localized alveolar osteitis (p = 0.015).conclusion : this study showed a significant patients compliance with post - extraction warm saline rinse, prophylactic antibiotics and analgesic and a corresponding significant reduction in the incidence of localised alveolar osteitis following intra - alveolar molar tooth extraction. this study emphasises the need to properly educate patients on the effect of compliance to various combination of post - extraction regimen. |
insulin resistance (ir) is currently one of the most important metabolic risk factors associated with cardiovascular disease, type 2 diabetes mellitus (t2 dm), and some phenotypes of metabolic syndrome (ms). as of today, several factors have been related to the progressive loss of tissue - targeted insulin effects including lifestyle behavior, environmental factors, prenatal reprogramming, nutritional patterns, physical activity, and ethnicity. in spite of its importance during pathogenesis and amplification of disease, there is still controversy regarding which ir evaluation method to apply. the gold standard for ir estimation is the hyperinsulinemic - euglycemic clamp technique proposed by defronzo., albeit several limitations when applied to larger populations, such as technical difficulties and high cost, rendered it unviable. therefore, mathematical models have been devised to measure ir in manner comparable to the hyperglycemic - euglycemic clamp. one of such models is the homeostasis model assessment, first published by matthews. in 1985, which proposed the original homa equation (fasting glucose fasting insulin/22.5), and a recalibrated formula by 1998 labeling it homa2-ir. this new model offered several advantages including the calculation of % sensitivity and % beta - cell, using not only fasting levels of glycemia and insulin but also peptide c. in 2004, the university of oxford launched the homa2 calculator, free software which renders a more precise and fast calculation of homa2-ir, determining insulin sensibility and beta - cell function within a range of 1300 ui / l for insulin and 20460 mg / dl for glucose, adjusting this model for hyperinsulinemic or hyperglycemic conditions, hepatic and peripheral ir, and circulating proinsulin [11, 13, 14 ]. although the homa model has been extensively used, a worldwide cut - off point has not been established, albeit its prerequisite has been strained by the mandatory requirement of population and ethnic specific cut - off points for metabolic indicators, homa - ir included. therefore, 2 approaches have been proposed to determine homa - ir cut - off points. the first one uses a certain percentile such as the 95th or the 75th as recommended by reaven in the first annual world congress on the insulin resistance syndrome. the second approach relies on construction of roc curves derived from a specific population in order to select a valid cut - off point according to sensitivity, specificity, and other indexes. given the importance of ir quantification during the evaluation of ms components in large populations studies and its requirement in obesity - burdened, physically inactive, and inflammation - prone communities such as ours, the purpose of this study is to determine the appropriate cut - off point for homa2-ir in a representative population sample from the city of maracaibo, venezuela. the sampling method was already published in the maracaibo city metabolic syndrome prevalence study cross - sectional proposal. briefly, population estimations for maracaibo city (the second largest city of venezuela) from the national institute of statistics were used (1,428,043 by 2007), the sample was calculated to be 1,986 individuals and the overall number of individuals was 2,230. a total of 244 subjects (12%) were added for oversampling, in order to increase accuracy of the estimates obtained from smaller subgroups from the overall sample [23, 24 ]. the city of maracaibo is divided into parishes and each of these was proportionally sampled in a multistage cluster method ; during the first stage, the cluster was represented by sectors from each of the 18 parishes, finally selecting 4 from each parish by simple random sample. in the second phase, the clusters were represented by city blocks within the sectors, in which they were selected by simple random sample using a random number generation tool. from an overall population, for the determination of the homa2-ir cut - off point, a reference population of 602 healthy individuals was selected based on the exclusion of subjects with the following conditions : obesity, ms, hypertension, type 1 diabetes mellitus, thyroid or hepatic disease, coronary artery disease, heart rhythm disorders or cerebrovascular disease, polycystic ovary syndrome, and consumption of medication which may influence fasting blood glucose or lipid profile. all the individuals enrolled in the study signed a written consent before physical examination and anamnesis and all procedures were approved by the ethics committee of the endocrine and metabolic diseases research center of the university of zulia, maracaibo, venezuela. the assessment of blood pressure was done using a calibrated mercury sphygmomanometer, with the patient previously rested (for a minimum of 15 minutes) in a sitting position with both feet touching the floor. the arm was positioned at heart level, and a properly sized cuff was used for the procedure. systolic blood pressure was determined at the first korotkoff sound, whereas diastolic blood pressure was determined at the fifth korotkoff sound. blood pressure values were determined twice, with an interval of at least 15 minutes, and the results were averaged. blood pressure classification was completed using the criteria proposed in the vii joint national committee (jnc-7). weight was assessed using a digital scale (tanita, tbf-310 gs body composition analyzer, tokyo, japan), while height was obtained with a calibrated rod ; the subjects were barefooted and wearing light clothing at all times. body mass index formula was applied to all individuals (weight / height) and categorization was done using the who classification. waist circumference (wc) was measured using calibrated measuring tape in accordance with the anatomical landmarks proposed by the usa national institutes of health protocol. overnight fasting determination of glucose, total cholesterol, triglycerides, and hdl - c was done with an automated analyzer (human gesellschaft fr biochemica und diagnostica mbh, germany) ; the intra - assay variation coefficient for the total cholesterol, tag, and hdl - c was 3%, 5%, and 5%, respectively. ldl - c and vldl - c levels were calculated applying the friedewald formula only if triglycerides were below 400 mg / dl ; if they were above the mentioned cut - off point, ldl - c concentrations were measured through lipoprotein electrophoresis and densitometry with a biorad gs-800 (biorad). insulin was determined using an ultrasensitive elisa double - sandwich method (drg instruments gmbh, germany, inc.). qualitative variables were expressed as absolute and relative frequencies, considering the results statistically significant when p < 0.05 in either the z test for proportions or the test when applied. no normally distributed quantitative variables were subjected to logarithmic transformation observing a normal distribution after geary test ; results were expressed as mean standard deviation. to determine differences between means, student 's t - test was applied (when comparing two groups) or one - way anova (when comparing three or more groups) complemented with the post hoc tukey test. data were analyzed using the statistical software for social sciences (spss version 20 for windows, chicago, il, usa). homa2-ir was calculated using the software supplied by the oxford centre for diabetes endocrinology and metabolism available at http://www.dtu.ox.ac.uk/homacalculator/index.php. in order to determine a proper cut - off point, a reference population was selected, of which the primary results were distributed in percentiles (5 - 25 - 50 - 75 - 95), with the 75th percentile chosen as the cut - off for homa2-ir based on recommendations by reaven. to further ascertain the homa2-ir cut - off, a receiving operating characteristic curve was constructed based on the aforementioned reference population and a selected metabolically unhealthy population of 379 subjects. this sick population was comprised of subjects complying with either or both of the following inclusion criteria : presence of obesity and presence of ms. these criteria yielded a preliminary group of 457 subjects, which was reduced to final sick sample of 379 after exclusion of subjects currently consuming medication which may influence glycemic or lipid profiles (such as hypoglycemic agents, insulin sensitizers, insulin, beta - blockers, or hydrochlorothiazide), as shown in figure 1. three separate roc curves were constructed, one for females, one for males, and one for merging both genders. the comparison between auc by sex was assessed using delong 's test. to establish the optimal cut - off for homa2-ir, the following indexes were used : youden 's index, the distance closest to roc (0.1), and positive likelihood ratio. a total of 2,026 subjects were studied, 52.1% of whom were female (n = 1056) and 47.9% were male (n = 970). overall arithmetic mean for homa2-ir was 2.21 1.42, with 2.18 1.37 and 2.23 1.47 for women and men, respectively ; p = 0.466. significant differences were observed between groups aged 2029 and 4049 years (2.03 1.34 versus 2.35 1.46, resp. ; p = 0.012), as well as 2029 and 5059 years (2.03 1.34 versus 2.34 1.44, resp. displays homa2-ir means according to gender, whereas figure 3(b) depicts these values by gender and age groups. statistical differences between genders were found within the groups aged 2029 years (women 2.25 1.48 versus men 1.87 1.20 ; p = 0.001) and 4049 years (women 2.19 1.36 versus men 2.57 1.56 ; p = 0.009). indeed, women with low weight had homa2-ir values of 1.53 0.92, while obese class iii females had 2.97 1.79. similarly, men with low weight had 1.03 0.44, while the obese class iii had 4.41 2.17. table 2 shows the p values for arithmetic mean bmi categories comparisons between men and women. when comparing homa2-ir means between females and males, differences were found within the normal weight category, where women obtained the highest results (1.51 0.96 versus 1.79 10.88, resp. ; p = 3.76 10), and within the obese class iii category, where the men had higher values (4.41 2.17 versus 2.97 1.79 ; p = 0.001). figure 5 exhibits homa2-ir according to wc quartiles for men and women, observing a progressive increase along the categories, with 1.81 0.99 in the 1st quartile and 2.86 1.90 in the 4th quartile for women and 1.48 0.88 for the 1st quartile and 3.10 1.56 for 4th quartile. differences between genders were found within the 1st quartile (p = 3.99 10) and 4th quartile (p = 0.017). the selected reference population (n = 602) were constituted by a healthy group of 301 women (48.6%) and 318 men (51.4%). following recommendations by reaven who proposed the p75th for determining homa2-ir cut - offs a preliminary value of 2.00 was selected for both men and women. when assessing by gender, the p75th value for women was 2.10, whereas men showed a p75th of 1.90. percentile distribution of homa2-ir values in reference population is shown in table 4. in order to further explore homa2-ir cut - off determination, the roc curve based on both males and females rendered a cut - off value of 1.95 (auc 0.801), with 75.3% sensitivity and 72.8% specificity. figure 6 shows the resulting roc curve for women, with a cut - off point of 1.95 (auc 0.748) with 72.5% sensitivity and 67.7% specificity ; the roc curve for men rendered a cut - off point of 1.95 (auc 0.846) with 77.9% sensitivity and 77.3% specificity. delong 's test shows nonsignificant differences between the auc of roc curves for men and women ; p = 0.265. based on the values of sensitivity and specificity, 1.95 was selected as the best homa2-ir cut - off value (table 5). as previously stated, ir has been associated with several metabolic disorders, including cardiovascular disease, t2 dm, ms, metabolic reprogramming during fetal life, and physical inactivity. such role has been fundamental in order to promote knowledge concerning pathogenesis of such diseases and to properly choose potential pharmacological targets to manage them. the current gold standard for the evaluation of insulin sensitivity is the glucose clamp technique. the latest methods are mathematical in concept, and one of them is the homa - ir equation proposed by matthews. and its upgraded version, homa2-ir, published by levy.. homa2-ir has been validated for latin american populations as seen in the brams project from brazil, a multicentric study which showed both homa - ir and homa2-ir to be applicable in epidemiological vigilance for ms and ir, with cut - off points of 2.3 for homa - ir and 1.4 for homa2-ir. moreover, garmendia. reported a homa - ir cut - off of 2.6 for elderly chilean subjects and buccini and wolftbal reported a homa - ir cut - off point of 2.64 and finally a 1.67 cut - point for homa2-ir in a small argentinean cohort (n = 208). despite the importance of ir in the development, progression, and end - organ damage in ms, t2 dm, and their comorbidities, there is no consensus regarding optimal cut - off values, particularly in our country. therefore, the purpose of this investigation was to determine an appropriate cut - off point for homa2-ir using roc curves. this approach in data analysis requires determination of suitable populations to serve as reference or control / healthy individuals, while the remaining individuals were sorted to obtain an appropriate sick population. both of these components are primary materials in the construction of the roc curve and the selection of cut - offs (19,32). the selection of the cut - off point for homa2-ir was performed through two approaches : (a) selection of p75 values, as recommended by reaven, and (b) construction of roc curves in a reference population. first, according to the percentile distribution of homa2-ir from the reference sample (n = 602), the resulting p75 was 2.00. then, after constructing roc curves, the selected cut - point was 1.95 with corresponding sensitivity of 71.8% and specificity of 77.8%. interestingly, these approaches rendered similar cut - offs, confirming and supporting one another, suggesting that reaven was right in recommending the p75 values as reference. when comparing our results to those from brazil, chile, and argentina, our cut - point is 0.30.6 points higher, which can be ascribed to sociodemographic and nutritional differences inherent to these populations. in effect, despite a tendency towards growing obesity prevalence currently entailing all of latin america, obesity figures appear to be higher in our country than in the other aforementioned territories, reinforcing the need for local intervals to evaluate insulin sensitivity. indeed, proper evaluation of cardiometabolic risk factors such as ir through the homa2-ir equation is one of the most important tools when assessing epidemiologic risk in a population, particularly in ours, which boasts alarming figures such as 68.1% of elevated bmi (25 kg / m) and 42.4% prevalence of ms. the san antonio heart study, one of the largest prospective studies undertaken in the united states, comparing cardiovascular risk factors in mexican - americans and non - hispanic whites, has reported that cardiovascular events increase as homa - ir quintiles elevate as well, even after adjustment for age, sex, and ethnic group resulting in an or of 2.52 (95% ci 1.464.36, p < 0.0001). these results are similar to those obtained from the verona diabetes complications study, which published that homa - ir is an independent predictor of cardiovascular disease in t2 dm subjects, shedding light once more on the imminent need for proper diagnosis and management of insulin resistance in primary and secondary prevention. at first glance, ir shows an increasing tendency according to age, with the highest peak found at midlife (figure 2) and predominantly in men over women (figure 3). interestingly, within the group aged 2029 years, women obtained higher ir values than men, related to higher levels of physical inactivity, which coincides with previous findings in our locality. moreover, ir increases as bmi and wc rise, being higher in women within the normal weight category, whereas it was higher in men within the obesity class iii category. these results demonstrate that ir in normal weight subjects is higher for women ; and in obese groups, ir is higher for men. this dichotomy could be attributed to visceral adipose tissue quality variance and adipose distribution. as indicated by previous research, the population of maracaibo has an alarmingly elevated prevalence of obesity, with 33.3% of the sample classified as obese and 34.8% as overweight, associated with 59.06% prevalence of physical inactivity and significant low grade inflammation. insulin resistance states have been associated with oxidized low - density lipoproteins in latino individuals, elevated levels of apob, and higher lipoprotein insulin resistance index suggesting association with lipoprotein particle size and cardiovascular risk and vascular markers of inflammation. moreover, vella. reported that insulin resistance surrogates, such as homa - ir, were associated with cardiovascular disease risk in hispanic normal weight women ; in this regard, our team previously published that as homa2-ir increased, so did cardiovascular risk calculated with a correction of the framingham - wilson equation, being highest in insulin resistant subjects. last, yet equally important, is the fact that it has been suggested that amerindian descendants have higher homa - ir indexes, which would suggest that all latino populations would have different ir results due to ethnicity influences, enhancing its role as a nonmodifiable cardiovascular risk factor. the selection of an appropriate population - specific cutoff is of great importance, not only because it enhances accuracy of diagnosis but also because it is adapted to the socioeconomic and genetic factors, especially when results are bound to be compared with other countries. as a matter of fact, our cut - off points are different than those found in other latino countries such as argentina, a country that also has a very unique genetic admixture. if genetics influences are as important as it would seem to be, then all metabolic variables and anthropometric measurements must be selected according to ethnicity and population [44, 46 ], validating the need for studies such as this one. in conclusion, we propose an optimal cut - off value of 2.00 for homa2-ir for the evaluation of ir by this mathematical method. this interval offers great sensitivity and specificity, sufficient for proper assessment of ir in the adult population of maracaibo. population - specific reference values are required for accurate risk assessment and preventive planning in regard to public health problems such as obesity, t2 dm, ms, and cardiovascular disease. | background. mathematical models such as homeostasis model assessment have gained popularity in the evaluation of insulin resistance (ir). the purpose of this study was to estimate the optimal cut - off point for homeostasis model assessment-2 insulin resistance (homa2-ir) in an adult population of maracaibo, venezuela. methods. descriptive, cross - sectional study with randomized, multistaged sampling included 2,026 adult individuals. ir was evaluated through homa2-ir calculation in 602 metabolically healthy individuals. for cut - off point estimation, two approaches were applied : homa2-ir percentile distribution and construction of roc curves using sensitivity and specificity for selection. results. homa2-ir arithmetic mean for the general population was 2.21 1.42, with 2.18 1.37 for women and 2.23 1.47 for men (p = 0.466). when calculating homa2-ir for the healthy reference population, the resulting p75 was 2.00. using roc curves, the selected cut - off point was 1.95, with an area under the curve of 0.801, sensibility of 75.3%, and specificity of 72.8%. conclusions. we propose an optimal cut - off point of 2.00 for homa2-ir, offering high sensitivity and specificity, sufficient for proper assessment of ir in the adult population of our city, maracaibo. the determination of population - specific cut - off points is needed to evaluate risk for public health problems, such as obesity and metabolic syndrome. |
acute bronchiolitis is a common seasonal lower respiratory tract infection (lrti) and a major cause of respiratory morbidity in the first year of life [13 ]. respiratory syncytial virus (rsv) is the most prevalent pathogen, responsible for up to 75% of bronchiolitis cases in infants during the epidemic season [1, 46 ]. it usually affects infants aged 80% of admissions involving infants aged < 6 months. however, certain groups are at a higher risk of severe disease requiring hospitalization [7, 9 ]. a large retrospective cohort study of all children aged <3 years hospitalized for rsv infections and enrolled in the tennessee medicaid program from july 1989 to june 1993 showed that rsv hospitalizations in the first 6 months of life occurred in 44/100 child - years in healthy infants and increased to 8094/100 child - years in infants born prematurely ; the hospitalization rates were directly correlated with decreasing gestational age, thus indicating that prematurity per se was associated with increased severity of rsv infections. the severity of rsv bronchiolitis is likely to be determined by a complex interplay between viral and host factors [3, 11 ]. independent clinical risk factors for severe rsv bronchiolitis and hospital admission in paediatric intensive care units (picus) comprise : young age at onset of the rsv season [1214 ] ; lower gestational age, especially birth at <33 weeks ' gestational age (wga) [7, 1218 ] ; history of neonatal respiratory distress syndrome [1618 ] ; low birthweight [11, 13 ] ; chronic lung disease [7, 10, 13, 15, 16 ] and congenital heart disease [7, 14, 17 ]. environmental individual risk factors promoting cross - infection especially crowding due to living with siblings and day - care attendance have been shown to correlate with a higher risk of severe bronchiolitis. however, the role of such factors is highly dependent on the geographical region as well as on socio - cultural peculiarities, and therefore their evaluation in a given setting is crucial for the accurate identification of specific populations at higher risk of severe disease. in france there are no prospective epidemiological data on the risk of hospitalization for bronchiolitis (rsv and all types) in very premature infants without bronchopulmonary (bpd), those with bpd being unanimously accepted as being at high risk for severe disease ; moreover, the country - specific environmental risk factors for rsv hospitalization in this population have not been previously explored. in this context, the castor study (comparison of the rate of hospitalization for rsv bronchiolitis between preterm infants born at 32 weeks ' gestational age or less without bronchopulmonary dysplasia and full - term infants) aimed to compare the hospitalization rates for severe rsv bronchiolitis in very preterm infants (<33 wga) without bpd vs. matched full - term infants, all aged < 6 months at the beginning of the 20082009 rsv season in france, and to evaluate the risk factors for severe disease in this population. a multicentre, ambispective, longitudinal, non - interventional cohort study was conducted in nine french regional perinatal networks. participating paediatricians and neonatologists were invited to identify and record in a registry all the preterm infants <33 wga without bpd born during the 6 months preceding the 20082009 rsv season in all the maternity units (public and private) belonging to the nine french regional perinatal networks participating in the study. an equal number of full - term infants (3941 wga) were recruited in these maternity units, and matched to preterm infants according to age (1 month), gender and birth region. to allow strong comparisons on follow - up, participating physicians were invited to include in the registry two full - term infants for each preterm infant. because the start date of the 20082009 rsv season (i.e. 29 september 2008) was determined during the course of the study, the birth period of the infants was extended from 1 may 2008 to 31 march 2008 in order to align with the inclusion criterion chronological age < 6 months at the beginning of the rsv season. the inclusions effectively started on 12 november 2008, and extended up to 31 january 2009 due to certain recruitment difficulties for full - term infants. however, the investigated period was the entire 20082009 rsv season (29 september 2008 to 30 april 2009), as the data on bronchiolitis hospitalizations from the beginning of the rsv season to inclusion date were collected retrospectively, and those regarding hospitalizations from inclusion date to the end of the rsv season (30 april 2009) were collected prospectively. physicians were selected in type 2 (neonatal intermediate care units) or type 3 (neonatal intensive care units) neonatal hospital centres belonging to the nine regional perinatal networks participating the study. infants were eligible if their chronological age was < 6 months at the beginning of the 20082009 rsv season, their parents were able to understand french and agreed to take part in the study and to be contacted by phone. those born at <33 wga without bpd (defined according to study protocol as oxygen dependency at 28 days of life) were eligible as preterm infants, and those born between 39 and 41 wga as full - term infants. excluded infants were those receiving or having received specific rsv prophylaxis, and infants whose life expectancy was < 6 months, with known immune deficiency or a serious or chronic illness that may have an impact on their health status, including severe congenital heart disease. the physicians had to match each eligible preterm infant to a full - term infant, according to age (1 month), gender and geographical location of infant 's home or birth in order to ensure comparable sociodemographic characteristics. infants were included in the study if all inclusion criteria were fulfilled / non - inclusion criteria were not fulfilled, and written participation and phone contact informed consents signed by the parents were received. the participating physicians were free to prescribe, treat and follow - up the included infants according to their usual practice ; hospital admission and management for bronchiolitis were based on their medical experience, and their compliance with the corresponding french recommendations. according to the national agency for accreditation and evaluation in health (anaes), the term bronchiolitis covers all forms of viral obstructive bronchial disease occurring in epidemics in infants aged 124 months, manifesting as dyspnoea with tachypnoea, restricted expiration, chest hyperinflation and respiratory distress potentially interfering with feeding ; auscultation is dominated by crepitant or subcrepitant rales, rapidly followed by sibilant rales and wheezing. rsv testing in case of hospitalization for bronchiolitis was not mandatory, but recommended for epidemiological purposes. the primary endpoint was to compare hospitalization rates for rsv - confirmed bronchiolitis between the group of preterm infants <33 wga without bpd and the group of matched full - term infants followed - up during the 20082009 rsv season in france. secondary endpoints included the comparison of hospitalization rates for all types of bronchiolitis (rsv confirmed or not) between the two groups, description of the sociodemographic and medical characteristics of hospitalized infants, description of the therapeutic care of infants hospitalized for rsv - confirmed bronchiolitis in both groups of infants, and evaluation of the risk factors for bronchiolitis hospitalization in the french setting. data on bronchiolitis hospitalizations from the beginning of the investigated rsv season (i.e. 29 september 2008) to inclusion (i.e. 12 november 2008) were collected retrospectively ; those regarding the hospitalizations from inclusion to the end of the study (i.e. 30 april 2009) were collected prospectively. the data were collected from the medical files of eligible infants, and during the follow - up period until the end of the rsv season (30 april 2009) via information recorded by the investigators and parents. physicians had to complete for each included infant, at inclusion and at end of study, a case report form with all the medical data regarding the admissions for bronchiolitis during the follow - up period. the parents were contacted at inclusion to collect sociodemographic data and then monthly during the follow - up period to collect data regarding hospitalizations for bronchiolitis. a hospitalization diary helped parents to remember hospitalization episodes, and to identify the managing practitioners in order to obtain further information if necessary. between two phone contacts, parents were able to report any hospital admission for bronchiolitis through a freephone number. statistical analyses were performed using sas software, version 9.2 (sas institute, usa). for comparison of hospitalization rates between preterm and full - term infants, or fisher 's exact tests were used. a level of significance of 5% zero - inflated poisson (zip) models were used to further explore the risk of hospitalization for bronchiolitis. this model was retained in order to take into account the high estimated proportion of non - events, and applied to evaluate the risk of hospitalization for bronchiolitis in the overall study population. the following covariates were first included in a univariate zip model : chronological age at the beginning of the rsv season (< 60, 6089, 90119, 120149, 150 days), gestational age, gender, breastfeeding, geographical location, environmental risk factors, multiple pregnancy, and intrauterine growth restriction (defined as birthweight below the 10th percentile of the corresponding standard) ; all the covariates that were significant at the threshold of 25% were then entered in a zip multivariate model. the least significant covariate in a model was dropped from the model and the process continued until all remaining covariates were significant at 5%. of the 679 infants (427 preterm infants <33 wg and 252 full - term infants) entered in the initial study registry, 548 infants (297 preterm, 251 full - term) were included in the overall study population, and 498 infants (249 infants in each group) in the matched study population as shown in figure 2. similar characteristics at inclusion were found in the matched and the overall study population (data not shown) the mean age at birth for preterm infants was 314 09 wga ; 94% were born at 3032 wga, and none was born at < 28 wga. at their first bronchiolitis hospitalization, preterm infants were slightly younger (58 20 months) than full - term infants (60 16 months). table 1.sociodemographic characteristics at inclusion (matched population)variablespreterm infants (n = 249)full - term infants (n = 249)p valuedemographic characteristicsage at beginning of 20082009 rsv season, months28 1629 1607633age at inclusion, months59 1860 1807251genderboys128 (514)127 (510)09286girls121 (486)122 (490)multiple pregnancy95 (382)1 (04)<00001birth weight, g1588 3053399 428<00001birth height, cm405 27501 17<00001at least one sibling161 (647)144 (578)00804hospital stay at birthtype of birth centretype 3201 (807)165 (663)00006type 240 (161)76 (305)length of stay, days396 11649 61<00001hospitalization in neonatology services249 (100)8 (32)<00001results are presented as mean s.d. or n (%) when appropriate.distinct hospital units dedicated to the post - natal management of preterm infants and/or newborns with an altered health status. sociodemographic characteristics at inclusion (matched population) results are presented as mean s.d. or distinct hospital units dedicated to the post - natal management of preterm infants and/or newborns with an altered health status. regarding birth hospitalization, all preterm infants compared to only 32% of full - term infants were hospitalized in neonatology services (table 1), and significantly more preterm infants required ventilatory support compared to full - term infants (698% vs. 04%, p < 00001) as shown in table 2. table 2.types of ventilation required during birth hospitalization (matched population)type of ventilationpreterm infants (n = 249)full - term infants (n = 249)p valuemv only14 (56)0 (0)<00001niv ventilation only91 (365)1 (04)mv and/or niv69 (277)0 (00)none67 (269)232 (932)mv, mechanical ventilation ; niv, non - invasive ventilation.results are presented as n (%).missing data included in calculation of percentages, but excluded in the statistical testing. types of ventilation required during birth hospitalization (matched population) mv, mechanical ventilation ; niv, non - invasive ventilation. results are presented as n (%). missing data included in calculation of percentages, but excluded in the statistical testing. preterm infants were significantly more exposed than full - term infants to antenatal smoking, and received significantly less breastfeeding ; conversely, they had a lower attendance at day - care centres (table 3). table 3.environmental risk factors for bronchiolitis at inclusion (matched population)environmental risk factorspreterm infants (n = 245)full - term infants (n = 249)p valuesmoking status of the mother during pregnancy yes66 (269)45 (181)00178smoking status of the family since birth yes23 (94)30 (120)03841number of siblings11 1109 0900147breastfeeding yes125 (510)165 (663)00006type of child careday - care centre3 (12)23 (92)<00001child minder47 (192)69 (277)family193 (788)138 (554)other0 (00)19 (76)results are presented as mean s.d. or n (%) when appropriate.missing data included in calculation of percentages, but excluded in the statistical testing. environmental risk factors for bronchiolitis at inclusion (matched population) results are presented as mean s.d. or n (%) when appropriate. missing data included in calculation of percentages, but excluded in the statistical testing. during the 20082009 rsv season, 35 preterm and five full - term infants were admitted for bronchiolitis irrespective of the aetiology, accounting for 41 and nine hospitalizations, respectively, as shown in table 4. table 4.bronchiolitis hospitalizations and respiratory syncytial virus (rsv) testing (matched population)preterm infants (n = 249)full - term infants (n = 249)hospitalized infants, n355total number of rsv tests, n (%) 27 (771)4 (80)rsv - positive tests, n164rsv - negative tests, n110missing rsv tests, n81hospitalizations for bronchiolitis, n419total number of rsv tests, n (%) 33 (805)7 (778)rsv - positive tests, n174rsv - negative tests, n163missing rsv tests, n82 bronchiolitis hospitalizations and respiratory syncytial virus (rsv) testing (matched population) the hospitalization rate was significantly higher in the preterm than in the full - term group (141% vs. 20%, p < 00001) ; preterm infants had a sevenfold increased risk of hospitalization for all types of bronchiolitis compared to matched full - term group [95% confidence interval (ci) 2791757 ]. there was no difference in hospitalization rates for all types bronchiolitis between preterm infants requiring ventilatory support during birth hospitalization compared to their non - ventilated counterparts (149% vs. 119%, p = 05821). the majority of infants (902% of preterm, 889% of full - term) required non - medicinal care perfusion or parenteral nutrition (350%), enteral nutrition with stomach tube (260%), oxygen therapy (440%), mechanical ventilation (50%), non - invasive ventilation (125%) and respiratory physiotherapy (540%) and all recovered. the majority of hospitalizations for bronchiolitis were rsv - tested (nasopharyngeal aspirate and viral immunofluorescence) as shown in table 4. seven infants (five preterm, two full - term) were hospitalized more than once for bronchiolitis (rsv - positive / rsv - negative / not tested) during the studied season. hospitalization rates for rsv bronchiolitis were significantly higher in the preterm group than in the full - term group as presented in figure 3 ; preterm infants had a fourfold increased risk of hospitalization for rsv bronchiolitis compared to the matched full - term group (95% ci 1361180). 3.hospitalizations for bronchiolitis [respiratory syncytial virus (rsv) and in general ] during the 20082009 rsv season (matched population). relative risk 700 (95% ci 2791757) ; relative risk 400 (95% ci 1361180). two approaches were considered in the sensitivity analysis to handle missing rsv tests : high hypothesis imputed missing rsv tests as positive tests. crossed hypothesis added a number of adjusted rsv hospitalizations to each study group. hospitalizations for bronchiolitis [respiratory syncytial virus (rsv) and in general ] during the 20082009 rsv season (matched population). relative risk 700 (95% ci 2791757) ; relative risk 400 (95% ci 1361180). two approaches were considered in the sensitivity analysis to handle missing rsv tests : high hypothesis imputed missing rsv tests as positive tests. crossed hypothesis added a number of adjusted rsv hospitalizations to each study group. these results were further confirmed with sensitivity analyses to handle missing rsv tests using a high hypothesis, i.e. imputing missing rsv tests as positive tests, as well as a crossed hypothesis, i.e. by adding to each study group a number of rsv hospitalizations obtained by adjusting the number of missing rsv tests in the group to the percentage of rsv - positive tests in the other group (fig. similar results were found in the overall study population, with a hospitalization rate for rsv - confirmed bronchiolitis significantly higher in preterm compared to full - term infants (57% vs. 16%, p = 0012), corresponding to a relative risk for rsv bronchiolitis hospitalization of 359 (95% ci 1221054) for preterm compared to full - term infants. preterm infants with rsv - confirmed bronchiolitis were mainly hospitalized in general paediatric (882%) or in neonatology (118%) units ; only a few (59%) preterm infants were admitted to picus. preterm infants hospitalized with rsv - confirmed bronchiolitis had longer episodes and longer hospital stays compared to those hospitalized with rsv - negative bronchiolitis ; the mean duration of episodes was 95 34 days for rsv - positive vs. 68 26 days for rsv - negative (p = 00183) bronchiolitis, and the mean length of hospital stay was 72 33 days for rsv - confirmed cases vs. 55 27 days for rsv - negative cases (p = 01901). the same trend was observed in the full - term group, without any significant differences between rsv - positive and rsv - negative cases (data not shown). in the preterm group, bronchiolitis hospitalizations that were tested for rsv appeared to be more severe compared to the non - tested cases as the preterm infants in the tested group had longer hospital stays and needed more non - medicinal care than the non - tested group (table 5). table 5.characteristics of respiratory syncytial virus (rsv)-tested and non rsv - tested bronchiolitis hospitalizations (matched very preterm group)characteristics of bronchiolitis hospitalizationstested for rsv (n = 33)not tested for rsv (n = 8)length of hospital stay (days)mean (s.d.)64 (31)24 (16)median5020min max201300050non - medicinal therapeutic care (n, %) yes31 (939)6 (750)no2 (61)2 (250)type of care (n, %) perfusion or parenteral nutrition11 (333)enteral nutrition with stomach tube10 (303)3 (375)oxygen therapy18 (545)1 (125)assisted breathing1 (30)non - invasive ventilation5 (152)respiratory physiotherapy19 (576)3 (375)for each hospitalization, several types of care could have been required. characteristics of respiratory syncytial virus (rsv)-tested and non rsv - tested bronchiolitis hospitalizations (matched very preterm group) for each hospitalization, several types of care could have been required. the multivariate zip model applied to explore the risk of hospitalization for bronchiolitis demonstrated a good fit to the data, and in particular did not show any overdispersion. the only explanatory variable kept in the logistic model component of the zip model was gestational age, with a positive parameter estimate (parameter estimate 259), indicating that full - term infants had a greater chance of not being hospitalized for bronchiolitis than preterm infants <33 wga. the poisson model component of the zip model, which reflects the risk of multiple hospitalizations, included gender of infant and having one or more siblings aged 2 years : the parameter estimates for both female infants and infants without siblings aged 2 years was lower than 0 indicating a lower risk of multiple hospitalizations for bronchiolitis in these infants (table 6). table 6.results of the final zero - inflated poisson (zip) model in the overall population (n = 548) : parameter estimates of both logistic model and poisson model component of the zip modelparameterestimate95% cip valuelogistic model componentintercept006121 to 11009253preterm / full - term infants (ref. results of the final zero - inflated poisson (zip) model in the overall population (n = 548) : parameter estimates of both logistic model and poisson model component of the zip model ci, confidence interval. the french castor study showed that very preterm infants (<33 wga) without bpd had a fourfold increased risk of hospitalization for rsv - confirmed bronchiolitis compared to matched full - term infants, all aged < 6 months at the beginning of the 20082009 rsv season, and confirmed these data in the overall study population. rsv testing using nasal swabs, recommended but not mandatory, was performed in nearly 80% of hospitalizations. rsv - positive bronchiolitis was found in one out of every two rsv - tested cases, and estimated in up to 725% of hospitalized bronchiolitis cases when considering a high hypothesis to count missing tests as rsv - positive. the decision to perform a rsv test on admission for bronchiolitis seemed most probably related to the severity of disease as tested cases had longer hospital stays and required more non - medicinal therapeutic care compared to non - tested cases. these results are consistent with the spanish data issued from a study conducted by the iris study group, where rsv testing was performed in 75% of admissions in preterm infants (<33 wga), with 66% of cases being rsv - positive. a recent literature review of the diagnosis and testing in bronchiolitis concluded that rsv testing, although commonly used to document the cause of bronchiolitis, rarely changes the clinical management or the outcomes of hospitalized infants. in their review on bronchiolitis, smyth & openshaw stated that there is no good evidence on the effectiveness of this approach, despite of the fact that many hospitals require routine rsv testing to allow infected infants to be cohorted together in order to reduce the risk of cross - infection. in this context of heterogeneous rsv detection practices, it appears important to evaluate the hospitalization rates for all types of bronchiolitis, rsv confirmed or not. all preterm infants included in the castor study were hospitalized in neonatology units after birth compared to only 3% of matched full - term infants ; moreover, the duration of birth hospitalization was nine times longer in the preterm group, reflecting an increased need for postnatal care. it should be noted that the recruitment of full - term infants was performed in type 3 centres, therefore favouring the inclusion of possibly more fragile infants, which could have diminished the observed differences in the risk of hospitalization for bronchiolitis between the very preterm group compared to the full - term group. the comparison of the environmental risk factors for bronchiolitis between preterm vs. full - term infants hospitalized for bronchiolitis in our study showed a higher prevalence of multiple pregnancies and antenatal smoking exposure in the preterm group ; this finding is consistent with previous international reports [7, 14, 23, 24 ]. however, the relatively small number of infants hospitalized for bronchiolitis in both groups could explain the lack of significant correlation with the risk of being hospitalized and/or of having multiple hospitalizations observed via the multivariate zip model in the present study. regarding child care, the protective attitude of french parents towards their preterm infants could have limited the bronchiolitis hospitalization rates in the preterm group of the present study, as these infants were mostly kept within the family, and therefore less exposed to viral cross - contamination. nevertheless, very preterm infants without bpd included in the castor study were four times more at risk of being hospitalized for rsv - confirmed bronchiolitis compared to their full - term counterparts. this finding is in line with data from the retrospective analysis by boyce., which found a 33-fold increased risk of hospitalization for rsv bronchiolitis (95% ci 2347) in preterm infants (29 to <33 wga) aged 612 months compared to full - term infants. comparable rsv hospitalization rates have been reported by stevens. based on the analysis of a large us retrospective cohort of 1029 very preterm infants (32 wga), of which 44% of those born at 3032 wga conversely, much higher hospitalization rates for respiratory disease have been reported by cunningham. in a cohort including 133 infants born at <33 wga that were discharged from the nicu department of a tertiary - care facility serving an 18-county region in central new york state. in this cohort, preterm infants aged <33 wga without bpd had a hospitalization rate of 25% vs. 141% hospitalization for all types of bronchiolitis in the castor preterm group, while similar hospitalization rates were reported in full - term subgroups from both studies (25% and 2%, respectively) ; this difference could be due to the different evaluation end - points, i.e. hospitalization for respiratory disease in the us cohort compared to hospitalization for bronchiolitis only in the present study. recent data from a swiss prospective cohort of 462 children aged <3 years hospitalized in intermediate or intensive care units for a rsv - related illness showed that only 2% of the very preterm infants (<33 wga) without bpd were admitted to these services compared to 64% hospitalized for rsv - confirmed bronchiolitis in our study ; the same trend was observed in the full - term groups, with 01% hospitalization rate in the swiss cohort vs. 16% in the french castor study. these differences are possibly due to country - dependent medical practices and hospitalization guidelines. in spite of these specificities, the median length of admission for rsv - confirmed bronchiolitis in the preterm group of our study (5 days, range 213 days) is comparable, for example, to the median length of stay for rsv - bronchiolitis (7 days, range 59 days) reported by carbonell - estrany. in spanish prematurity of <33 wga increased the risk of being hospitalized for severe bronchiolitis, whether rsv - confirmed or all aetiologies, but had no significant impact on the number of hospitalizations. several factors, including pulmonary and immune system immaturity, are likely to predispose preterm infants to severe lrti [7, 9, 27 ]. it has been suggested that healthy infants born prematurely have smaller sized airways relative to their lung volume and therefore persistent lower pulmonary function compared to infants born at term [2830 ] ; furthermore, rsv infection in premature infants was associated with abnormal lung function at follow - up [22, 27 ]. the castor study also showed that, in the french setting, male gender and the presence of siblings aged 2 years are independent risk factors for multiple bronchiolitis hospitalizations [12, 15 ]. as rsv testing was not mandatory, hospitalization rates for rsv bronchiolitis could have been underestimated in both groups of infants. furthermore, we can not exclude that certain preterm infants were not included because of the presence of associated environmental risk factors generating the physician 's decision to administer specific rsv prophylaxis to prevent severe rsv infections. moreover, the high proportion (67%) of full - term infants recruited in type 3 centres might have determined an inclusion bias by favouring the inclusion of more fragile full - term infants, thus leading to a potential overestimation of the bronchiolitis hospitalization rate in this group. all these limitations would probably diminish the observed differences in the hospitalization rates for bronchiolitis, rsv - confirmed and all types, between very preterm infants without bpd and their full - term counterparts ; in spite of this, our study revealed important differences between the cases and the controls. the castor study highlighted that, in the french setting, very preterm infants (<33 wga) without bpd are at significantly increased risk of hospitalization for severe bronchiolitis, rsv - confirmed and all aetiologies compared to matched full - term infants. the preterm group was also prone to more severe disease, as suggested by longer hospital stays and the requirement of more therapeutic care compared to the full - term group. moreover, we showed that in france, prematurity <33 wga was the only factor that significantly increased the risk of being hospitalized for bronchiolitis, and that the risk of multiple hospitalizations for bronchiolitis in the same infant significantly increased with male gender and the presence of siblings aged 2 years. as rsv bronchiolitis is an important healthcare issue, leading to significant morbidity in preterm infants with or without associated bpd, larger prospective studies are clearly needed to further explore the specific risk factors and outcomes in this high - risk population. this approach could improve the identification of those infants most likely to develop severe disease, and contribute to the improvement of their healthcare in the future. | summarythis study was conducted during the 20082009 respiratory syncytial virus (rsv) season in france to compare hospitalization rates for bronchiolitis (rsv - confirmed and all types) between very preterm infants (<33 weeks ' gestational age, wga) without bronchopulmonary dysplasia and full - term infants (3941 wga) matched for date of birth, gender and birth location, and to evaluate the country - specific risk factors for bronchiolitis hospitalization. data on hospitalizations were collected both retrospectively and prospectively for 498 matched infants (249 per group) aged < 6 months at the beginning of the rsv season. compared to full - term infants, preterm infants had a fourfold [95% confidence interval (ci) 1361180 ] and a sevenfold (95% ci 2791757) higher risk of being hospitalized for bronchiolitis, rsv - confirmed and all types, respectively. prematurity was the only factor that significantly increased the risk of being hospitalized for bronchiolitis. the risk of multiple hospitalizations for bronchiolitis in the same infant significantly increased with male gender and the presence of siblings aged 2 years. |
because life expectancy has increased worldwide in recent years, a considerable increase has occurred in the incidence of proximal femoral fractures complications with peritrochanteric fractures arise primarily from fixation rather than union or delayed union because the peritrochanteric area is made up of spongious bones. the aim of the surgery is to achieve early mobilization and to quickly return the patient to pre - surgery activity levels. various implants have been designed to facilitate fracture fixation, obtain early ambulation, and reduce the risk of complications in the treatment for peritrochanteric fractures [5, 6 ]. these implants can be divided into two groups : intramedullary and extramedullary [59 ]. to achieve rotational and angular stability, in 2004, the proximal femoral nail antirotation (pfna) device, one of the third - generation intramedullary implants (pfna ; synthes oberdorf, switzerland), was developed by the ao / asif group. pfna blades have been biomechanically proven to compact cancellous bone and achieve increased stability and thus to delay rotation and varus collapse. biomechanical tests have also indicated significantly higher cutout resistance in osteoporotic bone compared to other widely used screw systems [6, 1012 ]. the sliding hip screw has become the most widely used extramedullary implant in the treatment for hip fractures [6, 13, 14 ]. some investigators, however, have reported that this implant is not proper for unstable fractures, and these investigators have supported various alternative methods of fixation for these more difficult types of fractures [14, 15 ]. the dcs, an implant of extramedullary fixation, which was modified from the 95 fixed - angle plate by the ao / asif group, is much easier to apply in that location because its screw is cannulated [16, 17 ]. the purpose of this retrospective study was to compare the results of the dcs and the pfna in the treatment for unstable peritrochanteric fractures in patients older than 60. this observational study enrolled patients who were treated in our hospital with the dcs and the pfna for peritrochanteric fractures between january 2007 and december 2010. the inclusion criteria were radiologically diagnosed unstable peritrochanteric fractures (31-a2 and -a3 for ao / asif classification), age older than 60 years old, and an american society of anesthesiologists (asa) score of 14. the exclusion criteria were pathologic fractures, poor ambulation before the trauma, polytrauma, and severe concomitant medical conditions (asa 5). the patients underwent surgery 410 days (mean, 6 days) after admission. intramedullary fixation with the pfna system (synthes oberdorf, switzerland) was implemented in group a (n = 42). this group was composed of 42 patients with peritrochanteric fractures (ao classification : 31-a2 in 23 and 31-a3 in 19). group b (n = 37) underwent extramedullary fixation with the dcs system (synthes oberdorf, switzerland). this group consisted of 37 patients with peritrochanteric fractures (ao classification : 31-a2 in 21 and 31-a3 in 16). for all of the patients, background variables, including age, gender, associated comorbidities, and mechanism of trauma, were recorded. the number of units of blood transfused intraoperatively and postoperatively was recorded in each group (hct 10 varus / valgus and/or anteversion / retroversion). rehabilitation was started as early as possible after surgery, and the patients were allowed to bear as much weight as they could tolerate. all of the patients were regularly examined physically and radiographically after 6 weeks and at 3, 6, and 12 months after their operations. radiographs of the operated hip were obtained at each follow - up visit, and the position of the implant and extent of fracture union were noted. student s t tests were used to compare the two groups with regard to mean age, surgical time, mean follow - up duration, units of blood transfused, partial weight - bearing time, salvati wilson hip score, and consolidation time. analyses were performed to compare the groups with regard to gender, side of injury, mechanism of injury, associated comorbidities, ao fracture classification, mortality at 1-year follow - up, fracture reduction quality, and complications. student s t tests were used to compare the two groups with regard to mean age, surgical time, mean follow - up duration, units of blood transfused, partial weight - bearing time, salvati wilson hip score, and consolidation time. analyses were performed to compare the groups with regard to gender, side of injury, mechanism of injury, associated comorbidities, ao fracture classification, mortality at 1-year follow - up, fracture reduction quality, and complications. the etiological reasons, in order of incidence, for treatment were falls and traffic accidents, and fall frequency was not statistically significant (p = 0.39). the mean surgical time for patients treated with pfna was 57 min (range 3296 min) and was significantly lower than in those treated with dcs, in which the mean time was 87 (range 64178) min (p < 0.05). the mean salvati wilson hip score on final evaluation was 31 in the pfna group, and in the dcs group, it was 26 (p < 0.05). in the group treated with the pfna, the time for consolidation was significantly shorter compared to the dcs group (p < 0.05). in terms of associated comorbidities, no significant differences were seen between the two groups (p = 0.67) (table 1).table 1comparison of the main characteristics of the patients included in the study and the outcomes obtained using the pfna and the dcs devicespfna(group a ; n = 42)dcs(group b ; n = 37) p valuesgender : male / female17/2518/190.46age (years) : mean (range)77.02 7.8872.05 5.80<0.05side : right / left19/2317/200.95mechanism of injury simple fall at home34270.39 traffic accident810associated comorbidities hypertension1080.67 diabetes77 cardiovascular disease34 neurological disease33ao fracture classification a223210.85 a31916surgical time (min)57.69 17.4787.86 23.71<0.05mean follow - up period (months)20.67 5.3223.19 7.220.07blood transfused (units) (erythrocyte suspensions)0.21 0.421.78 1.08<0.05partial weight - bearing (days)7.28 3.9722,27 10.72<0.05mortality at 1-year follow - up460.37salvati wilson hip score (maximum points 40)31.04 4.6426.11 4.97<0.05consolidation time (weeks)15.71 5.4922.59 10.21<0.05 comparison of the main characteristics of the patients included in the study and the outcomes obtained using the pfna and the dcs devices fracture reduction was considered good or acceptable in 69 patients (37 pfna, 32 dcs) on postoperative radiographs. there were no significant differences between the quality of reduction for both implants and fracture types (p = 0.83) (table 2).table 2quality of fracture reduction and postoperative radiographic evaluationpfna (%) dcs (%) p valuefracture reduction quality (%) good73.970.20.83 acceptable14.216.3 poor11.913.5 quality of fracture reduction and postoperative radiographic evaluation the orthopedic and general postoperative complications are listed in table 3. no significant differences were seen between the two groups in terms of orthopedic or general complications (p = 0.10 and p = 0.57, respectively). the mortality rate at 1 year was 9.5 % in the pfna group, compared with 16.2 % in the dcs group. there was no statistically significant difference between the two groups in terms of the 1-year mortality rate (p = 0.37).table 3distribution of patients with complications according to the internal fixation devicespfna(n = 42, %) dcs(n = 37, %) p valuesorthopedic complications lateral migration of blade or screw210.1 cut - out21 nonunion01 infection13 implant failure02 reoperation24general complications symptomatic dvt120.57 decubitus01 pneumonia11 urinary infection21 distribution of patients with complications according to the internal fixation devices in group a, 37 patients experienced satisfactory reduction and fixation and were able to undertake early weight - bearing. in group b, 12 patients were able to bear weight early, although 32 patients experienced satisfactory reduction (figs. 1, 2).fig. 1seventy - six - year - old female patient sustaining an isolated and closed 31-a2 fracture at the left side (a) after a simple fall. postoperative x - ray after closed reduction and internal fixation with a pfna (b). final anteroposterior hip radiograph at the 12-month follow - up (c)fig. 2 a anteroposterior radiograph of the right hip of a 76-year - old female who fell in the street, revealing an 31-a3 peritrochanteric fracture. b post op radiographs after open reduction and extramedullary fixation with a dcs seventy - six - year - old female patient sustaining an isolated and closed 31-a2 fracture at the left side (a) after a simple fall. postoperative x - ray after closed reduction and internal fixation with a pfna (b). final anteroposterior hip radiograph at the 12-month follow - up (c) a anteroposterior radiograph of the right hip of a 76-year - old female who fell in the street, revealing an 31-a3 peritrochanteric fracture. failure rates due to complications are still considerable, although the implants and surgical techniques have improved greatly. the functional results might not be satisfactory because of failure to heal or failure of fixation, although a wide range of techniques are in use. sliding hip screws, as well as blade plates, dynamic condylar screws (dcs), and the formerly used intramedullary devices, have been found to be problematic. that intramedullary devices might be superior to plating systems in unstable proximal femoral fractures has been shown in biomechanical examinations. because it produces a small bending moment, the pfna system acts as an internal splint, and at the same time, it can bear a large axial load. along with this ability, the helical blade of the pfna system enhances its bone purchase in the femoral neck head. additionally, the blade prevents rotation or compaction of the proximal fragment by locking with the nail rotationally. these factors allow the patient to bear partial weight sooner after surgery [12, 21, 22 ]. another important advantage of the pfna technique is that it can be performed with minimal surgical invasion. lateral migration of proximal screws or helical blades is also a complication with this implant [11, 2325 ]. the dcs is an implant designed by the ao / asif group for use in proximal and distal femoral fractures. this device has been proved to have some technical advantages over the ao condylar blade plate. dcs plates provide the ability to produce a range, especially in the sagittal plan, of rotation of the proximal part of the lag screw. however, it is obvious that many complications have been observed after surgeries. the most important of these complications are devascularization, seen as a result of over - dissection, union delay, failure to unify, and infection [26, 27 ]. the deficiency and fatigue of the implant should also be considered [17, 28, 29 ]. in our study, it was observed that the time to partial weight - bearing on the associated extremity in the dcs group was significantly longer than in the pfna group (p < 0.05). in addition, the patients operated on with the dcs were more often advised to avoid sudden full weight - bearing, and because the fracture fixation was not considered stable enough, there was no satisfactory fracture impaction postoperatively [30, 31 ]. open reduction and intramedullary fixation have been suggested as the first choice, regardless of age, in intertrochanteric fractures in which the medial colon is intact. i m nails, with their biomechanical features, have come into prominence in some types of unstable peritrochanteric fractures [4, 22 ]. in a study by sadowski., ao type iii patients were examined, and it was found that implant deficiency occurred in one case in the pfna group. in our study, no significant differences between the two groups regarding orthopedic complications were detected. the dcs used in this technique was cheaper and more widely available in our country than other techniques. there are important stages in the technique, such as appropriate placement of the guide wire and slipping of the plate over the lag screw, and these stages can be simplified using the technique explained here. provided that the technique is performed correctly, the success rate is high. as such intramedullary fixation for proximal and distal femoral fracture fixations, although the pfna is the gold standard for the stabilization of the femoral neck and for most peritrochanteric fractures [11, 12 ]. the profits of intramedullary nailing are more commonly observed than with the extramedullary procedure, which often requires reoperation due to technical problems. in our series, femoral head or neck perforation the rates of femoral head perforation were found to be 1.4 % in a study by karapnar. and 1.2 % in a study by simmermaher., the rates of cutout were noted as 26.3 and 5 %, respectively. in our study, the reasons for cutout in the pfna group were related to technical failure. there are very few studies comparing intramedullary fixation with angular stable plates for the treatment for unstable fractures. as in many articles in the literature, sliding hip screw devices have been compared with the pfna in the treatment for all types of unstable intertrochanteric fractures [6, 8, 25 ]. in our study, there were limitations inherent in the methodology used because it was a retrospective, controlled study. the main objective of the management of elderly patients with peritrochanteric fractures is a successful return to safe mobility. in our study,. nevertheless, intraoperative parameters, such as simpler technique, minimal exposure, shorter surgical time, reduced blood loss, and postoperative functional parameters, demonstrated that the pfna is a more effective device for the management of peritrochanteric fractures, compared to the dcs. | purposethe aim of this study was to compare the results of intramedullary fixation with those of plate - screw fixation for peritrochanteric femoral fracture patients older than 60 years old.methodsthis article reports on a retrospective review of patients who had peritrochanteric femoral fractures and were treated with a 95 fixed - angle screw plate (dcs) or an intramedullary nailing system (pfna). patients with 79 fractures were enrolled in the study ; 47 of them were treated with the pfna system and 37 with the dcs. followed for at least 1 year, the treatment groups were compared by taking into consideration all demographic and trauma variables.resultsno significant differences were discovered between the two groups with regard to side of injury, mechanism of trauma, associated comorbidities, ao fracture classification, average follow - up duration, mortality, and fracture reduction quality at the 1-year follow - up. the average surgical time was significantly lower in the pfna group (57 min.) compared to the dcs group (87 min.). longer operative time was needed in the dcs group, and thus, greater blood loss occurred compared to the pfna group. the functional results of the pfna group were found to be significantly better than those of the dcs group.conclusionsowing to some advantages, such as minimal exposure, reduced operative blood loss, and the achievement of biological fixation, pfna is a better choice for the treatment for unstable peritrochanteric fractures. |
patients with signs suggestive of heart attack are instructed to call for an ambulance immediately after symptoms start. emergency medical services (ems) have the advantage of commencing important therapy such as aspirin, of alerting the emergency department and/or catheterization laboratory of a patient arriving with myocardial infarction (mi), and of being able to perform resuscitation should the mi portend cardiac arrest [2, 3 ]. still, studies show that only about 25 to 50% percent of chest pain patients call an ambulance when they have chest pain [4, 5 ]. it is known that patients with certain pathologies who present to the hospital via ems are more likely to be ill than their alternately transported counterparts. in trauma patients with headaches have been found to have a much higher likelihood of serious intracranial pathology when they present via ems. overall, patients with all complaints who come to the ed via ems are more likely to be acutely sick and severely injured than those who arrived by private transport. chest pain is a common complaint, with 6.4 million patients presenting to us eds each year. in order to prioritize patient evaluations, we wished to determine if patients who arrived to the ed via ems with undifferentiated chest pain were more likely to have a final diagnosis of mi than those who presented by alternate transportation. the study was conducted at a tertiary care, urban emergency department that sees approximately 39,000 adult and pediatric patients each year. midlevel providers supervised by these physicians include residents from internal medicine, surgery and gynecology, as well as physician assistants. the study was deemed exempt for formal review by our hospital s institutional review board. all adult patients ages 1899 who presented between 1 january 2006 and 7 july 2006 with a chief complaint that included chest pain were eligible for retrospective analysis. for patients with multiple visits, only the first visit for this complaint during the study period was included. patients who were transferred or who left without being seen or against medical advice were excluded. that is, all patients had detection of rise and/or fall of troponin i (with at least one value greater than our laboratory s reference range of 00.10 ng / ml) with evidence of myocardial ischemia being the symptom of chest pain. furthermore, patients who went urgently to cardiac catheterization and had at least one vessel with > 90% occlusion with a final clinical impression of myocardial infarction but in whom cardiac biomarkers were not followed were also considered to have suffered an mi. all emergency department records, including physician and nurse charts, are electronic in our department (edis, medhost, addison, tx). inpatient data are stored electronically as well (soarian, siemens medical solutions, malvern, pa). patients records were evaluated for mode of arrival, demographic information, co - morbidities, presentation vital signs, disposition and final diagnosis. data from the ed visit were extracted by one author (jtw) from the ed medical record. for all admitted patients, a second author (pb) flagged all patients with any documented elevated cardiac biomarkers and/or performance of cardiac catheterization from the inpatient medical record system. a third author (sgw) determined which of the remaining patients had suffered an acute mi based on the aforementioned criteria. all analyzed data were exported to a spreadsheet (excel, microsoft, redmond, wa) and analyzed with statistical analysis software (sas) version 9.0 (sas institute, inc., cary, nc). student s t - test was used to compare continuous variables, and chi - squared analysis was used to compare proportions. there were 690 visits for chest pain during the study period, representing 4% of the total ed census. a total of 39 visits were excluded because the patients left without being seen or were transferred. only the first chest pain visit for each individual during the study period was analyzed. in all the characteristics of the cohort are demonstrated in table 1. of the patients, 26.2% (157/599) arrived by ambulance ; 52.3% (313/599) of the patients were admitted to the hospital ; 5.8% (35/599) of the patients had a final diagnosis of myocardial infarction. of these patients, 32/35 (91.4%) had elevated troponin i with a clinical impression of mi. the remaining three patients went urgently to catheterization, had > 90% occlusion of at least one vessel, but their cardiac biomarkers were not followed. table 1 also demonstrates the comparisons between the ambulance and alternate transportation groups. table 1a comparison among the characteristics of the total cohort, and patients brought in by ambulance vs. alternate transportationntotal cohortambulance arrivalalternate transportpn = 599n = 157 (26.2%)n = 442 (73.8%)age(years, mean 95% ci, n)48.81.4 (599)51.92.7 (157)47.71.6 (442)p = 0.010sexp = 0.706female (%, n)44.6% (267)45.9% (72)44.1% (195)male (%, n)55.4% (332)54.1% (85)55.9% (247)racewhite (%, n)64.3% (385/599)66.9% (105/157)63.3% (280/442)p = 0.714black (%, n)18.0% (108/599)17.2% (27/157)18.3% (81/442)asian (%, n)8.9% (53/599)7.0% (11/157)9.5% (42/442)hispanic (%, n)6.0% (36/599)7.0% (11/157)5.7% (25/442)other (%, n)2.8% (17/599)1.0% (3/157)3.2% (14/157)insuranceprivate (%, n)41.7% (250/599)35.7% (56/157)43.9% (194/442)p = 0.038medicaid (%, n)25.4% (152/599)22.9% (36/157)26.2% (116/442)medicare (%, n)21.5% (129/599)29.3% (46/157)18.8% (83/442)self pay / other (%, n)11.4% (68/599)12.1% (19/157)11.1% (49/442)insuranceprivate / self / medicaid (%, n)78.4% (467/596)70.1% (108/154)81.2% (359/442)p = 0.004medicare (%, n)21.6% (129/596)29.9% (46/154)18.8% (83/442)time of arrival7:00 am-6:59 pm (%, n)70.6% (423/599)65.6% (103/157)72.4% (320/442)p = 0.1087:00 pm-6:59 am (%, n)29.4% (176/599)34.4% (54/157)27.6% (122/422)day of arrivalweekday (%, n)75.0% (449/599)79.6% (125/157)77.4% (342/442)p = 0.560weekend (%, n)25.0% (150/599)20.4% (32/157)22.6% (100/442)shortness of breathno (%, n)92.0% (551/599)94.9% (149/157)91.0% (402/442)p = 0.117yes (%, n)8.0% (48/599)5.1% (8/157)9.0% (40/442)history of cadno (%, n)76.3% (425/557)70.6% (101/143)78.3% (324/414)p = 0.064yes (%, n)23.7% (132/557)29.4% (42/143)21.7% (90/414)history of diabetesno (%, n)87.7% (490/559)81.9% (118/144)89.6% (372/415)p = 0.016yes (%, n)12.3% (69/559)18.1% (26/144)10.4% (43/415)history of hypertensionno (%, n)70.6% (397/562)61.0% (89/146)74.0% (308/416)p = 0.003yes (%, n)29.4% (165/562)39.0% (57/146)26.0% (108/416)history of hypercholesterolemiano (%, n)86.2% (483/560)83.3% (120/144)87.3% (363/416)p = 0.238yes (%, n)13.8% (77/560)16.7% (24/144)12.7% (53/416)smokerno (%, n)67.2% (379/564)67.6% (92/136)67.1% (287/428)p = 0.898yes (%, n)32.8% (185/564)32.4% (44/136)32.9% (141/428)systolic blood pressuremmhg, mean 95% ci, n134.51.7 (594)134.93.7 (154)134.32.0 (440)p = 0.802diastolic blood pressuremmhg, mean 95% ci, n75.01.2 (594)78.62.2 (154)73.71.4 (440)p < 0.001heart ratebpm, mean 95% ci, n83.01.5 (595)86.73.1 (154)81.71.7 (441)p = 0.004respiratory raterpm, mean 95% ci, n18.30.4 (573)18.51.2 (142)18.20.2 (431)p = 0.508temperaturec, mean 95% ci, n36.60.1 (535)36.70.1 (139)36.60.1 (396)p = 0.134oxygen saturationpercent, mean 95% ci, n97.90.2 (542)98.20.4 (141)97.80.2 (401)p = 0.018dispositiondischarged (%, n)47.7% (286/599)37.6% (59/157)51.4% (227/442)p = 0.003admitted (%, n)52.3% (313/599)62.4% (98/157)48.6% (215/442)final diagnosis of mino (%, n)94.2% (564/599)93.0% (146/157)94.6% (418/442)p = 0.469yes (%, n)5.8% (35/599)7.0% (11/157)5.4% (24/442) a comparison among the characteristics of the total cohort, and patients brought in by ambulance vs. alternate transportation when comparing the two groups, several significant differences were found. ambulance - transported patients were older in age (51.9 2.7 vs. 47.7 1.6 years, p = 0.010), more likely to have a higher heart rate (86.7 3.1 vs. 81.7 1.7 bpm, p = 0.004), and less likely to have a lower oxygen saturation on arrival (98.2 0.4 vs. 97.8 0.2 percent, p = 0.018). insurance status was an indicator of ambulance utilization, with 35.7% (46/129) of medicare patients vs. 23.1% (108/467) of patients with other insurance or self - pay [or 1.84 (95% ci 1.212.80), p = 0.005 ] utilizing ems for transfer. past medical histories of diabetes [31.9% (26/69) vs. 24.1% (118/490), or 1.91 (95% ci 1.123.24), p = 0.020 ] and hypertension [34.5% (57/165) vs. 22.4% (89/397), or 1.83 (95% ci 1.232.72), p ambulance - transported patients were significantly more likely to be admitted to the hospital than those who arrived with alternate transportation (62.4% vs. 48.6%, p = 0.003). a total of 11 of the 157 (7.0%) patients brought in by ambulance were diagnosed with myocardial infarction, while 24 of the 442 (5.4%) patients arriving by alternate transportation were diagnosed with myocardial infarction (p = 0.469). this equates to an odds ratio (95% ci) = 1.3 (0.62.7) of mi for patients who arrive via ambulance. only 31.4% (11/35) of patients who ultimately were diagnosed with mi arrived by ambulance. when a patient has chest pain, arrival to the emergency department by ems is clearly advantageous. a sub - analysis of the national registry of myocardial infarction 2 (nrmi2) study demonstrated that use of ems was associated with wider use of reperfusion therapies and faster time to either fibrinolytics or cardiac catheterization. likewise, the react trial showed that patients with chest pain who arrived to the hospital via private transportation often arrived more quickly than if they activated ems, but did not have the benefit of prehospital treatment and had longer times to reperfusion therapy. the newest suggestion that ems be able to obtain an electrocardiogram and bypass the emergency department to go directly to the catheterization laboratory to reduce door - to - balloon times in st - elevation mi patients is a further advantage of ems transport. motives of why people choose to use alternate transportation instead of an ambulance have been studied. a telephone survey demonstrated that 89% of respondents stated that they would activate ems with a suspected cardiac event, but only 23% actually did use the service. wait before going after speaking with an on - call physician or taking an antacid or aspirin at home were risk factors for decreased likelihood of ems use. the aforementioned nrmi2 study found that nonusers of ems were younger, more likely to be male and were lower risk on presentation. furthermore, racial and payer status differences were also detected, with blacks using ems more than whites, and patients with hmo insurance, the uninsured and those with medicaid more likely to use ems than those with private insurance. in our study patients who used ems were older and were more likely to have medicare than other insurance. we did not detect a difference in gender, or in the time of day or weekday vs. weekend arrival periods. even though patients who arrived by ambulance were more likely to be admitted to the hospital, rates of mi were not significantly different. the first limitation is that this was a retrospective study and therefore relies on the accuracy of data recorded on the medical record. second, we assumed that patients who were discharged did not have mi, though it is possible that some of these patients may have been erroneously discharged. there are several other potentially life - threatening causes of chest pain, from pneumothorax to aortic dissection to certain abdominal emergencies. it is not known if patients with these pathologies are more likely to present via ems than alternate transportation. additionally, it should be noted that the abstractors were not blinded to patient mode of transport and there was no measurement of inter - rater reliability among the abstractors in this study. most importantly, our study may not be powered sufficiently to find a truly statistically significant difference. we determined that our current power to detect the odds ratio is only 12.9%. maintaining a 2.8:1 ratio of alternate transportation to ambulance arrival for patients with chest pain as determined in our patient population, we would need to include a total of 9,000 patients (5,803 alternate transportation vs. 3197 ambulance arrival) to obtain sufficient power. our hope is that this study has raised an interesting question with large public health implications, and we believe that future multi - center trials are warranted. although we were able to detect differences in which patients with the complaint of chest pain utilize ems, we were not able to determine a difference in the final diagnosis of mi between those who arrive via ems or alternate transportation. for this reason, equal consideration and urgency should be given to these patients regardless of their mode of arrival to the ed. | aimsthis study aims to determine if patients who arrive by ambulance with a chief complaint of chest pain have a higher risk of myocardial infarction (mi) than those who arrive via alternate transportation.methodsall patients ages 1899 who presented to an urban academic ed between january 2006 and july 2006 with a chief complaint that included chest pain were eligible for retrospective analysis. patients who were transferred or who left without being seen or against medical advice were excluded. myocardial infarction was defined as patients who were admitted and who had elevated troponin i or went urgently to catheterization laboratory and had > 90% occlusion of a vessel, with a final clinical impression of mi.resultsthere were 690 visits for chest pain during the study period, representing 4% of total ed census. a total of 39 visits met exclusion criteria, and 37 patients had 52 repeat visits, leaving 599 unique patients included for analysis. mean age was 48.8 1.4 years (sd 17.7), 44.6% were female, and 35 patients (5.8%) were diagnosed with mi. in all, 157 patients (26.2%) arrived via ems. patients who arrived by ambulance did not have a significant difference in rate of mi when compared with alternate transportation [7.0% vs. 5.4%, or (95% ci) = 1.3 (0.62.7), p = 0.469 ]. only 31.4% (11/35) of patients who ultimately were diagnosed with mi arrived by ambulance.conclusionwe were unable to show a significant difference in rate of mi between patients who arrived via ambulance or private transportation. equal consideration and urgency should be given to both types of patients when they arrive at the ed. |
treatment of animals in this study was in accord with the guide for the care and use of laboratory animals, 8th edition. animals were housed at the new england regional primate center (nerpc ; southborough, ma), tulane national primate research center (tnprc ; covington, la), or bioqual (baltimore, md). the neprc protocol number for this study is 04420 and the animal welfare assurance number is a3431 - 01. the tnprc number for this study is 3497 and the animal welfare assurance number is a4499 - 01. animals were monitored daily for evidence of disease progression and changes in appetite or behavior, with clinical support administered under the direction of an attending veterinarian. nineteen rhesus macaques (macaca mulatta) were infected with siv mac251 (2 ng of siv - p27) intravenously (kindly provided by ronald desrosiers, university of miami ; table1). all animals were cd8-lymphocyte depleted using cm - t807, a human anti - cd8 antibody, administered subcutaneously (10 mg / kg) on day 6 post - infection (pi) and intravenously (5 mg / kg) on days 8 and 12 pi, as previously described.1 ten animals (n=4 late natalizumab treated, n=6 untreated) were sacrificed at similar time points with progression to aids (49 to 65 days postinfection [dpi ]). all cd8-lymphocyte - depleted animals had high viral load at peak viremia that remained elevated and was not different between early and late treated animals and controls. six early natalizumab - treated animals were sacrificed at 21 dpi and 3 untreated controls were sacrificed at 22 dpi. sections of left ventricular myocardium (hereafter referred to as cardiac tissue) were analyzed by a board - certified veterinary pathologist (a.d.m.), and scored based on degree of inflammation, fibrosis, and cardiomyocyte degeneration. sections were scored as either no significant findings (nsf), mild, moderate, or severe, based on degree of change in cardiac tissues, as previously described.28 animals used in this study and cardiac pathology nineteen animals were used in this study, housed at either the new england regional primate center (nerpc), tulane national primate research center (tnprc), or bioqual, as indicated. six animals began natalizumab treatment at the time of infection at 0 days postinfection (dpi) and were sacrificed at 21 dpi. four late natalizumab - treated animals began treatment at 28 dpi and were sacrificed at 49 dpi. three animals each for late untreated controls without cardiac pathology and with cardiac pathology were sacrificed at 56 to 65 dpi. pathology was assessed based on the degree of inflammation, fibrosis, and cardiomyocyte degeneration. to investigate whether blocking monocyte / macrophage traffic to the heart decreased siv - associated cardiac pathology, 10 randomly chosen, 200 fields of view were chosen and analyzed blindly by a veterinary pathologist. sections of cardiac tissue were scored based on the degree of change as having no significant findings (nsf), mild, moderate, or severe inflammation, fibrosis, and cardiomyocyte degeneration. the anti-4 integrin monoclonal antibody (natalizumab) was provided by biogen idec (cambridge, ma) in a sterile concentrated solution. natalizumab has specificity for the 4 subunit of 41 and 47 integrins expressed on surfaces of all leukocytes, except neutrophils.43 natalizumab was administered weekly for 3 weeks beginning on the day of infection (0 dpi ; n=6) or on 28 dpi (n=4), as previously described.42 this treatment regimen maintains high levels of natalizumab in serum of rhesus macaques during treatment.44 to study monocyte / macrophage traffic to the heart, animals were administered brdu (60 mg / kg) at indicated time points (table1), as previously described.42 numbers of macrophages and t lymphocytes present in formalin - fixed, paraffin - embedded tissues were determined by immunohistochemistry and cell counting. cardiac tissues were stained with antibodies against cd163 (1:250 ; abd serotec, kidlington, uk), cd68 (1:200 ; dako, carpinteria, ca), mac387 (1:100 ; dako) macrophages, and cd3 t lymphocytes (1:300 ; dako). the number of macrophages that traffic to cardiac tissues was determined using a mouse monoclonal brdu antibody (1:50), as previously described.28 twenty random, nonoverlapping, 200 microscopic fields of view were taken for each animal and the number of positive cells / mm calculated for each. data are represented as the average number of positive cells / mm from the 20 random fields. percent of collagen per tissue area used as a marker of fibrosis29 was measured using a modified massons trichrome stain kit (newcomer supply, middleton, wi), according to the manufacturer s recommendation. tissue sections were imaged using a zeiss axio imager m1 microscope (zeiss, oberkochen, germany) using plan - apochromat 20/0.8 korr objectives, as previously described.28,45 the area of red and blue dyes corresponding to cytoplasm and collagen, respectively, were measured to determine the percentage of total tissue area. statistical analyses were conducted using prism software (version 6.0 ; graphpad software inc., la jolla, ca). whitney t test with significance accepted at p 21 dpi) and that there are few macrophages present in untreated animals sacrificed at 21 dpi.28 in the current study, we found that natalizumab treatment beginning at 0 dpi resulted in decreased numbers of cd163 and cd68 macrophages, compared to uninfected controls, but cardiac pathology in early infection is minimal and most of the pathology occurred in the later stages of infection. when compared to untreated controls with cardiac pathology, late treated animals had significant decreases in the number of cd163 and cd68 macrophages. in fact, the numbers of macrophages in late natalizumab - treated animals were similar to untreated animals without cardiac pathology. additionally, we found a correlation between decreased macrophage numbers in natalizumab - treated animals with decreased cardiac fibrosis. overall, these data show that blocking monocyte / macrophage traffic to the heart alleviates hiv- and siv - associated cardiac pathology that resulted in reduced inflammation, fibrosis, and cardiomyocyte degeneration. though not significant, we found a trend of decreasing numbers of mac387 macrophages in the left ventricle of late treated animals, compared to untreated animals with cardiac pathology. the finding of a decrease in newly recruited macrophages is supported by our observation of fewer brdu - labeled macrophages (that traffic from the bone marrow), suggesting that macrophage traffic to the heart results in increased fibrosis. whereas previous research showed that natalizumab decreased traffic of cd3 t lymphocytes and mac387 to the brain and gut,42 in the current study, we did not find significant differences in the number of cd3 t lymphocytes or mac387 macrophages in cardiac tissues of siv - infected animals with or without natalizumab treatment. this possibly suggests that cd3 t lymphocytes and mac387 macrophages use different integrins to traffic to the heart than to the brain or gut ; however, we have previously shown that mac387 macrophages and not cd3 t lymphocytes correlate with increased fibrosis in cardiac tissues.28 our lack of finding a statistically significant reduction in the number mac387 macrophages may be owing to the relatively few numbers of those cells in cardiac tissues. previously, we have shown that the rate of monocyte / macrophage traffic to the heart is increased later in infection (after 48 dpi), as opposed to early infection.28 using brdu labeling, we found a trend of decreased traffic of monocytes / macrophages to the heart in late natalizumab - treated animals, compared to untreated animals that developed cardiac pathology. late natalizumab - treated animals had a similar rate of traffic of newly released monocytes / macrophages to the heart as untreated animals without cardiac pathology. this provides evidence that potentially blocking traffic of monocyte / macrophages later during siv infection can alleviate siv - associated cardiac pathology. although cart can decrease hiv to nondetectable levels in plasma, it does not necessarily target monocyte / macrophages that play a role in the development of cardiac pathology and cvd.32 chronic immune activation with hiv infection is posited to play a role in hiv - associated cardiovascular pathology. previous studies show that hiv - infected individuals have increased inflammation in the ascending aorta that correlates with levels of scd163 in plasma.55 increased inflammation in the aorta is also been linked to high - risk noncalcified plaques that are prone to rupture.8 18-fluorodeoxyglucose positron emission tomography imaging studies have demonstrated that such plaque areas are comprised of areas with accumulation of macrophages.8,56,57 whereas macrophage accumulation in the aorta and cardiac plaques are critical in hiv - associated cardiac disease, it is not surprising that also there is increased macrophage inflammation in cardiac tissues at the same time. unpublished data from our laboratory, using matched cardiac tissues (left ventricle) and aorta from hiv and hiv individuals, shows that, with hiv infection, there is an increased macrophage accumulation inflammation in ventricular tissues and the aorta. moreover, increased macrophage inflammation in cardiac tissues correlates with increased fibrosis, macrophage accumulation in the aorta, and increased aortic intima - media thickness. to date, there are few therapies that target macrophages specifically or indirectly to diminish hiv - associated cv pathology. therapeutic agents that have been successful in the treatment noncalcified cardiac plaque in hiv individuals include 3-hydroxy-3-methylglutaryl - coenzyme a reductase inhibitors (statins), which, fortuitously, have anti - inflammatory effects on macrophages.58,59 statin therapy in conjunction with cart reduced serum levels of inflammatory markers, including il-6, il-8, and tumor necrosis factor alpha, more so than cart alone.60 statins have similarly been used in monkeys, where they decreased the macrophage content in plaques in the abdominal aorta.34 a recent study in hiv - infected individuals showed that statins significantly decreased the volume on noncalcified plaques, but whether statin use in this study directly affected monocyte and/or macrophage activation and traffic was not studied.35 in rodent models of experimental autoimmune myocarditis, rosuvastatin - reduced numbers in macrophages, t lymphocytes, and multinucleated giant cells in the heart resulted in decreased numbers of apoptotic cardiomyocytes. the effects of statins on myocarditis with hiv infection have not been examined.61 other studies found that maraviroc treatment decreased chemotaxis of monocyte / macrophages, in vitro,62 but in clinical studies with advanced hiv, it did not affect the development of immune reconstitution inflammatory syndrome,63 maraviroc is used primarily to inhibit viral replication of r5-tropic hiv by blocking interactions between the virus and ccr5 on host cells.64,65 studies using maraviroc in siv - infected monkeys demonstrated fewer cd163 macrophages in the heart, but this could have been owing to a decreased cd163 expression (activation) on macrophages already present in the heart and not a decrease in inflammatory cells. all together, these experiments add further evidence to the role that monocyte / macrophages play in cardiac pathology with hiv and siv infection, and suggest that therapies blocking monocyte / macrophage traffic to the heart could diminish hiv - associated cardiac pathology. in this study, we showed that directly blocking monocyte / macrophage traffic to cardiac tissues with natalizumab successfully decreased the numbers of macrophages present in tissues. studies examining whether blocking traffic to vessels result in decreased high - risk vascular plaques with hiv infection are warranted. our data suggest that studies examining the efficacy of blocking monocyte / macrophage traffic, or directly targeting monocyte / macrophage activation as an adjunctive therapy with cart, should be examined with an aim to decrease hiv - associated cardiac pathology. this work was supported by the following national institutes of health (nih) grants : r01 ns040237 (williams) and r01 ns082116 (burdo). | backgroundcardiovascular disease (cvd), myocarditis and fibrosis are comorbidities of hiv+ individuals on durable antiretroviral therapy (art). although mechanisms for these vary, monocytes / macrophages are increasingly demonstrated to be key players.methods and resultswe directly blocked monocyte / macrophage traffic to the heart in an siv model of aids using an anti - alpha-4 integrin antibody (natalizumab). nineteen rhesus macaques were sivmac251 infected and cd8-lymphocyte depleted for the development of rapid aids. ten animals received natalizumab once a week, for 3 weeks, and were sacrificed 1 week later. six animals began treatment at the time of infection (early) and the remaining 4 began treatment 28 days post - infection (late), a time point we have previously established when significant cardiac inflammation occurs. nine animals were untreated controls ; of these, 3 were sacrificed early and 6 were sacrificed late. at necropsy, we found decreased siv - associated cardiac pathology in late natalizumab - treated animals, compared to untreated controls. early and late treatment resulted in significant reductions in numbers of cd163 + and cd68 + macrophages in cardiac tissues, compared to untreated controls, and a trend in decreasing numbers of newly recruited mac387 + and brdu+ (recruited) monocytes / macrophages. in late treated animals, decreased macrophage numbers in cardiac tissues correlated with decreased fibrosis. early and late treatment resulted in decreased cardiomyocyte damage.conclusionsthese data demonstrate a role for macrophages in the development of cardiac inflammation and fibrosis, and suggest that blocking monocyte / macrophage traffic to the heart can alleviate hiv- and siv - associated myocarditis and fibrosis. they underscore the importance of targeting macrophage activation and traffic as an adjunctive therapy in hiv infection. |
much attention is being paid these days to the importance of engaging patients and their caregivers in identifying and prioritizing research topics and throughout the conduct of the research itself.2 however, this trend toward user engagement is far broader than the individual patient or clinician. for over 30 years, the field of community - based participatory research has been refining methods for engaging community representatives in research design and execution.3,4,5 researchers focused on policy relevant questions have long recognized the importance of connecting with policymakers early on at the federal, state and local levels.6,7,8 increasingly, delivery system leaders are building embedded research units to apply rigorous hsr methods to the study of the operational questions system leaders have. what these user groups have in common is a desire to play a substantive role in setting the agenda for the research community as well as sometimes being involved in the research itself. however, doing this effectively will require significant changes in how many investigators approach their work which will entail changes in how research is funded and knowledge of which user engagement approach is effective for which research. academyhealth convenes the consumer patient researcher (cpr) roundtable as a collaborative forum to explore these issues and has recently published a report on the many phases of consumer / patient engagement in research.9 egems extends our work in this area and is an opportunity to share what the field is learning about truly engaging patients and other users in our research. recent investments in health information technology and comparative effectiveness research (cer) have provided substantial capital to make meaningful progress toward building an infrastructure (governance, data, methods, and training) capable of addressing many of the challenges of traditional study designs and data sources used in outcomes research and quality improvement (qi). of the $ 1.1 billion provided for cer in the american recovery and reinvestment act, approximately 38 percent10 supported infrastructure development (including data, methods, governance, and training). the prospect, drn, enhanced registry studies (representing a $ 100 million investment to build ecd infrastructure for multiple uses), as well as the edm forum are all funded through this mechanism and offer key lessons that are important for others to learn from if we are to accelerate cer and qi and use the knowledge to improve clinical care and patient outcomes. the proliferating numbers of research and qi networks are each confronting fundamental issues and challenges in all aspects of using ecd including access, governance, management, and linkages. while the lessons they are learning are not the stuff of traditional peer - reviewed journals, they do have tremendous scientific import. we need to exchange these lessons as rapidly as possible to benefit the growing community of researchers grappling with problems that, more often than not, are similar. our hsr community needs a platform like egems to make this happen. without this type of dissemination and sharing, researchers are doomed to keep re - learning the same lessons in study after study, network after network.11,12,13,14 the challenges of translating and disseminating hsr, cer, pcor, and qi are anything but new.15,16,17,18 what is new is the urgency of the need to change business as usual in health care. policymakers, employers, health system leaders, and patients have made it clear that costs can not continue to increase and that better quality and outcomes must be achieved. the research community can not stand apart from this demand : we need to deliver answers more quickly and effectively. to do so we need to learn together how to respond to the imperative to improve health and health care. to that end academyhealth is launching a new translation and dissemination institute to extend our capacity to have an impact. as a research community we have much to learn in the coming years for us to effectively seize our moment. egems is another new tool to help all of us accelerate this learning together. to borrow from ralph waldo emerson, learning is also a journey, not a destination, and egems can help us share our journeys. | the field of health services research faces significant challenges as it aims to address pressing issues of quality and cost in the us healthcare system. major advances in the availability of electronic clinical data (ecd) provide the opportunity to address questions that are important to the recipients, providers, and purchasers of health care. this is where egems has a role to play, meeting an expressed need in the scientific community by disseminating approaches and methods for using ecd. egems can help researchers address these important questions and consider strategies to further improve the us healthcare system. |
we defined a standard model for cost - effectiveness analysis in diagnostic imaging. comparing total 1-year health costs and benefits, cta is superior to mra. a strategy of combining cta and dsa was found to be the most cost - effective diagnostic approach. rupture of an intracranial aneurysm is a major cause of death and disability with an overall incidence of approximately 9 per 100,000. in the group of patients who survive the initial bleed, the risk of rebleeding without treatment is 12 % per day for the first 4 weeks with a 70 % case fatality rate [2, 3 ]. to prevent rebleeding from ruptured aneurysms, treatment is performed as soon as feasible in order to exclude the aneurysm from the arterial circulation [2, 4, 5 ]. treatment options are either neurosurgical clipping via craniotomy or endovascular aneurysm occlusion by detachable platinum coils. coiling is the preferred option in patients where both treatments are feasible [69 ]. total treatment costs were found to be slightly higher for surgical clipping compared with endovascular coiling [1013 ]. non - traumatic subarachnoid haemorrhage (sah) is caused in about 85 % of patients by a ruptured intracranial aneurysm. diagnostic imaging is required to fulfil two tasks : (1) detect the presence of intracerebral aneurysms ; (2) determine the most suitable treatment : endovascular coiling or surgical clipping. dsa is the reference standard for detecting aneurysms and for determining the feasibility of coiling. nevertheless this invasive and labour - intensive technique is relatively expensive and carries discomforts and potential risks [1517 ]. promising non - invasive and less costly diagnostic alternatives are computed tomographic angiography (cta) and magnetic resonance angiography (mra). although costs and characteristics of diagnostic tests and treatment have been reported, we found no studies determining the optimal diagnostic pathway in patients with suspected ruptured intracranial aneurysms. our study aimed to compare the cost - effectiveness of mra, cta and dsa in these patients. cost - effectiveness of diagnostic tests in follow - up of coiled aneurysms has previously been evaluated by schaafsma.. we previously assessed test characteristics and accuracy of mra (1.5 t) and cta in 75 patients with non - traumatic sah. patients were recruited consecutively between 2004 and 2006, and for all patients mra, cta and dsa had been performed. informed consent was given and this study protocol was approved by the institutional ethical committee. feasibility of coiling depended on the judgment of the interventionist based on dsa, or the actual suitability during a coiling attempt. for cta and mra two experienced neuroradiologists determined the presence of an aneurysm and judged suitability for endovascular treatment using common criteria, like dome - to - neck ratio and absence of branches originating from the base of the aneurysm. pooled sensitivity of cta for the detection of aneurysms was 91.5 % (95 % ci, 85.095.5) and specificity was 94.4 % (95 % ci, 79.099.0). pooled sensitivity of mra for the detection of aneurysms was 95.4 % (95 % ci, 89.898.1) and specificity was 83.3 % (95 % ci, 66.593.0). pooled sensitivity of cta in determining feasibility of endovascular coiling was 71.9 % (95 % ci, 59.082.1) and specificity was 75.4 % (95 % ci, 62.085.5). pooled sensitivity of mra in determining feasibility of endovascular coiling was 60.6 % (95 % ci, 48.271.7) and specificity was 81.4 % (95 % ci, 68.789.9). dsa as standard of reference was regarded to have a sensitivity and specificity for aneurysm detection and determination of best treatment of 100 %. a decision tree (using excel software) was developed to determine differences in health benefits and costs in patients with ruptured aneurysms for various diagnostic pathways. for patients with acute non - traumatic subarachnoid haemorrhage, the decision tree delivers different health states due to characteristics of diagnostic imaging and choice of therapy. health states were based on whether patients were alive and, when they were alive, whether they were well or disabled. more specifically, a cohort of patients with non - traumatic subarachnoid haemorrhage is divided over the different pathways in the decision tree, based on a set of probabilities that were derived from literature. this allowed us to synthesise evidence and thereby to evaluate health costs of diagnostic test and treatment as well as related quality of life and associated costs determined by diagnostic decision. a provider (or healthcare) perspective was used, which included only direct healthcare costs.fig. tp true - positive, fn false - negative, tn true - negative, fp false - positive test result. input parameters derived from table 1 markov decision tree basic model showing exemplary branch for cta. tp true - positive, fn false - negative, tn true - negative, fp false - positive test result. input parameters derived from table 1 in the basic model, patients underwent dsa, mra or cta. following our standard clinical practice, if no aneurysm was detected on cta or mra, an additional dsa study was performed. in whom an aneurysm was detected, feasibility of coiling of the aneurysm was determined. depending on the result of each test, either a coiling or clipping procedure was performed. in patients where false - positive feasibility of coiling was determined by cta or mra, angiography during the coiling procedure would show no feasibility and transfer to surgical clipping would have been performed. in case of false - negative determination of coiling, surgical clipping would have been performed, although coiling would have been feasible. in a scenario analysis we explored two alternative strategies. we therefore analysed whether it is cost - effective to add dsa to the cta and mra strategies only if an aneurysm is deemed not suitable for endovascular treatment. in this scenario, in each patient in whom an aneurysm was detected which was deemed not suitable for coiling by mra or cta, an additional dsa study was considered to be performed. sensitivity and specificity of the diagnostic tests for detection of aneurysms and determination of treatment possibility were taken as input parameters to the decision model. for the costs of dsa, mra and cta, standard prices from the dutch manual for cost research were used. these include expenditures for personnel, equipment, materials, maintenance, housing, cleaning, administration and overheads. total 1-year costs of surgical clipping and endovascular coiling were derived from a literature search for western countries. health outcome after 1 year of treatment was derived from the isat trial [6, 7 ]. input parameters for related utilities and costs [18, 21, 23 ], as well as health risk of dsa, are based on available literature [17, 24 ]. all costs were updated to 2010 by means of national price index figures and expressed in euros (1 = $ 1.32). standard discount rates of 1.5 % for effects and 4 % for costs according to dutch guidelines were used. table 1 shows the model input parameters and their sources.table 1model input parametersmodel parametermeanse / sd / rangedistributionsource discount rates cdr (cost discount rate)4 % fixed odr (outcome discount rate)1.5 % fixedtreatment independent parameters probabilities, (p) paneurysm in case of non - traumatic sah0.85fixedcosts, c () diagnostics cdsa725 fixed cmra252 fixed ccta197 fixed treatment ccoiling38,238 1,833 gamma cclipping31,739 2,503 gamma health state costs number of days in nursing home per year (d)365 costs per day in nursing home (euros / d)241 cdisability87,975 event costs cdeath2,741 [18, 23]utilities (u) uwell0.780.019beta[18, 22 ] uwell after sah0.720.650.80triangular[18, 22 ] udisabled0.250.210.30triangular[18, 22 ] udead0.00fixed[18, 22]clinical outcome probabilities probability of being well after clipping0.690.014beta probability of being disabled after clipping0.210.013beta pclipdead (probability of being dead after clipping)0.100.009beta pclipwell (probability of being well after survived clipping)0.77 probability of being well after coiling0.760.013beta probability of being disabled after coiling0.160.011beta pcoildead (probability of being dead after coiling)0.080.008beta pcoilwell (probability of being well after survived coiling)0.83 probability of being well after dsa0.9980.001beta[17, 24 ] probability of being disabled after dsa0.0020.001beta[17, 24 ] pdsadead (probability of being dead after dsa)0.000fixed[17, 24 ] pdsawell (probability of being well after survived dsa)0.998 probability of being dead / disabled after surgery without aneurysm psurgdead0.0250.004beta psurgdisab0.1320.008beta pcoiltp (probability coiling is feasible in true - positive aneurysm)0.5850.061beta pcoilfp (probability coiling is feasible in false - positive aneurysm)0fixedtreatment dependent parametersvaluese / sd / rangedistributionsource probabilities pmratpd (sensitivity of mra in diagnosing aneurysm)0.9540.018beta pmratnd (specificity of mra in diagnosing aneurysm)0.8330.061beta pctatpd (sensitivity of cta in diagnosing aneurysm)0.9150.024beta pctatnd (specificity of cta in diagnosing aneurysm)0.9440.038beta pmratpt (sensitivity of mra in determining whether coiling is feasible)0.6060.058beta pmratnt (specificity of mra in determining whether coiling is feasible)0.8140.050beta pctatpt (sensitivity of cta in determining whether coiling is feasible)0.7190.056beta pctatnt (specificity of cta in determining whether coiling is feasible)0.7540.057beta pdsatpd (sensitivity of dsa in diagnosing aneurysm)1fixed pdsatnd (specificity dsa in diagnosing aneurysm)1fixed pdsatpt (sensitivity of dsa in determining whether coiling is feasible)1fixed pdsatnt (specificity of dsa in determining whether coiling is feasible)1fixedrange presented for triangular distributionsdata based on study model input parameters range presented for triangular distributions data based on study we assumed that dsa, as the standard of reference, has a sensitivity and specificity of 100 % in detecting aneurysms and determining feasibility of coiling. furthermore, we assumed no significant gender- or age - related differences in outcome, as well as no influence due to aneurysm size or location. patients presenting with acute sah without presence of a ruptured aneurysm were assumed to have no other intracranial vascular pathology to be treated. in our model, we evaluated the outcome of diagnostic pathways based on the input parameters in table 1. in the base case analysis, the average costs and effects were calculated for a hypothetical cohort of 1,000 patients. we compared total 1-year costs of diagnostic test, treatment option and health state to 1-year health benefits in terms of quality - adjusted life - years (qalys). the alternative that yielded the highest number of qalys was considered to be most effective. this implies that for an intervention to be adopted, it has to be cost - effective compared with its next best alternative. if a strategy is less costly and more effective, it is superior to and dominates the alternative strategy. if a strategy is more costly and less effective than its alternative, it is dominated by the alternative. in case the strategy is more costly and more effective, or less costly and less effective than the alternative, incremental cost - effectiveness ratios (icers) were calculated from each model by dividing the incremental costs by the incremental qalys. the decision whether the strategy is deemed cost - effective then depends on how much society is willing to pay for a qaly gained. in the netherlands the informal societal willingness to pay (wtp) threshold level is 80,000 ($ 106,000). if the icer is lower than this level, we conclude that the strategy is cost - effective compared with the alternative. additionally, a scenario analysis was performed in which, for all patients, dsa was performed in case an aneurysm was visible but coiling was deemed not feasible. uncertainty regarding the model input parameters was explored with a probabilistic sensitivity analysis using monte carlo simulation [20, 28 ]. for this purpose we assigned distributions to all uncertain parameter. with monte carlo simulation, for each sample, the hypothetical cohort runs through the model based on these sampled probabilities, and costs and effects are derived. this results in 1,000 estimates of costs and effects for the hypothetical cohort, representing the uncertainty in the cost - effectiveness estimation. all assigned distributions are listed in table 1. to illustrate the results of the simulation, cost - effectiveness acceptability curves (ceacs) were calculated. ceacs show the probability that a strategy is cost - effective, given different values of willingness to pay for a qaly. additionally, univariate sensitivity analyses were performed for test characteristics and costs of dsa, mra and cta, as well as for costs of coiling and clipping, to determine association between these model input parameters and cost - effectiveness outcome. a decision tree (using excel software) was developed to determine differences in health benefits and costs in patients with ruptured aneurysms for various diagnostic pathways. for patients with acute non - traumatic subarachnoid haemorrhage, the decision tree delivers different health states due to characteristics of diagnostic imaging and choice of therapy. health states were based on whether patients were alive and, when they were alive, whether they were well or disabled. more specifically, a cohort of patients with non - traumatic subarachnoid haemorrhage is divided over the different pathways in the decision tree, based on a set of probabilities that were derived from literature. this allowed us to synthesise evidence and thereby to evaluate health costs of diagnostic test and treatment as well as related quality of life and associated costs determined by diagnostic decision. a provider (or healthcare) perspective was used, which included only direct healthcare costs.fig. tp true - positive, fn false - negative, tn true - negative, fp false - positive test result. input parameters derived from table 1 markov decision tree basic model showing exemplary branch for cta. tp true - positive, fn false - negative, tn true - negative, fp false - positive test result. in the basic model, patients underwent dsa, mra or cta. following our standard clinical practice, if no aneurysm was detected on cta or mra, an additional dsa study was performed. in whom an aneurysm was detected, feasibility of coiling of the aneurysm was determined. depending on the result of each test, either a coiling or clipping procedure was performed. in patients where false - positive feasibility of coiling was determined by cta or mra, angiography during the coiling procedure would show no feasibility and transfer to surgical clipping would have been performed. in case of false - negative determination of coiling, surgical clipping would have been performed, although coiling would have been feasible. in a scenario analysis we explored two alternative strategies. we therefore analysed whether it is cost - effective to add dsa to the cta and mra strategies only if an aneurysm is deemed not suitable for endovascular treatment. in this scenario, in each patient in whom an aneurysm was detected which was deemed not suitable for coiling by mra or cta, sensitivity and specificity of the diagnostic tests for detection of aneurysms and determination of treatment possibility were taken as input parameters to the decision model. for the costs of dsa, mra and cta, standard prices from the dutch manual for cost research these include expenditures for personnel, equipment, materials, maintenance, housing, cleaning, administration and overheads. total 1-year costs of surgical clipping and endovascular coiling were derived from a literature search for western countries. health outcome after 1 year of treatment was derived from the isat trial [6, 7 ]. input parameters for related utilities and costs [18, 21, 23 ], as well as health risk of dsa, are based on available literature [17, 24 ]. all costs were updated to 2010 by means of national price index figures and expressed in euros (1 = $ 1.32). standard discount rates of 1.5 % for effects and 4 % for costs according to dutch guidelines were used. table 1 shows the model input parameters and their sources.table 1model input parametersmodel parametermeanse / sd / rangedistributionsource discount rates cdr (cost discount rate)4 % fixed odr (outcome discount rate)1.5 % fixedtreatment independent parameters probabilities, (p) paneurysm in case of non - traumatic sah0.85fixedcosts, c () diagnostics cdsa725 fixed cmra252 fixed ccta197 fixed treatment ccoiling38,238 1,833 gamma cclipping31,739 2,503 gamma health state costs number of days in nursing home per year (d)365 costs per day in nursing home (euros / d)241 cdisability87,975 event costs cdeath2,741 [18, 23]utilities (u) uwell0.780.019beta[18, 22 ] uwell after sah0.720.650.80triangular[18, 22 ] udisabled0.250.210.30triangular[18, 22 ] udead0.00fixed[18, 22]clinical outcome probabilities probability of being well after clipping0.690.014beta probability of being disabled after clipping0.210.013beta pclipdead (probability of being dead after clipping)0.100.009beta pclipwell (probability of being well after survived clipping)0.77 probability of being well after coiling0.760.013beta probability of being disabled after coiling0.160.011beta pcoildead (probability of being dead after coiling)0.080.008beta pcoilwell (probability of being well after survived coiling)0.83 probability of being well after dsa0.9980.001beta[17, 24 ] probability of being disabled after dsa0.0020.001beta[17, 24 ] pdsadead (probability of being dead after dsa)0.000fixed[17, 24 ] pdsawell (probability of being well after survived dsa)0.998 probability of being dead / disabled after surgery without aneurysm psurgdead0.0250.004beta psurgdisab0.1320.008beta pcoiltp (probability coiling is feasible in true - positive aneurysm)0.5850.061beta pcoilfp (probability coiling is feasible in false - positive aneurysm)0fixedtreatment dependent parametersvaluese / sd / rangedistributionsource probabilities pmratpd (sensitivity of mra in diagnosing aneurysm)0.9540.018beta pmratnd (specificity of mra in diagnosing aneurysm)0.8330.061beta pctatpd (sensitivity of cta in diagnosing aneurysm)0.9150.024beta pctatnd (specificity of cta in diagnosing aneurysm)0.9440.038beta pmratpt (sensitivity of mra in determining whether coiling is feasible)0.6060.058beta pmratnt (specificity of mra in determining whether coiling is feasible)0.8140.050beta pctatpt (sensitivity of cta in determining whether coiling is feasible)0.7190.056beta pctatnt (specificity of cta in determining whether coiling is feasible)0.7540.057beta pdsatpd (sensitivity of dsa in diagnosing aneurysm)1fixed pdsatnd (specificity dsa in diagnosing aneurysm)1fixed pdsatpt (sensitivity of dsa in determining whether coiling is feasible)1fixed pdsatnt (specificity of dsa in determining whether coiling is feasible)1fixedrange presented for triangular distributionsdata based on study model input parameters range presented for triangular distributions data based on study we assumed that dsa, as the standard of reference, has a sensitivity and specificity of 100 % in detecting aneurysms and determining feasibility of coiling. furthermore, we assumed no significant gender- or age - related differences in outcome, as well as no influence due to aneurysm size or location. patients presenting with acute sah without presence of a ruptured aneurysm were assumed to have no other intracranial vascular pathology to be treated. in our model, we evaluated the outcome of diagnostic pathways based on the input parameters in table 1. in the base case analysis, we compared total 1-year costs of diagnostic test, treatment option and health state to 1-year health benefits in terms of quality - adjusted life - years (qalys). the alternative that yielded the highest number of qalys was considered to be most effective. this implies that for an intervention to be adopted, it has to be cost - effective compared with its next best alternative. if a strategy is less costly and more effective, it is superior to and dominates the alternative strategy. if a strategy is more costly and less effective than its alternative, it is dominated by the alternative. in case the strategy is more costly and more effective, or less costly and less effective than the alternative, incremental cost - effectiveness ratios (icers) were calculated from each model by dividing the incremental costs by the incremental qalys. the decision whether the strategy is deemed cost - effective then depends on how much society is willing to pay for a qaly gained. in the netherlands the informal societal willingness to pay (wtp) threshold level is 80,000 ($ 106,000). if the icer is lower than this level, we conclude that the strategy is cost - effective compared with the alternative. additionally, a scenario analysis was performed in which, for all patients, dsa was performed in case an aneurysm was visible but coiling was deemed not feasible. uncertainty regarding the model input parameters was explored with a probabilistic sensitivity analysis using monte carlo simulation [20, 28 ]. for this purpose we assigned distributions to all uncertain parameter. with monte carlo simulation, the hypothetical cohort runs through the model based on these sampled probabilities, and costs and effects are derived. this results in 1,000 estimates of costs and effects for the hypothetical cohort, representing the uncertainty in the cost - effectiveness estimation. all assigned distributions are listed in table 1. to illustrate the results of the simulation, cost - effectiveness acceptability curves (ceacs) were calculated. ceacs show the probability that a strategy is cost - effective, given different values of willingness to pay for a qaly. additionally, univariate sensitivity analyses were performed for test characteristics and costs of dsa, mra and cta, as well as for costs of coiling and clipping, to determine association between these model input parameters and cost - effectiveness outcome. comparing expected average 1-year health benefits, dsa was the most effective diagnostic option, yielding 0.6039 qalys (95 % ci, 0.57610.6327), followed by cta yielding 0.5983 qalys (95 % ci, 0.57040.6278) and mra yielding 0.5947 qalys (95 % ci, 0.56740.6237). the total expected 1-year health costs were lowest for dsa (39,808 ; 95 % ci, 37,18242,663), followed by cta (40,748 ; 95 % ci, 37,93743,831) and mra (41,814 ; 95 % ci, 38,73045,146). initial diagnostic costs were highest for dsa, while therapy and health state - related costs were lowest for dsa, resulting in dsa being the least costly diagnostic option. cta as diagnostic test resulted in 128 out of 1,000 patients being referred for clipping although coiling would have been feasible. with mra, 187 patients out of 1,000 were treated surgically, although endovascular treatment would have been feasible. table 2 shows costs and qalys for the three diagnostic tests.table 2related 1-year costs and qalys for mra, cta and dsa per patient in the basic modelmractadsacosts () qalyscosts () qalyscosts () qalysmra resp. cta / mra showing no aneurysm related 1-year costs and qalys for mra, cta and dsa per patient in the basic model average costs per patient for dsa performed in case cta / mra showing no aneurysm the strategy that included dsa for every patient for whom coiling was not deemed feasible on the basis of cta and mra, led to equal effectiveness for dsa, mra and cta (0.6039 qalys each). in this approach no patient was treated surgically unless coiling was no option. in this scenario, cta was estimated less costly, reaching a total average 1-year cost per patient of 39,767, and therefore cost - effective compared with mra and dsa, which reached costs of 39,851 and 39,808 respectively. table 3 shows total expected 1-year costs and qalys as well as incremental costs and qalys for diagnostic tests in the scenario analysis and basic model.table 3total one - year outcome and incremental cost - effectiveness ratio (icer) for basic and scenario modelcosts () (95 % ci)qalys(95 % ci)basic model dsa39,808(37,182 ; 42,663)0.6039(0.5761 ; 0.6327) cta40,748(37,937 ; 43,831)0.5983(0.5704 ; 0.6278) mra41,814(38,730 ; 45,146)0.5947(0.5674 ; 0.6237)scenario model dsa39,808(36,982 ; 42,414)0.6039(0.5771 ; 0.6333) cta39,767(36,903 ; 42,402)0.6039(0.5771 ; 0.6333) mra39,851(37,003 ; 42,486)0.6039(0.5771 ; 0.6333)incremental costs () (95 % ci)incremental qalys(95 % ci)icer (per qaly)comparatorbasic model dsa cta940(10 ; 2,122)0.006(0.003 ; 0.009)dominateddsa mra2,007(604 ; 3,767)0.009(0.005 ; 0.015)dominateddsascenario model dsa cta40(103 ; 20)0.000(0.000 ; 0.000)dominantdsa mra84(15 ; 98)0.000(0.000 ; 0.000)dominatedctaci confidence interval (calculated based on the probabilistic sensitivity analysis) total one - year outcome and incremental cost - effectiveness ratio (icer) for basic and scenario model ci confidence interval (calculated based on the probabilistic sensitivity analysis) in univariate sensitivity analyses we explored whether changing individual parameters altered the conclusions of our base case analysis. assuming equal treatment cost for coiling and clipping did not change the conclusions. furthermore, results remained stable for the assumption of higher costs of dsa up to factor 2.8. higher sensitivity and specificity for detection of aneurysms and determination of feasibility of coiling for cta and mra up to 96 % or a reduction of sensitivity and specificity for dsa to 90 % also yielded stable results. in the scenario analysis, the strategy where cta is followed by dsa remained the least costly strategy for a sensitivity of determination of coiling within the range of 60100 %. the probabilistic sensitivity analysis showed that in the basic model analysis, dsa had a 98100 % probability of being cost - effective. in the scenario analysis, cta had the highest probability of being cost - effective (91 %), followed by dsa (9 %) and mra (0 %). because in the scenario analysis effectiveness is equal for all strategies, ceacs are constant over the different values of willingness to pay for a qaly. comparing expected average 1-year health benefits, dsa was the most effective diagnostic option, yielding 0.6039 qalys (95 % ci, 0.57610.6327), followed by cta yielding 0.5983 qalys (95 % ci, 0.57040.6278) and mra yielding 0.5947 qalys (95 % ci, 0.56740.6237). the total expected 1-year health costs were lowest for dsa (39,808 ; 95 % ci, 37,18242,663), followed by cta (40,748 ; 95 % ci, 37,93743,831) and mra (41,814 ; 95 % ci, 38,73045,146). initial diagnostic costs were highest for dsa, while therapy and health state - related costs were lowest for dsa, resulting in dsa being the least costly diagnostic option. cta as diagnostic test resulted in 128 out of 1,000 patients being referred for clipping although coiling would have been feasible. with mra, 187 patients out of 1,000 were treated surgically, although endovascular treatment would have been feasible. table 2 shows costs and qalys for the three diagnostic tests.table 2related 1-year costs and qalys for mra, cta and dsa per patient in the basic modelmractadsacosts () qalyscosts () qalyscosts () qalysmra resp. cta / mra showing no aneurysm related 1-year costs and qalys for mra, cta and dsa per patient in the basic model average costs per patient for dsa performed in case cta / mra showing no aneurysm the strategy that included dsa for every patient for whom coiling was not deemed feasible on the basis of cta and mra, led to equal effectiveness for dsa, mra and cta (0.6039 qalys each). in this approach cta was estimated less costly, reaching a total average 1-year cost per patient of 39,767, and therefore cost - effective compared with mra and dsa, which reached costs of 39,851 and 39,808 respectively. table 3 shows total expected 1-year costs and qalys as well as incremental costs and qalys for diagnostic tests in the scenario analysis and basic model.table 3total one - year outcome and incremental cost - effectiveness ratio (icer) for basic and scenario modelcosts () (95 % ci)qalys(95 % ci)basic model dsa39,808(37,182 ; 42,663)0.6039(0.5761 ; 0.6327) cta40,748(37,937 ; 43,831)0.5983(0.5704 ; 0.6278) mra41,814(38,730 ; 45,146)0.5947(0.5674 ; 0.6237)scenario model dsa39,808(36,982 ; 42,414)0.6039(0.5771 ; 0.6333) cta39,767(36,903 ; 42,402)0.6039(0.5771 ; 0.6333) mra39,851(37,003 ; 42,486)0.6039(0.5771 ; 0.6333)incremental costs () (95 % ci)incremental qalys(95 % ci)icer (per qaly)comparatorbasic model dsa cta940(10 ; 2,122)0.006(0.003 ; 0.009)dominateddsa mra2,007(604 ; 3,767)0.009(0.005 ; 0.015)dominateddsascenario model dsa cta40(103 ; 20)0.000(0.000 ; 0.000)dominantdsa mra84(15 ; 98)0.000(0.000 ; 0.000)dominatedctaci confidence interval (calculated based on the probabilistic sensitivity analysis) total one - year outcome and incremental cost - effectiveness ratio (icer) for basic and scenario model ci confidence interval (calculated based on the probabilistic sensitivity analysis) in univariate sensitivity analyses we explored whether changing individual parameters altered the conclusions of our base case analysis. assuming equal treatment cost for coiling and clipping did not change the conclusions. furthermore, results remained stable for the assumption of higher costs of dsa up to factor 2.8. higher sensitivity and specificity for detection of aneurysms and determination of feasibility of coiling for cta and mra up to 96 % or a reduction of sensitivity and specificity for dsa to 90 % also yielded stable results. in the scenario analysis, the strategy where cta is followed by dsa remained the least costly strategy for a sensitivity of determination of coiling within the range of 60100 %. the probabilistic sensitivity analysis showed that in the basic model analysis, dsa had a 98100 % probability of being cost - effective. in the scenario analysis, cta had the highest probability of being cost - effective (91 %), followed by dsa (9 %) and mra (0 %). because in the scenario analysis effectiveness is equal for all strategies, ceacs are constant over the different values of willingness to pay for a qaly. when a patient presents with a suspected ruptured intracranial aneurysm, imaging is a challenge. due to high risk of case fatality in ruptured intracranial aneurysms, timely detection of a causative aneurysm and determination of appropriate treatment patients may be left untreated, or their aneurysms clipped surgically, or they receive endovascular treatment. in recent years, much research has been done to evaluate the best treatment options and to optimise the approach to imaging modalities. in particular, the role and necessity of dsa in this patient population has been widely discussed [3134 ]. in our study we compared the diagnostic work - up by cta, mra and dsa based on a cost - effectiveness approach. we found that dsa is more cost - effective than cta, which is in turn superior to mra. a combination strategy of cta followed by dsa if endovascular treatment is deemed not feasible was found to be as effective as dsa alone at slightly lower expected costs. probabilities of health outcome which we clustered from the isat trial are relatively stable in the long run and do not change our results. incomplete occlusion and refilling of the aneurysm is a complication occurring mainly in coiling which can cause rebleeding, resulting in a higher rate of follow - up imaging costs and re - interventions in the endovascular group. the rate of recanalisation is highest in the first months and decreases significantly over time [3538 ]. calculation of total costs for both treatment options included not only cost for material, intensive care and standard unit days and medication but also cost of follow - up imaging and re - intervention for the first year. taking the results from literature, we can assume that the validity of our data will be stable for the following years. our basic model results show that dsa is the most cost - effective imaging modality and is superior to both mra and cta. although initial test costs are about three times higher than those of mra and cta, optimal detection of aneurysms and determination of treatment in dsa yield lower overall 1-year costs per patient and the highest qalys. the morbidity of dsa, although low, was included as an input parameter in our model [17, 24 ]. nevertheless, we did not consider the potential influence of test - related short - term disutility described by swan.. depending on the severity grade of clinical presentation, the short - term discomfort undergoing dsa experienced by patients with sah might be of varying importance and influence to our model might be less compared with in elective imaging. we assumed dsa to be the standard of reference with sensitivity and specificity for detection and determination of treatment of 100 %. it is debatable whether sensitivity of dsa to cerebral aneurysms is 100 % in an acute setting. actual costs of dsa might be higher than in our model and likely differ in other countries. we therefore performed sensitivity analysis showing stable results for assumption of 90 % sensitivity and specificity in aneurysm detection and coilability, as well as costs up to 2,025 for dsa. given this range of diagnostic performance and costs, other researchers or decision - makers can assess transferability of the results to their specific situation and jurisdiction. differences between united states cost estimates and european cost estimates, in particular, may not change conclusions, as costs are proportionally higher in the united states. the test characteristics for cta and mra are based on our study performed with 75 patients. in literature, meta - analyses of test characteristics of cta as well as other studies show higher sensitivity and specificity for cta and mra in the detection of aneurysms and determination of treatment compared with our study [4345 ]. since our basic model results are stable in sensitivity analysis with assumption of 96 % sensitivity and specificity for aneurysm detection as well as determination of treatment for cta and mra, we can assume that our conclusion is valid in a broad range of diagnostic performance. in the scenario model sensitivity analysis, increasing sensitivity and specificity for cta and mra shows even increasing preference of our imaging strategy starting with non - invasive imaging compared with only dsa. comparing the two non - invasive imaging modalities, cta dominated mra in the basic model and thus was superior. this was mainly due to lower sensitivity in determination of feasibility of coiling in mra. beside its inferiority in cost - effectiveness, mra has limitations as first line imaging in acute settings, as it might be unavailable during night hours and not applicable in severe clinical presentation. our scenario model with initial cta followed by dsa in case an aneurysm is detected but deemed not suitable for coiling was found to be the most cost - effective imaging strategy overall. the slightly lower costs compared with only dsa result from the group of patients where on cta an aneurysm is detected which is deemed feasible for coiling. in this group endovascular treatment the average reduction in costs per patient is 40, which is marginal in comparison to the overall 1-year costs. in case of suspected sah, standard diagnostic imaging is non - contrast enhanced ct (nect), followed by examination of csf, obtained by lumbar puncture, if nect is negative. in case a sah is detected on nect, performing an additional cta study can be assumed to be less expensive than the total cta cost that we used as an input parameter in our model., we might assume that the actual cost advantage of the scenario strategy will likely be higher. there are heated discussions about whether or not to always perform dsa in patients with suspected ruptured intracranial aneurysms [4756 ]. first, dsa is both more effective, yielding higher qalys, and less costly in overall costs than cta and mra. dsa is therefore superior to the non - invasive imaging modalities and should play a key role in diagnostic work - up of non - traumatic sah. second, we analysed a scenario that combines invasive and non - invasive diagnostic tests. we found the less costly and therefore most efficient diagnostic approach overall to be a strategy, starting with cta as the first examination, followed by dsa if coiling is not deemed feasible. this means that every patient undergoes additional dsa unless an aneurysm has been detected and deemed suitable for endovascular coiling. this strategy yields the same amount of qalys compared with dsa alone in all patients, but results in lower average costs per patient. in comparison to diagnostic dsa, cta is easily available during night hours and can directly be performed on hospital admission. this further emphasises cta as a first - line imaging strategy, as it may fasten clinical treatment decisions and therefore help avoid rebleedings. besides representing the most cost - effective approach overall, we believe that the strategy of combining cta and dsa also represents the most practical approach in routine clinical practice. for patients with suspected sah, when cta shows no aneurysm or shows an aneurysm which is not suitable for coiling, an additional dsa study should be performed. for diagnostic work - up of suspected ruptured intracranial aneurysms, a combined strategy of cta followed by dsa if cta is negative or endovascular treatment deems not feasible is found to be as the most cost - effective approach overall. | objectivesintra - arterial digital subtraction angiography (dsa), magnetic resonance angiography (mra) and computed tomographic angiography (cta) are imaging modalities used for diagnostic work - up of non - traumatic subarachnoid haemorrhage. the aim of our study was to compare the cost - effectiveness of mra, dsa and cta in the first year after the bleed.methodsa decision model was used to calculate costs and benefits (in quality - adjusted life - years [qalys ]) that accrued to cohorts of 1,000 patients. costs and characteristics of diagnostic tests, therapy, patients quality of life and associated costs were respected. the diagnostic strategy with highest qalys and lowest costs was considered most cost-effective.resultsdsa was the most effective diagnostic option, yielding on average 0.6039 qalys (95 % ci, 0.57610.6327) per patient, followed by cta 0.5983 qalys (95 % ci, 0.57040.6278) and mra 0.5947 qalys (95 % ci, 0.56740.6237). cost was lowest for dsa (39,808 ; 95 % ci, 37,18242,663), followed by cta (40,748 ; 95 % ci, 37,93743,831) and mra (41,814 ; 95 % ci, 38,73045,146). a strategy of cta followed by dsa if cta was negative or coiling deemed not feasible, was as effective as dsa alone at average costs of 39,767 (95 % ci, 36,90342,402).conclusiona combined strategy of cta and dsa was found to be the most cost - effective diagnostic approach.main messages we defined a standard model for cost - effectiveness analysis in diagnostic imaging. comparing total 1-year health costs and benefits, cta is superior to mra. a strategy of combining cta and dsa was found to be the most cost - effective diagnostic approach. |
it is estimated that close to 4 million skin cancer diagnoses (including basal cell and squamous cell carcinomas) are made every year. melanoma (an aggressive form of skin cancer) is diagnosed in more than 70,000 persons every year, creating a high health and economic burden with an estimated annual cost of $ 3.5 billion. risk factors for skin cancer include sun sensitivity (sunburning easily, difficulty tanning), a history of excessive sun exposure, sunburns, use of artificial tanning, and a past history of skin cancer. most of skin cancer cases could be prevented by protecting the skin from excessive sun exposure and avoiding indoor tanning. results from an analysis of national data showed that the majority of the us population reported infrequent incidence of sun protection behaviors. characteristics of groups reporting lower incidence of sun protection include being young (under the age of 40), having a lower education level, being a smoker or a risky drinker, and being less sensitive to the sun. health research should focus on the identification of psychosocial and modifiable variables to promote sun protection among groups at higher risk for skin cancer and in the general population. even when it has been documented that the hispanic / latino (referred to as hispanic) population suffers from a disparity regarding certain cancers compared to non - hispanic whites (referred to as whites), the lifetime risk of developing skin cancer is higher among whites than other racial groups. for melanoma, it is higher among whites (2.9% in men, 1.9% in women) than in hispanics (0.52% in men, 0.51% in women) [1, 4 ]. a study conducted in miami showed that, among 3000 cases of nonmelanoma skin cancer reviewed, 60.1% were diagnosed in whites and 38.4% were diagnosed in hispanics. findings using the southeastern arizona skin cancer registry showed that the rates for nonmelanoma skin cancer in whites were approximately 11 times greater than rates for latinos. a case control study of nonmelanoma cancer diagnoses in hispanics (with whites as control) showed that 15.3% of latino patients reported recurrence of their malignancy as compared to 31.3% of controls. also, a lower proportion of latinos (34.0% versus 61.3% controls) had a current diagnosis or prior history of actinic keratosis. on the other hand, skin cancer has been associated with considerable morbidity and mortality in the hispanic population. compared with whites, hispanics are more likely to have advanced and thicker melanomas at diagnosis when compared with whites [816 ]. a greater percentage of melanomas occurred among hispanics in younger age groups (24.4% less than 40 years old) compared with blacks and whites, 15.8% and 14.3%, respectively. also, hispanics tend to report lower frequency of skin - related visits to dermatologists than their white counterparts. data obtained from cancer registries of puerto rico, new york, new jersey, and connecticut show that puerto ricans living in the us report higher melanoma rates than those residing in puerto rico. at the same time, there are variations in the behaviors reported by hispanics and non - hispanics. a systematic review examined the incidence of sun protection behaviors among hispanics in the us. while a slightly lower share of hispanics (9.529.9%) report usage of sunscreen either most of the time or always compared to 16.5%35.9% of whites, hispanics reported slightly higher rates of wearing hats either most of the time or always (23.925.0% versus 2020.7%). recent studies of sun protection behaviors show that around 53% of hispanics stay in shade, and around 20% use protective clothing when outside on a warm sunny day either most of the time or always [20, 21 ]. hispanics who are less acculturated report lower rates of sunscreen use than those who are more acculturated. it is critical to identify psychosocial and modifiable factors influencing skin cancer morbidity and mortality in hispanics in the us. the community preventive service task force reviewed skin cancer prevention evidence from a community guide systematic review published in 2004 combined with more recent evidence [22, 23 ]. the review found that education interventions in primary and middle schools (kindergarten8th grade), which include strategies to integrate parents, caregivers, and teachers, decrease sun exposure, sun protection, and formation of new moles. multicomponent, communitywide interventions including a combination of individual - directed strategies (e.g., activities to change the knowledge, attitudes, beliefs, or behaviors), mass media campaigns, and policy changes are recommended based on evidence of effectiveness in increasing sunscreen use, but results for effects on other protective behaviors are mixed. in addition, findings illustrate that other approaches, such as mass media alone, provider education and media - based education sessions in health care settings, and educational activities in high school and colleges, did not provide sufficient evidence to determine their applicability for skin cancer prevention. many of these studies were conducted outside of the us (i.e., australia and the united kingdom), but the task force suggests that findings are likely to be applicable to the us because results were similar across countries. various interventions and education initiatives in the recent past have targeted minorities with the intention of improving skin cancer, but these were not multicomponent initiatives [2426 ]. a group of hispanic women evaluated two educational videos to increase positive sun protection beliefs and behaviors. there was an effect in skin cancer risk awareness postintervention, and participants reported they preferred the video emphasizing the benefits of sun protection for skin cancer prevention more than the video emphasizing its effect on photoaging. little research has examined the association between sun protection behavioral outcomes and the health outcome of interest, that is, skin cancer incidence. more research is needed to verify the efficacy of multicomponent, communitywide interventions addressing the effect of sun protection attitudes, perceptions, beliefs, and behaviors on increasing sun protection. in addition, research should evaluate its effect on decreasing sunburns (short - term effect) and skin cancer incidence (long - term effect) in the general public and in subgroups at particular risk for skin cancer. this paper examines published studies that include health beliefs concerning skin cancer prevention and sun protection in hispanics. one example of a search strategy used with the pubmed database is ((skin cancer) and hispanic) and beliefs ; ((skin cancer) and latino) and beliefs ; ((sun protection) and hispanic) and beliefs ; ((sun protection) and latino) and beliefs. we decided to use broad search terms to make sure we would identify as many pertinent studies as possible. for our search, we decided to use the word hispanic and latino to indicate our population of interest, that is, us residents of mexican, cuban, puerto rican, central american, south american, and other spanish - speaking country origins. a search in pubmed demonstrated how research, with some exceptions (including us census data and self - report), interchangeably uses these terms and lacks stratification of the members of this group. a study by the pew hispanic center found that more than half hispanics (51%) have no preference for any of the two terms to describe their ethnicity. at the same time, the term hispanic was chosen to be used in this paper given that sun protection research applies this term more frequently compared with the term latino (see table 1 for list of the term(s) for ethnicity and raced used by each study included in this review). a manual secondary search of all bibliographies from relevant articles was performed to yield further relevant publications. we excluded studies conducted outside the us, as well as studies without data for hispanic participants on the report of sun protection beliefs. studies that compared the differences in sun protection beliefs between hispanics and non - hispanics were included as well. (1) the reports had to quantitatively examine and report (frequency, means, percentages, effect sizes, and/or odds ratio) sun protection beliefs in hispanic samples, including constructs such as skin cancer risk / susceptibility and severity / seriousness, and sun protection beliefs (barriers and benefits of sun protection and skin cancer risk). (2) (3) studies that reported the differences in sun protection beliefs between hispanics and other racial and ethnic groups were used. books, book chapters, meta - analyses, comments, and reviews were excluded. the first database searched was pubmed, followed by a search of psycinfo and cinahl. a total of 86 articles were identified from these databases, and 6 articles were identified from bibliographies, for a total of 92 records (see figure 1). a search of duplicates was conducted, leaving 61 records that were title- and abstract - screened and 18 records that were screened in full. the title and abstract screening step evaluated the title and the abstract of each of the 61 articles to determine whether the abstracts met the following criteria : (1) informed about sun protection beliefs, (2) informed about the sample used (humans), (3) suggested that the study included hispanics in the sample, (4) used english as publication language, and (5) indicated that the publication was peer - reviewed. as part of the full screening step, the manuscripts from the 18 abstracts were obtained and read in full to determine whether they met the eligibility criteria : (1) the records had to quantitatively examine and report sun protection beliefs in hispanics, (2) the number of hispanic participants in the sample had to be clearly specified, and (3) studies reporting differences in sun protection beliefs between hispanics and other racial and ethnic groups were included in the review. these manuscripts were read in full, and eleven were included in the final analysis (see figure 1). data on the author, year of publication, sample characteristics, methodology used, measures selected, and quantitative results from each of the articles were abstracted and evaluated. six studies included adult participants, and three studies included children and adolescents (students in middle school and high school). one study included both adolescents and adults, and one study did not report information regarding the inclusion of participants that were under 18 years old (participants were patients at a dermatology clinic). three studies excluded participants with history of skin cancer, and five studies reported data on sun protection behaviors. two studies had survey materials available in spanish, and one study was conducted entirely in spanish. most studies (n = 10) were published during the last decade (20042014). skin cancer seriousness (severity, worry) was a belief considered in three studies [2931 ]. results from a population study showed that hispanics and whites share similar levels of worry about skin cancer (p > 0.05). another study found that perceived skin cancer severity was associated with incidence of total body examination (i.e., a head - to - toe examination of the skin performed by a physician used to identify suspicious growths that may be cancer or growths that may develop into skin), but not with incidence of skin self - examination (i.e., a head - to - toe examination of the skin performed by the individual, not a physician) [30, 31 ]. most studies included in the review (n = 8) considered skin cancer susceptibility (or risk) beliefs. one study found that hispanics believe they have lower than average risk of developing skin cancer, and that their level of risk is lower when compared with whites. a third study also found lower perception of skin cancer risk when hispanics were compared with whites but the difference was not significant when participants were asked to compare their own likelihood of getting skin cancer compared with the risk of an average person of the same age. two studies reported similar scores on their skin cancer risk and photoaging (changes in skin appearance induced by sun exposure) concern measures but used different scales for the scores [30, 34 ]. participants were inclined to not agree or disagree (midrange score) with the following statement : the natural color of my skin protects me from the sun. after quantifying qualitative information from an all - female sample, it was found that less than half of the participants believe they can develop skin cancer. on the other hand, using a different sample as part of an experiment within the same study, participants reported an increase in skin cancer susceptibility of hispanics with fair skin and hispanics with dark skin (not their own susceptibility) after watching an educational video about sun protection behaviors. another study asked hispanics about the skin cancer susceptibility of people in darker skin types, and results showed that a slightly lower proportion of hispanics (78%) endorsed this statement compared with white (91%) and black (86%) participants. another study reported that perceived skin cancer risk is associated with skin self - examination. a strong effect size was reported for association between perceived peer norms for sun exposure and barriers to sun safety in hispanic middle school students. hispanics are more likely to believe that there is not much they can do to lower their risk of getting skin cancer and that there are too many recommendations to prevent this illness. it was also reported that more than half of hispanics believe that tanning makes people look more attractive and do not endorse the belief that tanning makes people older. one study showed that hispanics tend to marginally agree more with statements regarding sun protection benefits than barriers. participants also indicated what were the most important benefits and barriers to engage in sun protection behaviors, with avoid getting sunburn and not part of my daily routine as frequently endorsed statements. this study examined published reports of sun protection beliefs in hispanics, and we found eleven manuscripts that followed the established criteria. results suggest that low skin cancer susceptibility is commonly found in this population and that hispanics moderately perceive skin cancer as a serious health threat. results also suggest that assessments of sun protection barriers and benefits vary significantly by study. overall, findings illustrated that there are limited studies on psychosocial and modifiable factors that influence sun protection. many of the studies included in this review have limited sample size or used samples that do not represent the heterogeneous hispanic population (e.g., 70% of participants in one study were of mexican origin) [30, 31 ]. an evaluation of interventions designed to educate primary care physicians about skin cancer showed a lack of uniformity across interventions and outcome assessments, preventing the direct comparison of intervention efficacy and the dissemination of effective components. skin cancer can be prevented by practicing sun protection, but skin cancer disparities might be associated with the perceptions, knowledge, attitudes, and beliefs hispanics hold regarding skin cancer and sun protection. it has been found that individuals who express the benefits of sun protection are likely to report sun protection behaviors consistently more than those who communicate the barriers of sun protection [4042 ]. hispanics are more likely to believe there is little they can do to lower their chances of getting skin cancer, that there are so many recommendations about skin cancer prevention that they do not know which one to believe and believe that they are below average risk for skin cancer compared with whites. it is critical to understand the sets of beliefs that underlie sun protection among hispanics and improve health promotion initiatives to decrease sun protection disparities. most common types of skin cancer are squamous cell carcinoma and basal cell carcinoma (scc and bcc, resp. ; basal cell carcinoma diagnoses are more common in hispanics than squamous cell carcinoma and melanoma diagnoses [43, 44 ]. while person characteristics (e.g., light skin, sun sensitivity, and blistering sunburns early in life) and intermittent sun exposure are strong risk factors for the diagnosis of melanoma, both cumulative and intermittent sun exposure are the most common cause of basal cell carcinoma. in terms of presentation, melanoma usually involves sites not exposed to the sun, including palmar, plantar, and mucosal surfaces, and the lower extremities. areas such as the head and the neck regions seem to be more prone for basal cell carcinoma. literature in sun exposure at the workplace indicates an elevated risk for scc but is less conclusive for bcc. a population - based control - study among individuals diagnosed with invasive melanoma found that frequent sunscreen use when not planning to be in the sun during the last 20 years was strongly associated with lower likelihood of melanoma. also, those who reported use of sun protection (not sunscreen) were at lower risk of developing melanoma, even if its use was inconsistent. consistent with the compensation hypothesis of sunscreen use and increased sun exposure, optimal use of sunscreen spf+15 was associated with highest amount of sun exposure. research directly associating sun protection behaviors to decreased skin cancer risk is limited and inconsistent. the present study shows that research still struggles to investigate and understand the specific factors that might be associated with melanoma and nonmelanoma skin cancer incidence, and disparities in skin cancer. research should clarify the association between the disease, the target population, and the particular mechanisms to prevent the disease. previous research shows a moderate level of awareness about skin cancer risk factors and prevention behaviors among hispanics. using a qualitative approach, results illustrated that participants did not recognize possible indicators of skin cancer risk (e.g., painful sunburns). one study showed that more hispanics do not use sunscreen because they perceive themselves as dark skinned when compared with whites and asian / pacific islanders (29% versus 3% versus 11.4%, p < 0.05). results included in the present review emphasize the need for improved assessments of sun protection beliefs and to incorporate the evaluation of the meaning and significance hispanics give to sun protection : do they know what protective clothing is ? ; what do they think about using sunscreen all the time when out in the sun, including cloudy days ? ; would they wear a hat at all ? ; do they know how their skin reacts to sun exposure ? these are questions that future research should address if we want to report a more accurate analysis of sun protection. this review underscores the importance of developing culturally relevant, validated, and reliable measures of sun protection and skin cancer risk perception for hispanic adults, adolescents, and children. our search was limited to peer - reviewed journals, which generally publish studies with significant results. it was also limited to results on sun protection beliefs in hispanics living in the us. findings can not be generalized to studies conducted in other countries, and there is a possibility that important elements of sun protection were not captured in our review. a strength of this review is that it helped us realize that the full potentials of the assessment and applications of sun protection beliefs for the prevention of skin cancer in hispanics remain largely unverified and untested. this finding should open the doors to many research initiatives to promote health in a growing minority population and to identify and understand factors that contribute to disparities in the incidence and mortality of cancer. hispanics are a diverse group that exhibit differences in terms of sun protection behaviors, sun sensitivity, level of acculturation, country of origin, access to health services, and socioeconomic status. future research should develop comprehensive, culturally sensitive measures of sun protection beliefs, facilitators, and barriers. measures should be grounded in theory, research evidence, and ethnographic study. also, researchers must ensure that their recruitment strategy attains a more diverse sample than previous research. the national cancer institute states that overcoming cancer health disparities is one of the best opportunities we have for lessening the burden of cancer. it is goal of the institute to improve the understanding of the causes of cancer health disparities as a way to eliminate them. it is critical to identify modifiable factors that can reduce skin cancer morbidity and mortality disparities in the us. there is a need for informed, culturally sensitive measures to assess sun protection in the hispanic population in order to (1) directly inform the development of a study to investigate the ability of sun protection beliefs to predict the likelihood to engage in positive health outcomes (i.e., sun protection behaviors), (2) make informed assessments of the effect of sun protection on skin cancer morbidity and mortality, (3) contribute to the limited literature on sun protection in hispanics, (4) inform the development of targeted public health recommendations and initiatives to increase sun protection, and (5) make a contribution to the identification and understanding of experiences hispanics have regarding sun protection. | purpose. we reviewed the literature on sun protection beliefs in hispanics living in the united states to explore what challenges are faced by area of research. method. a review of pubmed, psycinfo, and cinahl databases was performed. studies were published in peer - reviewed journals (in all years available) and written in english. the search terms used were [skin cancer or sun protection ] and [latino or hispanic ] and beliefs. eligible papers were included in the final analysis after meeting the following inclusion criteria : (1) the records had to quantitatively examine and report sun protection beliefs in hispanics, (2) the number of hispanic participants in the sample had to be clearly specified, and (3) studies reporting differences in sun protection beliefs between hispanics and other racial and ethnic groups were included in the review. results. of the 92 articles identified, 11 met inclusion criteria and addressed sun protection beliefs regarding skin cancer seriousness and susceptibility, and benefits and barriers of sun protection and skin cancer risk behaviors. characteristics of studies and results were examined. conclusion. there is insufficient evidence to determine a pattern of sun protection beliefs among hispanics in the united states. more quality studies are needed which focus on sun protection beliefs in hispanics. |
the defects of the anterior oral cavity or the floor of the mouth may lead to significant functional and cosmetic problems. if the defect is under 2 cm in size, it may be effectively treated with primary closure. however, if it is larger, it may require local or distant flaps. tongue, buccal mucosa, or other regional flaps have been used to solve the problem. regional flaps are widely used to address the problem, but sometimes they may be too bulky for small defects. the nasolabial flap is a type c fasciocutaneous flap, based on the underlying angular artery. advantages of the flap are relatively easy and it is a short operative procedure compared to other reconstructive techniques, also the donor site morbidity is minimal. the flap has widely been used in the reconstruction of the lip and floor of the mouth, however, there are few reports on the usage of this method in the reconstruction of the anterior oral cavity. we hereby present this patient as a good example for the versatility of the nasolabial flap in anterior oral cavity defects. a 56-year - old woman presented with a scar in the right upper gingivobuccal sulcus, involving the right commissure of the mouth [figure 1a and b ]. in the history, she had a recurrent dental abscess after using a dental prosthesis, and she had a purulent wound on the right modiolus, involving the right upper gingivobuccal sulcus. she was previously operated twice for the same deformity elsewhere. in the first operation, a dermal fat graft was used. on account of the poor take of the graft, a second operation was performed combining the release of the contracture with a skin graft and lipofilling the depression in the soft tissue. (a, b) a 56-year - old woman presented with a scar in the right upper gingivobuccal sulcus, involving the right commissure of the mouth in our operation, after release of the scar, a nasolabial flap with a diameter of 2 cm was prepared on the same side [figure 2a ]. the flap was tunnelized to fill up the defect, just superior to the commissure [figure 2b ]. three months postoperatively, the appearance of the flap in the mouth was good and aesthetic, and the functional outcome was satisfactory [figure 3a and b ]. (a) during the operation, after release of the scar a nasolabial flap with a diameter of 2 cm was prepared on the same side. (b) the flap was tunnelized to fill up the defect, just superior to the commissure. the tunnel from the cheek skin through the buccal mucosa was made by blunt scissor dissection (a) early postoperative result at one month. (b) the appearance of the flap in the mouth was considered to be satisfactory the nasolabial flap has widely been used for an intermediate - sized defect of the cheek, upper and lower lip, palate, and especially, the anterior floor of the mouth. the main advantages of the flap in facial reconstruction are its accessibility, ease of dissection, good color match, and minimal donor site morbidity. evaluation of the depth of the sulcus is important for the mobility of the tongue, which is paramount in proper speech, therefore, integrity of the sulcus and sufficient reconstruction is necessary. for larger defects of the floor of the mouth, it may be necessary to use a submental flap, platysmamyocutaneous flap, buccal mucosa flap, or a free flap. however, especially in the case smaller - to - medium size defects that do not cross the midline, a nasolabial flap can be a sound option. in our patient, the nasolabial flap was chosen as the best option for reconstruction, because of the limited size of the defect. one of the advantages of the nasolabial flap is its good vascularity. the flap is reliable even after facial artery ligation. napolitano and mast have stressed the rich collateral blood supply of the cheek, deriving from the masseteric, buccal, infraorbital, and transverse facial arteries, and they have found the nasolabial flap to be quite reliable. conventional vestibuloplasty techniques require repositioning of the supportive musculature and adjacent mucosa, followed by resurfacing with a skin graft. three basic treatment philosophies exist in vestibuloplasty ; (1) submucosal vestibuloplasty, (2) secondary epithelialization vestibuloplasty, and (3) soft - tissue grafting vestibuloplasty. submucosal vestibuloplasty can be performed easily under local anesthesia, however, the outcomes are poor, because of the high incidence of relapse. the kazanjian vestibuloplasty, also known as the lip - switch, involves utilization of a bipedicle labial mucosal flap, to resurface the prepared alveolus to the depth of the neovestibule. this technique is used mainly in the anterior mandibular region, but can be applied to the anterior maxilla as well. a similar technique, the edlan vestibuloplasty, places an incision at the alveolus, and supraperiosteal dissection is performed anteriorly, preserving a pedicled mucosal flap at the level of the lip. this mucosal flap is used to resurface the labial surface of the vestibule, and the alveolar component is left to heal secondarily. the inherent problem with secondary epithelialization vestibuloplasty is the uninhibited wound contracture and relapse through loss of vestibular depth. understanding the principles of wound healing associated with this concept led to the development of soft - tissue grafting vestibuloplasty. contemporary reconstruction of the oral vestibule is performed using keratinized or non - keratinized mucosa or split - thickness skin grafts applied to a de - epithelialized alveolar ridge. in the present case, similar vestibuloplasties were attempted twice elsewhere, however, they failed to give a satisfactory result. we considered using a nasolabial flap in our patient, as the defect that occurred after release of the scar was not large. a possible disadvantage of the technique is that occasionally rotation of the hair - bearing tissue into the oral cavity is possible in the male. in conclusion, the inferiorly based nasolabial island flap was found to be a good method, providing thin and pliable soft tissue coverage. we recommend the utilization of the nasolabial flap in reconstruction of small - to - medium sized defects of the anterior oral cavity. | a 56-year - old woman with a recurrent depressed scar of the commissure, treated with a nasolabial island flap, is presented. on examination, the scar was located on the right modiolus involving the right upper gingivobuccal sulcus. a history of recurrent canine abscess was obtained. after excision of the scar and release of the vestibular fold, reconstruction of the defect was performed with a nasolabial island flap from the same side. the postoperative course was uneventful, with a good aesthetic and functional outcome. |
the incidence of cutaneous malignant melanoma (cmm) has been increasing at a steady rate in fair - skinned populations around the world for decades [112 ]. scientists are not certain why cmm has been steadily increasing over the decades, but strong intermittent uvb (290320 nm) exposures, especially sunburn episodes, evidently initiates cmm. the uva (321400 nm) passing through glass windows in offices and cars has been proposed to promote cmm. in support of those possibilities exists the paradox between indoor and outdoor worker 's uv exposures and their incidences of cmm. although outdoor workers get three to ten times the annual uv dose that indoor workers get [15, 16 ], they have similar or lower incidences of cmm. scientists think the increasing incidence of cmm is linear based on surveillance epidemiology and end results (seer) data in the usa that only dates back to 1973, but it may actually be exponential in the usa and in some other regions of the world. to understand what factor(s) may be responsible for the increasing incidence of cmm, one must know the temporal incidence for as many decades as possible. because whatever the causative agent is, or agents are, it must have entered or left our environment some time (1030 yrs) before the increasing trend was first documented back in 1935. thus, one can analyze the cmm incidence data for fair - skinned people around the world and plot it temporally by each country in the northern and southern hemispheres and by latitude. further analysis of r values can determine if the curves are linear or exponential. this paper will analyze the cmm incidences of fair - skinned populations all over the world, test if the increases are exponential or linear, and show that the increasing incidence decreases with increasing latitude until 50n, where it reverses and begins to increase with increasing latitude in northern europe. one can obtain cmm incidence 's throughout the decades from 1935 to 2007 for australia, new zealand, usa (except connecticut 19351940 and 1945 and new york state 1955), middle europe, canada, and northern europe from the international agency for research on cancer (iarc) [311 ]. the cmm incidence data from iarc was averaged from the following states / provinces / territories to get a mean value for each country at average latitude (table 1 and figure 1) : australia (30s ; range 19.542.5s)queensland (19.5s), western (24s), south (32s), new south wales (33s), capital territory (35.5s), victoria (36.5s), and tasmania (42.5s). usa (40n ; range 2047n)hawaii (20n), los angeles, california (34n), atlanta, georgia (34n), new mexico (34n), san francisco, california (38n), utah (39n), connecticut (41.5n), iowa (42n), michigan (43.5n), new york state (43n ; excludes new york city), and washington state (47n). middle europe (49n ; range 4652n)switzerland (46n), slovenia (46n), romania (46n), hungary (47.5n), slovakia (48.5n), france (48.5n ; bas - rhin), germany (49.5n ; saarland), all of poland (51n), the netherlands (52n), and all of england (52n). canada (52n ; range 4565n)nova scotia (45n), new brunswick (46.5n), prince edward 's island (46.5n), ontario (51n), newfoundland (53n), quebec (53n), alberta (54n), british columbia (54n), manitoba (54n), saskatchewan (54n), and northwest territories (65n). northern europe (60n ; range 5365n)ireland (53n), denmark (56n), scotland (57n), sweden (62n), iceland (63n), norway (64n), and finland (65n). figure 2 has the same cmm incidence data for the year 2000 as shown in figure 1 only plotted by latitude [111 ]. figures 3(a) and 3(b) show cmm incidence data from [111 ], which includes 10 registries, the same 9 registries as seer 9 along with los angeles. figure 4(a) cmm incidence data for the usa (19732007) is from the seer website at (http://seer.cancer.gov/faststats/selections.php). the data type is seer incidence, the statistic type is age - adjusted, the year range is 19752007 (seer 9), and the race / ethnicity is white (includes hispanics), for both sexes of all ages. seer 9 (white includes hispanic) compared to seer 17 (non - hispanic white) has 1 - 2/100,000 people lower incidence of cmm. the seer 9 registries are atlanta, georgia ; connecticut ; detroit, michigan ; hawaii ; iowa ; new mexico ; san francisco - oakland, california ; seattle - puget sound, washington ; utah. data are available for cases diagnosed from 1973 and later for these registries with the exception of seattle - puget sound and atlanta. the seattle - puget sound and atlanta registries joined the seer program in 1974 and 1975, respectively. seer 11 includes los angeles and san jose - monterey, california starting in 1992. figure 4(b) shows the same data as in figure 4(a) only extended back to 1940 using data from [111 ]. table 1 contains the averaged cmm incidences of fair - skinned populations around the world from 1940 to 2000 for each country or region of the world [111 ] : australia (~30s), new zealand (40s), usa (40n), middle europe (49n), canada (52n), and northern europe (60n). new zealand has the highest incidence of cmm closely followed by australia while middle europe has the lowest incidence. figure 1 shows a temporal plot of the cmm incidence from 1940 to 2000 [111 ]. note that australia, usa, middle europe, and canada all have exponential increases except new zealand and northern europe, which have linear increases. in fact, all the countries of northern europe have linear increases in cmm except iceland, which is exponential. figure 2 shows the incidence of cmm in 2000 decreases with increasing latitude up to 50n where it changes and begins to increase with increasing latitude in northern europe [111 ]. notice that the cmm incidence increases with decreasing latitude ; however, near 50n in northern europe the incidence begins to increase with increasing latitude. note that the cmm incidence data from 1960 to 2000 all show the same change near 50n (see table 1). figure 3 shows the cmm incidence data in the usa analyzed in different ways to know whether or not the increase is exponential or linear [111 ]. figure 3(a) shows how the cmm incidence data at 39n can be linear if we only average san francisco and utah together. the data at 43n is exponential because connecticut is included in that data set. figure 3(b) shows that when the connecticut cmm incidence data is included with the 39n data, it becomes exponential, while the data at 43n becomes linear. connecticut data extends a couple of decades further back in time (to 1935), which may be necessary to know if the trend is truly linear or exponential but might represent underestimates in cmm during that earlier time frame that would make the data appear to be exponential when it is really linear. figure 4(a) shows the seer 9 data from 1975 to 2005, which appears to be almost perfectly linear (r is 0.9954), possibly because it does not extend far enough into the past as the data presented in figures 1, 3(a), and 3(b). figure 4(b) shows the same seer 9 data in figure 4(a) only extended back in time to 1940 using references through. this changes the usa data from a linear (r is now 0.9189) to an exponential (r is 0.9755) increase in the incidence of cmm. the incidences of cmm in fair - skinned, indoor - working people have been increasing worldwide for decades (figure 1). the countries with the highest incidences per annual erythemally weighted uv dose are closest to the equator : australia (30s), new zealand (40s), and the usa (40n). the regions of the world with lowest fair - skinned incidences of cmm are in middle europe, around 49n ; however, the decreasing trend with increasing latitude changes around 50n so that canada (52n) and northern europe (60n) have higher incidences than middle europe (figure 2). in fact, northern europe at 60n has a higher incidence than canada at 52n and almost equates the incidence in the usa at 40n. the increasing incidence of cmm appears to be exponential in most regions except europe ; however, as analysis in figures 3(a) and 3(b) show, data in the usa extending to 1940 can make the cmm incidence appear to be exponential. the seer data from 1973 to 2007 suggests that the cmm incidence is linearly increasing in the usa (r is 0.9954, figure 4(a)). however, when the connecticut [1, 2 ], new york state, and iarc data [311 ] extend the seer data back to 1940, the incidence of cmm in the usa increases in an exponential manner (figure 4(b)), possibly indicating that longer periods are needed to know if the increase is truly linear or if it is really exponential. thus, we can not be completely certain if the increase is linear or exponential in other countries because it may be that the data has to be collected for five decades or more to be conclusive. whether or not the incidence of cmm is increasing linearly or exponentially does not change the fact that it is increasing at the alarming rate of about 4 - 5% per year. in order to slow or stop this increasing trend based on the temporal plot shown in figure 1, we know whatever started the increasing incidence of cmm either entered or left our environment before 1935, because that is when we have documented data for the first increases in cmm in the usa. fluorescent lights (mid-1940 's ;), sunscreens (late 1950 's for uvb absorbing and 1988 for uva and uvb absorbing ;), and tanning devices (1978 ;) all entered our environment after the increasing incidence of cmm was first documented in the usa back in 1935. thus, one should analyze what happened before 1935during the early 20th century to discover what may have really affected the incidence of cmm. in the early 20th century, people went against evolution by going indoors during the day to work, which drastically decreased their daily amount of cutaneous vitamin d3 and exposed them to only uva radiation passing through glass windows. the artificial uv barrier created by windows divided uvb from uva, so that the vitamin - d - making uvb wavelengths were excluded and only the vitamin - d - breaking and dna - mutating uva wavelengths [2325 ] were included in our indoor - working environment. possibly because this unnatural uv environment existed for decades in buildings and later in cars, cmm was promoted by uva, after being initiated by uvb sunburns and began to steadily increase in the mid-1930 's. along these lines of reasoning, we now also have the increasing incidence of cmm with increasing latitude above 50n (figure 2). people living above 50n go to the beach during the summer and get sunburned at lower latitudes to initiate cmm and then return home to northern latitudes that have primarily uva for most of the year to promote cmm. the higher latitudes also allow the sun to aim more often at a perpendicular angle to the window glass allowing more uva to pass through and directly expose people 's skin during their workday. in addition, above 50n there is little uvb to make cutaneous vitamin d3 most of the year. further, in the northern regions of the world (above 37n), a vitamin d3 winter occurs from at least november to february, which extends from october to march at higher latitudes, when the dose rate of uvb is too low to make any previtamin d3 even if an office worker goes outside during peak hours. on the other hand, uvb exposure during peak hours occurs to outdoor workers to some extent during their workweek, so that they can maintain adequate levels of vitamin d3 in their skin and blood (as 25-hydroxyvitamin d) for most of the year. note that the blood levels of vitamin d (measured as 25-hydroxyvitamin d in serum) in outdoor workers (gardeners), who get about five times the solar dose that indoor workers get, are about twice as high as indoor workers. the reason vitamin d is important for controlling melanoma is because it can be converted to the hormone calcitriol inside melanoma cells. calcitriol can control the growth [3032 ] and apoptotic cell death of responsive melanoma cells, while it also affects the immune system [34, 35 ] and inhibits tumor promotion, which may all be responsible for increasing the survival of melanoma patients who get regular, moderate sun exposures. thus, intermittent, strong uvb - induced sunburns may initiate cmm, while low concentrations of vitamin d3 in the skin and uva - induced dna damage may promote cmm. the incidence of cmm is increasing at an alarming rate around the world in fair - skinned, indoor - working populations, and may be increasing at an exponential rate. the cmm incidence decreases with increasing latitude up to ~50n where it changes and increases with increasing latitude. this inverse may occur because there is more uva relative to uvb for most of the year at higher latitudes compared to lower latitudes. if windows, allowing uva to enter our indoor - working environment and cars, are at least partly responsible for the increasing incidence of cmm, then uv filters can be applied to office and car windows to help reduce the rate of increase in the incidence of cmm worldwide. | the incidence of cutaneous malignant melanoma (cmm) has been increasing at a steady rate in fair - skinned populations around the world for decades. scientists are not certain why cmm has been steadily increasing, but strong, intermittent uvb (290320 nm) exposures, especially sunburn episodes, probably initiate, cmm, while uva (321400 nm) passing through glass windows in offices and cars probably promotes it. the cmm incidence may be increasing at an exponential rate around the world, but it definitely decreases with increasing latitude up to ~50n where it reverses and increases with the increasing latitude. the inversion in the incidence of cmm may occur because there is more uva relative to uvb for most of the year at higher latitudes. if windows, allowing uva to enter our indoor - working environment and cars, are at least partly responsible for the increasing incidence of cmm, then uv filters can be applied to reduce the rate of increase worldwide. |
past theoretical mechanisms have failed to explain the observed, postoperative hypothermia in laparoscopic patients. mismatch of tissue and inflation gas thermal capacities rule out any substantial global tissue temperature reductions during laparoscopic procedures. overlooked, however, is the possibility that severe hypothermia is due to local super - cooling of tissue caused by evaporation from tissue surfaces of peritoneal fluid water into the dry jet of insufflation gas. this mechanism is examined analytically and experimentally and is found to be real and significant. patient hypothermia during laparoscopic surgery is widely reported in recent literature. while this clinical condition and its undesirable consequences to the patient are well understood, the exact cause of the additional hypothermia due to laparoscopy is not clear. it is reported that the heat capacity effects of the co2 insufflation gas are not sufficient to cause physiologically significant, bulk tissue cooling. the question remains, therefore, what is the cause of a patient 's additional hypothermic response observed during laparoscopic surgery ? at least one possible causative factor could be severe cooling of local tissue surfaces through the evaporation of peritoneal fluid by the jet of dry insufflation gas (usually co2) impinging on the epithelial surface while the gas is being introduced to the peritoneal cavity through a trocar or other device, such as a veress needle. this study was designed to explore the possibility that such a mechanism could result in substantial cooling of surface tissue during the initial abdominal insufflation and/or subsequent insufflation during the laparoscopic procedure. a theoretical analysis was completed of the evaporative cooling effects caused by the simultaneous tissue - conductive, and gas - convective, jet heat and mass transport that reasonably could be expected to occur during typical insufflation. laboratory measurements of the temperature transient occurring when a dry co2 gas jet impinged on simulated and animal tissue material was obtained to verify the results of these theoretical calculations. the experimental studies included measurement of the effects of procedure variables : insufflation gas flow rates ; height 's of the gas jet ; tissue cooling - area size ; type of gas entry port ; tissue type ; and, most importantly, the thermal / humidity condition of the insufflation gas stream. table 1 summarizes the range of these variables for which theoretical and experimental results were obtained in this work. evaporation jet cooling of the epithelial tissue occurs when the end of a veress needle or trocar is positioned close to a tissue surface, as shown in figure 1. the insufflation gas exits from the end of the veress needle or trocar (the nozzle of figure 1) in a free vertical jet. when the gas jet reaches the epithelial surface (impingement surface of figure 1), the gas flow is redirected horizontally and flows radially away from the centerline of the entry port on the impingement surface (the stagnation point). because of a very rapid flow transition on the epithelial surface, high heat and mass transfer rates are generated in the area of the stagnation point with the maximum effect occurring at the stagnation point. when the insufflation gas is dry, as it is in all laparoscopic procedures, a large evaporation - driving force is generated in the resulting gas phase boundary layer, with velocity profile transitions, in very high evaporation rates around the stagnation point. the energy needed to evaporate fluid from peritoneal tissue surfaces is from jet evaporation and cools the surface at a rapid rate. how fast, how long, and how much tissue will be cooled and to what temperature is a complex function of the gas flow rate (f 1/m), the height of the gas injection device above the tissue (h mm), the dimension of the gas injection device (d, the diameter of the circular entrance port, or w, the width of the annular jet from a trocar), the size of the cooled tissue area (measured by r, the distance from the stagnation point for the circular jet, or x, the distance from the center of slot width w of an annular jet), the magnitude of the tissue 's thermal parameters of thermal conductivity and diffusivity and, most important, the relative humidity of the insufflation gas. the theoretical analysis and. experimental verification of the theoretical results were obtained through in vitro measurements of jet cooling of synthetic and animal tissue surfaces at various flow and geometry conditions known to influence the cooling rates. the rapid tissue cooling rates were measured with single and four thermocouple (t / c) configurations that detected tissue surface temperature at the stagnation point and with the four t / c configuration, at progressively larger distances (r) from the stagnation point (figure 1). temperatures were collected by computer data acquisition at rates of 5 to 15 points per second and stored for analysis and display. great care was taken during the experimental measurements with the apparatus seen in figure 1 to assure that the surrounding conditions of temperature (t) and humidity simulated conditions that are experienced in the human abdomen. much theoretical and experimental work has been done on heat and mass transfer into and from circular and slot gas jet streams. the mass transfer elements of these studies, however, have been limited to sublimation of a solid into a gas jet ; and gas jet evaporative mass transfer of a liquid surface has been studied very little. for this study, we have used the well known and reliable heat - mass transfer analog for convective flow, making it relatively simple to circumvent this lack of direct experimental correlations. referring to figure 1, the impingement surface of our model is wet with peritoneal fluid and is cooled by latent evaporative heat flux into the jet of dry co2 insufflation gas. the heat transfer flux due to this evaporative mass transfer is characterized by the equation, where he is the evaporative heat transfer coefficient, ps, the vapor pressure of the surface fluid (a function of surface temperature), and is the jet gas 's relative humidity (assumed zero for this analysis). as has been discussed, the heat and mass transfer coefficients needed in equation 1 vary considerably from a maximum at the stagnation point to decreasingly lower values at positions r (or x for a slot jet) away from the circular jet center line. the integral mean values for the transfer coefficients in these regions in terms of r and x ; the diameter, d, of the circular jet (or w, the width of the slot jet) ; and the height of the nozzle, h, from the impingement surface have previously been determined and is called the martin 's nusselt number. from the martin 's nusselt number correlations we obtained mean (with r or x) heat transfer coefficients, he. the classic heat - mass transfer analog is in the form where sh is the sherwood number, nu = nusselt number, sc = schmidt number, and pr is the prandtl number. equation 2, with sc = 0.49, pr = 0.75 for diffusion of water through co2, permitted the calculation of the mass transfer coefficients hd. the evaporative heat transfer coefficient, he of equation 1, was determined by the simple conversion of the mass transfer coefficient, hd, from concentration units to partial pressure units, with assumptions of ideal gases and the boundary layer mean temperatures and the known values of water 's heat of vaporization. this led to the following simple relationship : thus, with martin 's correlations for nusselt number as a function of entry port type (gas jet stream), size, orientation, flow conditions, and height, h, above the impingement surface, and with equations 13 described above, it is possible to determine the mean (between the stagnation point and the position r or x) heat flux due to evaporation for any set of flow and entry port configuration conditions. these values then become the critical boundary conditions for the unsteady state, tissue - conduction problem. the speed that peritoneal tissue will be cooled by evaporative jet cooling is governed by the jet 's evaporative surface heat flux, jh, e, the size of the jet, and the conduction characteristics of the tissue. to simplify the analysis, it was assumed that the very high heat fluxes caused by the evaporative gas jet would completely dominate tissue internal heating due to metabolism and perfusion near the tissue surface. additionally, it was assumed that the tissue dimensions would be large in comparison to that of the gas jet, for example, very large conduction pathways. under these conditions, the general transient heat conduction differential equation reduces to where, for our model, the reduced temperature, theta, is defined as and the boundary conditions for evaporative surface cooling, with c = je / k where k is the surface tissue 's thermal conductivity, are as follows : this classic transient - conduction problem has been solved by many. the solution for the general case, as well as the special situation of x = 0, or the impingement surface temperature of our model can be expressed as where, for our jet evaporation system, c = jh, e / k and solutions to equation 5 are complicated by the fact that the value for c = jh, e / k is itself a nonlinear function of surface temperature, t, through jh, e and its dependence on the vapor pressure term ps which, in turn, is a nonlinear function of surface temperature. this results in an equation 5 that is transcendental in t and must be solved numerically by iterative, non - constant interval, finite - difference techniques. figure 2 shows the results of an equation 5 calculation for the surface tissue temperature transient, caused by evaporative gas jet cooling of a circular area of 0.79 sq. the data of this figure are calculated for a veress needle height over the tissue of 5 mm and for insufflation gas flows of 1 to 11 liters per minute. a typical set of surface temperature cooling curves for evaporative jet cooling via a trocar gas injection device is shown in figure 3. as with the veress needle theoretical results, the effect of insufflation gas flow rate is indicated parametrically for co2 flows from 1 to 11 liters per minute. experimental measurements have been made to verify the results of the theoretical analysis of laparoscopic evaporative gas jet cooling. the first (phase 1) was a series of tests using a single thermocouple (t / c) measurement, at the stagnation point = t1, of the temperature transient detected in 3 mm thick sections of synthetic materials ; cellular polyurethane (cp) and woven rayonpolyester (wr). data were collected at a rate of five points a second for phase 1 measurements. a second series of tests (phase 2) consisted of measurements using cellular polyurethane and woven rayon, and 3 mm sections of animal tissue consisting of turkey or ham. a four thermocouple array, with the first t / c (t1), was located at the stagnation point, and the remaining t / cs (t2, t3, & t4) were spaced on a radial line 1, 2 and 4 mm from the first thermocouple, respectively. phase 2 temperature data were collected simultaneously from each t / c at a rate of approximately 15 points per second for each thermocouple. the t / cs for this series, as were those of phase 1, were small (0.2 mm diameter) sensors, with fast response times (time constant 0.6 sec), and positioned from below, at or just slightly below (0.2 mm) the impingement surface. a 1 mm, inside diameter, veress needle and a standard 10 mm trocar were used as the insufflation gas injection ports. these were positioned 2 to 5 mm above the tissue surfaces, and gas flows were varied between two and eight liters per minute. the experimental jet / impingement surface configuration of figure 1 was totally enclosed in a transparent container to provide an isolated environment that reproduced the gas space, temperature, and humidity conditions that the gas injection device and impingement surface experience during a laparoscopic procedure, (co2, 37c and = 100%). during all tests the tissue surface was irrigated with a temperature controlled sterile saline drip at 37c. figure 4 shows the results of phase 1 experimental tests of evaporative jet cooling at the stagnation point on a wet cellular polyurethane impingement surface at 37c. experimental temperature transient - cp material. the results of a typical phase 2 experimental test are seen in figure 5, which shows the surface temperature cooling transient for ham tissue when evaporatively cooled by a 1 mm circular jet at approximately 6 liters per minute flow rate. these data are typical of the measurements obtained at the flow rates and jet height tests of this study. figure 6 shows typical impingement surface cooling transients resulting from an approximately 6 liters per minute co2 flow rate from a 10 mm trocar. experimental temperature transients - thick ham. experimental temperature transients - thick ham. the effects of changes in insufflation gas flow rate and gas port height above the impingement surface on tissue temperature decrease with the initiation of evaporative surface cooling. the average of replicate runs of temperature measurements of thermocouple tc1 (the impingement point temperature), measured five seconds after initiation of the insufflation gas flow, is plotted against various gas flows. the parameters of figure 7 represent lines of constant height, h, of the tip of the injector above the tissue surface gas flow effects on tissue temperature after five seconds. to confirm that the rapid temperature drops demonstrated in the tissue tests of this study were caused primarily by evaporation of tissue - surface liquid, a series of experiments were carried out in which the insufflation gas was both heated (to 37c) and humidified (to > 95 relative humidity) prior to its introduction to the insufflation gas injection port. a typical result of these tests is shown in figure 8, where thermocouples tc1 through tc4 measured the tissue surface temperatures at various distances from the impingement point for approximately eight seconds after initiation of the gas flow rate of approximately 6 liters per minute. experimental temperature transients - thick ham. the results of the analytical modeling of evaporative jet cooling of tissue surfaces gives dramatic indications of rapid and significant cooling of these surfaces, as seen in figures 2 and 3. these results predicted that, for circular jets, tissue surface temperatures could drop to under 20c in less than three seconds after initiation of insufflation gas flow and cool a tissue area of more than 0.75 square centimeter. our modeling of this cooling effect showed serious tissue temperature drops for a wide range of delivery port types, which resulted from jet streaming of gas and, importantly, for jet heights (h) up to 1 cm. comparison of figures 2 and 3 demonstrates that the decrease of surface temperature is relatively independent of type of gas injection device, although the veress needle injector caused a somewhat faster tissue - cooling rate (1.2c / s) than the 10 mm trocar. results, however, from the trocar analysis, predicted that considerably more tissue surface area would experience the cooling effect (1.9 cm). phase 1 experimental testing (figure 4) showed very large cooling rates of 30c / s in the first second of gas flow ; larger, in fact, than cooling rates predicted by the analytical model. the temperature for these tests tended to reach an equilibrium level within two to five seconds of the initiation of the evaporative gas jet. this effect most certainly is due to the jet dispersing and evaporating the fluid in the region of the t / c. under these conditions, the slower evaporation rate could be offset by internal heat conduction to the tissue surface. phase 2 results (figures 57) confirm the theoretical prediction of the degree of evaporative cooling on wet tissue both qualitatively and quantitatively. at insufflation flow rates above 51/m, significant areas of tissue (> 2 cm) were cooled to temperatures less than 18c within six seconds of initiating gas flow. these cooling effects, as can be seen in figure 7, were relatively independent of the height, h, of the gas port site above the tissue surface. that these cooling effects are caused by evaporation of surface fluid into jets of dry co2 is demonstrated by the results of figure 8, where pre - heated and humidified insufflation gas jet streams with flow rates over 5 1/m caused less than 1c of local surface cooling. in some cases, local tissue was heated approximately 1c. these small temperature rises or drops experienced with the introduction of heated / humidified insufflation gas were caused by small temperature differences (positive or negative 1c) between the temperature of the incoming gas and the temperature of the injection device. the degree of tissue cooling, with either the veress needle or the trocar gas injector were found, in phase 2 experiments, to be independent of the types of tissue tested. the differences in maximum cooling rate and/or the minimum temperature reached by the tissue in the first five to seven seconds of evaporative jet cooling were not statistically significant for the different types of tissue tested. the crossing of tc1 and tc2 curves after two seconds of evaporative cooling as shown in figure 7, most likely is due to drying around the stagnation point. tissue heat conduction from the sub - surface regions toward the stagnation point (sp) would heat this region after most of the resident liquid is evaporated and its cooling effect decreased. the analytical modeling and experimental testing of this study show that substantial evaporative cooling occurs to extensive local regions of wet surface tissue when dry insufflation gas flows against its surface. cooling rates of 20c / s and local tissue temperature reductions of 20c were measured. these findings suggest that local neurological reaction and tissue damage are possible from such hypothermic events and that this can be overcome. clinical observations and improvement of peri- and postoperative patient hypothermia by heating and humidifying the gas during laparoscopy has been previously demonstrated. this study shows further that the effects of insufflation gas jet evaporative cooling during laparoscopy are completely eliminated by humidifying the insufflation gas stream. | background and objectives : explanations for laparoscopic - induced hypothermia fail to explain clinical observations. it is possible that water evaporation occurs from the jet stream of gas inflation resulting in tissue surface super - cooling leading to tissue damage and drying.methods:theoretical calculations based on thermal conductivity, mass transfer effects and heat flux considerations correlated closely with synthetic and tissue experiments. thermocouple measurements at a rate of 15 data points per second were performed.results:cooling rates of 10 to 25 degrees centigrade per second for high flow rates were found based on gas flow rate and effective size of gas delivery site. these rapid temperature drops extended beyond a 2 cm2 diameter.conclusions:evaporative cooling accounts for significant hypothermia. the cooling is dependent on the lack of water vapor in the gases currently used during laparoscopy. cooling rates are independent of height from tissue and geometry of delivery port. heating and hydrating the gas to a physiologic condition eliminates hypothermia and tissue dessication. |
some of these impacts are already being felt.13 health effects are among some of the major effects, for example, increased frequency and intensity of heat - waves and flooding, and more extended and changed distribution of disease vectors. in addition, health is indirectly impacted by many other factors that are themselves affected by climate change, such as water quality and quantity, ecosystems, food security, agriculture, transportation, energy production, and economic growth itself. also in europe, some health gains from climate change are anticipated, such as reduced winter cold - related deaths, and positive health and economic benefits of increased crop productivity in some geographical areas.1,2,4 however, in relation to climate change impacts, the 53 countries that make up the world health organization (who) european region should not be analyzed as a homogeneous group of nations. these countries vary enormously in relation to geography, climate, sociocultural environment, health system development, population health status, and economic level. hence, their vulnerability and capacity to respond to the health threats of climate change are widely divergent.5 mitigation measures are currently not being implemented fast enough to prevent substantial changes to the global climate over the next 100 years.1,2 hence, these new health threats will need adaptive responses to avert significant but as yet unpredictable health impacts with major implications for population welfare and economic indicators. in europe, many actions are ongoing at country as well as at regional levels.6 between 2004 and 2014, 22 of 53 who european region countries (or 15 of 28 european union countries) adopted national adaptation plan (nap) or strategies.7 the majority of these include assessment of health as a vulnerable sector. the main health actions planned include strengthening health systems, early warning systems, disaster preparedness, awareness - raising of citizens, and specific legislative changes for buildings and constructions to regulate heat in the internal environment. the european commitment to act of the fifth ministerial conference on environment and health has further set direction on the action to take in the health sector and beyond to avert the adverse health effects of climate change.8 as a response, a total of 26 european countries have conducted national health vulnerability assessments between 2001 and 2012.9 to respond effectively to the health effects of climate change, evidence is needed to justify action as well as support the selection, planning, and budgeting of preferred actions. additional spending in the health sector will need to demonstrate value for money through health economic studies in order to access sustained funding flows to climate change adaptation activities. therefore, the aim of this paper is to review the economic evidence relating to the health impacts and adaptive responses to climate change in the who european region, and to identify the most important evidence gaps to be filled and methodological issues to be addressed. the geographical focus of this review is on the 53 countries in the who european region.10 literature in the english language was obtained using a medline search for the years 19902012 and an internet search, and were included when they both (1) addressed health impacts of extreme weather events or effects of climate change on respiratory diseases and infectious diseases (vector borne, water related, and food borne) and (2) provided monetary estimation of the health impacts of climate change or the adaptation measures to protect health from climate change in europe, or both. all costs are presented in euro () in the year of the study ; when costs are presented in another currency, they are also converted to euro at the average exchange rate in the year of the study. based on the title and abstract reviewed for relevance, full articles were obtained. once a study was found to present quantitative estimates of health impact cost, health adaptation cost, or cost - effectiveness of interventions, the key results and methodological approaches were evaluated for inclusion or exclusion. these three types of studies have different primary purposes and are classified as follows : health impact cost studies (also referred to as damage cost studies) estimate the societal costs (or benefits) (eg, the costs of disease treatment, the costs of lost production because of disease, value of premature mortality) of the health impacts of climate change, valuing health impacts in monetary units. the principle objective is to provide aggregated economic impact numbers, which allow assessment of importance over time (such as comparing to gross domestic product (gdp)) as well as provide a comparison of economic impacts in money metric across sectors, based on which policy makers can prioritize sectors where adaptive measures are most needed. health adaptation cost studies estimate the costs of alternative measures to reduce, or avert altogether, the health impacts of climate change. the objective of health adaptation cost studies is to identify the expenditure required for specific health actions and thus enable realistic budgeting of fund - holding decision makers. health economic evaluation studies compare the costs and benefits of health adaptation measures, estimating a return on spending in the form of a cost - effectiveness ratio (such as cost per death averted) or a cost benefit ratio (monetary return per currency unit spent). the objective is to enable selection of efficient measures to protect the population s health, in comparison with other health protection options or uses of public resources. these three types of studies are related, and often draw on similar health and economic data. these outputs feed into other decision - making tools, such as multi - criteria analysis or policy analysis. in all, 40 relevant studies from europe were identified, covering the health damage costs (10 studies) or adaptation costs (5 additional studies) related to the health effects of climate change, and the efficiency of response measures for climate - sensitive diseases (25 additional studies). efficiency means an input output metric such as cost per death averted or cost benefit ratio. table 1 presents data on the health impact cost studies and adaptation cost studies where projections are available. studies that do not estimate health impact or adaptation costs attributed to climate change (but only of climate - sensitive diseases or conditions) are described in the text but excluded from the table, as they are not comparable. there were no economic evaluation studies identified that compared costs and benefits of response measures specific to the impacts of climate change. nevertheless, a range of economic studies on response measures to avoid climate - sensitive health effects was identified, even though they were developed not to reduce the effects of climate change but rather reduce the disease condition (with no reference to climate change). the literature search identified three studies estimating economic damages or savings resulting from multiple diseases4,11,12 and seven studies estimating the economic damages from single health risks associated with climate change (including heat - waves and salmonella) in europe. bosello use the general equilibrium global trade analysis project (gtap) model to estimate, among other impacts, the health - care costs of treating climate change - attributed cases and labor productivity impacts of six disease groups for each world region.11 for the european regions, the study included cardiovascular, respiratory, and diarrheal diseases for the year 2050. the paper does not report if it valued the future economic impacts in present values using discounting. the study predicts 176,000 net deaths avoided from higher temperatures, which are valued at a saving of 38 billion annually in the eu area, and 284,000 annual deaths avoided in former soviet union (fsu) countries valued at a saving of 4 billion. the significantly lower economic value in fsu countries for a higher number of deaths avoided is because the estimation of the value of life is based on gdp per capita, which is significantly lower in fsu countries. the net reductions in death in temperate regions in the northern hemisphere are due to the avoidance of cold - related cardiovascular death exceeding the increase in heat - related deaths. however, there is no assessment of winners and losers, by demographic or geographical group within each of the eight world regions. using a computable general equilibrium model to assess economy - wide impact of the global health effects, the study reports that the negative impact on gdp is greater than the sum of the costs associated with the three diseases because of their impacts on other economic activities. kovats estimate the welfare costs of heat mortality and salmonellosis cases in 27 eu countries.12 under the special report on emission scenarios (sres) a1b scenario (medium high emission trajectory, leading to central estimates of global average surface temperatures of around 34 c relative to pre - industrial levels), the authors use two alternative units of health impact to value the lives lost from these two risk factors : the number of life years lost because of premature mortality and the number of premature deaths. the economic value of a life year is derived from that of a premature death, and is a fraction of the latter. when valuing life years lost, the marginal impact of climate change alone is 0.8 billion by the 2020s, 2.8 billion by the 2050s, and 4.0 billion by the 2080s ; using the numbers of deaths, the marginal impact of climate change alone is 31 billion by the 2020s, 103 billion by the 2050s, and 147 billion by the 2080s. the 30-fold difference in valuation methodologies is accounted for by the fact that the two risk factors brought forward death by an average of just a few months. kovats also estimate the economic costs of additional cases of salmonella.12 under the a1b scenario and assuming a decreasing case rate, the estimated annual costs without adaptation because of climate change are 29.5 million / year by the 2020s (20112040), 46.4 million / year by the 2050s (20412070), and 48.9 million / year by the 2080s (20712100). if the case rate is held constant, the corresponding annual costs are 36, 68.4, and 88.8 million / year. additionally, the study estimates the costs of fatalities from coastal flooding, with a marginal impact of climate change costing 34 million by the 2020s (20112040), 122 million by the 2050s (20412070), and 720 million by the 2080s (20712100). the study projection of economic impacts of climate change in sectors of the european union based on bottom - up analysis (peseta) predicts almost 107,000 extra heat - related deaths per year in 20712100 for 27 eu member states under a global mean temperature increase of 3.9 c, compared to the baseline period 19611990.4 in 2080, the value of excess deaths is estimated at 50 billion annually (when valuing each excess death) and 118 billion (when valuing the loss of a year of life). these impacts are, however, likely to be balanced out by reduced cold - related deaths, with the greatest gains in northern europe and the united kingdom. for food - borne diseases, the peseta study estimates that the average annual number of temperature - related cases of salmonella may have increased by a total of almost 20,000 as a result of climate change in europe, leading to annual costs of 70140 million between the years 2011 and 2040, based on a cost per case of 3,500 and 7,000, respectively.4 these unit costs were based on a review of studies that ask potential beneficiaries what they would be willing to pay to avoid food - borne disease. under climate scenario a2, these cases and costs are predicted to double for the period 20712100. other studies estimate the climate change - attributable impact on single diseases in specific countries. for example, the application in the former yugoslav republic of macedonia of a who toolkit for the estimation of health and adaptation costs related to climate change focused on morbidity and mortality from heat - waves in the capital city, skopje.13 over a 5-year period from 2006 to 2010, the study estimated an annual average of 316 additional cases of cardiovascular disease and 344 additional cases of respiratory disease attributable to climate change, with 13 and 1 deaths resulting, respectively. the estimated average cost resulting over the 5-year period was 1.03 million per year or 2.5 per inhabitant of skopje. similarly in germany, hbler estimated the costs of heat - induced health effects in terms of hospital admissions ; but the greater impact is the impact of heat on work performance resulting in an estimated output loss of 0.10.5% of gdp.15 within the project climate change and adaptation strategies for human health in europe (ccash), a contingent valuation survey was carried out to estimate the benefits of reducing the risk of dying during heat - waves. in contingent valuation, a survey questionnaire builds theoretical scenarios to enable values to be obtained for situations that do not commonly arise in real - life, or can not easily be observed. the survey was administered to adults aged from 30 to 75 years in the czech republic and italy. for the city of rome, the monetized mortality damages of the heat - waves in the absence of planned adaptation programs was estimated to be 281 million for the year 2020 (in 2004 values).14 other studies estimate the costs of excess deaths from heat - waves, but do not estimate attributed costs to climate change, such as the 2003 heat - wave in the united kingdom that led to 2,157 excess deaths at a cost of 2.6 billion (3.6 billion) using valuation of a death at 1.2 million (1.7 million), or 32 million (45 million) using valuation of a saved year of life of 15,000 (21,000).21 in all, 1,650 excess hospital admissions were estimated to cost 15 million (21 million), at an upper threshold of 9,120 (12,770) per admission. likewise, the 2003 heat - wave in france was estimated to have caused 14,800 excess deaths from august 1 to 20, 2003, costing society more than 500 million using a value per life saved of 37,500.22 both studies assume an average of 1 year of life lost per deceased person given that the majority of excess deaths were of people 75 years and over. furthermore, the absence of a surge in health insurance expenses for the year 2003 in france led the authors to conclude that the increased hospitalizations from excess cases balanced out with hospitalizations averted because of excess deaths in the same year.22 for air pollution - related deaths, the attributed cost of acute and chronic mortality to climate change was estimated by the climate cost project at 125 billion per annum in 2050 for the 27 european union countries.16 these costs are dominated by premature death (86 billion), with the majority of the remaining accounted for by the cost of chronic bronchitis (17 billion), restricted activity days (lost productivity at 14 billion), and suffering from disease symptoms (at 9 billion). one - third (42 billion) of the overall economic impacts can be reduced by mitigation measures. three studies have been conducted that estimate health adaptation costs in europe two global multi - sectoral cost studies, by the world bank18 and the united nations framework convention on climate change (unfccc)23 and one global cost study focusing on the health service response alone.19 the multi - sectoral cost studies estimate costs of adapting to health impacts of climate change as part of other sector activities such as agriculture and water resources, as well as the health sector. the unfccc study, whose estimates are based on the methodology of the another cost study,19 is not presented here, as the study does not provide a cost breakdown for the european region. in the world bank s economics of adaptation to climate change study,18 the health sector costs include only the costs of treating and preventing diarrhea cases for europe and central asia from 2010 to 2050 at 2005 prices. the results are presented with future costs in present values using an annual discount rate of 5% and 0%. however, investments in several other sectors have important implications for health, such as the water sector and extreme events, and hence these are added to the health sector costs below to give health - related costs. two alternative global circulation models are used to predict future disease cases : the national centre for atmospheric research (ncar) community climate system model (ccsm)-3 model (termed the wet scenario) and the commonwealth scientific and industrial research organisation (csiro)-3 model (termed the infrastructure investment (estimated as the cost of health education, water supply, and sewers) is estimated to cost between us$300 (250) (csiro) and us$800 (670) (ncar) million annually. the adaptation cost for agriculture and fisheries (estimated as the cost to prevent climate change - attributed cases of malnutrition) is estimated at between us$470 (390) (csiro) and us$1,320 (1,100) (ncar) million annually. water storage and flood protection cost between us$300 million (250) (csiro) and us$2,600 (2,170) (ncar) million annually. preparing for extreme events involving education and training schemes for target populations is estimated to cost between us$500 (415) (csiro) and us$1,000 (830) (ncar) million annually. the overall health - related costs for europe and central asia are estimated at between us$1.57 billion (1.3 billion) (csiro) and us$5.72 billion (4.8 billion) (ncar). two other global studies of health adaptation costs are based on economic integrated assessment models. de bruin used the adapted regional integrated model of climate and the economy (ad - rice) model and estimated that health would be a very small total of adaptation costs for europe up to 2050.24 agrawala used the witch model (a world induced technical change hybrid model) and estimated that there will be net cost savings in disease treatment of 0.74 billion with a doubling of co2 concentrations for western and eastern europe combined.20 watkiss and taylor conclude that the estimates from both studies can only be considered illustrative because of the highly theoretical nature of these models, and their treatment of adaptation. in terms of water - borne diseases, as many as 17.5 million additional diarrhea cases have been estimated as attributed to climate change by ebi for the year 2030 for the who european region.19 based on emission reductions resulting in stabilization at 750 ppm carbon dioxide equivalent by 2210, and applying the unit costs of providing preventive services (immunization, and water and sanitation improvements) to avert these cases, the costs are estimated at us$217 million per year. the cost of preparing the heat - wave and health alert system (systme dalerte canicule et sant [sacs ]) in 2005 was calculated at 287,000 and the operating cost between june 1 and august 31 was calculated at 454,000, summing to a first year cost of 741,000.22 these costs cover mainly the additional human resource costs. compared to the estimated health costs of more than 500 million, including loss of human life at the value of 37,500 per year of lost life, this intervention cost is relatively small. for newly emerging infectious diseases in europe however, few actual vaccines are available on the market for vector - borne diseases. no available studies have estimated the costs of vaccinating the at - risk or high - risk populations in europe. health economic studies not only assess adaptation costs (above) but also compare these costs to health impacts and other outcomes in cost - effectiveness analysis (cea) or cost benefit analysis (cba).25 ideally, economic evaluation includes a comparative economic assessment of alternative policy options. the literature search revealed no studies that have specifically examined the costs and health effects of interventions specifically related to addressing the additional disease burden associated with climate change. however, several studies were found that assessed cost - effectiveness or cost benefit of health interventions targeting climate - sensitive diseases. these are most available in the areas of preventing food - borne diseases, preventing diarrhea through rotavirus vaccination and preventing or treating air quality - related conditions. however, as these studies were non - specific to climate change, they fell outside the initial systematic search criteria. therefore, the studies presented below illustrate the types of studies available, but they do not represent the entire published economic literature. these were included in this assessment as they provide indications for future research. in the area of heat - health early warning systems, while many european countries have heat - health plans,26 no studies were found from europe that compare the costs and health impacts of such systems. a study from philadelphia, united states of america, indicates the potential value for money of such systems. the philadelphia heat - health early warning system, initiated in 1995, was considered unique at the time because of its coordination between different public and private agencies, including mass media campaigns and community mobilization. at a value of us$4 million (5.4 million) per life saved, the gross benefits of the philadelphia heat - health warning system were in the order of us$468 million (626 million) over 4 years, or us$117 million (157 million) per year. the annual marginal costs of the system were estimated at us$115,000 (154,000), in addition to the costs of developing the system of us$60,000 (80,000).27,28 hence, the cost per life saved is very low at less than us$4,000 (5,350), indicating an efficient use of public funds. however, these costs are only marginal costs, and do not consider the redeployment of resources already paid for by public authorities, such as salaries and vehicles. in the area of food - borne disease prevention, quite a number of farm - level economic studies have been performed, focusing mainly on salmonella prevention. these studies generally find that disease prevention is cost - effective or economically viable (ie, benefits greater than costs). for example : dutch studies found hygiene interventions with relatively favorable cost utility ratio for salmonella reduction29,30 and campylobacter control.29,30 danish studies compare the economic performance of decontamination technologies at pork abattoirs in danish farms. one study estimates that the technologies might reduce salmonella from the present level of 2.2% to between 0.18% and 0.89%.31 a second study compares alternative approaches to salmonella reduction, and finds hot - water decontamination to be the only intervention with positive net present value.32 in finland, the benefits of the finnish salmonella control program were estimated to be four times the costs of the program.33 in the united kingdom, surveillance and early withdrawal of products contaminated with salmonella had benefits to the public sector of 3.5 times the cost, and benefits to society of 23 times the cost.34,35 economic studies on end - use food preparation studies are fewer. one study evaluated the potential cost - effectiveness of a disinfection program that targets high - risk food preparation activities in household kitchens in the united states of america, canada, and united kingdom.36 the average cost utility ratio in united kingdom was 86,341 (124,770) per quality - adjusted life - year (qaly) gained. a qaly is a positive measure of health, and is the equivalent of a year of life lived in full health. when targeting households with high - risk members (those less than 5 years of age, greater than 65 years of age, or immune compromised), the cost per qaly reduced to 28,158 (40,700). cardiovascular and respiratory diseases result from air pollution, which are exacerbated with higher temperatures. while the pathways of effects are indirect and complex, the purpose of this presentation is to explore studies that assess the damage costs of overall air pollution and the economics of various response measures. the economic literature on air quality - related disease burden includes a range of studies examining different interventions applied at different levels, from sector - specific studies to national studies to europe - wide studies.37,38 most studies value economic gains by aggregating the value of reduced premature deaths, lower health - care costs, and work days gained because of lower morbidity. few studies include other economic benefits, such as avoided damage to agriculture and ecosystems or avoided damage to infrastructure and public buildings from corrosive pollutants. cost ratios were as follows : the clean air for europe (cafe) program estimates a benefit cost ratio of between 6 and 19 for achieving air quality targets for europe.39 the united kingdom air quality strategy review estimates a benefit cost ratio of meeting eu standards of between 1.5 and 3.8 for low - intensity interventions and 0.9 and 2.3 for high - intensity interventions.40 the benefit cost ratio of air pollution control measures in various sectors in hungary varies from 3 in agriculture, to 5 in industry, to 6 in transportation and energy, to 16 in household interventions, and to 17 in the service sector.41 pollution emission reduction in the oil extraction industry in kazakhstan is estimated to have a benefit cost ratio of 5.7.42 the economic returns on investing in cycle networks in three cities of norway are between 3 and 14 times greater than the costs.43 reducing greenhouse gas emissions in europe by 20% in 2020 would improve life expectancy by 3.3 months and reduce health damage costs by 1229 billion.44 for curative or palliative care, several cost - effectiveness studies have been conducted on respiratory conditions, such as asthma interventions,45,46 allergic rhinitis testing methods,47 immunotherapy,48 and drugs.49,50 for example, the cost per qaly of treating grass allergen with grazax ranged between 12,930 and 18,263 in seven northern european countries.49 for waterborne diseases, rotavirus vaccination has been the subject of several economic evaluation studies across europe. the cost per qaly gained ranges from 21,900 to 35,076 in dutch children from 0 to 4 years old, using rotarix.51 in france, a routine universal rotavirus immunization program was estimated to cost 138,000 per qaly saved and avoid annually 89,000 cases of diarrhea, 10,500 hospitalizations, and 8 deaths.52 in finland, the cost per qaly gained was 25,218 for rotarix and 45,199 for rotateq, preventing annually 2,000 hospitalized cases and over 10,000 outpatient visits.53 the costs and benefits of improved provision of water supply and sanitation services were estimated by the who in who epidemiological strata b and c (mainly, non - eu and non - oecd countries) finding that the economic benefits (including health and time savings) were worth 20 times the costs of these services54 and costing us$9,500 (6,940) per disability - adjusted life - year (daly) averted.55 a daly is a negative measure of health, the other side of the coin to a qaly (where a health intervention leads to a daly being avoided while a qaly is gained), and with its own estimation approach. a hypothetical tick - borne encephalitis (tbe) vaccine for french troops stationed in the balkans was estimated to avert 121 cases of tbe at a program cost of 10.05 million, thus costing 83,000 per case prevented.56 based on estimated economic benefits of 4.37 million, the net costs were 5.68 million and hence not justifiable on the grounds of providing economic returns. for lyme disease, an economic study from the usa estimates the average cost per case averted to be us$4,466 (4,190).57 however, cost - effectiveness is highly variable, depending on the vaccine price, incidence, and probability of early detection and referral. hence, cross - border extrapolations should be made with care, adjusting for differences in key determinants. the literature search identified three studies estimating economic damages or savings resulting from multiple diseases4,11,12 and seven studies estimating the economic damages from single health risks associated with climate change (including heat - waves and salmonella) in europe. bosello use the general equilibrium global trade analysis project (gtap) model to estimate, among other impacts, the health - care costs of treating climate change - attributed cases and labor productivity impacts of six disease groups for each world region.11 for the european regions, the study included cardiovascular, respiratory, and diarrheal diseases for the year 2050. the paper does not report if it valued the future economic impacts in present values using discounting. the study predicts 176,000 net deaths avoided from higher temperatures, which are valued at a saving of 38 billion annually in the eu area, and 284,000 annual deaths avoided in former soviet union (fsu) countries valued at a saving of 4 billion. the significantly lower economic value in fsu countries for a higher number of deaths avoided is because the estimation of the value of life is based on gdp per capita, which is significantly lower in fsu countries. the net reductions in death in temperate regions in the northern hemisphere are due to the avoidance of cold - related cardiovascular death exceeding the increase in heat - related deaths. however, there is no assessment of winners and losers, by demographic or geographical group within each of the eight world regions. using a computable general equilibrium model to assess economy - wide impact of the global health effects, the study reports that the negative impact on gdp is greater than the sum of the costs associated with the three diseases because of their impacts on other economic activities. kovats estimate the welfare costs of heat mortality and salmonellosis cases in 27 eu countries.12 under the special report on emission scenarios (sres) a1b scenario (medium high emission trajectory, leading to central estimates of global average surface temperatures of around 34 c relative to pre - industrial levels), the authors use two alternative units of health impact to value the lives lost from these two risk factors : the number of life years lost because of premature mortality and the number of premature deaths. the economic value of a life year is derived from that of a premature death, and is a fraction of the latter. when valuing life years lost, the marginal impact of climate change alone is 0.8 billion by the 2020s, 2.8 billion by the 2050s, and 4.0 billion by the 2080s ; using the numbers of deaths, the marginal impact of climate change alone is 31 billion by the 2020s, 103 billion by the 2050s, and 147 billion by the 2080s. the 30-fold difference in valuation methodologies is accounted for by the fact that the two risk factors brought forward death by an average of just a few months. kovats also estimate the economic costs of additional cases of salmonella.12 under the a1b scenario and assuming a decreasing case rate, the estimated annual costs without adaptation because of climate change are 29.5 million / year by the 2020s (20112040), 46.4 million / year by the 2050s (20412070), and 48.9 million / year by the 2080s (20712100). if the case rate is held constant, the corresponding annual costs are 36, 68.4, and 88.8 million / year. additionally, the study estimates the costs of fatalities from coastal flooding, with a marginal impact of climate change costing 34 million by the 2020s (20112040), 122 million by the 2050s (20412070), and 720 million by the 2080s (20712100). the study projection of economic impacts of climate change in sectors of the european union based on bottom - up analysis (peseta) predicts almost 107,000 extra heat - related deaths per year in 20712100 for 27 eu member states under a global mean temperature increase of 3.9 c, compared to the baseline period 19611990.4 in 2080, the value of excess deaths is estimated at 50 billion annually (when valuing each excess death) and 118 billion (when valuing the loss of a year of life). these impacts are, however, likely to be balanced out by reduced cold - related deaths, with the greatest gains in northern europe and the united kingdom. for food - borne diseases, the peseta study estimates that the average annual number of temperature - related cases of salmonella may have increased by a total of almost 20,000 as a result of climate change in europe, leading to annual costs of 70140 million between the years 2011 and 2040, based on a cost per case of 3,500 and 7,000, respectively.4 these unit costs were based on a review of studies that ask potential beneficiaries what they would be willing to pay to avoid food - borne disease. under climate scenario a2, these cases and costs are predicted to double for the period 20712100. other studies estimate the climate change - attributable impact on single diseases in specific countries. for example, the application in the former yugoslav republic of macedonia of a who toolkit for the estimation of health and adaptation costs related to climate change focused on morbidity and mortality from heat - waves in the capital city, skopje.13 over a 5-year period from 2006 to 2010, the study estimated an annual average of 316 additional cases of cardiovascular disease and 344 additional cases of respiratory disease attributable to climate change, with 13 and 1 deaths resulting, respectively. the estimated average cost resulting over the 5-year period was 1.03 million per year or 2.5 per inhabitant of skopje. similarly in germany, hbler estimated the costs of heat - induced health effects in terms of hospital admissions ; but the greater impact is the impact of heat on work performance resulting in an estimated output loss of 0.10.5% of gdp.15 within the project climate change and adaptation strategies for human health in europe (ccash), a contingent valuation survey was carried out to estimate the benefits of reducing the risk of dying during heat - waves. in contingent valuation, a survey questionnaire builds theoretical scenarios to enable values to be obtained for situations that do not commonly arise in real - life, or can not easily be observed. the survey was administered to adults aged from 30 to 75 years in the czech republic and italy. for the city of rome, the monetized mortality damages of the heat - waves in the absence of planned adaptation programs was estimated to be 281 million for the year 2020 (in 2004 values).14 other studies estimate the costs of excess deaths from heat - waves, but do not estimate attributed costs to climate change, such as the 2003 heat - wave in the united kingdom that led to 2,157 excess deaths at a cost of 2.6 billion (3.6 billion) using valuation of a death at 1.2 million (1.7 million), or 32 million (45 million) using valuation of a saved year of life of 15,000 (21,000).21 in all, 1,650 excess hospital admissions were estimated to cost 15 million (21 million), at an upper threshold of 9,120 (12,770) per admission. likewise, the 2003 heat - wave in france was estimated to have caused 14,800 excess deaths from august 1 to 20, 2003, costing society more than 500 million using a value per life saved of 37,500.22 both studies assume an average of 1 year of life lost per deceased person given that the majority of excess deaths were of people 75 years and over. furthermore, the absence of a surge in health insurance expenses for the year 2003 in france led the authors to conclude that the increased hospitalizations from excess cases balanced out with hospitalizations averted because of excess deaths in the same year.22 for air pollution - related deaths, the attributed cost of acute and chronic mortality to climate change was estimated by the climate cost project at 125 billion per annum in 2050 for the 27 european union countries.16 these costs are dominated by premature death (86 billion), with the majority of the remaining accounted for by the cost of chronic bronchitis (17 billion), restricted activity days (lost productivity at 14 billion), and suffering from disease symptoms (at 9 billion). one - third (42 billion) of the overall economic impacts can be reduced by mitigation measures. three studies have been conducted that estimate health adaptation costs in europe two global multi - sectoral cost studies, by the world bank18 and the united nations framework convention on climate change (unfccc)23 and one global cost study focusing on the health service response alone.19 the multi - sectoral cost studies estimate costs of adapting to health impacts of climate change as part of other sector activities such as agriculture and water resources, as well as the health sector. the unfccc study, whose estimates are based on the methodology of the another cost study,19 is not presented here, as the study does not provide a cost breakdown for the european region. in the world bank s economics of adaptation to climate change study,18 the health sector costs include only the costs of treating and preventing diarrhea cases for europe and central asia from 2010 to 2050 at 2005 prices. the results are presented with future costs in present values using an annual discount rate of 5% and 0%. however, investments in several other sectors have important implications for health, such as the water sector and extreme events, and hence these are added to the health sector costs below to give health - related costs. two alternative global circulation models are used to predict future disease cases : the national centre for atmospheric research (ncar) community climate system model (ccsm)-3 model (termed the wet scenario) and the commonwealth scientific and industrial research organisation (csiro)-3 model (termed the infrastructure investment (estimated as the cost of health education, water supply, and sewers) is estimated to cost between us$300 (250) (csiro) and us$800 (670) (ncar) million annually. the adaptation cost for agriculture and fisheries (estimated as the cost to prevent climate change - attributed cases of malnutrition) is estimated at between us$470 (390) (csiro) and us$1,320 (1,100) (ncar) million annually. water storage and flood protection cost between us$300 million (250) (csiro) and us$2,600 (2,170) (ncar) million annually. preparing for extreme events involving education and training schemes for target populations is estimated to cost between us$500 (415) (csiro) and us$1,000 (830) (ncar) million annually. the overall health - related costs for europe and central asia are estimated at between us$1.57 billion (1.3 billion) (csiro) and us$5.72 billion (4.8 billion) (ncar). two other global studies of health adaptation costs are based on economic integrated assessment models. de bruin used the adapted regional integrated model of climate and the economy (ad - rice) model and estimated that health would be a very small total of adaptation costs for europe up to 2050.24 agrawala used the witch model (a world induced technical change hybrid model) and estimated that there will be net cost savings in disease treatment of 0.74 billion with a doubling of co2 concentrations for western and eastern europe combined.20 watkiss and taylor conclude that the estimates from both studies can only be considered illustrative because of the highly theoretical nature of these models, and their treatment of adaptation. in terms of water - borne diseases, as many as 17.5 million additional diarrhea cases have been estimated as attributed to climate change by ebi for the year 2030 for the who european region.19 based on emission reductions resulting in stabilization at 750 ppm carbon dioxide equivalent by 2210, and applying the unit costs of providing preventive services (immunization, and water and sanitation improvements) to avert these cases, the costs are estimated at us$217 million per year. the cost of preparing the heat - wave and health alert system (systme dalerte canicule et sant [sacs ]) in 2005 was calculated at 287,000 and the operating cost between june 1 and august 31 was calculated at 454,000, summing to a first year cost of 741,000.22 these costs cover mainly the additional human resource costs. compared to the estimated health costs of more than 500 million, including loss of human life at the value of 37,500 per year of lost life, this intervention cost is relatively small. for newly emerging infectious diseases in europe however, few actual vaccines are available on the market for vector - borne diseases. no available studies have estimated the costs of vaccinating the at - risk or high - risk populations in europe. health economic studies not only assess adaptation costs (above) but also compare these costs to health impacts and other outcomes in cost - effectiveness analysis (cea) or cost benefit analysis (cba).25 ideally, economic evaluation includes a comparative economic assessment of alternative policy options. the literature search revealed no studies that have specifically examined the costs and health effects of interventions specifically related to addressing the additional disease burden associated with climate change. however, several studies were found that assessed cost - effectiveness or cost benefit of health interventions targeting climate - sensitive diseases. these are most available in the areas of preventing food - borne diseases, preventing diarrhea through rotavirus vaccination and preventing or treating air quality - related conditions. however, as these studies were non - specific to climate change, they fell outside the initial systematic search criteria. therefore, the studies presented below illustrate the types of studies available, but they do not represent the entire published economic literature. these were included in this assessment as they provide indications for future research. in the area of heat - health early warning systems, while many european countries have heat - health plans,26 no studies were found from europe that compare the costs and health impacts of such systems. a study from philadelphia, united states of america, indicates the potential value for money of such systems. the philadelphia heat - health early warning system, initiated in 1995, was considered unique at the time because of its coordination between different public and private agencies, including mass media campaigns and community mobilization. at a value of us$4 million (5.4 million) per life saved, the gross benefits of the philadelphia heat - health warning system were in the order of us$468 million (626 million) over 4 years, or us$117 million (157 million) per year. the annual marginal costs of the system were estimated at us$115,000 (154,000), in addition to the costs of developing the system of us$60,000 (80,000).27,28 hence, the cost per life saved is very low at less than us$4,000 (5,350), indicating an efficient use of public funds. however, these costs are only marginal costs, and do not consider the redeployment of resources already paid for by public authorities, such as salaries and vehicles. in the area of food - borne disease prevention, quite a number of farm - level economic studies have been performed, focusing mainly on salmonella prevention. these studies generally find that disease prevention is cost - effective or economically viable (ie, benefits greater than costs). for example : dutch studies found hygiene interventions with relatively favorable cost utility ratio for salmonella reduction29,30 and campylobacter control.29,30 danish studies compare the economic performance of decontamination technologies at pork abattoirs in danish farms. one study estimates that the technologies might reduce salmonella from the present level of 2.2% to between 0.18% and 0.89%.31 a second study compares alternative approaches to salmonella reduction, and finds hot - water decontamination to be the only intervention with positive net present value.32 in finland, the benefits of the finnish salmonella control program were estimated to be four times the costs of the program.33 in the united kingdom, surveillance and early withdrawal of products contaminated with salmonella had benefits to the public sector of 3.5 times the cost, and benefits to society of 23 times the cost.34,35 economic studies on end - use food preparation studies are fewer. one study evaluated the potential cost - effectiveness of a disinfection program that targets high - risk food preparation activities in household kitchens in the united states of america, canada, and united kingdom.36 the average cost utility ratio in united kingdom was 86,341 (124,770) per quality - adjusted life - year (qaly) gained. a qaly is a positive measure of health, and is the equivalent of a year of life lived in full health. when targeting households with high - risk members (those less than 5 years of age, greater than 65 years of age, or immune compromised), the cost per qaly reduced to 28,158 (40,700). cardiovascular and respiratory diseases result from air pollution, which are exacerbated with higher temperatures. while the pathways of effects are indirect and complex, the purpose of this presentation is to explore studies that assess the damage costs of overall air pollution and the economics of various response measures. the economic literature on air quality - related disease burden includes a range of studies examining different interventions applied at different levels, from sector - specific studies to national studies to europe - wide studies.37,38 most studies value economic gains by aggregating the value of reduced premature deaths, lower health - care costs, and work days gained because of lower morbidity. few studies include other economic benefits, such as avoided damage to agriculture and ecosystems or avoided damage to infrastructure and public buildings from corrosive pollutants. cost ratios were as follows : the clean air for europe (cafe) program estimates a benefit cost ratio of between 6 and 19 for achieving air quality targets for europe.39 the united kingdom air quality strategy review estimates a benefit cost ratio of meeting eu standards of between 1.5 and 3.8 for low - intensity interventions and 0.9 and 2.3 for high - intensity interventions.40 the benefit cost ratio of air pollution control measures in various sectors in hungary varies from 3 in agriculture, to 5 in industry, to 6 in transportation and energy, to 16 in household interventions, and to 17 in the service sector.41 pollution emission reduction in the oil extraction industry in kazakhstan is estimated to have a benefit cost ratio of 5.7.42 the economic returns on investing in cycle networks in three cities of norway are between 3 and 14 times greater than the costs.43 reducing greenhouse gas emissions in europe by 20% in 2020 would improve life expectancy by 3.3 months and reduce health damage costs by 1229 billion.44 for curative or palliative care, several cost - effectiveness studies have been conducted on respiratory conditions, such as asthma interventions,45,46 allergic rhinitis testing methods,47 immunotherapy,48 and drugs.49,50 for example, the cost per qaly of treating grass allergen with grazax ranged between 12,930 and 18,263 in seven northern european countries.49 for waterborne diseases, rotavirus vaccination has been the subject of several economic evaluation studies across europe. the cost per qaly gained ranges from 21,900 to 35,076 in dutch children from 0 to 4 years old, using rotarix.51 in france, a routine universal rotavirus immunization program was estimated to cost 138,000 per qaly saved and avoid annually 89,000 cases of diarrhea, 10,500 hospitalizations, and 8 deaths.52 in finland, the cost per qaly gained was 25,218 for rotarix and 45,199 for rotateq, preventing annually 2,000 hospitalized cases and over 10,000 outpatient visits.53 the costs and benefits of improved provision of water supply and sanitation services were estimated by the who in who epidemiological strata b and c (mainly, non - eu and non - oecd countries) finding that the economic benefits (including health and time savings) were worth 20 times the costs of these services54 and costing us$9,500 (6,940) per disability - adjusted life - year (daly) averted.55 a daly is a negative measure of health, the other side of the coin to a qaly (where a health intervention leads to a daly being avoided while a qaly is gained), and with its own estimation approach. a hypothetical tick - borne encephalitis (tbe) vaccine for french troops stationed in the balkans was estimated to avert 121 cases of tbe at a program cost of 10.05 million, thus costing 83,000 per case prevented.56 based on estimated economic benefits of 4.37 million, the net costs were 5.68 million and hence not justifiable on the grounds of providing economic returns. for lyme disease, an economic study from the usa estimates the average cost per case averted to be us$4,466 (4,190).57 however, cost - effectiveness is highly variable, depending on the vaccine price, incidence, and probability of early detection and referral. hence, cross - border extrapolations should be made with care, adjusting for differences in key determinants. the presentation of the available economic evidence - base on health costs and health intervention efficiency related to climate change in europe shows major gaps in evidence, as well as limitations in the quality and usefulness of the existing studies. first, the few studies presented in this paper indicate that the economic evidence - base is incomplete and fragmented. there are few europe - wide economic studies that provide a comprehensive overview of the economics of climate change health impacts and response measures. likewise, there are even fewer peer - reviewed country or city - level studies that provide an adequate economic evidence - base to inform policy decisions. second, the lack of standardization of economic outcome measures is a serious constraint to the use of evidence by decision makers. as the literature review reveals, a range of economic outcomes are used, such as cost per daly averted, cost per qaly gained, cost per case averted, cost per death averted, net cost or net present value, and economic benefits per unit of money invested. indeed, the choice of different outcome measures is justified by the fact that different decision - making contexts require expression of efficiency in different units. interventions requiring public health funds tend to favor the use of cost per qaly gained or cost per daly averted. interventions requiring private investment or cost recovery from households, such as food - borne disease prevention or water and sanitation services, tend to show net present value or benefit cost ratio. however, the reviewed economic studies do not analyze the mix of financing sources that might be required to successfully implement the evaluated interventions. this is a particular gap given that the analysis of financing options provides a concrete link from academic studies to policy makers. third, widely varying climate models and economic methods and impacts were used in the studies reviewed, making it difficult to compare results between studies. there are a large number of climate models that vary in their specifications and precision. economic models include various types of general equilibrium model (where linkages between impacts in different sectors are quantitatively assessed), sector - specific estimates (with no linkage assessed between sectors), and also a mixture of the two such as the witch model.20 most studies only examine health service costs and savings, while other studies such as the world bank adaptation cost study broadened the intervention beyond health services.18 health effects included in health impact models vary some focus on only the negative effects such as heat - related health impacts or salmonellosis, while others present net effects by including the positive health effects associated with climate change in the european region such as fewer cold - related deaths. for example, premature mortality is valued in terms of both a saved life and a saved life year. valuing a saved life year usually leads to lower economic values than saved lives, especially for the elderly population. furthermore, some studies only measure morbidity costs (mainly health - care costs) or some measure only mortality costs, while others include both morbidity and mortality costs. in general, when they are both included, mortality costs outweigh morbidity costs by several times. given that mortality valuations are based on value of statistical life, and not actual financial transactions to reduce health risks, the major share of overall welfare gain from health protection measures is of a non - financial nature. cost comparisons between studies are further impeded by the fact that the baseline year is often different, the years covered by the study are different, and some studies estimate future impacts in current values using discounting, while others do not. good practice is to present results under different scenarios to show sensitivity of results to different assumptions or study scope and to support comparison. fourth, the number of studies that assess the health effects of climate change is very few. while general relationships and trends are predicted with increasing confidence as models are refined and data quality improves (eg, on changes in temperature and precipitation), the health impact numbers are still not known. the only eu - wide health economic estimates are on selected temperature - related disease burdens on salmonella and diarrheal disease cases. given the variation in climate models and impact assessment frameworks used, combined with uncertainty in economic values, the resulting economic outcomes would have very wide confidence intervals or ranges if sensitivity analysis was conducted using alternative data inputs. this makes it extremely difficult to understand the range of likely economic impact or intervention efficiency, and thus reduces the strength of policy recommendation possible. further, current available health vulnerability assessments have not been used to assess the economic costs. fifth, the long - time horizons involved in climate change make it important to clarify what is the baseline scenario. for example, should the costs of future measures take into account future expected health sector developments that affect underlying disease vulnerability and hence adaptation responses ? this point is particularly relevant for the lower income countries of europe where there is a greater adaptation deficit. whether future health investments are labeled as development investments or adaptation investments given that investments in climate change adaptation will increasingly become part and parcel of a country s development process, especially for lower income countries, the development baseline approach adopted by the world bank study, for example, will underestimate the actual costs of adapting to climate change.18 based on this review, methodological guidelines specific to the economics of health and climate changes are needed to stimulate more economic research and provide standardization of approaches. more recently, the who regional office for europe developed detailed methods on how to assess impacts and adaptation costs, which hopefully will be helpful to further shape this agenda.13 further, more information and data are necessary on the health impacts of climate change, potential future developments, adaptation options, and the monetary requirements in the health sector and beyond. at the moment, the few studies available indicate simply that more resources need to be allocated in countries with the highest health impacts and with the least resilience to withstand climate change. despite the evidence gaps, investing in health protection measures will pay economic dividends in terms of saved treatment costs, gained workforce productivity, as well as the very large social and welfare value associated with saved lives. current approaches focus mainly on a near - term perspective with a lower risk no regrets policies, rather than a longer term perspective. a monitoring and surveillance system targeted to detect the health effects of climate change would help to identify emerging health risks so that timely action can be taken, as well as would generate data to be used in assessments and economic analysis. evidence plays a more important role than it currently does in guiding health and policies that cut across different sectors. this fact is generally recognized across eu member states, for example, where health systems regulate what health interventions are applied through strict health technology assessment procedures and other evidence - to - policy initiatives.58 however, preventive policies environmental health policies more specifically have a significantly weaker evidence - base than curative and drug - related procedures in the health community. more evidence is required to show the health - protecting effect of safe environments (eg, urban areas, homes, workplaces) as well as the intangible health and non - health impacts. to support the generation of this evidence, methodological guidelines specific to the economics of health and climate change are needed to stimulate more economic research and provide standardization of approach. the first step has been done by the recent economic tool developed by the who regional office for europe to guide health damage and adaptation studies.13 this will help to stimulate health and scientific authorities to consider approaches to utilizing existing health economic studies based on recalculating results from common methods using local input values, and adjusting to current prices. access to the underlying data and tools remains a challenge, in particular when health effects estimations are not available or detailed health service utilization costs are required. significant health gains can be achieved through interventions that are not primarily under the control of the health sector, but are embedded in action at, for example, the urban or regional level.. economic analyses in areas such as air pollution, nutrition, and transport policy generally show the importance of health outcomes. the new health 2020 aims at promoting health in all policies.5961 collaboration with other ministries and public agencies, in order to ensure that health is not overlooked in any policy that can potentially harm or benefit health, is embedded in the expo convention and the strategic impact assessment, however not always carried out. economics can be used as a common language to make the case for intervention and hence bring others on board. to achieve this, a standardized approach, increased transparency of methods and interests, and a more common presentation from economic studies are needed. | backgroundin responding to the health impacts of climate change, economic evidence and tools inform decision makers of the efficiency of alternative health policies and interventions. in a time when sweeping budget cuts are affecting all tiers of government, economic evidence on health protection from climate change spending enables comparison with other public spending.methodsthe review included 53 countries of the world health organization (who) european region. literature was obtained using a medline and internet search of key terms in published reports and peer - reviewed literature, and from institutions working on health and climate change. articles were included if they provided economic estimation of the health impacts of climate change or adaptation measures to protect health from climate change in the who european region. economic studies are classified under health impact cost, health adaptation cost, and health economic evaluation (comparing both costs and impacts).resultsa total of 40 relevant studies from europe were identified, covering the health damage or adaptation costs related to the health effects of climate change and response measures to climate - sensitive diseases. no economic evaluation studies were identified of response measures specific to the impacts of climate change. existing studies vary in terms of the economic outcomes measured and the methods for evaluation of health benefits. the lack of robust health impact data underlying economic studies significantly affects the availability and precision of economic studies.conclusionseconomic evidence in european countries on the costs of and response to climate - sensitive diseases is extremely limited and fragmented. further studies are urgently needed that examine health impacts and the costs and efficiency of alternative responses to climate - sensitive health conditions, in particular extreme weather events (other than heat) and potential emerging diseases and other conditions threatening europe. |
the purpose of this study was to test the hypothesis that particulate matter < or = 10 microns in aerodynamic diameter (pm10) particles have the ability to generate free radical activity at their surface. we collected pm10 filters from the edinburgh, united kingdom, enhanced urban network sampling site, removed particles from the filter, and tested their ability to cause free radical damage to supercoiled plasmid dna. we found that the pm10 particles did cause damage to the dna that was mediated by hydroxyl radicals, as shown by inhibition of the injury with mannitol. the pm10-associated hydroxyl radical activity was confirmed using a high - performance liquid chromatography - based assay to measure the hydroxyl radical adduct of salicylic acid. desferrioxamine abolished the hydroxyl radical - mediated injury, which suggests that iron was involved. analysis of pm10 filters confirmed the presence of large amounts of iron and leaching studies confirmed that the pm10 samples could release substantial amounts of fe(iii) and lesser amounts of fe(ii). to investigate the size of the particles involved in the hydroxyl radical injury, we centrifuged the suspension of pm10 to clarity, tested the clear supernatant, and found that it had all of the suspension activity. we conclude, therefore, that the free radical activity is derived either from a fraction that is not centrifugeable on a bench centrifuge, or that the radical generating system is released into solution.imagesfigure 1. |
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the most astounding events in the reproduction of oviparous (egg - laying) species are the growth and development of the female germ cell. the events leading to a mature oocyte in a wide variety of species are very similar. oocytes derive from mitotic cells called oogonia, which develop from primordial germ cells migrating into the ovary during early embryogenesis. the cells arrest in prophase of division i of meiosis when the chromosomes take on a these meiotically arrested female germ cells, termed primary oocytes, enter growth periods whose length is species - dependent. during this growth, primary oocytes acquire all components of the machinery subsequently required for embryo development, including ribosomes, trnas, cytoplasmic organelles, mrnas, and early yolk. in the female domesticated chicken (gallus gallus domesticus) and likely in all birds, fertilization - competent oocytes develop continuously in follicles of the left hand ovary (the right hand ovary is obliterated during embryogenesis). chicken oocytes grow in three phases : first, for several months, numerous microscopically small oocytes increase in size to 2 - 3 mm diameter, which still lack the typical yellow yolk ; second, several of these oocytes continue to grow slowly, and third, at reaching a diameter of 5 - 6 mm, an estimated 75% of these oocytes are destined for atresia (i.e., resorption), and one the remaining oocytes enters the last, very rapid growth phase, reaches a diameter of ca. this final stage is characterized by a dramatic 7-day growth spurt during which the oocyte extracts from the circulation plasma - borne components amounting to up to 14 ml of yolk. the yolk is comprised of lipid imported in the form of lipoproteins, mainly very low density lipoprotein (vldl) and vitellogenin (vtg), which contribute ca. 5 g of triglycerides and 230 mg of cholesterol to the yolk mass. lipoproteins and most of the additional minor yolk precursors are synthesized in and secreted from the liver and taken up by the growing oocytes via endocytotic processes involving specific cell - surface receptors. ovulation of the largest oocyte occurs every 25 hour and triggers the beginnning of the rapid growth phase of the next oocyte in line (laid as egg 7 - 8 days later), establishing a size- and time - related hierarchy of preovulatory follicles. if the hen has been inseminated, fertilization of the ovulated oocyte occurs in the uppermost portion of the oviduct. during the next 25 hour of migration through the lumen of the oviduct, structural components stabilizing the oocyte proper, egg white proteins, water, egg membranes, and the shell are deposited around the oocyte to form the laid egg. although the yolk appears homogeneous, it actually is a rather complex and precisely compartmentalized structure. from the germinal vesicle (the visible white spot on the oocyte surface), the latebra, a pear - shaped yolk - free cytoplasmic region, extends radially into the center of the oocyte. a medial cross - section reveals 7 concentric shells of yellow yolk (likely related to the 7 days of massive yolk deposition), which are separated by layers of oocytic cytoplasm that are connected to the latebra, thereby forming a continuous cytoplasmic compartment. the cytoskeletal elements and regulatory mechanisms that generate this structural organization, which may be unique to avian oocytes, are not known in any detail. as will be described in this review, most of the contents of the oocyte are imported by the action of multifunctional oocyte - specific receptors belonging to the ldl receptor (ldlr) gene family. both close and distant relatives of this family have been identified in the chicken, which thus has been established as a prime model to investigate the molecular genetics of these important receptors. molecular information on any of the proteins involved in oocyte growth and systemic lipoprotein transport in the laying hen was lacking until the mid-1980 's, although the presence of bona - fide lipoprotein particles in yolk was highly suggestive of receptor - mediated processes for yolk lipoprotein deposition. indeed, since then our studies on yolk precursor transport in the laying hen have not only provided proof for this concept, but also have revealed surprising new aspects of lipoprotein receptor biology with relevance to other species. it is now clear that a 95-kda protein in the plasma membrane of the oocyte binds both major yolk lipoproteins, vldl and vtg. this receptor protein reacts with antibodies to mammalian ldlrs and recognizes apolipoprotein (apo) e, an apo produced by mammals, but not by birds. these properties predicted that the oocyte receptor for vldl and vtg is a homologue of mammalian ldl receptors which recognize apob and apoe. the ldlr superfamily is defined by common structural elements with a high degree of sequence identity (70%-100%) between the molecules harbouring them, and in a wide range of species. their conserved sequences likely have evolved from an ancestral gene by duplication and/or exon shuffling events. the most typical of these elements are the so - called la repeats, which are tandemly arranged in clusters and form the ligand binding domains of ldlr family members. the classical ldlrs are characterized by the presence of seven la repeats in their ligand binding domains, whereas the vldlrs contain a cluster of eight la repeats. molecular cloning of the 95-kda oocyte protein indeed revealed an eight - repeat ligand binding domain which was confirmed to bind apoe despite the fact that apoe is absent from the chicken genome. thus, in order to distinguish this chicken oocyte receptor from mammalian vldrs, we subsequently termed it lr8 (ldl receptor relative with 8 la repeats). the gene specifying lr8 is located on the galline sex chromosome z. as discussed below, in addition to the high evolutionary conservation of eight - repeat receptors, the absence in chicken oocyte lr8 of a serine- and threonine - rich domain carrying o - linked carbohydrate chains appears significant. whereas the spectrum of physiological functions of mammalian vldlrs is not yet fully delineated, the role of chicken lr8 is documented by both biochemical and genetic evidence. lr8 mediates a key step in the reproductive effort of the hen, i.e., normal oocyte growth via yolk deposition. this conclusion can be drawn from the fact that functional absence of lr8 blocks oocytes from entering into the rapid growth phase. this phenotype is observed in a rare chicken strain carrying a single mutation at the vldlr locus (on the sex chromosome z, see above) termed restricted ovulator (r / o) strain. furthermore, as a consequence of the failure to deposit vldl and vtg, which are produced at normal levels, into their oocytes, the mutant females develop severe hyperlipidemia and features of atherosclerosis. carrier roosters (genotype, vldlr / vldlr) have normal lipid metabolism, as expected from our finding that lr8 is expressed almost exclusively in oocytes. thus, -/vldlr females, which in fact represent a model for an oocyte - specific receptor defect leading to familial hypercholesterolemia, are sterile due to non - laying. in addition to vldl and vtg, the receptor was shown to bind riboflavin - binding protein complexed with vtg and clusterin (apolipoprotein j ;), components which fail to accumulate in the yolk of the atretic r / o oocytes. the r / o allele of vldlr carries a point mutation (g to c substitution) resulting in the replacement of cysteine-682 in the extracellular domain of lr8, located outside the binding domain, with a serine residue. interestingly, the first ever delineated mutation in the human ldlr gene causing familial hypercholesterolemia occurred exactly in the equivalent position. the disruption of a disulfide bond by the loss of the cysteine residue due to this mutation was shown to cause protein misfolding accompanied by rapid intracellular degradation of the altered receptor molecules. in last consequence, the mutant lr8 protein does not reach the plasma membrane of oocytes of r / o hens, and thus is unable to mediate the uptake of serum - derived yolk precursor molecules. further investigations of the lipoprotein receptor system of hens revealed that those tissues which express the vldlr in mammals, i.e., heart, skeletal muscle, brain, and adipose tissue, but not the liver, also express lr8 in the chicken, albeit at very low levels (approx. interestingly, the structures of the major vldlr isoforms in mammals and the chicken lr8 differ by the presence (in mammals) and absence (in chicken oocytes) of the o - linked sugar domain (see above), respectively. the absence or presence of this domain of approximately 60 amino acids arises by differential splicing of the precursor mrna. thus, we performed detailed studies on the expression of lr8 splice variants in the chicken (for simplicity, the longer form is termed lr8 +, and the shorter one, lr8-). it was shown that somatic cells and tissues, in particular granulosa cells, heart, and skeletal muscle express predominantly lr8 +, while the oocyte express only lr8-. these results in the laying hen demonstrate that the oocytic lr8- is a multifunctional receptor for the transport of lipoproteins and other components required for embryonic growth. it is entirely possible that this holds true for the vldlr in other tissues, including the mammalian ovary. lr8 +, on the other hand, likely performs analogous functions in mammals and oviparous species, as they express this isoform in the same tissues. the molecular and functional properties of lr8 strengthen the hypothesis that it is the product of an ancient gene that has retained the ability to interact with many ligands of younger ldlr superfamilymembers. in this context, vtg, absent from mammals, and apoe, not found in birds, have been suggested to be functional analogues, as they show common biochemical properties and regions of sequence similarities. thus, triglyceride - rich particles, the likely physiological substrate for mammalian vldlrs, could be transported into metabolically active tissues (such as muscle, where receptors are abundant), while in avian oocytes the uptake of vtg, vldl, and other hepatically synthesized yolk precursors by lr8 provides nutrients and energy for the developing embryo. the most astounding events in the reproduction of oviparous (egg - laying) species are the growth and development of the female germ cell. the events leading to a mature oocyte in a wide variety of species are very similar. oocytes derive from mitotic cells called oogonia, which develop from primordial germ cells migrating into the ovary during early embryogenesis. the cells arrest in prophase of division i of meiosis when the chromosomes take on a these meiotically arrested female germ cells, termed primary oocytes, enter growth periods whose length is species - dependent. during this growth, primary oocytes acquire all components of the machinery subsequently required for embryo development, including ribosomes, trnas, cytoplasmic organelles, mrnas, and early yolk. in the female domesticated chicken (gallus gallus domesticus) and likely in all birds, fertilization - competent oocytes develop continuously in follicles of the left hand ovary (the right hand ovary is obliterated during embryogenesis). chicken oocytes grow in three phases : first, for several months, numerous microscopically small oocytes increase in size to 2 - 3 mm diameter, which still lack the typical yellow yolk ; second, several of these oocytes continue to grow slowly, and third, at reaching a diameter of 5 - 6 mm, an estimated 75% of these oocytes are destined for atresia (i.e., resorption), and one the remaining oocytes enters the last, very rapid growth phase, reaches a diameter of ca. this final stage is characterized by a dramatic 7-day growth spurt during which the oocyte extracts from the circulation plasma - borne components amounting to up to 14 ml of yolk. the yolk is comprised of lipid imported in the form of lipoproteins, mainly very low density lipoprotein (vldl) and vitellogenin (vtg), which contribute ca. 5 g of triglycerides and 230 mg of cholesterol to the yolk mass. lipoproteins and most of the additional minor yolk precursors are synthesized in and secreted from the liver and taken up by the growing oocytes via endocytotic processes involving specific cell - surface receptors. ovulation of the largest oocyte occurs every 25 hour and triggers the beginnning of the rapid growth phase of the next oocyte in line (laid as egg 7 - 8 days later), establishing a size- and time - related hierarchy of preovulatory follicles. if the hen has been inseminated, fertilization of the ovulated oocyte occurs in the uppermost portion of the oviduct. during the next 25 hour of migration through the lumen of the oviduct, structural components stabilizing the oocyte proper, egg white proteins, water, egg membranes, and the shell are deposited around the oocyte to form the laid egg. although the yolk appears homogeneous, it actually is a rather complex and precisely compartmentalized structure. from the germinal vesicle (the visible white spot on the oocyte surface), the latebra, a pear - shaped yolk - free cytoplasmic region, extends radially into the center of the oocyte. a medial cross - section reveals 7 concentric shells of yellow yolk (likely related to the 7 days of massive yolk deposition), which are separated by layers of oocytic cytoplasm that are connected to the latebra, thereby forming a continuous cytoplasmic compartment. the cytoskeletal elements and regulatory mechanisms that generate this structural organization, which may be unique to avian oocytes, are not known in any detail. as will be described in this review, most of the contents of the oocyte are imported by the action of multifunctional oocyte - specific receptors belonging to the ldl receptor (ldlr) gene family. both close and distant relatives of this family have been identified in the chicken, which thus has been established as a prime model to investigate the molecular genetics of these important receptors. molecular information on any of the proteins involved in oocyte growth and systemic lipoprotein transport in the laying hen was lacking until the mid-1980 's, although the presence of bona - fide lipoprotein particles in yolk was highly suggestive of receptor - mediated processes for yolk lipoprotein deposition. indeed, since then our studies on yolk precursor transport in the laying hen have not only provided proof for this concept, but also have revealed surprising new aspects of lipoprotein receptor biology with relevance to other species. it is now clear that a 95-kda protein in the plasma membrane of the oocyte binds both major yolk lipoproteins, vldl and vtg. this receptor protein reacts with antibodies to mammalian ldlrs and recognizes apolipoprotein (apo) e, an apo produced by mammals, but not by birds. these properties predicted that the oocyte receptor for vldl and vtg is a homologue of mammalian ldl receptors which recognize apob and apoe. the ldlr superfamily is defined by common structural elements with a high degree of sequence identity (70%-100%) between the molecules harbouring them, and in a wide range of species. their conserved sequences likely have evolved from an ancestral gene by duplication and/or exon shuffling events. the most typical of these elements are the so - called la repeats, which are tandemly arranged in clusters and form the ligand binding domains of ldlr family members. the classical ldlrs are characterized by the presence of seven la repeats in their ligand binding domains, whereas the vldlrs contain a cluster of eight la repeats. molecular cloning of the 95-kda oocyte protein indeed revealed an eight - repeat ligand binding domain which was confirmed to bind apoe despite the fact that apoe is absent from the chicken genome. thus, in order to distinguish this chicken oocyte receptor from mammalian vldrs, we subsequently termed it lr8 (ldl receptor relative with 8 la repeats). the gene specifying lr8 is located on the galline sex chromosome z. as discussed below, in addition to the high evolutionary conservation of eight - repeat receptors, the absence in chicken oocyte lr8 of a serine- and threonine - rich domain carrying o - linked carbohydrate chains appears significant. important functional insights into vldrs were gained from further studies in the chicken. whereas the spectrum of physiological functions of mammalian vldlrs is not yet fully delineated, lr8 mediates a key step in the reproductive effort of the hen, i.e., normal oocyte growth via yolk deposition. this conclusion can be drawn from the fact that functional absence of lr8 blocks oocytes from entering into the rapid growth phase. this phenotype is observed in a rare chicken strain carrying a single mutation at the vldlr locus (on the sex chromosome z, see above) termed restricted ovulator (r / o) strain. furthermore, as a consequence of the failure to deposit vldl and vtg, which are produced at normal levels, into their oocytes, the mutant females develop severe hyperlipidemia and features of atherosclerosis. carrier roosters (genotype, vldlr / vldlr) have normal lipid metabolism, as expected from our finding that lr8 is expressed almost exclusively in oocytes. thus, -/vldlr females, which in fact represent a model for an oocyte - specific receptor defect leading to familial hypercholesterolemia, are sterile due to non - laying. in addition to vldl and vtg, the receptor was shown to bind riboflavin - binding protein complexed with vtg and clusterin (apolipoprotein j ;), components which fail to accumulate in the yolk of the atretic r / o oocytes. the r / o allele of vldlr carries a point mutation (g to c substitution) resulting in the replacement of cysteine-682 in the extracellular domain of lr8, located outside the binding domain, with a serine residue. interestingly, the first ever delineated mutation in the human ldlr gene causing familial hypercholesterolemia occurred exactly in the equivalent position. the disruption of a disulfide bond by the loss of the cysteine residue due to this mutation was shown to cause protein misfolding accompanied by rapid intracellular degradation of the altered receptor molecules. in last consequence, the mutant lr8 protein does not reach the plasma membrane of oocytes of r / o hens, and thus is unable to mediate the uptake of serum - derived yolk precursor molecules. further investigations of the lipoprotein receptor system of hens revealed that those tissues which express the vldlr in mammals, i.e., heart, skeletal muscle, brain, and adipose tissue, but not the liver, also express lr8 in the chicken, albeit at very low levels (approx. interestingly, the structures of the major vldlr isoforms in mammals and the chicken lr8 differ by the presence (in mammals) and absence (in chicken oocytes) of the o - linked sugar domain (see above), respectively. the absence or presence of this domain of approximately 60 amino acids arises by differential splicing of the precursor mrna. thus, we performed detailed studies on the expression of lr8 splice variants in the chicken (for simplicity, the longer form is termed lr8 +, and the shorter one, lr8-). it was shown that somatic cells and tissues, in particular granulosa cells, heart, and skeletal muscle express predominantly lr8 +, while the oocyte express only lr8-. these results in the laying hen demonstrate that the oocytic lr8- is a multifunctional receptor for the transport of lipoproteins and other components required for embryonic growth. it is entirely possible that this holds true for the vldlr in other tissues, including the mammalian ovary. lr8 +, on the other hand, likely performs analogous functions in mammals and oviparous species, as they express this isoform in the same tissues. the molecular and functional properties of lr8 strengthen the hypothesis that it is the product of an ancient gene that has retained the ability to interact with many ligands of younger ldlr superfamilymembers. in this context, vtg, absent from mammals, and apoe, not found in birds, have been suggested to be functional analogues, as they show common biochemical properties and regions of sequence similarities. thus, triglyceride - rich particles, the likely physiological substrate for mammalian vldlrs, could be transported into metabolically active tissues (such as muscle, where receptors are abundant), while in avian oocytes the uptake of vtg, vldl, and other hepatically synthesized yolk precursors by lr8 provides nutrients and energy for the developing embryo. the above described findings are relevant to one of the crucial requirements for successful reproduction, i.e., the generation of mature, fertilization - competent oocytes., the features of a mechanism assuring normal growth and development of embryos in egg - laying (oviparous) species is described. in general, normal development of the embryo depends on the adequate supply with nutrients from maternal sources. when the mammalian placenta has become established, it provides the maternal - fetal interface mediating nutrient transfer between the maternal and embryonic circulation. however, before the placental circulation becomes fully functional during neural tube closure, the mammalian so - called visceral yolk sac (ys) is essential in providing nutrition to the developing embryo. in oviparous species, such as the chicken, the functionally equivalent interface is provided by the ys, which encloses the yolk that originally has been taken up and stored by the oocyte, as described in section chicken oocyte growth. the yolk, the almost exclusive source of nutrients for the developing embryo, contains macromolecular complexes comprising lipids, proteins, vitamins, minerals, and other essential micronutrients. at the onset of gastrulation, the formation of the mammalian visceral ys as well as the chicken ys is initiated ; when completed the yss are composed of cells derived from all three germ layers. avian ys formation is a highly coordinated growth process in which rapidly dividing ectodermal cells spread from the embryo proper to ultimately cover the entire yolk compartment. trailing the migrating front of the ectodermal layer, and between the yolk surface and the ectoderm, the ys s endodermal epithelial cells (eecs) proliferate and follow the ectodermal cells to form a tight epithelial layer, which remains in close contact with the yolk over the entire period of embryo development. finally, tightly associated with the basal aspect of the eecs, cells derived from the so - called splanchnic mesoderm migrate into the space between the ectodermal and endodermal layers and form a network of capillaries in tight contact with the neighboring two layers. thereby, the three germ layer cell types generate the fully functional ys, which is capable of mediating the targeted transfer of yolk - derived nutrient components to the embryonic circulation, as described below. during much of its growth, the chicken ys consists of three morphologically and functionally different regions (for an overview, see fig.1). these are termed (i) area pellucida, the central area of the early blastoderm which is considered part of the embryo proper ; (ii) area vasculosa, which forms the major, vascularized part of the nascent ys ; (iii) area vitellina, the ys 's non - vascularized portion localized at the growth front. ultrastructural studies have provided information on the dynamics of the ys s transformation during the phenotypic changes of the eecs upon transition of the area vitellina to the area vasculosa. during this transition, the apical face of eecs acquires the typical characteristics of polarized epithelial cells with numerous villi, invaginations, and coated pits. taken together, these and other observations support the notion that the eecs in the area vasculosa are the most active yolk - absorbing cells in the entire ys. three regions of the growing ys (area pellucida not shown) can be distinguished, each schematically represented by one eec and associated cells. the 3 regions are the area vitellina (left), a transition zone (center), and the area vasculosa (right). the area vitellina eecs lack endocytic receptors and nonspecifically phagocytose yolk components while they migrate along the yolk surface underneath the ectoderm. due to the presence of plin2 on the area vitellina ld surface (red circle around lds in area vitellina), the lipids of the lds, particularly triglycerides, are not available for extensive lipidation of apoa - i, and therefore the predominant secreted lipoprotein particles have a high density (hdl). in the transition zone, characterized by migration of mesenchymal progenitor cells into the interstitium between ecto- and endoderm, receptors begin to be produced, phagocytotic yolk uptake continues, and as plin2 levels drop, some ld lipids become available for increased lipoprotein synthesis and secretion. in the area vasculosa, blood vessel formation is coordinated with the enhanced production and localization of endocytic receptors with specificity for selected yolk components to the apical aspect of eecs, the disappearance of plin2, and the expression of genes specifying mtp and additional apolipoproteins. the lipids liberated by lipolysis from lds, and components derived from uptake and lysosomal processing of lipoproteins such as yvldl and other yolk macromolecules are utilized for the assembly of lipoproteins containing newly synthesized apoa - i, apob, and apoa - v, i.e., vldl - like and hdl - like particles which become efficiently secreted for transfer into the adjacent blood vessels, thereby supplying the embryo with nutrients generated by transformation of oocytic yolk in the eecs (adapted from bauer.). while morphological aspects of differentiation of ys cells and the acquisition of functional features in the course of ys maturation have been well characterized, very little is known about the components and regulation of the mechanisms that accomplish the complex process of nutrient uptake from yolk into the eecs and subsequent transfer to the embryo. previous gene expression profiling of area vasculosa eecs from 2 - 4 d old chick embryos revealed the enrichment of several transcripts for enzymes likely involved in lipid metabolism and for proteins possibly important for embryonic growth in these cells. however, candidate genes whose products may be functionally involved in the uptake of yolk nutrients did not emerge from this analysis. therefore, we have used the growing chicken ys to identify the processes and molecules that provide it with the ability to transfer nutrients from the yolk to the embryo. the rationale for these investigations results from the questions (i) whether the mature chicken ys resembles the mammalian visceral ys, a tissue essential to embryo nutrition prior to the onset of placental circulation, and (ii) at which stage of ys development the tissue becomes competent for nutrient transport. to answer these questions, the characteristic phenotypic differentiation of eecs during the area vitellina / area vasculosa transition, which accompanies the ys s vascularization, was exploited to identify and characterize proteins involved in the uptake of lipoprotein particles and other nutrient precursors from the yolk into the eecs. we showed that, subsequent to uptake and degradation of lipoproteins and other macromolecules from the yolk, the eecs synthesize lipoproteins de - novo that differ in composition from those in yolk and secrete them for targeting to the embryonic circulation. importantly, the expression patterns of key molecules for nutrient transfer, such as endocytic receptor complexes, apolipoproteins, and lipid droplet - associated proteins, differ between eecs of the area vasculosa and the area vitellina. one of the most significant observations was that plin2 (also known as adrp) is found in the area vitellina, but not vasculosa. plin2 has been shown to reduce the lipid droplet association of adipose triglyceride lipase and to slow triacylglycerol turnover (see conclusion and perspective). thus, the findings support a model (fig.1) in which the vascularized portion of the ys is its functionally active region, whereas the area vitellina, a tissue comparable with the extraembryonic endoderm in early rodent embryos appears to have primarily lipid storage functions. the specific genes and their products involved in nutrient transfer by the ys were identified in stepwise fashion. first, since the epithelium of the mature mammalian visceral ys expresses certain genes that have hitherto not been studied in the chicken, we established that these and other key components are indeed expressed in the fully developed avian ys. importantly, the multiligand receptors cubilin and ldlr related protein 2 (lrp2 or megalin), as well as amnionless, which together form a trimeric endocytosis - competent complex, are produced only by the eec layer of the area vasculosa, and not area vitellina. this receptor triad has been established as essential for nutrient transport by the mammalian visceral ys. mutations in any of the three genes or blocking their function by antibodies compromise normal embryonic development in rodents, especially affecting neural tube and brain formation. cubilin and lrp2 recognize a vast number of diverse ligands, many of which are yolk constituents, such as protein - bound vitamins, hormones, and a range of lipoproteins. furthermore, we have previously reported that the ys s eecs also express lr8, which is capable of internalizing apob - containing lipoproteins such as vldl, as well as vtg and clusterin, thus expanding the list of receptors involved in the uptake of yolk precursor macromolecules into the eecs. similar to receptor expression, only the area vasculosa eecs produce mtp and secrete apoa - i, apob, and apoa - v as protein moieties of newly synthesized lipoproteins targeted for ultimate uptake and utilization by the embryo. in contrast, the eecs from the area vitellina produce almost exclusively hdl - like, apoa - i - containing lipoprotein particles. the above described findings are relevant to one of the crucial requirements for successful reproduction, i.e., the generation of mature, fertilization - competent oocytes., the features of a mechanism assuring normal growth and development of embryos in egg - laying (oviparous) species is described. in general, normal development of the embryo depends on the adequate supply with nutrients from maternal sources. when the mammalian placenta has become established, it provides the maternal - fetal interface mediating nutrient transfer between the maternal and embryonic circulation. however, before the placental circulation becomes fully functional during neural tube closure, the mammalian so - called visceral yolk sac (ys) is essential in providing nutrition to the developing embryo. in oviparous species, such as the chicken, the functionally equivalent interface is provided by the ys, which encloses the yolk that originally has been taken up and stored by the oocyte, as described in section chicken oocyte growth. the yolk, the almost exclusive source of nutrients for the developing embryo, contains macromolecular complexes comprising lipids, proteins, vitamins, minerals, and other essential micronutrients. at the onset of gastrulation, the formation of the mammalian visceral ys as well as the chicken ys is initiated ; when completed the yss are composed of cells derived from all three germ layers. avian ys formation is a highly coordinated growth process in which rapidly dividing ectodermal cells spread from the embryo proper to ultimately cover the entire yolk compartment. trailing the migrating front of the ectodermal layer, and between the yolk surface and the ectoderm, the ys s endodermal epithelial cells (eecs) proliferate and follow the ectodermal cells to form a tight epithelial layer, which remains in close contact with the yolk over the entire period of embryo development. finally, tightly associated with the basal aspect of the eecs, cells derived from the so - called splanchnic mesoderm migrate into the space between the ectodermal and endodermal layers and form a network of capillaries in tight contact with the neighboring two layers. thereby, the three germ layer cell types generate the fully functional ys, which is capable of mediating the targeted transfer of yolk - derived nutrient components to the embryonic circulation, as described below. during much of its growth, the chicken ys consists of three morphologically and functionally different regions (for an overview, see fig.1). these are termed (i) area pellucida, the central area of the early blastoderm which is considered part of the embryo proper ; (ii) area vasculosa, which forms the major, vascularized part of the nascent ys ; (iii) area vitellina, the ys 's non - vascularized portion localized at the growth front. ultrastructural studies have provided information on the dynamics of the ys s transformation during the phenotypic changes of the eecs upon transition of the area vitellina to the area vasculosa. during this transition, the apical face of eecs acquires the typical characteristics of polarized epithelial cells with numerous villi, invaginations, and coated pits. taken together, these and other observations support the notion that the eecs in the area vasculosa are the most active yolk - absorbing cells in the entire ys. three regions of the growing ys (area pellucida not shown) can be distinguished, each schematically represented by one eec and associated cells. the 3 regions are the area vitellina (left), a transition zone (center), and the area vasculosa (right). the area vitellina eecs lack endocytic receptors and nonspecifically phagocytose yolk components while they migrate along the yolk surface underneath the ectoderm. due to the presence of plin2 on the area vitellina ld surface (red circle around lds in area vitellina), the lipids of the lds, particularly triglycerides, are not available for extensive lipidation of apoa - i, and therefore the predominant secreted lipoprotein particles have a high density (hdl). in the transition zone, characterized by migration of mesenchymal progenitor cells into the interstitium between ecto- and endoderm, receptors begin to be produced, phagocytotic yolk uptake continues, and as plin2 levels drop, some ld lipids become available for increased lipoprotein synthesis and secretion. in the area vasculosa, blood vessel formation is coordinated with the enhanced production and localization of endocytic receptors with specificity for selected yolk components to the apical aspect of eecs, the disappearance of plin2, and the expression of genes specifying mtp and additional apolipoproteins. the lipids liberated by lipolysis from lds, and components derived from uptake and lysosomal processing of lipoproteins such as yvldl and other yolk macromolecules are utilized for the assembly of lipoproteins containing newly synthesized apoa - i, apob, and apoa - v, i.e., vldl - like and hdl - like particles which become efficiently secreted for transfer into the adjacent blood vessels, thereby supplying the embryo with nutrients generated by transformation of oocytic yolk in the eecs (adapted from bauer.). while morphological aspects of differentiation of ys cells and the acquisition of functional features in the course of ys maturation have been well characterized, very little is known about the components and regulation of the mechanisms that accomplish the complex process of nutrient uptake from yolk into the eecs and subsequent transfer to the embryo. previous gene expression profiling of area vasculosa eecs from 2 - 4 d old chick embryos revealed the enrichment of several transcripts for enzymes likely involved in lipid metabolism and for proteins possibly important for embryonic growth in these cells. however, candidate genes whose products may be functionally involved in the uptake of yolk nutrients did not emerge from this analysis. therefore, we have used the growing chicken ys to identify the processes and molecules that provide it with the ability to transfer nutrients from the yolk to the embryo. the rationale for these investigations results from the questions (i) whether the mature chicken ys resembles the mammalian visceral ys, a tissue essential to embryo nutrition prior to the onset of placental circulation, and (ii) at which stage of ys development the tissue becomes competent for nutrient transport. to answer these questions, the characteristic phenotypic differentiation of eecs during the area vitellina / area vasculosa transition, which accompanies the ys s vascularization, was exploited to identify and characterize proteins involved in the uptake of lipoprotein particles and other nutrient precursors from the yolk into the eecs. we showed that, subsequent to uptake and degradation of lipoproteins and other macromolecules from the yolk, the eecs synthesize lipoproteins de - novo that differ in composition from those in yolk and secrete them for targeting to the embryonic circulation. importantly, the expression patterns of key molecules for nutrient transfer, such as endocytic receptor complexes, apolipoproteins, and lipid droplet - associated proteins, differ between eecs of the area vasculosa and the area vitellina. one of the most significant observations was that plin2 (also known as adrp) is found in the area vitellina, but not vasculosa. plin2 has been shown to reduce the lipid droplet association of adipose triglyceride lipase and to slow triacylglycerol turnover (see conclusion and perspective). thus, the findings support a model (fig.1) in which the vascularized portion of the ys is its functionally active region, whereas the area vitellina, a tissue comparable with the extraembryonic endoderm in early rodent embryos appears to have primarily lipid storage functions. the specific genes and their products involved in nutrient transfer by the ys were identified in stepwise fashion. first, since the epithelium of the mature mammalian visceral ys expresses certain genes that have hitherto not been studied in the chicken, we established that these and other key components are indeed expressed in the fully developed avian ys. importantly, the multiligand receptors cubilin and ldlr related protein 2 (lrp2 or megalin), as well as amnionless, which together form a trimeric endocytosis - competent complex, are produced only by the eec layer of the area vasculosa, and not area vitellina. this receptor triad has been established as essential for nutrient transport by the mammalian visceral ys. mutations in any of the three genes or blocking their function by antibodies compromise normal embryonic development in rodents, especially affecting neural tube and brain formation. cubilin and lrp2 recognize a vast number of diverse ligands, many of which are yolk constituents, such as protein - bound vitamins, hormones, and a range of lipoproteins. furthermore, we have previously reported that the ys s eecs also express lr8, which is capable of internalizing apob - containing lipoproteins such as vldl, as well as vtg and clusterin, thus expanding the list of receptors involved in the uptake of yolk precursor macromolecules into the eecs. similar to receptor expression, only the area vasculosa eecs produce mtp and secrete apoa - i, apob, and apoa - v as protein moieties of newly synthesized lipoproteins targeted for ultimate uptake and utilization by the embryo. in contrast, the eecs from the area vitellina produce almost exclusively hdl - like, apoa - i - containing lipoprotein particles. taken together, these data support a model for differentiation of the developing chicken ys as a result of coordination of vascularization and acquisition of function, as outlined in fig. 1. a key feature of this process at the molecular level is the switch of the eecs from a storage type to a metabolically highly active type of cells in synchrony with the acquisition of vasculature. this conversion is characterized by the onset of increased production of endocytic yolk protein receptors, accessory proteins, and apolipoproteins in addition to apoa - i, as well as by the secretion of lipoproteins by eecs in the neo - vascularized region of the growing ys. furthermore, in contrast to the eecs of the area vasculosa, which synthesize and secrete (apo)lipoproteins at high rates, the requirement for lipids stored in lipid droplets (lds) in the eecs of the area vitellina (which produce low levels of lipoproteins) is expected to be low. this important difference might be regulated by proteins that play a role in the stability of lds, i.e., via reducing the susceptibility of lds to lipolysis and thus availability of substrate for lipoprotein synthesis. one of the known proteins that promotes the accumulation and growth of lds in cells by compromising lipolysis is plin2 (fig.1). indeed, plin2 knock - out mice show elevated vldl secretion rates and a decrease in hepatic lipid content, and knockdown of plin2 in mca - rh7777 hepatoma cells results in increased vldl secretion. the absence of plin2 from the area vasculosa eecs, but its high levels in the area vitellina, where lipoproteins containing apob are hardly produced at all, indicates that plin2 performs a so far unknown regulatory function in lipid metabolism of the developing chicken ys. these results support the notion that a crosstalk between the processes of eec differentiation and of vasculogenesis is essential to ys function and thus, for embryo development. | abstractstudies of receptor - mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. a prime example for the power of comparative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipoprotein receptors in somatic cells and oocytes are the key to oocyte growth. in avian species, yolk precursor transport from the hen 's liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes. oocytes grow from a diameter of 5 mm to 2.5 - 3 cm in only 7 days, and the yolk sac transfers nutrients from the yolk stored in the mature oocyte to the embryo within just 2 weeks. the underlying key transport mechanism is receptor - mediated endocytosis of macromolecules, i.e., of hepatically synthesized yolk precursors for oocyte growth, and of mature yolk components for embryo nutrition, respectively. recently, the receptors involved, as well as the role of lipoprotein synthesis in the yolk sac have been identified. as outlined here, lipoprotein degradation / resynthesis cycles and the expression of lipoprotein receptors are not only coordinated with the establishment of the follicular architecture embedding the oocyte, but also with the generation of the yolk sac vasculature essential for nutrient transfer to the embryo. |
the human epidermal growth factor receptor (her) family of receptors plays a central role in the pathogenesis of several human cancers. they regulate cell growth, survival, and differentiation via multiple signal transduction pathways and participate in cellular proliferation and differentiation. the family is made up of four main members : her-1, her-2, her-3, and her-4, also called erbb1, erbb2, erbb3, and erbb4, respectively. all four her receptors comprise a cysteine - rich extracellular ligand binding site, a transmembrane lipophilic segment, and an intracellular domain with tyrosine kinase catalytic activity. epidermal growth factor receptor (egfr, erbb1, and her1)the first receptor tyrosine kinase, was discovered by carpenter and coworkers at vanderbilt university, usa, in 1978. erbb stands for its origin in the erb - b gene responsible for avian erythroblastosis virus. the neu oncogene (also known as her2, erbb2, or p185) was discovered by a group of scientists at massachusetts institute of technology, rockefeller, and harvard university [4, 5 ]. the her2 receptor is a 1255 amino acid, 185 kd transmembrane glycoprotein located at the long arm of human chromosome 17 (17q12). her2 is expressed in many tissues and its major role in these tissues is to facilitate excessive / uncontrolled cell growth and tumorigenesis [79 ]. the her receptors exist as monomers on the cell surface. upon ligands binding to their extracellular domains, her2 has no known direct activating ligand and may be in an activated state constitutively or become active upon heterodimerization with other family members such as her1 and her3. homo- or heterodimerization results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways, principally the mitogen - activated protein kinase (mapk), phosphatidylinositol-4,5-bisphosphate 3-kinase (pi3k), and protein kinase c (pkc) resulting in cell proliferation, survival, differentiation, angiogenesis, and invasion. heterodimers generate more potent signals than homodimers, and those containing her2 have a particularly high ligand binding and signaling potency as her2 exists in an open conformation making it the dimerization partner of choice among the family members. the her2-her3 heterodimer is the most potent stimulator of downstream pathways, particularly the pi3k / akt, a master regulator of cell growth and survival. moreover, her2 dimerization promotes the mislocalization and rapid degradation of cell - cycle inhibitor p27 protein leading to cell - cycle progression [7, 10, 11 ]. her2 can also be activated by complexing with other membrane receptors such as insulin - like growth factor receptor 1. figure 1 shows the main transduction pathways regulated by the four her family members egfr, her2, her3, and her4. most of the studies on her2 have been carried out in breast cancer, after it was found to induce mammary carcinogenesis in vitro and in vivo. amplification or overexpression of the her2 gene occurs in approximately 1530% of breast cancers. with increasing understanding of her2 biology, it has now been recognized that her2 overexpression occurs in other forms of cancers also such as stomach, ovary, uterine serous endometrial carcinoma, colon, bladder, lung, uterine cervix, head and neck, and esophagus [17, 18 ]. apart from its role in development of various cancers, it has also been intensely evaluated as a therapeutic target. the aim of this review is to update the role of her2 in various cancers. her2 is overexpressed in 1530% of invasive breast cancers, which has both prognostic and predictive implications. breast cancers can have up to 2550 copies of the her2 gene, and up to 40100-fold increase in her2 protein resulting in 2 million receptors expressed at the tumor cell surface. even estrogen, working via the nongenomic activity of estrogen receptor (er) outside the nucleus, has been shown to activate her2 signaling. an aberrant form of her2 (known as p95), lacking the extracellular domain, is found in some breast cancers. p95 is constitutively active and causes resistance to trastuzumab which requires the extracellular domain of her2 for binding. for the same reason, p95 is not detected by antibodies that target the extracellular domain [21, 22 ]. her2 gene amplification is associated with shorter disease - free and overall survival in breast cancer. slamon. established the prognostic significance of her2 amplification in 189 human breast cancers. amplification of her2 gene was found to be a significant predictor of both overall survival (p 2.5) did significantly worse than those with lower her2 amplification (ratio 2.02.5). the mutations targeted never or light smokers, oriental ethnicity, and female gender [51, 52 ]. in invasive urothelial bladder carcinomas, amplification and/or overexpression range from 23% to 80% for overexpression and from 0% to 32% for amplification [53, 54 ]. however, clinical trials using her2 directed therapies in lung and bladder cancers reported disappointing clinical benefits [55, 56 ]. although several methods for her2 testing have been developed, approximately 20% of current her2 testing may be inaccurate. therefore, the american society of clinical oncology (asco) and the college of american pathologists (cap) have recommended guidelines in her2 testing to ensure accuracy. the two methods currently approved for her2 testing are immunohistochemistry (ihc) and fluorescence in situ hybridization (fish). her2 status should be determined in all patients with invasive breast cancer on the basis of 1 or more test results. breast cancer specimens should initially undergo her2 testing by a validated immunohistochemistry (ihc) assay for her2 protein expression. the scoring method for her2 expression is based on the cell membrane staining pattern and is as follows:3 + : positive her2 expression, uniform intense membrane staining of more than 30% of invasive tumor cells;2 + : equivocal for her2 protein expression, complete membrane staining that is either nonuniform or weak in intensity but has circumferential distribution in at least 10% of cells;0 or 1 + : negative for her2 protein expression.breast cancer specimens with equivocal ihc should undergo validation fluorescence in situ hybridization (fish). the interpretation for her2 fish testing (her2-to - cep17 ratio and gene copy number) is as follows : positive her2 amplification : fish ratio higher than 2.2 or her2 gene copy greater than 6.0;equivocal her2 amplification : fish ratio of 1.82.2 or her2 gene copy of 4.06.0;negative her2 amplification : fish ratio lower than 1.8 or her2 gene copy less than 4.0.figure 2 shows her2 analysis by ihc and fish on breast tumor tissue. 3 + : positive her2 expression, uniform intense membrane staining of more than 30% of invasive tumor cells ; 2 + : equivocal for her2 protein expression, complete membrane staining that is either nonuniform or weak in intensity but has circumferential distribution in at least 10% of cells ; 0 or 1 + : negative for her2 protein expression. positive her2 amplification : fish ratio higher than 2.2 or her2 gene copy greater than 6.0 ; equivocal her2 amplification : fish ratio of 1.82.2 or her2 gene copy of 4.06.0 ; negative her2 amplification : fish ratio lower than 1.8 or her2 gene copy less than 4.0. gastric cancer. in gastric cancers, heterogeneity of the her2 genotype can lead to discrepancies in the results from ihc and fish testing. tumor heterogeneity was seen in roughly 4.8% of samples with moderate or strong her2 staining and was higher than what was experienced in breast cancer (1.4%). incomplete basolateral membrane her2 ihc staining is also more common in gastric cancer than in breast cancer. this is due to the higher frequency of glandular formations that occur in gastric tissue. in gastric tissue, the basolateral membrane is stained, not the luminal membrane resulting in the heterogeneity. currently, there are no asco / cap approved her2 testing guidelines for gastric cancer. table 1 shows consensus panel recommendations on her2 scoring for gastric / esophageal cancer [60, 61 ]. the national comprehensive cancer network (nccn) guidelines panel recommended that less than 3 + overexpression of her2-neu by ihc should be additionally examined by fish or other in situ hybridization methods. gastric cancers with her2 ihc overexpression of 3 + or fish positive are considered positive and thus be treated with trastuzumab. thus, her2 3 + or fish + /her2 ihc 1 +, fish + /her2 ihc 2 +, fish + /her2 ihc 3 + gastric cancer patients should be treated with trastuzumab. her2 has been successfully targeted in breast cancer and gastric / gastroesophageal cancers. in ovarian cancer trastuzumab is a monoclonal antibody that binds to domain iv of the extracellular segment of the her2 receptor. proposed mechanisms of trastuzumab actions include (1) inhibition of her2 shedding, (2) inhibition of pi3k - akt pathway, (3) attenuation of cell signalling, (4) antibody - dependent cellular cytotoxicity, and (5) inhibition of tumor angiogenesis. trastuzumab was approved as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of women with node - positive, her2 overexpressing breast cancer. the approval was based on evidence of a significant prolongation in disease - free survival in women receiving trastuzumab and chemotherapy compared to those receiving chemotherapy alone. table 2 shows five pivotal trials involving more than 10,000 women which demonstrated that one year of trastuzumab therapy provided significant clinical benefit [6366 ]. these trials demonstrated that inclusion of trastuzumab produces roughly a 50% improvement in disease - free survival and 33% improvement in overall survival, regardless of the chemotherapy regimen or sequence of trastuzumab delivery. in the metastatic her2 breast cancer also, trastuzumab is recommended in the first - line setting. in a phase iii trial, trastuzumab plus chemotherapy was associated with a significant improvement in time to disease progression, objective response rate, and 1-year survival compared with chemotherapy alone. trastuzumab was approved in combination with cisplatin and a fluoropyrimidine, for the treatment of patients with her2 overexpressing metastatic gastric or gastroesophageal (ge) junction adenocarcinoma who have not received prior treatment for metastatic disease. the pivotal toga (trastuzumab for gastric cancer) trial demonstrated the median survival of 13.1 months for patients receiving trastuzumab and chemotherapy and 11.7 months for patients receiving chemotherapy alone. trastuzumab was found to be most effective in prolonging survival in patients with her2 ihc 3 + tumors as compared to patients with ihc 2 + tumors. trastuzumab is recommended at a dose of 4 mg / kg followed by 2 mg / kg weekly for breast cancer and 8 mg / kg followed by 6 mg / kg q3 weekly for gastric / gastroesophageal cancer. the duration of therapy is one year in adjuvant setting for breast cancer and till disease progression for metastatic breast, gastric, and gastroesophageal cancer. the most common adverse effects seen with trastuzumab are fever, vomiting, infusion reactions, diarrhea, headache, fatigue, rash, neutropenia, and anemia. the most serious adverse effects include cardiomyopathy, pulmonary toxicity, infusion reactions, and febrile neutropenia. left ventricular ejection fraction (lvef) should be evaluated in all patients prior to and during treatment with trastuzumab. lapatinib is an orally active dual tyrosine kinase inhibitor which interrupts the her2 and epidermal growth factor receptor (egfr) pathways. lapatinib is approved in combination therapy with capecitabine for her2 overexpressing advanced and metastatic breast cancer patients who have received prior therapy including an anthracycline, a taxane, and trastuzumab. this was based on a study that demonstrated delay in time to disease progression when lapatinib was used in combination with capecitabine. the risk of disease progression was reduced by 51%, and the combination therapy was not associated with increases in toxic side effects. lapatinib is recommended at a dose of 1250 mg po qday on days 121 continuously in combination with capecitabine (2000 mg / m/day po divided q12hr) on days 114 in a repeating 21-day cycle. lapatinib is also approved in combination with leterozole for the treatment of postmenopausal women with hormone receptor and her2 receptor positive metastatic breast cancers. the addition of lapatinib to letrozole is well tolerated and leads to a significantly greater progression free survival, overall response rate, and clinical benefit rate than with letrozole alone. the most common adverse effects with lapatinib are diarrhea, anemia, hand - foot syndrome, liver dysfunction, nausea, rash, and neutropenia. pertuzumab is a humanized monoclonal antibody that blocks the activation of the her2 receptor by hindering dimerization. it is approved in combination with trastuzumab and docetaxel in her2-positive metastatic breast cancer patients previously not treated with hormone therapy or chemotherapy. the approval of pertuzumab was based on results from the clinical evaluation of pertuzumab and trastuzumab (cleopatra) trial. the trial compared first - line trastuzumab plus docetaxel (plus placebo) to trastuzumab plus docetaxel plus pertuzumab in her2-positive metastatic breast cancer. results from the study showed an average increase in progression - free survival of 6.1 months in patients receiving pertuzumab in addition to trastuzumab and docetaxel with minimal to no increase in cardiac toxic effects. pertuzumab is also approved for use as neoadjuvant treatment in combination with trastuzumab and docetaxel for patients with her2-positive, locally advanced, inflammatory, or early stage breast cancer. this was based on a randomized trial in which 39.3% of patients treated with pertuzumab, trastuzumab, and docetaxel achieved a pathologic complete response (pcr) compared with 21.5% of patients treated with trastuzumab and docetaxel at the time of surgery. the recommended dose of pertuzumab is 840 mg initial dose followed by 420 mg every 3 weeks administered as an intravenous infusion over 30 to 60 minutes. the adverse effects seen with pertuzumab are alopecia, diarrhea, nausea, neutropenia, and cardiomyopathy. ado - trastuzumab emtansine is an antibody - drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent mertansine (dm1). ado - trastuzumab offers a novel mechanism for overcoming trastuzumab resistance by exploiting trastuzumab to target the cytotoxic activity of dm1 to her2 overexpressing cells. ado - trastuzumab is approved as a single agent for treatment of her2-positive, metastatic breast cancer in patients who have already received trastuzumab and a taxane either separately or in combination. approval was based on results from emilia trial which compared ado - trastuzumab to lapatinib plus capecitabine. the study showed a significantly prolonged progression - free survival and overall survival with less toxicity than lapatinib plus capecitabine. the recommended dose of ado - trastuzumab is 3.6 mg / kg iv infusion q3 weeks until disease progression or unacceptable toxicity. the most common adverse effects include fatigue, nausea, musculoskeletal pain, thrombocytopenia, headache, transaminitis, constipation, and peripheral neuropathy. the serious adverse effects include liver failure, hepatic encephalopathy, nodular regenerative hyperplasia, cardiac dysfunction, and interstitial lung disease. a phase ii open label study in locally advanced breast cancer (labc) showed a 16-week progression - free survival rate of 75% in 36 trastuzumab - naive patients and 51% in previously treated disease. diarrhea was the most common grade 3/4 toxic effect (21%) in this study. phase iii evaluation of neratinib is ongoing in adjuvant trastuzumab - pretreated early - stage breast cancer. afatinib is an oral, irreversible inhibitor targeting egfr / her1, her2, and her4. results from a phase ii study of afatinib for her2-positive mbc progressing posttrastuzumab (n = 41) showed 4 partial responses among 35 assessable patients. the most common all - grade treatment - related aes included diarrhea (90.2%) and rash (65.9%). lux - breast 1 is an ongoing phase iii study of vinorelbine plus either afatinib or trastuzumab for her2-positive mbc in patients who failed one trastuzumab - containing regimen as first - line treatment of mbc or as adjuvant therapy. her2 has served as a prognostic and predictive biomarker in breast and gastric / gastroesophageal cancers. therapies directed against her2 have revolutionized the treatment of her2 overexpressing breast and gastric cancers and improved the clinical outcome. although her2 overexpression was also found to correlate with poor outcome in other cancers, her2 directed therapies provided disappointed results. various novel her2 directed agents alone or in combination are under investigation and in near future we will be expecting more varied implications of her2 directed therapies. till more robust data on the prognostic significance of her2 in other cancers is available, her2 testing and her2 directed therapies are recommended in only breast and gastric / gastroesophageal cancers. | human epidermal growth factor receptor 2 (her2) is a member of the epidermal growth factor receptor family having tyrosine kinase activity. dimerization of the receptor results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways leading to cell proliferation and tumorigenesis. amplification or overexpression of her2 occurs in approximately 1530% of breast cancers and 1030% of gastric / gastroesophageal cancers and serves as a prognostic and predictive biomarker. her2 overexpression has also been seen in other cancers like ovary, endometrium, bladder, lung, colon, and head and neck. the introduction of her2 directed therapies has dramatically influenced the outcome of patients with her2 positive breast and gastric / gastroesophageal cancers ; however, the results have been proved disappointing in other her2 overexpressing cancers. this review discusses the role of her2 in various cancers and therapeutic modalities available targeting her2. |
formaldehyde is a strong antibacterial and preservative that is utilized for controlling microbial growths in water containing solutions and organic materials. formaldehyde is a gaseous and colorless molecule at room temperature and it is readily soluble in water. this compound is a strong antibacterial and very low cost preservative so producers use it in liquid detergents and cosmetic products instead of other preservatives. the hazard of formaldehyde has been reported which found enough evidence for the mutagenicity and carcinogenicity of this compound [1, 2 ]. it is an irritating agent to the respiratory zone and eye which can cause allergic contact dermatitis or eczema (drying and reddening of the skin) at high concentration. the formaldehyde and formaldehyde releasing preservative is widely used in surfactants, dishwashing liquids, cosmetic products particularly hair shampoos, and care products such as hair straightening products. toxicology of formaldehyde has been widely discussed and studies have indicated the highest risk for individuals with a daily contact with this substance [4, 5 ]. the use of formaldehyde as a preservative is allowed to a maximum concentration of 0.2% in detergent and cosmetic products (with the exception of nail polish for which a concentration up to 5% is allowed). due to allergic potential of formaldehyde, liquid detergents and cosmetic products contain formaldehyde when there is a minimum content of 0.05% free of this substance [6, 7 ]. because of the above reasons and the influence of formaldehyde in human bodies, various methods have been developed for determination of formaldehyde in some detergents and cosmetic products including high performance liquid chromatography (hplc) [811 ], headspace solid phase microextraction by gas chromatography (gc), and isotope dilution mass spectrometry [12, 13 ], fluorimetry, and spectrophotometry [15, 16 ]. various reagents have been proposed for derivatization of formaldehyde by spectrophotometry [1721 ] and also fluorimetry [2226 ]. however, spectrophotometric method is not sensitive enough for the analysis of real samples and sometimes subject to numerous interferences which are serious problems. examined several novel reagents for detection of formaldehyde based on the hantzsch reaction which were benzoylacetone, n - methyl acetoacetamide, n - acetoacetyl - o - toluidine, and acetoacetanilide (aaa) used in fluorimetry. in this study, the proposed method is based on the hantzsch reaction, which involves the cyclization between 2-methyl acetoacetanilide and formaldehyde in the presence of ammonium acetate. the derivatization reaction of formaldehyde with 2-methyl acetoacetanilide is shown in scheme s1 (see supplementary material available online at http://dx.doi.org/10.1155/2016/1720530). the aim of this study is applying fluorescence spectroscopy to routine determination of formaldehyde in detergents and cosmetic products. fluorescence spectroscopy as an analytical technique has the advantage of selectivity and sensitivity in comparison with other spectroscopic methods. although this method may be efficient in some experiments, it is not effective while there are several independent variables and interaction influences affecting the responding factors. design of experiments (doe) is a powerful technique used for discovering a set of variables (or factors) which are most important to the process or experiment and then determining what levels of these factors must be kept to optimize the process performance. optimization strategy sets several experiments that can determine all factors and probable interactions between these independent variables. when the responses are influenced by several factors, response surface methodology (rsm) is a useful tool of modeling with a collection of statistical and mathematical techniques. the box - behnken design is a second - order design under rsm that requires 3 levels of each factor ; it is reliable, less laborious, efficient, cost - effective, providing sufficient data on the influence variables and decreasing the number of experimental trials and overall experiment error with a minimum number of experiments. stock solution of 2-methyl acetoacetanilide (0.3 m) was prepared by dissolving 5.73 g of 2-methyl acetoacetanilide (aldrich pure chemicals, germany) in 50 ml of ethanol and diluting it to 100 ml with distillation water. an ammonium acetate stock solution was prepared by dissolving 54.00 g of ammonium acetate (merck pure chemicals, germany) in the water and diluting it to 100 ml with the purified water. the ph of ammonium acetate was adjusted as 7.5 by addition of 0.1 m naoh. a 0.10 m standard solution of formaldehyde was prepared by diluting 0.9 ml of 30.0% formaldehyde solution to 100 ml with distillation water, followed by an accurate concentration determination using the iodometric method before analysis since formaldehyde is volatile. formaldehyde reacts with potassium iodide solution and iodine is back - titrated with standard solution of sodium thiosulphate in the presence of starch indicator. the working standard solutions were daily prepared by accurate dilution of the standard stock solution. for interference testing, the following compounds were used : sodium chloride, iron (iii) nitrate, nickel, lead and cadmium (ii) nitrate, calcium and magnesium (ii) chloride, benzaldehyde, propionaldehyde, and acetaldehyde. a photoluminescence spectrometer ls55 (perkinelmer, uk) was employed for spectral measurements. a ph meter model 691 (metrohm, swiss), heater stirrer (gerhardt, germany), and analytical balance model r160p (sartorius, germany) were applied. hnmr spectra were recorded on bruker avance-400 mhz spectrometers in the presence of tetra methyl silane as internal standard. box - behnken design contains numbers of design points and a repeated center point for calculating of error. box - behnken design does not contain an embedded factorial design like the central composite design and instead of design points there are the midpoints and it requires 3 levels (1, 0, and + 1) for each factor. the box - behnken design can be considered a highly fractionalized three - level factorial design where the treatment combinations are the midpoints of edges of factor levels and the center point. these designs are rotatable (or nearly rotatable) and require three levels each under study factors. box - behnken design can fit full quadratic response surface models that it does not contain an embedded factorial or fractional factorial design and does not contain axial points so all design points are sure to be within safe operating limits. in this design, the treatment combinations are at the midpoints of edges of the process space and at the center. the advantages of the box - behnken design over other response surface designs are as follows : (a) it needs fewer experiments than central composite design (less points) ; (b) in contrast to central composite designs, it has only three levels ; (c) it is easier to arrange and interpret than other designs ; (d) it avoids combined factor extremes since midpoints of edges of factors are always used. in this work, because of derivatization reaction of formaldehyde depending on five factors in three levels, we employed the response surface method and box - behnken design to optimize the independent factors that are effective on the response. in order to reduce the effects of unexplained variability in the real responses due to irrelevant parameters, the experiments were done in randomized order. then, a second - order quadratic equation was fitted to the data by multiple regression procedure. the generalized response surface model for a five - factor system is shown by (1)yi=0+1x1+2x2+3x3+4x4+5x5+11x12+22x22+33x32+44x42+55x52+12x1x2+13x1x3+14x1x4+15x1x5+23x2x3+24x2x4+25x2x5+34x3x4+35x3x5+45x4x5,where yi is the predicted response, xi values (i = 1, 2, 3, 4, and 5) are the independent variables, 0 is the intercept (constant), 1, 2, 3, 4, and 5 are linear coefficients, 11, 22, 33, 44, and 55 are squared coefficients, 12, 13, 14, 15, 23, 24, 25, 34, 35, and 45 are interactions coefficients. in this work, five studied factors consist of reaction time (x1), % v / v ethanol (x2), ammonium acetate concentration (x3), 2-methyl acetoacetanilde concentration (x4), and temperature (x5) on the analytical signal. minitab14 as a statistical software package was used for data processing, studying linear terms, squared terms, and interaction between the variables. a box - behnken design was applied to estimate the correlation of the five independent factors, with three levels for each factor. in this study, the name of independent effective factors and their coded levels scheme of box - behnken design are shown in tables 1 and s1, respectively. the optimum values were calculated for effective parameters on derivatization reaction for 50 gkg of formaldehyde. the optimum value of each variable was calculated through codes and their corresponding values to be encoded (actual) with minitab14 software (table 2). additionally, the optimum response values of each factor can be obtained by the graphical analysis of the surface related to each equation obtained by minitab software (figures s1 and s2). the effects of efficient parameters, regression coefficients, and the associated standard errors for derivatization reaction of formaldehyde are shown in table s2. the excitation and emission spectra were obtained with 2-methyl acetoacetanilide in the presence and in the absence of formaldehyde. the maximum wavelengths of excitation and emission were 360 and 460 nm, respectively. figure 1 shows maximum excitation and emission wavelength for product of formaldehyde derivatization with 2-methyl acetoacetanilide. the calibration curve for determination of formaldehyde was constructed under the optimum condition summarized in table 2. for fluorometric measurements, emission spectra of 020 10 m of formaldehyde were recorded (figure 2). the fluorescence response was linear in the formaldehyde concentration range of 0.3320 10 m (160 gkg). the equation of the calibration graph for 0.3320 10 m was expressed as y = 40.12x + 42.12, where y is the fluorescence intensity and x is the formaldehyde concentration, with a correlation coefficient of 0.997. as hnmr spectroscopy is a perfect method for characterization of chemicals, this method was used for characterization of the hantzsch product. all protons of hantzsch product were correctly determined in the hnmr : = 2.4 ppm : 12h (4 ch3). according to the hnmr data (figure s3), we conclude that the hantzsch product was successfully synthesized and purified. in the hnmr spectra, the signals of methyl groups joined to the double bond and benzene ring appear as signals at = 2.4 and 2.27 ppm, respectively. the nh protons related to amino group appears in = 5.16 ppm (figure s4), while nh signal related to amino groups is shown in 7.86 and 7.87 ppm, respectively. the signals about = 7.07.5 ppm assigned by proton of ch - ch of aromatic rings in the product spectrum (figure s5). the limit of detection is calculated as the concentration corresponding to three times of the baseline noise (s / n = 3). the proposed methodology was applied for the analysis of iranian brands of liquid detergents and cosmetic products. some of these products contained detectable amounts of formaldehyde that were higher than the maximum allowed concentration of 0.2%. for evaluation of the precision and accuracy of the proposed method, spiking experiments were performed. in table 4, we spiked 50 mgkg of formaldehyde to surfactant, hair shampoo, body shampoo, and hand cleaner which have no free formaldehyde. after spiking experiments, recovery factors were then calculated according to the following equation (the results are shown in table 4):(2)recovery factor=100crecoveredctrue, where crecovered is the measured concentration after adding known concentration of the analyte to the real sample and ctrue is the expected concentration. in table 5, we spiked 3, 5, and 8 gkg of formaldehyde to dishwashing liquid 1 and hair shampoo 1 that contain formaldehyde. after spiking experiments, recovery factor and relative standard errors the use of formaldehyde as a preservative in liquid detergents such as dishwashing liquids and cosmetic products such as hair and body shampoos and shower gel is allowed to a maximum concentration of 0.2%, that is, 600 mgkg. considerable decreasing in the content of formaldehyde occurs after a few months in detergents and cosmetic products. the effect of various spectral interferences possibly present in the real detergent samples was investigated. detergents and cosmetic products such as hair and body shampoos contain water (about 70%), surfactants as a cleaning and foaming agent (about 25%), and additives (about 5%) such as colors, fragrances, and other compounds. three - dimensional florescence spectrum of a shampoo sample was recorded after derivatization of formaldehyde (figure s6). to generate three - dimensional fluorescence, excitation wavelengths between 200 and 400 nm with 10 nm intervals and emission wavelengths between 250 and 500 nm with 0.5 nm intervals were used. the tolerable concentrations are defined as the concentration of foreign species causing less than 5% relative error. the influences of foreign species were examined by adding a certain amount of species in 1.0 10 m hcho solution. the hantzsch reactions with some diketones analogues reagents are usually slow and therefore detection reactions must be carried out at higher temperature than room temperature. the effect of reaction temperature on signal intensity in reaction of 2-methyl acetoacetanilide with formaldehyde was examined by varying it from 20 to 60c. the results obtained are shown in figure s7 that, in the temperature over 30, the fluorescence intensity is decreased. the experimental design results show that 27c is the optimum temperature ; thus, above 28c, the intensity gradually decreased with increasing temperature. however, for convenient operation, 25c (room temperature) was selected. in the reaction of formaldehyde with the 2-methyl acetoacetanilide reagents, the preliminary examination showed that the fluorescence intensity of product of formaldehyde with 2-methyl acetoacetanilide in acetate buffer was much higher than that in phosphate buffer. replacement of ammonia by ammonium acetate allowed the efficient synthesis of hantzsch 's compounds under mild conditions. the effect of ph on the sensitivity was investigated in the range of ph 5.08.0 using ammonium acetate as buffers. the results obtained are shown in figure s8, which indicates that, in the ph range over 6.57.5, the fluorescence intensity is increased, and above ph 7.5, the fluorescence intensity becomes decreased. from these results, the ph of ammonium acetate was adjusted as 7.5. despite the official regulation, high contents of formaldehyde in detergent and cosmetic products have been reported in iranian brand of these products but producers have labeled their products formaldehyde free. our results confirmed that the risk of cosmetic and detergent formulations with formaldehyde above 0.2% is not negligible, as these products may facilitate considerable exposure of formaldehyde for consumers. after spiking experiments, comparison of the predicted concentrations and recoveries provided by the proposed method shows a good predictive ability towards the spiked liquid detergents and cosmetic products for determination of formaldehyde with 2-methyl acetoacetanilide by photoluminescence method. the stability of formaldehyde has been determined in dishwashing liquid 1 and hair shampoo 1. the media of cosmetic and detergent products contain water and organic compounds such as surfactants and additive compounds such as colors and fragrances that bacteria could grow easily. it is evident from table 6 which considerable decreasing occurs in free formaldehyde content after a few months of controlling microbial growths in water containing solutions and organic material. the effect of various spectral interferences possibly present in the real detergent samples was investigated. three - dimensional florescence spectrum of a shampoo sample was recorded after derivatization of formaldehyde (figure s6). in figure s6, there is no overlap spectrum between compound in shampoo and product of formaldehyde derivatization. the results are not acceptable if there is spectral overlap between product of formaldehyde derivatization and other compounds in real samples. in order to remove spectral overlap, second - order calibration such as three - way methods these algorithms can be applied with great success to identify and quantify overlapped fluorophores. due to the unique solution of three - way methods (e.g., parallel factor analysis) nickel, lead, and cadmium did not affect up to 100-fold excess over formaldehyde did not interfere. iron (iii) interfered with determination of formaldehyde at a concentration ratio more than 50. these results indicate that the proposed method has good selectivity for determination of formaldehyde (table 7). in this research, a rapid, simple, and selective method was developed for determination of formaldehyde in detergents and cosmetic products. by spiking known concentration of formaldehyde to the real samples, the accuracy of the proposed methods was validated and recoveries of the spiked value were calculated. the proposed method minimizes an additional extraction step and consumption of expensive hazardous and environmentally unfriendly organic solvents and allows saving time compared with liquid and gas chromatography methods. in addition, the expanses of gc / ms systems are high, operation system is not easy and unavailable in many laboratories, and a separation step was needed to remove the possible matrix effect of detergents and cosmetic products samples. the reaction of formaldehyde with 2-methyl acetoacetanilide was done in room temperature in comparison with other regents such as acetylacetone or fluoral p that need high temperature and longer reaction time. the proposed methodology was applied for the analysis of different iranian commercial brands of liquid detergents and cosmetic products. some brands of these products have measurable amounts of formaldehyde that were higher than the maximum allowed concentration of 0.2%. the obtained investigation suggests that consumer and user information are required to warn about the highest risks of using detergents and cosmetic products with high formaldehyde content because of allergenic potential and evidence for carcinogenicity of this substance. the results in table 8 show comparison between the proposed method and other methods. | formaldehyde is commonly used in detergents and cosmetic products as antibacterial agent and preservative. this substance is unfavorable for human health because it is known to be toxic for humans and causes irritation of eyes and skins. the toxicology studies of this compound indicate risk of detergents and cosmetic formulations with a minimum content of 0.05% free formaldehyde. therefore, determination of formaldehyde as quality control parameter is very important. in this study, a photoluminescence method was achieved by using 2-methyl acetoacetanilide. also, the box - behnken design was applied for optimization of hantzsch reaction for formaldehyde derivatization. the investigated factors (variables) were temperature, % v / v ethanol, reaction time, ammonium acetate, and 2-methyl acetoacetanilide concentration. the linear range was obtained from 0.3320 107 m (160 gkg1) and the limit of detection (lod) was 0.12 gkg1. the proposed method was applied for the analysis of iranian brands of liquid detergents and cosmetic products. the formaldehyde content of these products was found to be in the range of 0.033.88%. some brands of these products had higher concentration than the maximum allowed concentration of 0.2%. high recoveries (96.15%104.82%) for the spiked dishwashing liquid and hair shampoo indicate the proposed method is proper for the assessment of formaldehyde in detergents and cosmetic products. the proposed methodology has some advantages compared with the previous methods such as being rapid, without the necessity of applying separation, low cost, and the fact that the derivatization reaction is carried out at room temperature without any heating system. |
advances in endoscopy over the last decade have provided the endoscopic surgeon an armamentarium of tools that have shifted the field from a purely diagnostic modality to a therapeutic one. one illustration of this progress is endoscopic decompression of malignant colorectal obstruction with self - expanding stents, a procedure that has gained wide acceptance as an alternative to surgical diversion or excision, especially in the setting of metastatic disease or in poor surgical candidates. stenting of benign colorectal conditions has been described, but its role has been limited due to technical issues and lack of long - term data on permanent metal stents for nonmalignant disease. however, with the recent introduction of nonmetal esophageal stents, there is a growing interest and some experience with their role in the treatment of benign conditions, including postoperative complications following esophageal or gastric surgery. in this article, i describe the technical aspects and results of endoscopic stenting in 2 patients with acute postoperative anastomotic complications following colorectal surgery. he was discharged home on the third postoperative day but readmitted to the hospital a week later with abdominal distention, nausea, and vomiting. a computed tomography of the abdomen at the time of admission revealed colonic obstruction at the anastomosis with surrounding inflammatory changes without evidence of intraabdominal abscess (figure 1). he was managed with nasogastric tube decompression, bowel rest, and intravenous peripheral nutrition. the bowel obstruction persisted, and an operation was advised, but the patient inquired about alternative options. the colorectal surgery service was consulted. at the time of consultation, the patient 's vital signs were stable and he was afebrile. his abdomen was significantly distended without peritoneal signs., the patient consented to endoscopic stenting with possible laparotomy, colonic resection, and fecal diversion. the bleeding persisted, and following transfusion of 8 units of packed red blood cells, the patient underwent emergent abdominal colectomy with ileorectal anastomosis. her postoperative course was significant for persistent bowel obstruction managed with bowel rest, nasogastric tube decompression, intravenous antibiotics, and fluids. computed tomography performed on postoperative day 10 revealed an anastomotic obstruction with a leak, perianastomotic inflammatory changes, and free air under the diaphgram. her vital signs showed sinus tachycardia with a heart rate of 115 beats per minute, and a temperature of 38.5 degrees celsius. her abdomen was significantly distended, and she had localized peritoneal signs in the lower quadrants. her laboratory findings included a white blood count 12.1 10/l, hemoglobin 8.9 g / dl, and albumin of 1.8 g / dl.the patient was advised to undergo laparotomy with diverting ileostomy, but her family inquired about alternative options with the hope of avoiding a stoma. endoscopic intervention was discussed, and the patient consented to endoscopic stenting with possible laparotomy and fecal diversion. he was discharged home on the third postoperative day but readmitted to the hospital a week later with abdominal distention, nausea, and vomiting. a computed tomography of the abdomen at the time of admission revealed colonic obstruction at the anastomosis with surrounding inflammatory changes without evidence of intraabdominal abscess (figure 1). he was managed with nasogastric tube decompression, bowel rest, and intravenous peripheral nutrition. the bowel obstruction persisted, and an operation was advised, but the patient inquired about alternative options. the colorectal surgery service was consulted. at the time of consultation, the patient 's vital signs were stable and he was afebrile. his abdomen was significantly distended without peritoneal signs., the patient consented to endoscopic stenting with possible laparotomy, colonic resection, and fecal diversion. the bleeding persisted, and following transfusion of 8 units of packed red blood cells, the patient underwent emergent abdominal colectomy with ileorectal anastomosis. her postoperative course was significant for persistent bowel obstruction managed with bowel rest, nasogastric tube decompression, intravenous antibiotics, and fluids. computed tomography performed on postoperative day 10 revealed an anastomotic obstruction with a leak, perianastomotic inflammatory changes, and free air under the diaphgram. her vital signs showed sinus tachycardia with a heart rate of 115 beats per minute, and a temperature of 38.5 degrees celsius. her abdomen was significantly distended, and she had localized peritoneal signs in the lower quadrants. her laboratory findings included a white blood count 12.1 10/l, hemoglobin 8.9 g / dl, and albumin of 1.8 g / dl.the patient was advised to undergo laparotomy with diverting ileostomy, but her family inquired about alternative options with the hope of avoiding a stoma. endoscopic intervention was discussed, and the patient consented to endoscopic stenting with possible laparotomy and fecal diversion. an edematous and obstructed anastomosis was noted in patient 1 at 60 cm from the anal verge (figure 2). in patient 2, there was dehiscence of approximately 40% of the anastomosis with an associated abscess cavity (figure 3). under endoscopic and fluoroscopic guidance, the anastomosis was carefully traversed in both patients by using an amplatz super stiff wire (0.09652 cm 260 cm) (boston scientific, natick, maryland). the endoscope was withdrawn keeping the wire in place, and the delivery device of the polyflex stent (120 mm, 18x23 mm diameter) (boston scientific, natick, maryland) was advanced over the wire under fluoroscopic and endoscopic guidance. in patient 1, the adult flexible sigmoidoscope was used to straighten the sigmoid loop to allow safe advancement of the rigid delivery device. the stent was centered across the anastomosis, and the position was verified by fluoroscopy with the flexible endoscope tip at the distal aspect of the stent. the stent was successfully deployed in both patients, and the delivery device and endoscope were withdrawn without difficulty (figure 4). endoscopic appearance of anastomotic leak with cavity in patient # 2 (dehiscence on left, the bowel lumen with wire on the right). patient 1 had spontaneous decompression of his colonic obstruction with passage of flatus and bowel movement the day of the procedure. the nasogastric tube was removed, and a liquid diet was initiated the following day. repeat colonoscopy 8 months later revealed a patent, nonstrictured anastomosis (figure 5). patient 2 had spontaneous decompression of her obstruction the day of her procedure but 2 days later redeveloped obstructive symptoms. repeat stenting was performed, and the distal aspect of the new stent was clipped to the rectal mucosa by using 4 resolution clips (boston scientific, natick, maryland) to minimize migration. the patient 's condition improved, and she was started on a liquid diet ; however, 10 days following her second stent, her symptoms recurred. stenting was repeated, and the distal aspect of the stent was secured to the rectum by using triclip clips (wilson - cook medical, winston - salem, north carolina) (figure 6). the obstructive symptoms resolved, and the patient was started on a liquid diet that was advanced to a regular diet. she was discharged from the hospital on postoperative day 28. except for a urinary tract infection treated on an outpatient basis, she had a full recovery. repeat endoscopy 3 months later demonstrated a patent and healed anastomosis without evidence of stricture (figure 7). endoscopic view of healed and patent ileorectal anastomosis 3 months poststenting in patient # 2. although uncommon, acute anastomotic complications following colorectal resection do occur and are distressing to the patient and the surgeon. they include bleeding, obstruction, or leakage that often results in reoperative surgery in the early postoperative period. reoperative intervention in such a setting carries increased morbidity, prolonged recovery, and may lead to fecal diversion, committing the patient to additional operations to close the stoma. endoscopic stenting can effectively palliate malignant colorectal obstruction in patients with metastatic disease or in patients who are poor surgical candidates due to significant medical comorbidities. however, stenting for benign colorectal disorders has not been widely practiced or advocated due to technical limitations, lack of instrumentation, and absence of long - term data on its effectiveness and the safety of metal stents. furthermore, numerous operative interventions are available to successfully address the needs of most patients with benign colorectal disorders. but with the recent introduction of covered metal and nonmetal esophageal stents, there has been interest in exploring their role in the management of benign gastrointestinal conditions or postoperative complications following gastric or esophageal surgery. eubanks and colleagues recently reported their experience with off - label use of polyester or silicone covered esophageal stents in 19 patients with gastrojejunal anastomotic complications following bariatric surgery. the stenting was undertaken for acute leaks in 11 patients, chronic gastrocutaneous fistulas in 2, and stricture in 6. immediate symptomatic improvement was noted in 90% of their patients, including 91% of the patients with leaks and 84% of patients with strictures. during a mean follow - up of 3.6 months, resolution of the anastomotic complications a total of 34 stents were used in the 19 patients with an endoscopic reintervention rate of 42%. the most common complication was stent migration, which occurred in 58% of stent placement. while most patients with stent migration were treated endoscopically, 3 required laparoscopic extraction of the stent from the distal small intestine. in another study from germany, kauer and endoscopic reintervention to treat stent - related complications was performed in 50% of patients. no patient required surgical intervention. during the study period, 5 patients underwent endoscopic stent removal after full healing, 3 had spontanenous passage of the stent per anus, and 2 patients died with the stent in place from nonstent related causes. a recent study from karbowski and colleagues in seattle reported their experience with the polyflex esophageal stent in a variety of esophageal conditions. the polyflex stent is a self - expanding and removable stent composed of a polyester infrastructure with silicone covering. currently, it is approved in the united states for esophageal insertion. in their study, a total of 37 stents were placed in 30 patients with esophageal conditions. indications for stenting included esophageal fistula in 7, anastomotic stricture in 5, acute esophageal perforations in 3, acute postoperative leak in 1, and other causes in 14. endoscopic stenting for malignant colorectal disease is an option for patients with metastatic disease or those who are poor surgical candidates. the 2 patients described in this report illustrate that endoscopic stenting can be helpful in patients with acute postoperative complications after colorectal resection. while the standard of care in 2009 is operative procedure when an intervention is needed under such circumstance, an endoscopic approach may successfully treat the anastomotic complication and avoid the morbidity and recovery of another laparotomy and the potential need for fecal diversion. however, it is important to note that there is a lack of data in the literature on the short- and long - term outcome of the interventions described in this report. furthermore, the polyflex stent is fda approved for esophageal indications and was used off - label in this study due to the lack of other commercially available stents to treat benign colorectal conditions. however, the patients ' request to seek an alternative to operative intervention and possible stoma raised the possibility of an endoscopic option. but only after assessing the patients ' full understanding that endoscopic stenting for their conditions is experimental at this stage and after obtaining full informed consent was the procedure undertaken. fortunately, this nonconventional approach resolved the anastomotic complications in both patients but also demonstrated the challenges noted by the above studies in terms of risk of migration and the need for multiple endoscopic reinterventions. the risk of stent migration and need for reintervention in the setting of benign disease is higher than when stents are used for malignant obstruction. in the case of the second patient, the use of a triclip (wilson - cook medical, winston - salem, north carolina) clip to secure the distal end of the stent to the bowel wall helped prevent further migration. the use of triclip in this setting was off - label, and there is currently no data in the literature to support such a use. to my knowledge, currently no other means area available to properly anchor a temporary stent to the bowel. future technologic development may yield stent devices that are less prone to migration or ancillary fixation clips that help fixation to the colorectal wall. some postoperative anastomotic complications, traditionally managed operatively, may be amenable to endoscopic interventions, as illustrated by the 2 cases in this report. hopefully, future technological advances will increase the armamentarium of tools and enable endoscopic surgeons to tackle a broader spectrum of colorectal conditions with endoluminal procedures. | acute postoperative anastomotic complications following colorectal resection include leak and obstruction. often an operation is necessary to treat these complications. the role of endoluminal procedures to treat these complications has been limited. this article illustrates that such an approach is technically feasible and can be used to treat some colorectal anastomotic complications. |
prostate cancer represents the most frequent non - cutaneous neoplasia in males, corresponding to 40% of the cases, being the second death cause due to malignant tumors in men. the incidence as much as the mortality increases exponentially after 50 years of age. this type of neoplasia can develop peculiar patterns of evolution, presenting, in many cases, precocious relapse and metastasis. of a total of patients who have received curative treatment for the localized prostate cancer, 30% relapse. the most frequent is the bone metastasis, generally multiple, mainly reaching the vertebral column, shoulder joints and sacroiliac bone. among these patients, bone metastasis in the mouth is extremely rare, and represents 1% of all malignant mouth neoplasias. the incidence is 80 to 90% in the mandible, mainly in the molar region and it always sign the spreading of the cancer illness. it originates from primary malignant neoplasias located mainly in the breast, prostate, lung, thyroid and kidney. however, the occurrence of prostate and kidneys as primary fields for jaw 's metastasis through blood pathways are considered extremely rare. among the bone metastases affecting the oral cavity the main clinical manifestations may include pain, swelling, tooth loss, bleeding, trismus, paresthesia sensation (often associated with inferior alveolar nerve involvement) and epistaxis. included in the differential diagnosis of metastatic peripheral lesions on the gingiva or alveolar ridge are pyogenic granuloma, peripheral giant cell granuloma and, possibly, peripheral ossifying fibroma or ulcerated fibroma. the difficulty of clinical identification and differentiation between primary lesions and metastatic lesions of the jaw make the biopsy with histopathological analysis (combined with immunohistochemistry) mandatory for diagnosis. besides representing great importance for public health, this issue becomes fundamental for a multidisciplinary evaluation of the patient. male patient, white, 54 years old, was admitted in the oral and maxillofacial trauma and surgery team at hospital de base de bauru (bauru base hospital), in march 2009. the patient 's main complaint was pain in the left mandibular region above the median line and paresthesia of the anatomical areas sensitized by the left alveolar inferior nerve, branch of the mandibular nerve and trigeminal nerve. the first signal related by the patient was a pathology originated from a root treatment about one year before, which developed a mandibular abscess clinically treated. once healed from this abscess, chronic pain persisted in half mandible associated with localized paresthesia. because of the posterior absence of dental symptoms even as mandibular pain and face paresthesia, the clinical dentist referred the patient to the one who suggested the diagnosis of trigeminal neuralgia, establishing treatment with carbamazepin 200 mg and gabapentin 300 mg, both once a day, that lasted about 1 year or more. the patient 's medical history revealed pharmacologically controlled diabetes and prostate acinar adenocarcinoma gleason score 7 (3 + 4), with initial psa score on 4.78. this latter treated in 2005 by means of radical prostatectomy performed in january 2006 (pt3n0m0), having follow - up procedures with urology, but in a random and intermittent manner, a history not explored or valued by neurology. physical examination revealed left mandibular bulging in the buccolingual aspect and tooth mobility involving bulging region extending to the opposite canine tooth associated with a slight gums erythema, but without symptoms compatible with periodontal disease. in addition, we observed the absence of pathognomonic signs and symptoms of classic trigeminal neuralgia, showing no intermittent pain, neither specific localized pain spots (trigger - points). based on this clinical symptomatology, we established a hypothesis of diagnosis as osteomyelitis and/or cancer, not yet discarding the possibility of atypical neuralgia of the trigeminal nerve (figure 1). clinical aspect of the patient. it is possible to see the bulging involving left buccolingual region of premolars and molars. source : oral and maxillofacial surgery team bauru base hospital association through a radiography exam, it was possible to define a mild, diffuse radiolucency in the left mandibular body. the computed tomography scan showed an anatomical structure alteration with osteolysis areas in the premolars, molars and in the left region of the chin (figures 2 and 3). source : oral and maxillofacial surgery team bauru base hospital association computed tomography showing areas of osteolysis in the left mandibular body and symphysis region. source : oral and maxillofacial surgery team bauru base hospital association because of that, we carried out an incisional biopsy and culture / antibiogram of sensitivity of the affected area. the results showed adenocarcinoma (clear cell type) with necrosis areas and infiltrating bone and soft tissue. the tumor had no communication between the internal and external environment, without skin or mucosa fistula, confirmed by whole - body bone scintigraphy with special interest in the mandibular region, where increased uptake of the radiocontrast agent was observed, as well as in many areas, such as the right clavicle and scapula, humerus, ribs, spine, sternum, hip bone and femur (right / left) (figure 4). whole - body bone scintigraphy, increased uptake of the radiocontrast agent observed in the mandibular region, right clavicle and scapula, humerus, ribs, spine, sternum, hip bone and femur (right / left). source : oral and maxillofacial surgery team bauru base hospital association after confirming a definitive diagnosis, the patient was taken to the oncology clinic, where in may 2009 was admitted to the clinical oncology department with a metastatic bone disease diagnosis (prostate acinar adenocarcinoma) confirmed by three different methods : bone scintigraphy, incisional biopsy and immunohistochemical analysis of mandibular lesions. the treatment consisted of the use of goserelin acetate (3.6 mg / month) and bisphosphonate (clodronate, 4 ampoules / month). the patient was evaluated as having good disease control and good tolerance to treatment for some months, when disseminated neoplasm and pulmonary involvement led to a lethal outcome, with the patient 's death in less than a year. prostate cancer is a silent disease ; the findings show that the prevalence of histological malignancy exceeds the clinically manifest disease in approximately eight times. according to the american cancer society, for early cancer detection in individuals without symptoms, it is recommended the digital rectal exam and psa dosage annually for individuals over age 50 or 45 for men belonging to risk groups. the psa is a kallikrein family protease, produced by the prostate epithelium, whose function is to solubilize the sperm after ejaculation. the accepted level as the upper limit of normal psa is 4 ng / ml. the psa has a great clinical utility ; it serves for early detection of prostate cancer, cancer staging, prognostic assessment and therapeutical response control. in addition to the psa level in case of established disease, it is necessary to evaluate the prostate cancer advancement by image exams [transrectal ultrasound, bone scintigraphy, chest radiography, computed tomography of the abdomen and pelvis, magnetic nuclear resonance of the abdomen and pelvis, and positron emission tomography (pet) ] to define the appropriate therapeutical strategy. in order to classify neoplastic levels, some methods are used to analyze the histological grade (gleason score), tumor volume and stage at the diagnosis time. when the primary tumor volume is less than 3 ml, metastasis rarely occurs. when the present volume is greater than 12 ml metastasis is almost always present. according to mcneal(1992), only tumors larger than 1 cm in diameter (volume of 0.5 ml) are capable of extracapsular extension. the lymph nodes or seminal vesicles spread are usually more common in tumors larger than 2 cm in diameter. the gleason system has been introduced to aid on the tumor evolution prediction and the pathological staging by transrectal biopsy or of the surgical specimen itself. the lesions with gleason score 2 - 4 (well differentiated) have a more indolent behavior, whereas the ones scoring 7 - 10 (undifferentiated) are aggressive, responding poorly to various treatment options. the clinical staging of tumors, tnm classification, has been adopted to characterize tumors, helping in choosing the most appropriate therapy and to estimating the patients ' survival chances. the patients ' selection for specific treatments is usually done through the risk of tumor recurrence and dissemination subdivision. among the therapeutical modalities there are clinical observation, surgery, radiotherapy, chemotherapy, hormonal therapy and combination therapy. (1990) in a period of 35 years, reported 1008 patients with craniofacial malignancies, found only five patients (0.5%) with metastatic tumors to the jaw, from different primary sites. (2003), identified adenocarcinoma as the primary histological type responsible for gnathic bone metastases, starting from different locations of primary tumors, confirming the reported case. the four most common tumor sites that metastasize to the man jaws, in descending order of frequency, are lung, prostate, kidney and liver and, in women, breast, adrenal gland, female genital organs (uterus, cervix, ovaries) and colorectum. in the first decade of life, tumors of the adrenal gland (neuroblastoma) most frequently spread to the jaws and in the second decade, bone primaries prevail (osteosarcoma, ewing's / pnet). most patients with oral metastasis, generally have the primary cancer well diagnosed, confirming the reported case in which the primary tumor was treated years before by radical prostatectomy and the patient was still on medical care during the gnathic metastasis. in 25 - 35% of cases, the presence of metastasis on the head and neck is the first sign of disseminated malignant disease, and in about 70% of cases, metastatic lesions are diagnosed in conjunction with the primary focus. sometimes, however, the diagnosis of the primary lesion may be difficult or even impossible. multidisciplinary work is recommended in the care of patients with malignant neoplasms or neurological disorder of doubtful identification in order to provide diagnosis, planning and treatment that offer the most favorable prognosis. (1999) conducted a survey of 390 oral cancer cases, of which 22 cases (5.6%) were metastatic tumors on gnathic bones from other regions, being prostate gland the primary site of these tumors, as occurred in our case. in cases of metastatic bone illness, the pain and pathological fractures are prevalent manifestations, although the condition is asymptomatic in some patients. in this case, the patient reported the presence of chronic pain in hemi mandible, including a localized paresthesia, symptoms that were initially treated as trigeminal neuralgia. however, by conducting special examinations, we obtained the diagnosis of metastatic adenocarcinoma prostate cancer in the lung and bone, involving, in addition to the cited organs, the mandibular region. bone tumor involvement of the oral region is higher compared to the soft tissue. metastatic disease in the jaws may also affect the adjacent soft tissues and present as dental or periodontal infection. when the metastasis is located in the jaw, the primary neoplasm is more likely to develop bone metastases, especially in the molar region. it is believed that the greatest amount of cancellous bone present in the region is the reason for the higher frequency of involvement when compared to other facial bones, as trabecular bone presents growth factors that improve the neoplasia spread, confirming the reported case in which bone metastasis reached the region of the body and the mandibular symphysis. the unilateral paresthesia type, as occurred in this case, hypoesthesia or anesthesia of the lower lip, known as numb chin syndrome with or without the presence of pain, may be the initial clinical manifestation of a significant percentage that manifests on the jaw. alterations in sensitivity involving the lower lip, even without significant radiological changes, should alert the expert to think about the possibility of an initial metastasis. for an accurate diagnosis and treatment of this rare condition, detailed and careful evaluation of the clinical picture, associated with a high degree of suspicion, is needed, requiring a multidisciplinary approach to the case. the prognosis of these patients is reserved for the pathophysiology of the primary tumor. in addition, bone metastases are associated with advanced disease and in some cases may be involved in malignant lesions - which may lead the patient to death, as reported in this case. the radiation treatment can be considered curative regarding single oral metastases whose primary focus is clearly identified and which responded well to therapy. however, most authors consider an unfavorable prognosis for most cases of this oral cancer. the cases that may have a better indication are those with localized metastases, with no other identified metastasis and controlled primary disease, which does not occur in our case, since the examination of bone scintigraphy identified multiple metastatic tumors distributed throughout the body, palliative therapy being considered the best alternative. chemotherapy is indicated only in metastatic disease refractory to hormonal treatment, as in cases of predominance of hormone - independent cell clones or progressive increase in serum psa. the use of bisphosphonates, as reported in the present case, is justified in the presence of bone metastases and when refractory to hormonal treatment, despite the risk of complications related to these drugs, such as osteonecrosis. there is an interesting indication to the use of these drugs as this therapy provides control of the neoplastic disease and also a relative patient survival chance. in advanced cases, the quality of life, despite the patients having no chance of cure for the disease, is the main objective, providing less pain and discomfort. metastatic lesions are related to the advanced stage of the disease and usually with a high degree of histological aggressiveness. this fact explains the difficulty of curative treatment, and these injuries are usually classified as reserved and dark prognosis. it is important to emphasize the relevance of detailed clinical examination and multidisciplinary evaluation in order to establish the differential diagnosis and so the correct treatment. | prostate cancer represents the most frequent non - cutaneous neoplasia in males. this type of neoplasia can develop peculiar patterns of evolution, presenting, in many cases, precocious relapses and metastasis. bone metastasis in the mouth is extremely rare, and represents 1% of all malignant mouth neoplasias. the aim of the present study is to report a clinical case of bone metastasis in the mandibular region associated with a tumoral prostate adenocarcinoma, as well as to discuss connected aspects about diagnosis, prognosis and integrated treatment of this condition. |
trichotillomania consists of the recurrent compulsive habit of pulling out of one 's own hair, resulting in a perceptible hair loss pattern that frequently is associated with other psychiatric processes, and social or functional impairment. it can be a self - limiting symptom but in most cases is a chronic disorder with frequent remissions and relapses. therapeutical management is difficult and behavioral therapy, psychotherapy, hypnosis, or pharmacological treatment has been used without satisfactory results. recently, n - acetylcysteine has been proposed as an effective alternative in the treatment of this disorder. the first patient was a 23-year - old woman that attended at our outpatient dermatology department with partial alopecia of her eyebrows, eyelashes, and frontal hairline of the scalp. the disorder was related with the death of her mother during childhood. on physical examination, patchy alopecia in eyebrows, eyelashes and in the frontal area was noted, with somewhat artificial appearance and presence of hairs with different length [figure 1a ]. in the past n - acetylcysteine 1,200 mg / day was started and complete regrowth occurred in the frontal area within the first two months of treatment, which maintained until the 6-month follow - up period [figure 1b ]. the second patient was a 19-year - old woman with an irresistible urge to pull out of her hair since the age of 9. she presented diffuse hair loss affecting the entire scalp with broken hairs and hairs with different lengths on dermatoscopic examination [figure 2a ]. complete regrowth was observed after introducing n - acetylcisteine 1,200 mg / day during 3 months [figure 2b ]. no adverse events with the medication (a) patchy alopecia in frontal area with presence of hairs with different length ; (b) complete regrowth in the frontal area after two month treatment with n - acetylcysteine dermoscopic examination before (a) and after (b) three month treatment with n - acetylcysteine 1,200 mg / day trichotillomania is a traumatic alopecia caused by the patient himself by pulling out of the hair with the aim of tearing it off. the incidence it 's not known with exactitude, but it is estimated that affects between 0.6 - 1% of the population. it can appear in any hair - bearing area, but the most frequent locations are the scalp and eyebrows, while the eyelashes are exceptionally affected. the alopecia presents with an artificial pattern, with circular or lineal forms, and fractured hairs at different lengths. the disorder can be associated with trichofagia, onichofagia and other self - inflicted cutaneous mutilations, as well as trichobezoar. it depends on the beginning - age of the disorder and of the possible associated co - morbidities. in any case is important to establish a good doctor - patient relationship, to inform and advice about the nature of the disorder and recommend a psychiatric evaluation. between the available pharmacological treatments for trichotillomania, tricyclic antidepressant clomipramine has shown to be effective, but often the patient adherence is poor and the results are modest. also, selective serotoninergic receptor reuptake inhibitors (ssris) have been considered as a first line treatment for this disorder but there is no evidence that supports its benefits. furthermore, some agents, such as naltrexone or neuroleptics, like pimozide, have been used in combination with other alternatives. habit reversal training is the most effective behavioral therapy, and should be associated with pharmacological treatment. recently several reports have indicated that n - acetylcysteine, a glutamate modulator, could be effective in reducing symptoms of trichotillomania. it acts by restoring the extracellular glutamate concentration in the nucleus accumbens, decreasing its levels, which seem responsible for the pathogenesis of compulsive behaviors, and therefore, trichotillomania. conducted an aleatorized double - blind clinical trial, comparing n - acetylcysteine with placebo, with doses ranging between 1200 and 2400 mg per day and have observed that it was more effective and safe than placebo, and that also produced better results than other pharmacological alternatives, with no reported adverse events. nevertheless, the promising results n - acetylcysteine is showing further studies need to be conducted to establish the appropriate treatment regimen and to evaluate it long - term efficacy in improving this chronic condition. | trichotillomania is as medical condition caused by the patient himself by pulling out of is own hair, resulting in a perceptible hair loss pattern that frequently is associated with other psychiatric processes. generally has a chronic course in most patients, and a challenging therapeutical management. there are several available options for is treatment, but the clinical response is not satisfactory in many patients. recently, n - acetylcisteine, a glutamate modulator, has shown efficacy in the treatment of trichotillomania and other compulsive behaviors, and is considered a new alternative in the management of this condition. we describe two patients with trichotillomania successfully treated with n - acetylcysteine. nevertheless, further studies need to be conducted to establish the appropriate treatment regimen and to evaluate it long - term efficacy in improving this chronic condition. |
obesity is known to be associated with cardiovascular outcomes [13 ] and with elevated levels of several cardiometabolic risk factors (crfs), including blood pressure and several metabolic factors. the association between obesity and several crfs has already been observed in children and adolescents both cross - sectionally [57 ] and longitudinally [810 ], but the data are limited. furthermore, intervention studies through lifestyle interventions, and bariatric surgery in children show that a reduction of body weight among obese children results in reduced levels of several crfs, such as blood pressure [1315 ], lipids [1618 ], and insulin. there are several methods to measure adiposity in children, including bmi, waist circumference, or the waist - to - hip ratio. there is continued debate on which indicator best predicts cardiovascular risk but several studies suggest that body mass index (bmi) can adequately predict cardiovascular risk in adults, adolescents, and young adults [9, 21, 22 ]. there is, however, a relative scarcity of studies that have examined the relation between obesity and crfs in population - based samples of children and adolescents (i.e., outside of the clinical setting). furthermore, most such studies in children and adolescents have relied on a cross - sectional design, although a few did have a prospective design, but most such studies were conducted in high - income countries [8, 9 ]. furthermore, only a few studies have simultaneously compared the relative strength of the associations between obesity and a broad set of crfs including both physiological risk factors (e.g., blood pressure) and biological markers (e.g., blood lipids, glucose, and uric acid) in youths so that the associations between obesity and different crfs can be directly compared. in this study, we examined both the cross - sectional and longitudinal relationships between overweight and several crfs in a population - based sample of adolescents and young adults. data on crfs were derived from a cohort study of children followed since their birth to examine the association between maternal fish consumption and the children 's neurological development [23, 24 ]. in this cohort study, bmi and crfs were measured for the first time on the seventh follow - up visit at the age of 19 - 20 years. we then linked the crf data with bmi values measured at the age of 1215 years previously obtained during a routine screening program in all schools of the country [25, 26 ]. the main objectives of the present study were to directly compare the associations between obesity and several crfs, to compare the associations based on cross - sectional or longitudinal designs, and to examine the contribution of current and past weight statuses in the prediction of current crf levels. the republic of seychelles is a rapidly developing small island state in the indian ocean located east of kenya. a cohort of 779 children was enrolled at the age of 6 months as part of the seychelles child development study (scds). the scds was undertaken to assess the association between pre - natal exposure to methyl mercury from fish consumption and neurocognitive development in a sample of children representative of the general population. bmi and a broad set of cardiometabolic risk factors were measured for the first time in the seventh follow - up visit in 2008 - 2009 when the participants were aged 19 - 20 years (age range : 18.720.5 ; mean age : 19.5 ; sd : 0.5). bmi at the age of 1215 years was derived from a routine school - based surveillance program. bmi was measured when children were in the 7th and 10th grades and were aged approximately 12.7 years for children in the 7th grade (sd : 0.4 ; range : 11.613.4) and 13.9 years for children in the 10th grade (sd : 0.6 ; range : 12.115.9). the school screening program was conducted in all public and private schools in seychelles under the auspices of the ministries of health and education. the methods and results of this screening program have been published previously [2527 ]. linkage of bmi in the school - based screening program with crfs measured at the age of 19 - 20 years within the scds study was based on the national identification number that is available for all seychelles citizens. we had previously linked data from the scds with data from the school screening program in order to examine the relationship between exposure to prenatal mercury (scds) and blood pressure at the age of 1215 years (school screening program). informed consent was obtained from the subject and caregiver of every participant for both the scds and the school screening program. the school screening program and the scds were approved by the research and ethical committee of the ministry of health, victoria, seychelles. in addition, the scds was approved by the human subjects review board at the university of rochester, rochester, ny, usa. briefly, weight was measured without shoes and in light garments by trained school nurses in the schools, during regular school hours (8 am2:30 pm), using electronic scales (seca 870, hamburg, germany). height was measured with a fixed stadiometer (seca 208). the same weight scales and stadiometers were available in all schools and the instruments were regularly checked for accuracy by the screening nurses and further checks were preformed at least once per year by the manager of the screening program. bmi was calculated as weight (kg) divided by height (meters) squared. we used the average of the two values of bmi measured at ages 12 and 15 years, to reflect bmi in early adolescence. at the age of 19 - 20 years, all measurements took place between 8 a.m. and 10 a.m. at the scds study center. weight was measured with a precision electronic scale (seca 870) and height was measured with a fixed stadiometer (seca 208). three readings were measured by a nurse, on the left arm, and using an appropriately sized cuff. readings were taken at intervals of at least 1 minute between the bp readings after the participant had been sitting for at least 5 minutes. systolic bp (sbp) and diastolic bp (dbp) were based on the average of the three readings. the methods used to measure venous blood taken at the age of 19 - 20 years have been described previously. glucose, total cholesterol, hdl - cholesterol, triglycerides, and uric acid were measured at the central laboratory of the main hospital in seychelles (seychelles hospital), using standard enzymatic methods (konelab t series reagents, thermo scientific) with a thermo konelab 30 automatic analyzer (konelab corp., ldl - cholesterol was calculated using the friedewald formula. among the 423 adolescents who had complete data on crfs and bmi at the age of 19 - 20 years in the sdcs (77% of 549 children seen at the age of 19 - 20 years), 390 (92%) also had data on bmi at the age of 1215 years ; hence, this study includes 390 participants. analyses were conducted separately in males and females because of substantial sex differences in the distribution of several variables, including bmi and several crfs, and the possibility that the relations between bmi and crfs may differ according to sex. in order to examine the associations between bmi and crfs, we considered three categories of body weight : (1) low bmi was defined as a bmi below the median values for age and sex (both at age of 19 - 20 years and at age of 1215 years) ; (2) elevated bmi was defined as bmi above the age- and sex - specific median values of bmi and below the age- and sex - specific bmi cut - off values for overweight ; and (3) overweight. overweight at age of 1215 years was defined according to the age- and sex - specific iotf criteria of overweight. overweight in persons aged 18 years and above follows guidelines in adults and are defined as a bmi of 25 or above. in both adolescents and young adults our cut - off values for overweight we did not distinguish between overweight and obesity because of an insufficient number of participants. we also conducted analyses examining the relationship between crfs and joint categories of body weight at both age of 1215 years and at age of 19 - 20 years (e.g., high - high, low - low, high - low, low - high) but the sample sizes in some categories (high - low, and low - high) were too small for meaningful statistical inference (results are available from the authors). we tabulated the mean values and 95% confidence intervals of crfs according to these three categories of bmi separately in males and females. in order to directly compare the magnitude of the differences of the mean values of crfs between these three bmi categories, we plotted the percent changes between values in the elevated weight and overweight categories compared to participants with low body weight. we also provide results in terms of odds ratios of elevated levels of crfs according to the three defined categories of bmi, at age of 1215 years or at age of 19 - 20 years, as determined by logistic regression. regression models were not adjusted for other covariates, since the aim of the study was to examine the performance of bmi to predict crfs. we defined dichotomized categories of crfs in youths aged 19 - 20 years based on recent guidelines for subjects aged 1821 years. however, because only 4% of subjects had triglycerides 1.3 mmol / l, we used a lower cutoff (1.0 there is no widely used cutoff for serum uric acid in adolescents or young adults so we defined elevated levels as levels above the sex - specific 80th percentile. the cut - off values for the dichotomized categories of crfs appear in table 3. we also examined the linear relationship between crfs and bmi (used as a continuous variable) for both the crosssectional (bmi and crfs measured at the age of 19 - 20 years) and longitudinal (bmi measured at the age of 1215 years and crfs measured at the age of 19 - 20 years) associations using linear regression, separately in males and females. we also examined regression analysis models that included both bmi at the age of 1215 years and bmi at the age of 19 - 20 years to determine how well the bmi predicted the defined outcomes at each age (i.e., to examine the association between crfs and bmi at age of 19 - 20 years given a certain bmi at age of 1215 years and, inversely, the association between crfs and bmi at age of 1215 years given a certain bmi at age of 19 - 20 years). to permit a direct comparison of the results based on cross - sectional and longitudinal analyses, we used standardized regression coefficients, which express change in the considered outcomes associated with a 1 standard deviation change in the exposure (bmi). all analyses were done among the 390 children who had complete data at age of 1215 years and at age of 19 - 20 years. table 1 presents the mean values and standard deviations of bmi and crfs as well as the prevalence of the defined categories of bmi in participants aged 1215 years and aged 19 - 20 years, separately in males and females. the proportion of participants who were overweight in the school evaluations was 18.9% among males and 19.5% among females aged 1215 years. at age of 19 - 20 years, 12.6% of males and 27.9% of females were overweight (this also includes obesity). bmi and several crfs differed substantially between males and females at both the age of 1215 years and at the age of 19 - 20 years. the spearman correlation coefficients between bmi at age of 1215 years and bmi at age 19 - 20 years were 0.69 (p < 0.001) in males and 0.83 (p < 0.001) in females, which suggests a high degree of tracking of bmi over age. the distribution of the crfs at the age of 19 - 20 years according to our three defined bmi categories at the age of 1215 years and at the age of 19 - 20 years is presented in table 2. a significant positive linear trend was found between categories of bmi and most crfs at both ages. associations did not reach statistical significance for triglycerides and glucose in males and ldl - cholesterol, hdl - cholesterol and triglycerides in females at age of 1215 years and glucose in males at age 19 - 20 years. figure 1 shows the percent changes in mean crfs levels in males and females comparing the categories elevated bmi and overweight with bmi below the median. overall, these changes were quite similar whether based on the longitudinal or cross - sectional analyses. however, the cross - sectional associations tended to be slightly larger than the longitudinal ones. the magnitude of the percent changes tended to be larger for several metabolic markers than for bp. however, the associations for the metabolic crfs were not always statistically significant, which is consistent with larger variability (e.g., larger sd) for metabolic crfs than for bp (table 1). table 3 shows the odds ratios (ors) for the presence of elevated crfs in participants with elevated bmi and with overweight compared to participants with low bmi. comparing participants with overweight versus participants with low bmi, these ors were as high as 4.0 (95% ci : 1.510.4) for ldl - cholesterol among males aged 1215 years and 8.0 (95% ci : 2.328.6) for triglycerides among males aged 19 - 20 years. similar to the results from the stratified (table 2) and linear regression (table 3) analyses, the magnitude of the associations based on dichotomous categories of crfs (i.e., ors for the presence of elevated crfs versus non elevated crfs) in relation to bmi categories was generally not different whether the results were derived from longitudinal analysis (bmi measured at the age of 1215 years and crfs measured at the age of 19 - 20 years) or cross - sectional analysis (bmi and crfs both measured at the age of 19 - 20 years), with some exceptions. table 4 shows the standardized linear coefficients derived from linear regression between crfs measured at the age of 19 - 20 years and bmi measured either at the age of 1215 years or at the age of 19 - 20 years, in males and females separately. most of the associations were highly significant for most crfs at both the earlier and later ages. this is in line with what was found for stratified analysis (table 2, figure 1). the associations tended to be slightly stronger for the cross - sectional associations between bmi and crfs measured at 19 - 20 years than for the longitudinal associations (bmi measured at the age of 1215 years and crfs measured at the age of 19 - 20 years). consistent with results of stratified analysis (table 2), no associations were found for glucose in males (with bmi measured at any age) as well as for ldl - cholesterol and hdl - cholesterol in females when bmi was measured at the age of 1215 years. in analyses including bmi measured at both ages, only bmi measured at the age of 19 - 20 years was significantly associated with crfs, but not bmi measured at the age of 1215 years. the results may be sensitive to the variability in the measurements of the exposure variable and reflect that bmi was measured more accurately during the research evaluation at 19 - 20 years (highly standardized protocol with few observers and scales). however, the standard deviations of bmi were not substantially different whether bmi was measured when participants were aged 1215 years or 19 - 20 years. these results suggest that current bmi matters more than past bmi when predicting current levels of crfs. we found that elevated levels of bp and several metabolic risk factors measured at the age of 19 - 20 years are predicted by either current bmi or bmi levels measured several years earlier (age of 1215 years). furthermore, when relating crfs at the age of 19 - 20 years toboth current and past bmi levels, only current levels were significantly associated with crfs. this suggests that a life course approach to weight control is needed but that current weight status is particularly important in order to limit impact of adiposity on crfs. this is the first study to examine both the cross - sectional and longitudinal associations between obesity and a broad panel of crfs in a population - based sample of adolescents and young adults in the african region. our findings of strong associations between obesity and several crfs in adolescents and young adults are consistent with earlier studies and holds true whether analyses are cross - sectional [57 ] or longitudinal [810 ]. the crfs most consistently associated with obesity in youth include high blood pressure, dyslipidemia, hyperinsulinemia, and insulin resistance [9, 10, 32 ]. what our study adds, is that a large impact of obesity on several crfs can be observed both cross - sectionally and longitudinally in adolescents and young adults in a country in the africa region. these data provide further useful evidence for the need to address obesity and its impact on crfs in youth in the african region. the increase in mean levels of crfs in a dose - response manner across categories of bmi is consistent with previous studies showing a strong association and clustering of obesity with crfs in young people. it is interesting to note, from our stratified analysis (table 2), that the relation between bmi and mean levels crfs seemed to be linear (i.e., an effect was also seen with the intermediate elevated category) in the case of bp, while the impact of bmi on several metabolic crfs was most apparent only in the highest bmi category (i.e., overweight, which also includes obesity). an implication of these findings is that interventions targeting the entire population are useful for blood pressure control at the population level while abnormal levels of biological crfs might be better addressed by targeting obese adolescents and young adults. the question of whether bmi or different obesity indicators would perform differently in their ability to predict crfs is still ongoing. based on data of participants aged 19 - 20 years (scds), we previously showed that bmi was at least as adequate as several other adiposity markers to predict mean levels of crfs. we found in this study that the magnitude of the associations between overweight and crfs was important, with odds ratios of elevated levels of crfs associated with overweight being larger than 2 - 3 for most crfs. these results are consistent with findings in other studies among young people, for example, the bogalusa heart study in the usa, in which a bmi larger than the 95th percentile was associated with odds ratios of 2.4, 4.5, 3.0, 7.1, and 12.1 for raised diastolic bp, systolic bp, ldl - cholesterol, triglycerides, and insulin concentration, respectively, and 3.4 for low hdl cholesterol. overweight was also related strongly to lipid markers among children aged 611 years in qatar. of note, we found strong associations between overweight in adolescents and young adults and virtually all components of the metabolic syndrome. the magnitude of the observed associations and the broad scope in terms of the many factors involved stress the importance of weight control programs in youth. it is interesting to note that the association between bmi and crfs was almost as strong in our study when bmi was measured 46 years before measuring the crf outcomes (longitudinal design) as when they were measured at the same time (cross - sectional design). analyses based on the longitudinal design strengthen the possibility that elevated body weight is causally associated with elevated levels of crfs. confounding or reverse causation is unlikely to account for these findings (e.g., high bp or dyslipidemia is unlikely to be the cause of obesity). it is remarkable, however, that the longitudinal associations between bmi and crfs were quite strong in comparison to the cross - sectional associations. this reinforces the conclusion that overweight is very likely an important issue for cardiovascular health and that weight control needs be addressed early in life. the finding that only bmi at age of 19 - 20 years remained a strong predictor of crfs in a regression model including both bmi at age of 1215 years and bmi at age of 19 - 20 years suggests that current bmi is particularly important in relation to the prediction of blood pressure and metabolic risk factors. conclusions must, however, be carefully drawn on these results in view of possible methodological issues, for example, the possibility that that bmi was measured with less precision at age of 1215 years in the context of a routine screening than at age of 19 - 20 years in a study with highly standardized methods. nonetheless, this finding (i.e. that crfs are strongly associated with current bmi but not with bmi measured several years earlier when considering both weight statuses together) implies the important role of current versus past bmi in relation to current crfs levels. this is welcome news since current weight, not past weight, can possibly be addressed through interventions. however, overweight tends to track over age, and children who were overweight when they were younger are largely the same children who are overweight when they are older. this is confirmed by the spearman correlation coefficient of bmi at age of 1215 years and at age of 19 - 20 years. the larger importance of current versus several longitudinal studies of both past and current weights have shown that obesity in childhood has only limited impact when assessing cardiovascular health in adulthood [33, 34 ], but other studies have found an impact of obesity in young people on both risk factors [9, 10 ] and actual cvd outcomes [3537 ] in adulthood. overall, our findings that crfs are associated with current and past bmi, and that bmi tracks over time, support a life course approach to obesity including both population - based interventions aimed at preventing the occurrence of overweight in the population at all ages and individual - based interventions targeting overweight among adolescents and young adults. the observation of higher uric acid blood levels in males compared to females was expected and consistent with the uricosuric effect of estrogens in females, which confers a protective effect on several metabolic crfs. a higher sbp in males compared to females may be related to males being generally taller since height is an important determinant of bp in adolescents and young adults. we found a significant association between bmi and glucose only in females even though males had higher blood glucose levels than females. a possible explanation for the observed sex differences could be the greater adiposity, in particular fat mass, in females than in males. the influence of abdominal fat, which is particularly active metabolically, may not be fully captured by the measurement of bmi and might be a key determinant of some of the observed sex differences. our study has several strengths, including the population - based sample ; a fairly large sample size for a study including blood markers among healthy adolescents and young adults ; a broad panel of crf markers considering the study included healthy participants ; analyses based on both longitudinal and cross - sectional designs ; and the presence of a minimal number of potential confounders, comorbid conditions, and related treatments at this early age. accuracy of bmi measurements at the age of 1215 years may be less than optimal in the context of routine school screening programs. in addition, although participants were asked to fast, they may not have been all fasting when blood was collected at the age of 19 - 20 years. a larger sample size would also have been useful to assess the associations separately among overweight and obese children and in order to generate different cohorts with sufficient numbers of children who either gained or lost weight between age of 1215 and age of 19 - 20 and the subsequent effects on crfs at the age of 19 - 20 years. in conclusion, we found strong associations between adolescents and young adults who were overweight and several crfs in both cross - sectional and longitudinal analyses. these findings indicate that a life course approach to weight control with interventions as early as possible is warranted but that weight control even at a later age is equally or perhaps more efficacious in reducing cardiovascular risk. these findings in a country of the african region extend previous similar findings in high - income countries and highlight the adverse consequences of obesity on crfs in adolescents and young adults in all regions. | we assessed the association between several cardiometabolic risk factors (crfs) (blood pressure, ldl - cholesterol, hdl - cholesterol, triglycerides, uric acid, and glucose) in 390 young adults aged 19 - 20 years in seychelles (indian ocean, africa) and body mass index (bmi) measured either at the same time (cross - sectional analysis) or at the age of 1215 years (longitudinal analysis). bmi tracked markedly between age of 1215 and age of 19 - 20. bmi was strongly associated with all considered crfs in both cross - sectional and longitudinal analyses, with some exceptions. comparing overweight participants with those having a bmi below the age - specific median, the odds ratios for high blood pressure were 5.4/4.7 (male / female) cross - sectionally and 2.5/3.9 longitudinally (p < 0.05). significant associations were also found for most other crfs, with some exceptions. in linear regression analysis including both bmi at age of 1215 and bmi at age of 19 - 20, only bmi at age of 19 - 20 remained significantly associated with most crfs. we conclude that crfs are predicted strongly by either current or past bmi levels in adolescents and young adults in this population. the observation that only current bmi remained associated with crfs when including past and current levels together suggests that weight control at a later age may be effective in reducing crfs in overweight children irrespective of past weight status. |
tumor necrosis factor (tnf) was isolated and cloned 25 years ago [1, 2 ]. this molecule became the prototype of a growing familyof related proteins called the tnf superfamily (tnfsf) that share common features. most members of the family are synthesized as type ii transmembrane proteins and share a common structural motif, the tnf homology domain (thd), that mediates self - trimerization and receptor binding [3, 4 ]. the extracellular domain can be cleaved by specific proteases to generate soluble cytokines. most are type i transmembrane glycoproteins and are characterized by the presence of extracellular cysteine - rich domains. tnfrsf proteins are usually membrane bound, but some also exhibit a soluble form. similarly to tnfsf ligands, most tnfsf ligands bind to a single receptor ; some bind to more than one, and there is evidence of crosstalk between receptors for different ligands. ligand activation of tnfrsf members modulates cell proliferation, survival, differentiation, and apoptosis. such cellular events participate in a broad array of biological processes such as inflammation, fibrosis, the immune response, and tissue repair. tnfsf and tnfrsf proteins have been targeted therapeutically, and several drugs and biologicals are approved for use in inflammatory and autoimmune diseases. cumulative experimental evidence supports a role of the tnfsf / tnfrsf members in kidney injury outlined in table 1. many tnfsf cytokines, including tnf, fasl, trail, and tweak may activate the nf - kappab family of transcription factors. nf - kappab dna - binding complexes are homo- or hetero - dimers of five rel proteins : nf - kappab1 (p50, generated from p105), nf - kappab2 (p52, generated from p100), rela (p65), relb, and c - rel. the nuclear translocation and dna binding of nf - kappab occurs by two main pathways. classical or canonical nf - kappab activation is a rapid and transient response to a wide range of stimuli whose main effector is rela / p50. the alternative or noncanonical nf - kappab pathway is a more delayed response to a smaller range of stimuli resulting in nik activation and dna binding of relb / p52 complexes. tnfsf / tnfrsf members mediate different functions, in different tissues that depend on the surrounding milieu. unraveling their complex and pleiotropic actions will be essential for their use as therapeutic targets. tnf (tnfsf2) was the first member of the family to be implicated in the pathogenesis of kidney injury. tnf is expressed, synthesized, and released by infiltrating macrophages and by intrinsic kidney cells, namely, endothelial, mesangial, glomerular, and tubular epithelial cells. in vivo, the tnf expression pattern seems to be related to the primary kidney compartment injured. tnfr1 is present in normal glomeruli and is upregulated on infiltrating leukocytes in response to renal injury. tnfr2 is usually not expressed in normal kidney and is upregulated in tubular cells in response to renal injury. these receptors induce different and possibly opposing functions in inflammation and immunity, and the differential contribution of tnfr1- and tnfr2-mediated tnf signaling in renal lesions has only recently started to be explored [11, 16 ]. increasing evidence has implicated tnf as a major participant in the pathogenesis of kidney injury, promoting inflammation, apoptosis, and accumulation of extracellular matrix, reducing glomerular blood flow and damaging the glomerular permeability barrier with development of albuminuria [14, 1722 ]. the pathogenic role of tnf as well as the potential benefits of modulating tnf activity has been shown in models of immune complex - mediated glomerulonephritis, lupus nephritis, antineutrophil cytoplasmic antibodies (anca-) associated glomerulonephritis, minimal change disease, diabetic nephropathy (dn), acute kidney injury (aki), obstructive uropathy, and kidney allograft rejection [14, 15, 19, 21, 2326 ]. tnfr1 or tnfr2 deficiency protects mice from cisplatin - induced aki [27, 28 ] and obstructive uropathy. however, tnf also has immunosuppressive functions, depending on the surrounding milieu, the timing of the inflammatory response, and the differential interaction with its receptors. thus, tnfr1 deficiency enhances disease in mrl - lpr / lpr lupus mice, while tnfr2 deficiency confers protection from autoimmune renal injury [31, 32 ]. in 1995 first candidates for (anti - tnf strategies) trials will be. rapidly progressive glomerulonephritis and vasculitis. in 2010, emerging clinical data suggest a potential benefit of tnf antagonism in lupus nephritis [33, 34 ] and wegener 's granulomatosis [35, 36 ]. however, overall experience with different tnf formulations in vasculitis is inconclusive, and questions remain on the optimal combination of immunosuppressive drugs and specific subgroups of patients that might benefit [3740 ]. moreover, tnf blockade has been associated with the emergence of autoantibodies and lupus syndromes [41, 42 ] and with the development of infection, particularly reactivation of tuberculosis [43, 44 ]. the net effect of tnf actions depends on the balance between the proinflammatory and immunosuppressive functions, and current efforts are focusing on the selective inhibition of its deleterious actions. fas (apo-1/cd95/tnfrsf6) is a 45-kda type i membrane receptor containing an intracellular death domain (dd). fas is engaged by fas ligand (fasl / tnfsf6), a 3640-kda type ii membrane tnfsf member. metalloprotease - mediated soluble fasl (sfasl) shedding from membrane - bound fasl (mfasl) as well as decoy receptors modulates the system [4648 ]. thus, mfasl induces apoptosis more efficiently than sfasl [49, 50 ]. fas activation triggers apoptosis through recruitment and activation of caspase-8 by the adaptor protein, fas - associated protein with dead domain (fadd). nonapoptotic effects, such as proliferation, cell differentiation and inflammation, are also triggered in a range of cell types [5153 ]. fasl and fas play a critical role in modulating the immune response, including the peripheral deletion of autoimmune cells, activation - induced t cell death, and t cell - mediated cytotoxicity, thereby guarding against autoimmunity and tumor development. the fas receptor is constitutively expressed by mesangial and tubular cells, podocytes, and fibroblasts and is upregulated by noxious stimulus and inflammation [5457 ]. potential sources of renal fasl include infiltrating leukocytes and intrinsic renal cells, mainly tubular, but also mesangial, endothelial, and fibroblastic cells. activation of nf - kappab upregulates fasl in cultured mesangial cells exposed to inflammatory mediators and in hiv - associated nephropathy podocytes. fas and fasl are segregated from each other to different cellular compartments in kidney tubular cells : fas is restricted to the basolateral surface, while fasl is sequestered to an intracellular compartment and, to a lesser extent, the apical surface. this segregation may prevent autocrine / paracrine cell death, but is lost upon disruption of tight junctions by physical injury, ischemia, or proinflammatory cytokines. the fasl - fas system participates in renal injury, regulating renal cell apoptosis and the immune and inflammatory responses [54, 59, 65 ]. however, tubular cells are resistant to fas - dependent apoptosis under basal conditions, despite the constitutive, low - level fas expression [18, 59, 67 ]. activation of these low amounts of fas receptors results in jnk activation, not apoptosis, in renal tubular cells. other inflammatory mediators upregulating fas are required to prime tubular cells to undergo fasl - induced apoptosis [59, 69 ] (figure 1). these facts underscore the importance of the extracellular microenvironment to define cell fate in response to fas / fasl. renal cell fasl promotes apoptosis of lymphoid cells, potentially modulating the immune and inflammatory response. consistent with novel roles as a mediator of cell stress or chronic inflammation, fasl activates nf - kappab and the expression of proinflammatory cytokines [52, 70 ]. moreover, fas stimulation upregulates alpha(v)beta (8) integrin on tubular cells, relating fas to cell migration and fibrosis. fas agonists induce glomerular cell apoptosis and glomerular injury characterized by proteinuria and hematuria. in vivo, fas / fasl signaling has been implicated in tubular cell apoptosis in experimental ischemic injury, endotoxemia, transplant rejection, chronic kidney disease [69, 75 ], tubulointerstitial injury of obstructive uropathy, and focal segmental glomerulosclerosis [77, 78 ]. apoptosis of glomerular and tubular cells has also been linked to fas / fasl expression in hypertensive renal disease [79, 80 ], hiv - associated nephropathy, and human proliferative lupus nephritis. mice with genetically disrupted fasl / fas systems (b6 lpr / lpr mice) or these treated with small interfering rna targeting fas are protected from tubular cell injury during ischemia - reperfusion [72, 82, 83 ] and cisplatin - induced aki. loss - of - function mutations on fas (lpr / lpr) or fasl (gld / gld) on the mrl background result in lymphoproliferation, autoimmunity, and lupus - like glomerulonephritis. the autoimmune milieu appears to be the main inducer of injury, as kidney removal from the autoimmune (lpr / lpr) environment significantly reduces inflammation, and wild - type or lpr / lpr kidney grafts transplanted to lpr / lpr recipients display similar inflammation. moreover, in the course of lupus nephritis fas deficiency does not protect from renal disease or from tubular cell apoptosis. fas and fasl may be important for resolution of inflammation, promoting apoptosis of infiltrating lymphocytes as shown in b6 lpr / lpr mice and b6 gld / gld mice. in addition, in fasl - defective mice (gld / gld), fas agonists decrease renal injury, probably by limiting autoimmunity. the role of fas / fasl in renal transplantation is ambiguous : fasl gene transfer prolonged rat renal allograft survival, probably by inducting cytotoxicity in alloreactive t cells. in other studies, the absence of donor kidney fas (lpr) or fasl (gld) did not impact on histological lesions or apoptosis [58, 89 ] although it improved mice survival and kidney function. a gene - targeted murine model exploring the relative importance of mfasl and sfasl demonstrated that mfasl is essential for cytotoxic activity, while sfasl appeared to promote autoimmunity through nonapoptotic actions, namely nf - kappab activation. mice that lacked sfasl (mfasl intact) appeared normal, while mice lacking mfasl (sfasl intact) had higher nf - kappab activation and developed a lupus - like autoimmune kidney disease more severe than gld / gld mice (which lack sfasl and mfasl). tnf - related apoptosis - inducing ligand (trail) was originally identified by two independent groups as the third member of the tnf superfamily to induce apoptosis [90, 91 ]. trail is a type ii transmembrane protein of 281 and 291 amino acids in the humans and mice, respectively, with an expected molecular mass of 3335 kda. membrane - bound trail can be cleaved from the cell surface to form a soluble trimeric ligand that retains the proapoptotic activity. trail is normally expressed in many human tissues including kidney, suggesting that trail must not be cytotoxic to most tissues in vivo under normal physiological conditions [91, 92 ]. however, when normal cells are immersed in an inflammatory environment, data from knockout mice suggest that trail may induce parenchymal cell apoptosis. two additional alternative splice variants of trail in human cells lacking either exon 3 (trail - beta) or exons 2 and 3 (trail - gamma) had been described. the lack of apoptotic activity in both isoforms and an alternative splicing in response to cytokine stimulation add complexity to the system. five receptors for trail have been described in humans ; four membrane - bound and one soluble receptor. of the membrane - bound receptors, trail receptor 1 (trail - r1, apo-2, dr4) and trail receptor 2 (trail - r2, dr5) contain an intact intracellular dd which is required for apoptosis induction. trail receptor 3 (trail - r3, dcr1) has a glycosylphosphatidylinositol membrane anchor and lacks an intracellular domain, and trail receptor 4 (trail - r4, dcr2) contains a truncated dd. the latter may function as decoy receptors or be involved in nonapoptotic signaling [97, 98 ]. osteoprotegerin is a soluble receptor without cytoplasmic or transmembrane domains, first described as a bone remodeling regulator. osteoprotegerin is a decoy receptor for the tnfsf cytokine receptor activator of nf - kappab ligand (rankl) and for trail [99, 100 ]. however, recent studies support the biological relevance of the osteoprotegerin / trail interaction in different in vitro cell systems [102105 ]. further studies to unravel the relation between trail, osteoprotegerin, and rankl could illuminate potential cross - regulatory mechanisms. different combinations of trail and chemotherapeutic drugs or the use of agonistic anti - trailr1 or r2 antibodies shows promising results in the treatment of renal carcinoma [107, 108 ]. however, trail also has nonapoptotic functions, such as prosurvival and proliferative effects [109112 ]. in normal kidney, trail trail - r1 has a similar pattern of expression to trail, while trail - r2 is additionally expressed in henle 's loop. trail - r3 expression was not detected in the normal kidney, and there are no reports regarding renal tissue expression of trail - r4. no kidney pathology has been reported in trail knockout mice, suggesting that trail is not required for normal kidney development and physiology. transcriptomics disclosed increased trail and osteoprotegerin expression in human dn that correlated with parameters of kidney injury. interestingly, in dn there was de novo glomerular trail expression and increased tubular staining. inflammatory cytokines, such as tnf, interferon- (inf-), and macrophage migration inhibitory factor (mif), induce trail expression in tubular cells [59, 116 ]. a high - glucose medium, characteristic of diabetes, sensitized tubular cells and podocytes to the proapoptotic effect of trail. although it is difficult to extrapolate from cell culture studies to the in vivo situation, the low level of apoptosis induced by trail in cultured tubular cells is consistent with the slow loss of renal function, over years, characteristic of dn. trail blockade in murine models of autoimmune diabetes (type i diabetes) led to an increased incidence and severity of disease [117119 ]. thus, depending on the type of diabetes and on the disease stage, trail can have a dual role either as an immune modulator or as a regulator of renal cell survival. while many tnfsf ligands bind to multiple receptors, only a single signaling receptor for tweak (tweakr) has been confirmed [121, 122 ]. tweakr was identical to the previously characterized human fibroblast growth factor - inducible 14 (fn14) receptor. initial reports that the tnfrsf protein death receptor 3 (dr3) was the tweak receptor were not confirmed in subsequent studies [125, 126 ]. current knowledge suggests that tweak and fn14 might play a role in several processes relevant to kidney damage such as regulation of survival / proliferation of kidney cells and their ability to regenerate in response to aggression and the regulation of the inflammatory response. the potential sources of tweak in the kidney include infiltrating monocytes and t lymphocytes, tubular cells, and mesangial cells. human and murine mesangial cells, podocytes, and tubular cells express fn14 and are responsive to tweak [129, 130 ]. the process of tweak binding and activation of the fn14 receptor has proliferative, proapoptotic, and proinflammatory actions in renal cells that depend on cell type and the microenvironment (figure 1). tweak, as other tnfsf members, can either induce apoptosis or proliferation depending on the experimental conditions (figure 1). tweak increased the proliferation, cell number, and cyclin d1 expression of quiescent cultured tubular cells. tweak also induced proliferation in mesangial cells and podocytes [129, 131 ]. tweak - induced tubular cell proliferation is enhanced in the presence of survival factors from serum which increase fn14 expression. tweak - induced tubular cell proliferation was prevented by inhibitors of mitogen - activated protein kinases and by the nf - kappab inhibitor parthenolide. several tnfsf cytokines, such as fasl, tnf, and trail, induce apoptosis in stressed renal cells [62, 113 ]. however, in the presence of inflammatory cytokines (tnf and inf), tweak induced apoptosis in tubular cells through the activation of the fn14 receptor, caspases, and mitochondria involvement. tnf or inf alone increased fn14 expression but neither was sensitized tweak - induced cell death. this, together with a more intense proliferative response, but not cell death, when fn14 is upregulated by serum, suggests that fn14 upregulation, per se, does not determine the type of response to tweak. further, less characterized intracellular changes are required to determine the lethal or proliferative response of tubular cells to tweak. interestingly, a pan - caspase inhibitor prevented tweak / tnf / inf-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. in tubular cells nf - kappab activation was associated with degradation of ikappab - alpha, nuclear translocation of rela, and early (36 h) increased mrna and protein expression of the chemokines monocyte chemotactic protein-1 (mcp-1) and rantes. parthenolide, which prevents ikappab - alpha degradation, inhibited tweak - induced nf - kappab activation and prevented the expression of mcp-1 and rantes on tubular cells. tweak also induced the expression of inflammatory mediators in glomerular mesangial cells through nf - kappab activation and in podocytes. in addition, tweak induces nik - mediated, noncanonical nf - kappab activation in tubular cells, characterized by late nuclear translocation of relb / nf - kappab2 dna - binding complexes [134, 135 ]. the delayed tweak - inducted upregulation of the ccl21 and ccl19 chemokines was under noncanonical nf - kappab control and was not observed in cells stimulated with tnf. fn14 receptor is the mediator of both the proliferative and the apoptotic effects of tweak, and the cell response is modulated by the cell microenvironment : in the presence of proinflammatory cytokines, tweak potentiates cell death while in the presence of serum tweak has the opposite effect, proliferation. given the multifunctional nature of tweak / fn14, only in vivo functional studies in specific diseases will clarify their role. in lupus proliferative nephritis, tweak / fn14 are upregulated and tweak contributes to mesangial cell proliferation or apoptosis [129, 136 ]. this is a situation characterized by tubular cell proliferation in the absence of tubular injury or increased expression of inflammatory cytokines. lower tubular cell proliferation was observed in the remnant kidney of tweak knockout mice compared with wild - type mice. moreover, administration of exogenous tweak to uninephrectomized wild - type mice further increased renal cell proliferation. is followed by compensatory tubular cell proliferation taking place in an inflammatory environment that leads to recovery. prophylactic treatment with anti - tweak antibodies decreased inflammation and the rates of apoptosis and tubular cell proliferation during aki [131, 133 ]. studies with tweak - deficient mice confirmed a role of tweak in tubular cell apoptosis as well as in proliferation during aki. these data are consistent with the proapoptotic action of tweak in an inflammatory milieu in cultured tubular cells. since renal function was improved by anti - tweak strategies and there was no delay in recovery, it was hypothesized that the reduced tubular cell proliferation during aki observed in anti - tweak - treated animals reflected the lesser degree of initial injury, rather than a requirement for tweak for compensatory post - aki tubular proliferation. multiple lines of evidence indicate the involvement of different tnfsf cytokines, including tnf, fasl, trail, and tweak in the pathogenesis of renal injury. however, there is an insufficient understanding of the cooperation between cytokines in the complex in vivo environment. this information is important for the design of multipronged approaches aimed at targeting several members of the family in order to maximize benefit and minimize side effects. | members of the tnf superfamily participate in kidney disease. tumor necrosis factor (tnf) and fas ligand regulate renal cell survival and inflammation, and therapeutic targeting improves the outcome of experimental renal injury. tnf - related apoptosis - inducing ligand (trail and its potential decoy receptor osteoprotegerin are the two most upregulated death - related genes in human diabetic nephropathy. trail activates nf - kappab in tubular cells and promotes apoptosis in tubular cells and podocytes, especially in a high - glucose environment. by contrast, osteoprotegerin plays a protective role against trail - induced apoptosis. another family member, tnf - like weak inducer of apoptosis (tweak induces inflammation and tubular cell death or proliferation, depending on the microenvironment. while tnf only activates canonical nf - kappab signaling, tweak promotes both canonical and noncanonical nf - kappab activation in tubular cells, regulating different inflammatory responses. tweak promotes the secretion of mcp-1 and rantes through nf - kappab rela - containing complexes and upregulates ccl21 and ccl19 expression through nf - kappab inducing kinase (nik-) dependent relb / nf - kappab2 complexes. in vivo tweak promotes postnephrectomy compensatory renal cell proliferation in a noninflammatory milieu. however, in the inflammatory milieu of acute kidney injury, tweak promotes tubular cell death and inflammation. therapeutic targeting of tnf superfamily cytokines, including multipronged approaches targeting several cytokines should be further explored. |
anterior cruciate ligament (acl) reconstruction is one of the most common orthopedic procedures. however, despite very satisfactory clinical outcomes [1, 2 ], the surgeon often detects the persistence of a certain rotatory instability, even in cases of reconstructions with no detectable intra- or post - operative complications, and regardless of the type of graft used. the cause of this phenomenon is not completely understood yet, and the goal of many researchers is to be able to finally reconstruct a knee with the original rotational stability. in this paper the main function of the acl is to control tibial anterior translation with a secondary effect on knee internal rotation [4, 5 ]. both acl functions are possible due to the presence of other articular structures which are linked to the acl in maintaining knee stability. it is well documented how the acl is made of two different bundles, the antero - medial (am) and the postero - lateral (pl), with different specific functions, but working synergically so that they can not be considered as separate structures. one of the main aspects of acl anatomy, in regard to surgical reconstruction, is the exact position of its femoral and tibial insertion. the femoral insertion has been the most commonly studied since the first attempts at open intra - articular reconstructions, because it is thought to be the most difficult to reproduce due to its very posterior positioning on the medial surface of the external femoral condyle. it was common opinion that, despite the graft choice, the positioning of the graft could never be posterior enough, so many surgeons tried to perform an over the top technique ; however, it is now appreciated that the native acl femoral insertion site is located along osseous landmarks on the posterior aspect of the medial wall of the lateral femoral condyle, termed the lateral intercondylar and bifurcate ridges. the lateral intercondylar ridge corresponds to the feature termed the resident ridge reported by clancy.. identification of this ridge when present has been shown to be an accurate and reliable method of locating the native acl insertion site and the true entry point of the femoral tunnel. diagnosis of acl rupture is basically clinical, and is usually performed with the lachman and the pivot - shift tests, whose sensibility and specificity have been widely documented. the pivot - shift test assesses both tibial translation and rotation, reproducing the most common traumatic mechanism of rupture of the ligament. moreover, the pivot - shift test better correlates with a patient s disability. for this reason it is thought to be the most reliable test for detecting rotatory instability of the knee following an acl tear. the pivot - shift is usually graded in three degrees (grade i : glide ; grade ii : jerk ; grade iii : subluxation), but it is highly dependent on the ability and experience of the operator. since the importance of this test is in evaluating both acl deficiency and the effects of different surgical techniques of reconstruction, many authors have proposed various methods of objectively evaluating and quantitatively measuring the test, such as the use of a navigation device (fig. 1), mechanical or electromagnetic tool, or conventional radiological or magnetic resonance dynamic methods [1117 ]. however, none of these methods have become widespread among surgeons and many of them remain operator - dependent. as a matter of fact the pivot - shift test is still the most popular test for assessing the rotatory instability of the knee after an acl rupture and reconstruction.fig. 1the pivot - shift phenomenon as detected by a navigator : tibial antero - posterior diagram and rotation in a knee with intact acl (a) and in an acl - deficient knee (b) the pivot - shift phenomenon as detected by a navigator : tibial antero - posterior diagram and rotation in a knee with intact acl (a) and in an acl - deficient knee (b) the relationship between acl lesions and the resulting rotatory instability of the knee has mainly been studied on cadaver knees. in fact, in in vivo studies where the instability was compared to the contralateral healthy knee, we can never assume that the lesion of the acl was the only damage that occurred in the knee as a consequence of the initial trauma or as a result of progressive stretching of other structures due to the lack of the torn acl. in a recent study we performed on ten cadaver knees, we evaluated internal rotator instability after sectioning either component of the acl, showing that an isolated section of the pl bundle did not produce an increase in internal rotator instability ; on the contrary, the section of the entire acl did produce an increase in internal instability in nine cases. in our trials, only after a complete section of the entire acl did we detect a + pivot - shift (glide) in three cases and a + + pivot - shift (jerk) in seven cases ; a + + + pivot - shift (subluxation) was never detected after isolated complete section of the acl. however, in regard to rotatory instability, acl lesions, and the pivot - shift test, we should remember that the pivot - shift test assesses rotatory instability, and in particular, according to hughston, the antero - lateral instability, whose origin is more complicated than a simple result of acl lesion. and feagin. stated that even though the pivot - shift is related to an acl tear, it is significantly increased by concomitant lesions of the external compartment and in particular of the middle one - third of the lateral capsular ligament actually, in our study cited above on cadaver knees we detected a significant increase in rotatory instability along with a + + + pivot - shift in all the cases in which, after tearing the entire acl, even a partial lesion (1 cm) of the alftl was performed. even more recently, mushal. showed, also with the use of a navigation system, a significant increase of the anterior tibial translation in the external compartment during the pivot - shift maneuver in acl deficient knees after external meniscectomy. the same group of researchers had previously shown that the anterior translation of the external part of the tibia was strictly correlated to the pivot - shift score. using a robotic / ufs testing system. in conclusion, we can state that, while an isolated lesion of the pl bundle does not seem to increase internal rotation, lesion of the entire acl produces a slight increase of internal rotation but, most of all, a different pattern of rotation whose fulcrum moves from the central pivot of the joint to the medial collateral ligament (mcl), with following increase of the anterior translation of the external tibial plateau. the pivot - shift test, in assessing this particular biomechanical pattern of instability, is related not only to the lesion of the acl, but it is strongly influenced by associated lesions, in particular those of the lateral compartment (alftl, lcl, lateral meniscus). even though acl reconstruction is a very widely used operation with a very satisfactory success rate which allows the majority of active patients involved in sports activities to resume their pre - operative levels, the persistence of a certain rotatory joint laxity might produce subsequent meniscal or chondral damage leading to a clear degenerative arthritic disease. even though the pivot - shift assessment has always been part of the evaluation scoring scales used, it has often been underestimated ; only within the last few years has this topic come back to scientific attention thanks to freddi fu s studies on the functional anatomy of the acl, on its two distinct bundles and, most of all, on failure of the single - bundle reconstruction to restore exact joint stability especially in regard to internal rotation. as a result, many surgeons, with the aim of improving their success rates, have in turn started using the double - bundle reconstruction of the acl. in recent years, and in particular in the first years of the third millennium, many clinical and laboratory studies have been published on the advantages of reconstruction of the two bundles, thus supporting fu s theory [2527 ]. in particular, clinical studies with a minimum follow - up of 2 years in which double - bundle reconstructions were used showed better results than the single - bundle, both in terms of knee stability and in terms of recurrence rate. moreover, other studies, such as the one by robinson. on cadaver knees with the use of a navigation system, further provided objective data about how, in acl reconstructions, the am and pl bundles act differently in stabilizing the knee, particularly during the pivot - shift test, where the pl bundle is important in controlling not only anterior laxity toward knee extension, but also the rotational component. in net contrast with what was reported by the above mentioned authors, in a similar study we performed on cadaver knees with the use of a navigation system, we found that the further addition of the pl bundle to an am single - bundle reconstruction did not provide any additional stability to the knee in regard to internal rotation. how might these different results be explained since the methodologies of the studies were similar ? the explanation may likely be in the different surgical way of reconstructing the am bundle : in fact, in robinson s study, the am bundle was positioned slightly more anterior and vertical than in our study, whereas we tried to place the femoral insertion, approached with an out - in technique, more horizontal and as posterior as possible, thus in accordance with the actual anatomy of the acl. this is a very important topic which deserves deeper examination. since the beginning of the reconstruction of the acl with open techniques, it was mandatory for the surgeon to scrupulously respect the anatomy of the acl with a very posterior positioning of the femoral insertion. since this goal could not be reached by drilling the femoral condyle from the articular joint, surgeons started using the out - in technique. after a few years, with the advent of arthroscopic techniques, the majority of surgeons preferred to perform less invasive techniques (single - incision techniques), performing trans - tibial femoral tunnel drilling, often resulting in a non - anatomical positioning of the acl. it seems as if, despite the advantages provided by the scope, the arthroscopists preferred mini - invasiveness over respect of the anatomy and function. however, since rotatory instability is a complex phenomenon not simply dependent on the acl rupture or the anatomical reconstruction, other hypotheses have been proposed to correct this type of instability. among these, an important aspect is represented by the peripheral plasties [30, 31 ], whose biomechanical role in controlling rotational instability and the pivot - shift has been widely proven, even in recent studies published by our group : internal rotation was better restored in cases in which the anatomic acl reconstruction was performed along with a peripheral plasty than in cases treated with a double - bundle technique. did not reach the same conclusions as us : however, they did not put any tension on the lateral sling, thus losing a big part of the efficacy of the technique itself. even more controversial is the clinical effectiveness of the peripheral plasties in long - term follow - up, even though zaffagnini., in assessing three groups of patients treated with hamstrings (stg), bone - patellar - tendon - bone (bptb) and st plus peripheral plasty, obtained the best results in the third group. in our experience we found peripheral plasty useful in patients with severe rotatory instability (+ + + pivot - shift), in women and in cases of revision. a complete lesion of the acl often causes a rotatory instability of the knee detectable with the pivot - shift test. during this complex phenomenon the tibia tends to internally rotate with respect to the femur, thus changing its rotational fulcrum which moves medially closer to the mcl, with following anterior translation of the external tibial plateau. the severity of the pivot - shift, commonly scored in three degrees, essentially depends on the amount of constitutional tibial rotation and on the presence of concomitant associated lesions, such as the alftl and the external meniscus. in order to obtain the best rotatory stability, the acl reconstruction must be performed accurately, reproducing its anatomical positions (either single- or double - bundle). peripheral plasties may contribute to better control of rotator instability and may be indicated in selected types of patients. | although acl reconstructions provide satisfactory clinical results nowadays, regardless of the type of graft or the surgical technique used (out - in vs in - out or single- vs double - bundle), the residual rotatory instability which is often detected at clinical follow - ups is still a matter of concern among surgeons. in this paper we try to analyze all the aspects which might contribute to this phenomenon by summarizing the biomechanical functions of the two bundles of the acl, and by evaluating all the other factors strictly related to the rotatory instability of a reconstructed knee, such as the anatomical positioning of the single- or double - bundle new acl, or the importance of a valid lateral compartment (lcl, altfl). clinical, biomechanical and cadaver studies are discussed in order to contribute to better understanding of the origin of post - operative residual rotatory instability. |
land plants host rich and diverse microbial communities in the thin layer of soil adhering to the roots, i.e., the rhizosphere, and within the root tissues, designated rhizosphere and root microbiota, respectively (bulgarelli., 2013). roots secrete a plethora of photosynthesis - derived organic compounds to the rhizosphere (dakora and phillips, 2002). this process, known as rhizodeposition, has been proposed as the major mechanism that enables plants to sustain their microbiota (jones., 2009). in turn, members of the rhizosphere and root microbiota provide beneficial services to their host, such as indirect pathogen protection and enhanced mineral acquisition from surrounding soil for plant growth (bulgarelli. thus, the dissection of the molecular mechanisms underlying plant - microbe community associations at the root - soil interface will be a crucial step toward the rational exploitation of the microbiota for agricultural purposes. recent studies performed using the model plant arabidopsis thaliana revealed that the soil type and, to a minor extent, the host genotype shape root microbiota profiles (bulgarelli., 2012 ; lundberg., the structure of the microbial communities thriving at the root - soil interface appears to be resilient to host evolutionary changes, as indicated by a largely conserved composition of the root bacterial microbiota in a. thaliana and related species that spans 35 ma of divergence within the family brassicaceae (schlaeppi., 2014). however, it is unclear whether microbiota divergence is greater in host species belonging to other plant families and whether the process of domestication, which gave rise to modern cultivated plants (abbo., 2014) and which can not be studied in a. thaliana, has left a human footprint of selection on crop - associated microbiota. barley (hordeum vulgare) is the fourth - most cultivated cereal worldwide (newton., 2011) and one of the earliest cereals consumed by humans, with evidence of presence of wild barley (hordeum vulgare ssp. spontaneum) in human diets dating back to 17,000 bc (kislev., 1992). barley was one of the first plants subjected to domestication, which culminated 10,000 years ago when the cultivation of domesticated barley (hordeum vulgare ssp. anthropic pressure on barley evolution continued through diversification, which progressively differentiated early domesticated plants into several genetically distinct accessions whose area of cultivation radiated from the middle east to the rest of the globe (comadran., 2012). nowadays, wild and cultivated barley accessions still coexist, providing an excellent experimental framework to investigate the structure and the evolution of the microbiota associated with a cultivated plant. here, we used an amplicon pyrosequencing survey of the bacterial 16s rrna gene and combined it with state - of - the - art metagenomics and computational biology approaches to investigate the structure and functions of the bacterial microbiota thriving at the barley root - soil interface. we found evidence for positive selection being exerted on a significant proportion of the proteins encoded by root - associated microbes, with a bias for cellular components mediating microbe - plant and microbe - microbe interactions. we have grown barley accessions in soil substrates collected from a research field located in golm, near berlin (bulgarelli., 2012), under controlled environmental conditions (experimental procedures). we subjected total dna preparations from 6 bulk soil, 18 rhizosphere, and 18 root samples to selective amplification of the prokaryotic 16s rrna gene with pcr primers encompassing the hypervariable regions v5-v6-v7 (schlaeppi., 2014), and we generated 691,822 pyrosequencing reads. after in silico depletion of error - containing sequences, and chimeras as well as sequencing reads assigned to plant mitochondria, we identified 1,374 prokaryotic operational taxonomic units (otus) at 97% sequence similarity (database s1 ; experimental procedures). taxonomic classification of the otu - representative sequences to phylum level highlighted that actinobacteria, bacteroidetes, and proteobacteria largely dominate the barley rhizosphere and root communities, where 88% and 96% of the pyrosequencing reads, respectively, were assigned to these three phyla. of note, other members of the soil biota, such as firmicutes and chloroflexi, were virtually excluded from the plant - associated assemblages (figure 1). the enrichment of members of the phylum bacteroidetes significantly discriminated rhizosphere and root samples from bulk soil samples irrespective of the accession tested (moderated t test, false discovery rate - adjusted [fdr ], p value 15 and no ambiguous bases were retained for the analysis. chimeras were identified using the gold reference database (http://drive5.com/uchime/gold.fa), and otus were defined at 97% sequence identity. otu - representative sequences were taxonomically classified using the rdp classifier (wang., 2007) trained on the greengenes reference database. the resulting otu table was used to determine taxonomic relative abundances and subsequent statistical analyses of alpha- and beta - diversity (see supplemental experimental procedures). paired - end illumina reads were subjected to trimming, filtering, and quality control using a combination of custom scripts and the clc workbench v5.5.1 and assembled using soapdenovo (heger and holm, 2000). a small fraction of the partially assembled metagenome samples (on average 3.02% of the reads) was mapped to the annotated barley genomic sequences, and the corresponding contigs or singleton reads were removed (table s2 ; supplemental experimental procedures). we used taxator - tk (drge., 2014) to taxonomically classify the partially assembled metagenome sequences (including unassembled singleton reads) using the ncbi database as a reference. coding sequences were predicted using metagenemark (zhu., 2010) and annotated using matches to hmm (hmmer v3.0) profiles to the tigrfam (haft., 2013) and pfam (punta., 2012) databases as well as a k - mer - based matching using the seed (edwards., 2012) api and server scripts. to test for a significant enrichment of functional categories in the root - associated bins relative to the remaining bins, we assumed a correspondence at the family level between metagenome bins and root- and rhizosphere - enriched otus (rr_otus) of these families found in the amplicon survey. to search for signatures of positive selection we first employed hmmer to obtain multiple sequence alignments (msas) of orthologous sequences found in the metagenome samples. from each msa, we calculated neighbor - joining trees and used them to infer ds and dn changes. clusters of residues with significant signs of positive selection were calculated using a sliding window approach. a detailed description of the methods and tools used for the analysis of the metagenome is available in the supplemental experimental procedures. | summarythe microbial communities inhabiting the root interior of healthy plants, as well as the rhizosphere, which consists of soil particles firmly attached to roots, engage in symbiotic associations with their host. to investigate the structural and functional diversification among these communities, we employed a combination of 16s rrna gene profiling and shotgun metagenome analysis of the microbiota associated with wild and domesticated accessions of barley (hordeum vulgare). bacterial families comamonadaceae, flavobacteriaceae, and rhizobiaceae dominate the barley root - enriched microbiota. host genotype has a small, but significant, effect on the diversity of root - associated bacterial communities, possibly representing a footprint of barley domestication. traits related to pathogenesis, secretion, phage interactions, and nutrient mobilization are enriched in the barley root - associated microbiota. strikingly, protein families assigned to these same traits showed evidence of positive selection. our results indicate that the combined action of microbe - microbe and host - microbe interactions drives microbiota differentiation at the root - soil interface. |
self - poisoning with pesticide accounts for nearly one - third of the world 's suicides. over the last few decades, agricultural pesticides have become major cause of self - poisoning in rural areas of the developing world due to their easy availability. while organophosphorus compounds cause most self - poisoning deaths in southern and central india, aluminium phosphide causes most deaths in northern india. although accidental poisoning with hydrogen cyanamide is known, reports of self - poisoning with the same are limited. we present a case of 22-year - old male who succumbed to hydrogen cyanamide following self - poisoning. awareness of the incident is necessary to provide an early and aggressive management as there is no specific antidote for the same. a 22-year - old male was brought in with alleged history of consuming approximately 150 ml of hydrogen cyanamide (dormex - active ingredient : 520 g / l of cyanamide) about 2 hours prior to hospitalization. initial assessment revealed glasgow coma scale (gcs) score of 7/15 (e2, m4, v1), pulse of 110 beats / min, blood pressure of 90/60 mmhg, respiratory rate of 30/min, and oxygen concentration of 88% (98% with 6l oxygen inhalation by mask) in supine position. chest x - ray showed minimal pleural effusion, otherwise normal, and the electrocardiograph showed sinus tachycardia [figure 1 ]. computerised tomography (ct) brain and echocardiography were done as a part of initial assessment and were normal. ultrasonography of abdomen revealed bilateral grade i nephropathy, mild ascitis, and minimal right pleural effusion. comprehensive toxicology profile including serum pseudocholinesterase was normal and benzodiazepines and barbiturates were not detected on urine examination. baseline and follow - up investigation reports of the patient arterial blood gas analysis and the correction given for the metabolic acidosis at different timeline during patient 's hospitalization electrocardiograph of the patient showing sinus tachycardia patient was given gastric lavage and fluid management was done according to cvp. as the patient was drowsy and responding only to painful stimulus, he was electively intubated and mechanically ventilated (simv mode ; fio2 - 60% ; peep-5 cm h2o). sodium bicarbonate (25 meq / h intravenously) was given to correct the metabolic acidosis and the dosage was adjusted by repeated blood gas analysis [table 2 ]. dopamine was started at 5 g / kg / min, followed by noradrenaline 5 g / kg / min and dobutamine at 2g / kg / min, which were gradually stepped up to their maximum dose (dopamine 15 g / kg / min, noradrenaline 20 g / kg / min, dobutamine 10 g / kg / min). cardio pulmonary resuscitation was started and atropine (1.2 mg iv) and adrenaline (1 mg iv) were given. atropine (0.6 mg iv) and adrenaline (1 mg iv) were repeated every 5 minutes over next one hour. in spite of all these measures, although insecticide poisoning has been reported worldwide time and again, there are limited reports of poisoning with a plant growth regulator - hydrogen cyanamide. hydrogen cyanamide is an active ingredient of dormex (degussa ag, trostberg, germany), which is used as a plant growth regulator designed to stimulate more uniform bud - break following dormancy, resulting in more uniform flowering and maturity at harvest. the department of pesticide regulation of the california environmental protection agency have studied the toxic effects of hydrogen cyanamide and concluded that hydrogen cyanamide causes adverse effects in the liver, thyroid, kidney, ovaries, and testes of laboratory animals. it further stated that in the absence of additional data to the contrary, hydrogen cyanamide has the potential to cause similar effects in humans. in their study, approximately 40% of oral dose was recovered in the urine in the first 24 hours. the remainder of oral doses was excreted in the feces or exhaled as carbon dioxide. the adverse health effects from contact of hydrogen cyanamide include severe irritation and ulceration of the eyes, skin, and respiratory tract. animal studies have indicated that at cellular level, cyanamide is activated by catalase, which in turn causes catalase inhibition resulting in uncoupling of oxidation and phosphorylation and inhibition of adenosine nucleotide synthesis. the substance also inhibits aldehyde dehydrogenase and can produce disulfiram - like syndrome (e.g., vomiting, parasympathetic hyperactivity, dyspnoea, hypotension, and confusion) when exposure coincides with alcohol use. cederbaum and dicker showed that ethanol prevented inhibition of catalase if given before or at the same time as cyanamide, suggesting that ethanol may protect against the activation of cyanamide by catalase. the application of these principles in treating acute cyanamide toxicity is an option although there are no supporting studies for the same. there are reports of cutaneous reaction to exposure to hydrogen cyanamide in india and worldwide. ingestion of hydrogen cyanamide has been reported in a 44-year - old male from ragusa, italy when he unintentionally ingested the product that had been placed in a plastic water bottle in refrigerator. following consumption, he became seriously ill with third degree shock, coma, miosis, and hepatic necrosis and required care in an intensive care unit. in our patient, we found similar manifestation of toxicity due to hydrogen cyanamide. also, persistent metabolic acidosis and hypotension with lactic acidosis was noted. since there is no known antidote for this compound, patient was managed symptomatically. poisoning with plant growth regulator has been described from italy, usa, and india. majority of these reports were incidents of adverse effects following the occupational or accidental exposure to the chemical. the present case is probably the first reporting hydrogen cyanamide ingestion with a suicidal intent, leading on to persistent metabolic acidosis, encephalopathy, and refractory shock with a fatal outcome. | case reports of acute and chronic exposure to hydrogen cyanamide (dormex) have been reported but mainly as a result of occupational or accidental exposure and without any mortality. we report a case of acute hydrogen cyanamide poisoning in a young male due to suicidal intent. the patient was managed under intensive care with all the standard protocols for detoxification. however, in spite of aggressive management, patient could not be rescued. an extensive literature search did not yield any similar case reports. hence, we report this case to the medical community to be aware of the entity. |
tissue engineering is a new field that allows the combination of engineering, biology, and material methods for developing new techniques with potential to create tissues and organs. the ability of networked three - dimensional structure to elicit altered cell behaviors, including cell adhesion, has raised heightened interest in the scaffold materials for various biomedical applications, including orthopedic repair and regeneration. cells in vitro usually do not reproduce in a three - dimensional fashion unless being allowed to grow on scaffolding. the scaffolds should have appropriate characteristics such as pore size, shape, and mechanical properties to enable cells to grow in every dimension. the cells have to be able to attach, migrate, proliferate, and differentiate into various organs on the scaffold. several engineered tissue grafts have been developed for the reconstruction of the injured hard and soft tissues. used lindenmayer systems, an elegant fractal - based language algorithm framework, in designing vasculature networks that could potentially be incorporated in hydrogel scaffolds like pegda. the reason for using pegda over other materials is that pegda is 3d networked structures that can be manufactured easily to allow for the cell growth at higher depth using photolithograph process. photolithography is a process which is commonly used in microfabrication to produce the desired scaffolds with a high level of detail and precision. it has been found to be a valid method to manufacture multiple - layer scaffolds for allowing the constructions of channels within the scaffold to better distribute nutrients to the cells. study also found that polyethylene glycol diacrylate (pegda) tissue scaffolds having thickness higher than 1 mm were shown to have limited applications as a three - dimensional cell culture device due to the inability of cells to survive within the scaffolds. without access to adequate nutrients, cells placed deep within the pegda tissue construct having thickness higher than 1 mm die out, leading to nonuniform tissue regeneration. photopolymerization system is usually comprised of three major parts : (1) a uv light source, (2) mold, and (3) a polymer solution. the role of the mold is to allow the pegda to polymerize in the desired shape. tissue scaffolds, with nutrient conduit networks, need to be designed with intricate architecture, porosity, pore size and shape, and interconnectivity in order to provide the required structural strength, transport nutrients, and the microenvironment for cell and tissue ingrowth. by selecting the appropriate unit cell interior structures, structural properties such as the mechanical strength, ductility, and permeability and biological activities such as cell viability, degradation, and tissue generation of pegda structure the relationship between the interior nutrient conduit network structure and biomechanical properties (mechanical and biological properties) of pegda is not understood yet. knowledge of the biomechanical properties of the networked pegda constructs with respect to the photoinitiator (pi) concentration, temperature, and incubation time is also necessary for adequate design and effective use of pegda for tissue engineering constructs. to understand the effect of nutrient conduit networks on the pegda biomechanical performances, this study compared the failure stress of pegda flat dumbbell - shaped mold with nutrient conduit networks from the displacement controlled tension tests. different photoinitiator concentration affects the materials properties due to the difference of crosslink density due to the difference of amount of free radicals created by the pi during the uv photopolymerization process. in addition, pegda hydrogels were susceptible to time and temperature dependent degradation, which in general negatively affect the mechanical strength. the failure assessments of the pegda molds were conducted in this study as a function of the pi concentration, temperature, and time. in addition, the cell viability assessments of with and without nutrient conduit networks cylindrical pegda molds were conducted as a function of the pi concentration. flat dumbbell shaped pegda tension test samples with nutrient conduit networks at the gauge section were designed and fabricated by uv - photo polymerization process. pegda solution was added to different concentrations of pi (0.2% and 0.6%) solution to make the specimen. tension tests were conducted on those samples at different temperatures (23c and 37c) and incubation time (0 and 7 days) after the fabrication of the specimen. human dp147 mesenchymal fibroblasts cells were encapsulated within pegda hydrogel having nutrient conduit networks during the photopolymerization of the pegda. cell viability experiments analyzed effects of conduit networks and photoinitiator concentration after 7 days of cell culture on the hydrogels. two solutions, pegda (mn = 700 ; sigma - aldrich) with the phosphate buffer solution (pbs) solvents, and the photoinitiator (pi), alpha - alpha - dimethoxy - alpha - phenylacetophenone (mw = 256.35 g / mol ; sigma - aldrich) with the 1-vinyl-2-pyrrolidone (mw = 111.14 g / mol ; fluka) solvents, were used to fabricate the gel solutions. pbs was used instead of water in this study, since pbs is better biological solvent than water when preparing cell encapsulating pegda gel. three samples were made for each experimental group of samples to evaluate the different concentrations of photoinitiator (0.2% and 0.6%) temperatures (23c and 37c) and incubation time (0 and 7 days) effect on the tension failure stresses of the samples. the reason for selecting 0.2% and 0.6% concentrations of photoinitiator for pegda solution was that in our earlier studies cells were successfully grown in pegda structures with significant cell viability differences with those concentrations of photoinitiator. figure 1 shows the steps used for the preparation of the mold, specimen, and mechanical tests. fields landing, ca) mold (figure 1--(a)) was fabricated to make astm e855 - 90 standard flat dumbbell - shaped for mechanical experiments. two additional abs plastic pieces (figure 1--(b and c)), fabricated using dimension elite 3d printer (stratasys, inc.), were assembled with the silicon mold to fabricate nutrient conduit networked pegda specimen. three pieces of mold were used in this study to generate same nutrient conduit networked pegda specimen for all group of test samples, while only silicon mold was used to prepare pegda samples without nutrient conduit network. each plastic piece has an array of holes (diameter : ~2 mm and spacing : ~1 mm). the bottom piece has 14 (7 2) holes, whereas the side piece has 21 (7 3) holes. a total amount of 35 pins (0.8 mm diameter) were inserted through these holes in the gauge section of the flat dumbbell - shaped mold as shown in figure 1-. the purpose of the pins is to produce an array of nutrient conduit network channels at the gauge section of the flat dumbbell - shaped pegda sample after uv polarization of pegda solution in the mold (figure 1-). an open - ended sterile cylindrical borosilicate glass tube was used to prepare mold for cell viability tests samples. one side of the open - ended tube was cured on silicon rubber disc to prepare cylindrical shaped hydrogels on the tube. the glass tube functioned as hydrogel housing as well as providing a support for pins to create nutrient architecture channels on hydrogel inside the tube. in addition, the glass tube provided a novel way to acquire thin section of hydrogel samples for viability assays. to create networked channeled pegda hydrogels thin stainless steel pins were carefully placed into the open glass end and secured into the silicon disc (figure 2). the 20 wt% pegda solution was produced by mixing 2 ml of pegda with 8 ml of pbs. the pi solution was produced by mixing 0.3 g of pi with 1 ml of solvent in dark room to prevent premature crosslinking. the 0.2 wt% and 0.6 wt% pi gel solution was produced by mixing 4 l and 12 l of pi with 2 ml of pegda solution, respectively. the solution was poured in the custom - made mold to cure the mixture in a flat dumbbell - shaped gel. the solution was polymerized by exposure to 365 nm long wave uv (b-100sp ultraviolet lamp, uvp, llc) light. in general, the biocompatibility of pegda hydrogel depends on complete polymerization of pegda solution while using minimal concentration of photoinitiator. the exposure to uv light causes photoinitiators to generate free radicals that initiate the polymerization to form the hydrogel. since mazzoccoli study found that 20 wt% and 40 wt% pegda having 0.6 wt% pi is biocompatible, therefore, this study used 0.2 wt% and 0.6 wt% pi gel solution to evaluate the pi concentration effect on failure stress of pegda samples. due to the short - term uv exposure (3 to 5 minutes), photopolymerization is generally considered as a safe method to encapsulate cells. since encapsulation of cells is the main purpose of creating nutrient conduit networked in pegda gels, therefore, uv light was exposed for 3 min for all specimens to form the hydrogels and get the failure stresses of the samples. the flexible silicon mold with hydrogel specimen was disassembled from the solid plastic pieces (figure 1-). dp147 dermal fibroblast cells, used in hydrogels, were acquired using standard techniques and protocols for culture and isolation. cells used for culture were incubated at standard conditions, 37c and 5% co2, in tissue culture dishes with dulbecco 's modified eagle 's medium (dmem) containing 10% fetal bovine serum (fbs) and 1% antibacterial / antimicrobial (abam). cells for hydrogels were isolated by removing nutrient media, washing with dpbs, and adding trypsin to break up cell tissues and suspend in media for counting. suspended cells were counted three times with a hemocytometer and light microscope and averaged. after counting the average number of cells per volume cell suspensions were centrifuged for 5 minutes until cell pellets formed, separating cells from the media. liquid was suctioned from cell pellet in test tube followed by adding the two hydrogel solutions and mixing thoroughly directly before uv curing. cultured human dp147 fibroblasts, for hydrogel seeding, were trypsinized and counted to add to hydrogel solutions. the desired hydrogel mixtures were added to the cell pellet and vortexed to ensure thorough mixing. cell infused pegda solution was photopolymerized by uv light. under the aseptic conditions of a biological safety cabinet hood, custom - made molds, for networked and nonnetworked hydrogels, shown in figure 1 were placed in covered 35 (mm) tissue dishes for sterile curing. hydrogel cell solutions were pipetted into a mold and cured in layers under the lamp. excess liquid was removed under hood after each layer cured, and the following layers were added and photopolymerized. for networked hydrogels, caution was taken during removal of secured pins, not to damage the delicate structures. finished hydrogel samples were rinsed twice in dpbs to remove the noncured hydrogel liquid solution. next cured hydrogels were directly placed in new tissue culture dishes containing nutrient media and incubated at standard conditions of 37 degrees celsius and 5% co2. nutrient media were removed and replenished every three days during incubation period. a custom - made tension test setup was designed and fabricated for determining the tensile failure stresses of the specimens at room (laboratory) and physiological (nuaire nu-4750 incubator) temperatures. the complete test setup for conducting mechanical tests of pegda samples at room temperature is shown in figure 1-. the specimens were placed in the holders in an unstressed state. cover plates, same sizes as the holders, were placed above the specimen to restrict upward movement of the specimen. a precision actuator (newport lta - hl) was used in the setup to extend the specimens at a rate of 0.01 mm / sec until failure of specimen. force was measured using 1 lb load cell (futek lrm200) consistently throughout extension. displacement data were recorded simultaneously by a user written labview program 10.0 (national instruments) from the load cell and actuator, respectively. for conducting mechanical tests of pegda samples at physiological temperatures (37c), an electric connection was developed using the utility side access port of the incubator (nuaire nu-4750) to operate the actuator and load cell inside incubator (figure 1-), while doing data acquisition outside the incubator. a sample after failure is shown in figure 1-. stress was calculated by the magnitude of the force divided by the cross - sectional area (~5.5 mm ~7.5 mm) at the center of the gage length. gage length (~15.7 mm) for the specimen was determined as the distance between the holders at the initiation of positive load to the specimen. stress was calculated by the magnitude of the displacement after the initiation of load force divided by the gage length. viability of cells infused in pegda hydrogels was assessed using the invitrogen live / dead viability / cytotoxicity kit, for mammalian cells (molecular probes, invitrogen), and the fluorescent microscopy techniques. two probes, calcein am, and ethidium homodimer-1 (ethd-1) were used in the assay. two different bandpass filters were chosen for the individual probes resulting in two fluorescent images, red and green. a digital camera, attached to the uv microscope, and computer imaging software captured, saved, and merged the (10x) magnified images. the final third image, combined red and green, consisted of two merged saved images. viability was calculated from the merged images by counting the number of live green cells and dead red cells. the equation for hydrogel cell viability ((number of live cells) 100%/(number of live and dead cells)) was applied to each merged image. multiple assay samples were collected from each hydrogel during culture to show the percent change in cell viability over the hydrogel incubation period. after the curing, thin sample sections of the incubated hydrogel were collected for hydrogel cell viability at 7 days. figures 3(a) and 3(b) show the sectioning of cell imbedded hydrogel specimen samples, enclosed in glass tube housing for separated slices by scalpel, for analysis of hydrogel cell viability. thin even sections ranging from 0.5 to 1.0 (mm) were sampled from the incubated hydrogels with difficulty. acquiring desired sample sections with certain dimensions for the viability without damaging the hydrogel structure was a difficult task. this process was improved by designing a prototype device (figure 3(a)) to hold the hydrogel in the glass tube securely. once the glass was secured the built - in micrometer could be turned to move the hydrogel out of the glass housing in desired increments for samples. after sectioning the sampled hydrogel was rinsed with dpbs and placed back in normal incubation conditions. next, the viability assay live / dead solution was pipetted onto sample sections, in 35 (mm) tissue culture dishes. sampled sections were covered with aluminum foil and incubated for 75 minutes. after the incubation period, sections were rinsed twice with dpbs, to remove excess stain, and placed on microscope slides that emerged in dpbs to prevent dehydration and remove unwanted liquids. samples were assayed and viewed with two fluorescent microscope filters to produce an image for viability analysis. statistical analyses were performed using student 's t - test for the different groups of specimen using microsoft excel 2000 statistical analysis toolkit. figure 4 compares the stress - strain curves between pegda hydrogels at variable pi concentrations (0.2% and 0.6%) and test temperatures (23c and 37c). the stress - strain response of all specimens is characterized as long elastic response, followed by a negligible inelastic region and then stable descending response. this is reasonable since the conduit networks create three - dimensional voids on the samples. figure 5 and table 1 showed a significant difference of maximum failure stress between the various pegda samples fabricated in this study due to photoinitiator (pi) concentration, incubation time, and temperature applied to the specimen during testing. this result clearly shows the higher pi concentration significantly increased the mechanical integrality of the pegda gel. this result can be explained with the fact that increasing pi concentration of the samples increased the crosslink density of the polymer matrix due to a larger number of reactive diacrylate groups, which, in turn, increased the failure stress of experimental samples. there is a thermodynamic relationship between the modulus (slope of stress and strain curve) and the crosslink density of a given polymer, where the modulus is directly proportional to changes in the crosslink density. in each concentration, longer incubation (7 days versus 0 days) time and higher temperature (37c versus 23c) decreased the maximum failure stress of the pegda samples. this happens due to the fact that pegda undergoes small but significant degradation in vitro in pbs buffer solution as found by xin.. this study suggested that failure stress of pegda samples was highly dependent on the amount of the pi concentration and the methods by which it was processed. the failure stress of the nutrient conduit networked pegda samples is significantly lower than the natural liver tissue tensile stress of 232 kpa and breaking stress of 451 kpa. higher concentration of pi can be used to increase the failure stress of the pegda based tissue engineering scaffolds as liver implant materials. in our earlier studies, it was found that cell viability was not as high in pegda scaffolds. the cell viability was found higher for a concentration of pi of 0.2% than 0.6% for without network conduit pegda structure, whereas, in this study, it was found that higher concentration of photoinitiator contributes to toughening the hydrogels, increasing their failure strength. pegda tissue constructs had the strongest maximum failure strength when they are at room temperature compared to body temperature. also pegda hydrogel was found to lose strength when tested a week after production under incubation conductions. more network conduit channels for cells to be exposed to media nutrient flow should increase the cell viability. the samples may have been exposed to high heat causing damage in the curing process during the fabrication of the scaffold. variations on curing times, adding collagen, and cell seeding encapsulation prior to curing could improve the strength in addition as suggested to increase the cell viability. cells were successfully grown in uv crosslinked pegda hydrogel structures with significant viability differences in networked architecture compared to nonnetworked architecture pegda samples for both pegda gels having 0.2% and 0.6 wt% pi as shown in figure 6. statistical significant differences of cell viability between 0.2% and 0.6% pi concentration pegda samples were found after 7 days of incubation times for both channel and w / o channel pegda samples (p values < 0.05). viability results were not as high as expected when compared to 80% fibroblast viability at 14 days observed by other research, although network conduit channels exposing cells to nutrient flow demonstrated increased viability. the difference of cell viability results is due to the fact that pegda molecular weight and/or uv light intensity in the curing process was different from the previous authors. variations on curing times and uv intensity, lowering of pi concentration, adding nutrients to hydrogel solution, increasing the cell seeding density, and substituting higher molecular weights of pegda could improve the cell viability of pegda hydrogel. this study is limited to determine the effect of photoinitiator concentration, temperature, and incubation time on the mechanical and cell viability properties of network conduit channel pegda. the chemical properties (e.g., degree of polymerization and polymer spectra), surface characteristics (e.g., scanning electron microscope), and physical properties (e.g., swelling ratio, density) are beyond the scope of this study. the authors used same test setup and condition to prepare the different test samples. since the sample preparation for different groups of sample is identical, therefore, the study assumes the chemical, surface, and physical properties of different test samples groups are identical. in the future, microsize network conduit channel will be created on pegda by adding layered degradable fiber mat inside the pegda gel. the effect of photoinitiator concentration, temperature, and incubation time on the mechanical and cell viability properties on such produced pegda samples will be determined and will be compared with the macrosize network conduit channeled pegda samples results. tensile failure assessment of nutrient conduit networked pegda was conducted as a function of incubation time, test temperature, and photoinitiator concentration. this study concludes that the maximum failure stress of pegda can be increased significantly by the degree of photocrosslinking concentration, although significant decrease of failure stress occurs within 7 days of incubation time and at 37c incubation temperature. cell assay results demonstrated networked pegda hydrogels possessed increased viability compared to nonnetworked and decreased viability with increased photoinitiator concentrations. further research using higher molecular weights of pegda, improved designs for networked molds, a device for attaining thin uniform assay samples, and the infusion of nutrients in hydrogels could increase the cell viability during incubation. nutrient conduit networked pegda formed hydrogels can be tailored with adequate mechanical properties for various cell - based tissue engineering needs. | nutrient conduit networks can be introduced within the polyethylene glycol diacrylate (pegda) tissue construct to enable cells to survive in the scaffold. nutrient conduit networks can be created on pegda by macrochannel to nanochannel fabrication techniques. such networks can influence the mechanical and cell activities of pegda scaffold. there is no study conducted to evaluate the effect of nutrient conduit networks on the maximum tensile stress and cell activities of the tissue scaffold. the study aimed to explore the influence of the network architecture on the maximum tensile stress of pegda scaffold and compared with the nonnetworked pegda scaffold. our study found that there are 1.78 and 2.23 times decrease of maximum tensile stress due to the introduction of nutrient conduit networks to the pegda scaffold at 23c and 37c temperature conditions, respectively. this study also found statistically significant effect of network architecture, pi concentration, temperature, and wait time on the maximum failure stress of pegda samples (p value < 0.05). cell viability results demonstrated that networked pegda hydrogels possessed increased viability compared to nonnetworked and decreased viability with increased photoinitiator concentrations. the results of this study can be used for the design of pegda scaffold with macrosize nutrient conduit network channels. |
cardiovascular diseases (cvd) are the major cause of death globally, with myocardial infarction (mi) being one of the main causes of mortality. after mi, the damaged myocardium releases inflammatory signals that trigger a cascade of cellular processes in order to repair damaged tissue, leading to the formation of scar tissue and left ventricular (lv) dysfunction [2, 3 ]. our laboratory has characterised the therapeutic properties of the insulin - like growth factor-1 ea (igf-1ea) propeptide during wound healing / regeneration and pathological inflammation. the igf-1 gene is encoded in 70 kb of genomic dna distributed over six exons and five introns [4, 5 ]. use of alternative start codons generates proteins with n - terminal variability while different exon use at the 3 end generates multiple c - terminal extension - peptides, termed e - peptides. the most predominant is a 35-amino - acid - long e - peptide, termed ea, alternating with a far less abundant e - peptide termed eb or mechanogrowth factor (mgf) [6, 7 ]. the e - peptides control local igf-1 bioavailability by adhering strongly to the extracellular matrix (ecm), retaining the propeptides locally and preventing their release into the circulation. expressed as a cardiomyocyte - specific transgene or delivered locally to the mouse heart, igf-1ea improves functional recovery after cardiac injury [9, 10 ] ; however the underlying mechanisms are not fully understood. tissue restructuring after infarction involves the breakdown of the ecm by proteolytic enzymes, mainly the matrix metalloproteinases (mmp) mmp-2 and mmp-9, balanced by interaction with tissue inhibitors of metalloproteinases (timps). initially a temporary matrix is formed, primarily composed of collagen type iii (col i3), providing a scaffold for replacement cells and structural integrity to the heart, thereby reducing the risk of lv dilation and rupture [12, 13 ]. this is later replaced by collagen type i (col i1) which will constitute the permanent ecm. col i1 confers tensile strength and resistance to stretch and deformation, while col i3 confers compliance. their balance determines cardiac tissue stiffness with increased col i3 to col i1 ratio generating a more resilient left ventricle [13, 14 ]. innate immune cells recruited to the injured myocardium from the blood include neutrophils, monocytes, macrophages, and dendritic cells [1517 ]. they play a prominent role in remodelling, producing the mmps that break down the matrix, synthesising new ecm components, and activating fibroblasts to myofibroblasts which will later in the inflammatory process be the main producers of matrix proteins. the tissue microenvironment at a given time after mi influences the recruitment of immune cells as well as their phenotypic and functional properties. this is especially relevant for the macrophage population which undergoes a time - dependent shift between inflammatory and reparative functions [3, 18 ]. at early time points after injury, the majority of macrophages produce inflammatory cytokines and reactive oxygen species including interleukin- (il-) 1, il-12, mmp-9, and nitric oxide. these are termed inflammatory or m1-polarised macrophages, which express high levels of ly6c and function to recruit more inflammatory cells and phagocytose cellular debris and produce growth factors. in contrast to the inflammatory macrophages, these cells, many of which express cd206, are involved in the suppression of inflammation due to high production of il-10 and tgf- [3, 19 ] and assist in the progression from inflammation to repair. they also perform reparative roles promoting cell growth, angiogenesis, and remodelling of the ecm. additionally, different monocyte populations can be distinguished by ly6c in the mouse and may preferentially give rise to inflammatory versus reparative macrophages. we have previously shown that igf-1ea and its mature circulating form igf-1 can modulate immune responses and suppress pathological inflammation by inducing regulatory cytokines and immune cell types [20, 21 ]. in the heart, igf-1ea increased expression of il-10 after cardiotoxin injury and decreased levels of il-1 suggesting that a shift in immune cell populations may also occur in the heart. in the present study, we investigated whether there was a difference in the immune cell dynamics after mi in transgenic igf-1ea hearts and whether this had a carry - on effect on tissue remodelling. myocardial infarction by permanent left coronary artery occlusion was induced in wild - type (wt) and mhc.igf-1ea male mice which were 8 to 12 weeks old. the heart was exposed and the left coronary artery was ligated using an 8.0 mm non - absorbable suture (ethicon - johnson & johnson, usa) below the left atrium to produce an ischemic region of 2030% of the left ventricle area. the chest cavity and skin were sutured with 6.0 mm silk sutures (ethicon - johnson & johnson, usa). they were housed in individually ventilated cages in temperature - controlled facilities on a 12-hour light / dark cycle on standard diet. all mouse procedures were approved by the imperial college london ethical committee and were in accordance with national and international regulations (uk home office project license 70/7589). echocardiographic measurements were taken using a high - frequency ultrasound system vevo 770 (visualsonics, inc., canada) with a 30 mhz linear transducer and recorded images mice were anesthetised with 1 - 2% isofluorane, and the anesthetic flow rate was adjusted to maintain heart rate of approximately 450 50 beats per minute. furthermore, warmed ultrasound gel and a heating platform were used to maintain body temperature at 37 0.5c to minimise variation between mice. this analysis was performed at basal level, 1, 3, 4, 7, and 28 days after mi to evaluate left ventricle cardiac function, chamber dimensions, and infarct size. samples were fixed in paraformaldehyde (4% in pbs) for 48 h, washed in pbs, dehydrated, and embedded in paraffin wax. five - micron - thick sections were stained with celestine blue for 5 minutes, washed in tap water, and then incubated in haematoxylin for 5 minutes. slides were then incubated with acid fuchsin for 5 minutes, rinsed in distilled water, incubated in phosphomolybdic acid (1%), and then rinsed in distilled water before staining with methyl blue for 2 - 3 minutes. slides were dehydrated in ascending concentrations of ethanol, cleared in xylene, and mounted in dpx (vwr, uk). to analyse neutrophils, monocytes, macrophages, and dendritic cells, a single cell suspension was prepared from hearts before or at various time points after mi (days 1, 3, 5, 7, and 28 after operation). the hearts were mechanically dissociated using surgical scissors and subsequently treated with a 1x hank 's balanced salt solution (hbss) (invitrogen, usa), enzymatic dissociation buffer containing 0.1 mg / ml liberase th research grade (roche diagnostics, uk), 50 g / ml of dnasei (roche diagnostics, uk), 10 mm hepes (invitrogen, usa), and 30 mm taurine (sigma, uk) for 4 cycles of 10 min at 37c. after each 10 min incubation cycle, the cells were collected and filtered using a 70 m cell strainer (bd pharmingen, usa) and an equal volume of ice cold 1x hbss containing 10 mm hepes, 30 mm taurine (sigma, uk), and 20% fetal bovine serum (fbs) (ge healthcare, usa) was added to the enzyme dissociation buffer. the cells were pelleted at 320 g for 7 min at 4c and washed with the 1x hbss media solution containing 20% fbs as described above. under these isolation conditions, adult cardiomyocytes are predominately lysed, as the enzymatic dissociation buffer is toxic to these large, fragile cells. an aliquot of the cell suspension was used to quantify the cell concentration / ml using a beckman coulter vi - cell xr cell counter (beckman coulter, high wycombe, uk). isolated cells were incubated in a 1x dulbecco 's modified eagle medium (dmem) solution (gibco, life technologies, uk) containing 2% fbs and 10 mm hepes for 30 min, on ice, in the dark, with the following primary antibodies : cd11b - pe (bd biosciences ; catalogue 553311), f4 - 80-biotin (ebioscience ; catalogue 13 - 4801 - 85), cd45-apc - cy7 (biolegend ; catalogue 103116), cd206-percp - cy5.5 (biolegend ; catalogue 141716), ly6c - apc (ebioscience ; catalogue 17 - 5932 - 82), and ly6g - alexafluor700 (biolegend ; catalogue 127622). samples stained with biotin - labelled primary antibody were incubated with a streptavidin - pe - cy7 (ebioscience ; catalogue 25 - 4317 - 82) secondary antibody for 30 min, on ice, in the dark, with the 1x dmem media solution as mentioned above. the samples were washed and resuspended in fresh 1x dmem media solution with 1.5 m sytox blue (invitrogen. usa) dead cell stain and refiltered using 5 ml, 35 m filter cap tubes (bd falcon) just prior to sample acquisition. flow cytometric cell sorting was performed using a bd facsariai cell sorter (bd biosciences, oxford, uk) equipped with a 355 nm uv laser, a 405 nm violet laser, a 488 nm blue laser, a 561 nm yellow - green laser, and a 640 nm red laser. the antibody cocktail fluorescence minus one (fmo) controls were used as gating controls for analyses to distinguish positive from negative fluorescence signal (see supplementary 46 in supplementary material available online at http://dx.doi.org/10.1155/2015/484357). total leukocytes (cd45 +), neutrophils (cd45 +, cd11b+, f4/80, cd11c, and ly6g+), and monocytes (cd45 +, cd11b+, cd11c, ly6g, and f4/80) were analysed. macrophages were defined as cd45 +, cd11b+, cd11c, ly6g, and f4/80 + and further characterised on the basis of ly6c and cd206 expression (i.e., ly6c / cd206 (inflammatory macrophages) and ly6c / cd206 (reparative macrophages)). dendritic cells were defined as cd45 +, cd11b+, f4/80, cd11c+, and ly6g. flow jo software (version 9. samples from the infarct and remote (non - infarct) myocardium were placed in a 1.5 ml tube and homogenised in trizol (invitrogen, usa) reagent using a rotor - stator homogeniser (polytron pt 2500 e). rna was pelleted, air - dried, and resuspended in dnase / rnase free water and the yield quantified using nanodrop (thermo scientific, usa) at 260 nm. one microgram of rna was reverse - transcribed into cdna using the quantitect reverse transcription kit (qiagen, crawley, uk). usa) was performed on an abi 7900ht sequence detection system (applied biosystems, carlsbad, ca, usa). the probes used were igf-1ea (mm00710307_m1), il-10 (mm00439614_m1), il-1 (mm00434228_m1), ccl2 (mm00441242_m1), ccl5 (mm01302427_m1), tgf (mm03024053_m1), collagen i1 (mm00801666_g1), collagen i3 (mm01254476_m1), lox (mm00495386_m1), mmp2 (mm00439498_m1), mmp9 (mm00442991_m1), timp1 (mm00441818_m1), timp2 (mm004418225_m1), actin, alpha 1, skeletal muscle, acta, (mm00808218_g1), atrial natriuretic peptide, and anp (mm01255747_g1). gene expression was determined as fold induction over uninjured hearts after normalising to the reference gene, gapdh. two - tailed student 's t - test was performed to compare wt and mhc.igf-1ea mice at selected time points after mi. data were analysed with graphpad - prism 5.0 (graphpad software, inc., www.graphpad.com), and differences were considered statistically significant at p < 0.05. endogenous igf-1ea expression in wt hearts was measured 1, 3, 5, 7, and 28 days after mi in both ischemic and remote (nonischemic) regions. igf-1ea levels increased in both the ischemic and remote regions (figure 1(a)) with a substantially stronger induction in the ischemic area (8-fold over uninjured levels). these results indicate that, similar to other organs, endogenous igf1-ea expression is upregulated after cardiac tissue damage [23, 24 ]. in mhc.igf-1ea hearts, the baseline expression of transgenic igf-1ea was much higher than the expression of endogenous igf-1ea in wt hearts (average 286-fold) at all experimental time points (supplementary 1a and 1b). although far exceeding the expression of endogenous igf-1ea, the mhc.igf-1ea transgenic mice provide a suitable model of igf-1ea at supraphysiological concentrations of possible therapeutic relevance. previously our group showed an improvement in cardiac function in mhc.igf-1ea compared to wt mice one month after mi. to pinpoint the start of functional improvement we extended this analysis and performed echocardiography before and 1, 3, 5, 7, and 28 days after mi (supplementary table 1). one day following mi, both groups displayed a reduction in ejection fraction (ef) ; however, mhc.igf-1ea hearts showed significant improvement in left ventricular ef by day 7 after mi (figure 1(b) and supplementary table 1). left ventricular end systolic / diastolic volumes significantly increased after mi in wt hearts, indicating left ventricular dilation, while the mhc.igf-1ea hearts did not display any such signs (figure 1(c) and supplementary table 1). in support of the functional data, the expression of molecular markers for cardiac damage such as actin - alpha 1 skeletal muscle (acta) and atrial natriuretic peptide (anp) was significantly reduced in mhc.igf-1ea compared to wt mice 28 days after mi (figures 1(d) and 1(e)). the peak of endogenous igf-1ea in the ischemic region of wt mice by day 7, along with the improvement in cardiac function in mhc.igf-1ea as early as day 7, indicates that igf-1ea signalling at early time points is key for cardiac repair. three days after mi, wt and mhc.igf-1ea hearts displayed infarcts of equal size, quantified by masson 's trichrome staining (figure 2(a)). however, by 28 days after infarction, mhc.igf-1ea hearts exhibited smaller scar areas compared to wt (figures 2(b), 2(c) and 2(d)), as previously reported. this was observed as a reduction in scar length but increased scar thickness, consistent with reduced infarct expansion. as a molecular measurement of fibrosis, we quantified tgf- mrna levels which were significantly lower in mhc.igf-1ea than in wt mice after mi (figure 2(e)). we therefore measured expression of genes involved in ecm turnover and synthesis, mmp-2, mmp-9, timp-1, timp-2, col i1, col i3, and lysyl oxidase (lox) at 1, 3, 5, 7, and 28 days after mi. in wt hearts, mmp-9 was upregulated 3 days after mi, followed by mmp-2 at day 7 (figures 2(f) and 2(g)). interestingly, neither mmp-2 nor mmp-9 was significantly upregulated in mhc.igf-1ea hearts at any time point. inhibitors of matrix degradation, timp-1 and timp-2, had similar expression in wt and mhc.igf-1ea hearts at all time points except for day 7 when timp-2 was significantly upregulated in mhc.igf-1ea hearts compared to wt (figures 2(h) and 2(i)). taken together, this supports the idea of a net overall reduction in matrix breakdown, which may contribute to the reduced fibrosis observed in mhc.igf-1ea hearts. we next analysed the composition of the newly synthesised matrix by measuring mrna expression of the most abundant cardiac ecm collagen, col i1 and col i3, as well as the collagen cross - linker, lox, which increases matrix stiffness. upregulation of both collagen types was detected by day 3, peaking at day 7 (figures 2(j) and 2(k)). at this time point, wt hearts expressed significantly more of both collagen types than mhc.igf-1ea hearts. we also noted a difference in the ratio of the two collagen types ; mhc.igf-1ea hearts had a reduced col i1/col i3 ratio compared to wt, which was significant at days 3, 7, and 28 (figure 2(l)). at the peak of collagen upregulation, lox was also upregulated in wt hearts, yet this was not observed in mhc.igf-1ea hearts (figure 2(m)). these results indicate that igf-1ea overexpression reduces ecm turnover after mi and alters the composition of the matrix, with preferential expression of col i3 over col i1 and less cross - linking, which likely alters the mechanical properties of the fibrotic area. we therefore compared the inflammatory status of the mhc.igf-1ea and wt hearts, monitoring the production of key immune genes il-1 expression of the inflammatory cytokine il-1 was rapidly induced upon injury in wt hearts (66-fold over uninjured) yet was not upregulated as strongly in mhc.igf-1ea hearts (19-fold over uninjured ; figure 3(a)). similarly the strong upregulation of mcp-1 in wt hearts was not observed in mhc.igf-1ea hearts (figure 3(b)). in contrast to il-1 and mcp-1, this immunosuppressive cytokine was upregulated 3-fold higher in mhc.igf-1ea hearts compared to wt controls. although a second peak of il-10 mrna at 7 days was comparable in both wt and mhc.igf-1ea hearts 7 days after mi (figure 3(c)). ccl5, which is involved in the recruitment of ly6c monocytes [26, 27 ], was upregulated in mhc.igf-1ea hearts 7 days after mi (figure 3(d)). these data suggest an early bias towards a less inflammatory environment potentially modulating the recruitment of monocytes in mhc.igf-1ea hearts after myocardial infraction. to document accumulation of the main innate immune cell populations involved in cardiac inflammation after infarct, single cell suspensions were prepared from whole mouse hearts and analysed by flow cytometry. all cell populations described in this work were identified using the gating strategy shown in supplementary figures 36. in wt and mhc.igf-1ea hearts, the total number of infiltrating leukocytes (cd45 +) gradually increased after mi, both reaching comparable peak numbers at day 5 (figure 4(a)). however at the earlier 3-day time point, mhc.igf-1ea hearts contained 49% less leukocytes per milligram of tissue than wt hearts. in examining specific immune cell populations, this difference was partly explained by a 75% reduction in neutrophils (cd45 +, cd11b+, f4/80, cd11c, and ly6g+ ; 186 versus 46 cells / mg of tissue, figure 4(b)) and a 67% reduction in monocytes (cd45 +, cd11b+, cd11c, ly6g, and f4/80 ; 191 versus 62 cells / mg of heart, figure 4(c)) ; however it was mostly due to reduced presence of macrophages (cd45 +, cd11b+, cd11c, ly6g, and f4/80 + ; 58%, 2276 versus 949 cells / mg of heart, figure 4(f)). these data agree with the reduced mcp-1 expression observed in mhc.igf-1ea hearts (figure 3(b)), as it is the principal chemokine involved in monocyte recruitment [26, 28 ]. the ly6c surface marker distinguishes two different subsets of monocytes [2, 3 ]. analysis of the ly6c (cd45 +, cd11b+, cd11c, ly6g, f4/80, and ly6c) and ly6c (cd45 +, cd11b+, cd11c, ly6g, f4/80, and ly6c) populations revealed that the reduction of total monocyte numbers at day 3 was attributable to a 20% decrease of the ly6c population (102 versus 32 cells / mg of heart), whereas the ly6c population was not significantly different in wt compared to mhc.igf-1ea (figures 4(d) and 4(e)) hearts. to differentiate between inflammatory and reparative macrophage populations, we used the markers ly6c and cd206. for this work, only cells that were either ly6c+/cd206 or ly6c/cd206 + were analysed, although we noted a double positive cell population (i.e., cd206 +, ly6c+). however no changes were observed over time between wt and mhc.igf-1ea for the double positive population (supplementary 2). both ly6c+ inflammatory macrophage (cd45 +, cd11b+, cd11c, ly6g, f4/80 +, and ly6c+/cd206) and cd206 + reparative macrophage (cd45 +, cd11b+, cd11c, ly6g, f4/80 +, and ly6c/cd206 +) normalised cell numbers were reduced by 71% and 48%, respectively, in mhc.igf-1ea hearts at the day 3 time point (522 versus 153 and 596 versus 310 cells / mg of tissue of heart, resp. ; figures 4(g) and 4(h)) ; however this was significant only for the ly6c+ population. by day 7 after mi, macrophage dynamics changed and we observed a greater number of total macrophages in mhc.igf-1ea hearts compared to wt, which was mainly due to a 155% preferential increase in the cd206 + population (575 versus 1468 cells / mg heart, figure 4(h)). dendritic cells (cd45 +, cd11b+, f4/80, cd11c+, and ly6g) were increased by 48% in mhc.igf-1ea hearts compared to wt 5 days after mi (168 versus 248 cells / mg heart ; figure 4(i)). in summary, cardiac - restricted expression of an igf-1ea transgene limited the early accumulation of innate immune cells at day 3 after mi, with a bias towards the reduction of inflammatory myeloid populations rather than regenerative populations. this reduction corresponds with the lower expression of myeloid chemokines and the less inflammatory milieu observed in the mhc.igf-1ea hearts. previous work in our lab showed that local expression of igf-1ea promoted functional restoration after mi and observed reduced infarct expansion, thinning, and dilation of the left ventricular wall. we now demonstrate transcriptional modulation of key ecm remodelling genes in the igf-1ea hearts associated with tempering of the inflammatory myeloid cell response. increased mmp expression after injury has been implicated in contributing to scar destabilisation, as transgenic animal model knockouts of either mmp-2 or mmp-9 have been shown to attenuate lv dilation, rupture, and impairment of cardiac function [29, 30 ]. while mmp-2 and mmp-9 mrna expression levels were upregulated in injured wt hearts, this increase was not observed in mhc.igf-1ea hearts. in line with reduced matrix breakdown, mrna expression of the mmp inhibitor, timp-2, thus igf-1ea may prevent adverse cardiac remodelling, in part, by modulating transcription of mmps / timps. the production of new matrix components was also altered by the presence of the igf-1ea transgene with an overall reduction in collagen synthesis, confirmed in histological stains, and a bias towards expression of col i3 over col i1. in mi patients, turnover of cardiac extracellular matrix can be assessed by using circulating collagen peptides as blood biomarkers and high type i collagen is associated with adverse clinical outcome. a prospective multicentre study further showed that a low type iii / type i collagen ratio especially at 1 month after mi is predictive of detrimental left ventricular remodelling as well as cardiovascular deaths and hospitalisation cases for heart failure. changes in collagen ratios are known to affect the strength and tensile properties of the ecm. it would be interesting to determine whether these properties are modified in the mhc.igf-1ea hearts. igf-1ea could promote the changes in collagen deposition by directly acting on fibroblasts as it promotes both their proliferation and activation to myofibroblasts [34, 35 ]. alternatively, igf-1ea may influence accumulation and activation of immune cells present at the infarct which in turn regulate myofibroblast activation. indeed, we observed modulation of the inflammatory process in mhc.igf-1ea hearts, with less monocyte (ly6c) infiltration into the injured myocardium. this effect is associated with reduced expression of the monocyte chemoattractant mcp-1, although no significant changes were observed in the ly6c monocyte population in infarcted hearts, possibly due to ccl5 upregulation. it is interesting that while complete depletion of monocytes at any stage of repair leads to poor recovery, a more subtle modulation of the monocyte population in the mhc.igf-1ea hearts is associated with improved heart function. studies abrogating monocyte recruitment using a selective ccr2 inhibitor have resulted in reduced il-1, il-6, mcp-1, and tnf levels. igf-1ea influences macrophage polarisation in skeletal muscle and we similarly found reduced ly6c monocyte and ly6c+ macrophage normalised cell numbers while cd206 + macrophage numbers were increased by day 7, suggesting that igf-1ea promoted a quick progression to the reparative phase of repair by modulating macrophage phenotype. in the mhc.igf-1ea hearts we observed a decrease in mcp-1, which is the chemokine for ccr2, recently shown to distinguish infiltrating monocytes from the resident macrophage population which has a different embryological origin and expresses cd206 [3941 ]. it is therefore possible that, in our mhc.igf-1ea transgenic mouse model, igf-1ea reduces the infiltration of inflammatory ly6c monocytes by preventing upregulation of mcp-1 while still allowing for expansion of the resident macrophage population. in summary, we show that the mhc.igf-1ea mouse model can modulate several associated aspects of the cellular repair process after mi, including immune cell recruitment, cytokine expression, and matrix turnover. all of these changes occur within the first 7 days after infarct, at which time a functional improvement can already be measured in mhc.igf-1ea hearts compared to wt controls. these data provide new insights into the mechanism of igf-1ea and suggest that early immunomodulation is key to successful cardiac repair after injury. | strategies to limit damage and improve repair after myocardial infarct remain a major therapeutic goal in cardiology. our previous studies have shown that constitutive expression of a locally acting insulin - like growth factor-1 ea (igf-1ea) propeptide promotes functional restoration after cardiac injury associated with decreased scar formation. in the current study, we investigated the underlying molecular and cellular mechanisms behind the enhanced functional recovery. we observed improved cardiac function in mice overexpressing cardiac - specific igf-1ea as early as day 7 after myocardial infarction. analysis of gene transcription revealed that supplemental igf-1ea regulated expression of key metalloproteinases (mmp-2 and mmp-9), their inhibitors (timp-1 and timp-2), and collagen types (col 11 and col 13) in the first week after injury. infiltration of inflammatory cells, which direct the remodelling process, was also altered ; in particular there was a notable reduction in inflammatory ly6c+ monocytes at day 3 and an increase in anti - inflammatory cd206 + macrophages at day 7. taken together, these results indicate that the igf-1ea transgene shifts the balance of innate immune cell populations early after infarction, favouring a reduction in inflammatory myeloid cells. this correlates with reduced extracellular matrix remodelling and changes in collagen composition that may confer enhanced scar elasticity and improved cardiac function. |
the systemic inflammatory response syndrome can be self - limited or can progress to severe sepsis and septic shock. pathogens or their products, such as lipopolysaccharide (lps), play an important role in the process. upon lps / toll - like receptor 4 (tlr4) activation, immune cells can produce proinflammatory cytokines, such as tumor necrosis factor- (tnf-), interleukin-6 (il-6), and high mobility group box-1 (hmgb1), overwhelming production of which might result in immunological and inflammatory diseases. one of the most severe examples is septic shock. despite significant advances in the understanding of the molecular and cellular mechanisms of septic shock, it is still one of the most frequent and serious problems confronting clinicians in the managements. identification and characterization of the negative regulator of lps / tlr signaling attracts much attention in recent years. some intracellular negative regulators such as interleukin-1 receptor - associated kinase - m (irak - m), suppressor of cytokine signaling 1 (socs1), a20, ship1, mixed - lineage kinase 4 (mlk4), dok1/2, and rp105 have been identified to inhibit tlr4 signaling [48 ]. recently, the effects of both synthetic and endogenous cannabinoids upon the immune system have acquired a great interest. cannabinoid receptor 1 (cb1r) expresses primarily in central nervous system, which is associated with the psychoactive effects of cannabinoids. cannabinoid receptor 2 (cb2r) expresses primarily by immune cells, which mainly mediates anti - inflammatory actions [9, 10 ]. cb2r agonists have potential utility as anti - inflammatory drugs for the treatment of many disease conditions, such as multiple sclerosis, rheumatoid arthritis, and autoimmune uveoretinitis [1113 ]. these findings further make the cb2r an attractive therapeutic target in sepsis or septic shock. however, in the experimental sepsis, there are conflicting results regarding the effects of cb2r activation. it is reported that cb2r knockout mice following cecal - ligation - and - puncture- (clp-) induced sepsis had a higher mortality and cb2r agonist improved survival of wild - type mice. while in another study cannabinoid antagonist am 281 was reported to reduce mortality rate after clp in rats, while very recently, lehmann and his colleagues found that cb2r activation reduced intestinal leukocyte recruitment and inflammation in rat acute sepsis models. these controversial results leave this issue ambiguous. the specific contribution of cb2r to sepsis needs to be further explored. macrophages are activated early in response to immune challenge and are major players in both innate and adaptive immunity. adult patients dying of sepsis - induced multiple organ failure were found to have lymphocyte depletion and apoptosis. antigen - dependent t - cell activation influences survival in a murine model of sepsis. however, whether macrophages and lymphocytes are the cellular target of cb2r in sepsis and the corresponding molecular mechanisms still remains undefined. 1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-(2-(morpholin-4-yl)ethyl)-1h - indole (gw405833) is a selective cb2r agonist, which has been proved to elicit efficacious antihyperalgesic effects against neuropathic and inflammatory pain in rat model. whiteside and his colleagues using cb2r knockout mice proved that gw405833 produces an antihyperalgesic activity through cb2r. here, gw405833 was firstly used as a selective cb2r agonist to evaluate the role of cb2r in sepsis shock. the present study was designed to investigate the possibility for cb2r to be a new therapy target for the treatment of sepsis in vivo and further clarify the cellular and molecular mechanisms in vitro. gw405833, lps, and concanavalin a (cona) were purchased from sigma - aldrich (st. louis, mo, usa). dulbecco 's modified eagle medium (dmem), fetal calf serum (fcs), and phosphate - buffered saline (pbs) free of ca and mg were obtained from life technologies (gibco, ca, usa). elisa kits for mouse il-6 and tnf- were obtained from r&d systems (minneapolis, mn, usa). recombinant rabbit - polyclonal antibodies to nuclear factor - kappa b (nf-b) p65 and monoclonal antibodies to glyceraldehyde - phosphate dehydrogenase (gapdh) were purchased from santa cruz biotechnology (santa cruz, ca, usa). primary antibodies against extracellular signal - regulated kinase 1/2 (erk1/2), phospho - erk1/2, signal transducer and activator of transcription 3 (stat3), phospho - stat3, and inhibitor - kappa b alpha (ib) were purchased from cell signaling technology (boston, ma, usa). irdye - conjugated donkey anti - rabbit igg and goat anti - mouse igg were purchased from rockland immunochemicals inc. alexa fluor 565-conjugated donkey anti - rabbit igg was purchased from life technologies (carlsbad, ca, usa). male c57bl/6j mice (8 weeks old) were obtained from slrc laboratory animal co., ltd. cb2r gene knockout (cb2r) mice were purchased from jackson laboratory (bar harbor, maine, usa) and expanded under specific pathogen - free conditions in laboratory animal centre of second military medical university. all animals were fed standard mouse chow and water freely and maintained under constant conditions (temperature : 2025c ; humidity : 40%60% ; light / dark cycle : 12 h). all procedures were conducted in accordance with the university guideline and approved by ethical committee for animal care and the use of laboratory animals of second military medical university. male cb2r mice (weigh 1822 g) and the wild - type littermates (cb2r) were challenged with lps in saline (15 mg / kg) intraperitoneally. survival rate was recorded every 1 h after lps injection for 24 h. male c57bl/6j mice (weigh 1822 g) were injected with different doses of gw405833 intraperitoneally half an hour in advance and then challenged with lps (30 mg / kg). survival rate was recorded for 72 h. for cb2r and cb2r mice, serum was obtained 3 hours after lps (5 mg / kg) intraperitoneally injection. for c57bl/6j mice, serum was collected 6 hours after drug administration followed by lps (5 mg / kg) injection. serum level of il-6, tnf-, or hmgb1 was determined according to the manufacture 's instruction. for splenocytes, 500 l / well cell suspension were seeded in 24-well plate, and cona was added at a final concentration of 5 g / ml immediately followed by gw405833 treatment for 24 h. samples were centrifuged at 1,2000 g for 5 min to obtain the supernatants. for peritoneal macrophages, lps (1 ng / ml) with or without gw405833 were added into culture medium. mixed splenocytes were separated using lymphocytes separation medium (ez - sep mouse 1x) under the manufacturer 's introduction. briefly, isolated spleens were grinded with a syringe piston and passed through 200-mesh nylon net to obtain homogeneous cells suspending in the lymphocytes separation medium. after centrifugation (800 g for 30 min), the layer of lymph cells was transferred into another new 15 ml centrifuge tubes, washed with pbs for three times and resuspended in complete rpmi 1640 medium containing 10% fcs, 100 u / ml of penicillin, 100 g / ml streptomycin, and 100 g / ml amphotericin b. the cells were counted and seeded in 96- or 24-well plate at about 1 10/ml for subsequent procedure. injection of sterilized broth culture (1 ml) to cb2r or cb2r mice as described previously. the cells were washed twice with pbs, resuspended in dmem containing 10% fcs, and seeded at a density of 13 10/ml in 6-well or 24-well plates. two four hours later, the culture medium was replaced to remove the nonadherent cells and then incubated at 37c in a humidified 5% co2 atmosphere overnight for the subsequent procedures. splenocytes were seeded into 96-well plate at 1 10 cell / ml in 100 l complete rpmi containing cona (5 g / ml) with / without gw405833 and cultured for 24 h in a humidified, 5% co2 atmosphere at 37c. cell proliferation was measured as previously described using cck-8 purchased from dojindo laboratories (kumamoto, japan). briefly, 10 l of cck-8 reagent was added to each well 4 h in advance and the absorbance at 450 nm was determined by elisa plate reader (multiskan mk3, labsystems, finland). peritoneal macrophages were treated with vehicle or lps (1 ng / ml) in the presence or absence of gw405833 (10 m) for 30 min and then fixed and penetrated with 4% paraformaldehyde containing 0.3% triton x-100 for 15 min. cells were blocked with 5% bovine serum albumin for 30 min and incubated with primary anti - p65 antibody for 2 h at room temperature followed by incubating with secondary alexa fluor 565-conjugated donkey anti - rabbit igg for 1 h. at last cells were counterstained with 4,6-diamidino-2-phenylindole (dapi) for 3 min. the lysates were fractionated by tris - glycine buffered 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and incubated overnight at 4c with antibodies against stat3 (1/1,000 dilution), phospho - stat3 (1/2,000 dilution), erk1/2 (1/1,000 dilution), phospho - erk1/2 (1/2,000 dilution), ib (1/1,000 dilution), or gapdh (1/10,000 dilution). after washing, membranes were incubated with irdye - conjugated secondary antibodies (1/5,000 dilution) and then scanned using odyssey infrared imaging system (li - cor, usa). one - way analysis of variance (anova) with tukey 's post - test for multiple comparisons was used to compare gw405833 treatment groups with vehicle group for levels of inflammatory factors in serum and supernatants. to investigate the role of cb2r in endotoxic shock, cb2r and cb2r mice were challenged with a lethal dose of lps (15 mg / kg), and survival rate was observed. as shown in figure 1(a), cb2r mice had a significantly higher survival rate compared with mice lacking cb2r (86.7% versus 46.7%, resp.). consistent with this observation, serum levels of tnf-, il-6, and hmgbi were also significantly higher in the cb2r deficient mice as compared with the wt control group at 3 hours after 5 mg / kg of lps administration (figures 1(b)1(d)), indicating that the genetic deletion of cb2r resulted in increased susceptibility to infection. to evaluate the role of pharmacological activation of cb2r in sepsis, gw405833, a selective agonist of cb2r, was applied to the lethal dose of lps - treated mice. after injection of 30 mg / kg lps, mice began to die at 6 hours and no survivor was found at the end of 72 hours. although 3 mg / kg gw405833 shows a little but not significant improvement in survival rate, 10 mg / kg gw405833 obviously increased the survival rate of mice (26.7%, figure 1(e)). consistently, 10 mg / kg gw405833 markedly decreased the serum levels of tnf-, il-6, and hmgbi at 6 hours after 5 mg / kg lps injection (figures 1(f)1(h)). cona was used to stimulate t subtype of splenocytes to proliferate and differentiate. as shown in figure 2(a), gw405833 dose - dependently inhibits con - a - triggered splenocytes proliferation. the inhibition ratio of gw405833 (10 m) on cona - induced splenocytes proliferation was reduced from 40.6% to 24.2% (figure 2(b)), which suggested that cb2r partially mediated the inhibitory role of gw405833 in splenocytes proliferation. we also observed the effect of gw405833 on the splenocytes release of th1 cytokine tnf- and th2 cytokine il-6. gw405833 significantly decreased the capacity of cona - stimulated splenocytes to release tnf- and il-6 in a dose - dependent manner (figures 3(a) and 3(b)). however, this role was partially blocked when cb2r was knocked out. as shown in figure 3(c), the inhibition ratio of gw405833 (10 m) on cona - triggered il-6 release of splenocytes was reduced from 56.2% to 31.32%. the inhibitory action of gw405833 on tnf- production was reduced modestly but significantly by knockout of cb2r (32.38% to 24.34%, figure 3(d)), indicating that cb2r partially mediated the inhibitory effects of gw405833 on cytokines release of splenocytes. dose - dependently inhibits the production of il-6, tnf-, and hmgb1 in lps - triggered peritoneal macrophages from cb2r mice (figures 4(a)4(c)), with remarkable effect in il-6 and hmgb1 production but a weaker one in tnf- production. however, these inhibitions were completely abolished in peritoneal macrophages from cb2r mice (figures 4(d)4(f)). the stimulation of tlr4 by lps can activate the myd88-independent pathway resulting in the activation of nf-b and mapk cascades. the nf-b signaling pathway is critical in the pathogenesis of sepsis shock [27, 28 ]. briefly, upstream signals lead to phosphorylation and ubiquitin - dependent degradation of ib or ib and translocation of p65-related dimmers into the nucleus followed by subsequent gene transcription. there also are reports that erk1/2 [2931 ] and stat3 [32, 33 ] as upstream regulator participating in the activation of nf-b signaling pathway in sepsis. we therefore further explored the effect of cb2r activation on the lps / tlr4 signal pathway. classically, lps (1 ng / ml) triggered phosphorylation of stat3 and erk1/2, induced the degradation of ib, and promoted the translocation of nf-b p65 from the cytoplasm into the nucleus. study emphasis will be put on the drugs which can decrease the death in patients with sepsis. lps was a toxic component of the outer membrane of gram - negative bacteria and high dose of lps challenge in animal can induce a rapid response which resembles septic shock in clinical. here, we evaluated the role of cb2r in lps - induced acute experimental sepsis model. these studies use a genetic loss and pharmacological gain of cb2r to suggest that cb2r provide a protective role in response to sepsis, indicating that the cb2r represents a possible therapeutic target for the treatment of sepsis. previous studies showed that the cb2r appears to produce both immunoenhancing and immunosuppressing effects during sepsis depending upon the cell type examined and severity of injury inflicted [1417 ]. our studies presented here showed that cb2r activation produced immunosuppressing effects no matter in lps - triggered macrophages or cona - triggered splenocytes, which is consistent with a previous report that the inhibition of lps mediated no release by win55212 was mediated by cbr2 in murine macrophages. furthermore, no matter in the lethal dose of lps - induced septic shock or in the low dose of lps - induced endotoxemia, cb2r activation by gw405833 showed a protective role, which increased the survival rate and decreased the serum proinflammatory cytokines levels. it is widely reported that the endocannabinoid system is upregulated during sepsis, although a recent study reported that lps downregulated cb2r expression in peritoneal macrophages. in the sera of patients and animals suffering from sepsis, the concentrations of endogenous ligands of cbr (2-ag and anandamide) were elevated [37, 38 ]. several studies reported that endocannabinoids can modulate the release of proinflammatory mediators via cb2r - related pathways. particularly, 2-ag inhibits cytokine production in lps - treated murine macrophages and il-2 secretion in activated murine splenocytes. our studies found that deletion of cb2r resulted in being more vulnerable to death after lps challenge, indicating that endocannabinoids system might mediate anti - inflammatory actions through cb2r. proinflammatory cytokines such as tnf-, il-6, and hmgb1, as released from macrophages, further augment systemic inflammation [23, 43, 44 ]. cells of adaptive immune system, such as nave t cells, proliferate to generate effecter cells, which in turn liberate distinct cytokine profiles. our results demonstrated that cb2r is essential for the inhibitory role gw405833 in the production of proinflammatory cytokines in lps - triggered macrophages but just partially mediates role of gw405833 in splenocytes proliferation and cytokines release. reasons for this phenomenon may result from the expression of cb2r in macrophages being more abundant than that in t cells [9, 10 ], and also provide a clue that the possibility of the existence of other subtype cannabinoid receptors in t cells. so far, there are a few candidates reported previously, such as transient receptor potential vanilloid 1 (trpv1), orphan receptor gpr55 [47, 48 ], and marine cyanobacterial fatty acid amides, which might mediate the role of gw405833 besides cb2r in splenocytes. anyway, it can not be denied that cb2r is the predominant mediator for the function of gw405833 in sepsis, and compared to t cells, macrophages may contribute more in the cellular target of this process. the cb2r was reported to signal through a g - protein coupled receptor linked to a gi protein, which reduces intracellular camp levels by decreasing adenylyl cyclase activity. in the present study, we found that gw405833 could attenuate the lps - triggered phosphorylation of erk1/2 and stat3 and block lps - induced degradation of ib and translocation of p65 in peritoneal macrophages, suggesting possible crosstalk between tlr4 signal pathway and camp pathway. in addition, johammes tschop reported that in clp - treated cb2r mice, p38 mapk activation is decreased, while cb2r agonist increases p38 mapk activation in clp - treated cb2r mice. although data presented here show that the absence of cb2r is critical for sepsis, the signal pathway that mediates the protection of cb2r in sepsis is still not enough. taken together, our results show that cb2r plays an important protective role in acute experimental sepsis. cb2r agonist, gw405833, could decrease mortality and proinflammatory cytokines production in lps - challenged mice, which mainly targets t cells as well as macrophages via inhibiting lps - trigged signal pathway. these results also indicate that cb2r is a potential therapeutic target for the treatment of sepsis. | the systemic inflammatory response syndrome can be self - limited or can progress to severe sepsis and septic shock. despite significant advances in the understanding of the molecular and cellular mechanisms of septic shock, it is still one of the most frequent and serious problems confronting clinicians in the treatments. and the effects of cannabinoid receptor 2 (cb2r) on the sepsis still remain undefined. the present study was aimed to explore the role and mechanism of cb2r in acute sepsis model of mice. here, we found that mice were more vulnerable for lipopolysaccharide- (lps-) induced death and inflammation after cb2r deletion (cb2r/). cb2r agonist, gw405833, could significantly extend the survival rate and decrease serum proinflammatory cytokines in lps - treated mice. gw405833 dose - dependently inhibits proinflammatory cytokines release in splenocytes and peritoneal macrophages as well as splenocytes proliferation, and these effects were partly abolished in cb2r/ splenocytes but completely abolished in cb2r/ peritoneal macrophages. further studies showed that gw405833 inhibits lps - induced phosphorylation of erk1/2 and stat3 and blocks ib degradation and nf-b p65 nuclear translocation in macrophages. all data together showed that cb2r provides a protection and is a potential therapeutic target for the sepsis. |
colorectal cancer (crc) is a major medical and public health challenge that develops via a series of genetic and epigenetic changes. these alterations result in the transformation of normal mucosa to a premalignant polyp, and ultimately to a tumor (13). at least three different molecular pathways have been postulated as main players in crc : chromosomal instability (cin), microsatellite instability (msi) and cpg island methylator phenotype (cimp) (4). cin is the most common cause of genomic instability in crc and is responsible for approximately 6570% of sporadic crc (5). cin, or classic adenoma - to - carcinoma pathway, is characterized by an imbalance in chromosome number (aneuploidy), chromosomal genomic amplifications, and a high frequency of loh, which has been determined through a series of mutations in tumor suppressor genes, such as apc and p53, and oncogenes, such as kras (6). the most common genetic alterations are mutations in apc and kras genes (7). a very small percentage of chromosomal instability tumors are inherited and arise secondary to germline mutations in the apc gene (familial adenomatous polyposis ; less than 1% of crcs) or the mutyh gene (mutyh - associated polyposis ; 1% of crcs) (8). the second pathway, microsatellite instability (msi), is observed in 15% of crcs and also most of these tumors are sporadic, in which damaged dna mismatch repair (mmr) enzymes contribute to acquire copy number variants in repeat sequences of microsatellites. this mechanism is identified by a test for msi, which categorizes each tumor as msi - high (msi - h), msi - low (msi - l), or microsatellite stable, based on evaluating the size of multiple microsatellites (9). the last pathway is characterized by epigenetic alterations, resulting in changes in gene expression or function without changing the dna sequence of that particular gene (5). for example, methylation of cpg islands in distinct promoter sites can lead to the silencing of critical tumor suppressor genes. the resulting tumors are termed to have the cpg island methylator phenotype, or cimp (5). cimp tumors have been closely correlated with mutations in the braf oncogene (4, 1012). several papers have documented in serrated polyps (sessile serrated adenoma, traditional serrated adenoma, and hyperplastic polyp) a high frequency of braf mutations, and a low frequency of kras mutations, and in conventional adenomas, a low frequency of braf mutations and a high frequency of kras mutations. this observation provides further data to support the hypothesis that serrated polyps are precursor lesions of cimp+ crcs, which have a high frequency of braf mutations and a low frequency of kras mutations (13, 14). mutations in the kras and braf genes may be observed in ras / braf / mek / erk pathway (mapk signaling) (15). together, these observations result in a growing clinical importance of braf mutation in crc patients (16). the braf gene is composed of 18 exons, and the major common mutation is found in exon 15 at nucleotide position 1799, accounting for more than 90% of all mutations. this thymine to adenine transversion within codon 600 leads to substitution of valine by glutamic acid at the amino acid level. the other commonly mutated are exon 11, codon 468 and exon 15, codon 596 (17). recently, braf mutation testing has been utilized into routine clinical laboratories because of its beneficial operation in differentiating sporadic crc from hereditary in msi tumors, determination of clinical prognosis, and prediction of response to drug therapy (18). hereditary non - polyposis colorectal cancer (hnpcc), also called lynch syndrome, is characterized by msi and is the most common hereditary colon cancer, accounting for 2 - 6% of all colorectal cancer burden (19). available data has provided evidence that, family history alone is unreliable for identifying hnpcc cases. so tumor screening methods such as immunohistochemistry (ihc), genetic testing for mutations and microsatellite instability (msi) should be considered for identifying individuals with hnpcc. there are different diagrams for evaluation of ls using msi, ihc and genetic testing. every approach has benefits and drawbacks and may depend on provider preferences and institutional resources. of course each test will miss about 5 - 15 percent of all hnpcc cases. in this context, accumulative data show that the events leading to hnpcc is an inherited mutation of dna mismatch repair (mmr) gene, mainly mlh1 or msh2, which account for approximately 90% of the known mutations to date. subsequent, mutations in msh6 account for almost 10% of the cases and bottommost, mutations in pms2 have been also reported in a few cases (2022). relative to the panel of msi markers, 80 - 91% of mlh1 and msh2 mutations and 55 - 77% of msh6 and pms2 mutations will be detected by msi testing. however, msi in sporadic colorectal cancer is most often associated with hypermethylation of the mlh1 gene promoter (23). the braf v600e mutation is often correlated with this sporadic mlh1 promoter methylation and this mutation has not been detected in tumors that arise from individuals with a germline mutation in mmr. thus, it has been proposed that when ihc reveals absent mlh1, evaluated of braf mutation may avoid unnecessary further genetic testing for identifying tumors as a result of ls. braf mutation screening may indentify hnpcc in msi - h tumors, although it may not be applicable in the case of pms2 mutation carriers (2428). according to a study by loughrey., the braf v600e mutation has been reported as a germline mutation in 17 of 40 (42%) tumours showing loss of mlh1 protein expression by immunohistochemistry, but only in patients with sporadic crc. the authors recommend the incorporation of braf v600e mutation testing into the laboratory algorithm for pre - screening patients with suspected hnpcc, whose crcs show loss of expression of mlh1 (28) : if a braf v600e mutation is present, no further testing for hnpcc would be warranted. also another study showed that detection of the braf v600e mutation in a colorectal msi - h tumor rules out of the presence of a germline mutation in either the mlh1 or msh2 gene (25). the result of this study showed that the braf - v600e hotspot mutation was found in 40% (82/206) of the sporadic msi - h tumors analysed, but in none of the 111 tested hnpcc tumors. thus screening crc for braf v600e mutation is a reliable, fast, and low cost strategy that simplifies genetic testing for hnpcc (25). the concurrent use of msi testing, mmr protein ihc and braf mutation analysis would detect almost all mmr - deficient crc (29). a suggested algorithm incorporating somatic braf v600e mutation testing of tumor tissue into the investigation of suspected hnpcc. 2. algorithm for genetic testing in colorectal cancer, following the microsatellite instability (msi) route, when the samples are first tested for microsatellite instability, next for mutated braf, and finally for expression of mismatch repair enzymes by immunohistochemistry (ihc). algorithm for genetic testing in colorectal cancer when the samples are first tested for expression of mismatch repair enzymes by immunohistochemistry (ihc), next for mutated braf, and finally checking mmr in full sequence recently, papers demonstrated that information about braf mutation status, like for kras, is useful to help select efficacious therapy, especially when selecting systemic chemotherapy (30). in this context, many papers showed that, the patients harboring kras mutation do not respond to anti - epidermal growth factor receptor (egfr) therapies, but many patients with wild type kras also do not respond to this therapy (4). it has been suggested that other mutations, such as braf and pik3ca, have a critical role in those cases (31). braf testing is suggested in crcs that are negative for kras mutation when the patient is being pondered for anti - egfr therapy. colucci. in 2010 showed that the efficacy of anti - egfr mab is confined to patients with wild - type kras, whereas no mutations in any of the patients were detected in the braf gene (32). the efficacy of braf mutation on cetuximab or panitumumab response was also evaluated by cellular models of crc (30). according to an italian paper, 53% of the patients (110 out of 209) were considered as potentially non - responders to anti - egfr therapy because of kras, braf or pi3k mutations (33). it is now recognized that anti - egfr mab therapy should only be used in patients whose tumors express wild - type kras. furthermore, braf, pten, and pi3k are emerging as future potential predictive markers of response. however, further clinical studies are warranted to define the role of these biomarkers (34). an algorithm that includes testing for kras and braf mutation for the selection of patients for anti - egfr therapy is shown in fig. 3. algorithm for genetic testing in colorectal cancer when the patient is a candidate for anti - egfr therapy. the last potential use of braf mutation testing is for prognosis of crc (18). in an interesting paper, kalady. documented that braf mutation is associated with distinct clinical characteristics as mutant tumors were characterized by female sex, advanced age, proximal colon location, poor differentiation, msi, and importantly, worse clinical prognosis for the patient (4). many studies suggest that the association between braf mutation and crc survival may differ by some factors. documented that poor clinical prognosis associated with braf mutation was limited to cases diagnosed at ages < 50, and in another study samowitz. reported that this poor clinical outcome in crc were microsatellite stable and not msi - h cases (35, 36). fari a - sarasqueta. in 2010 reported that the v600e braf mutation confers a worse prognosis in stage ii and stage iii colon cancer patients independently of disease phase and therapy (37). in one interesting paper, teng. in 2012 showed that braf mutation is an independent prognostic biomarker in patients with liver metastases after metastasectomy (38). suggest that, the worst prognosis associated with braf mutation may in part be disannulled by a high cimp status and the good prognosis associated with msi - high status is partly weakened by a mutated braf status (39). there are few data on braf and kras mutation in iran (40, 41). in one valuable study, tumor samples from 182 iranian colorectal cancer patients (170 sporadic cases and 12 hnpcc cases) were screened for kras mutations at codons 12, 13 and 61 by sequencing analysis (40). kras mutations were observed in 68/182 (37.4%) cases, which is slightly lower as compared to the outcome of a study on an italian population (33). mutation frequencies were similar in hnpcc - associated, sporadic msi - h and sporadic microsatellite - stable (mss) tumors (40). another study was done by shemirani. and showed that probably the profile of kras mutations in tumors is not entirely compatible with the pattern of mutations in polyps (41). montazer haghighi. investigated 78 patients and determined with the pentaplex panel of mononucleotide repeats that 21 patients (26.9%) had tumors that were msi - h, 11 patients (14.1%) were msi - l and 46 patients (59%) were mss. there were no statistically significant different between msi - h, msi - l and mss regarding clinical features, pathology or family history of cancer in the patients (42). however, naghibalhosseini. reported high frequency of genes promoter methylation, but lack of braf mutation among 110 unselected of sporadic patients (43). it is conclude that studies are warranted to determine the prevalence of braf mutation in different site of iran to examine their impact on prognosis and response to targeted treatment. therefore, we suggest to do clinical studies on the correlation between braf mutation and ethnic background. the results will show if the prevalence of braf mutations in crc differs by ethnic background and also whether ethnic background has influence on clinical prognosis or response to drug treatment. in terms of prognosis, an emerging body of literature describes worse clinical prognosis and decreased response to therapy for patients with braf mutant tumors. braf mutation has been reported to have a worse prognosis in mss tumors, but there is little information regarding the effect of braf mutations on msi - h tumors. similar to patients with kras mutations, patients with metastatic tumors that are being considered for anti - egfr therapies should be tested for braf mutations as well. because kras is more frequently mutated than braf we propose studies in crc in iran on the clinical impact of kras and braf mutation, especially considering ethnic background. | knowledge about the clinical significance of v - raf murine sarcoma viral oncogene homolog b1 (braf) mutations in colorectal cancer (crc) is growing. braf encodes a protein kinase involved with intracellular signaling and cell division. the gene product is a downstream effector of kirsten ras 1(kras) within the ras / raf / mapk cellular signaling pathway. evidence suggests that braf mutations, like kras mutations, result in uncontrolled, non growth factor - dependent cellular proliferation. similar to the rationale that kras mutation precludes effective treatment with anti - egfr drugs. recently, braf mutation testing has been introduced into routine clinical laboratories because its significance has become clearer in terms of effect on pathogenesis of crc, utility in differentiating sporadic crc from lynch syndrome (ls), prognosis, and potential for predicting patient outcome in response to targeted drug therapy. in this review we describe the impact of braf mutations for these aspects. |
with the recent advancement of medical technologies and enhancement of patients awareness of medical services, the necessity for therapists to improve the quality of their services is consistently increasing1. however, the basic scholastic ability of those desiring to be admitted into training schools is decreasing every year, resulting in decreases in the quality of therapists, particularly their abilities, and this is regarded as a challenge to be addressed2. as of 2010, the majority of the members of the japanese physical therapy association were young therapists in their 20s or 30s, and this suggests the possibility of insufficient provision of education due to lack of supervising therapists, leading to a decrease in the quality of therapists. in 2005, the minimum pre - graduation education goal specified in the physical therapy education guidelines was changed from becoming able to perform basic physical therapy to becoming able to perform basic physical therapy with some advice and supervision4. furthermore, according to a survey to examine the status of clinical services provided by novice physical therapists immediately after graduation, such therapists independently implement their duties only on limited occasions, and need advice from supervisors3. based on this, physical therapists immediately after certification possibly lack sufficient clinical abilities, requiring postgraduate education, such as that provided by staff at facilities they belong to and participation in training seminars, as essential approaches. in other professional areas, for example, in physician education, the provision of 2 years of postgraduate clinical training has become compulsory, and the goals of such training have been determined5. postgraduate training goals and guidelines on supervision have also been established in nursing education6. based on the results of qualitative studies examining behavioral goals that must be met by physical therapists before graduation from training schools and those for clinical supervisors, clinical evaluation scales have been developed in the united states, and studies on them have been reported7, 8. the american physical therapy association has set professionalism - related goals as core values, and evaluation tables corresponding to them are available5, 9. in some reports, it has been recommended that final goals should be shown to promote self - directed learning based on adult learning theories10 ; for continuous provision of specialized education in particular, goal setting is necessary. however, goals for therapists after certification have not yet been determined. in addition, the decreases in the quality of therapists have recently been so serious that supervision is needed for novice therapists in extensive areas, such as basic attitudes, therapeutic skills, and clinical practice - related thoughts. the authors previously conducted interviews and questionnaires in therapists with experience supervising other therapists to examine the abilities necessary to independently implement duties as a therapist11 ; in these surveys, semistructured interviews were conducted to extract such abilities using a qualitative, inductive study method12, which were followed by 2 self - administered questionnaires, adopting the delphi technique, to determine highly needed abilities for independent implementation of duties. subsequently, based on the results, a clinical ability evaluation table (evaluation table) was developed, and its reliability was confirmed (table 1table 1.postgraduate evaluation table for physical and occupational therapistsevaluation itemsbasic attitudesdressing appropriately as a member of societyusing appropriate language as a member of societyadhering to appointed times and deadlinescomplying with rules in the workplaceunderstanding the role and duties of a therapist as a team memberadopting appropriate actions in consideration of the role of the therapist as a team membercontributing to the improvement of coordination as a team memberefficiently performing duties to complete them within working hoursappropriately understanding and considering confidentiality and personal information managementperforming appropriate infection control measures (including washing hands)performing appropriate equipment management (before and after use)performing treatment with a sense of responsibilityappropriately managing the his / her own physical condition and schedule and avoiding interference with his / her dutiesappropriately implementing reporting, communication, and consultation procedures (developing and expressing his / her own thoughts) at all timesidentifying problems that are difficult to independently addressconsulting about problems that are difficult to independently address with appropriate persons in appropriate situationsseriously accepting and addressing issues noted by a supervisor or his / her own failuresdeveloping positive attitudes and making efforts to achieve knowledge and skillsperforming treatment and duties based on learned outcomes and experiencetherapeutic skillsadopting appropriate measures, such as life - saving techniques, to manage sudden changes in patients conditionsappropriately dealing with individual patients in consideration of their symptomsusing appropriate verbal or nonverbal communication methods for individual patientsshowing empathy when communicating with patients in consideration of their psychological conditionsappropriately listening to patients and their families to clarify their needshaving the medical knowledge necessary for a therapistselecting appropriate evaluation items for individual patientsperforming vital (blood pressure and heart rate) measurements, according to each situationappropriately (and accurately and efficiently) conducting medical interviews with patientsappropriately (and accurately and efficiently) examining reflexesappropriately (and accurately and efficiently) conducting orthopedic examinationappropriately (and accurately and efficiently) evaluating painappropriately (and accurately and efficiently) evaluating coordinationappropriately (and accurately and efficiently) evaluating muscle toneappropriately (and accurately and efficiently) measuring range of motionappropriately (and accurately and efficiently) evaluating muscle strengthappropriately (and accurately and efficiently) conducting sensory examinationappropriately (and accurately and efficiently) performing morphometryappropriately (and accurately and efficiently) evaluating the motor function of patients with paralysis (using the sias and brunnstrom stage test)appropriately (and accurately and efficiently) evaluating activities of daily living (using instruments, such as the fim and barthel index)clinical practice - related thoughtsclarifying individual patients general characteristicsidentifying individual patients possible risks based on the results of examinationlogically examining the causes of problems in movements or activities of daily livingdeveloping treatment programs to achieve goals (also referring to literature)safely implementing treatment programssafely handling treatment devicesappropriately managing risks related to medical accidents, such as tube removal and bleedingappropriately managing risks related to fallsproviding appropriate range - of - motion trainingproviding appropriate muscle - strengthening trainingproviding appropriate assistance and guidance for the maintenance of sitting positionsproviding appropriate assistance and guidance for the maintenance of standing positionsproviding appropriate assistance and guidance for standing from a seatproviding appropriate assistance and guidance for transferproviding appropriate assistance and guidance for gait trainingcontinuously evaluating (and observing) patients in the progress of treatmentratings. 4=being able to accurately understand and adopt appropriate actions without supervision. 3=being able to accurately understand and adopt appropriate actions under monitoring and supervision. 2=being able to understand and adopt appropriate actions to a certain extent under monitoring and supervision. this paper reports the results of a study that used this evaluation table to examine the contents of supervision needed by novice therapists for ability development. 2=being able to understand and adopt appropriate actions to a certain extent under monitoring and supervision. 1=professionally incompetent. therapists working in hospitals were asked to evaluate the clinical abilities (basic attitudes, therapeutic skills, and clinical practice - related thoughts) of those under their supervision, using the evaluation table, which had been developed with 55 items extracted as abilities necessary for therapists to independently implement their duties. on evaluation, a 5-point rating method was adopted (total score : 0 to 220) : 0=being inappropriate for implementation ; 1=being unable to understand or adopt appropriate actions even under monitoring or supervision ; 2=being able to understand and adopt appropriate actions to a certain extent under monitoring and supervision ; 3=being able to accurately understand and adopt appropriate actions under monitoring and supervision ; and 4=being able to accurately understand and adopt appropriate actions without supervision. therapists who belonged to 4 medical facilities, which were located in the tokai area, and consented to cooperate with the study were studied. one and 4 of the study facilities specialized in acute and acute to post - acute care (including outpatient and visiting rehabilitation services), respectively. the inclusion criteria were as follows : (1) therapists targeted for evaluation : those with experience of less than 3 years after certification ; and (2) supervisors : those with 3 or more years of experience who were supervising targeted therapists daily. as ethical considerations, the participants were provided with oral or written explanations regarding this study, and their participation in it was regarded as their consent. for statistical analysis, predictive analytics software (pasw) statistics 18.0 was used. scores for items related to basic attitudes (10 ; 76 points), therapeutic skills (20 ; 80), and clinical practice - related thoughts (16 ; 64) were converted to percentage. to compare the means of experience - based groups (1 year group, 29 therapists with 01 year of clinical experience ; 2 years group : 21 therapists with 12 years of clinical experience ; and 3 years group : 9 therapists with 23 years of clinical experience), one - way analysis of variance and tukey - kramer multiple comparisons were performed. this study was conducted with the approval of the ethics committee of fujita health university (13 - 254). on comparison of scores for the 55 evaluation items focusing on clinical experience, there were no significant differences in those for basic attitudes - related items among the 3 groups, while those for therapeutic skills - related items markedly varied between the 1 and 3 years groups. in scores for clinical practice - related thoughts - related items, significant differences were observed between the 1 and 3 years groups and between the 2 and 3 years groups. on comparison of scores among the items in each group, there were marked differences between those related to basic attitudes and clinical practice - related thoughts in the 1 and 2 years groups. on comparison of scores among basic attitudes - related items, significant differences were observed between appropriately implementing reporting, communication, and consultation procedures and identifying problems that are difficult to independently address (table 2table 2.comparison of scores for postgraduate evaluation items (related to basic attitudes, therapeutic skills, and clinical practice - related thoughts) among experience - based groups1 yearn=292 yearsn=213 yearsn=91 year vs. 2 years1 year vs. 3 years2 years vs. 3 yearsbasic attitudes87.711.391.77.291.57.9therapeutic skills81.515.389.59.093.96.7clinical practice - related thoughts74.813.282.313.794.44.3basic attitudes vs therapeutic skillsbasic attitudes vs clinical practice - related thoughtstherapeutic skills vs clinical practice - related thoughtsp<0.05. - related items, there were marked differences among adopting appropriate measures to manage sudden changes in patients conditions, appropriately dealing with individual patients in consideration of their symptoms, appropriately listening to patients and their families to clarify their needs, having medical knowledge necessary for a therapist, and appropriately evaluating muscle tone. on comparison of scores among clinical practice - related thoughts - related items, significant differences were observed among clarifying individual patients general characteristics, identifying individual patients possible risks, logically examining the causes of problems in movements or activities of daily living, developing treatment programs to achieve goals, appropriately managing risks related to medical accidents, providing appropriate range - of - motion training, providing appropriate assistance and guidance for the maintenance of standing positions, standing from a seat, transfer, and gait training, and continuously evaluating patients in the progress of treatment(table 3table 3.comparison of scores among the post - graduate evaluation items (related to basic attitudes, therapeutic skills, and clinical practice - related thoughts) in each groupevaluation items1 yearn=292 yearsn=213 yearsn=91 year vs. 2 years1 year vs. 3 years2 years vs. 3 yearsbasic attitudesdressing appropriately as a member of society96.611.098.85.591.712.5using appropriate language as a member of society94.014.498.85.597.28.3adhering to appointed times and deadlines92.216.596.412.091.717.7complying with rules in the workplace94.014.498.85.591.712.5understanding the role and duties of a therapist as a team member86.217.190.516.791.712.5adopting appropriate actions in consideration of the role of the therapist as a team member85.319.589.312.791.712.5contributing to the improvement of coordination as a team member88.815.891.714.491.712.5efficiently performing duties to complete them within working hours81.922.182.122.686.118.2appropriately understanding and considering confidentiality and personal information management94.812.396.412.097.28.3performing appropriate infection control measures (including washing hands)98.36.496.49.0100.00.0performing appropriate equipment management (before and after use)96.68.8100.00.097.28.3performing treatment with a sense of responsibility86.218.490.516.797.28.3appropriately managing the his / her own physical condition and schedule and avoiding interference with his / her duties93.114.896.49.094.411.0appropriately implementing reporting, communication, and consultation procedures (developing and expressing his / her own thoughts) at all times75.920.689.316.983.317.7identifying problems that are difficult to independently address70.721.284.516.786.113.2consulting about problems that are difficult to independently address with appropriate persons in appropriate situations76.724.082.117.986.113.2seriously accepting and addressing issues noted by a supervisor or his / her own failures88.818.494.010.991.717.7developing positive attitudes and making efforts to achieve knowledge and skills84.519.483.319.983.321.7performing treatment and duties based on learned outcomes and experience82.820.283.316.588.918.2therapeutic skillsadopting appropriate measures, such as life - saving techniques, to manage sudden changes in patients conditions60.315.777.420.883.317.7appropriately dealing with individual patients in consideration of their symptoms68.118.875.019.491.712.5using appropriate verbal or nonverbal communication methods for individual patients80.219.386.917.091.712.5showing empathy when communicating with patients in consideration of their psychological conditions81.019.784.516.786.118.2appropriately listening to patients and their families to clarify their needs75.020.084.518.594.411.0having the medical knowledge necessary for a therapist64.715.776.218.588.913.2selecting appropriate evaluation items for individual patients78.420.884.512.488.913.2performing vital (blood pressure and heart rate) measurements, according to each situation94.012.897.67.594.411.0appropriately (and accurately and efficiently) conducting medical interviews with patients82.819.088.117.097.28.3appropriately (and accurately and efficiently) examining reflexes91.418.097.67.597.28.3appropriately (and accurately and efficiently) conducting orthopedic examination85.319.589.318.791.712.5appropriately (and accurately and efficiently) evaluating pain81.022.886.917.091.712.5appropriately (and accurately and efficiently) evaluating coordination84.520.595.210.194.411.0appropriately (and accurately and efficiently) evaluating muscle tone81.922.194.010.9100.00.0appropriately (and accurately and efficiently) measuring range of motion88.818.497.67.5100.00.0appropriately (and accurately and efficiently) evaluating muscle strength87.120.797.67.5100.00.0appropriately (and accurately and efficiently) conducting sensory examination87.920.796.49.097.28.3appropriately (and accurately and efficiently) performing morphometry87.920.796.49.097.28.3appropriately (and accurately and efficiently) evaluating the motor function of patients with paralysis (using the sias and brunnstrom stage test)86.219.692.914.097.28.3appropriately (and accurately and efficiently) evaluating activities of daily living (using instruments, such as the fim and barthel index)82.821.291.712.194.411.0clinical practice - related thoughtsclarifying individual patients general characteristics75.921.681.017.594.411.0identifying individual patients possible risks based on the results of examination69.017.276.216.791.712.5logically examining the causes of problems in movements or activities of daily living63.815.872.617.586.113.2developing treatment programs to achieve goals (also referring to literature)64.712.579.818.791.712.5safely implementing treatment programs83.615.385.716.994.411.0safely handling treatment devices89.714.292.914.097.28.3appropriately managing risks related to medical accidents, such as tube removal and bleeding77.620.486.918.797.28.3appropriately managing risks related to falls78.420.886.917.094.411.0providing appropriate range - of - motion training80.219.391.714.4100.00.0providing appropriate muscle - strengthening training86.220.790.514.7100.00.0providing appropriate assistance and guidance for the maintenance of sitting positions80.222.585.716.997.28.3providing appropriate assistance and guidance for the maintenance of standing positions73.320.083.318.397.28.3providing appropriate assistance and guidance for standing from a seat70.716.578.618.297.28.3providing appropriate assistance and guidance for transfer68.113.278.616.491.712.5providing appropriate assistance and guidance for gait training62.114.471.418.288.913.2continuously evaluating (and observing) patients in the progress of treatment73.321.175.017.791.712.5 criteria : ratings. 4=being able to accurately understand and adopt appropriate actions without supervision. 2=being able to understand and adopt appropriate actions to a certain extent under monitoring and supervision. criteria : ratings. 4=being able to accurately understand and adopt appropriate actions without supervision. 3=being able to accurately understand and adopt appropriate actions under monitoring and supervision. 2=being able to understand and adopt appropriate actions to a certain extent under monitoring and supervision. 1=professionally incompetent. in japan s super - aging society, the numbers of certified physical and occupational therapists are rapidly increasing to meet increased social demands for rehabilitation. under these circumstances, it is necessary to give more importance to postgraduate education in workplaces, in addition to improving school education systems. school education should enable students to sufficiently learn about items necessary to achieve more specialized knowledge and skills after graduation rather than about the application of skills that should be focused on in postgraduate education. in short, systems for teaching staff, clinical supervisors, and therapists in charge of postgraduate education to comprehensively provide standardized skill education before and after graduation13,14,15,16,17 ; it is particularly important to set education goals. in education for those specializing in medicine and medical services, education goals are frequently classified into 3 domains, cognitive, emotional, and psychomotor, based on taxonomy for the setting of educational goals18. therapists with experience in supervising other staff members need to develop not only knowledge to simply implement therapy - related duties, perspectives on clinical practice, and techniques to conduct therapy evaluation but also appropriate attitudes as members of society, as well as a broad range of abilities, such as those related to self - management and self - education for continuous improvement. in the present study, novice therapists basic attitudes, therapeutic skills, and clinical practice - related thoughts were evaluated using an original clinical ability evaluation table to compare their clinical abilities. the results revealed that the longer the clinical experience, the higher the scores for these items. for example, those for clinical practice - related thoughts were significantly higher in the 3 years group compared with the 1 and 2 years groups, confirming the influence of differences in clinical experience on therapists specialties. regarding the definition of each evaluation domain, basic attitudes covered abilities needed to work in clinical environments or as a member of society, self - management abilities required when working for a system, and self - education abilities required to continuously improve skills as a specialist, as mentioned in the japanese physical therapy association s ethics code19. therapeutic skills were regarded as information collection skills, such as those used to communicate with patients and in evaluation techniques necessary for therapists. similarly, clinical practice - related thoughts were defined as processes such as integrating patient information obtained through therapy evaluation and examination results to identify problems, develop treatment programs, conduct reevaluation, and revise treatment programs. on comparison of the evaluation items among the experience - based groups, the 3 years group showed significantly higher scores than the 1 and 2 years groups for therapeutic skills- and clinical practice - related thoughts - related items, both of which represented contents indicating improvement through clinical experience. the items for which the 1 year group showed markedly lower scores compared with the remaining groups were adopting appropriate measures to manage sudden changes in patients conditions, having medical knowledge necessary for a therapist, and developing treatment programs to achieve goals ; these items covered the contents of supervision needed by a relatively large number of novices. on the other hand, the proportion of those showing high scores for some items, such as appropriately evaluating muscle tone, providing appropriate range - of - motion training, and providing appropriate assistance and guidance for transfer, which represent the contents of training for students that are, repeatedly provided at training schools or facilities, was 90% or higher in the 3 years group, while it was limited to 70% or lower in the 1 year group, indicating a high likelihood that basic therapeutic skills are mastered after employment. this may be explained by the influence of improvements in technical skills, such as utilizing shifts in the center of gravity, rather than the arm strength and bringing patients potential abilities out to the fullest as transfer techniques, through experience. based on these results, which demonstrate that novice therapists need supervision in diverse areas from basic abilities to those requiring accumulated experience, it may be appropriate for educators to provide technical education regarding skills that, which are achievable for students in the early stages in consideration of applied movements. also, education for novices should be provided with importance attached to abilities influenced by clinical experience. on comparison of scores among the evaluation items in each group, scores for basic attitudes - related items were significantly higher than those for therapeutic skills - related items in the 1 and 2 years groups., the process of reasoning from the recognition and interpretation of information, development and revision of diverse hypotheses, and decision - making to reevaluation after intervention is defined20. this may generally correspond to clinical practice - related thoughts extracted in the present study, indicating cognitive processes necessary for therapists to provide intervention for targets. compared to with basic attitudes, clinical practice - related thoughts consist of more complex processes, and therefore, it may be necessary to establish educational systems that enable novice therapists with 1 or 2 years of experience to develop appropriate clinical practice - related thoughts. regarding study limitations, this study did not include mid - career therapists with four or more years of experience, so the extracted contents of supervision needed in clinical environments were limited to novices. further studies should be conducted with a broader range of therapists to examine abilities necessary for managers and clinical supervisors, with the aim of nurturing them. | [purpose ] this study examined the contents of supervision needed by novice therapists to develop clinical abilities, focusing on their clinical experience and using an original evaluation table. [subjects and methods ] an evaluation of clinical abilities basic attitudes, therapeutic skills, and clinical practice - related thoughts was conducted in 29, 21, and 9 therapists with clinical experience of 01 (1 year group), 12 (2 years group), and 23 (3 years group) years, respectively. [results ] there were no significant differences among the 3 groups in basic attitudes. therapeutic skills markedly varied between the 1 and 3 years groups. in clinical practice - related thoughts, significant differences were observed between the 1 and 3 years groups and between the 2 and 3 years groups. [conclusion ] it may be appropriate for educators to provide technical education regarding skills that are achievable for students in the early stages in consideration of applied movements. also, education for novices should be provided with importance attached to abilities influenced by clinical experience. |
mntrier disease (md) is rare that is involved in both the small bowel and entire colon. we describe a case of a 76-year - old male patient whose clinical presentations include intermittent diarrhea, epigastric pain, nausea, vomiting, asitia, and weight loss. an endoscopy was performed showing a large number of irregular forms and different sizes of polypoid lesions in the gastrointestinal tract, which is rare for md. herein, this case was diagnosed as md, mainly dependent on endoscopic evaluation, typical clinical symptoms, and histopathological examination of biopsy. as this patient was also infected with helicobacter pylori, the eradication of h pylori was administered. meanwhile, a high - protein diet was enjoined, the aforementioned patient 's symptoms were alleviated evidently after 1 month. although the etiology of md remained undetermined, we showed that eradication of h pylori in this case might contribute to the disease remission. mntrier disease (md), a hypoproteinemic hypertrophic gastropathy, presents typical clinical symptoms including nausea, vomiting, diarrhea, epigastric pain, weight loss, malnutrition, fatigue, and peripheral edema due to hypoalbuminemia. although the definite etiology of md in adults still remains unknown, it often coexists with some specific infections, such as cytomegalovirus, helicobacter pylori, herpes virus, human immunodeficiency virus, mycoplasma as well as nonspecific ulcerative colitis. nevertheless, md patients can show no remission when specific therapies are employed in these disorders. in this case, we showed that md was involved in the stomach, small intestine, and entire colon, which rarely happened. this study was approved by the ethics committee of tongji hospital, tongji medical college, huazhong university of science and technology. a 76-year - old male patient was admitted to tongji hospital (wuhan, hubei province, china). he presented with intermittent diarrhea, epigastric pain, nausea, vomiting, asitia, and weight loss. g / l), which the normal range of the index is from 35 to 52 previous medical history included hypertension ; laparoscopic cholecystectomy, common bile duct exploration, and t tube drainage ; endoscopic retrograde cholangiopancreatography for the obstruction of the common bile duct. the tumor markers test including alpha - fetoprotein, carcinoembryonic antigen, cancer antigen125, and cancer antigen19 - 9 was negative. the gastroscopy results showed protrusive lesions in the body and the fundus of the stomach (fig. 1c), and it was the same to the whole small intestine that identified by capsule endoscopy. the colonoscopy was also performed because of the diarrhea, revealing diffuse elevations of various sizes, which was obvious in the cecum and ascending colon (fig. (a) view showing the gastric body ; (b) view of the pyloric antrum ; (c) picture of the duodenum ; (d) picture exhibiting the ascending colon ; and (e) histology of specimen from the gastric antrum acquired by emr (hematoxylin and eosin staining). a histology of biopsy specimens acquired by endoscopic mucosal resection showed marked foveolar epithelial hyperplasia and no epithelial atypia, elongated foveolar epithelium, interstitial edema, and cystic dilation of foveolar glands without increase of inflammatory cells infiltration (fig. therefore, eradication therapy for h pylori was administered, which was composed of pantoprazole (standard dose, b.i.d.), amoxicillin (1000 mg, b.i.d.), and clarithromycin (500 mg, b.i.d.) for 10 days after 1 month, the above patient 's symptoms were alleviated evidently, the hypoalbuminemia had been improved (32.7 g / ml), and his body weight also increased. the telephone follow - up was conducted 3 months afterward, and the patient still did not feel discomfortable. md is an uncommon condition characterized by protein - losing and hypertrophic gastropathy. in adults (mean age at diagnosis is 55), the disorder of md usually presents with an insidious onset and a progressive clinical course. the definitive etiology of md is still unknown, but h pylori infection is believed to have some relation with it. md should be suspected in cases of upper gastrointestinal tract symptoms and hypertrophied gastric mucosa with or without h pylori or hypoalbuminemia. before md is diagnosed, some other hypertrophic gastropathies, including lymphoma, polyposis, and suspected gastric malignancies, should discriminate from it. further, it must be based on a comprehensive collection of data concerning clinical, endoscopic, laboratory, and histopathological findings. md is generally shown by huge gastric mucosal folds in the body and fundus, with antral sparing. however, there are some reports showing that md affects the entire stomach, the duodenum, and the small bowel. herein, we describe a patient who bears h pylori infection and md, and this disorder is involved not only in stomach, but also in both the small bowel and total colon. however, as for the colon, the lesion is especially obvious in the cecum and ascending colon. h pylori eradication treatment was initiated and provided therapeutic benefit to this patient. above all, in order to establish the correct diagnosis of md, the histological findings, endoscopic and clinical features all should be necessary. since the knowledge regarding its pathogenesis and effective therapeutic management has been lack so far, md should be paid more attention to with particular treatment and ruled out of other similar diseases. | abstractintroduction : mntrier disease (md) is rare that is involved in both the small bowel and entire colon.the main symptoms and the important clinical findings : we describe a case of a 76-year - old male patient whose clinical presentations include intermittent diarrhea, epigastric pain, nausea, vomiting, asitia, and weight loss. an endoscopy was performed showing a large number of irregular forms and different sizes of polypoid lesions in the gastrointestinal tract, which is rare for md.the main diagnoses, therapeutics interventions, and outcomes : herein, this case was diagnosed as md, mainly dependent on endoscopic evaluation, typical clinical symptoms, and histopathological examination of biopsy. as this patient was also infected with helicobacter pylori, the eradication of h pylori was administered. meanwhile, a high - protein diet was enjoined, the aforementioned patient 's symptoms were alleviated evidently after 1 month.conclusion:although the etiology of md remained undetermined, we showed that eradication of h pylori in this case might contribute to the disease remission. this study enlarged the present understanding of md. |
mucopolysaccharidosis type vi (mps vi) is an autosomal recessive, hereditary lysosomal storage disorder (lsd) caused by a deficiency in arylsulfatase b (ars - b ; n - acetylgalactosamine-4-sulfatase) enzyme activity. this condition, also known as maroteaux lamy syndrome, results in the progressive accumulation of glycosaminoglycans (gags), especially dermatan and chondroitin sulfates in lysosomes that cause mild to severe symptoms including skeletal and joint deformities, enlarged spleen and liver, growth deceleration and death. swift and early confirmatory diagnosis is crucial to prevent serious irreversible damage, particularly since enzyme replacement therapy with galsulfase (naglazyme) has shown significant retardation of symptoms,,,. existing methodology for diagnosis includes analysis of lysosomal gags in urine and enzyme activity measurements in blood leukocytes and fibroblast cultures, followed by mutational analysis,. we have previously developed fluorometric microtiter plate - based bench top dbs assays for mps i and ii,. these assays are robust, accurate and have a rapid turn - around time, thus reducing the time for diagnosis. here we describe the development and validation of a similar fluorometric dbs assay to identify mps vi. sodium acetate (anhydrous), lead acetate trihydrate, sodium hydroxide, glacial acetic acid, glycine, 4-methylumbelliferyl sulfate (4-mus), 4-methylumbelliferone sodium salt (4-mu) and corning 96-well - black, flat - bottom, microtiter plates were all obtained from sigma aldrich corp. stock buffer solution of 50 mmol / l sodium acetate was prepared and stored at 4 c after adjusting ph to 5.0 with 2 m acetic acid solution. mmol / l in sodium acetate buffer (ph 5.0), and stored at 20 c in 1 ml aliquots. extraction buffer stock was prepared by dissolving lead acetate trihydrate in 50 mmol / l sodium acetate to a concentration of 15 this buffer stock was further diluted in water to a final concentration of 6 mmol / l lead acetate in 20 mmol / l sodium acetate for extracting the enzyme from the dbs. stop buffer stock was prepared by dissolving glycine in water to a final concentration of 0.5 mol / l with ph adjusted to 10.3 using 1 m sodium hydroxide. this 0.5 mol / l glycine - sodium hydroxide stock was diluted in molecular biology grade water to a final concentration of 85 mmol / l, to provide the working stop buffer for terminating the enzyme reaction. de - identified dried blood spots (n = 78) obtained from the duke biochemical genetics laboratory (bgl) were used to establish the normal range for ars - b enzyme activity. mps vi patient dbs (n = 10), diagnosed through leukocyte enzyme assay and/or molecular sequencing, were de - identified and kindly provided by greenwood genetic center (ggc) under their irb protocol. these samples were used to establish the ars - b enzyme activity range for affected patients. clinical assays were performed according to standard operation procedures developed at the duke biochemical genetics laboratory. briefly, one 3.2 mm punch from a dbs card (3 l of blood) was added to 200 l of extraction buffer in a microfuge tube and incubated at room temperature (rt ; 2025 c) for 2 h on a rocker. 40 l aliquots of the dbs extract were transferred to four duplicate wells of the 96-well microtiter plate. 40 l 4-mus substrate solution was added to two duplicate reaction wells, while the remaining two wells served as duplicate sample blanks (no substrate). the plate was sealed with adhesive aluminum foil and incubated at 37 c for 20 h along with the remaining 4-mus solution in a tube. the reaction was subsequently terminated by adding 220 l of stop buffer (85 mmol / l glycine - naoh) to all wells. the remaining 4-mus substrate was added to the duplicate blank wells to measure background fluorescence from the undigested substrate. the microplates were read using a benchtop fluorometer (tecan, austria) and the mean fluorescence of the duplicate blank wells was subtracted from that of the reaction wells to calculate the relative fluorescence units (rfu) for each sample. the rfu was then plotted against a freshly prepared 4mu standard curve and converted to enzyme activity units, expressed in pmol / punchh, based on the standard curve. a set of eight normal control dbs were assayed five times within one plate to determine the intra - day assay variability. ten normal control spots were assayed individually on six different days to establish the inter - day assay variability. two different analysts tested the same set of ten dbs to determine operator variability as part of the validation process. in addition, dbs extracts were serially diluted (1:5, 1:10, 1:25, 1:50, 1:100 and 1:200) to establish the least detectable activity measurement (limit of detection). stability of ars - b enzyme activity in dbs under different storage conditions was also analyzed. a set of ten individual dbs were stored at 20 c, 4 c and 22 c (rt) for 7 days, 30 c for 6 h and 37 c for 2 h before measuring enzyme activity. storage at 30 c and 37 c was intended to mimic short, high temperature transportation times as in a delivery truck or samples being held transiently during shipping and receiving in warm climate regions. specificity of the ars - b enzyme activity assay was determined by testing the dbs samples from patients with other known lsds (mps i, mps ii, mps iva, gaucher, fabry and pompe). sodium acetate (anhydrous), lead acetate trihydrate, sodium hydroxide, glacial acetic acid, glycine, 4-methylumbelliferyl sulfate (4-mus), 4-methylumbelliferone sodium salt (4-mu) and corning 96-well - black, flat - bottom, microtiter plates were all obtained from sigma aldrich corp. stock buffer solution of 50 mmol / l sodium acetate was prepared and stored at 4 c after adjusting ph to 5.0 with 2 m acetic acid solution. mmol / l in sodium acetate buffer (ph 5.0), and stored at 20 c in 1 ml aliquots. extraction buffer stock was prepared by dissolving lead acetate trihydrate in 50 mmol / l sodium acetate to a concentration of 15 this buffer stock was further diluted in water to a final concentration of 6 mmol / l lead acetate in 20 mmol / l sodium acetate for extracting the enzyme from the dbs. stop buffer stock was prepared by dissolving glycine in water to a final concentration of 0.5 mol / l with ph adjusted to 10.3 using 1 m sodium hydroxide. this 0.5 mol / l glycine - sodium hydroxide stock was diluted in molecular biology grade water to a final concentration of 85 mmol / l, to provide the working stop buffer for terminating the enzyme reaction. de - identified dried blood spots (n = 78) obtained from the duke biochemical genetics laboratory (bgl) were used to establish the normal range for ars - b enzyme activity. mps vi patient dbs (n = 10), diagnosed through leukocyte enzyme assay and/or molecular sequencing, were de - identified and kindly provided by greenwood genetic center (ggc) under their irb protocol. these samples were used to establish the ars - b enzyme activity range for affected patients. clinical assays were performed according to standard operation procedures developed at the duke biochemical genetics laboratory. briefly, one 3.2 mm punch from a dbs card (3 l of blood) was added to 200 l of extraction buffer in a microfuge tube and incubated at room temperature (rt ; 2025 c) for 2 h on a rocker. 40 l aliquots of the dbs extract were transferred to four duplicate wells of the 96-well microtiter plate. 40 l 4-mus substrate solution was added to two duplicate reaction wells, while the remaining two wells served as duplicate sample blanks (no substrate). the plate was sealed with adhesive aluminum foil and incubated at 37 c for 20 h along with the remaining 4-mus solution in a tube. the reaction was subsequently terminated by adding 220 l of stop buffer (85 mmol / l glycine - naoh) to all wells. the remaining 4-mus substrate was added to the duplicate blank wells to measure background fluorescence from the undigested substrate. the microplates were read using a benchtop fluorometer (tecan, austria) and the mean fluorescence of the duplicate blank wells was subtracted from that of the reaction wells to calculate the relative fluorescence units (rfu) for each sample. the rfu was then plotted against a freshly prepared 4mu standard curve and converted to enzyme activity units, expressed in pmol / punchh, based on the standard curve. a set of eight normal control dbs were assayed five times within one plate to determine the intra - day assay variability. ten normal control spots were assayed individually on six different days to establish the inter - day assay variability. two different analysts tested the same set of ten dbs to determine operator variability as part of the validation process. in addition, dbs extracts were serially diluted (1:5, 1:10, 1:25, 1:50, 1:100 and 1:200) to establish the least detectable activity measurement (limit of detection). stability of ars - b enzyme activity in dbs under different storage conditions was also analyzed. a set of ten individual dbs were stored at 20 c, 4 c and 22 c (rt) for 7 days, 30 c for 6 h and 37 c for 2 h before measuring enzyme activity. storage at 30 c and 37 c was intended to mimic short, high temperature transportation times as in a delivery truck or samples being held transiently during shipping and receiving in warm climate regions. specificity of the ars - b enzyme activity assay was determined by testing the dbs samples from patients with other known lsds (mps i, mps ii, mps iva, gaucher, fabry and pompe). 1a shows the relative ars - b enzyme activity in the dbs tested from normal and known mps vi patients. the duke - bgl control dbs (gray circles) showed an enzyme activity range of 21.6103.6 pmol / punchh. the calculated mean standard deviation values were 49.3 15.6 pmol / punchh. the mps vi - affected dbs (black squares) showed enzyme activity ranging between 0.78.9 pmol / punchh with a mean value of 4.8 2.8 (mean std. dev.). the ars - b deficient dbs activity levels were clearly separated and distinguishable from those of normal control spots (p < 0.0001, tukey 's t - test). stability experiments carried out to measure ars - b activity over various lengths of times and temperatures using ten dbs samples ranged from 28.638.7 pmol / punchhour and showed that the enzyme activity was relatively stable, with no significant difference under the various conditions tested (fig. storage at the higher temperatures (30 c & 37 c) for shorter durations was tested because dbs may often be exposed to such conditions during transportation, temporary storage and handling in certain situations. the inter - day (n = 10) and intra - day (n = 8) variations (% cv) of the fluorometric assay remained within acceptable limits (< 16.05% and < 13.88%, respectively), as was the operator variation (n = 10 ; cv < 14.75%), demonstrating the robustness and reproducibility of this new microplate assay. the assay was sensitive enough to detect enzymatic activity in normal control dbs extracts at 1:25 dilution (data not shown). this assay was also shown to be specific to mps vi patients as dbs from patients with other lsds (mps i, mps ii, mps iva, gaucher, fabry and pompe) showed ars - b activities in the normal ranges (22.870.4 pmol / punchh) (fig. 2). the use of minimally invasive dbs samples for diagnosis of mps vi is advantageous due to the ease of sample procurement and shipping compared to the existing diagnostic methods that require cultured skin fibroblast cells or venous blood draw for leukocyte isolation. dbss can be conveniently shipped through the mail to distant clinical laboratories for testing since the enzyme activity is demonstrably stable under a range of storage conditions, including those that may be encountered in hot climates. these advantages make this dbs - based fluorescent microplate assay desirable as a screening tool to identify patients with mps vi. it is, however, recommended to confirm the diagnosis using another method (enzyme activity in leukocytes or fibroblasts) or by molecular analysis, if possible. we have developed and validated a new dried blood spot - based microplate assay using a fluorescent substrate for measuring arylsulfatase b activity to diagnose patients with mps vi. this assay is robust, reproducible and more convenient than the existing, more labor - intensive assay techniques that require fibroblast cultures or blood leukocytes. | mucopolysaccharidosis type vi or maroteaux lamy syndrome is an autosomal recessive lysosomal storage disorder caused by deficiency of arylsulfatase b (ars - b) enzyme activity. it results in mild to severe multi - organ system failure from accumulation of undigested glycosaminoglycans (gags) ; dermatan sulfate and chondroitin-4-sulfate. we have developed a single - step enzyme assay using a fluorescent substrate and dried blood spots to measure ars - b activity to identify disease patients. this assay is robust, reproducible, specific and convenient to perform. |
we present a case of a 17 year old boy with coccidiomycosis osteomyelitis of the right first toe. we discuss the epidemiology of disease caused by coccidioides immitis, the patient s risk factors and hypothesize how these risk factors interacted. this young man s case serves to illustrate the importance of considering a fungal etiology and the role of comorbid conditions on coccidiomycosis. a 17 year - old male presented to the emergency department (ed) at our california central valley community hospital with pain and swelling of his right first toe. the pain had started two weeks prior to presentation ; the swelling and redness had been present for only one week. his only significant past medical history was diabetes insipidus (di) diagnosed at the age of 3. he had gone to a local urgent care center where they noticed an ingrown toe nail, and supposed the infection was due that, removed part of the nail and discharged him on oral trimethoprim / sulfamethoxazol. during the next few days the pain, swelling, and redness increased. he returned to the urgent care center where he was administered ceftriaxone 1 g intramuscularly and was referred to the emergency department (ed) for x - rays, which revealed erosive changes of the right great toe distal phalanx, suggestive of osteomyelitis [fig. 1].fig. 1x - ray of right foot : erosive changes of the great toe distal phalanx suggesting osteomyelitis.fig. 1 x - ray of right foot : erosive changes of the great toe distal phalanx suggesting osteomyelitis. laboratory studies showed an elevated erythrocyte sedimentation rate (esr) of 130, although the complete blood count was only minimally abnormal with a white cell count of 12,260 cells / microliter. he also had an elevated c - reactive protein (crp) at 133.0 mg / l. a magnetic resonance imagining (mri) scan with contrast showed acute osteomyelitis of the toe [fig. 2mri of right foot with and without contrast : there is abnormal marrow signal intensity throughout the distal phalanx of the great toe. findings compatible with acute osteomyelitis.fig. 2 mri of right foot with and without contrast : there is abnormal marrow signal intensity throughout the distal phalanx of the great toe. day two he was taken to the operating room for partial distal hallux amputation ; wound samples were sent for culture. he suffered no operative complications. on hospital day three, wound culture showed fungi. coccidiomycosis was considered the most likely fungal infection, given that the patient had been born and raised in the central valley of california. he was started on 400 mg of fluconazole by mouth, twice daily. following the surgery, his white count trended down to 8250, esr down to 89, and crp to 104.7. blood serology and coccidioidomycosis titers were sent to an outside lab, at uc davis medical center. the dna probe came back positive for coccidioides immitis on day five of hospital admission. immunodiffusion was positive for igg and igm complement fixation titer was 1:64, raising concern for dissemination. the patient was discharged home five days after admission on fluconazole 800 mg by mouth daily. he continued to follow up with the infectious disease clinic and remained on long - term azole therapy to treat coccidiomycosis osteomyelitis. coccidiomycosis, also known as valley fever, is named after the san joaquin (central valley) of california. it is caused by coccidiodes immitis, a fungus endemic to the southwestern united states, mexico, and parts of central and south america,,. inhalation of the spores from soil - dwelling, dimorphic fungi c. immitis usually is asymptomatic, but if symptomatic, most commonly causes pulmonary symptoms. pulmonary coccidioidomycosis is commonly misdiagnosed as bacterial community acquired pneumonia. clinical examination along with a thorough history is vital in many cases to determine diagnosis. there are no pathognomonic chest x - ray findings to indicate an infection with c. immitis. most people living in the central valley show serological evidence of past infection, however the majority remain asymptomatic or have had a mild, self - limiting respiratory disease,. of those who become almost all the morbidity and mortality associated with coccidioidomycosis is due to the chronic manifestations rather than to the acute respiratory infections. extra - pulmonary manifestations such as skin, brain, or bone involvement are rare but can be fatal. disseminated disease can cause meningitis, osteomyelitis, arthritis, and other soft - tissue infections. when the musculoskeletal system is involved, coccidioidomycosis prefers the axial skeleton, with joint involvement as the next most common target. it has been hypothesized that at least some coccidioidomycosis bone infections arise through extension from adjacent soft tissues rather than from hematogenous spread. the most common radiographic finding with bony involvement includes osteolytic lesions, either with punched - out, well - circumscribed borders or demonstrating a permeative (or moth - eaten) appearance. mri, along with ct scan, has been shown to be helpful in evaluating the extent of soft tissue damage and bone erosion, along with determining any abscess formation. obesity, especially morbid obesity (bmi 35 kg / m), may be associated with immune suppression possibly due to the suppression of autophagy. furthermore, dendritic cells, which are key immune response cells, are significantly decreased in obesity. with a bmi of 51, diabetes insipidus (di) is a disease characterized by excessive thirst due to secretion of dilute urine. di, by itself, is not an immunosuppressing disease ; however there is one case report of di in a patient with common variable immunodeficiency, although it is unclear whether there is a link between the two diseases. we found no case reports or studies mentioning osteomyelitis occurring in patients with diabetes insipidus. primary cutaneous coccidioidomycosis was also considered as the patient did not show any respiratory symptoms. however, it seemed unlikely as primary cutaneous infection is extremely rare, with about 25 cases previously reported in literature since 1926. primary cutaneous infection results from direct traumatic inoculation of the organism into the skin by an external source and typically manifests as a painless, indurated nodule with ulceration. diagnostic criteria for primary cutaneous coccidioidomycosis included : absence of pulmonary disease, clear evidence of traumatic inoculation, incubation period of 1 to 3 weeks, a chancriform lesion with a painless, ulcerated nodule or plaque, a negative or low complement fixation reaction, and spontaneous healing after some weeks,. we present this case of a young man who presented like a typical case of bacterial osteomyelitis, which, due to his co - morbidity of obesity, was initially assumed to be bacterial. he was ultimately diagnosed with coccidioidomycosis osteomyelitis, was treated appropriately, and discharged home after an uneventful short stay at the hospital. this research did not receive any specific grant from funding agencies in the public, commercial, or not - for - profit sectors. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. | we report a case of a 17-year - old male who presented with pain in his right first toe. his pain and swelling had worsened and x - rays of his foot revealed erosive changes of the great toe distal phalanx suggesting possible osteomyelitis. his co - morbidities were morbid obesity and diabetes insipidus. he was admitted to the hospital, blood cultures were drawn, and he was started on vancomycin for presumed bacterial osteomyelitis. he underwent incision and drainage of the fluctuant abscess of the toe, where a culture of the wound was taken. preliminary results grew fungi. being located in an endemic area, he was started on anti - fungal treatment for presumed disseminated coccidioidomycosis ; culture was positive for coccidiodes immitis. he also had serology positive for coccidioidomycosis titers. he had uneventful hospital stay and was discharged on long - term oral antifungal therapy. |
balance dysfunction is one of the main factors leading to impaired mobility and postural control, influences walking after discharge and in the community - dwelling elderly, and also impacts balance control in daily life (brosseau., 1996 ; wee., 1999 previous studies have shown that more than 33% of people with chronic balance dysfunction have an increased risk of falling in their daily lives (colledge., 2002), and the cause of more than half of older people 's accidental death was significantly correlated with falling and balance dysfunction (agrawal., 2009). some balance ability training methods have demonstrated postural control and mobility improvement (yavuzer., 2006 ; lo., 2012), but the effectiveness of existing interventions is limited because patients tend not to be fully involved and adhere to the training protocol. with the development of technology, rehabilitation facilities and technology that can study the simulated real environment are increasingly used to restore motor function and address balance dysfunction (bower., 2014 ; mcewen., 2014) virtual reality technology is developed on a computer hardware and software environment that enables immersed users to generate visual, audio, and haptic feedback, and obtain an interactive experience in the three - dimensional visual space, which strives to give users the impression of a real environment. the virtual real environment is created by virtual reality technology with a focus on three characteristics : autonomy, interaction and sense of being. patients who have perceived deficiencies and motor dysfunction can use virtual reality technology during their rehabilitation training, to produce the greatest recovery in their impaired motor function. consequently, following this treatment, patients might be able to achieve full self - care, and virtual reality training might allow some of them to work independently, improving their quality of life (berra, 2004). balance control relies on the central nervous system at multiple levels ; some studies have shown that exploring the virtual real environment activates the cortical and sub - cortical regions (nancy., 1980 ; bolton., 2012). in this current review, the possible central control mechanism of virtual reality technology as well as its clinical application will be introduced. generally, balancing the body is thought to be achieved by the coordination of three major systems, including visual, vestibular and proprioceptive sensation. previous studies have shown that the prefrontal cortex is one of the most important brain areas in controlling human balance (nancy, 1980 ; virk., 2006 ; walker. they provided information about the position and motion of the head with respect to the surroundings, and based on information in the visual surroundings provided by visual cues. the main role of the proprioceptive and somatosensory system is to sense the distributed tactile input stimuli at the neural level, and to provide a relationship between limb position and the central nervous system (figure 1). the vestibular system, located in the inner ear, is used to control and perceive the motion and position of the head in space (virk., 2006). the function of these systems reduced after the brain is impaired. when one or two of these sensory input systems are dysfunctional, the allocation of different gains to the sensory inputs can be exploited to compensate for the impaired systems (nancy, 1980 ; walker., 2010). (2006) proposed that such an adaptation can take place in everyday life or in a virtual environment. by using a virtual environment to train the eye - head movement, balance in older people can be improved and occupational falls are minimized. a simplified representation of the human motion control loop. a desired body state directs the controller within the central nervous system generating motor commands that drive the muscles of the body. external sources like electric current (d) can stimulate the body (a). the actual state of the body is registered by (among other things) the vestibular apparatus and the visual system (b). the signals from these sensors are processed by the central nervous system (c) for comparison with the desired body state. adapted from bos. behavioral experiences including an enriched environment, physical activity and spatial learning substantially improve sensorimotor function outcome after brain injury following ischemia (johansson. virtual reality systems have been shown by many studies to provide similar real - world training effects, such as in the intensity and repetition referred to as constraint - induced movement therapy, body weight support treadmill training and allow patients with stroke to modify their neural organization and promote neuroplastic changes (viau., 2004 ; therefore, we hope to explore the possible mechanism of how virtual reality promotes balance rehabilitation after brain injury in terms of visual, vestibular and proprioceptive feedback. recent studies have shown that balance control in standing is a complex sensorimotor action based on automatic and reflexive spinal programs under the influence of several distinct and separate supra - spinal centers in the brainstem, cerebellum and cortex (drew., 2004). multimodal dance training over 4 weeks could activate the region of the prefrontal cortex (tachibana., 2011). some experiments have allowed patients to play and learn spatial navigation in mazes (ayaz., 2011), and activation could always be found in brain regions in our preliminary study of virtual reality training of stroke patients in sysu using an xbox 360 (figure 2). although the mechanism is not clear, these results suggest a corresponding relationship between the prefrontal cortex and spatial orientation ability. a further study from miller suggests that during the incremental swing balance task, when unpredictable and external postural perturbations were applied to healthy standing participants, the prefrontal cortex would bilaterally recruit both visual and proprioceptive information to adaptively modify postural instability in response to changes in the virtual environment to complete the action goal (miller., 2001). another study by basso moro also demonstrated that, when healthy participants performed an incremental swing balance task in a semi - immersive virtual reality environment, the effect of oxygenation increased in the prefrontal cortex of both hemispheres (basso., 2014). (2005) recorded the ankle movement of twelve young healthy participants using force plates, eeg and emg as they performed oscillatory and discrete postural movements in the anterior and posterior directions. a statistically significant difference in the amplitude of all three components of movement - related cortical potentials, with respect to the baseline at the front - central electrode sites was found in the eeg data. they also demonstrated that the frontal region may play an important role in postural equilibrium, likes moving continuously in different directions, or only in the forward direction. areas activated during an active foot movement task by a patient with stroke at the first affiliated hospital, sun yat - sen university, china. the two functional mri images of preliminary experiments are supplied by the motor recovery laboratory in the department of rehabilitation, showing active tasks of the right paretic foot before (a) and after (b) virtual reality training. during walking some studies show that when adjusting walking speed on the treadmill (suzuki., 2004) and during the recovery process following ataxic stroke (mihara., 2007), the prefrontal cortex participates in the control of locomotion. basso used functional near - infrared spectroscopy to study changes in regional activation in the frontal cortices in terms of hemoglobin oxygenation in the brain 's vascular system during walking at 3 and 5 km / h and running at 9 km / h on a treadmill. the density of oxygenated hemoglobin increased, but that of deoxygenated hemoglobin was not found in the frontal cortices. moreover, other studies found a positive correlation between presence and parietal brain activation, and a negative correlation between presence and frontal brain activation during interactive virtual reality training (mihara., 2008). we can see that alongside the prefrontal cortex, the parietal cortex makes a contribution to the control of balance. through investigating the hemodynamics of patients who had cortical activation induced by postural perturbation, mihara found that there was a strong relationship between continuous activation during ataxic gait and the compensatory mechanisms for ataxic gait after infarct stroke. in conclusion, there are widespread cortical network activations, including the prefrontal, premotor, supplementary motor, and parietal cortical areas in both hemispheres, which have a significant impact on postural control in post - stroke hemiplegic patients (kober. the simulated real - world scenarios give balance dysfunction patients more and stronger information input than the real world to ensure that they relearn their coordination and sense of balance. the fidelity of virtual reality may play an important role in the effectiveness of recruiting neural circuits and the delivery of desirable outcomes at the functional level. the structure of the brain can be enhanced using visual feedback in virtual reality to augment interconnected, distributed cortical regions. it is suggested that visual information can provide a potent signal for the reorganization of sensorimotor circuits (lewis., 2000 ; lewis., 2005 ; stepniewska., 2005). patients with stroke can use visual feedback to adjust the body 's center of gravity, and to achieve the goal of controlling the body state (srivastava.,, the multi - sensory virtual reality feedback loop can strengthen the control of balance. (1998) conducted some animal experiments on monkeys, cats and other animals, and showed that visual experience and action seem to be the keys to functional recovery of vestibular function deficiency (lacour. a large number of clinical trials support this view. in a study by webster. (2010), a virtual environment was created to help people control the mobility of their wheelchairs, and participants had to navigate through a virtual obstacle course. after treatment, compared with participants who did not have training on the virtual course, the experimental group showed a decreased rate of wheelchair accidents and falls and a better performance on obstacle courses (webster., 2001). to study the influence of virtual reality technology on the recovery of balance function following chronic stroke, belinda and cho used different kinds of video games to stimulate patients balance, coordination, strength and upper limb coordination, and the results showed that all patients had significantly improvement in balance function (cho., 2012 ; michalski., 2012). some other studies have suggested that middle cerebral artery infarcts in the right hemisphere might have a relationship with increased dependence on vision, and non - infarct brain regions are recruited to control postural sway. it is also suggested that the control of lateral posture following a right hemisphere infarct requires more visual input than in a healthy participant (manor., 2010). at present, virtual reality use in vestibular rehabilitation is a relatively new concept, and there are not many studies on the relationship between virtual reality and balance in the vestibular field. common vestibular function disorders may be caused by abnormalities in the vestibulo - ocular reflex. viirre suggested that the vestibulo - ocular reflex could be adapted in virtual reality simulations with an increase in vestibulo - ocular reflex gain, and virtual reality could be used to increase the rate of adaptation by specifically adapting scenes to a person 's capabilities, thereby facilitating their recovery (viirre., 2000). miles study suggested that the key to adjusting the vestibular system is the retinal slip, the movement of a visual image across the retina, which is a powerful signal that induces the adaptation of vestibular responses. the primary rationale for using virtual reality for vestibular rehabilitation is that realistic visual environments may enhance adaptation by causing retinal slip (miles., 1980). (2001) studied people with uncompensated unilateral peripheral vestibular dysfunction using a customized exercise program or a machine - base optokinetic stimulation exercise program. preliminary findings suggest that both exercise programs could improve vestibular dysfunction, yet the machine - base group demonstrated that physical therapy involving conflicting visual environments might be more effective than a customized program alone or a program that includes only cawthorne - cooksey exercises. virtual reality scenes may promote rehabilitation more effectively than optokinetic - based therapies, since there is an ability to finely control the virtual scene (pavlou., 2001). to discover the potential of virtual reality for vestibular disorders, in a small sample investigation, wbitney asked two vestibular disorder patients and three healthy people to stand while viewing a sinusoidal waveform on a force plate. the results suggested that virtual reality was a valid way to perform vestibular rehabilitation (whitney., 2002). nevertheless, because of the small number of participants, the accuracy of the study needs to be validated with further data collection. another theory to explain the effect of virtual reality on the vestibular system is habituation therapy. the patients may feel uncomfortable at the beginning, and then a sensory mismatch is sent to the brain and promotes compensation for and adaptation to labyrinthine disorders (norre., 1989). ninety consecutive patients with hemiplegic involvement following a single cerebrovascular accident were recruited to assess the relative importance of factors affecting balance, and it was found that balance function and mobility post - stroke were both strongly influenced by proprioception (keenan., 1984). proprioceptive training affects mobility and balance function by altering how proprioceptive receptors input information, and improves the control of the musculoskeletal motion system and balance function in stroke patients. some studies have demonstrated that proprioceptive neuromuscular facilitation promotes neuromuscular control and motor function recovery by stimulating proprioceptive stimuli, such as stretching the limbs, joint compression, traction and so on. in addition, the use of spiral diagonal movement patterns can stimulate the joint and muscle proprioceptors. by training the proprioceptive system, the responsiveness of muscles can be enhanced and the recovery of neuromuscular and motor function can be stimulated (pan., 2011). furthermore, virtual reality training could improve standing balance in stroke patients, including normal and abnormal proprioceptive function (song., 2014). but compared with proprioceptive neuromuscular facilitation technology, virtual reality training exercises can not only train the limbs, but also train the body by enriching proprioceptive information input. (2011) trained 31 patients using the pro - kin training system and found that the score on the berg balance scale in these patients was significantly increased after training, which indicates that proprioception training played an active role in improving balance function in stroke patients. compared with conventional therapies (physiotherapy, occupational therapy, or other exercises), virtual reality technology has unique advantages in the field of rehabilitation. first, it makes treatment more interesting and brings patients more enthusiasm, and the sports favored by the patients in daily life can be used as a training program in a virtual reality system (figure 3). second, patients can undergo familiar training in a wheelchair or sitting in a chair as well as standing or walking, so there is no need for stabilized posture control, rehabilitation difficulty is reduced, and the safety of the rehabilitation exercise is increased. third, the game system is cheap and easy to carry, and can be easily operated in the hospital, at home or in the community. virtual training with bilateral limb movement in the motor recovery laboratory, department of rehabilitation, the first affiliated hospital, sun yat - sen university, china. ding. (2013) used a simple computer game involving virtual reality - based constraint - induced movement therapy training for balance dysfunction in three chronic stroke survivors, which improved dynamic stability and weight symmetry by encouraging them to use their paretic leg with more effort. the patients received motion signals from virtual avatars in virtual reality as well as augmenting the control gain. measured by functional gait assessment, the berg balance scale and a walking speed test, horlings and coworkers confirmed that virtual reality balance training could provide more realistic proprioceptive and visual input, and improved the patient 's reaction time, postural stability, balance and walking function effectively (bisson, 2007 ; horlings., 2009 ; singh., 2012). therefore, virtual reality balance games can be used as a potential and useful tool to train adults with balance dysfunction (schwesig., 2011). virtual environments are used in many areas, such as in entertainment, training situations and medicine, including the rehabilitation of behavioral and neurological dysfunction. the use of virtual reality technology in the functional rehabilitation of various debilitating diseases not only provides patients with a very authentic virtual environment and an immersive experience, but also greatly improves their participation enthusiasm and initiation in rehabilitation. compared with conventional rehabilitation therapy, there is a more significant improvement in motor function and activities of daily living when treated using virtual reality (bryanton. virtual reality therapy is an effective therapy in stroke, spinal cord injury, cerebral palsy and other motor function deficient diseases. wii games combined with virtual reality technology have been used to treat balance dysfunction. in clinical trials, deutsch and colleagues randomly allocated stroke patients into a wii exercise training group and a conventional training group, and the experiment demonstrated that the wii group exhibited a higher degree of functional recovery of balance and the patients had higher enthusiasm when they performed the virtual reality exercises (deutsch., 2009). in another study, children with spastic cp completed ankle selective motor control exercises using a virtual reality exercise system and conventional exercises. the results showed that greater fun and enjoyment were expressed during the virtual reality exercises, and children completed more repetitions of conventional exercises. furthermore, the range of motion and hold time in the stretched position were greater during virtual reality exercises. these data suggest that using virtual reality to train or guide exercises may improve exercise compliance and raise exercise effectiveness (bryanton., 2006). however, a randomized controlled trial by eser. (2008) did not find a statistically significant difference between virtual reality therapy and conventional therapy in terms of lower extremity motor recovery, mobility or activity level. there is no doubt that the intervention time for equilibrium dysfunction rehabilitation is one of the most critical factors in functional recovery after stroke, which has important research value. at present, many studies about virtual reality training mainly focus on the clinical effects ; the mechanisms, especially of neural reorganization and neuroplasticity, and which regions are mainly activated at cortical and sub - cortical levels, are still not clear. in addition, how virtual reality training influences the individual involves the central nervous system at multiple levels. virtual reality therapy is mostly used in the chronic phase of stroke rehabilitation (lacour., 1976 ; however, the early stages are the prime time of exercise for balance recovery in patients with balance dysfunction. according to the literature, sufficient and early rehabilitation training can greatly improve the prognosis of patients with stroke, and reduce complications (langhorne. 2012), improve motor function of the hemiplegic limb (li., 2011), promote their skills (craig., 2010) and improve their quality of life (tyedin., 2010). therefore, the early phase of brain injury rehabilitation is the most important time in the recovery of patients with stroke. however, during this phase the patients are often too paretic to do training, an important reason for which is that they have a lack of cortical stimulation. therefore, using virtual reality in the early phase of brain injury could be feasible. in addition, to explore the safety and therapeutic effects and search for the most suitable intervention time point for balance recovery, virtual reality exercise training should be used for acute and subacute patients, which is a necessary and valuable research direction. meanwhile many studies (flynn., 2007 ; cikajlo., 2012 ; pluchino., 2012) have shown that virtual reality exercise training is likely to be an effective therapy for patients with balance dysfunction at home in the future. but there are still many problems to solve, such as the evaluation criteria for the treatment and the interactive performance of the virtual reality system. in addition, what kind of model of virtual reality should be chosen, how long the use should be and what precautions should be taken need to be carefully considered when performing virtual reality training. there is still no clear reference index at present. the next stage of the study of virtual reality technology applied to the rehabilitation of balance function needs to explore the mechanism of the integrated central and peripheral nervous system, visual, vestibular and proprioceptive sensation, and more detailed clinical techniques. establishing a quantitative standard of virtual reality therapy furthermore, the most appropriate way of applying virtual reality intervention and suitable intervention intensity according to different patients brain function mechanisms should be determined. in these ways, functional recovery of stroke patients will be promoted and the operational feasibility of virtual reality will be improved by formulating better plans, and the technology can be widely used in the family in the future. in conclusion, virtual reality technology based on the study of the mechanisms of functional rehabilitation and its application following stroke has wide space to develop. | virtual reality is a new technology that simulates a three - dimensional virtual world on a computer and enables the generation of visual, audio, and haptic feedback for the full immersion of users. users can interact with and observe objects in three - dimensional visual space without limitation. at present, virtual reality training has been widely used in rehabilitation therapy for balance dysfunction. this paper summarizes related articles and other articles suggesting that virtual reality training can improve balance dysfunction in patients after neurological diseases. when patients perform virtual reality training, the prefrontal, parietal cortical areas and other motor cortical networks are activated. these activations may be involved in the reconstruction of neurons in the cerebral cortex. growing evidence from clinical studies reveals that virtual reality training improves the neurological function of patients with spinal cord injury, cerebral palsy and other neurological impairments. these findings suggest that virtual reality training can activate the cerebral cortex and improve the spatial orientation capacity of patients, thus facilitating the cortex to control balance and increase motion function. |
educators can readily integrate brain - related topics into subjects where standards may not explicitly include neuroscience content. in many states, an exploration of how sensory organs, such as the eyes and ears, work with the brain serves as an excellent jumping - off point for further discussion of brain science, especially for young learners who enter the classroom with some awareness of how the senses help them relate to the world around them. one very easy way of bringing the brain into a lesson on the senses involves optical illusions. students find classic illusions, such as the old woman or the young woman, and activities like thaumatropes enthralling. when we look at an object, we see it as a single item, but our brain processes visual information based on submodalities like shape, size, color, and movement. certain cells respond to specific stimuli such as color or orientation, and all of the information comes together to form the bigger picture of what we see. if the human brain had to process all of the sensory stimuli a person encounters, the information would become overwhelming, so the brain uses shortcuts. in the old woman or the young woman illusion, the shortcut involves making the stimuli into a face, because that is what the stimuli most closely resemble, although the image is ambiguous. based on our individual past experiences, it is easier for the brain to see either a young woman or an old woman. given the complexity of this system, it is no surprise that scientists are still learning about sensory processing. examples like illusions provide an opportunity to make the simple point that visual processing is never complete and the brain works hard to make sense of perceptual information. visual and auditory illusions help scientists learn about normal sensory processing ; just mentioning that the brain is involved in the use of our senses helps open students eyes to how the brain works. in much the same way that the brain processes visual or auditory information, it must also process scent, or olfactory, information. generally speaking, based on the pattern of activation, the brain can identify the substances and provide information about where we might have encountered similar odorants in the past. this strong connection between memory and the senses can be used in language - arts classrooms. for instance, a teacher might ask students to smell an object that evokes a strong emotional memory, and then write down this memory in the form of a poem. the teacher would briefly explain the link between the amygdala, memory, and our sense of smell while encouraging creativity and writing skills. research has demonstrated that the amygdala, a structure found in the temporal lobe, plays a significant role in emotion and it is also thought to be involved with some types of memory. interestingly, the amygdala has many synaptic connections with the structures responsible for processing olfactory information. this explains why when some of us smell apple pie, for instance, we not only remember thanksgiving with our families, but also actually feel calm and happy. with younger children, using words like amygdala or visual processing may not be suitable, but by using neuroscience - related activities and explaining them at a level appropriate for the age group, educators can lay the groundwork for future neuroscience learning. many famous neuroscientists have focused their work on subjects such as how the brain interprets artistic material like music or literature. for instance, in musicophilia, renowned neurologist oliver sacks explores the power of music through the lens of neuroscience. books and articles that blend science with literature illustrate the connection between the brain and other areas of study, and teachers can also use them as assigned readings in the classroom. books like sacks the man who mistook his wife for a hat : and other clinical tales or v.s. ramachandran and sandra blakeslee s phantoms in the brain : probing the mysteries of the human mind include chapters that can be tailored to different subject areas and are short enough to maintain student interest. most educational content areas include literacy as a major component, and utilizing books like these, either as homework or as in - class readings, ensures that the standards are being met while at the same time engaging student interest and highlighting neuroscience content. instead of assigning reading on the history of music or the biography of a famous musician, for instance, educators can ask students to read a chapter on how the brain understands music. many of the famous writers, artists, and musicians included in classroom curricula suffered from mental illnesses. students are drawn to these personal struggles, and by addressing them in depth, educators can create a discussion of different types of mental illnesses, how scientists treat these illnesses, and why mental illness and creativity seem to have some sort of connection.1 teachers can utilize the strong connection between neuroscience and other subject areas to boost scientific literacy. some students find certain topics in neuroscience, such as neurotransmitters, very abstract. by tying in other subject areas, especially through hands - on techniques they can easily turn neurons into an art project by using pipe cleaners and other materials to model different structures, or into an exercise in physical education by asking students to use their arms as axons and dendrites to pass a ball that serves as a neurotransmitter. making connections to popular activities like sports and films students often ask, when am i ever going to use this ? using examples relevant to students lives makes the integration of neuroscience content more meaningful. an educator could ask students what parts of the brain allow them to kick a soccer ball, or with the increase in popularity of 3d movies, could have students research how the brain allows us to see in three dimensions. students routinely learn that they must wear bicycle helmets, stay away from drugs, and eat properly, but they are not always taught how helmets, drugs, and nutrition can affect brain function. by making clearer connections to material already being taught, educators can increase students understanding of the brain. for older students, presenting brain scans from people who have used drugs or suffered brain trauma make the brain - health connection more evident. for younger students, creating brain hats can help illustrate both the importance of protecting the brain and fundamental ideas such as cortical localization of function. this basic concept states that while some structures may have roles that overlap, and some structures may do multiple jobs, in general there is a division of labor in the brain. understanding this basic idea primes students for deeper exploration of neuroanatomy. there are many brain - hat templates available on the internet ; educators can create paper hats that students label with the various parts or functions of the brain. for young learners, one could simply put a picture of an eye in the back of the brain hat rather than use words like occipital lobe or visual - processing center. by having students label the hats this way and then wear them, an educator can ask students to consider what would happen if they fell off their bikes and hit their heads in different areas. in addition to creating connections with other subject areas and making use of topics of interest to students, educators can use technology to bring neuroscience into the classroom. many outstanding web sites and software programs bring neuroanatomy into the classroom without the use of scalpels or probes. for example, genes to cognition online2 allows students to explore topics in neuroscience in a dynamic and self - directed way, while links on the dana foundation s brainy kids3 site let students observe dissections or digitally probe a brain. there are also programs and web sites that allow students to hear the firing of actual action potentials or manipulate brain scans (see sidebar). rather than lecturing, educators can use these tools to allow for hands - on exploration of the brain using active teaching techniques. several similarities exist between the brain and a computer, and an increasing number of technology teachers use brain / computer comparisons to liven up their curricula. nerves can be compared to wires since, to some extent, both carry information in the form of electrical impulses. our sensory receptor cells share certain similarities with the sensors in computers, but unlike a computer, the brain communicates in part by using chemical neurotransmitters. by prompting students to find similarities and differences between technology and the brain, educators can give students the opportunity to learn more about both topics. videos of classic neuroscience studies are available online and lend themselves to use in the classroom. by accessing and watching the videos, children not only practice computer skills and gain a better understanding of how a computer functions, but they also learn about the brain and how we study it. while most students think brain - science research can only take place in a lab using brain - imaging technology, neuroscientists recruit participants and carry out valid research using web sites that classrooms can access. students can take part in brain - science studies and get a better understanding of what exactly a neuroscientist does. students can participate in actual experiments related to topics such as facial recognition or spatial memory.4 experimental design is part of middle- and high - school curricula, and real brain - science studies naturally lend themselves to a discussion of how scientists create experiments. all of these resources make it easier than ever before to help educators become comfortable with brain - related content. often, a separation between scientists and the lay public makes it difficult to implement strategies based on research. in a recent article on neuroeducation, hardiman and denckla assert, one source of this apparent disconnect is the human tendency to view research findings through the lens of a specific discipline.5 now, however, there are sites that interpret research into lay terms. by making these resources available online, educators can become more comfortable with content and stay up - to - date with breakthroughs in brain science. in turn educators can make stronger connections between neuroscience and learning in terms of study skills and classroom practices. these techniques include strategies for remembering content, such as devising mnemonics or chunking items. chunking aids in memory because our brains organize items into networks for more efficient storage and retrieval. our memory is limited, and by chunking items, or grouping them, the brain is essentially creating a smaller number of items that contain more information. when reciting a phone number, for example, we usually speak the numbers in groups, saying 555 - 555 - 5555, not 5555555555. in effect, our brains store three multiple - digit numbers as opposed to ten single - digit numbers. this grouping leads to easier storage and retrieval. by making clear how these techniques help us remember, teachers can use everyday classroom occurrences to create brain - science - literate students additionally, students can become more aware of their study habits and can adjust accordingly. school administrators and others encourage educators to create lesson plans that include creative, hands - on activities that present content in many different modalities and make use of the brain s plasticity to create connections. students brains can change and adapt based on experiences and stimuli the students encounter in their environments. the brain continually reorganizes neural pathways, and repeated experiences, such as practice, can increase or create synaptic connections. additionally, some brains respond to different types of stimuli better than they do to other types. by using different modalities and reinforcing information, educators can create learning environments that take advantage of the brain s amazing changeability as well as the differences in student brains. the educator can explain to the class why he or she uses both hands - on learning and visual or auditory repetition of the subject, and again use normal classroom practices as teachable moments. attention is always a subject of interest in the classroom. in a study available from the visual cognition lab at the university of illinois, researchers instructed participants to count the number of times a ball got passed among a group of players.6 many times, the participants focused so intently on the ball that they did not notice a person in a gorilla suit pass through the group. by using an example like this, educators can introduce students to a classic neuroscience study while making the class more aware of the idea that attention takes work, and we often miss things right in front of us. discussing common signs of mental fatigue can help students learn when to take breaks, which ultimately results in more studying. the tip - of - the - tongue phenomenon provides one example of the importance of taking breaks. when the phenomenon occurs, we struggle to find a word but have the overwhelming feeling that we will retrieve it at any second. in reality, if we stop trying to think of the word and let our conscious brains rest, we have a better chance of retrieving the thought. even though we are not actively trying to remember the word, the brain is able to keep working on the problem. rather than taking a break, however, most people struggle to think of the word or phrase immediately. by using relevant examples like this universal phenomenon, educators can take the opportunity to explain basic neuroscience principles, such as brain fatigue, to further student awareness of brain function and at the same time improve study habits. educators can incorporate readings from scientific publications into their lesson plans to encourage students to think about how they learn. students have a natural curiosity about the brain and are drawn to articles that discuss both normal and abnormal brain functioning. by taking advantage of this innate interest, educators can introduce students at the high - school level to the skill of reading a scientific paper while at the same time including neuroscience content. as the field of neuroscience continues to grow, so does the link between brain science and education. educators may already use or want to use many of the strategies suggested in this article, but find themselves restricted by a lack of resources or brain - related standards in their curricula, or by their unfamiliarity with brain - science content. but by taking small steps and making stronger connections between neuroscience and what they already teach, educators can make great strides in increasing their students understanding of how we learn and perform. new resources and technologies make accessing and understanding brain - science content easier than ever before, and this ease can translate into classroom practice. there are now programs that match researchers with educators to disseminate information. as part of a 2009 summer institute, harvard university s mind, brain, and education program kurt fischer, the program s director, states, educators have a strong desire to make a connection between biology and scientific findings. to build appropriate connections with biology, we educators must make sure that research is conducted responsibly and that we are training people to truly understand the connection between brain science and the world of education.7 in addition to an increased dialogue among researchers and educators, we may start to see standards that include more brain - science components. as we learn more about how the brain functions and malfunctions, it will become increasingly important for students to have a mastery of brain science. as our scientific knowledge base increases, brain science will likely take its rightful place alongside the other sciences in the classroom as an essential component of the curriculum. therefore, educators must take an active role in creating students fluent in the language and practices of neuroscience. genes to cognition online : students can explore topics in neuroscience in a dynamic and fluid way. brainy kids : this dana foundation web site provides resources and activities ranging from virtual dissections to interactive games. test my brain : researchers recruit participants via this web site, allowing students to participate in and learn about actual neuroscience studies. university of illinois visual cognition lab : this site provides videos of classic neuroscience studies on topics like inattentional blindness. brain hat template : educators can use this hat to aid students in learning the different parts and functions of the brain. neuroscience for kids : this site has many great resources, including recordings of action potentials. the whole brain atlas : this site associated with harvard medical allows students to manipulate brain scans. the scientist and scientific american : these magazines have interesting articles on current topics in neuroscience for classroom use with older students. language arts scent poemsreadings from science magazines and journals readings from science magazines and journals arts and music pipe cleaner neuronsoptical illusionsreadings connecting the arts and neuroscience readings connecting the arts and neuroscience health, nutrition, and fitness brain hatsbrain food activitykinesthetic models of synaptic transmissionbrain dissection kinesthetic models of synaptic transmission technology videos of classic neuroscience studiesmodels comparing computers and the brainvirtual dissection web - explorationonline neuroscience studies videos of classic neuroscience studies models comparing computers and the brain virtual dissection web - exploration online neuroscience studies genes to cognition online : students can explore topics in neuroscience in a dynamic and fluid way. brainy kids : this dana foundation web site provides resources and activities ranging from virtual dissections to interactive games. test my brain : researchers recruit participants via this web site, allowing students to participate in and learn about actual neuroscience studies. university of illinois visual cognition lab : this site provides videos of classic neuroscience studies on topics like inattentional blindness. brain hat template : educators can use this hat to aid students in learning the different parts and functions of the brain. neuroscience for kids : this site has many great resources, including recordings of action potentials. the whole brain atlas : this site associated with harvard medical allows students to manipulate brain scans. the scientist and scientific american : these magazines have interesting articles on current topics in neuroscience for classroom use with older students. language arts scent poemsreadings from science magazines and journals readings from science magazines and journals arts and music pipe cleaner neuronsoptical illusionsreadings connecting the arts and neuroscience readings connecting the arts and neuroscience health, nutrition, and fitness brain hatsbrain food activitykinesthetic models of synaptic transmissionbrain dissection kinesthetic models of synaptic transmission technology videos of classic neuroscience studiesmodels comparing computers and the brainvirtual dissection web - explorationonline neuroscience studies videos of classic neuroscience studies models comparing computers and the brain virtual dissection web - exploration online neuroscience studies | editor s note : there are many simple ways to incorporate neuroscience into the k-12 classroom, even when the subject is not explicitly part of the curriculum. here, michaela labriole, a science instructor at the new york hall of science, provides tangible examples of how teachers can encourage brain - science literacy in students at a time when growing knowledge of the brain is shaping our understanding of how to best foster learning. |
the annotation of conserved domain footprints on protein sequences often serves as the first step toward characterizing protein function in silico. protein domains may be viewed as units in the molecular evolution of proteins and can be organized into an evolutionary classification. the set of protein domains characterized so far appears to describe no more than a few thousand superfamilies, where members of each superfamily are related to each other by common descent. ncbi 's conserved domain database (cdd) attempts to collate that set and to organize related domain models in a hierarchical fashion, meant to reflect major ancient gene duplication events and subsequent functional diversification. computational annotation of protein function is generally obtained via sequence similarity : once a close neighbor with known function has been identified, its annotation is copied to the sequence with unknown function. this strategy may work very well in functionally homogeneous families and when applied only for very close neighbors or suspected orthologs, but it is doomed to fail often when domain or protein families are sufficiently diverse and when no close neighbors with known function are available. to this end, the cdd (1) provides a strategy toward a more accurate assessment of such neighbor relationships, similar to approaches termed cdd acknowledges that protein domain families may be very diverse and that they may contain sets of related subfamilies. of these, only few may have been characterized experimentally, and within this set function may have diverged considerably. while it may be possible, and certainly efficient, to represent such a set of subfamilies with just a single family model, that model could only provide very generic annotation. in cdd curation, we attempt to detect evidence for duplication and functional divergence in domain families by means of phylogenetic analysis. we record the resulting subfamily structure as a set of explicit models, but limit the analysis to ancient duplication events several hundred million years in the past, as judged by the taxonomic distribution of protein sequences with particular domain subfamily footprints. cdd provides a search tool employing reverse position - specific blast (rps blast), where query sequences are compared to databases of position - specific score matrices (pssms), and e - values are obtained in much the same way as in the widely used psi - blast application (3). when cdd is scanned with protein query sequences, a region on a query may pick up more than one overlapping footprint from a set of related models. one of those models provides the best score or lowest e - value, but that alone may not be sufficient to indicate that the query sequence is a bona fide member of the corresponding subfamily. since the cdd collection also contains imported models, which have not been curated at ncbi, search results may present a mixture of curated models (accessions starting with cd..) and un - curated models (accessions starting with pfam, smart or by default, overlapping domain hits are sorted by e - value, but curated models are listed first, if their e - values exceed a secondary significance threshold of 1e-05. default displays are presented in a concise fashion, where domain hits that overlap with the top - ranked domain hits are hidden. cdtree allows users to examine the results of simple phylogenetic analysis on the sequences from a curated domain hierarchy, and view their query sequence in the context of such a phylogenetic sequence tree. figures 1 and 2 demonstrate how one might use a web browser, the curated cdd resource and the cdtree application to obtain confidence for the transfer of annotation from a domain model to a particular protein sequence. a cartoon depicting domain model footprints on a protein sequence can be obtained by following the conserved domains link from an entrez protein search results page, or by submitting a live cd - search (4) request (figure 1a). a particular domain annotation is examined in detail by clicking on the corresponding item in the graphical display (figure 1b). this generates a cd summary page (figure 1c) which lists descriptive information and a multiple sequence alignment including the user query sequence (not shown). if the domain model has been curated at ncbi (cdd accessions starting with cd..), it also lists conserved features that have been recorded by ncbi curators, displays a sequence cluster tree diagram for the particular family and indicates its position in a hierarchy of related domains. a button labeled interactive display with cdtree (figure 1d) launches cdtree. (a) pre - computed domain annotation is retrieved for a protein sequence in the entrez database, gi|47123187 from xenopus laevis. the graphical annotation can be obtained by following the conserved domains link on the entrez document summary for gi|47123187. by default, graphical domain summaries hide redundant information from the user. by clicking on the red balloon (b), representing a conserved domain footprint for the model cd02907, the user launches a summary view of that domain model, which also preserves information about the (query) sequence of interest (c). a section labeled links (data not shown), for example, provides links to all protein sequences in entrez that match the current domain model, to references in pubmed and entrez books, and to the original source of the curated family, which may be a model imported from outside databases such as pfam. clicking on the button labeled interactive display with cdtree (d) launches cdtree on the user 's computer as a local application, which retrieves its data via the web - browser. the cdtree view corresponding to this example cdtree default display, as launched from a web browser, showing a curated domain hierarchy with an embedded user query sequence. a protein sequence found in ncbi 's entrez database was inspected for the presence of conserved domains. the user has followed links from one of the domain footprints annotated on the model, and inspected a particular domain model, cd02907. from the cd summary page, the main window (a) presents the organization of the conserved domain hierarchy, as already visible on the cd summary page (figure 1). in this case, children. the sequence tree shown in panel (c) provides evidence for this particular subfamily structure. groups of branches rendered in the same color correspond to alignment rows that have been assigned to a particular subgroup. sequence trees are always calculated from the curated cd alignments ; in this particular example, the distance data have been obtained from pair - wise alignment scores. pair - wise scores are subtracted from the highest observed pair - wise score to yield distances, and the distance units plotted here correspond to blosum62 scores. by default, a taxonomy viewer window is opened as well (b). users may select and highlight whole branches in the sequence tree view and examine corresponding highlights in the taxonomy viewer, to understand the taxonomic scope of particular subfamilies, or select / highlight taxa in the taxonomy viewer and examine their distribution in the sequence tree. in this example, a user query sequence has been added to one of the models, cd02907. cd02907 gave the best scoring hit in a database search for a particular region of the user 's query sequence. in the sequence tree display, it appears that the user query is a typical member of this particular subfamily, as it clusters tightly with all the other members, and therefore transfer of annotation from the model to the sequence - or functional inference - may be appropriate. cdtree is a helper application for the web browser and must be downloaded and installed on the user 's computer. cdtree functions as a viewer for curated protein domain hierarchies ; it retrieves data for display via the web browser. it uses a separate program, cn3d (5), to view 3d structure and to display and edit multiple alignments of protein structure and sequence. cdtree requires a recent version of cn3d, version v4.2, which is contained in the cdtree installation package. fa2cd, which can be used to convert fasta - formatted multiple sequence alignments into models stored in the cd format, so that they can be imported into cdtree. cdtree also allows the de novo buildup of alignment models starting from single protein sequences. more details can be found on the cdtree home page (see table 1). when the user launches cdtree from a cd summary page, cdtree displays the contents of the curated domain or domain hierarchy, and serves as a viewer for the evidence supporting a particular subfamily structure. by default, cdtree displays a sequence tree view, a taxonomy view and a hierarchy overview. the sequence tree has been pre - computed and is contained in the data sent by the server. when a query sequence has been submitted to cd - search and a matching cdd model has been identified, launching cdtree from its cd summary page will cause the user 's query sequence to be added to the model and cdtree will recalculate a sequence tree. potentially, this allows users to distinguish between several cases : (i) query sequences may be bona fide members of clusters which curators have explicitly declared subfamilies, and which may carry specific functional annotation, as in the case illustrated in figures 1 and 2 ; (ii) query sequences may be bona fide members of subfamilies which do not (yet) carry specific functional annotation ; (iii) query sequences may be members of clusters, which curators have not declared subfamilies, as they may not have seen enough evidence for a subfamily at the time of curation ; (iv) query sequences may be outliers and not cluster with any particular group of sequences in the curated hierarchy. only the first scenario listed above may allow the transfer of specific functional annotation from the model to the query. parent model may be more appropriate, assuming that the parent model provides annotation that is valid for all or the majority of members in a superfamily. cdd can be searched directly as part of ncbi 's entrez database and query system (6), where it is listed as the domains database. entries in cdd are cross - linked reciprocally to ncbi taxonomy, citations in pubmed, and to protein sequences in entrez. links to protein sequences reflect the results of pre - computed rps blast searches and are updated on a daily basis and stored in the cdart database (7). the current version of cdd, version v2.09, contains a total of 12 422 models, of which 2494 have been curated at ncbi. of these curated models, < 300 are solitary domain models, while the rest are organized into hierarchies. 5252 models have been obtained from pfam (version 11) (8), 575 have been obtained from smart (9) and 4101 have been derived from the cog collection (10). together, these models cover about 69% of non - identical protein sequences in ncbi 's entrez protein database. the full set of models as imported from pfam, smart, cog and kog(9), are available in separate search databases, although not all of them have been indexed in entrez, since lineage - specific models with limited taxonomic scope, as well as largely redundant models, have been filtered out. the size of the cdd model collection, details with respect to versions of external databases mirrored in cdd, and the control of redundancy may change over time, as we attempt to provide a resource that is more comprehensive as well as efficient. expert curation of cdd is an ongoing effort, and we plan to eventually replace a majority of imported models with hierarchies curated at ncbi. | the conserved domain database (cdd) is part of ncbi 's entrez database system and serves as a primary resource for the annotation of conserved domain footprints on protein sequences in entrez. entrez 's global query interface can be accessed at and will search cdd and many other databases. domain annotation for proteins in entrez has been pre - computed and is readily available in the form of conserved domain links. novel protein sequences can be scanned against cdd using the cd - search service ; this service searches databases of cdd - derived profile models with protein sequence queries using blast heuristics, at. protein query sequences submitted to ncbi 's protein blast search service are scanned for conserved domain signatures by default. the cdd collection contains models imported from pfam, smart and cog, as well as domain models curated at ncbi. ncbi curated models are organized into hierarchies of domains related by common descent. here we report on the status of the curation effort and present a novel helper application, cdtree, which enables users of the cdd resource to examine curated hierarchies. more importantly, cdd and cdtree used in concert, serve as a powerful tool in protein classification, as they allow users to analyze protein sequences in the context of domain family hierarchies. |
at the end of 2007, a smaller cdna fragment than the expected size was observed from a fetal piglet when a diagnostic reverse transcription pcr (rt - pcr) was performed to amplify the unique genetic marker of the highly pathogenic prrsv, indicating that a novel prrsv variant was found. this strain, designated em2007, was subsequently isolated and the full - length genomic sequence was determined. the genome of em2007 was 15,272 bp, including the poly(a) tail (genbank accession no. eu262603), and shared 87.6% and 57.9% sequence identity with vr-2332 and lv, respectively, indicating that em2007 belongs to the north american genotype. the nsp2 gene of em2007 was 2,736 bp and encoded 912 aa, with a unique continuous deletion of 68 aa at positions 499566, relative to strain vr-2332 (technical appendix). this unique deletion is substantially different from previous prrsv isolates with deletions in nsp2 (3,711). to establish the genetic relationships of em2007, we constructed phylogenetic trees using the neighbor - joining method based on the full - length genome. results showed that em2007 formed a minor branch, which was located in the middle of 2 clusters represented by ch-1a (the first prrsv isolated in china in 1996) and jxa1 (the highly pathogenic prrsv isolated in china in 2006), respectively (data not shown). we also compared the sequence identity of individual em2007 orfs with representative prrsv isolates and found that all orfs have highest identity (> 92%) with ch-1r (an attenuated vaccine strain used in china), except for nsp2 (80.2%). because recombinations have been reported in prrsv in previous studies (6), we speculated that em2007 is a mosaic. to test our hypothesis first, simplot, which calculates and plots the percent identity of a query sequence against a panel of reference sequences in sliding windows (12), was performed using em2007 as a query. based on a set of complete genome sequences, including 56 chinese prrsvs isolated during 1996 - 2008, three representative north american strains (vr-2332, mn184b, and p129), and 2 attenuated vaccine strains (respprrs, ch-1r) (the origin of all strains is listed in the table), the simplot graph clearly showed that em2007 was generally closer to ch-1r than to any other strain. however, there were 3 narrow zones showing disproportionately low levels of similarity between the 2 strains compared to other regions (figure 1, panel a). notably, the 3 narrow zones of em2007 had high levels of similarity with wuh1 (a highly pathogenic prrsv, isolated in wuhan, china in 2006). these results indicated that em2007 is a possible recombinant and ch-1r and wuh1 are 2 putative parental - like strains. recombination was further analyzed by bootscan, a program for the detection of recombination events, and the genetic algorithm for recombination detection (gard) (13). six potential recombination breakpoints, with maximal, were found (figure 1, panel b), indicating that 3 recombination events have taken place within em2007. two recombination fragments (1,4572,312 and 3,2454,584) are located in nsp2 ; the third (8,1959,168) is located in nsp9. recombination event analyses of the em2007 strain of porcine reproductive and respiratory syndrome virus (prrsv). analysis made use of a sliding window of 200 bases and a step size of 20 bases. the y - axis shows the percentage similarity between the selected prrsv sequences and the query sequence. the y - axis shows the percentage of permutated trees using a sliding window of 600 bases and a step size of 20 bases. red vertical lines and numbers indicate the recombination breakpoints identified by the genetic algorithm for recombination detection (gard). numbers corresponding to ch-1r, wuh1, and jxa1 indicate the quantity of informative sites in 7 zones defined by 6 recombination breakpoints, respectively. phylogenetic trees of nucleotide sequences of each recombination region defined by gard, including flanking regions, were further reconstructed by the neighbor - joining method. a large discrepancy (p<0.001, by shimodaira - hasegawa test) between phylogenetic trees inferred for each recombination region in addition, a retrospective survey found that the fetal piglet from which em2007 was isolated was from a farm in wuhan, china, and ch-1r was used on this farm to control prrs, indicating the potential for recombination between ch-1r and wuh1. this evidence further supported the possibility that em2007 is a natural recombinant between ch-1r and wuh1. to test the virulence of em2007, 40-day - old prrsv - free piglets (9 piglets in each group) were inoculated intramuscularly with 10 mean tissue cultures infectious doses/2 ml of em2007, ch-1a, and wuh1, respectively. two piglets from each group were euthanized and necropsied at 7 and 10 days postinoculation, and organs including lung, brain, spleen, kidney, liver, intestines, and lymph nodes were collected for viral load analyses and histopathologic examinations. the remaining 5 piglets in each group were observed for 21 days to evaluate death rates. results showed that piglets inoculated with ch-1a experienced only temporary fever and mild respiratory symptoms. obvious clinical signs, including inappetence, lethargy, high and continuous fever, red discolorations in the body, and blue ears were observed in piglets inoculated with wuh1 (figure 2, panel a). furthermore, severe interstitial pneumonia (technical appendix) and nonsuppurative encephalitis cases were also observed 7 and 10 days postinoculation (figure 2, panel b). piglets inoculated with em2007 also showed similar clinical signs to those seen in the wuh1 group. however, the interstitial pneumonia and nonsuppurative encephalitis were mild and no deaths occurred throughout the experimental period in em2007 group. the results of viremia and viral load also indicated that em2007 was more mild than wuh1, but of substantially higher virulence than ch-1a (data not shown). pathogenicity comparison among the em2007, ch-1a, and wuh1 strains of porcine reproductive and respiratory syndrome virus (prrsv). forty - day - old piglets (9 piglets in each group) free of prrsv were inoculated intramuscularly with 10 mean tissue culture infectious doses/2 ml of em2007, ch-1a, wuh1, respectively. two piglets from each group were euthanized and necropsied at 7 and 10 days postinoculation (dpi) for viral load analyses and histopathologic examinations. mean rectal temperature (a) and survival rate (b) of each group were recorded for 21 dpi. em2007, a prrsv variant with a unique continuous deletion of 68 aa in nsp2, was isolated in china. this variant is a natural recombinant between an attenuated prrsv vaccine strain ch-1r and a highly pathogenic prrsv strain, wuh1. animal experiments demonstrated that while em2007 has higher virulence than ch-1a, the parental strain of ch-1r, it is attenuated relative to wuh1. previous studies have shown that genetic recombination occurs between attenuated vaccine strains of prrsv grown together in culture (14). this study demonstrates for the first time that natural recombination can occur between vaccine and field strains, suggesting that live vaccines have the capacity to shape prrsv evolution by homologous recombination with circulating virus. | em2007, a porcine reproductive and respiratory syndrome virus (prrsv) variant with a unique 68 aa deletion in nsp2, was recently isolated in china. phylogenetic and molecular evolutionary analyses indicated that em2007 is a natural recombinant between a vaccine strain of prrsv and circulating virus. we also tested its pathogenicity in piglets. |
malaria remains the world 's most important tropical parasitic disease and one of the major public health challenges in the poorest countries of the world, particularly in sub - saharan africa, as the prospect of an effective vaccine remains uncertain. although some african countries have increased their spending in health along with the support from the global fund to fight aids, tb, and malaria (gfatm) and other partners, development assistance has been routed largely through public channels, whereas affected individuals seek treatment mostly through the private sector. consequently, the rising cost of medical services and the increasing trends of drug resistance raise critical public health concerns, as this constrains the provision of adequate health care in countries where the disease is endemic. evidence on plasmodium falciparum resistance to chloroquine (cq) between 1980s and early 2000 prompted most countries where malaria is endemic to revise their treatment guidelines, most of them replacing it with sulfadoxine - pyrime (sp) [2, 3 ]. in tanzania, cq was officially replaced with sp in 2001, apparently as an interim policy. however, sp resistance became widespread shortly after its adoption as first - line treatment, because its resistance had already been established in tanzania and the neighbouring countries [58 ], and also, because it had been used as the second - line treatment for malaria in tanzania until 2001, when it became the first - line treatment drug. its long half - life could also have contributed to the observed rapid development of resistance. most malaria endemic countries replaced sp with artemisinin - based combination therapy (act) between the year 2002 and 2008. tanzania replaced sp with artemether + lumefantrine (alu) as the firstline national antimalarial treatment in 2006. treatment of malaria in tanzania is typically guided by official recommendations from the ministry of health and social welfare (mohsw) regarding drugs of choice for various situations. treatment refers to the drug officially recommended as the drug of first choice for the treatment of uncomplicated malaria. treatment refers to the drug officially recommended as an alternative primarily to be used for treatment of patients in whom the first - line treatment failed to clear the infection and other select patients (such as those who are hypersensitive to the first - line treatment). third - line treatment typically refers to the drug recommended for severely ill patients (a rescue drug). in practice, few treatment failures are recognized, and patients are often moved directly from first to third - line treatment, consequently, little second - line drug is used compared to the first - line drug. sp replaced cq as the recommended first - line treatment on the tanzanian mainland in august 2001, after 18 years of its use as a second - line treatment since 1983. policy change is both an expensive and difficult process, and many endemic countries are already faced with difficult decisions on how to replace ineffective antimalarial drugs in use with more efficacious, but also more costly alternatives. one of the greatest challenges in this decision - making process is the fear that resistance will develop rapidly to the replacement drug, initiating an endless cycle of drug replacement and escalating costs. studies conducted one year after adoption of sp as the first - line antimalarial treatment policy in tanzania indicate that inadequate evidence - based education and sensitization of health workers together with negative media reports negatively impacted the adoption of the new policy [4, 10, 11 ]. theory predicts that we can protect the efficacy of future antimalarials by changing treatment practice or drug formulation, but the potential success of such interventions rests upon their impact on drug pressure in the field. mathematical models predict that drug coverage / extensive drug usage is the primary determinant of drug pressure, and the driving force behind the evolution of drug resistance [14, 15 ]. research findings have established that resistance to cq and sp results from accumulation of multiple mutations in the respective target gene(s). once formed, these mutations spread within and between continents [8, 1618 ]. we have systematically followed the rate of selection of sp resistance alleles in a one - year prior and five years after adoption of sp as the first - line antimalarial treatment in tanzania. from the molecular genetics perspective, we show that the trend displayed by resistant alleles together with linkage disequilibrium between double dhps and triple dhfr resistant alleles correlates with published reports of media [4, 10, 11 ] and health worker 's influence on policy adoption. community surveys were conducted during july, august, and september of 2000, 2001, 2002, 2004, 2005, and 2006 in kilombero (population. the surveys were part of a large combination therapy pilot implementation programme in tanzania, the interdisciplinary monitoring programme for antimalarial combination therapy (impact - tz). for the purpose of the study, kilombero and ulanga districts were treated as a single district, because population movement between these two districts is high and the study population spans the border region. plasmodium falciparum malaria transmission in the study area is intense (with an estimated entomological inoculation rate of 367 infectious bites per person per year and perennial with some seasonal fluctuation). a total of 24,212 adults and children belonging to randomly selected households participated in the study. a finger - prick blood sample for blood slide and filter paper bloodspot were collected from each individual in the household. the filter paper bloodspots were air - dried and put in self - sealing plastic bags with desiccant and stored on dry area at room temperature. bloodspots from microscopically positive subjects were selected and preserved at room temperature for molecular genotyping. prior to the first national antimalarial policy change in tanzania in august 2001, the first - line treatment drug was cq, sp was the second - line, while quinine (q) was the third - line treatment. the policy change replaced cq with sp as first - line treatment and sp was replaced with amodiaquine (aq) as second - line treatment, while q continued serving as the third - line treatment. issues surrounding the 2001 policy change are detailed in. the policy decision was supported by research evidence indicating an intolerable parasite resistance to cq and clinical cq treatment failure rates of above 52%, as compared to sp and aq failure rate of about 9.5% and less than 4.6%, respectively. research also indicated that since sp was also facing rising resistance trend, the need for a more effective drug was indispensable, but for an interim 510-year period, it was justifiable to recommend sp that was relatively more cost effective than cq and aq. the government launched the policy change considering that studies (ethically approved by the ministry of health) on therapeutic efficacy and cost effectiveness of artemisinin drug combination therapies were underway. nevertheless, the process of communicating research results and recommendations to policy - making authorities involved critical debates between policy makers and researchers, among the researchers themselves and between the researchers and general practitioners, the speculative media reports on sp side effects and reservations by the general public concerning the rationale for policy change, when to change, and to which drug of choice. the second national antimalarial policy change was implemented in 2006, whereby artemether and lumefantrine (alu) replaced sp as the first - line treatment. however, the policy remained silent about the second - line and the third - line treatments although in practice, q is used as both the second - line and rescue drug, because when a patient on alu therapy fails to clear parasite, he / she is moved straight to q. overall, while the dynamics surrounding the government 's policy to replace cq with sp in 2001 have been well documented, there is very little published data on the process of change from sp to alu. the evidence supporting the change came from a number of in vitro and in vivo studies showing unacceptable levels of parasite resistance to sp and the impact - tanzania studies which piloted the use of act in rural tanzania. scientific and ethical clearance was granted from the medical research council of the national institute for medical research in tanzania, the centers for disease control and prevention, usa, and the london school of hygiene and tropical medicine. a section of the dried blood spot filter paper was excised using a sterile blade or scissors and soaked in a 1 ml of 0.5% saponin in 1x phosphate buffered saline (pbs) overnight in a 96-deepwell plate. the segment was then washed twice in 1 ml of 1x pbs and was finally boiled for 8 min in 100 l pcr quality water with 50 l of 20% chelex suspension (ph 9.5). nested pcr was used to amplify a 594 base pair (bp) fragment of dhfr and a 711 bp fragment of dhps each containing the sequence, where mutations are found. pcr was performed in 96 well plates with 25-l pcr reaction volumes containing final concentrations of 0.25 m oligonucleotide primers, 2 mm mgcl2, 250 m each deoxyribonucleotide triphosphate (dntps), and 1x taq polymerase. 1 l of dna template was used in the outer (primary) pcr reaction mixture for dhfr and dhps amplifications. for the inner (secondary) the outer dhfr pcr products were diluted three fold before a 1 l was introduced into the inner pcr reaction mixtures. the amplified pcr products were screened for dhfr and dhps sequence variants at 10 loci, where single nucleotide polymorphisms (snps) are known. the sequence changes (and the amino acid substitutions they code for) are summarised in table 1. pcr products were spotted in a 12 by 8-grid and cross linked onto nylon membranes and probed for sequence polymorphisms by hybridisation to specific oligonucleotide probes described previously. for analysis of samples collected in 2000, the visualization of hybridised digoxygenin labelled probes on membranes was performed by the alkaline phosphatase - catalysed breakdown of the cspd substrate (roche boehringer mannheim, mannheim, germany) and visualised by exposure on hyperfilm - ecl (amersham pharmacia biotech, little chalfont, buckinghamshire, united kingdom), according to boehringer mannheim recommendations and previously described in. for analysis of samples collected in 2001 and 2002, the probed blots were visualised using ecf substrate and detection using a phosphoimager (storm). inspection of autoradiographic films was carried out by light box illumination, while the phosphoimager output was recorded through viewing of digitally captured images of chemiflourescent signal. the change in the method by which probe hybridisation signal was visualised did not affect the results in any way since the probes and hybridisation conditions were unchanged. the stringency and specificity of the hybridisation process was confirmed by inspection of a series of 4 controls with a known single genotype variant sequence. a snp was considered to be present in the pcr product when the intensity of signal was higher than that of the background. the blots were scored independently by two people. in our analysis, we aimed to establish the relative abundance of different point mutation haplotypes at dhfr and dhps. since bloodstage p. falciparum is haploid, this is very straightforward when an infection consists of a single genotype, because only one form of sequence at every snp locus is seen. when infections are composed of multiple genotypes a mixture of different sequence variants occur making the inference of point mutation haplotypes within that infection more difficult. the presence, absence, and relative abundance of hybridisation signals for every probe were recorded at each locus. a sample was considered to have a single haplotype when only one sequence variant was found at each locus. blood samples were categorised as having a single, a majority or a mixture of sequence at every snp locus. majority and mixed genotype infections were differentiated according to the relative intensity of signal. to determine the relative abundance of different point mutation haplotypes in the parasite population, only one haplotype was counted from each infection and those mixed infections, where haplotypes could not be resolved were omitted from the calculation of haplotype frequencies. hence, frequency data is based upon a subset of isolates which were unmixed or had a predominating majority haplotype. a breakdown of the proportions of isolates which were successfully pcr amplified and genotyped as single, majority or mixed haplotype infections is given in table 2. statistical comparison of allele frequencies at dhfr and dhps in the various sites was carried out using chi - squared analysis in stata version 9.2. the calculation of binomial exact 95% confidence intervals was carried out using stata version 9.2. 24,212 people were sampled and 4,513 asymptomatic infections identified. dna was extracted from the 4,513 p. falciparum positive bloodspots and pcr of dhfr and dhps performed once, giving a combined rate of pcr amplification success of 69% for both genes the amplified products were screened for all the variant sequences described in table 1. out of the 2,906 isolates which amplified for dhfr, 2,185 (75%) were single or majority genotype infections and the point mutation haplotypes could easily be resolved. of the 2,953 samples which amplified for dhps, 2,392 (81%) were single or majority genotype with resolvable haplotypes. the point mutations found in the dhfr gene were n51i, c59r, and s108n as reported elsewhere. they were found in the following haplotypic arrangements cncsi, cncni, cnrni, cicni, and cirni which are reported to be common throughout eastern africa, tanzania, malawi, kenya [5, 23 ] and uganda [24, 25 ]. five mutations were found at dhps ; s436a, s436f, s436c, a437 g, and k540e as reported elsewhere. these were found in nine distinct haplotypic arrangements ; five of which (sakaa, aakaa, sgeaa, sgkaa, and saeaa) were described previously in isolates from east africa [5, 7, 2325 ] while the remaining four (cakaa, fakaa, aaeaa, and faeaa) were found in extremely low frequency and have not been reported before, presumably because of their rarity. the dhfr cirni haplotype and the dhps sgeaa haplotype have the greatest significance for sp efficacy ; their frequency in the six successive surveys displayed three distinct phases as shown in figures 1 and 2. changes occurring under the cq policy (phase 1 : 2000 to 2001) are markedly different to those occurring under the sp policy (phase 2 : 2001 - 2002). likewise, the frequency of the dhps double mutant a437 g k540e (shown in figure 2) did not change significantly in phase 1, between 2000 and 2001 (p.497). contrastingly, there was a highly significant change in phase 2, between 2001 and 2002 (p.0001) (95% ci). in phase 3, between 2002 and 2006 both the dhfr triple and the dhps double mutant alleles showed a marked retarded rate of selection of the two unlinked resistant alleles. in a further subset of samples, where dhfr and dhps haplotypes were both conclusively resolved, it was possible to measure the frequency of two locus genotypes. in figure 3, the frequency of the triple dhfr + double dhps genotype for the six successive surveys is presented. as for the case of the dhfr triple and the dhps double mutant alleles, the frequency was around 0.05 in phase one and there was no change between 2000 and 2001 (p.824) but a remarkable 5 fold increase to frequency of 0.28 occurred in phase two between 2001 and 2002 (p.0001). in phase three, this two locus genotype (the triple dhfr + double dhps) was characterised by a marked reduction of the rate of selection between 2002 to 2006. as well as the highly resistant dhps a437 g k540e double mutant, a number of single 436 mutant alleles were recorded. among these, by far the most common was the s436a, which was consistently found at frequencies of 10%20% in all six successive surveys. figure 2 shows the frequencies of the sensitive s436a single mutant and the a437 g k540e double mutant alleles through time. from these data, it is clear that the increased frequency of the double mutant allele displaced the sensitive allele, which decreased nonsignificantly in 2000 - 2001 (p.791) and significantly in 2001 - 2002 (p.003). interestingly, the frequency of the a436a allele remained static in all time points in both districts. the effect of drug policy changes on the frequency of sensitive and double mutant dhfr alleles is shown in figure 1. the increase of the triple mutant allele acted to displace sensitive alleles which showed a substantial but non significant decline in phase one (2000 - 2001) and phase three (20022006) (p.352) and a highly significant decline in phase two (2001 - 2002) (p.0001). the double mutant dhfr alleles, which confer intermediate levels of resistance in vitro, neither increased nor decreased, remaining at a frequency of around 10% in both districts throughout all surveys. to examine the effect of simultaneous selection by pyrimethamine on dhfr and sulphadoxine on dhps, we looked at two - locus genotypes sampled from kilombero / ulanga in the six successive surveys. taking the subset of samples for which point mutation haplotypes could be unequivocally resolved for both genes, we compared the observed with expected frequencies generated from contingency tables. no significant linkage disequilibrium was found between dhfr and dhps loci in either 2000 (d ' = 0.5333, p.3307) or 2001 (d ' = 0.0417, p.75339) indicating that the drug selection pressure was insufficient to overcome the force of recombination. however, a highly significant ld (d ' = 0.3633, p.00001) was observed in 2002 indicating that the drug selection pressure was already sufficiently higher to overcome the effect of recombination. the degree of significance of the ld started to show a decline in 2004 (d ' = 0.2948, p.00001) and 2005 (d ' = 0.2595, p.00001), and by 2006, there was no more significant ld (d ' = 0.0814, p.2686) (table 3). this indicates that there was a gradual decline in drug pressure between 2002 and 2006. this study has generated results of six - year followup of dhfr and dhps genetic changes in the p. falciparum in two rural districts of south eastern tanzania. a high throughput sequence specific olignucleotide method was used to genotype point mutations present at the sp resistance genes, the dhfr and dhps in population samples of p. falciparum infections collected annually from year 2000 to 2006 in kilombero / ulanga. significant increase of the frequencies of resistant dhfr and dhps haplotypes was observed after the change of the national antimalarial guidelines from first - line cq to first - line sp. clearly, changes of drug use policies implemented during the conduct of this study had impact on the genetic determinants of sp resistance. the study has demonstrated that the influence of the national antimalarial treatment policy change on the genetic composition of the malaria parasite population was profound. dhfr and dhps allele frequency did not change significantly during 12 months (year 2000 to 2001) of cq as first - line and sp as second - line, but after the switch to sp as first - line treatment (year 2001 to 2002), the frequency of the triple mutant dhfr allele increased from 31%52%, while the frequency of the double mutant dhps allele increased 3-fold. a combination of these alleles is predictive of in vivo failure of sp treatment [5, 6, 27 ], and the rapid increase of this genotype from 5%29% between 2001 - 2002 suggests that the outlook for sp efficacy in this region was deteriorating. in fact, this observation was borne out by the results of sp treatment efficacy monitoring in south - eastern tanzania during 2003 which found that 49% of sp treatments failed by day 28. due to this high rate of sp resistance, national policy decision was switched to the recommended first - line treatment to artemether and lumefantrine in 2006. the observed escalation of sp resistance alleles is attributed to the policy change which brought about immediate shift in treatment practice, characterised by increased demand of sp both in the public and private sector leading to intense sp drug pressure. again, because the change of policy was nation wide, the same sp pressure was likely applying to the districts surrounding the study area, narrowing the likelihood of significant higher selection and hence flow of resistant parasites from the neighbouring districts to the study area. prior to the introduction of sp as first - line therapy (between 2000 and 2001), sp was widely available for self treatment via purchase from pharmacies, shops as well as through formal health facilities as the second - line treatment. the sp drug pressure exerted during this period was only sufficient to maintain dhfr and dhps resistance alleles at constant levels. by contrast, following the introduction of sp as first - line in 2001, there was dramatic increase in sp use leading to intense sp pressure and escalation of dhfr and dhps resistance alleles. although sp is currently being replaced as a first - line drug for treatment of malaria in tanzania and much of the rest of africa, it will continue to be used in intermittent preventive treatment of infants and pregnant women. the drug pressure applied when sp is used solely in intermittent programmes of treatment in infants and pregnant women will probably more closely resemble the situation observed between 2000 - 2001, when sp was second - line treatment than during the period it was used as the first - line treatment. the consequences of more restricted use for future efficacy of sp are therefore two fold. firstly, rates of resistance allele frequency change are likely to stabilise as a result of reduction of sp selection pressure. secondly, the reduction of selection is predicted to disassociate the triple mutant dhfr allele and double mutant dhps allele which will reduce the frequency of highly pyrimethamine and sulphadoxine resistant parasites in the p. falciparum population at large. direct observation such as plasma drug levels in the patient gives the best direct measure of drug selection in the population and it would be interesting to examine this in comparison to the ld measures. ld on the other hand is known to give a good in - direct indication of sp selection pressure, and hence, the lack of significant ld in kilombero / ulanga population in 2000 and 2001 when cq was the first - line treatment conforms to expectations. in 2002, however, the significant ld emerged indicating intensification of drug pressure following country wide distribution and use of the sp as the new first - line treatment. contrary to our expectations, just one year of sp use as first - line treatment, a noticeable decline in the rate of increase of resistant dhfr and dhps alleles along with decreasing significance of the ld was observed and continued in the rest of surveys. by 2006 the ld was no longer significant indicating that the sp drug selection strength was insufficient to keep the two alleles, the triple dhfr and double dhps alleles in combination. to understand the reason for the three phase pattern of progression of sp resistance alleles observed in this study, we retrospectively reviewed the process of policy change in tanzania in 2001, from cq to sp first - line treatment. in order to explain the underlying cause of reduction of sp selection strength just one year after sp first - line adoption in tanzania, we retrospectively reviewed the process of policy change from cq to sp first line. the study also examined retrospectively the public perception and the role of media in informing the public, who are the end point consumers of the policy. accordingly, the national task force on antimalarial drug policy was formulated in may 1999 as a subcommittee of the national malaria advisory committee (nmac) in consultation with the east african network for monitoring antimalarial treatments (eanmat) and who country office in dar es salaam. on 23 july 1999, the task force developed a three - page summary, drawing on evidence from clinical trials in the sentinel sites. it was recommended that the decision to change the policy should be interim because of increasing evidence of high sp resistance in various parts of the country such as muheza and kilombero districts. after the presentation of the new policy by the task force, a series of news papers and some private radio stations began to inform the public that cq was no longer a recommended drug for malaria treatment and that the government was considering replacing it with a new drug. this information caused public concern and debates erupted in different parts of the country about the rationality for the change [4, 10, 11 ]. those involved were the general public, the research community, traders, the pharmaceutical industry, and health - care providers in both the public and private health facilities. in an attempt to maintain public confidence, the government through the ministry of health gave out a press release that indicated its stand concerning the treatment guidelines to be followed while strategies were underway to make an appropriate decision. anecdotal evidence indicates that many health professionals were unaware of the extent of resistance to cq and did not perceive an urgent need for change. while who recommends change to an alternative drug when the treatment failure rates with the first - line drug reaches 25%, evidence from different sentinel sites in the country indicated that up to the time of policy change, cq treatment failure rate had already reached 52% (ranging 28%72%), 9.5% for sp (ranging 6%32%), while treatment failure to other drugs such as aq and q was less than 4.6% (ranging from 3.5%6%). more criticism ensued between the ministry of health, the local newspapers and the research community on the effectiveness of cq. while the public were advised to remain patient until the government gathered sufficient evidence, researchers continued to disseminate information indicating high levels of resistance and suggesting for replacement of cq with a more suitable drug. the government 's official announcement of the policy change came out of the media in 2000 although the actual implementation officially started on 1st august 2001. clearly, there were big differences between different actors which spurred a great deal of challenges in the long term uptake and support of the new policy down to the communities. a study conducted in 2002 (one year of policy adoption) reported widespread fear and negative perceptions about the use of sp. lack of educational campaign from the ministry of health and domination of the media with reports of adverse sp reaction, led to a growing trend of lack of confidence in sp use as demonstrated by the public [10, 11 ]. the trend of events in the policy adoption and implementation described here corroborates well with the genetic events described in figures 1 and 2. within the first year of policy implementation, usage of sp was highest (intense selection pressure with significant positive ld in the population). however, as adverse reaction to sp continued to mount, and were exaggerated by the media, the drug gradually lost popularity. it is also very possible that as sp resistance intensified over time, an increasing number of sp users reverted to alternative antimalarials again reducing sp selection pressure. the national policy change brought about an immediate shift in treatment practice and this in turn had a highly significant impact on drug pressure. this shows that even in rural areas where access to treatment is imperfect and treatment coverage relatively low, first - line sp selection is sufficiently strong to rapidly change the genetic composition of the parasite population in one year. furthermore, selection applied was strong enough to overcome recombination pressure and create linkage disequilibrium between the unlinked genetic determinants of resistance to the two co - administered drugs (sulfadoxine and pyrimethamine), showing that recombination is not a barrier to the evolution of multidrug resistance in high endemicity settings. the data presented here show that both the media and health workers play a key role to modify the political technical and social values of policy adoption success. the need to consider when and how research information should be communicated between different stake holders is very crucial and should enable avoiding similar dilemmas in future. this study was funded through an interagency agreement between the united states agency for international development (usaid) and cdc and a cooperative agreement between cdc and the ifakara health research and development center (ihrdc). usaid did not participate in the design, collection, analysis, or interpretation of the data, in the writing of the paper, or in the decision to submit for publication. | drug resistance negatively impacts malaria treatments, making treatment policy revision unavoidable. so far, studies relating sociopolitical and technical issues on policy change with malaria parasite genetic change are lacking. we have quantified the effect of malaria treatment policy on drug pressure and the influence of the media, policy makers, and health worker relationship on parasite population genetic change in kilombro / ulanga district. cross - sectional surveys of asymptomatic infections conducted before, during and after the switch from chloroquine to sulphadoxine / pyrimethamine were used for genetic analysis of sp resistance genes in 4,513 asymptomatic infections identified, and their frequency change was compared with retrospective study of the documented process of policy change. highly significant changes of dhfr and dhps resistance alleles occurred within one year of switch to sp first line, followed by a decline of their rate of selection caused by reduction of sp usage, as a result of negative media reports on sp usage and lack of adequate preparations. |
on december 8, 2014, a 78-year - old man (patient 1) from gbarnga (bong county), liberia, was admitted to the bong county ebola treatment unit (etu), where test results were positive for ebola by reverse transcription pcr. he reported recent travel to monrovia (montserrado county), where he cared for his 32-year - old son, a health care worker who died from an acute illness. on december 9, another son of patient 1 (patient 2, 39 years of age), who lived in monrovia, had fever, headache, and malaise and sought care at hospital a in bong county. he did not report contact with patient 1, nor did he report that he provided care for his sick brother in monrovia. on december 10, hematemesis developed, and the patient was transferred to the bong county etu and treated for laboratory - confirmed evd. all contacts were initially symptom free and were quarantined at a local holding center for 21-day monitoring. no contacts in monrovia were reported by patients 1 or 2. on december 16, bong county health officials were notified that a 15-year - old girl (patient 3) with fever, subconjunctival hemorrhage, and thrush was at hospital a. she had traveled 4 hours by taxi from monrovia to be near her ill grandfather and father (patients 1 and 2) and did not report exposure to evd patients or contacts in monrovia. she was admitted to the etu, and evd was confirmed. the next day, 4 additional family members who traveled by taxi from monrovia were stopped at a roadside monitoring station in gbarnga. all had fever and nonhemorrhagic symptoms and were transferred by ambulance to the etu for evaluation ; 2 family members (patients 4 and 5) had positive test results for evd. the 2 family members whose results were negative for evd, along with the taxi driver and a nonfamilial passenger, were transferred to a local holding center for 21-day monitoring. contact investigations for patients 4 and 5 revealed no new contacts in monrovia, but the patients reported that they resided in the same house in monrovia with patients 2 and 3, who were receiving treatment in bong county. because family members with evd had recently arrived from monrovia and were being treated in bong county, yet sources of infection and additional contacts were uncertain, bong county requested that montserrado county health officials conduct an investigation to identify patients and contacts at the monrovia address so that potential evd patients could be isolated and monitored. the monrovia investigation revealed that patients 13 had contact with patient 1 s ill son, who was designated the putative source - patient (patient 0). fever, headache, joint pain, and abdominal pain developed in patient 0 on november 14, 2014, and he was cared for at home by his family for 7 days while his symptoms worsened. although the patient and family members were aware of the evd epidemic, they did not think patient 0 had evd because he had no vomiting, diarrhea, and hemorrhagic symptoms ; they believed he had a spiritual illness. on november 21, he was taken to a church with the hope that he would be healed through prayer. he died there on november 24, and his body was carried to his residence for mourning and burial preparation. because all unexplained deaths were presumed to be ebola related, an evd burial crew retrieved his body for cremation the following day, despite resistance from the family and only after persuasion by local community leaders. after the body was removed, the home was sprayed with disinfectant, and the mattress, clothes, and other personal items used by patient 0 were burned. an attempt was made to identify additional contacts ; however, the family was reluctant to cooperate with health officials and reported being angry about the cremation of patient 0 and destruction of property, although these practices were routine at that time for controlling evd in monrovia. the family in monrovia began cooperating with montserrado county health officials 3 weeks later, on december 18, after learning that 5 family members (patients 15) had evd and after being provided with new mattresses and a small ration of food. at this time, they revealed 2 previously unreported symptomatic family members (patients 7 and 8). as of january 11, 2015, a total of 10 cases were included in this cluster. eight (80%) patients in this cluster were not identified as contacts before their evd diagnosis, and 4 (40%) sought care outside the county where they resided (table ; figure). timeline (a) and transmission diagram (b) of ebola virus disease cluster, bong and montserrado counties, liberia, november identifying sources of infection for index patients and tracing contacts are major components of evd prevention and control efforts (3), yet carrying out these policies is challenging when those ill with evd do not reveal the names of possible sources or contacts who could have been exposed to disease. detection delays and ineffective contact tracing occurred in this cluster in part because the family believed that the mandatory cremation and property destruction taken as public health actions in monrovia harmed more than helped. consequently, some family members sought care in bong county, riding 4 hours in a taxi from their home in monrovia, a distance of 197 kilometers. furthermore, family members were reluctant to reveal contact names in monrovia and initially concealed knowledge of symptomatic persons. this cluster may have been prevented if patient 0, presumably infected at the clinic where he worked, had been trained in infection control procedures and had access to personal protective equipment. additional exposures and subsequent infections could have been prevented if he had been identified earlier as a suspected evd patient, if testing had been performed on his body, if the results had been reported to the family, and if the monrovia contacts had been followed daily to identify, isolate, and treat symptomatic persons. had contact tracing identified patients 13 as patient 0 s contacts and isolated them immediately after symptoms developed, 6 cases of evd (in patients 49) and 4 deaths (patients 4, 5, 7, and 8) might have been prevented. rapid implementation of contact tracing to prevent disease transmission and increased coordination and communication between jurisdictions are critical to control of evd. these efforts can identify case - patients who may have entered the community from another jurisdiction (to better understand importation and transmission patterns) and improve case finding and contact tracing to ensure that no cases are missed (8,9). the effectiveness of these efforts depends on trust between public health officials and the communities they serve. | lack of trust in government - supported services after the death of a health care worker with symptoms of ebola resulted in ongoing ebola transmission in 2 liberia counties. ebola transmission was facilitated by attempts to avoid cremation of the deceased patient and delays in identifying and monitoring contacts. |
glycosyltransferases (gtrs) are enzymes that carry out diverse biological functions by catalyzing transfer of activated sugars to varied acceptor molecules, like proteins, nucleic acids, saccharides, lipids or small molecules (13). among the various classes of gtrs, one group of particular importance is the family of gtrs that differentially glycosylate the secondary metabolite aglycone during the late stages of biosynthesis to produce biologically active antibiotics. the site of glycosylation, nature of the sugar and the number of sugars are known to affect the efficacy of the antibiotic (4,5). the ndp - sugar substrates for antibiotic gtrs are typically tdp - hexoses, with the hexoses in either d- or l - configuration (6). a variety of functional modifications of the deoxyhexoses can result in enormous variations in donor substrates. similarly there can be a vast repertoire of acceptor substrates, as these aglycones can be polyketides or nonribosomal peptides with enormous variations in their chemical structure. therefore, understanding the donor / acceptor specificity of gtrs is crucial for the rational design of novel antibiotics by the reprogramming of their biosynthetic process. the relaxed substrate specificity of some gtrs from vancomycin, chloroeremomycin and elloramycin pathway and mutational experiments on gtrs from urdamycin pathway have been successfully exploited to generate new compounds by using unnatural substrates (712). however, for the successful engineering of gtrs with altered recognition properties, it is essential to have powerful bioinformatics tools that can correlate the sequence of the gtrs to the chemical structures of their substrates and provide guidelines for various genetic manipulation studies. such tools can also help in predicting substrate specificities of a large number of uncharacterized gtrs found in newly sequenced genomes. we have recently used a knowledge - based approach to develop such in silico tools (1315) for analyzing polyketide synthases (pks) and nonribosomal peptide synthetases (nrps). apart from helping in experimental characterization of several proteins in mycobacterium tuberculosis from pks / nrps family (16,17), these tools have also been successfully used to reprogram the pdim biosynthesis pathway to produce an altered metabolite (18). in principle, a similar knowledge - based approach can be used for identifying specificity determining residues of gtrs involved in the biosynthesis of secondary metabolites. recently available crystal structures of few antibiotic gtrs (1921) indicate that, despite the divergence in their primary sequence they adopt the same structural fold. in view of the conserved structural fold, the structures for various gtrs with known substrates can be modeled using threading approach and putative substrate binding residues can give insights into the structural basis of their substrate recognition. hence, we have carried out a comprehensive analysis of the sequence and structural features of various experimentally characterized gtrs, and based on the results of this analysis, we have developed, searchgtr, software for correlating sequences of gtrs to their substrate specificity. in this work, we describe methods for developing searchgtr, its various features and results from benchmarking. searchgtr uses a knowledge - based approach to carry out various predictions by comparing the query sequence with gtrs of known specificity. hence, an essential task in the development of this tool involved compilation of sequences of experimentally characterized gtrs in the form of a searchable database, gtrdb. figure 1 illustrates the organization of the database. the compilation of gtrdb involved extracting sequences of gtrs glycosylating various antibiotics and identifying their donor and acceptor specificity through exhaustive literature survey. gtrdb is a compilation of 102 annotated gtr sequences from 52 different natural product biosynthetic gene clusters. the database gives the chemical structure of the antibiotic and a variety of information on the gtrs involved in its biosynthesis. for each gtr, gtrdb stores only the primary sequence in fasta format, name of the source organism, identifiers for other databases like genbank (22), swiss - prot (23), cazy (3) etc. using these identifiers, links are provided to respective databases for additional details on these proteins. gtrdb also provides link to the literature which describes the experimental characterization of the corresponding gtr. for each gtr if unnatural substrates are known for a specific gtr, they are listed and a link is also provided to the corresponding literature. for deriving the homology relationships between various gtrs in the database, gtrdb stores both global and local alignments of each gtr with all other gtrs. in order to derive information about the structural features of various gtrs, each gtr sequence has been aligned, using a local version of threader program (24), with structural templates from the antibiotic gtr family available in pdb (25), namely 1iir, 1rrv and 1pn3. the structures of antibiotic gtrs show bi - domain architecture with the n- and c - terminal domains containing a majority of residues binding to acceptor and donor respectively. hence, the n- and c - terminal domains and linkers for each gtr have been identified from their threading alignments with 1rrv. out of the three crystal structures of antibiotic gtrs, 1rrv and 1pn3 represent gtrs in complex with donor as well as acceptor substrates. using the ligplot interactions from pdbsum database (26), 23 acceptor binding residues (abr) and 15 donor binding residues (dbr) were identified for each gtr from their respective threading alignments with 1rrv. similarly, 18 abr and 16 dbr were identified for each gtr using 1pn3 as the template. it may be noted that despite the high degree of sequence and structural similarity between 1rrv and 1pn3, identical acceptor substrate binds to them in different orientations and only a very small number of amino acids are common between the abr of 1rrv and 1pn3. it has been proposed that vancomycin gtrs achieve their regioselectivity for glycosylation by employing these two different binding modes (20,21). in view of the limited number of structures for gtrs in the complex with the substrates, prediction of the sbr for the gtrs in gtrdb thus, the sbr identified by using 1rrv and 1pn3 as templates only help to give a consensus view of the approximate location of the substrate binding pocket. a more accurate prediction of the substrate binding pocket is possible only if additional crystal structures for members of other antibiotic gtr families are available. the program consists of two major components, the backend database gtrdb, which have well - annotated gtr sequences and the query module searchgtr, which allows analysis of an uncharacterized gtr sequence. it is aligned with all the 102 gtrs in the gtrdb using both local alignment program blast as well as needleman and wunsch global alignment programs. sequence alignments are carried out using blosum62 scoring matrix, e - value cut off of 0.000001 and default values of gap initiation and extension penalties. after selecting a particular homologous sequence, the user can view its local / global alignment with the query. the page showing the alignment also provides a link to the gtrdb, which gives other details on the aligned gtr sequence, specifically the chemical structures of its donor and acceptor. this information on donor and acceptor substrates of the best matching gtr can provide important clues about possible substrates for the query gtr. using the best matching gtr from gtrdb and its precomputed threading alignment with 1rrv, searchgtr identifies the n- and c - terminal domains and linkers in the query sequence. these are then depicted in a pictorial format in a pop up window (figure 2). the numbers below the image of the domain specify its start and end positions on the query sequence. if there are parts of gtr which are overhangs on the n - terminal or c - terminal domains, they are shown as n - tail and c - tail, respectively. the fasta sequence of the domain or linker or tail can be viewed by clicking on the respective images. the program identifies putative sbr in the query sequence from the threading alignment of its best match with 1rrv or 1pn3 (figure 2). the user is given the option to choose either 1rrv or 1pn3 from a pull down menu as template for identifying the sbr. links are provided to the pdbsum site for viewing the interactions between the protein and the ligand in the template structure using the program ligplot. as can be seen from figure 2, for the structural template 1rrv/1pn3, the amino acids interacting with the ligand via hydrogen bonds and hydrophobic contacts are highlighted in different colors. however, no such coloring scheme is used for the query or the best match as equivalent residues are expected to have dissimilar amino acids depending on the chemical structure of the donor / acceptor substrates. in order to facilitate correlation between the residues in the substrate binding pocket and substrate specificity, the program provides a link to the chemical structure of the acceptor / donor substrate of the best matching gtr. additionally, searchgtr provides an option for comparing the query sequence with the experimentally characterized gtrs in terms of their substrate (acceptor / donor) binding pocket residues alone. the sbr of the query, identified based on either 1rrv or 1pn3 as the template, is compared with the corresponding library of sbr from gtrs with known donor and acceptor. the query sbr are pair - wise compared with sbr of all gtrs in gtrdb and each position is scored using the blosum62 matrix. the results are displayed as a sorted list of the best matching acceptor / donor - binding residues from gtrs in gtrdb along with their donor / acceptor specificities. this allows a one to one comparison of amino acids at each position of donor / acceptor binding pocket. furthermore, the program also has options for the alignment of the query sequence with any specific gtr in gtrdb by selecting them based on the type of donor substrate, acceptor substrate or type of antibiotic. global alignments are carried out using a local version of needleman and wunsch programs from the emboss package (27). searchgtr uses a knowledge - based approach to carry out various predictions by comparing the query sequence with gtrs of known specificity. hence, an essential task in the development of this tool involved compilation of sequences of experimentally characterized gtrs in the form of a searchable database, gtrdb. figure 1 illustrates the organization of the database. the compilation of gtrdb involved extracting sequences of gtrs glycosylating various antibiotics and identifying their donor and acceptor specificity through exhaustive literature survey. gtrdb is a compilation of 102 annotated gtr sequences from 52 different natural product biosynthetic gene clusters. the database gives the chemical structure of the antibiotic and a variety of information on the gtrs involved in its biosynthesis. for each gtr, gtrdb stores only the primary sequence in fasta format, name of the source organism, identifiers for other databases like genbank (22), swiss - prot (23), cazy (3) etc. using these identifiers, links are provided to respective databases for additional details on these proteins. gtrdb also provides link to the literature which describes the experimental characterization of the corresponding gtr. for each gtr if unnatural substrates are known for a specific gtr, they are listed and a link is also provided to the corresponding literature. for deriving the homology relationships between various gtrs in the database, gtrdb stores both global and local alignments of each gtr with all other gtrs. in order to derive information about the structural features of various gtrs, each gtr sequence has been aligned, using a local version of threader program (24), with structural templates from the antibiotic gtr family available in pdb (25), namely 1iir, 1rrv and 1pn3. the structures of antibiotic gtrs show bi - domain architecture with the n- and c - terminal domains containing a majority of residues binding to acceptor and donor respectively. hence, the n- and c - terminal domains and linkers for each gtr have been identified from their threading alignments with 1rrv. out of the three crystal structures of antibiotic gtrs, 1rrv and 1pn3 represent gtrs in complex with donor as well as acceptor substrates. using the ligplot interactions from pdbsum database (26), 23 acceptor binding residues (abr) and 15 donor binding residues (dbr) were identified for each gtr from their respective threading alignments with 1rrv. similarly, 18 abr and 16 dbr were identified for each gtr using 1pn3 as the template. it may be noted that despite the high degree of sequence and structural similarity between 1rrv and 1pn3, identical acceptor substrate binds to them in different orientations and only a very small number of amino acids are common between the abr of 1rrv and 1pn3. it has been proposed that vancomycin gtrs achieve their regioselectivity for glycosylation by employing these two different binding modes (20,21). in view of the limited number of structures for gtrs in the complex with the substrates, prediction of the sbr for the gtrs in gtrdb thus, the sbr identified by using 1rrv and 1pn3 as templates only help to give a consensus view of the approximate location of the substrate binding pocket. a more accurate prediction of the substrate binding pocket is possible only if additional crystal structures for members of other antibiotic gtr families are available. the program consists of two major components, the backend database gtrdb, which have well - annotated gtr sequences and the query module searchgtr, which allows analysis of an uncharacterized gtr sequence. it is aligned with all the 102 gtrs in the gtrdb using both local alignment program blast as well as needleman and wunsch global alignment programs. sequence alignments are carried out using blosum62 scoring matrix, e - value cut off of 0.000001 and default values of gap initiation and extension penalties. after selecting a particular homologous sequence, the user can view its local / global alignment with the query. the page showing the alignment also provides a link to the gtrdb, which gives other details on the aligned gtr sequence, specifically the chemical structures of its donor and acceptor. this information on donor and acceptor substrates of the best matching gtr can provide important clues about possible substrates for the query gtr. using the best matching gtr from gtrdb and its precomputed threading alignment with 1rrv, searchgtr identifies the n- and c - terminal domains and linkers in the query sequence. these are then depicted in a pictorial format in a pop up window (figure 2). the numbers below the image of the domain specify its start and end positions on the query sequence. if there are parts of gtr which are overhangs on the n - terminal or c - terminal domains, they are shown as n - tail and c - tail, respectively. the fasta sequence of the domain or linker or tail can be viewed by clicking on the respective images. the program identifies putative sbr in the query sequence from the threading alignment of its best match with 1rrv or 1pn3 (figure 2). the user is given the option to choose either 1rrv or 1pn3 from a pull down menu as template for identifying the sbr. links are provided to the pdbsum site for viewing the interactions between the protein and the ligand in the template structure using the program ligplot. as can be seen from figure 2, for the structural template 1rrv/1pn3, the amino acids interacting with the ligand via hydrogen bonds and hydrophobic contacts are highlighted in different colors. however, no such coloring scheme is used for the query or the best match as equivalent residues are expected to have dissimilar amino acids depending on the chemical structure of the donor / acceptor substrates. in order to facilitate correlation between the residues in the substrate binding pocket and substrate specificity, the program provides a link to the chemical structure of the acceptor / donor substrate of the best matching gtr. additionally, searchgtr provides an option for comparing the query sequence with the experimentally characterized gtrs in terms of their substrate (acceptor / donor) binding pocket residues alone. the sbr of the query, identified based on either 1rrv or 1pn3 as the template, is compared with the corresponding library of sbr from gtrs with known donor and acceptor. the query sbr are pair - wise compared with sbr of all gtrs in gtrdb and each position is scored using the blosum62 matrix. the results are displayed as a sorted list of the best matching acceptor / donor - binding residues from gtrs in gtrdb along with their donor / acceptor specificities. this allows a one to one comparison of amino acids at each position of donor / acceptor binding pocket. furthermore, the program also has options for the alignment of the query sequence with any specific gtr in gtrdb by selecting them based on the type of donor substrate, acceptor substrate or type of antibiotic. global alignments are carried out using a local version of needleman and wunsch programs from the emboss package (27). in order to benchmark the searchgtr program, predictions were carried out for the 102 gtrs of known specificity using a jackknife - type approach. the 52 antibiotics in gtrdb were grouped into 20 acceptor families based on the structural similarity of the acceptor aglycone core, e.g. the vancomycin group, the anthracycline group, the orthosomycin group, etc. substrate specificity was assigned to the query gtr based on the best match predicted by searchgtr. if the best match was from the same acceptor family as the query gtr, it was considered a correct prediction. for example, if the gtr from erythromycin transferring mycarose is used as query sequence, its closest homolog is the gtr which transfers mycarose in analogous position in megalomicin. in view of the very high degree of structural similarity of the acceptor cores of erythromycin and megalomicin this type of jackknife test was carried out for all 102 gtrs in gtrdb and the correct acceptor family could be predicted for 72 gtrs. however, 9 out of 102 gtrs belong to acceptor families containing single members only ; thus their acceptor family can not be predicted by our knowledge - based approach. similar analysis also indicated that searchgtr can correctly predict donor group in 45 out of 74 gtrs of known donor specificity, thereby giving a prediction accuracy of 61%. in a separate analysis, the nr database of ncbi was searched to identify experimentally uncharacterized gtrs, whose specificity could be predicted with a reasonable confidence level by our program. out of the 806 proteins extracted from the nr database, 19 sequences showed high sequence similarity (> 40% identity) to the known gtrs in the gtrdb. therefore, the results from in silico analysis by searchgtr program can aid experimental characterization of these proteins. the other proteins from this set of 806 show a relatively lower level of sequence similarity with known gtrs in gtrdb, so the prediction of substrate specificity for them may not be reliable. however, out of these 806 sequences, 111 proteins have been annotated as hypothetical proteins in the nr database, even though they show statistically significant sequence similarity with known gtrs in gtrdb. thus, searchgtr could also aid in the annotation of gtrs. in order to benchmark the prediction accuracy of sbr by searchgtr, we attempted to predict the dbr for 1rrv using 1pn3 as template and vice versa. it was found that 14 of the 15 known dbr could be predicted for 1rrv using 1pn3 as template. on the other hand, by using 1rrv as template 14 out 16 dbr could be identified for 1pn3. these two gtrs belong to the same antibiotic family and share a sequence identity of 55%. we also tested dbr prediction accuracy of searchgtr using murg structure, which adopts a gt - b fold but is not an antibiotic glycosyltransferase. for the structure of murg - donor substrate complex (1nlm), searchgtr could identify 6 out of the 12 donor binding residues correctly, using 1rrv or 1pn3 as template. it may be noted that the sequence identity between 1nlm and 1rrv (or 1pn3) is only 20%. even by the structural superposition of the donor binding domains of 1rrv (or 1pn3) and 1nlm only 7 of the 12 donor binding residues can be identified. hence, even for a difficult test case like murg, the performance of searchgtr is reasonably good. the eventual inclusion of additional structural templates would help in further improving the prediction accuracy. searchgtr is an interactive web server for the analysis of gtrs involved in natural product biosynthesis. it has options for carrying out a variety of detailed analyses on gtrs of known substrate specificity. it allows identification of homologous sequences, depiction of domains and linkers and extraction of putative donor / acceptor binding residues. as the program allows comparison of amino acids lining the substrate binding pocket, it can provide clues for altering donor / acceptor selectivity of gtrs by site- directed mutagenesis experiments. apart from its utility in the rational design of novel antibiotics, searchgtr can also help in in silico identification of substrates for various uncharacterized gtrs in newly sequenced genomes. benchmarking a set of gtrs of known substrate specificity indicate that the program can predict the acceptor specificity of antibiotic gtrs with an accuracy of 77% based on whole sequence comparisons. in view of the enormous diversity in the chemical structure of the antibiotic gtrs, even at this level of accuracy our analysis indicates that searchgtr fails to identify the correct substrates by comparison of whole sequences if the sequence similarity between the query sequence and the gtrs of known specificity is very low. it is possible that the prediction accuracy in such cases of low homology may be improved if active site residues alone are used for substrate prediction instead of whole sequences. even though searchgtr has options for comparing gtrs using only active site residues, the current version of the program extracts the putative active site residues using crystal structures of two gtrs (1rrv/1pn3) belonging to the vancomycin group of antibiotics. in view of the structural diversities of the acceptor group, it is possible that the mode of acceptor binding may be different for different groups. elucidation of crystal structures of acceptor complexes for members of other antibiotic families will help in better identification of their binding pocket. the links to other databases are shown in orange. a screenshot from searchgtr showing extraction of domains and linker and identification of putative acceptor binding residues (abr) using 1rrv as template. the abr of the structural template, best match and query are depicted in tabular format. the page provides link to the chemical structure of the donor / acceptor of best match and ligplot interactions for structural template. | searchgtr is a web - based software for the analysis of glycosyltransferases (gtrs) involved in the biosynthesis of a variety of pharmaceutically important compounds like adriamycin, erythromycin, vancomycin etc. this software has been developed based on a comprehensive analysis of sequence / structural features of 102 gtrs of known specificity from 52 natural product biosynthetic gene clusters. searchgtr is a powerful tool that correlates sequences of gtrs to the chemical structures of their corresponding substrates. this software indicates the donor / acceptor specificity and also identifies putative substrate binding residues. in addition, it provides interfaces to other public databases like genbank, swiss - prot, cazy, pdb, pdbsum and pubmed for extracting various information on gtrs homologous to the query sequence. searchgtr would provide new dimension to our previously developed bioinformatics tool nrps - pks. together, these tools facilitate comprehensive computational analysis of proteins involved in biosynthesis of aglycone core and its downstream glycosylations. apart from presenting opportunities for rational design of novel natural products, these tools would assist in the identification of biosynthetic products of secondary metabolite gene clusters found in newly sequenced genomes. searchgtr can be accessed at. |
staphylococcus aureus (s. aureus) is one of the most prevalent pathogens that cause both community and nosocomial acquired infections and can produce a wide variety of diseases from skin surface infections, such as folliculitis and furunculosis, to life threating conditions, such as endocarditis, pneumonia, and septicemia [13 ]. the expression of many virulence factors in s. aureus is under control of the agr system and to date four major agr types in s. aureus have been recognized [4, 5 ]. resistance to methicillin is due to meca gene that is part of the staphylococcal cassette chromosome. this gene encodes the protein pbp2a (protein binding to penicillin) that inhibits the action of -lactam antibiotics. sccmec elements have been classified into eight different types (i viii) and some of them are divided further into subtypes [6, 7 ]. the increasing antibiotic resistance in this bacterium is a major concern that underlines the importance of the use of efficient typing methods for monitoring and limiting the spread of epidemic clones between hospitals [1, 8, 9 ]. of the various molecular methods, pfge, due to its high differentiation potential, was considered the gold standard for strain typing of s. aureus. however since it is time consuming, expensive, complicated, and difficult to standardize among different laboratories, dna sequence - based methods have become increasingly popular during the recent years [10, 11 ]. genetic analysis of strain types of s. aureus can be performed by spa sequence typing. the spa gene (approximately 2150 bp) is composed of three regions, namely, the fc protein, the x region, and the c terminal. the spa typing is based on sequencing of the polymorphic x region of protein a and depends on pcr amplification of this hypervariable region. the x region is composed of a variable number of 24 base pair repeats which may differ by spontaneous mutation or deletion and duplication of the repeats. each repeat is attributed to one alpha - numerical code and the spa type is derived from the order of specific repeats [1214 ]. it can be useful in describing the natural population of s. aureus strains as well as in outbreak investigations. however sometimes similar or related spa types are located in different clonal lineage which limits the discriminating power of this method. the purpose of this study was to determine the prevalence of antibiotic resistant s. aureus isolates and the use of spa, agr, and sccmec typing to determine the dominant types in iran. fifty isolates of s. aureus were collected from clinical samples of patients who referred to isfahan 's alzahra hospital (iran) from january to may 2010. these isolates were obtained from different clinical sources including wound, blood, urine, and sputum. s. aureus identification was performed by standard tests including gram staining, catalase, dnase, mannitol fermentation, slide, and tube coagulase. thereafter they were classified as community - acquired (ca - mrsa) or hospital - acquired (ha - mrsa) based on the patients recorded data. the susceptibility of s. aureus isolates to antimicrobial agents was determined by the disk diffusion method according to the guidelines of the clinical and laboratory standards institute (clsi). the antibiotics utilized were as follows : vancomycin, tetracycline, gentamicin, clindamycin, ciprofloxacin, rifampin, cefoxitin, levofloxacin, and cotrimoxazol. s. aureus strain atcc25923 was used as a control strain for the quality control of antibiotic susceptibility testing. dna extraction was performed from all isolates using fermentas dna kit in accordance with the manufacturer 's protocol. pcr was performed for the detection of meca gene using primers displayed in table 1. pcr conditions were as follows : initial denaturation, 94c for 5 min, 40 cycles of denaturation at 94c for 30 s, annealing at 57c for 45 s, and extension at 72c for 30 s and final extension at 72c for 5 min. sccmec typing for 9 isolates resistant to methicillin (mrsa) was determined by multiplex pcr method. the pcr protocol comprised an initial denaturation step at 94c for 4 min followed by 30 cycles of denaturation at 94c for 30 s, annealing step at 55c for 30 s, and extension at 72c for 60 s and a final extension step at 72c for 4 min. agr typing was also performed on all isolates of s. aureus using primers shown in table 1. pcr conditions were as follows : initial denaturation at 94c for 5 min followed by 40 cycles of denaturation at 94c for 40 s, annealing at 60c for 40 s, and extension at 72c for 60 s and a final extension at 72c for 5 min. the polymorphic x region of the spa gene was amplified from all s. aureus isolates using the spa primers exhibited in table 1. all sequencing reactions were performed at bioneer (korea) and then the data were analyzed using mega 4 software. in the present study, from january to may 2010, 50 isolates of s. aureus from various clinical specimens including wound (38%), septicemia (26%), uti (18%), pneumonia (10%), and others (2%) were collected from alzahra hospital in isfahan. in this study, s. aureus resistance to tetracycline, cotrimoxazol, cefoxitin, clindamycin, ciprofloxacin, gentamicin, levofloxacin, rifampin, and vancomycin was 36%, 22%, 18%, 12%, 12%, 10%, 6%, 6%, and 0%, respectively. in our study, presence of meca gene in all isolates were evaluated by susceptibility test and then confirmed by pcr. of the 50 s. aureus isolates, 8 (16%) were mrsa and meca positive. to determine the type of mrsa isolates, sccmec typing was performed in which 4 (44.4%) were found to be sccmec type iv, 2 (22.2%) were sccmec type iii, and 2 (22.2%) were sccmec type i. agr typing results revealed that 45 (90%) isolates were agr type i, 2 (4%) were agr type iii, and 3 (6%) were nontypeable. ultimately, typing of 50 isolates of s. aureus was performed using spa typing method. genotyping of spa gene revealed 22 different spa types with spa type t7688 (26%) being the most frequent and other types with the following frequencies : t304 (10%), t037 (8%), t005 (8%), t230 (8%), t024 (6%), and t4892 (4%). spa types including t352, t8015, t937, t138, t436, t439, t045, t2790, t084, t1741, t267, t021, t7685, t5005, and t275 were found only once among the isolates (table 2). s. aureus, particularly methicillin - resistant staphylococcus aureus (mrsa), is one of the most common causes of infection both in the community and in hospitals. due to its diverse pathogenicity and high antibiotic resistance, molecular typing of this bacterium is therefore essential to determine the origin of the strains, its clonal relations, and for epidemiological investigations, playing an important role in the prevention and treatment of infections. in this study, we investigated the antibiotic resistance of s. aureus to tetracycline, cefoxitin, clindamycin, ciprofloxacin, gentamycin, cotrimoxazol, levofloxacin, rifampin, and vancomycin using disk diffusion method. resistance of s. aureus to antibiotics was as follows : tetracycline 36%, cefoxitin 18%, clindamycin and ciprofloxacin 12%, gentamicin 10%, cotrimoxazol 22%, levofloxacin and rifampin 6%, and 0% to vancomycin. resistance rates found in this study were lower than the global average [5, 17, 18 ]. several different phenotypic and genotypic methods can be employed for classifying strains used in epidemiological investigations, and for detection and monitoring nosocomial outbreaks. in the current study, we used sccmec, agr, and spa typing for this purpose [18, 19 ]. mrsa classification requires a thorough understanding of their genetic structure as well as detection of all sccmec types and carriers of the meca gene. sccmec typing provides important information about the movable genetic components responsible for resistance to methicillin and it is a marker for differentiation between ha - mrsa and ca - mrsa strains [6, 9, 19 ]. sccmec typing was performed on mrsa isolates in which 4 (44.4%) were sccmec type iv (2 of them were related to t037 spa type and agr types i and iii, and the other two isolates were t325 and t005 spa types and agr type i), 2 (22.2%) were sccmec type iii (related to t037 and t138 spa types and agr type i), and 2 (22.2%) were sccmec type i (related to t005 and t304 spa types and agr type i). the high frequency of sccmec types iv compared to other sccmec types may be due to their small size that facilitates their spread among s. aureus strains. in our study, 3 isolates with sccmec type iv belonged to ha - mrsa, whereas types iv and v were shown to belong to ca - mrsa. as expected, in this study we have shown 4 isolates related to sccmec i and iii which belong to ha - mrsa. in agreement with previous reports from iran, the majority 45(90%) of the isolates were agr i, 3 (6%) were agr iii, and 2 (4%) were nontypeable [4, 5 ] similar to many previous studies, the agr type iv was absent in our study [2, 20 ]. spa typing is an effective molecular typing technique based on sequencing of only single locus of s. aureus and has advantages such as rapidity, ease, and convenience of interpreting the results and exchangeability of results among laboratories and creates a global database based on spa typing for national and international control of s. aureus. the use of spa typing in our study revealed 22 different spa types where spa type t7688 (26%) was the most frequent followed by spa type t304. its global prevalence has been shown to be 0.32% in different countries including austria, belgium, canada, denmark, finland, france, gabon, germany, iceland, sweden, switzerland, united arab emirates, united kingdom, united states, lebanon, netherlands, norway, south africa, and spain (http://spaserver.ridom.de), whereas the frequency of this type was higher (10%) in our study. in addition the frequency of spa t304 has been found to be higher than that reported in previous studies in iran. spa types t037, t005, and t230 have been isolated from different parts of the world [1, 8, 21 ]. however, in our study each one was 8% and spa types t024 and t4892 were 6% and 4%, respectively. other spa types including t325, t267, t021, t275, t7685, t045, t005, t439, t138, t937, t436, t8015, t325, t084, t1741, and t5005 were found only once among the isolates. in a study by winiewska. the prevalent spa types in poland were reported to be t003, t151, and t008. neela. showed that spa type t037 and sccmeciii were prevalent in malaysia. found spa types t044 and t037 and sccmec iv as the prevalent types in lebanon. the use of molecular typing in spain revealed spa types t067 and t002, agr ii, and sccmec iv to be dominant. in a study conducted by emaneini. overall in the current study, spa typing showed 100% type ability. an interesting point to note was the dominance of spa type t7688 which has been only reported from iran to date. moreover the spa types of t084 and t037 are associated with the top ten spa types worldwide between mrsa and mssa isolates in which t037 is one of the prevalent types in asian countries. it is our understanding that the current results will aid in the characterization of s. aureus in neighboring countries. we suggest the use of additional typing methods such as burp and mlst to overcome the limitation of a single locus - based molecular typing (spa typing). | background. staphylococcus aureus (s. aureus) is one of the most common pathogens that cause hospital- and community - acquired infections in the world. the use of molecular typing methods is essential for determining the origin of the strains, their clonal relations, and also in epidemiological investigations. the purpose of this study was to determine the prevalence of antibiotic resistant s. aureus isolates and using spa, agr, and sccmec typing to determine the dominant types in iran. material and method. fifty isolates of s. aureus were collected from january to may 2010. s. aureus identification was performed by biochemical tests. disk diffusion method was employed to assess the sensitivity of s. aureus strains to antibiotics and then genetic analysis of bacteria was performed using sccmec, agr, and spa typing. results. s. aureus resistance to tetracycline, cefoxitin, clindamycin, ciprofloxacin, gentamicin, cot : cotrimoxazole, levofloxacin, rifampin, and vancomycin were found to be 36%, 18%, 12%, 12%, 22%, 6%, 6%, and 0%, respectively. the results of this study showed that 16% of the isolates were resistant to methicillin (mrsa) and the majority of isolates were ssc mec type iv. in addition spa and agr typing revealed agr typei and spa type t7688 to be the most predominant. conclusion. in this study, spa typing showed 100% reliability and the t7688 spa type had a frequency of 26% compared to the frequency of 0.0% in the ridom spaserver. the frequency of t304 spa type was higher than the global average. |
a 55-year - old man in good general health had pain and intermittent locking of the left knee of two months duration. magnetic resonance imaging (mri) showed a displaced bucket - handle tear of the lateral meniscus and so arthroscopic lateral meniscectomy was performed. increasing pain and swelling in the left popliteal fossa developed postoperatively over the following five days. a 65-ml hematoma was aspired and repeat arthroscopy of the knee detected only remnants of blood. the same symptomatology was displayed four days later and so the patient was referred for endovascular management. selective arteriography was performed via the right femoral artery, and a 4-fr catheter was placed into the left proximal popliteal artery. after contrast injection (30 ml at 8 ml / sec ; iopromida, shering, madrid, spain), a pseudoaneurysm measuring 2.51.60.9 mm and arising from the lateral inferior genicular artery was detected ; there was early venous drainage via the hypertrophied genicular veins, forming an arteriovenous (av) fistula (fig. 1). a hydrophilic 3-fr catheter (terumo, leuven, belgium) was selectively advanced over a guide wire into the injured genicular artery. the pseudoaneurysm was embolized with two small (2 mm in diameter, 20 mm in length) fibered platinum coils (cook europe, bjaeverskov, denmark). follow - up angiography demonstrated complete occlusion of the av fistula, with no filling of the pseudoaneurysm (fig. clinically, the patient became free of symptoms a few days after the embolization and further clinical follow - up at six months was uneventful. vascular injuries arising from artroscopy of the knee are very rare, and several large series have reported an incidence of less than 1% (1, 2). most vascular injuries involve the popliteal artery or vein, or both (2). pseudoaneurysms of the popliteal and lateral genicular arteries after knee surgery are rare and only a few, isolated case reports have been published (3 - 6). pseudoaneurysms are more likely to form when a vessel is incompletely divided and blood dissects into the surrounding soft tissues. the susceptibility of the popliteal artery and its branches to injury during arthroscopic meniscectomy is due to several factors, including the employed surgical technique, and the operator 's experience and knowledge of their anatomic location (3, 4). the popliteal artery is close to the posterior capsule of the joint and it is moved forward during knee flexion. however, visualization of this region during knee arthroscopy is limited and any injury is often not immediately recognized (4). nevertheless, the formation of combined pseudoaneurysm and av fistula after knee arthorscopy is extremely rare. only two such cases have been previously reported on, and both of these occurred in the popliteal artery (5, 6). reported a case of av fistula of the lateral superior and inferior geniculate arteries, and the etiology of this was unclear (7). in our case, the mechanism to injury was probably direct trauma by cutting all three layers of both the artery and vein with scissors, which resulted in an av fistula. alternatively, hematoma formation around the artery and vein with degradation of the enclosed vessels may have also resulted in a combined pseudoaneurysm with av fistula. when a patient has popliteal swelling, mass, bruit, thrill, recurrent hemarthosis, pain, calf edema, and/or a neurologic deficit after a knee arthroscopy, the possibility of a vascular injury should be considered early on in the diagnostic process (2, 4). the diagnosis can be confirmed by conventional arteriography, as in our case, by color doppler sonography, or with using three - dimensional ct or mri arteriography techniques (4 - 8). surgical exploration with vessel ligation is the " gold standard " of treatment because it preserves distal flow if collateral circulation has formed (3, 5 - 8). other treatment options include correction of the vascular tear by vascular patch or direct suture (8). sonographically guided compression to occlude the postcatherterization pseudoaneurysm of the femoral and brachial arteries has been reported on (9). however, with the advances in selective endovascular techniques, percutaneous embolization has been successfully performed for the management of popliteal pseudoaneurysms and also for the delayed combined pseudoaneurysm and av fistula of the anterior tibial artery after an open tibial fracture (10). in our case, we embolized the lesion with using microcoils to definitely occlude the pseudoaneurysm and av fistula. this case demonstrates that superselective percutaneous angiographic embolization with coils offers the advantages of a minimally invasive approach (no surgical incision, a reduced risk of infection and a shortened hospital stay) ; therefore, this technique may represent an effective and safe therapeutic alternative for this uncommon complication of knee arthroscopy. in summary, we report here on a unique case of a lateral inferior genicular artery pseudoaneurysm with concomitant arteriovenous fistula as a complication of knee arthroscopy. | arthroscopic meniscectomy of the knee is generally a safe and effective procedure with a low rate of vascular complications. we report here on a unique case of a 55-year - old man with a lateral inferior genicular artery pseudoaneurysm and a concomitant arteriovenous fistula that developed after arthroscopic meniscectomy ; this was successfully treated with selective angiographic embolization. this case illustrates the effectiveness of an endovascular approach as a minimally invasive treatment for this uncommon complication that occurs after an arthroscopic procedure. |
charcot arthropathy was first described in 1868 by jean martin charcot as a progressive and destructive joint disease,. although in the years described neurosyphilis was the most frequent cause, at the present diabetes mellitus is the most common etiology for this disease. incidence among the patients with diabetes is 7.5% and the disease most commonly involves the feet and ankles. it is known that the 6% of the charcot arthropathy in diabetes affects knee joint,. its pathophysiology is explained with sensorineural, autonomic and motor dysfunction leading to instability, osteopenia, and micro trauma. polyneuropathy, syphilis, syrengomyelia and chronic alcoholism are the main causes of the disease in the literature. however, charcot arthropathy can be seen after some spinal cord pathologies, there is no literature known as charcot knee arthropathy as a complication of spinal stenosis surgery. in this study a 62 years old women had a spinal stenosis surgery at 2010 with a posterior instrumentation (figs. 1, 2). at the 1 year follow up an mri of the patient s left knee showed inflammatory changes compatible with a charcot knee. patient had been out of follow up for 4 years and did not receive any treatment. when patient first presented there were an increased varus and valgus instability with hyperextension laxity in her left knee joint. x - rays also showed excessive bone loss at the medial plateau of the tibia (figs. 3, 4). there were no sings of oedema, swelling and erythema on inspection. according to the patient s medical history it was obvious in hindsight that the patient had been charcot arthropathy and the disease was already beyond the fragmentation and coalescence stages at that moment. an immediate emg study showed us the l3-s1 roots had a severe chronic polyneuropathy with total sensorineural loss.fig. 4lateral x - ray of the knee joint at the end stage of the disease.fig. lateral x - ray of the knee joint at the end stage of the disease. patient also had an ulcerative wound at the left heel which diagnosed as charcot foot because of the polyneuropathy and that wound was treated at 2006 with surgery. based on the medical history, orthopaedic and neurologic examination and the radiologic sings we believe that this patient developed a neuropathic arthropathy at her left knee after a spinal stenosis surgery. we preferred conservative treatment for the patient and suggested a custom made hinged knee brace for a daily use,, (fig. follow up we have seen that the patient was walking comfortably with crutches while using the brace. patient was consulted with the physical therapy and rehabilitation department for the use of bisphosphonates and medical treatment as well.fig. 5clinic and functional pictures of the patient, at present.fig it is usually seen at patients with diabetes mellitus as a long - term complication with or without polyneuropathy. furthermore the literature shows us that after spinal canal pathology or even after spinal anaesthesia procedure charcot arthropathy can be seen. incidence of the involvement of the knee is less than the distal joints such as foot and ankle. for that reason, spinal procedures should be applied with extra caution on the patients with polyneuropathy or any neuropathic arthropathy. it should be remembered that it is possible to encounter unexpected complications such as proximally migration of the level of sensorineural loss and progression of the actual disease after spinal procedures of these patients. written informed consent was obtained from the patient for publication of this case report and accompanying images. | highlightswe report here a case of a 62 years old patient with charcot arthropathy at her left knee developed one year after spinal stenosis surgery.the patient s knee joint was already beyond the fragmentation and coalescence stages at the moment of physical examination.unsuccessful spinal surgery affected polyneuropathy and migrated the level of the sensorineural loss proximally.elevated level of sensorineural loss resulted in charcot knee joint in a short period of time. |
traumatic achilles tendon tears are frequent occurrences in lower - limb trauma, involving young, active patients. to better understand the etiology and etiopathogenesis of tendon tears, a comprehensive model of the achilles tendon behavior during rupture - risky daily situations and motions analyzing the biomechanics of the achilles tendon requires in fact a dissection of the main kinematic events during a running sequence. the achilles tendon structure allows it to transmit significant forces from muscle to bone, behaving like a hyperelastic material past a certain longitudinal strain, but shows a limited resistance to shearing and compression loads. at rest, tendon fibers appear wavy, whilst during strain they become taut, behaving like an elastic material up to 2% elongation ; past that, they behave like a hyperelastic material. partial tears develop at 4% elongation and beyond, while complete tears usually occur at 8% elongation and beyond. the forces acting on the foot can be measured experimentally with the aid of a force plate, a device that utilises newton s third law. by recording the ground reaction force (grf), we can determine the forces that act on the achilles tendon while running, in particular those acting on the tendon - calcaneus jonction. a running sequence is much more complex compared to a walking one, completely lacking in bilateral support and having an intermediate phase of flight, when the body is completely airborne. the achilles tendon behaves like an interface between muscle and bone during such a sequence, establishing the so called calcaneus - tendon - sural triceps system. if we consider the bony surface as completely inelastic, the forces developed by the triceps surae input a maximum load on the tendon s insertion. understanding and predicting these forces during the ground contact phase of a running step is paramount to better understanding the impact of running on the achilles tendon. to record the ground reaction force (grf), a kistler force plate was used, with a recording frequency of 2.5 khz ; a 23-years - old human subject with a weight of 77 kg, a height of 182 cm and with no prior tendon injuries and no prior specific physical training was asked to run over the force plate 30 times. the collected data were filtered and statistically analysed, ignoring any force past 5 sds (standard deviations), rendering a force cloud that was sorted in regards to time : it became readily apparent that the median (red line) is a rough, intuitive image of the running function model. a median regression function applied to the force cloud yields a clearer image : in order to elaborate and compare a mathematical model to an empirical one, we chose the running sequence that was closest to the median regression curve. although one could easily use the regression function as a model itself, we felt that a mathematical inference model was much more useful to appreciate the biomechanics of the achilles tendon. given that f = m, where f stands for force, m for mass and is the gravitational pull (9.81 m / s), f for our subject amounts to 755.3 n. the area below the curve is in fact impulse (i), which can be calculated using the riemann sums : where fk is the force recorded at the moment tk, with t=.384 and t = tktk1 being constant (0.004 s). an impulse of i=360.31 ns results. this can be used to compare the accuracy of our mathematical model to the empirical data. a first modeling attempt is to try and approximate the function with the help of a second degree polynomial, f(t) = at+bt+c, the first term being negative because the function is concave. the impulse is obtained through imposing limits f(0)=o and f(t)=0, thus the initial equation becoming f(t) = a(ttt) = at(tt), with only a unknown. impulse becomes : empirical data shows that the maximal value of the function is satisfied at the middle of the interval, f(t2)=fmax and f(t2)=0, which is actually obvious as we purposedly chose to model using a 2 degree function. and impulse i=2fmax t3, we can calculate the impulse using maximal force (known empirically). thus, t=0.384 and maximal force is 1,711.66 n, the resulting impulse i=438.18 ns, which is close, but not sufficiently so the empirically devised impulse. using body weight to model the function can be useful, as in mathematical terms, body weight (bw) can be described with the median of the function (during a sprinting step, the entire body weight is shifted from one foot to another, thus during a whole step the entirety of the weight is supported) : where t1 and t2 represent the crossing of the curve by the line that expresses the median of the function. thus, bw can be described as : an obvious relation between weight and impulse is described, but as we can see by using previous results, bw=2fmax3, which in turn means fmax=32bw. using a fourth order polynomial : f(t) = at + bt + ct + dt + f, where t[0, t ], with the limits f(0) = 0, f(t)=0, f(0)=0, f(t)=0 and f(t2)=fmax, leads us to : f(t)=16fmaxt4t2(t - t)2, which results in fmax=158bw, a rather accurate description. in the end, by comparing the different methods of approximation, we can observe that a known maximal force approximation seems to be the most accurate : obtaining a perfectly accurate model of the step in a running sequence is a daunting task if attempted through mathematical means alone ; the higher the order of the polynomial, the more accurate the modeling gets, but this continues ad infinitum, because it is done by approximation. it needs not to be perfect to serve a predictive purpose though : having established a satisfactory model and developing a quick, repeatable and easy way to provide it is a sufficient goal if one seeks to understand the behaviour of the foot in a running sequence and through it, the strain and loads incurred on the achilles tendon calcaneus joint. strictly from a mathematical point of view, a fourth order polynomial seems to be sufficient to model running ; a further increase in order will only slim the curve of the approximation, making the transition to the peak force steeper while useful to extract a more accurate impulse and therefore load, it diminishes the inequality of force distribution, opposite to the empirical data gathered that reveals the fact that the running step is in fact a walking step in a much shorter time span. a high order polynomial approximation function suggests that the step is a sprinting step, where ground contact is achieved only on the forefront of the foot, toe region and distal metatarsal region, foregoing the calcaneus landing altogether. it is probably more accurate to divide running into light running (which was the object of the study) and sprinting, where the foot behaves differently. as for inaccuracy sources, recording noise when collecting data can probably be considered the main source of error, while the presence of medio - lateral and anterio - posterior forces unaccounted for during data collection and filtering could come in second place. when modeling a foot that has a distinctly altered biomechanics due to malformations, recent surgery, pain, neuromotor injuries etc., care should be taken in ignoring the concurrent forces ; in a healthy foot these forces are negligible in a running sequence. building an accurate model of running allows to identify key kinematic moments and events that impact the foot and specifically, the ankle with its main muscle / bone system that is the triceps surae achilles tendon the heel region in general and the achilles tendon in particular bears loads equivalent to twice and sometimes more than twice the body weight during running. moreover, the impulse generated by these loads is distributed unequally throughout the running sequence, creating high - strain events for the achilles tendon. the most important and impacting force during running is the vertical traction force (as described, through newton s third law, by the ground reaction force). by establishing and describing the relationship between body weight, ground reaction force and impulse in each step of the running sequence, we can model the entire kinematic chain. a polynomial model, although rather intricate, can provide a useful tool in predicting and assessing the behavior of everyday movements, in this particular case during sprinting and running. by recording the movement, one can simply extrapolate and compare between results from healthy individuals or between similar tendon healing rates in traumatic tendon injuries ; pathological motions can be detected as well as abnormal loads or prolonged loading times which can suggest unfit tendons or slow functional healing. one must remember that while the vertical traction force is the main actor while running, medio - lateral and anterio - posterior forces can be detected, even though minimal, they can impact the achilles tendon and the overall motion, if for some reason their value is greatly increased through pathological movement or antalgic motions. the achilles tendon is poorly equipped to deal with shear and compression forces, which can be cause for re - rupture. evaluating these forces | the following study attempts to elaborate a model of the achilles tendon while in the process of running, specifically during a step that is part of a running sequence. data are collected with the help of a force plate and then is processed and modeled to serve as a starting point and comparison to a mathematical model using polynomial functions.the data collected were filtered to diminish recording of noise and an empirical model was established. mathematical models using second order and fourth order polynomials were employed, as well as an approximation using known maximal force. the increase in the accuracy of modeling was determined as the order of the polynomial function increased.achieving an accurate predictor function is essential in understanding the biomechanics of the achilles tendon. |
the combination of genetic mutations and epigenetic modifications that are peculiar to all tumors generate antigens that t and b lymphocytes can use to specifically recognize tumor cells (jamal - hanjani., it is increasingly clear that t lymphocytes recognizing tumor - derived peptides presented by major histocompatibility complex (mhc) molecules play a central role in immunotherapy and in conventional chemo - radiotherapy of cancer (galluzzi., 2015). in fact, anti - tumor t cell responses arise in cancer patients but are disabled upon tumor progression by suppressive mechanisms triggered by the interplay between malignant cells and the tumor microenvironment (munn and bronte, 2016). the tumor - dependent immunosuppressive mechanisms depend on the integrated action of infiltrating leukocytes and lymphocytes that upregulate a range of modulatory molecules, collectively called immune checkpoints, whose function is only partially characterized (pardoll, 2012). therefore, the search for agonists of co - stimulatory complexes or antagonists of inhibitory molecules to potentiate antigen - specific t cell responses is a primary goal of current anti - tumor research (sharma and allison, 2015, zitvogel., 2013). indeed, clinical trials have unequivocally shown that the blockade of immune checkpoints unleashes the spontaneous anti - tumor immune responses in such a powerful way that it has created a paradigm shift in cancer therapy (ledziska., 2015, topalian., 2015). among the immune checkpoints targeted by blocking strategies, anti - ctla-4 monoclonal antibodies (mab) show remarkable success in metastatic melanoma, and more recently in non - small - cell lung cancer, prostate cancer, renal cell carcinoma, urothelial carcinoma, and ovarian cancer (carthon., 2010, hodi., 2010, however, the fraction of patients that do not respond remains high, prompting a deeper investigation of the mechanisms underpinning the modulation of immune responses by tumors. recent experimental evidence shows that anti - ctla-4 mab efficacy depends on fcr - mediated depletion of cd4 regulatory t cells (treg cells) within the tumor microenvironment (peggs., 2009, selby., 2013, simpson., 2013 treg cells, which are physiologically engaged in the maintenance of immunological self - tolerance and immune homeostasis (josefowicz., 2012, sakaguchi., 2008), are potent suppressors of effector cells and are found at high frequencies in various types of cancers (fridman., 2012, treg cells adapt their transcriptional program to the various cytokines to which they are exposed in the inflammatory milieu (campbell and koch, 2011). this versatility is controlled by transcription factors generally associated with the differentiation of other effector cd4 t cell subsets, resulting in various treg cell populations with unique features and immunomodulatory functions (duhen. moreover, treg cells infiltrating non - lymphoid tissues are reported to exhibit unique phenotypes and transcriptional signatures, because they can display functions beyond their well - established suppressive roles, such as metabolic modulation in adipose tissue (cipolletta., 2012) or regulation of tissue repair in skeletal muscle (burzyn., 2013) and in lung tissue (arpaia., 2015) treg cell depletion has been reported to increase anti - tumor specific immune responses and to reduce tumor burden (marabelle., 2013, teng., 2010, walter., 2012). although promising clinical results have been achieved with treg cell depleting strategies, some relevant issues are to be addressed, for a safer, more effective, and wider clinical application of these therapies. first, severe autoimmunity can occur following systemic treg cells depletion (nishikawa and sakaguchi, 2010), which could be avoided if selective depletion of tumor infiltrating treg cells were feasible., treg cells share with effector lymphocytes most of the molecules targeted for therapy, which can possibly deplete also the tumor - specific effector cells. therefore, the molecular characterization of treg cells at different tumor sites should help to better define therapeutic targets through a better description of their signature molecules and of the network that regulates treg cell functions in the tumor microenvironment. non - small - cell lung cancer (nsclc) and colorectal cancer (crc) are the two most frequent cancers in both genders (torre., 2015). nsclc has the worst prognosis due to its high mortality rate even in early stages. although crc survival rate is highly dependent on the tumor stage at diagnosis, about 50% of patients will progress to metastatic cancer (gonzalez - pons and cruz - correa, 2015). both tumors have been targeted with therapies based on monoclonal antibodies to checkpoint inhibitors, but the outcomes have been different. while remarkable clinical success has been obtained in nsclc, evidence of durable response in crc is scarce with the exception of mismatch repair - deficient crc lesions (jacobs., 2015,. here we provide a comprehensive transcriptome analysis of human cd4 treg cells and effector cells (th1 and th17) infiltrating nsclc or crc and their matched normal tissues. we defined molecular signatures of tumor - infiltrating treg cells in these two cancer types and confirmed the relevance of these signatures by single - cell analyses. these data could help a better understanding of treg functional role at tumor sites and pave the way to the identification of therapeutic targets for more specific and safer modulation of treg cells in cancer therapy. to assess the gene expression landscape of tumor infiltrating cd4 t cells, we isolated different cd4 lymphocytes subsets from two different tumors, nsclc and crc, from the adjacent normal tissues, and from peripheral blood samples. from all these tissues, we purified by flow cytometry (figure 1a and s1a and s1b) cd4 treg (36 samples from 18 individuals), th1 (30 samples from 21 individuals), and th17 (22 samples from 14 individuals) cells (table 1 and table s1). to assess treg cell function, we tested their suppressor activity and showed that treg cells infiltrating either type of tumor tissues have a remarkably stronger suppressive activity in vitro compared to treg cells isolated from the adjacent normal tissue and peripheral blood of the same patients (figure 1b). the polyadenylated rna fraction extracted from the sorted cd4 treg, th1, and th17 cells was then analyzed by paired - end rna sequencing obtaining about 4 billion mapped reads first, we interrogated rna - sequencing data of cd4 t cells infiltrating both crc and nsclc and their matched normal tissues, to quantitate mrna expression of known immune checkpoints and their ligands. second, we analyzed rna - seq data of crc and nsclc, as well as of normal colon and lung samples. we found that several immune checkpoints and their ligands transcripts were strikingly upregulated in tumor infiltrating treg cells compared to both normal tissue and peripheral blood - derived treg cells, as well as to t and b lymphocyte subsets purified from peripheral blood mononuclear cells (pbmcs) (figures 1c and s1c and table s5). our findings highlight the specific expression patterns of immune checkpoints and their ligands in tumor infiltrating treg and effector cells and suggest that their functional relevance should be investigated directly at tumor sites. we then asked whether tumor infiltrating treg cells could be defined by specific gene - expression patterns. first, in order to capture the overall similarity between the tumor infiltrating lymphocytes, we performed a principal components analysis (pca) on the whole transcriptomes. tumor - infiltrating treg cells purified from crc and nsclc tissues clustered together and were clearly separated from th1 and th17 cells purified from crc and nsclc tissues (figures s2a and s2b). pca showed a distinct grouping of treg cells purified from different sites ; in fact, separation along the first principal component (pc1) clearly divided peripheral blood treg cells from tissue infiltrating treg cells (figure 2a), whereas normal - tissue and tumor - tissue infiltrating treg cells are mostly divided by the second component (pc2). these findings indicate that tumor - infiltrating treg cells have specific expression patterns compared not only to other cd4 t cell subsets but also compared to treg cells isolated from normal tissues. in order to identify genes that are preferentially expressed in tumor - infiltrating lymphocytes, we performed self - organizing maps (som) analyses that provide a powerful way to define coordinated gene - expression patterns that are visualized in spatial proximity in a 2d mosaic grid heatmap (wirth., 2012). in this way, we analyzed 7,763 genes that were differentially expressed between the different cd4 t cell subsets purified from pbmcs and tumor tissues (deseq2 package ; fdr 10 fkpm) tumor treg cell signature genes (figures 3a, s3a and s3b). notably, we found that the vast majority (75 over 79 ; 95%) of the tumor - infiltrating treg cell signatures were co - expressed with bona fide treg cell markers (i.e., foxp3 and il2ra) (figure 3b). the percentage of co - expression between these treg cell markers and the 79 genes selected among the tumor - infiltrating - treg - cell signature genes ranged between 81% of tigit and 0.59% of cga (figure 3b). the expression of treg signature genes in the rna - seq of the whole treg cell population correlated with the percentage of single cells expressing the different genes (figure 3c). in order to reduce the drop - out effect of the single cell data (i.e., events in which a transcript is detected in one cell but not in another one because the transcript is missed during the reverse - transcription step) (kharchenko., 2014), we defined a threshold (median value t = 8.4%) based on the expression distribution for each transcript and discarded genes below this threshold (see the supplemental experimental procedures). the forty - five signature transcripts of tumor infiltrating treg cells detected above this threshold were in most cases significantly overexpressed in treg cells from both tumors (39 over 45, 87% ; wilcoxon mann whitney test p < 0.05) or in one tumor type (43 over 45, 96% ; figure 3d). homogeneity of the purified tissue infiltrating treg cells can be affected by the carry - over of cells from other lymphocyte subsets. to quantitate this possible contamination, the single cell rt - qpcr analyses of treg cells was performed including markers specific for other lymphocytes subsets (i.e., th1, th2, th17, tfh, cd8 t cells, b cells) (figure s3c). our data showed that only a very low fraction of the purified single cells displayed markers of lymphocytes subsets different from treg cells (figure s3c). the overlap between the signature genes in the crc and nsclc infiltrating treg cells (figure 2d) prompted us to assess whether this signature were also enriched in treg cells infiltrating other tumors. rna was thus extracted from treg cells infiltrating breast cancer, gastric cancer, brain metastasis of nsclc, and liver metastasis of crc. we found by rt - qpcr that tumor infiltrating treg signatures genes were mostly upregulated also in these tumors (figure 3e). overall these data show that the tumor - infiltrating treg cell signature genes are co - expressed at single cell level with foxp3 and il2ra and that several primary and metastatic human tumors express the tumor - infiltrating treg cell signature. we then assessed at the single cell level by flow cytometry the protein expression of ten representative signature genes present in crc and nsclc infiltrating treg cells, adjacent normal tissues, and patients pbmcs. of the ten proteins, two were proteins (ox40 and tigit) whose relevance for treg cells biology has been demonstrated (joller. 2013), seven are proteins (batf, ccr8, cd30, il-1r2, il-21r, pdl-1, and pdl-2) whose expression has never been described in tumor - infiltrating treg cells, and one protein, 4 - 1bb, is a co - stimulatory receptor expressed on several hematopoietic cells, whose expression on treg cells has been shown to mark antigen - activated cells (schoenbrunn., 2012). our findings showed that all these proteins were upregulated (figure 4a), to different extent, in tumor infiltrating treg cells compared to the treg cells resident in normal tissues. given the increasing interest in the pd1 - pdls axis as targets for tumor immunotherapy, we assessed the effect of antibodies against pdl-1 and pdl-2 on the suppressive function of tumor - infiltrating treg cells toward effector cd4 t cell proliferation in vitro. we found that preincubation of tumor infiltrating treg cells with monoclonal antibodies against pdl-1 or pdl-2 reduced their suppressive activity as demonstrated by the increased proliferation of effector cd4 t cells (figure 4b). altogether, our data show there is a molecular signature of tumor infiltrating treg cells, which can be detected both at the mrna and at the protein levels. in an attempt to correlate our findings with clinical outcome, we asked whether the expression of the tumor - treg signature transcripts correlated with disease prognosis in crc and nsclc patients. we therefore interrogated for expression of treg signature genes transcriptomic datasets obtained from resected tumor tissues of a cohort of 177 crc patients (gse17536 ; smith., 2010) and of a cohort of 263 nsclc patients (gse41271 ; sato., 2013) and correlated high and low gene expression with the 5-year survival data. among those genes whose expression is highly enriched in tumor - infiltrating treg cells, we selected layn, mageh1, and ccr8 that are the three genes more selectively expressed (figure s5a). to normalize for differences in t cell densities within the resected tumor tissues, we used the ratio between expression of the selected signature genes and cd3 g. we found that high expression of the three signature genes is in all cases correlated with a significantly reduced survival (figure 5a). we also observed that expressions of the three signature genes increased with tumor staging of crc patients (figure 5b). in conclusion, high expression in the whole - tumor samples of three genes (layn, mageh1, and ccr8) that are specifically and highly expressed in tumor infiltrating treg cells correlates with a poor prognosis in both nsclc and crc patients. diversity of tumor - infiltrating treg cells should be fully elucidated to understand their functional relevance and prognostic significance in different types of cancer and to possibly improve the therapeutic efficacy of treg cell modulation through the selective depletion of tumor infiltrating treg cells. the transcriptome analysis we performed on crc- and nsclc - infiltrating t cells showed that tumor - infiltrating treg cells are different from both circulating and normal tissue - infiltrating tregs, suggesting that the tumor microenvironment influences specific gene expression in treg cells. our findings further support the view that treg cells from different tissues are instructed by environmental factors to display different gene - expression profiles (panduro., 2016). indeed the list of signature genes includes a number of molecules that are consistently upregulated in tumor - infiltrating treg cells isolated from different tumor types, and these signature genes would have not been identified if we had not profiled specifically tumor infiltrating treg cells. the number of genes highly expressed in tumor infiltrating cells, as defined by differential expression and som analyses, was significantly higher in treg than in th17 and th1 cells, suggesting that treg cells are more susceptible than other t cell subsets to external cues they are exposed to in tumor tissues. we found that tumor - infiltrating - treg signature genes are not only largely shared between crc- and nsclc - infiltrating cells but are also conserved in breast and gastric cancers, as well as in crc and nsclc metastatic tumors (in liver and brain, respectively) suggesting that expression of these genes is a common feature of tumor infiltrating treg cells that might correlate with treg cell - specific function within the tumor microenvironment. although our knowledge on the function of immune checkpoints on lymphocytes is still incomplete, agonist or antagonist monoclonal antibodies targeting checkpoints are in clinical development. we have found that some of these checkpoints (such as gitr, ox40, tigit, lag-3, and tim-3) and some of their ligands (such as ox40lg, galectin-9, cd70) are upregulated also in tumor - infiltrating treg cells, and this fact should be taken into account in interpreting clinical results with checkpoint inhibitors. indeed, it is likely that assessment of the expression of checkpoints and of their ligands on the various subsets of tumor infiltrating lymphocytes will help to elucidate conflicting results and provide the rationale for combination therapies. therefore, expression pattern of checkpoints should be evaluated both in tumor - infiltrating lymphocytes and in tumor cells. single - cell analysis on selected tumor treg signature genes confirmed the whole transcriptomic data and provided information on the expression frequency of these genes. tumor - infiltrating treg cells express with high frequency genes that are associated with increased suppressor activity, such as the well characterized ox40, ctla4, and gitr. moreover, there were a number of interesting and less expected genes the specific expression of which was validated also at the protein level. for example, il-1r2 upregulation could be another mechanism that tumor resident treg cells employ to dampen anti - tumor immune responses through the neutralization of il-1 function on effector cells. pd - l1 and pd - l2 expression has been recently reported on activated t cells or apcs (boussiotis., 2014, but, to the best of our knowledge, neither pd - l2 nor pd - l1 expression has ever been reported in treg cells, and our finding that they are overexpressed in tumor infiltrating treg cells adds an additional level of complexity to the pd1 - pd - ls immunomodulatory axis within the tumor microenvironment. batf is a transcription factor that has been mainly associated to th17 development and cd8 t cells differentiation (murphy., 2013). our findings revealed that batf transcript is upregulated in tumor - infiltrating treg cells more than in tumor infiltrating th17 cells (figure s4). expression of batf in cd8 t cells is induced by il-21 (xin., 2015), and we found that il21r is highly expressed in tumor - infiltrating treg cells (figure 4). we showed that tumor - infiltrating treg cells express high amounts of 4 - 1bb (cd137) a marker of tcr - mediated activation (schoenbrunn., 2012) and have shown they display very high suppressor function on effector t cell proliferation. it could be that expression of the signature genes correlated with the enhanced suppressive ability and so contributed to the establishment of a strong immunosuppressive environment at tumor sites. a corollary to our findings would have that increased number of treg cells in the tumor environment should associate with a worst clinical outcome. in fact, when layn, mageh1, and ccr8 (which represent three of the most enriched genes in tumor - infiltrating treg cells) are highly detected in whole - tumor samples there is a significant worsening of the 5-year survival of both crc and nsclc patients. although, the functional roles in treg cells of layn, a transmembrane protein with homology to c - type lectin (borowsky and hynes, 1998), and of mageh1, a member of the melanoma antigen gene family (weon and potts, 2015), are unknown, the high expression of the chemokine receptor ccr8 is instead intriguing. indeed, ccl18, the ligand of ccr8 (islam., 2013), is highly expressed in different tumors including nsclc (chen., 2011, the high specificity of ccr8 expression on tumor - infiltrating treg cells suggests it could be an interesting therapeutic target to inhibit treg cells trafficking to tumor sites, without disturbing recruitment of other effector t cells that do not express ccr8. considerable efforts have been recently put in the development of sophisticated bioinformatics approaches that exploit lymphocyte gene - expression data to understand the immune - modulatory networks at tumor sites, to predict clinical responses to immune - therapies, and to define therapeutic targets (bindea. the data we present here represent a comprehensive rna - sequencing analysis performed on tumor - infiltrating human cd4 treg, th1, and th17 cells. our findings highlight the relevance of assessing gene - expression patterns of lymphocyte at tumor - sites and suggest that generation of more transcriptomic data of tumor - infiltrating lymphocyte subsets purified from different cancer types might contribute to a better understanding of the dynamics underlying immune modulation in the tumor microenvironment. moreover, our data represent a resource to generate and validate hypotheses that will increase our knowledge on tumor - infiltrating treg cell biology and should lead to the identification of therapeutic targets. primary human lung or colorectal tumors and non - neoplastic counterparts were obtained from 15 and 14 patients, respectively. patients records clinicopathological staging, tumor histotype, and grade are listed in table s1. informed consent was obtained from all patients, and the study was approved by the institutional review board of the fondazione irccs ca granda (approval n.30/2014). nsclc specimens were cut into pieces and single - cell suspensions were prepared by using the tumor dissociation kit, human and the gentlemacs dissociator (miltenyi biotech cat. cell suspensions were than isolated by ficoll - hypaque density - gradient centrifugation (amersham bioscience). crc specimens were cut into pieces, incubated in 1 mm edta (sigma - aldrich) for 50 min at 37c, and then incubated in type d collagenase solution 0.5 mg / ml (roche diagnostic) for 4 hr at 37c. t cell fractions were recovered after fractionation on a four - step gradient consisting of 100%, 60%, 40%, and 30% percoll solutions (pharmacia). cd4 t cell subsets were purified by flow cytomtery sorting using the following fluorochrome conjugated antibodies : anti - cd4 apc / cy7 (clone okt4), anti - cd27 pacific blue (clone m - t271), anti - il7r pe (clone mb15 - 18c9), anti - cd25 pe / cy7 (clone bc96), anti - cxcr3 pe / cy5 (clone 1c6/cxcr3), anti - ccr6 apc (clone g034e3), and anti - ccr5 fitc (clone j418f1) using a facsaria ii (bd). libraries for illumina sequencing were constructed from 50 ng of total rna with the illumina truseq rna sample preparation kit v2. paired - end sequencing (2 125) was then performed on an illumina hiseq 2500. raw.fastq files were analyzed using fastqc v0.11.3, and adaptor removal was performed using cutadapt 1.8. trimming was performed on raw reads using trimmomatic : standard parameters for phred33 encoding were used. reads mapping to the reference genome (grch38) was performed on quality - checked and trimmed reads using star 2.4.1c. the overlap of reads with annotation features found in the reference.gtf was calculated using ht - seq v0.6.1. the output computed for each sample (raw read counts) was then used as input for deseq2 analysis. rlog, and normalized counts were used to perform and visualize principal component analysis (pca) results (using deseq2 s plotpca function). differential expression analyses of tumor - infiltrating cd4 treg, th1, and th17 subsets versus cd4 treg, th1, and th17 from pbmc were performed using deseq2. regulated genes were selected for subsequent analyses if their expression values were found to exceed the threshold of 0.05 fdr (benjamini - hochberg correction). expression values of genes selected in the previous differential expression step were z - score normalized and supplied in input to the automated pipeline for som training and analysis. genes from regulated spots in the bidimensional output space were selected according to fdr threshold (< 0.1) at group - level. expression values of genes assigned to regulated spots extracted from the opossom output were subject to correlation analysis using model vectors to further refine the results and genes having expression profiles with p < 0.05 were discarded from further analysis and signature definition. a go enrichment analysis was performed for biological process terms associated with genes assigned to upregulated spots in the som bidimensional space using david. treg cells from crc and nsclc were isolated as previously described (see also table s1). single cells were captured on a microfluidic chip on the c1 system (fluidigm) and whole - transcriptome amplified cdna was prepared on chip using the smarter ultra low rna kit (clontech). for qpcr experiments, harvested cdna from single cells was pre - amplified using the same pool of taqman gene expression assays to be used for qpcr. single - cell gene expression experiments were performed using the 96 96 quantitative pcr (qpcr) dynamicarray microfluidic chips (fluidigm) on a biomark real - time pcr reader following manufacturer s instructions. a list of the 78 taqman assays used in this study is provided in supplemental experimental procedures. co - expression analysis has been performed by considering both crc and nsclc samples and genes for which co - expression with foxp3 and il2ra was null were discarded for the subsequent analysis. the violin color gradient represents the percentage of cells that are expressing the gene of interest. a non - parametric test (mann - whitney p < 0.05) has been performed on the selected genes by comparing tumor versus peripheral blood samples (see also supplemental experimental procedures). surface markers were directly stained with the following fluorochrome - conjugated antibodies and analyzed by flow cytometry : anti - cd4 (okt4), anti - pd1-lg2 (cl24f.10c12), anti - cd127 (clone rdr5), anti - cd25 (clone 4e3), anti-4 - 1bb (clone 4b4), anti - ccr8 (biolegend clone l263g8), anti cd30 (ebioscience, clone ber - h2), anti - pd - l1 (biolegend clone 29e.2a3), anti- tigit (ebioscience, clone mbsa43), anti - il1r2 (r and d clone 34141), il21r (biolegend clone 2g1-k12), and anti - ox40 (biolegend clone ber - act35). foxp3 and batf intracellular staining was performed with anti - foxp3 antibody (clone 236a / e7), anti - batf (clone mbm7c7), and expression analyzed by flow cytometry. (cfse)-labeled responders cd4 naive t cells from healthy donors were cocultured with different effector to target (e / t) ratios with unlabeled cd127cd25cd4 t cells sorted from tils or pbmcs of patients with crc or nsclc, using facs aria ii (bd biosciences), in the presence of cd11ccd1cdentritic cells as antigen - presenting cells and anti - cd3 (okt3) mab. some suppression assays were also performed with tumor treg cells that were preincubated with the following antibodies (at a final concentration of 20 g / ml) : anti - human pd - l1 (biolegend clone 29e.2 a 3), anti - human pd - l2 (biolegend clone mih18), and anti - human functional grade as isotype control (ebioscience clone mbsa43). the kaplan - meier analysis (km) was used to compare the high and low expression of the tumor - treg signature transcripts either crc (gse17536, n = 177) and nsclc (gse41271, n = 263) patients. m.d., a.a., g.r., and p.g. designed and performed the main experiments, analyzed the data, and contributed to the preparation of the manuscript. set up all the bioinformatics pipelines, performed the bioinformatics analyses, and contributed to the preparation of the manuscript. | summarytumor - infiltrating regulatory t lymphocytes (treg) can suppress effector t cells specific for tumor antigens. deeper molecular definitions of tumor - infiltrating - lymphocytes could thus offer therapeutic opportunities. transcriptomes of t helper 1 (th1), th17, and treg cells infiltrating colorectal or non - small - cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single - cell level. we found that tumor - infiltrating treg cells were highly suppressive, upregulated several immune - checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (il1r2), programmed death (pd)-1 ligand1, pd-1 ligand2, and ccr8 chemokine, which were not previously described on treg cells. remarkably, high expression in whole - tumor samples of treg cell signature genes, such as layn, mageh1, or ccr8, correlated with poor prognosis. our findings provide insights into the molecular identity and functions of human tumor - infiltrating treg cells and define potential targets for tumor immunotherapy. |
generalized vitiligo (gv) is the most common depigmentation disorder, in which acquired multifocal patches of white skin and overlying hair result from loss of melanocytes in the involved areas (figure 1). the prevalence of gv is approximately 0.5% in various populations, with an average age of onset at about 24 years and occurring with approximately equal frequency in males and females. the concordance of gv in monozygotic twin - pairs is approximately 23%, and epidemiological evidence indicates that gv is a complex trait involving multiple genes and unknown environmental factors. most current evidence supports an autoimmune basis of disease, though the triggers of the autoimmune response remain unknown. among european - derived white individuals with gv, about 15% to 25% have at least one additional concomitant autoimmune disease, particularly autoimmune thyroid disease (hashimoto 's thyroiditis and graves ' disease), pernicious anemia, rheumatoid arthritis, psoriasis, type 1 diabetes, addison 's disease and systemic lupus erythematosus ; these diseases also occur at increased frequencies in first - degree relatives of patients with gv, whether or not those relatives have gv themselves. together, these findings indicate that patients with gv and their close relatives have inherited susceptibility to this specific diathesis of autoimmune diseases, mediated by shared susceptibility genes. candidate gene association studies and gene expression analyses have produced a long list of gv candidate genes, of which only hla (human leukocyte antigen) and ptpn22 (protein tyrosine phosphatase, non - receptor type 22) have had consistent support from multiple studies ; most of the rest are likely to represent false positives. two additional genes, nalp1 (now nlrp1 ; nlr family, pyrin domain containing 1) and xbp1 (x - box binding protein 1), were first mapped by unbiased genome - wide linkage analyses and subsequently were identified by positionally targeted genetic association studies ; both of these genes have subsequently been replicated by multiple studies. these four confirmed gv susceptibility loci - hla, ptpn22, nlrp1 and xbp1 - all encode important immunoregulatory proteins, lending support to the autoimmune hypothesis of gv pathogenesis. recently, three different genome - wide association studies (gwass) of gv have been reported : two from european - derived white populations [9 - 11 ] and one from china. together, these studies identified a total of 17 confirmed gv susceptibility loci, yielding major insights into pathways of disease pathogenesis and overall strongly supporting an autoimmune basis for typical gv. the first gwas, of a founder population in an isolated romanian village with a high prevalence of gv and other autoimmune diseases, detected association at chromosome 6qter near iddm8, which is a type 1 diabetes - rheumatoid arthritis locus in the vicinity of smoc2 (sparc - related modular calcium binding 2). the second gwas, also carried out in european - derived white individuals, identified a total of 13 susceptibility loci for gv, including hla class i (specifically, hla - a0201), hla class ii, ptpn22, rere (arginine - glutamic acid dipeptide repeats), foxp1 (forkhead box p1), lpp (lim domain containing preferred translocation partner in lipoma), ccr6 (chemokine (c - c motif) receptor 6), il2ra (interleukin 2 receptor,), tyr, gzmb (granzyme b), nlrp1, ubash3a (ubiquitin - associated and sh3 domain containing a) and c1qtnf6 (c1q and tumor necrosis factor related protein 6). moreover, subsequent re - analysis of this genome - wide dataset to specifically test association of 33 biological candidate genes previously implicated in gv identified three additional gv susceptibility loci : tslp (thymic stromal lymphopoietin), xbp1 and foxp3 (forkhead box p3). the third gwa study, carried out in a chinese population, also identified gv susceptibility loci in the hla class i and class iii regions, and likewise detected association with ccr6. moreover, the gv - associated snp in the ccr6 region is the same in european - derived white individuals and chinese people, suggesting that these two populations may share a single causal allele, and is located only 1.44 mb from the previous gv - associated snp in the smoc2 region, suggesting the possibility that these two signals might be related. virtually all of the confirmed gv susceptibility loci encode known immunoregulatory proteins, and many have been associated with genetic susceptibility to other autoimmune diseases that are epidemiologically linked to gv (figure 2). circles indicate loci associated with susceptibility to a given autoimmune disease : yellow, shared risk alleles ; orange, opposite risk alleles at same snp ; white, secondary association due to primary association with autoimmune disease epidemiologically associated with generalized vitiligo. the one exception among the susceptibility loci associated with gv is tyr, which encodes tyrosinase, the key enzyme of melanin biosynthesis in melanocytes. however, in gv even tyr may act primarily to modulate recognition of the melanocyte target cell by the immune system. beyond its role in pigmentation, regard, gv is thus analogous to type 1 diabetes and autoimmune thyroid disease, in that genetic susceptibility to disease involves genes that encode key specialized intracellular components of the autoimmune target cell types and that constitute major autoantigens for the corresponding disease (gv : tyr, tyrosinase ; type 1 diabetes : ins, insulin ; autoimmune thyroid disease : tg, thyroglobulin). for gv, the causal tyr susceptibility variant appears to be the major (arg) allele of rs1126809, a common non - synonymous (arg402gln) polymorphism that has a minor allele frequency of 0.22 to 0.40 in european - derived white individuals. this polymorphism is rare in other populations, which is why it was not detected in the chinese gwas, even though tyr may well play a role in gv pathogenesis in all populations. in contrast, the minor (gln) allele, which is protective with respect to gv, is associated with susceptibility to malignant melanoma in european - derived white individuals. thus, from the standpoint of genetic susceptibility, the tyr arg402gln polymorphism represents an inverse relationship between gv and malignant melanoma. much of the biology that is likely to underlie this inverse relationship is already known, largely from extensive studies on melanoma patients, in whom gv may develop during the course of treatment and is an auspicious prognostic sign. tyrosinase is a major antigen presented to the immune system on the surface of melanocytes and melanoma cells by hla class i molecules, principally hla - a0201, which itself is a major gv risk allele. indeed, hla - a0201 and tyr 402arg exhibit significant genetic interaction in promoting gv susceptibility, reflecting a corresponding biological interaction. one of the important class - restricted epitopes presented by hla - a0201 is a specific modified tyrosinase peptide : ymdgtmsqv. however, the tyr 402gln variant results in an unstable polypeptide that is retained in the endoplasmic reticulum and degraded, thereby reducing the amount of tyrosinase peptide available for presentation on the cell surface. moreover, presentation of this tyrosinase peptide by hla - a0201 requires the posttranslational modification of residue 371asn to asp, via a mechanism that is probably inefficient in the tyr 402gln polypeptide. tyrosinase is thus an important signal by which the immune system recognizes melanocytes, and tyrosinase-402arg is likely to make a greater contribution than tyrosinase-402gly to immune surveillance (and thus protection) against malignant melanoma and to susceptibility to gv, whereas tyrosinase-402gln is associated with lower susceptibility to gv but greater risk of melanoma. indeed, the odds ratio for vitiligo susceptibility is 2.5 in tyr 402arg homozygotes compared to 402gln homozygotes. interestingly, two of the other gv susceptibility loci, il2ra and gzmb, encode proteins involved in differentiation and effector functions of cytotoxic t lymphocytes (ctls) that mediate melanocyte killing in gv and perhaps also participate in immune surveillance for melanoma cells. thus, at least four of the gv genome - wide association signals - hla class i (hla - a0201), tyr 402arg, il2ra and gzmb - may be part of a pathway by which ctls recognize and ultimately kill melanocytes in patients with gv, and perhaps also protect against incipient melanomas. these observations open several doors to future studies. in gv, it has been known for some time that patients have circulating skin - homing melanocyte - specific ctls. however, the molecular epitopes recognized by these melanocyte - specific ctls have not yet been identified. at least in gv patients carrying hla - a0201, one of these ctl epitopes might be the modified tyrosinase peptide ymdgtmsqv. furthermore, it is not yet known whether susceptibility to gv is generic with respect to hla - a0201 (which is quite common), or whether it is particular to specific subtypes, and whether those subtypes conversely confer the greatest protection against melanoma. recent gwass have yielded substantial progress in identifying genes involved in risk of gv, with 17 loci now confirmed (hla class i, hla class ii, hla class iii, ptpn22, rere, foxp1, lpp, tslp, ccr6, il2ra, tyr, gzmb, nlrp1, ubash3a, xbp1, c1qtnf6, and foxp3) : 16 in european - derived white individuals and four in chinese people, and, for a few genes, in both. nevertheless, the 16 loci identified in european - derived white individuals together account for only 10% of the total genetic risk of gv in that group, indicating that additional loci probably remain to be discovered, with a few common and perhaps numerous rare variants accounting for disease risk at each locus. essentially all of the confirmed gv susceptibility genes regulate function of the immune system, and many have also been associated with other autoimmune diseases, highlighting shared pathways of autoimmune susceptibility among these diseases. furthermore, findings for both hla - a and tyr suggest an inverse relationship between susceptibility to gv and susceptibility to malignant melanoma, with genetic interaction that reflects underlying biochemical and functional interaction between the corresponding proteins. the overall picture indicates that genetic variation at hla - a0201 and tyr interacts to modulate immune surveillance against malignant melanoma, with heightened surveillance predisposing to gv and protecting against melanoma, and reduced surveillance protecting against gv but predisposing to melanoma. this biological relationship may also explain the frequent occurrence of gv in patients treated for melanoma, in whom development of this autoimmune phenotype constitutes a relatively favorable prognostic sign. while these discoveries underscore the autoimmune nature of gv, they do not offer specific clues as to the environmental triggers that may initiate the autoimmune response. gv is a disease of the skin, the organ that is the first point of contact for one 's interaction with the external environment, and which is highly accessible for analysis of that interaction. thus, identification of gv susceptibility genes may enable identification of individuals at high genetic risk, enabling relatively direct analysis of potentially causal gene - environment interactions, both retrospectively in patients with relatively recent disease onset, and prospectively in individuals who are at high genetic risk. ctl : cytotoxic t lymphocyte ; gv : generalized vitiligo ; gwas : genome - wide association study ; snp : single - nucleotide polymorphism. dr richard a spritz is professor of pediatrics and director of the human medical genetics program at the university of colorado school of medicine. the work of dr spritz in the field of human genetics began in the ' pre - molecular ' era, and in the mid-1970s he took part in the first cloning of human genes and identification of the first human disease gene mutation. over the past 30 years, dr spritz and his colleagues have studied the genes involved in causing many different human diseases, including hemoglobin disorders, albinism and other skin diseases, autoimmune diseases such as vitiligo and thyroid disease, and cleft lip / palate, and he has published over 200 scientific papers on these investigations. most recently, dr spritz led an international team that carried out the first genome - wide association study for generalized vitiligo. this work was supported in part by grants r01 ar45584 and r01 ar056292 from the national institutes of health. | generalized vitiligo (gv) is the most common pigmentation disease, in which white spots of skin and overlying hair result from loss of melanocytes from the involved regions. gv is a complex disease involving both genetic predisposition and unknown environmental triggers. whereas various pathogenetic mechanisms have been suggested, most evidence supports an autoimmune basis for this disease. recently, three different genome - wide association studies of gv have been reported, identifying a total of 17 confirmed gv susceptibility loci. almost all of these genes encode immunoregulatory proteins, together highlighting pathways by which melanocytes might be recognized and killed. moreover, the biological interaction between two of these gv susceptibility genes, hla - a and tyr (encoding tyrosinase), points to an apparent inverse relationship between susceptibility to gv versus malignant melanoma, suggesting that gv may result, in part, from dysregulation of normal processes of immune surveillance against melanoma. |
the problem of alcohol misuse in russia is immense ; but nonetheless there is a tendency to exaggerate it, which is evident for inside observers. such exaggeration tends to veil shortcomings of the health care system with responsibility shifted onto the patients, that is, self - inflicted diseases caused by excessive alcohol consumption. the aim of this report is to draw attention to the above - mentioned and other problems related to the alcohol consumption in russia, not clearly perceptible from the literature, e.g. toxicity of some legally sold alcoholic beverages. this report is based on a review of literature and observations by the author during the period 1970 - 2014. predictable increase of alcohol consumption after the anti - alcohol campaign facilitated the economical reforms of the early 1990s : workers and some intelligentsia did not oppose privatizations of state - owned enterprises partly due to their drunkenness, involvement in workplace theft and use of equipment for profit, which was often tolerated by the management at that and earlier time. last time, a gradual change of the alcohol consumption pattern in russia has been noticed : less heavy binge drinking of vodka, fortified wine and surrogates ; more moderate consumption of beer. the problem of alcohol misuse in russia is immense ; but nonetheless there is a tendency to exaggerate it, which is evident for inside observers. such exaggeration tends to veil shortcomings of the health care system, with responsibility for the relatively low life expectancy shifted onto the patients, that is, supposedly self - inflicted diseases caused by excessive alcohol consumption. during the anti - alcohol campaign (1985 - 88), a widespread consumption of non - beverage alcohol was observed : perfumery and technical fluids such as window - cleaner, which caused up to severe intoxications. considering the large scale of the window cleaner sales in some places e.g. in siberia, it was knowingly tolerated by authorities. the alcohol consumption predictably increased after the anti - alcohol campaign. following the abolition of the state alcohol monopoly in 1992, the country was flooded by legally sold alcohol of poor quality (1) ; more details are in (2, 3). it was reported that about a half of the cases of lethal intoxication by alcohol - containing fluids during the 1990s were caused in some areas by legally sold beverages, while in many lethal cases a relatively low blood alcohol concentration was detected (4). the aim of this report was to draw attention to the problems related to the alcohol consumption in russia : indirect misplacement of responsibility for the relatively low life expectancy and high mortality onto the patients, who supposedly suffered from self - inflicted diseases caused by excessive alcohol consumption ; instable quality and toxicity of some legally sold alcoholic beverages, which have sometimes caused poisonings even after consumption in moderate doses ; offences and crime against alcoholics and people with alcohol - related dementia, sometimes aimed at appropriation of their apartments, houses and other property ; violation of their human rights and humiliating treatment by some employers and civil servants. apart from a review of the literature, this report has been based on the observations by the author, who entered the i.m. sechenov medical academy (named institute at that time) in the year 1973, later practiced at the same and other academic and clinical institutions in moscow. the time of observation of the alcohol - consuming milieu included the period 1970 - 2014. however, those in russia have not shown any significant growth since the middle of the 20th century (5). in the author s opinion, one of the reasons has been stagnation in the field of health care (6). in the early 1990s, at the same time, the average life expectancy at birth decreased considerably, especially in men : by 1993 it had slumped to about 59 years (7). medication costs for outpatient treatment are not covered by the compulsory medical insurance in russia. modern therapy of chronic diseases such as hypertension and diabetes mellitus is hardly available on a regular basis for many people. irregular treatment of hypertension is now as before a problem in the former su (8), obviously contributing to the higher cardio- and cerebrovascular mortality. there are indications that the cause - effect relationship between the alcohol consumption, cardio- and cerebrovascular mortality e.g. in (9 - 14) was exaggerated. at the same time, the mortality from cardio- and cerebrovascular diseases has obviously been overestimated (6). according to the author s observations during the 1980 - 90s, cardiovascular diseases were often overdiagnosed post mortem in unclear cases. if a cause of death was unclear, it has often been written on death certificates : ischemic heart disease with cardiac insufficiency or a similar formulation. a tendency of false - positive post mortem diagnosis of cardiovascular diseases existed also for people dying at home without autopsy. stroke as a cause of death has sometimes been diagnosed in patients with neurological symptoms, which in reality could have been caused by intoxication with substances other than ethanol. quality decrease in pathology and in the health care in general during the 1990s coincided with the increase in cardiovascular mortality (5, 10) ; at the same time, the stroke mortality jumped dramatically (9). overdiagnosis coupled with insufficient medical attention is obviously an important cause of the increase in the registered cardio- and cerebrovascular mortality. the alcohol consumption, increased after the anti - alcohol campaign, certainly contributed to the enhanced mortality. however, exaggeration of the causative role of alcohol befogs other preventable causes of the higher mortality. statements like alcohol accounts for most of the large fluctuations in russian mortality, and alcohol and tobacco account for the large difference in adult mortality between russia and western europe (12) veil another cause of the difference : insufficient availability and quality of health care. the following citation is illuminative in this connection : increases and decreases in mortality (have been) related to cardiovascular diseases (cvd), particularly to other forms of acute and chronic ischemia and atherosclerotic heart disease, but not to myocardial infarction, the proportion of which in russian cvd - related mortality is extremely low (11). the reason is evident : the diagnosis of myocardial infarction is usually made on the basis of clear clinical and/or pathological criteria, whereas atherosclerotic heart disease has often been diagnosed post mortem unsupported by evidence. on the contrary to myocardial infarction, gross features of a cerebral infarction can be imitated destroying brain tissue e.g. by a junior pathologist or postgraduate student not inclined or unable (due to unavailability of toxicological tests etc.) to search for a cause of death. there were cases of false - positive post mortem diagnosis of cerebral infarctions at an academic institution, which has probably happened more frequently in hospitals with no supervision over the quality of post mortem examinations. furthermore, the term alcoholic cardiomyopathy was sometimes used post mortem without sufficient evidence. in a large autopsy study, alcohol abusers were diagnosed with cardiomyopathy predominantly of moderate degree ; while in the majority of alcoholics (registered as such with substance abuse prevention and treatment centers) (172 cases) the cardiomyopathy was graded as pronounced (14). according to a study (13), clinically significant cardiomyopathy had been diagnosed in approximately 50% of habitual drinkers consuming in equivalent 60 - 80 ml pure alcohol / day or more. cause and effect of the alcohol misuse are sometimes mixed up : for example, the images of malformations of teeth or feet are presented as manifestations or sequels of alcoholism (13) ; in fact, however, people with curable malformations, often remaining without help in russia, can be psychologically predisposed to alcoholism. cardiomyopathy was claimed to be a frequent cause of sudden death of alcoholics (14), which in some cases could have been caused by undiagnosed and untreated diseases, intoxications etc. the tendency to exaggerate the cause - effect relationship between alcohol and the cardiovascular morbidity / mortality is relatively new in russia. an earlier epidemiological study reported that the rates of cardiovascular diseases including hypertension were not significantly higher among excessively drinking men compared to the male population in general (15). the curves of alcohol consumption and stroke mortality follow each other (9) ; but the estimation method of alcohol consumption is questionable : the harm indicator series used was alcohol psychosis incidence rate because this indicator depends almost entirely on alcohol consumption (9). the incidence of alcohol psychoses may reflect the alcohol consumption in the countries with a stable quality of alcoholic beverages but not in russia, where the quality of legally sold alcohol worsened during and especially after the anti - alcohol campaign. it would be reasonable to assume that psychosis - like conditions can be induced not only by ethanol but also by other substances in poor - quality alcoholic beverages. we observed marked mental confusion after a moderate consumption of fortified wines purchased in shops. discussing alcohol - related diseases, epidemiologic data indicate a higher frequency of alcohol - induced acute pancreatitis in geographical areas where surrogates or homemade alcoholic beverages are easily available (16). such beverages contain, in addition to ethanol, considerable admixtures of higher alcohols (e.g. propanol and butanol) and other contaminants (aldehydes, esters etc.), whose potency to induce pancreatic damage is insufficiently known (16). the main conclusion of the article (17) is that unfavorable mixture of higher overall level of alcohol consumption and binge drinking pattern is an important contributor to the pancreatitis mortality rate in russian federation. this is obviously true ; however, as discussed here in the next paragraph, the overall alcohol consumption in russia tends to decrease, and the heavy binge drinking pattern is visibly in decline. poor quality of alcoholic beverages should be pointed out among preventable causes of the relatively high frequency of acute pancreatitis in russia (17). exaggeration of the problem of heavy binge drinking tends to misplace responsibility for the health - related risks onto the drinkers, supposed to choose this risky pattern of alcohol consumption. according to razvodovsky (9), epidemiological evidence suggests that binge drinking is an important determinant of high stroke mortality rate in russia. it should be commented that heavy binge drinking has been visibly decreasing since approximately the year 2000 or even earlier time, especially in large cities. during the soviet time and shortly thereafter, many heavily drunk individuals could be observed in the streets, public gardens, long - distance trains etc. today, there are not so many drunks even among marginalized people. along with the changing pattern, there is a general tendency of alcohol consumption decrease since approximately the last decade (18) ; for an observer this tendency appears to be marked. wine and vodka have been partly replaced by beer (18), which has also contributed to a decline in the heavy binge drinking pattern. many young people drink today one to several cans of beer during an evening but not a bottle of vodka with beer for three or two persons, as it used to be during the 1970 - 80s. fortified wines or their imitations (containing 17 - 20 % of alcohol) were also massively consumed during the 1970 - 80s, while a 0.75 liter bottle per person was a usual dose ; more details are in (3). the causes of the gradual change of the drinking habits have been discussed previously : more responsible way of life under the market economy, intimidation and crime against alcoholics and people with alcohol - related dementia, aimed at appropriation of their apartments, houses or other property (3, 19). furthermore, in some papers on consumption of non - beverage alcohol (20), the readers attention is diverted to technical fluids (household chemicals, disinfectants, window - cleaners) and moonshine (samogon). it should be commented that the relative price of vodka / average salary was approximately 5 times higher in 1985 prior to the anti - alcohol campaign compared to the year 2010 (3). high consumption of non - beverage alcohol would be improbable in conditions of low prices for legally sold alcoholic beverages and their easy availability. it is in accordance with the author s observations that consumption of alcohol - containing technical fluids, lotions etc. there are obvious motives to exaggerate the consumption of non - beverage alcohol : to disguise the issue of toxicity of some legally sold alcoholic products. the predictable increase in alcohol consumption after the anti - alcohol campaign obviously facilitated the economical reforms i.e. privatization of the state - owned enterprises during the 1990s. workers and some intelligentsia did not oppose privatizations of governmental enterprises, occurring not always in accordance with the law, due to their drunkenness, involvement in workplace theft, use of equipment and consumables for profit, theft of technical or medicinal alcohol etc., which was tolerated by the management. the management themselves were known and seen to purloin alcohol in scientific and medical institutions. furthermore, there has been a stereotype, partly preserved from the soviet time : post - graduate students and doctoral candidates came to moscow and other centers from different parts of the country. some of them have been prepared to pay for literature reviews, preparation of specimens, etc. certain doctors planning emigration accomplished their dissertations under time pressure. among others, invasive methods such as endoscopy, organ biopsy or surgical operations this kind of research involved also alcohol abusers (21 - 23). in conclusion, a society should care for its vulnerable members, including aged citizens suffering of alcoholism and alcohol - related dementia. | contextthe problem of alcohol misuse in russia is immense ; but nonetheless there is a tendency to exaggerate it, which is evident for inside observers. such exaggeration tends to veil shortcomings of the health care system with responsibility shifted onto the patients, that is, self - inflicted diseases caused by excessive alcohol consumption. the aim of this report is to draw attention to the above - mentioned and other problems related to the alcohol consumption in russia, not clearly perceptible from the literature, e.g. toxicity of some legally sold alcoholic beverages.evidence acquisitionthis report is based on a review of literature and observations by the author during the period 1970 - 2014.resultspredictable increase of alcohol consumption after the anti - alcohol campaign facilitated the economical reforms of the early 1990s : workers and some intelligentsia did not oppose privatizations of state - owned enterprises partly due to their drunkenness, involvement in workplace theft and use of equipment for profit, which was often tolerated by the management at that and earlier time.conclusionslast time, a gradual change of the alcohol consumption pattern in russia has been noticed : less heavy binge drinking of vodka, fortified wine and surrogates ; more moderate consumption of beer. |
morphological studies using ct and mr imaging and physiological imaging using pet and spect scanning have played a pivotal role in defining landmarks used to manage primary brain tumors clinically. however, many questions regarding the care of patients with cerebral gliomas remain unanswered. recent studies have shown that proton magnetic resonance spectroscopy (1hmrs) can substantially improve the non - invasive categorization of human brain tumors, especially for gliomas. the recent emphasis on the utilization of 1hmrs (coupled to routine mri techniques) in the evaluation of tumors has arisen because it provides greater information concerning tumor activity and characterization of the tumor tissue than is possible with standard mri techniques alone. moreover, since standard neuroimaging methods can not reliably distinguish radiation necrosis from tumor recurrence, 1hmrs may prove to be a highly beneficial modality in the post - irradiation care of patients with brain gliomas. this paper will review the current status of proton mr spectroscopy with emphasis on its clinical utility in the diagnosis of active tumor processes of gliomas and its use in planning surgical and radiation therapy interventions and in monitoring tumor treatment. there are two types of spectroscopic imaging, namely, single - voxel and multivoxel mr spectroscopy. press (point - resolved spectroscopy) and steam (stimulated echo acquisition mode) are the two types of sequences used for single - voxel spectroscopy. multiple volume mr spectroscopy is also referred to as either chemical shift imaging (csi) or spectroscopic imaging. it is a method for obtaining spectroscopic information from multiple adjacent volumes over a large volume of interest. it is essentially similar to single - voxel spectroscopy except that the defined volume is normally a large slab. spectral patterns or specific metabolite intensities can be overlaid onto gray - scale mr images either to compare changes in spectra from adjacent voxels or to obtain a distribution pattern of a particular metabolite within the tissue examined. for instance, in the case of focal lesions, spectra from a tumorous lesion can be compared with spectra from normal tissue, then specific metabolite distributions over the region can be obtained. metabolites which can be identified on a standard brain proton mr spectroscopy include n - acetyl aspartate (naa), choline (cho), creatine (cr), myo - inositol (mi), glutamate and glutamine compounds (glu - n). naa is a marker for neuronal density and viability and therefore is decreased in all disease processes in which there is death of the neurons or replacement of neurons by other cells. this peak is composed of resonances from cr with contributions from creatine phosphate, gamma - aminobutyric acid, lysine and glutathione. the peak is assigned at 3.03 ppm and serves as a marker for energy - dependent systems in the brain cells. the cho peak is assigned at 3.2 ppm and contains contributions from glycerophosphocholine, phosphocholine and phosphatidylcholine. it reflects the metabolism of cellular membrane turnover and therefore is increased in all processes leading to hypercellularity. glu - n peak, resonating at 2.32.5 ppm, is detected in more tumors than controls, and a rise of glu - n peak may relate to a role of glutamate as an excitotoxin in accelerated cell proliferation of malignant brain tumors. it is assigned at 1.32 ppm and when detected indicates the presence of anerobic or nonoxidative metabolism, e.g. in abscess or necrosis. prospective grading of primary cerebral gliomas is a hazardous endeavor but with significant clinical benefit. the treatment and prognosis of different grades of tumors are different and can potentially affect the clinical outcome. conventional mr imaging provides important information regarding contrast material enhancement, perienhancement edema, distant tumor foci, hemorrhage, necrosis, mass effect, and so on, which are all helpful in characterizing tumor aggressiveness and hence tumor grade. however, often a high - grade glioma may be mistaken for a low - grade glioma when it demonstrates minimal edema, no contrast material enhancement, no necrosis, and no mass effect. moreover, large cerebral gliomas are often histopathologically heterogeneous and may have components of varying grades of malignancy within them. therefore, accurate preoperative grading of gliomas and planning of adequate treatment strategies are often difficult with conventional mr imaging alone. in addition, conventional mr imaging does not provide reliable information on tumor physiology such as metabolism, micronecrosis, or cellularity, all of which are also important in determining tumor grade. the addition of complementary biochemical information, as provided by 1hmrs imaging, could lead to further advances in the determination of the tumor grade of gliomas. specifically, an elevation in cho with depression of naa is a reliable indicator of tumor. there is extensive literature demonstrating the metabolite ratios of cho /cr, naa /cr, and mi /cr and the presence of lipids and lactate to be useful in grading tumors and predicting tumor malignancy. the recent finding of a direct correlation between cho and cellular proliferative activity provides objective confirmation of the potential of mr spectroscopy in predicting tumor grade. the presence of necrosis is one important distinction between anaplastic astrocytomas (grade iii) and glioblastoma multiforme (grade iv). the lipid peak arises predominantly from fatty acyl moieties that are relatively mobile and probably no longer confined to membrane phospholipids. therefore, lipids do correlate with necrosis in high - grade glioma and so may also be useful in differentiating glioma grades. meanwhile, it has been confirmed by previous studies that the relative increase in choline present in most high - grade gliomas is due to the increase in membrane synthesis and accelerated cell proliferation. however, high cho peak in a tumoral region is characteristic of rapidly growing tumors rather than unique for gliomas. the rise of mi peak in tumoral region is possibly related to gliosis and poor myelination because mi has been labeled as a breakdown product of myelin. theoretically, increased mi levels in the tumoral region are more likely to be present in high - grade gliomas though this is still controversial and needs further consideration. in addition, glu - n peak, resonating at 2.32.5 ppm, may be detected in more high - grade tumors than low - grade ones, and this has been confirmed by recent studies. this may relate to a possible role of glutamate as an excitotoxin in accelerated cell proliferation of malignant brain tumors. therefore, excessive accumulation of glutamate in a tumoral region may be an indicator of poor prognosis for malignant gliomas, though glu - n peak alone can not differentiate between high - grade and low - grade gliomas. however, accurate detection is difficult due to the proximity of the glu - n peak to the dominant naa resonance in this region. recently, several 1hmrs studies have been published looking at the peritumoral region of gliomas. the determination of the peritumoral region is critically important for the proper planning of treatment and enables the physician to anticipate the course of the disease. the peritumoral region (the so - called uncertain zone) is peritumoral tissue that appeared morphologically normal on routine mr images but may be infiltrated by tumor as determined by histologic analysis. it is assumed that malignant gliomas are not strictly focal lesions but rather are characterized by intracerebral dissemination of malignant glial cells along the myelinized axons and blood vessels or through the subarachnoid space. the presence of peritumoral region is a characteristic of high - grade glioma owing to its remarkable invasiveness. therefore, both high cho and glu - n peaks in peritumoral regions are more valuable indicators for high - grade gliomas with a poor prognosis compared with those in tumoral regions, although its clinical utility warrants further investigation in a larger study. in addition, using only conventional mr images, overestimation or underestimation of tumor size occurs in at least 40% of cases ; while 1hmrs study of the peritumoral region of gliomas may be used to map tumor margins and extension, and provide an insight as to the location most likely to yield the most representative tumor specimen. distinguishing radiation necrosis from tumor recurrence is a key aspect of post - radiation follow - up in patients with malignant gliomas. radiation necrosis occurs 312 months following radiation treatment and can be associated with exuberant gliosis. on ct scan or mri, contrast enhancement can be seen in both radiation necrosis and recurrent tumor due to disruption of the blood therefore, radiation necrosis may be indistinguishable from recurrent or residual tumor by imaging alone ; it is difficult to use these imaging methods to evaluate the viability or the proliferation activity of a tumor. the results of various 1hmrs studies of tumor treatment effects indicated a decrease in naa in both tumor and radiation necrosis consistent with the loss of functioning neurons in both pathologies ; however, an increase in choline in tumor recurrence (reflecting the increased cellularity of malignant tissues) compares to the decreased choline associated with necrosis. higher glucose utilization rates for lesions in which lactate is detected usually indicate high - grade gliomas. the various biochemical components provided strong evidence for the presence of tumor since increased choline and a lactate peak would not be expected in necrotic tissue. cho is the most important metabolite for the distinction between tumor and radiation damage, particularly if a pre - treatment choline value is obtained. a persistent or newly arisen distinct choline accumulation indicates residual or recurrent tumor after radiation therapy. 1hmrs is able to diagnose tumor recurrence early and unambiguously in cases where focal choline accumulation is detected. meanwhile, cho concentration is thought to be a useful marker for the evaluation of both early and late post - radiation response. delayed cerebral necrosis (dn) is a significant risk for brain tumor patients treated with high - dose irradiation. the primary diagnostic information for differentiating dn from tumor lay in the normalized mrs peak areas for cho and cr : a marked depression of the intracellular metabolite peaks from cho, cr, and naa indicates dn, and median to high cho with easily visible cr metabolite peaks is labeled progressive / recurrent tumor. radiation response is usually observed as a reduction of cho and cr levels and an increase in lipid levels, typically within 6 months of treatment. complete necrosis is manifested spectroscopically as an absence of all metabolites ; increases in cho correlated with poor radiologic response and suggested tumor recurrence. multivoxel spectroscopy and spectroscopic imaging delineate areas of tumor, radiation - induced changes and viable brain. successful therapy is heralded by a progressive decrease in level of choline and modest increases in naa. the author wishes to thank chongpeng sun, md, for his valuable advice and support. | in vivo proton magnetic resonance spectroscopy (1hmrs) can substantially improve the non - invasive categorization of human brain tumors, especially for gliomas. it provides greater information concerning tumor activity and characterization of the tumor tissue than is possible with mri techniques alone. moreover, 1hmrs may ultimately prove to be a highly beneficial modality in the post - irradiation care of patients with brain gliomas. this paper reviews the current status of 1hmrs with the emphasis on its clinical utility in the diagnosis of active tumor processes of gliomas, and its use in planning surgical and radiation therapy interventions and monitoring tumor treatment paradigms. |
angiosarcoma is a malignant neoplasm of endothelial cells that begins in the lining of the blood vessel wall. although angiosarcoma of the breast is a rare entity, the breast is a favorable site for it. primary angiosarcoma arises within the breast parenchyma, the overlying skin, and subcutaneous tissue. in contrast, secondary angiosarcoma of the breast is commonly associated with radiation therapy in patients who have undergone breast - conserving surgery for another type of primary neoplasm of the breast. these secondary angiosarcomas occur several years after the initial treatment, are often associated with lymphedema, and involve the skin. primary angiosarcomas account for fewer than 0.04% of all malignant breast neoplasms, and tend to occur in younger patients with no prior history of malignancy. the prognosis is poor for patients with primary angiosarcomas as compared with invasive ductal carcinomas. angiogenesis, believed to be strongly influenced by vascular endothelial growth factor (vegf), is crucial in the pathogenesis of these tumors. the histologic grade of primary angiosarcoma of the breast plays an important role in the prediction of outcomes. because high - grade primary breast sarcomas behave like extremity sarcomas, some authors have advocated using the same treatment protocol : surgical resection followed by adjuvant chemotherapy and radiotherapy. because of its rarity, the clinical course of primary breast angiosarcoma remains unclear, although metastases involving lung, liver, and other organs have been reported. here we present the clinical course of a 47-year - old female with primary angiosarcoma of the breast which showed recurrence to the chest wall and fatal lung complication with the review of literature. a 47-year - old female presented with a lump in the left breast in september 2009. sonography demonstrated an irregular, spiculated, 1.7 cm hypoechoic nodule in the upper inner quadrant of the left breast, accompanied by skin thickening and edematous change (fig. a core needle biopsy identified an atypical angiomatous lesion, and the diagnosis of angiosarcoma was confirmed by positive results for factor viii - related antigen and cd34. because the patient had no history of previous radiation therapy or surgery, we classified the tumor as a primary angiosarcoma. on dynamic magnetic resonance imaging (mri), 1). a positron emission tomography (pet)/computed tomography (ct) scan confirmed an area increased fluorodeoxyglucose uptake in the upper inner quadrant of the affected breast with faint uptake by the lymph nodes in the left axilla, but grossly, there is a 3.0 1.5 1.4 cm sized ill defined dark brown hemorrhagic spongy mass in the upper outer quadrant of breast. the epithelioid areas are made up of large rounded cells of high nuclear grade which are arranged in sheets or rudimentary vascular channels (fig. these neoplastic channels are irregular in shape, freely intercommunicating with one another in a sinusoidal fashion. the mass shows high mitotic count (14/10 hpf), high nuclear grade and necrosis, the diagnosis is angiosarcoma, grade 3 in world health organization classification. there is no lymph node metastasis. on immunohistochemical staining, the patient was treated with adjuvant chemotherapy of doxorubicin (60 mg / m) and ifosfamide (5,000 mg / m) every 3 weeks for six cycles from november 2009 until february 2010. following this, the patient underwent radiation therapy on the left chest wall with 50 gy for 5.5 weeks. we decided on follow - up 6 months later due to the possibility of local recurrence. in january 2011, the patient returned complaining of dyspnea that had started 1 week prior. a chest x - ray showed a large pleural effusion in the left side (fig. a chest tube was placed to drain the effusion and 3,600 ml of dark, sanguineous fluid was emptied. on histological examination, there was no definite malignant lesion but a marked proliferation of vascular endothelial cells, which was thought to be a reactive change. during this admission, the patient 's initial platelet count was normal, but she developed a gradual onset of thrombocytopenia that persisted until discharge (2 months later) despite platelet transfusions. effusion decreased gradually and chest tube was removed, but the patient 's condition was not yet improved (fig. chest x - ray and ct showed pulmonary hemorrhage in the left side and pleural effusion in the right side (fig. a chest tube was placed to drain the effusion and sanguineous output was noted. the patient was anemic and again remained thrombocytopenic in spite of transfusion. after diagnosis of recurrent angiosarcoma, we tried combination treatment with bevacizumab and paclitaxel with informed consent. after the first treatment, the patient 's respiratory symptoms improved, but after five days, her respiratory status again deteriorated. angiosarcomas are rare malignant tumors that arise from vascular endothelial cells. in general, they develop in the soft tissue and skin. patients with primary angiosarcoma of the breast present with a palpable mass mri will reveal a heterogeneous mass with low signal intensity on t1-weighted images and high signal intensity on t2-weighted images due to the vascular endothelial origin of the tumor. immunohistochemical stains for factor viii - related antigen and cd34 are helpful to confirm the diagnosis of angiosarcoma. in this case, on initial core needle biopsy, immunohistochemical examinations were positive for the expression of factor viii - related antigen and confirmed the diagnosis of angiosarcoma. grade 1 (low grade) tumors consist of vascular channels invading the breast fat and parenchyma with little or no endothelial proliferation. grade 2 (intermediate grade) tumors contain solid neoplastic vascular growth and papillary endothelial components. grade 3 (high - grade) tumors present as sarcomatous growths with necrosis and hemorrhage. in the case the clinical course for this patient was very difficult due to the recurrence of tumor and complicated by an intractable thrombocytopenia. this platelet consumption may have been due to defective synthesis of prostaglandin i2 by neovascular endothelium or to the release of a platelet aggregating substance by tumor cells. endothelial cells express several kinds of cell adhesion molecules, including icam-1, vcam-1, and pecam-1, but in this case, we were not able to evaluate for these molecules. such an evaluation may have been useful to confirm that the patient 's thrombocytopenia was due to the angiosarcoma. uncontrolled thrombocytopenia in this case is similar with the consumptive coagulopathy seen in kasabach - meritt syndrome, which is characterized by platelet sequestration and consumption of clotting factors within the vascular bed of giant angiomatous naevi. the widespread skin lesion, which was diagnosed as angiosarcoma had been observed in the left chest wall. so, we deemed that the uncontrolled thrombocytopenia was caused by persistent platelet consumption in the large skin lesion. in high - grade angiosarcoma, doxorubicin - based chemotherapy has been the mainstay of treatment for high - grade soft tissue sarcomas, and while adjuvant chemotherapy is recommended for grade 3 tumors, little benefit has been reported for grade 1 or grade 2 tumors. bevacizumab is a humanized monoclonal immunoglobulin g1 antibody that targets vegf, prevents binding of vegf to receptors on vascular endothelial cells, and blocks tumor angiogenesis. it has demonstrated efficacy as a first line treatment of metastatic breast cancer, especially in combination with paclitaxel and docetaxel. phase ii trials evaluating the efficacy bevacizumab with paclitaxel for angiosarcoma are currently under way. on the basis of this trial, we tried bevacizumab treatment after informed consent. the prognosis is poor in patients with primary breast angiosarcoma compared with other types of breast carcinoma and is dependent on tumor grade. disease - free survival 5 years after initial treatment is 76% with grade 1 tumors and falls to 15% with grade 3 tumors. disease recurrence is more than 15 years with grade 1 tumors, but in patients with grade 3 tumors, the time to recurrence averages 15 months. in this case, the patient had a grade 3 tumor and the time to disease recurrence was about 18 months. metastases occur most frequently to the lung, bone, liver, and chest wall. although not confirmed by biopsy, this patient had evidence of metastasis to the lung. she developed dyspnea, hemoptysis, and episodes of pleural hemorrhage beginning about 18 months after initial treatment and persisting until death. the hemoptysis is considered to have been caused by alveolar hemorrhage secondary to the suspected pulmonary metastasis, rather than spontaneous bleeding secondary to the thrombocytopenia. because primary angiosarcoma of the breast is so aggressive and rare, there is currently no consensus data for standard treatment or clinical course. we have detailed our experience with a primary breast angiosarcoma that failed to respond to multimodality treatment of surgical resection and adjuvant chemotherapy and radiotherapy. | primary angiosarcomas of the breast are rare malignancy that account for fewer than 0.04% of all malignant breast tumors. the prognosis is poor. surgery is the first line of treatment for angiosarcoma. adjuvant chemotherapy and radiotherapy have been tried, but their efficacy remains controversial. here we present the case of a 47-year - old woman with a palpable left breast mass that was diagnosed as a primary angiosarcoma. the patient underwent modified radical mastectomy with adjuvant chemotherapy and radiotherapy. postoperatively, eighteen months later, the angiosarcoma recurred. the patient returned complaining of dyspnea and hemoptysis and was found to have a large pleural effusion. she developed a gradual onset of thrombocytopenia that persisted despite platelet transfusions. finally, the patient died of respiratory failure secondary to pulmonary hemorrhage. |
total hip arthroplasty (tha) is one of the most commonly performed surgical procedures in the us, with an estimated 148,000 discharges among individuals aged 4564 years and 168,000 aged above 65 years in 2010.1 the most common reasons leading to tha are pain and decreased quality of life resulting from osteoarthritis.24 utilization of tha is projected to grow by 174% between 2005 and 2030 and will continue to grow substantially in the future because of an aging population and the obesity epidemic.2,5,6 a previous study found that low muscle strength was common in patients with poor physical functioning after tha : 73% of the 78 tha patients who complained of poor functionality after tha had muscle weakness.7 muscle atrophy / weakness (maw) has been found to persist for up to 2 years after hip replacement.8 however, no study has examined whether patients with or without maw have different demographic and clinical characteristics. although previous studies have used extensive real - world data to examine the utilization trend over time and outcomes among patients with tha, few have focused on the costs associated with tha.2,3,911 only one study over the past 10 years reported the annual costs before and after total joint replacement surgery in a commercially insured population with osteoarthritis, but that study did not distinguish between knee and hip arthroplasty.12 no study to date has examined the associations between maw and health care utilization and costs among patients with tha. using a large us claims database from 2005 to 2010, the present study compared the demographics, comorbid medical conditions, and health care costs and utilization among thr patients with and without maw. populations both younger than 65 years, and 65 years and older were included and were examined separately. results from the present study, which uses the most recent data, may advance the understanding of the burden of tha and how maw impacts health care costs and utilization. the truven marketscan commercial and medicare supplemental insurance databases (truven health analytics, ann arbor, mi, usa) were analyzed for this study. over 30 million enrollees from the commercial database (commercial population) were covered by approximately 100 payers that include large employers, health plans, government organizations, and public organizations. about 3 million enrollees in the medicare supplemental databases (medicare population) were 65 years and older with both medicare coverage as well as employer - sponsored supplemental insurance. these databases contain claims across inpatient, outpatient, and prescription drug services, as well as enrollment status, health plan type, and demographic characteristics such as age, gender, and region of residence. medical service claims contain details of health service encounters such as date and place of service, provider type, plan- and patient - paid amounts, and international classification of diseases, 9th revision, clinical modification (icd-9-cm) diagnoses and procedure codes. pharmacy claims contain information on dispensed medications including the national drug code, dispensed date, quantity, days supplied, and plan- and patient - paid amounts. all records can be linked via unique and encrypted personal identifiers to provide a longitudinal view of the health care services received. only individuals who had an inpatient stay associated with tha (icd-9-cm procedure code 81.51 or current procedural terminology codes 27130 and 27132) between january 1, 2006 and september 30, 2009 were identified. patients receiving a prior tha procedure from 1 year through 7 days before the index date were excluded ; all patients included were aged 50 to 64 years for the commercial population and at least 65 years old for the medicare population as of the index date. finally, to assess the health care resource utilization and costs in the 1 year before and 1 year after the procedure, all included patients must have continuous coverage from the 12 months before the index date (the pre - index period) through the 12 months after the discharge date recorded on the index hospital stay (the postindex period). because medicare and commercial plans have different reimbursement procedures and payment rates, patients in the commercial and medicare databases were analyzed separately. commercially insured individuals in the truven marketscan database include employees and their dependents who purchased insurance coverage from private health plans via their employers. coverage and cost sharing (eg, deductibles and copay) vary from plan to plan. medicare, regulated and funded by the us government, provides insurance coverage for aged (65 years old or older) and disabled persons. studies have suggested that medicare generally has a lower reimbursement rate than commercial plans for the same procedure or service.13,14 because health care costs are the main study outcome, separate analyses on commercially insured and medicare patients minimize the bias introduced by these different payment rates. within each population, each patient was further classified into three mutually exclusive cohorts : pre - maw, post - maw, and no - maw. the pre - maw cohort included patients with medical claims associated with maw (icd-9-cm codes 335.1x, 335.21, 359.xx, 728.2x, or 728.87 ; see detailed descriptions in the supplementary materials) during the 12 months before the index hospitalization. the post - maw cohort contained patients whose first maw claim was dated during or within the 12 months after the index hospitalization. the no - maw cohort included patients without any maw claim over the entire study period. the cohorts were created based on the assumption that the pre - maw cohort included individuals who had pre - existing maw due to hip problems or other health conditions before receiving tha, and the post - maw cohort included individuals who developed maw after the tha. patient demographic characteristics included age, gender, region of residence, and type of insurance coverage. the proportion of patients with comorbid osteoarthritis, osteoporosis, and rheumatoid arthritis was estimated. the burden of chronic disease was approximated using an adapted charlson comorbidity index (cci), which creates a score based on the presence or absence of 23 chronic conditions identified from claims during the pre - index period.15 characteristics of the index hospitalization were assessed including the length of stay, total cost, and the patients discharge status (ie, discharge to a skilled nursing facility [snf ], home, inpatient rehabilitation facility, short - term hospital, other facility, and other alive status). health care resource utilization incurred during the pre- and post - index periods was assessed, including the proportion of any use and frequency of hospital stays, physician office visits, outpatient hospital visits, and emergency department (ed) visits. any use of snf, inpatient rehabilitation facility, home health care, physical therapy, or occupational therapy over the post - index period was also compared across cohorts. direct medical costs were estimated as the sum of health plan paid amount of all medical and pharmacy claims that dated during the pre- and post - index periods, and compared across maw cohorts. major cost components (ie, inpatient, outpatient, and pharmacy costs) were estimated and compared across maw cohorts as well. all costs were adjusted to 2011 us dollars using the medical care component of the consumer price index.16 patient demographic characteristics, comorbidities, health care resource use, and costs were summarized and compared among pre - maw, post - maw, and no - maw cohorts, with no - maw as the reference group. chi - squared tests were used to detect statistically significant differences between cohorts for categorical variables (eg, gender, region of residence, health plan type, comorbidities, discharge status of index stay, and proportion with resource use) ; student s t - test were used to assess differences in age, and the wilcoxon rank - sum tests were used for cci scores, length of index hospital stay, number of medical encounters by settings (ie, hospitalizations, physician office visits, outpatient hospital visits, and ed visits), and associated health care costs incurred during the pre- and post - index periods. generalized linear regressions with log link and gamma distributions were performed to assess the association between maw cohorts and health care costs during the 12-month post - index period controlling for patient age, gender, region of residence, comorbid medical conditions, cci score categories, pre - index period health care resource use (ie, any hospitalization, ed, or outpatient hospital visit), and the discharge status of the index hospitalization. the adjusted cost difference for pre - maw and post - maw cohorts as compared with the no - maw cohort was estimated by computing the difference of predicted costs by holding the covariates constant while changing the values of maw cohort variables.17 for costs with many zero values (at least 10% of the patients), two - part models were then performed to estimate the marginal effects of maw on health care costs. a logistic regression model was first applied to estimate the probability of having nonzero health care costs.18 a generalized linear model was used in the second stage to estimate the health care costs among patients with nonzero costs. the estimated costs were calculated through multiplying the predicted probability generated from the logistic regression by the estimated conditional costs from the generalized linear model. the adjusted difference in health care costs of pre - maw and post - maw cohorts compared with the no - maw cohort were calculated in the same way when costs were estimated via generalized linear regressions with log link and gamma distributions. the 2.5 and 97.5 percentile of the 1,000 estimated marginal difference estimates by bootstrapping the two - part model with 1,000 iterations were used to construct the 95% confidence interval (ci) for statistical significance.19 the association between maw status and the probability of having any all - cause and a hip replacement / revision - related hospitalization in the post - index period was examined via logistic regression. additional explanatory variables included age, gender, us region of residence, comorbidities, cci score categories, preperiod resource use, and discharge status of the index hospitalization. the odds ratio (or) and 95% ci for each explanatory variable were reported. all analyses were conducted using sas 9.2 (sas institute, inc, cary, nc, usa) ; p - values < 0.05 were considered to be statistically significant. nearly 57,000 commercially insured individuals received tha between january 1, 2006 and september 30, 2009 (table 1) ; among them, more than half (n = 28,662) had at least 12-month continuous enrollment before and after the index hospitalization with tha and did not have claims for tha in 7 days to 1 year before the index hospitalization. of the remaining patients, 23,127 were between 50 and 64 years old and were included in the commercially insured analytical sample. of the 30,597 medicare beneficiaries who had tha between january 1, 2006 and september 30, 2009, 19,820 had an at - least 12-month continuous enrollment before and after the index hospitalization with tha, and did not have claims for tha in 7 days to 1 year before the index hospitalization. among them, 19,607 were 65 years or older at the time of the tha and were included in the medicare analytical sample. of the selected individuals, 1.4% of the commercial population had maw diagnoses prior to tha (pre - maw) and 5.1% during or after their tha (post - maw). similar results were seen in the medicare population, with 1.7% of individuals in the pre - maw and 4.6% in the post - maw cohorts, respectively. in both study populations, pre- and post - maw patients were significantly more likely to be female and older (except for commercial post - maw cohort) compared with patients in the no - maw cohort. the majority of the commercial patients resided in the south, whereas the majority of the medicare patients were in the midwest region. more than half of the commercially insured patients were insured via preferred provider organizations, but most medicare patients had comprehensive coverage. the pre - maw cohort in both the commercially insured and medicare populations had a lower prevalence of osteoarthritis, but a higher prevalence of osteoporosis compared with the no - maw cohort (all p < 0.05). patients in the pre- and post - maw cohorts had significantly higher cci scores and a longer length of stay during the tha index hospitalization than did the no - maw cohort (all p < 0.05) in both the commercial and medicare populations. total costs associated with the index hospitalization were higher in the post - maw cohort than in the no - maw cohort (both p < 0.05), but they were similar between the pre - maw and no - maw cohorts in both populations. more than three - quarters of patients in the commercially insured population were discharged home, whereas close to two - thirds of the patients in the medicare population were discharged home, followed in frequency by discharge to snf and short - term hospitals. table 2 presents the health care utilization over the 12 month pre- and post - index periods in both the commercially insured and medicare populations. in general, the pre - maw and post - maw cohorts had significantly higher health care resource utilization during the pre- and post - index periods than did the no - maw cohort, including any hospital stay, total hospital days, any mean number of outpatient hospital visits, and the mean number of ed visits. pre - maw and post - maw cohorts were also significantly more likely to use snf, inpatient rehabilitation, home health, physical therapy, and occupational therapy during the postindex period than did the no - maw cohort. over 67% of the commercially - insured patients used physical therapy, 17% to 23% used occupational therapy, and 10% to 18% had a snf stay over the 12-month post - index period. among the medicare patients, 30% to 53% had a snf stay, 37% to 54% had a physical therapy visit, and 11% to 22% had used occupational therapy. among commercially insured patients, the pre - maw ($ 35,022) and post - maw ($ 15,011) cohorts had higher mean total costs than the no - maw patients ($ 12,952 ; both p < 0.05) over the pre - index period, with over 50% of the costs resulting from outpatient services (table 3). total costs increased from the pre- to post - index period the most for the post - maw cohort, followed by the no - maw cohort, but remained similar for the pre - maw cohort. during the post - index period, pre - maw patients had the highest total costs ($ 33,847) followed by the post - maw ($ 27,904) and no - maw cohorts ($ 17,049). similar trends were observed in the medicare population. after adjusting for patient demographic and clinical characteristics, commercial patients in both the pre- and post - maw cohorts had significantly higher adjusted postindex total costs than those in the no - maw cohort (adjusted cost difference : $ 6,697 and $ 8,594, respectively ; both p < 0.05) (figure 1). most of the total cost differences were attributed to the difference in inpatient costs ($ 5,681 and $ 6,121, respectively ; both p < 0.05), followed by outpatient costs ($ 2,006 and $ 3,425, respectively ; both p < 0.05). similarly, the pre- and post - maw cohorts in the medicare population had significantly higher total costs than the no - maw cohort (adjusted difference : $ 7,271 and $ 11,170, respectively ; both p < 0.05). however, most of this variation was due to the difference in outpatient costs ($ 4,167 and $ 7,138 respectively ; both p < 0.05). using logistic regression to control for cross - cohort differences in demographics, clinical, and economic characteristics, patients in the pre - maw and post - maw cohorts were more likely to have had a subsequent hospitalization during the 12-month post - index period than the no - maw cohort (table 4). specifically, compared with no - maw patients, commercially insured patients in the pre - maw and post - maw cohorts were 66% (or 1.66 ; 95% ci 1.312.11) and 57% (or 1.57 ; 95% ci 1.381.80) more likely to have any hospitalization than those in the no - maw cohort, respectively. a 28% higher risk for the pre - maw cohort (not statistically significant) and 90% (statistically significant) for the post - maw cohort were observed in the medicare population. other factors associated with a higher risk of any hospitalization include a higher cci score (.1), comorbid rheumatoid arthritis and osteoporosis, being discharged to snf, and prior inpatient and ed use. when assessing hip replacement - related hospitalization, a higher risk was observed only for the pre - maw (or 1.76 ; 95% ci 1:322.36) and post - maw (or 1.48 ; 95% ci 1.251.75) cohorts in the commercially insured population. the number of total hip arthroplasties performed in the us has increased substantially in the past decade, and the number will continue to rise in the future.5 by year 2030, kurtz have predicted that over a half million thas will be performed during that year. the associated health care costs are also predicted to increase substantially.20 with the increased demand and costs associated with tha, it is important to understand the health care resource utilization and costs associated with tha and to identify subgroups of high - cost patients. to the best of our knowledge, this retrospective database study of both commercially insured and medicare patients is the first to suggest that maw is associated with higher health care utilization and costs among individuals who underwent tha. in particular, patients with maw before receiving tha had the highest level of health care resource utilization and costs during the 12 months before the procedure among the three cohorts, and patients who developed maw during or after the tha had the most substantial increase of health care costs and the highest health care costs during the 12 months after tha. two studies have examined younger patients undergoing tha.10,12 however, one study is limited in terms of generalizability because it was conducted among veterans. the other study combined total knee and hip arthroplasty ; therefore, it did not report results specifically for tha. the present study is, to the best of the authors knowledge, the only study that uses the most recent data to provide patient characteristics, health care costs, and utilization for a patient population under 65 years of age undergoing tha. consistent with findings from previous studies that examined tha in the medicare population, the average age of the medicare beneficiaries in the current study was mid-70s, and over 60% of patients were female.9,11 the length of hospital stay associated with tha was also similar to that in previous studies. the present study not only provided more detailed information on health care costs and utilization among medicare patients after undergoing tha, but also confirmed that patients with maw (before or after tha surgery) had higher health care resource use and costs than those without maw. therefore, health care providers may need to pay more attention to the high - cost subgroups when considering tha. patients in both the pre - maw and post - maw cohorts examined in this study were older and more likely to be female than those without maw. as people age, muscle tends to be lost as part of the aging process. studies also suggest that women are at a higher risk of developing maw than men because women, in general, have less muscle mass and strength and lose muscle mass faster during menopause.2123 individuals undergoing tha who had maw also had poorer overall health (as reflected by a higher average cci score) ; this is consistent with existing clinical studies that suggest maw often exists in individuals with disabling chronic conditions, such as chronic obstructive pulmonary disease, heart failure, and osteoporosis.2430 therefore, patients with overall poor health or many comorbidities may need to be further evaluated for maw. previous publications have found that maw is reversible in many patients with timely and appropriate treatment as the development of maw has been mainly attributed to long - term inactivity, biological changes resulting from aging or disease, and inadequate nutrition, and can be treated with a combination of appropriate exercise, nutrition, and pharmacological treatment.27,3133 treating maw is especially important among patients receiving tha because maw has been linked to impaired functional status, delayed rehabilitation, and increased risk of complications, such as joint instability and pain.7,8,34 patients with maw in the present study also had significantly higher health care utilization and costs than did those without maw. appropriate management of maw among patients undergoing tha may lead to improved patient functioning, better quality of life, and potentially to cost savings. the identification of maw was based on diagnostic codes in the medical claims and may be underreported in the administrative claims due to unrecorded diagnosis. that can lead to including misclassified patients in the no - maw cohort. such misclassification is likely to bias the estimate of health care costs associated with maw. emergency or elective hip arthroplasty were not differentiated in this study, because such information is absent in administrative claims data. because the proportion with elective surgery across the maw cohorts is unknown, it is difficult to assess the impact of elective or emergency surgery on the study conclusions. no eligible patients included in the study received a prior hip arthroplasty in the previous 12 months, and all must have had at least 1 year of continuous enrollment before and after the index tha hospitalization. therefore, findings derived from the current study may not be generalizable to other populations. it is also plausible that there were unobservable confounders that were not adjusted, which could result in biased estimates. among us patients undergoing tha, those with maw had higher health care resource use and costs as compared to those without maw. commercially insured patients with maw were also more likely to have any all - cause, as well as replacement - related, subsequent hospitalization than those without maw. icd-9 codes for muscle atrophy / weakness abbreviation : icd-9, international classification of diseases, ninth revision. | this study analyzed administrative claims by a us population with commercial or medicare supplemental insurance to compare demographics, comorbid medical conditions, and health care utilization and costs among patients undergoing total hip arthroplasty (tha) with and without muscle atrophy / weakness (maw). patients were classified into three cohorts : having maw during the 12 months previous to tha (pre - maw) ; having maw during or over the 12 months after tha (post - maw) ; or no maw claim (no - maw). in total, 19,607 medicare and 23,127 commercially insured patients were examined. controlling for cross - cohort differences, both pre - maw and post - maw commercial cohorts had significantly higher total costs ($ 6,697 and $ 8,594, in usd respectively) and higher risk of all - cause hospitalization (odds ratios, 1.66 and 1.57, respectively) than the no - maw cohort (all p < 0.05) during the 1-year follow - up. similar trends were observed in the medicare population. |
alcohol abusers and patients with alcoholic liver disease (ald) usually suffer negative consequences from drinking such as significant financial burden, unemployment, loss of family, accidental injury, or death. alcoholism is a physical dependence that includes impaired control, craving, development of tolerance, and development of withdrawal symptoms on abstinence. ald is a spectrum that ranges from fatty liver to alcoholic steatohepatitis (ash) and eventually cirrhosis. simple hepatic steatosis is the commonest histological finding and occurs in 90% of heavy drinkers but is rapidly reversible with abstinence. alcoholic hepatitis or ash occurs in up to 35% of heavy drinkers and is usually a precursor of cirrhosis. epidemiological data suggest that a threshold of 80 g of daily alcohol in a male and 2040 g in a female for an average of 10 to 12 years is necessary for causing significant alcohol - induced liver injury [3, 4 ]. however, only a minority of individuals who consume alcohol in excess develop significant ald. synergistic factors such as chronic hepatitis c, obesity, and genetic factors may accelerate the development of ald even at lower doses of alcohol consumption. there may be low - grade fever, jaundice, leukocytosis, and mild elevation of transaminases. histological features of ash include the presence of parenchymal necrosis, mallory bodies, and a perivenular neutrophilic infiltrate. other features that are commonly present include bridging necrosis, fatty changes, bile duct proliferation, cholestasis, and perivenular fibrosis. liver biopsy as a means of prognostication in alcoholic hepatitis has mostly been replaced with less invasive scoring systems. patients with severe alcoholic hepatitis can have clinical presentation almost similar to those with decompensated cirrhosis, and it may become difficult to establish if such patients have associated cirrhosis or not. but histologically, the majority of patients with severe alcoholic hepatitis have either significant fibrosis or cirrhosis liver. and alcoholic hepatitis with underlying cirrhosis is one of the most important causes of acute on chronic liver failure (aclf). the maddrey discriminant function (df) score remains the most commonly used predictive model and was developed to facilitate the assessment of response in a clinical trial of corticosteroids in patients with alcoholic hepatitis. modified df is calculated as = 4.6 (prolongation of prothrombin time in seconds) + serum bilirubin (mg / dl). a modified df score > 32 in the presence of hepatic encephalopathy predicts > 50% mortality within 28 days in patients with alcoholic hepatitis [7, 8 ]. however, fatal outcomes have also been known to occur in patients with modified df score 2,000 kcal daily) with an isocaloric standard oral diet in 35 randomly allocated, severely malnourished, cirrhotic patients. in - hospital the second study which compared enteral feeding with steroids in 71 patients with acute, severe ash found that there was no difference in mortality between the groups during the 28-day treatment period. however, the mortality rate was lower in the enterally fed group in the year following treatment. in summary, nutritional supplementation could have a role in the improvement of medium - term to long - term survival in patients with severe ash. the american college of gastroenterology guidelines recommend 1.2 to 1.5 g / kg of protein and 35 to 40 kcal / kg of body weight for patients with ald. glucocorticoids are the most intensely studied and yet most hotly debated treatment for acute alcoholic hepatitis. the rationale for glucocorticoid use is to block cytotoxic and inflammatory pathways in alcoholic hepatitis. glucocorticoids have been shown to decrease proinflammatory cytokines and intercellular cell adhesion molecule 1 expression and inhibit neutrophil activation and have demonstrated short - term histological improvement in patients with alcoholic hepatitis. results from trials of glucocorticoids for ald are variable and depend on the nature of the trial and the group of patients recruited as the study population. even among glucocorticoids trials with beneficial results, enrolled subjects were heterogeneous with variable definitions of randomization and blinding and without homogeneous inclusion or exclusion criteria. steroid use in alcoholic hepatitis raises the risk of infection in an already immunocompromised host. some trials have demonstrated higher mortality in the glucocorticoid group compared to the placebo group [2326 ]. associated with this higher mortality, a greater incidence of fungal infections among patients receiving glucocorticoids has been reported by some authors. a meta - analysis on this subject, published in 1990, demonstrated a protective effect of glucocorticoids in high quality trials. this was especially so in studies that excluded patients with gastrointestinal bleeding but included those with hepatic encephalopathy. but another meta - analysis by christensen and gluud found no benefit once they attempted to control for confounders. a subsequent reanalysis of the same 3 randomized, controlled trials in christiansen and gluud 's meta - analysis, which pooled raw data from more than 200 patients with modified df 32, found a 28-day survival benefit of glucocorticoids (85%) versus placebo (65%). in patients with modified df 32, treatment with glucocorticoids improved short - term (28-day) survival, with mortality decreasing from 35% in controls to 15% with steroids. conversely, patients with modified df 90% survival rate without steroids. the number of patients who needed to be treated to save 1 patient was 5. another meta - analysis of 15 trials with 721 randomized patients reported that the evidence in favor of glucocorticoids was based on heterogeneous trials of low quality. patients were classified as complete responders (lille score 0.16 ; 35th percentile), partial responders (lille score 0.160.56 ; 35th70th percentile), and null responders (lille 0.56 ; 70th percentile). 28-day survival was strongly associated with these groupings (91% versus 79% versus 53%, p < 0.0001). corticosteroids had a significant effect on 28-day survival in complete responders and in partial responders but not in null responders. the long - term benefit of steroids is difficult to assess as the various trials had differing follow - up periods, and unless the patient abstains from alcohol completely, alcoholic hepatitis is likely to recur. the survival benefit of corticosteroid therapy has not been found to persist beyond 1 year. despite having 13 randomized controlled trials and 6 meta - analyses of steroids as a treatment for ash, concerns over their use continue. although corticosteroids are probably beneficial in patients with severe disease, mortality on treatment remains high, particularly when renal impairment is present, and treatment is contraindicated in a relatively large number of patients with concomitant infection and gastrointestinal bleeding. elevated tumor necrosis factor alpha (tnf) levels have been found to be predictive of poor survival in patients with alcoholic hepatitis. pentoxifylline is a nonselective phosphor - di - esterase inhibitor and a tnf suppressor. in 1991, a study of pentoxifylline for severe alcoholic hepatitis (df 32) reported a reduction in the development of hepatorenal syndrome and mortality in comparison with placebo. a subsequent study of 101 patients from the same center supported the earlier findings, demonstrating a 40% reduction in mortality in comparison with placebo. however, in this study, there was no demonstrable improvement in routine liver function tests or liver histology and the better survival was predominantly due to decreased incidence of hepatorenal syndrome. in another study, 29 patients who did not respond to corticosteroids (identified by an absence of an early decline in bilirubin) were switched to pentoxifylline for 28 days and compared to 58 other matched patients who persisted with corticosteroid therapy. thus, although some data suggest a benefit with pentoxifylline in alcoholic hepatitis, it is unclear whether its benefit extends beyond possibly preventing hepatorenal syndrome. infliximab is an anti - tnf mouse / human chimeric antibody and has been extensively studied in alcoholic hepatitis. early reports were encouraging, demonstrating improved survival rates, improved maddrey scores, or improved laboratory parameters. however, the largest randomized, controlled trial to date, which enrolled 36 patients and compared a combination of prednisolone (40 mg / day) and infliximab (10 mg / kg 3 times per week in weeks 0, 2, and 4) to prednisolone and placebo in alcoholic hepatitis was terminated prematurely. more deaths had occurred in the group treated with prednisolone and infliximab. whether this risk was related to the higher doses of infliximab used, more ill patients being recruited in this trial in comparison with earlier studies, the combined use of prednisolone and infliximab, or the possible unsuitability of infliximab for the treatment of alcoholic hepatitis is still debatable. a small, uncontrolled study evaluated the use of etanercept in 13 patients with moderate to severe alcoholic hepatitis. two patients died within 32 days, and 3 developed serious adverse events (infection, gastrointestinal bleeding, and hepatorenal syndrome) mandating withdrawal of the drug. a more recent multicenter, randomized, double - blind, placebo - controlled study of etanercept in 48 patients with severe alcoholic hepatitis (defined as meld 15) found no difference in the 1-month mortality rates in the 2 groups on an intention - to - treat analysis. alarmingly, the 6-month mortality in the etanercept group was significantly higher in comparison with the controls. these results have considerably dampened the enthusiasm for using anti - tnf agents in patients with severe ash. antioxidants have been tried in alcoholic hepatitis as oxidative stress is a key factor in its pathogenesis. phillips., who compared corticosteroids to an antioxidant cocktail (-carotene, vitamins c and e, selenium, methionine, allopurinol, desferrioxamine, and n - acetylcysteine), reported inferior survival rates in comparison with corticosteroids at 30 days. the active group received a loading dose of n - acetylcysteine (150 mg / kg followed by 100 mg / kg / day for 1 week) and daily doses of vitamins a and e, biotin, selenium, zinc, manganese, copper, magnesium, folic acid, and coenzyme q for 6 months. a more recent study of 87 patients with severe alcoholic hepatitis (defined as modified maddrey df 32) randomized patients to receive either corticosteroids with n - acetylcysteine infusion for 5 days or corticosteroids alone. although patients in the n - acetylcysteine group had better 1-month survivals, this effect did not persist at 3 and 6 months. s - adenosyl methionine (same) is a precursor of glutathione that theoretically might be protective in alcohol - induced liver injury. in a cochrane database review of 9 randomized trials that combined a heterogeneous sample of 434 patients with ald. only 1 trial of 62 patients deemed to have adequate methodology and outcome reporting and good quality suggested benefit (improved survival and delay to liver transplantation) with 2 years of same treatment for child 's class a and b alcoholic cirrhosis. in patients with severe ash, the development of renal failure is associated with a survival of less than 10% even with intensive management. the most significant advance in the management of patients with advanced liver disease over the past decade has been the introduction of albumin infusions combined with splanchnic vasoconstrictor agents for patients with hepatorenal syndrome. although no randomized trials have specifically examined this form of therapy in patients with ash, the previously reported high mortality in ash patients with hepatorenal syndrome suggests that albumin infusions combined with splanchnic vasoconstrictor agents would have a significant and beneficial effect on patient survival. orthotopic liver transplantation (olt) remains the definitive therapy for decompensated cirrhosis due to ald despite continued alcohol abstinence. short - term (1- to 7-year) graft survival and patient survival remain at par with, if not superior to, survival with non - ald if the patient remains abstinent. bellamy. reported that in 123 patients who were transplanted, patient survival at 1, 5, and 7 years was 84%, 72%, and 64%, respectively. graft survival was 81%, 66%, and 50%, respectively, over the same period. the 1- and 5-year patient and graft survival rates for all patients with cirrhosis were 86.9%/73.4% and 82.4%/67.4%, respectively. however, olt for ald patients continues to fuel controversy, including issues of recidivism, potentially poor compliance with postoperative care, and inherent biases against alcoholics, such as concern about using scarce organs for what is often perceived to be a self - inflicted disease.. 's prospective study of alcoholic recipients found that 22% had used some alcohol by the end of the first year post - olt and 42% had by 5 years, of whom 26% had a binge drinking pattern. such a wide range of relapse rates may stem from varying definitions of recidivism and methods of eliciting alcohol consumption data. most studies addressing recidivism in the past 20 years have used the any use definition of alcohol relapse. however 's report of patients who had returned to drinking revealed no decreased compliance with other medical care, including immunosuppressant therapy. stratified relapsers into slips and harmful drinking, revealing significantly worse 5- and 10-year survival rates (69.5% and 20%) among harmful drinkers versus abstainers (90.3% and 81.5%). a major focus in determining candidacy for liver transplantation in ald has been identifying factors to predict posttransplant recidivism. other medical and social variables can be associated with high relapse rates, including tobacco consumption, noncompliance to follow - up clinic visits, and mental illness. a 2008 meta - analysis of 50 studies looking at predictors of recidivism found 3 significant, albeit modest, variables : a poor social support system, a family history of alcohol abuse / dependence, and pretransplant abstinence of 6 months or less. studies over the years have provided convincing data for and against the 6-month abstinence requirement. lucey. suggested that this 6-month period of abstinence would allow the native liver to recover with medical management and possibly obviate transplantation. however, this minimal period of abstinence is sometimes waived if the patient is deemed too ill to survive beyond 6 months without a liver transplant. one study showed that recovery in decompensated alcoholic cirrhosis by alcohol abstinence can be predicted within 3 months of abstinence by the monitoring of clinical signs via the child - pugh scoring system (serum bilirubin, albumin, international normalized ratio, ascites, and hepatic encephalopathy). this study by veldt. found that although such improvement in liver function can take place within 3 months of abstinence, some abstinent patients die within 6 months ; this has led some authors to suggest reducing the period of abstinence from 6 to 3 months. yet, abstinence less than 12 months was recently identified as a significant risk factor for relapse in a large retrospective study of olt recipients. finally, an increasing concern of late is the high risk for de novo malignancies in long - term survivors transplanted for ald. although posttransplantation lymphoproliferative disorder and nonmelanoma skin cancer remain the most common malignancies after liver transplantation, the incidence of esophageal cancer is significantly increased in patients with alcohol as the etiology of end - stage liver disease.. 's prospective study showed a significantly higher incidence of malignancies in patients with ald compared to those with non - ald etiologies. they also detected squamous cell carcinoma of the oropharynx or esophagus only in recipients transplanted for ald. risk factors undoubtedly include the cumulative effects of alcohol and, in most cases, smoking with posttransplant immunosuppression. thus, regular ear, nose, and throat examinations appear justified in patients transplanted for ald. | alcoholic liver disease (ald) is a spectrum ranging from simple hepatic steatosis to alcoholic hepatitis and cirrhosis. patients with severe alcoholic hepatitis can have clinical presentation almost similar to those with decompensated cirrhosis. scoring with models like maddrey discriminant function, a model for end - stage liver disease, glasgow alcoholic hepatitis score, and lille model are helpful in prognosticating patients with ald. one of the first therapeutic goals in ald is to induce alcohol withdrawal with psychotherapy or drugs. most studies have shown that nutritional therapy improves liver function and histology in patients with ald. the rationale for using glucocorticoids is to block cytotoxic and inflammatory pathways in patients with severe alcoholic hepatitis. pentoxifylline, a tumor necrosis factor alpha (tnf) suppressor, and infliximab, an anti - tnf mouse / human chimeric antibody, has been extensively studied in patients with alcoholic hepatitis. liver transplantation remains the definitive therapy for decompensated cirrhosis / alcoholic hepatitis despite the issues of recidivism, poor compliance with postoperative care, and being a self - inflicted disease. |
risk of death and major complications after surgery is impressively low today in the general surgical patient population : less than 1% of all patients undergoing surgery die during the same hospital admission. despite this low overall risk of death, mortality in some subgroups of patients for example, in patients undergoing major abdominal surgery, the presence of more than one clinical risk factor of surgical complications may increase the postoperative mortality three - to four - fold. similarly, prolongation of hospitalisation after surgery due to any complication increases the mortality several fold. it is, therefore, not surprising that various systems have been developed to identify patients at increased risk of peri- and postoperative mortality and morbidity. examples of such tools include the asa classification, the possum scoring system (in diverse versions), the shoemaker criteria for high risk, and goldman 's cardiac risk index, just to name a few [3 - 6 ]. what is much more surprising is how little effort has been invested in developing tools to reduce the risk of peri- and postoperative complications in well - defined patient groups at high risk, and thus how little success has been achieved in this area. in this issue of critical care, publish two reports related to predicting and improving outcome in high - risk surgical patients. in the era of large, multicentre, randomised, controlled trials, the efforts of the research group established by dr david bennett at st george 's hospital, london, represent an alternative approach. instead of testing attractive clinical concepts in multicentre, randomised, controlled trials as soon as possible, these researchers have been very persistent and systematic in testing the concept of goal - directed haemodynamic management in high - risk surgery in their single - centre setting. they have largely adopted the original strategy presented by dr william shoemaker in the 1980s, using predefined targets of oxygen delivery, first applying the pulmonary artery catheter and now pulse power / lithium dilution - based cardiac output monitoring. dr bennett 's group started with feasibility and risk analysis studies, then progressed to randomised, controlled intervention studies and health economic analyses, and also applied the results in their daily clinical practice. the st george 's group, and groups interacting with them, have repeatedly demonstrated that, in the single - centre setting, various outcome measures (mortality, morbidity, hospital length of stay and costs) can be reduced with goal - directed haemodynamic management. boyd. demonstrated a reduction in mortality from 23% to 6% with oxygen transport - guided treatment in patients fulfilling the shoemaker criteria for high - risk surgery in major abdominal surgery. demonstrated reduced morbidity and length of stay in hip fracture patients when perioperative fluid management was driven by stroke volume monitoring. wilson. showed major reductions in mortality and morbidity with peri- and postoperative oxygen transport - guided treatment in patients undergoing major abdominal or vascular surgery.. showed reduced length of stay and morbidity with both central venous pressure- and stroke volume - guided perioperative treatments in patients with proximal femur fracture. mckendry. showed in cardiac surgery patients that haemodynamic management driven by stroke volume postoperatively reduced the length of hospital stay. demonstrates reduced morbidity and length of hospital stay in high - risk patients undergoing major, predominantly vascular or abdominal surgery when receiving oxygen - delivery - driven goal - directed management based on lithium indicator dilution and pulse power cardiac output. no difference in mortality was observed between the goal - directed management and the control group, and the mortality was substantially lower than that of the control group in the study by boyd. importantly, the management of the control group was also strictly protocolised, based on central venous pressure - driven fluid challenges. this series of single - centre studies with impressive positive results is in sharp contrast to the negative results of the multicenter study by sandham., where patients undergoing major surgery were randomised to receive pulmonary artery catheter with oxygen transport - driven guidelines for peri- and postoperative haemodynamic management versus conventional management the major limitations of the sandham trial have already been discussed in this journal in detail, and will not be repeated here. perhaps the most important difference is that all the successful single - centre trials have used strict treatment protocols, whereas sandham. used guidelines. the successful single - centre studies also have to be interpreted in the context of the specific institutions where they are performed. the risks associated with surgery are multifactorial, and the same high - risk criteria applied in different institutions and to different case mixes may reveal very different patient profiles. applying the same high - risk criteria as boyd. in a multicentre trial, we observed a mortality of 16% versus the 23% observed by boyd. furthermore, patients with only one risk factor had a mortality of 4%, whereas those with two or more risk factors had a mortality of 20%. the single - centre trials utilize their investigators ' intimate 2 knowledge of the strengths and weaknesses of the care processes and infrastructures of their institutions, and of the risk profiles and logistics of the whole production line. these issues are very difficult to address in a multicentre trial without far - reaching standardisation. does a postoperative treatment protocol have any chance of improving outcomes if pre- and perioperative management have been optimised ? an understanding of these interactions is vital for planning multicentre trials of goal - directed management. without considering these factors, powerful treatment concepts may be considered futile, when in fact the cause of futility may lie elsewhere. in their second study, show that low perioperative central venous saturation is associated with an increased risk of postoperative complications. this finding should also be viewed in the context of the particular institution, case mix, and treatment process. before planning interventional multicentre trials based on the use of central venous saturation, it is advisable to ensure that the predictive value of central venous saturation in the participating centres and in their case mix remains, and that the patient population at high risk in those centres can be identified. the clinic of intensive care medicine, university hospital bern and university of bern, has or has had research, education and consulting contracts with edwards lifesciences. | although various systems have been developed to identify patients at increased risk of peri- and postoperative mortality and morbidity, little effort has been made in developing tools to reduce this risk. in this issue of critical care, pearse. publish two reports related to predicting and improving outcome in high - risk surgical patients. rather than conducting large, multicentre, randomised, controlled trials, the research group at st george 's hospital in london has persistently and systematically tested the concept of goal - directed haemodynamic management in high risk surgery in their single - centre setting. their results have been impressive, demonstrating that in this setting, various outcome measures can be reduced with goal - directed haemodynamic management. the impressive positive results of the pearse studies contrast sharply with the negative results of multicentre studies, such as that of sandham. one reason may be that, like several other successful single - centre trials, pearse. used strict treatment protocols rather than guidelines. in addition, single - centre studies utilize their investigators ' knowledge of their patients ' risk profiles and familiarity with the care processes and infrastructures of their institutions. an understanding of the organisational and case - mix aspects of pre-, peri - and post - operative management is vital for planning multicentre trials of goal - directed management. |
non - rheumatic degenerative aortic stenosis is the most frequent type of acquired valvular heart disease in europe and north america and its prevalence averages from 2% to 7% in people above 65 years of age 1 - 4. degenerative aortic stenosis and coronary artery disease (cad) share many similarities, being chronic, gradually progressive inflammatory disorders. traditional cardiovascular risk factors are associated with the development of both these conditions 5 - 7. according to early clinical studies, only 40% of patients with severe aortic stenosis have significant cad and aortic stenosis is absent in the majority of cad patients, which suggests different pathogenesis of these diseases 8,9. additionally, the prevalence of internal carotid artery stenosis (icas) 50% in patients with degenerative aortic stenosis was higher in subjects with coexisting cad 10. there is a paucity of reports on determinants of both coronary and carotid atherosclerosis in patients with degenerative aortic stenosis. our aim was to investigate the prevalence of coronary and internal carotid atherosclerosis in relation to demographic and cardiovascular risk profile among patients with severe degenerative aortic stenosis undergoing diagnostic coronary angiography and carotid ultrasonography in our center. we retrospectively collected data on 145 consecutive patients with severe degenerative aortic stenosis who were admitted to our tertiary care center between january 2003 and october 2012 and underwent elective coronary angiography and carotid ultrasonography during the index hospitalization. patients with a non - degenerative etiology of aortic stenosis and subjects with bicuspid or unicuspid aortic valve were excluded from the registry. coronary angiography was performed as a part of a standard diagnostic procedure on the basis of classical indications : before elective aortic valve replacement and/or due to symptoms suggestive of myocardial ischemia. demographic and clinical data, including traditional risk factors (arterial hypertension, hypercholesterolemia, diabetes mellitus, smoking) and past medical history (cerebrovascular incident, coronary revascularization) were registered. biochemical data included serum creatinine, total cholesterol, low - density lipoprotein cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c) and triglycerides measured after an overnight fast during the index hospitalization. estimated glomerular filtration rate (egfr) was calculated by the simplified equation developed by the modification of diet in renal disease (mdrd) study group 11. the ethics committee of our university was notified about the registry and no objection was raised. we recorded mean and maximal transaortic valve pressure gradients, left ventricular ejection fraction ; aortic valve area (ava) was estimated by means of the continuity equation. severe aortic stenosis in echocardiography was defined as a mean transaortic gradient > 40 mmhg or a calculated ava 5.2 mmol / l during index hospitalization or diagnosed previously and/or medically treated ; - diabetes mellitus : fasting venous plasma glucose 7 mmol / l confirmed by repeated testing or casual plasma glucose 11.1 mmol / l during index hospitalization or diagnosis established before admission ; - smoking : self - reported regular smoking habit. continuous variables were presented as mean and standard deviation (sd) or median and range. the patients were divided into 2 groups according to the prevalence of significant cad or relevant internal carotid artery stenosis (icas). significant cad was defined as obstructive cad (according to the above mentioned criterion) or a history of coronary artery bypass grafting or percutaneous coronary intervention. relevant icas corresponded to a stenosis of 50% in at least one internal carotid artery. intergroup differences were calculated by a 2-sided student 's t test or the mann - whitney 's u test for continuous data and the chi test or fisher 's exact test for proportions. patients ' characteristics related to the prevalence of significant cad or relevant icas were identified by logistic regression and odds ratios (or) with 95% confidence intervals (ci) for predictor variables have been shown. or represents a multiplicative rise in the odds of a patient having significant cad or relevant icas associated with a change in the explanatory variable. in case of continuous variables, or is calculated for each one - unit increment in the predictor variable, whereas or for dichotomous categorical data reflects an increase in the odds of prevalent cad or icas in the patients exposed to a factor of interest compared to a reference group without this exposure. as preliminary data analysis indicated that the effect of age on the prevalence of cad or icas could be modified by gender, we created an interaction term that was set to 1 in women with an over - median age (> 76 years) being equal to 0 in the remainder study subjects. in order to evaluate the interaction, the interaction term was entered into the regression model in addition to age and gender. additionally, the interaction was depicted by calculating or of cad prevalence for 3 subgroups of subjects created according to gender and age with the reference to women aged > 76 years. this latter approach was not applied for icas because of a small number of subjects with this condition (n=22). independent predictors of the prevalence of relevant cad or icas were identified by multiple logistic regression with backward stepwise variable selection. when building the model, the values of p - to - enter and p - to - remove were set at 0.10 and 0.15, respectively. the goodness - of - fit of the final regression equation was confirmed by the hosmer - lemeshow test. continuous variables were presented as mean and standard deviation (sd) or median and range. the patients were divided into 2 groups according to the prevalence of significant cad or relevant internal carotid artery stenosis (icas). significant cad was defined as obstructive cad (according to the above mentioned criterion) or a history of coronary artery bypass grafting or percutaneous coronary intervention. relevant icas corresponded to a stenosis of 50% in at least one internal carotid artery. intergroup differences were calculated by a 2-sided student 's t test or the mann - whitney 's u test for continuous data and the chi test or fisher 's exact test for proportions. patients ' characteristics related to the prevalence of significant cad or relevant icas were identified by logistic regression and odds ratios (or) with 95% confidence intervals (ci) for predictor variables have been shown. or represents a multiplicative rise in the odds of a patient having significant cad or relevant icas associated with a change in the explanatory variable. in case of continuous variables, or is calculated for each one - unit increment in the predictor variable, whereas or for dichotomous categorical data reflects an increase in the odds of prevalent cad or icas in the patients exposed to a factor of interest compared to a reference group without this exposure. as preliminary data analysis indicated that the effect of age on the prevalence of cad or icas could be modified by gender, we created an interaction term that was set to 1 in women with an over - median age (> 76 years) being equal to 0 in the remainder study subjects. in order to evaluate the interaction, the interaction term was entered into the regression model in addition to age and gender. additionally, the interaction was depicted by calculating or of cad prevalence for 3 subgroups of subjects created according to gender and age with the reference to women aged > 76 years. this latter approach was not applied for icas because of a small number of subjects with this condition (n=22). independent predictors of the prevalence of relevant cad or icas were identified by multiple logistic regression with backward stepwise variable selection. when building the model, the values of p - to - enter and p - to - remove were set at 0.10 and 0.15, respectively. the goodness - of - fit of the final regression equation was confirmed by the hosmer - lemeshow test. the registry encompassed data of 145 patients (77 men, 53%) aged 49 - 91 years (mean 75 9 years). the women enrolled to the registry were significantly older than men (77.4 9.1 vs. 72.2 9.0 years, p 76 years compared to women aged 76 years (48% vs. 54%) (table 5). a similar pattern was observed for respective proportions of patients with icas, being 2-fold higher in men with an over - median age with the reference to those with a below - median age (28 vs. 14%), but over 2-fold lower in older versus younger women (7% vs. 17%) (table 5). by logistic regression analysis, the interactions between age and gender were confirmed by a significant effect of the age - gender interaction term on cad prevalence (or, 0.58 [95% ci, 0.38 - 0.88 ], p=0.01), which was equivalent to a decrease in the odds of prevalent cad in women with an over - median age. an analogous association was observed for the proportion of icas, albeit slightly below the level of statistical significance (or, 0.63 [0.38 - 1.02 ], p=0.06 for the interaction term). with the reference to women aged > 76 years, or of cad prevalence was significantly increased in men aged > 76 years (or, 3.03 [1.55 - 5.92 ], p=0.001), being only slightly higher in younger women (or, 1.14 [0.69 - 1.89 ], p=0.61) and younger men (or, 1.16 [0.77 - 1.74 ], p=0.48). the strength of these interactions was maintained after multivariate adjustment (cad : or, 0.54 [0.35 - 0.84 ], p=0.006 ; icas : 0.60 [0.35 - 1.02 ], p=0.06 for the age - gender interaction term). in addition to the interaction term, the following covariates were retained in final equations by backward stepwise logistic regression analysis : the number of risk factors (cad : or per increment of one, 2.15 [1.33 - 3.46 ], p=0.002 ; icas : or, 2.47 [1.33 - 4.57 ], p=0.004), egfr (cad : or per rise of 10 ml / min per 1.73 m, 0.81 [0.70 - 0.95 ], p=0.007) and female gender (cad : or, 0.71 [0.47 - 1.06 ], p=0.09 ; icas : or, 0.59 [0.33 - 1.05 ], p=0.07). the goodness - of - fit of the final regression models for the prevalence of cad and icas was validated (p=0.91 and 0.58, respectively, by the hosmer - lemeshow test). our salient finding was a significant effect of gender on the relation between age and the prevalence of angiographic cad in patients with severe degenerative aortic stenosis. in contrast to men, the prevalence of cad did not increase in women aged over 76 years compared to women with a below - median age (76 years), being even slightly lower. the age - gender interaction in terms of cad appears counterintuitive because the prevalence of angiographic cad increases with age in men and women 12. that this interaction retained statistical significance after adjustment for atherosclerotic risk factors and tended to be present - albeit slightly below the level of statistical significance - for relevant icas, suggests an underlying mechanism related to the severity of atherosclerosis, yet other than a different risk profile. a hypothetical explanation for the differential association of cad and icas with age in men and women may be a survival bias. according to this concept, women who suffered from both severe aortic stenosis and relevant coronary or internal carotid atherosclerosis could have been exposed to an excessive risk of death due to coronary or cerebrovascular events, which might have abolished the age - dependent increase in the prevalence of cad and icas in women with aortic stenosis. the limitation of the hypothetical effect to women with aortic stenosis and coexisting cad or icas is consistent with an almost 2-fold higher percentage of women as a whole among our patients with an over - median age, in agreement with a 9-year higher average length of life for women compared to men in our country (80.9 vs. 72.4 years) 13. we observed a joint contribution of recognized atherosclerotic risk factors to the development of coronary and carotid atherosclerosis in patients with severe degenerative aortic stenosis, which confirms earlier observations. reported a higher percentage of diabetes and smoking habit in subjects with degenerative aortic stenosis and concomitant icas, however, they also described an independent effect of coexistent cad on icas, which was not found in the present study 10. a possible explanation of this inconsistency could be different patients ' characteristics because they included 104 subjects aged 63 8 years with moderate - to - severe aortic stenosis 10. performed an angiographic case - control study in which 523 patients referred for elective diagnostic left heart catheterization because of severe aortic stenosis were compared to controls pair - matched for age, sex and cad prevalence 14. in patients with severe aortic stenosis the number of established risk factors predicted the presence of cad, similar to our data, nevertheless, in the absence of cad none of the risk factors was significantly more frequent in aortic stenosis compared to those with a normal aortic valve 14. this finding precipitated the conclusion that classical cardiovascular risk factors could be associated with degenerative aortic stenosis purely on the basis of its association with cad 14, which appears in disagreement with earlier suggestions that degenerative aortic valve disease was associated with similar factors to atherosclerotic risk factors as demonstrated in 5,201 subjects aged 65 years enrolled in the cardiovascular health study 6. unfortunately, as we analyzed only data of patients with aortic stenosis, our study is not able to clarify this controversy. on the other hand, goland. 15 observed a significantly increased prevalence of hypercholesterolemia in 45 subjects aged 75 10 years with isolated severe degenerative aortic stenosis without angiographic cad compared to 54 sex- and age - matched controls without aortic stenosis or cad. therefore, further investigations are required to identify additional factors which protect against the development of degenerative aortic stenosis despite the presence of atherosclerotic risk factors. first, this was a retrospective, non - blinded observational study in a relatively low number of subjects and all data were collected from discharge letters and hospital records as source documentation. due to a retrospective study design, the final dataset might have been incomplete, although we made every effort to ensure that all consecutive patients meeting the inclusion criteria had been included into the registry. second, the progress in resolution and quality of ultrasound imaging over past 10 years could have influenced the precision of the measurements, nonetheless, cardiac and carotid ultrasound were performed by experienced sonographers. third, egfr was calculated from the highest level of serum creatinine measured during the index hospitalization, nevertheless, time points of blood sampling were not standardized. finally, the evaluation of coronary and carotid stenoses was based mostly on visual assessment and a quantitative analysis was not performed by an independent core laboratory. first, this was a retrospective, non - blinded observational study in a relatively low number of subjects and all data were collected from discharge letters and hospital records as source documentation. due to a retrospective study design, the final dataset might have been incomplete, although we made every effort to ensure that all consecutive patients meeting the inclusion criteria had been included into the registry. second, the progress in resolution and quality of ultrasound imaging over past 10 years could have influenced the precision of the measurements, nonetheless, cardiac and carotid ultrasound were performed by experienced sonographers. third, egfr was calculated from the highest level of serum creatinine measured during the index hospitalization, nevertheless, time points of blood sampling were not standardized. finally, the evaluation of coronary and carotid stenoses was based mostly on visual assessment and a quantitative analysis was not performed by an independent core laboratory. | background. patients with degenerative aortic stenosis (as) exhibit elevated prevalence of coronary artery disease (cad) and internal carotid artery stenosis (icas). our aim was to investigate prevalence of significant cad and icas in relation to demographic and cardiovascular risk profile among patients with severe degenerative as.methods. we studied 145 consecutive patients (77 men and 68 women) aged 49 - 91 years (median, 76) with severe degenerative as who underwent coronary angiography and carotid ultrasonography in our tertiary care center. the patients were divided into two groups according to the presence of either significant cad (n=86) or icas (n=22).results. the prevalence of significant cad or icas was higher with increasing number of traditional risk factors (hypertension, hypercholesterolemia, diabetes, smoking habit) and decreasing renal function. we found interactions between age and gender in terms of cad (p=0.01) and icas (p=0.06), which was confirmed by multivariate approach. with the reference to men with a below - median age, the prevalence of cad or icas increased in men aged > 76 years (89% vs. 55% and 28% vs. 14%, respectively), whereas the respective percentages were lower in older vs. younger women (48% vs. 54% and 7% vs. 17%).conclusions. in severe degenerative as gender modulates the association of age with coronary and carotid atherosclerosis with its lower prevalence in women aged > 76 years compared to their younger counterparts. this may result from a hypothetical survival bias, i.e., an excessive risk of death in very elderly women with severe as and coexisting relevant coronary or carotid atherosclerosis. |
subdural hematomas are a common posttraumatic finding and are caused by tearing of cortical bridging veins. on most occasions, the evacuated specimen consists of clotted blood, macrophages (if subacute), with collagen, neovascularization, and focal hemosiderin (if chronic). we present a case of a subdural hematoma with foci of hematopoietic components hematopoietic elements identified outside of the bone marrow are defined as extramedullary hematopoiesis (emh). this 59-year - old man had a past medical history of diabetes, hypertension, and pancreatitis. he sustained a fall from a 12-foot ladder in december, 2009. at that time, he was diagnosed with subarachnoid hemorrhage in the right interhemispheric fissure, a right minimal frontal bone fracture involving the frontal sinus, a right frontal contusion and a right open femur fracture. no operational interventions were given to the patient for his skull and facial fractures since the fractures were minimally displaced. about a month later, he was discharged to a local rehabilitation center and was continuously treated with anticoagulants for femoral deep vein thrombosis. shortly thereafter, he presented to the emergency department with altered metal status after a fall from bed. computed tomography of the brain showed a right acute upon chronic subdural hematoma. microscopic examination of the hematoma demonstrated neovascularized tissue containing both acute and chronic inflammation and hemosiderin deposition consistent with a subdural membrane. remarkably, there were numerous microscopic nodules of hyperchromatic, small, round cells scattered diffusely in the hematoma (figure 1(a)), which were positive for glycophorin (figure 1(b)), an erythrocyte precursor marker. the cells of interest were negative for the b - cell marker cd20, endothelial cell marker cd34 and were mostly negative for the t - cell marker cd3. it is generally believed that emh is a compensatory process associated with anemia and other bone marrow diseases which leads to chronic hematopoietic deficiency. intracranial emh is a rare event and is most frequently associated with thalassemia and myelofibrosis. the first report of emh in a subdural hematoma was published in 1966 in a 4-month old infant who was very anemic and presented with an enlarged head with a persistent subdural hematoma. nucleated red blood cells were identified within the hematoma, but not in the peripheral blood, and they disappeared shortly thereafter even with the continuous presence of the subdural hematoma. the authors proposed that the occurrence of subdural emh in this case was either a congenital anomaly or a secondary reaction to the child 's anemia. there have been only a few subsequent reports of patients presenting with subdural hematoma - associated emh [35 ]. none of these reports included patients with a clinical history of skull fracture as was seen in our case. the clinical significance of the emh in subdural hematoma is thought to be of no importance as concluded in one previous report. when the bone marrow space is inadequate as a result of myelofibrosis or bone metastasis, emh occurs as a compensatory phenomenon with increased numbers of circulating hematopoietic stem cells. another theory is the myelostimulatory theory, which proposes that emh presents in fetal hematopoietic sites under stimulation of unknown myelostimulatory factors. koch. also hypothesized another possible explanation of emh, the redirected differentiation theory. they proposed that stem cells of different tissue types may differentiate into hematopoietic stem cells when induced by unknown circulating factors in response to anemia or other hematologic disorders. this phenomenon has been observed in lung tissue following bone fracture or cardiac surgery and in the presacral area following a sacrum fracture. it is presumed that the origin of emh in these cases is from bone marrow embolism. in the present case, the patient had no underlying hematopoietic disorder and developed a subdural hematoma following head trauma and a skull fracture. it is highly possible that a small nidus of marrow hematopoietic cells migrated from the fracture site and leaked into the subdural space with resultant emh within the hematoma. interestingly, subdural hemorrhage / hematoma can be a secondary process of emh in patients with hematopoietic disorders [8, 9 ]. however, the clusters of nucleated red blood cells can present a diagnostic pitfall for surgical pathologists as the common differential diagnoses of groups of small round cells with hyperchromatic nuclei and scant cytoplasm in the subdural space include metastatic malignant tumors, such as small cell carcinoma, melanoma, and/or lymphoma. however, negative immunostains for epithelial, neuroendocrine, mesenchymal, and lymphoid markers help to rule out these possibilities. the final diagnosis of emh can easily be confirmed by staining the small round cell clusters which are immunoreactive for the erythrocyte marker glycophorin. in summary, we present a rare case of emh in a subdural hematoma in a patient with head trauma and a skull fracture, but no evidence of an underlying myeloproliferative disorder. this case supports the hypothesis of bone marrow embolism as the origin of emh. in addition, we report this case to help to minimize the chance of misdiagnosis when surgical pathologists encounter emh at an unanticipated site or under unexpected circumstances. | we present a case of a 59-year - old man who was found to have clusters of hyperchromatic, small, round nucleated cells within a subdural hematoma removed after a skull fracture. immunohistochemistry study confirmed that the cells were hematopoietic components predominantly composed of normoblasts. in this paper, we describe the clinical and pathological findings. a brief review of published information on extramedullary hematopoiesis in subdural hematoma and the mechanisms of pathogenesis are also discussed. while extramedullary hematopoiesis is seen anecdotally by neuropathologists in chronic subdural hematomas, only a few cases are documented in the literature. furthermore, extramedullary hematopoiesis in subdural hematoma can pose a diagnostic challenge for general pathologists who encounter subdural hematoma evacuations seldom in their surgical pathology practices. |
a 35-year - old man, a visitor from papua new guinea, arrived at the darnley island health centre in march 2000, complaining of headache and symptoms of fever. he began a course of oral quinine for suspected malaria but remained ill and was transferred to thursday island hospital. the day after admission he was still febrile and was given timentin (ticarcillin plus clavulinic acid), gentamicin, and doxycycline. polymerase chain reaction for scrub typhus positive (by using primers for 56-kda antigen gene). a 19-year - old patient (patient 2) was transferred from murray island to the thursday island hospital in may 2000. he had been ill for 12 days with fever, frontal headache, and intermittent vomiting. he had a documented temperature of 40.1c before admission to hospital and had been treated symptomatically with aspirin and metoclopromide. he had not left murray island for several months and had never traveled to papua new guinea. he had slight conjunctival injection and mild tenderness in the right upper quadrant of his abdomen. a typical black eschar approximately 0.5 cm in diameter was found on his upper right thigh with associated tender right inguinal lymphadenopathy. tests indicated a normal leukocyte count (9.8 x 10/l), thrombocytopenia (platelet count 79,000/l), increased creatinine (0.14 mmol / l), and abnormal liver function tests (-glutamyl transferase 55 u / l, normal < 50 ; alanine aminotransferase 280 u / l, normal < 45 ; aspartate aminotransferase 223 u / l, normal < 40). he received doxycyline, became afebrile after 48 hours, and made an uncomplicated recovery in august 2000, a 29-year - old man (patient 3) arrived at the primary health center on thursday island with a 5-day history of fevers, sweats, lethargy, and headache. as a field worker with the local electricity authority, he had visited several of the outer torres strait islands (yorke, darnley, mabuaig, yam, badu, and stephen) in the 3 weeks before becoming ill. these visits included a 3-day trip to darnley island 2 weeks before his fevers began. some of his work involved clearing vegetation from the fence lines of the local power plants. on examination he was not treated with antibiotics, and his fever settled spontaneously during the 3 weeks. a 28-year - old woman (patient 4) arrived at darnley island health centre in january 2001 with her 9-year - old son (patient 5) ; both complained of being ill for approximately 1 week. the child also complained of vomiting and was noted to have a dry cough with some rhinorrhea. the next day, when seen at the visiting doctors clinic, both mother and son were given oral quinine to treat possible malaria. the mother had a typical black eschar on her left breast (figure 1) with associated regional lymphadenopathy. oral doxycycline was administered, and the fever resolved over 3 days. her son was treated with azithromycin (250 mg a day for 3 days) and became afebrile within 24 hours. eschar on the breast of a patient with scrub typhus during an outbreak on darnley island, torres strait. in the next 3 weeks, four more patients (one adult [patient 7 ], two teenagers [patients 6 and 8 ], and one 5-year - old child [patient 9 ]) arrived at darnley island health centre with nonspecific febrile illnesses associated with headache. two had eschars (one located on the scrotum and another on the scalp) with associated regional lymphadenopathy. patient 6 was successfully treated with azithromycin (1,000 mg as a single dose). patient 9 had been given a course of oral and intramuscular penicillin before scrub typhus was suspected and remained ill with fevers and abdominal pain (with right upper quadrant tenderness). he was transferred to the hospital 2 weeks after being initially seen but was afebrile on admission and remained well with no additional treatment. an additional case of scrub typhus (patient 10) was documented in april 2001. after recognition of this cluster of scrub typhus cases, a doctor and indigenous health worker traveled to darnley island to interview patients and educate the local community about the disease. the six patients had not left darnley island in the 6 weeks before becoming ill. the patients lived on different parts of the island, and no common area was visited before illness onset. patient 4 was an exception and had harvested mangoes in an area of jungle close to her house where her son (patient 5) played. the transmission of scrub typhus on darnley island probably occurred in the densely vegetated areas close to most domestic dwellings on the island. a public meeting was held, which focused on increasing the general awareness of the signs and symptoms of scrub typhus and the importance of early diagnosis and treatment. aspects of personal protection against mite bites were also stressed, including wearing protective boots and trousers and using insect and mite repellants when going into potential areas of transmission. after recognition of this cluster of scrub typhus cases, a doctor and indigenous health worker traveled to darnley island to interview patients and educate the local community about the disease. the six patients had not left darnley island in the 6 weeks before becoming ill. the patients lived on different parts of the island, and no common area was visited before illness onset. patient 4 was an exception and had harvested mangoes in an area of jungle close to her house where her son (patient 5) played. the transmission of scrub typhus on darnley island probably occurred in the densely vegetated areas close to most domestic dwellings on the island. a public meeting was held, which focused on increasing the general awareness of the signs and symptoms of scrub typhus and the importance of early diagnosis and treatment. aspects of personal protection against mite bites were also stressed, including wearing protective boots and trousers and using insect and mite repellants when going into potential areas of transmission. the torres strait islands (figure 2) lie between cape york peninsula in queensland and papua new guinea. approximately half of the people live on or near thursday island, which is the main commercial and government center. a 38-bed general hospital is located there. the other half of the population lives on the 15 outer island communities that stretch between the australian and papua new guinea mainland. health centers are located on each of these islands and are staffed by remote area nurses, indigenous healthcare workers, or both. map of torres strait islands showing areas of scrub typhus transmission, darnley and murray islands, torres strait, 20002001. the cases of scrub typhus infection we describe are not the first to be described from the torres strait islands. scrub typhus was previously reported in the 1950s, with a single case from hammond island (near thursday island), and another in a diver from a lugger near darnley island (2). we describe at least two islands where transmission of scrub typhus occurred in the torres strait during the wet seasons of 2000 and 2001. another patient lived on thursday island but traveled to several outer islands in the weeks before becoming ill and probably acquired his infection on darnley island. the occurrence of six cases of scrub typhus on darnley island over a 3-week period is important, representing the infection of nearly 2% of the total population (375) (6). rainfall for the region was well above average in november and december 2000, the beginning of the wet season (7). the resulting dense vegetation would have provided favorable conditions for the transmission of disease. we believe that o. tsutsugamushi has probably existed in the region for many decades, rather than having been recently introduced, and climatic conditions favored the transmission to humans in the early part of 2001. a 9-year - old boy and 14-year - old girl responded well to treatment with azithromycin. azithromycin has been used successfully in the treatment of scrub typhus in a small number of pregnant women (8), but to our knowledge these are the first published cases of its clinical use in children with the disease. | scrub typhus, caused by orientia tsutsugamushi, occurs throughout southeast asia. we descript ten cases that occurred in the torres strait islands of northern australia during 2000 and 2001. preceding heavy rain may have contributed to the outbreak. the successful use of azithromycin in two pediatric patients is also reported. |
familial mediterranean fever (fmf) is an autosomal recessive inherited disorder [1, 2 ]. it is common among mediterranean populations (jews, arabs, turks, and armenians). fmf is characterized by recurrent fever and inflammation of serous membranes, leading to abdominal pain, joint pain, and chest pain. the most important complication of fmf is amyloidosis, which eventually leads to kidney failure. symptoms of the disease usually occur during the first decade of life in more than 80% of patients. the highest prevalence of the disease among the sephardic jews and the armenians has been reported. fmf seems to be more common in males with the prevalence rate 1.5 to 2 times. its prevalence is one in 2,000 to one in 100, according to different studies. mefv gene, responsible for the disease, located on chromosome 16 p (13.3) codes synthesis of a protein called pyrin. ongoing and recurrent inflammation leads to amyloid production and deposition of it in vital organs, especially the kidneys. the appearance of homozygous mutations was significantly associated with severe clinical presentations of the fmf [10, 12 ]. this mutation is related to disease onset at an early age and more prevalence of arthritis and progression to amyloidosis. m680i is common among armenians and turks and is associated with more severe form of the relapsing fever in early childhood which may be the only presentation of familial mediterranean fever. in 90% of the patients disease is begun before 20 years of age. the most commonly involved joints include hip, knees, elbows, and wrists [1416 ]. protracted febrile myalgia syndrome (pfms) is presented with prolonged fever and an increase of esr and leukocytosis. it usually takes 6 to 8 weeks and respond to treatment with prednisolone [17, 18 ]. the m694v mutation associated with a higher incidence of pfms. pericarditis and tamponade, myocarditis, and cardiac amyloidosis are the most common cardiovascular presentation in fmf [21, 22 ]. the nonamyloidosis renal involvement has been reported in 22% of patients and include temporary or permanent hematuria, proteinuria, acute pyelonephritis, interstitial nephritis, and glomerulonephritis due to coexisting disease [23, 24 ]. it is a pathognomonic finding in fmf which usually fades within 7248 hours ; fever and leukocytosis may accompany the rash. the incidence of rash is 3%46% and is usually associated with m694v mutation ; sometimes erysipelas rash and fever are the only sign of fmf. acute scrotal swelling, a painful inflammation of the testis, in a small percentage of children with fmf, has been reported. in arabs infertility in patients with fmf has no difference with the general population [27, 28 ]. recurrent aseptic meningitis can rarely occur in fmf ; these attacks are prevented after treatment with colchicine. familial mediterranean fever may be associated with different systemic inflammatory diseases including behcet, polyarteritis nodosa and henoch - schonlein purpura, spondyloarthropathy, multiple sclerosis [31, 32 ], and inflammatory bowel diseases [33, 34 ]. on the basis of clinical findings tel - hashomer criteria have been described to be fmf diagnosis [11, 35 ]. there is no specific standard laboratory test for fmf ; nowadays compound mefv gene mutations in hemo- or heterozygote forms confirm the diagnosis of fmf. this study included all patients with familial mediterranean fever in fmf clinic at bouali hospital and fmf registration center in iran (http://www.fmfiran.ir/). 422 patients were enrolled in this study ; all patients had fmf according to tel - hashomer criteria and/or at least two mefv gene mutations. 19 patients were excluded because of unavailability and study was conducted on 403 patients with fmf. common mefv genes mutations (e148q, p369s, f479l, m680i (g / c), m680i (g / a), i692del, m694v, m694i, k695r, v726a, a744s, and r761h) have been analyzed in 239 patient. the youngest patient was 1.5 years old and the oldest one was 76 years old. the average age of onset of symptoms was 9.75 years and the first decade of life was the most common age range which was in 128 patients (31.8%). 228 patients (56.6%) were males and 333 patients were living in urban area. the main complaint of patients was abdominal pain, in 200 cases (49.6%), and abdominal pain plus fever in 67 (16.6%) patients was the second main complaint. interval between attacks was 36.5 29.6 days and the mean duration of each episode was 43.3 34.5 hours. the most common homozygous mutation was m694v - m694v (7.1%) and compound heterozygous mutation was m694v - v726a (10.46%) and monoheterozygous mutation was e148q (8.7%). with regard to the time between onset of symptoms and diagnosis of fmf in 207 patients (52.3%) this period was more than 3 years. colchicine administration in 89.9% patients had good results and in 7.2% patients showed moderate response while 2.9% patients had poor response. the most common colchicine side effect was diarrhea in 4.7% of patients. in our series two cases had erysipelas - like skin rash with negative mefv results which indicate the rarity of this feature in this area. we had two siblings with idiopathic hepatitis and massive ascites who were homozygous for m694v gene. many studies have shown male preponderance in fmf [38, 40, 41 ]. in sackesen in yilmaz study 90% of patients had fever, 95% of patients had abdominal pain, and 19% of patients had erroneous appendectomy. in a study on 229 patients, 115 were females and 68% of patients had mefv gene mutations. high fever in 94% of patients and abdominal pain in 83% were the main symptoms. in ebrahimi - fakhari. fever in 78% of patients, peritonitis symptoms in 95%, pleuritis in 59%, arthritis in 32%, arthralgia in 60%, erysipelas - like skin rash in 23%, vasculitis in 8% have been reported. in dundar 's report 49.6% of patients had no mefv mutations, 26.41% were heterozygous, 15.29% were compound, and 8.60% were homozygous mutation. in balci the genetic expression of this study is similar to arabs and somewhat to armenians. in this study, the common combined heterozygous mutation was m694v - v726a with 10.46%, m694v - m694v with 7.1%, and m680i - v726a with 6.27%. the most common genotype of patients was m694v - m694v in different studies [38, 40, 41 ] ; this genotype was in second rate of our results. in this study m694v mutation with 18.3% and compound mutation v726a - m694v with 12.7% were common mutations in male, while m694v mutation with 24.5% and m694v - m694v with 10.7% were the common genotype in female. rare mutations were observed in this study such as k695r and f479l with 0.2% frequency. in manna report rare mutations were k695r with 0.67%, m694i with 1.04%, and r761h with 1.91%. in yilmaz study the r761s in 1% and v726a in 3% of patients were rare mutations. the bonyadi study showed these rare mutations : a744s in 0.5%, m694i in 2.2%, f479l in 1.3%, p369s in 1.3%, e167d in 1.2%, and r408q in 1.2%. in our study there was one case of amyloidosis who presented with resistant nephrotic syndrome and m694i - m694i mutations. some studies have shown that the m694v mutation is associated with a severe form of the disease. this study showed that m694v - m694v mutations are more common in patients with presentation during first decade of life, while in those with disease presentation during second decade or later genotype v726a - m694v was common. two patients presented with the complaint of recurrent orchitis and compound heterozygous mutations m694v - v726a and m694v - e148q, and two female siblings had massive ascites, abdominal pain, and idiopathic hepatitis with homozygous mutation m697v ; this association has been reported in the literature. this study showed that first decade of life is the most common age in disease presentation of fmf ; abdominal pain is the main symptom ; m694v and m694v - v726a are the most common alleles and compound genotype. our genotype is similar to arabs and in some way armenians and erysipelas - like skin rash is not common feature in our patients. | background. familial mediterranean fever (fmf) is a periodic ar autoinflammatory disorder. this comprehensive study describes fmf in iran as a country near mediterranean area. materials and methods. from the country fmf registration center 403 patients according to tel - hashomer criteria enrolled this study, 239 patients had mefv gene mutations analyses. data, if needed, was analyzed by spss v20. results. 175 patients (43.4%) were female and 228 patients (56.6%) were male. the mean age was 21.3 years. abdominal pain was in 93.3% patients and 88.1% had fever. abdominal pain was the main complaint of patients in (49.6%). the mean interval between attacks was 36.5 29.6 days and the mean duration of every episodes was 43.3 34.5 hours. 15.1% of patients had positive family history and 12.7% had previous surgery ; in 52.3% of patients delay in diagnosis was more than three years. 12 common mefv gene mutations were analyzed, 21.33% were without mutations, 39.7% had compound heterozygote, 25.52% showed heterozygous, and 13.38% showed homozygous results. the most common compound genotype was m694v - v726a (% 10.46) and in alleles m694v (% 20.9) and v726a (% 12.7) were the most frequent mutations, respectively. conclusion. m694v was the most common mutation, and the most common compound genotype was m694v - v726a. our genotype results are similar to arabs and in some way to armenians, erysipelas - like skin lesions are not common in this area, and clinical criteria are the preferred methods in diagnosis of fmf. |
stroke is the main cause of death in portugal and the main cause of disability on elderly people. this situation has raised great concern and several initiatives have been put in place to deal with the problem. the unidades de acidentes vasculares cerebrais (uavc) (stroke unit) were launched in 2001, aiming at reducing length of stay in acute care hospitals, functional incapacity and post - stroke complications, the number of patients in need of home nursing or specialized inpatient long - term care, as well as facilitating reintegration in family and society, namely return to workplace. the study is based on deep literature review and clinical experience in specialized care for stroke patients in portugal. until the launch of uavcs, a patient suffering from stroke would be admitted to a servio de neurologia (neurology service) or to a servio de medicina interna (internal medicine service), probably in the hospital closest to the occurrence and care would be oriented to the acute phase. these units integrate health professionals especially trained for these situations and apply diagnostic and therapeutic procedures according to protocols that follow the most recent international recommendations. one of the most relevant practices adopted is physiotherapy care that promotes early mobilization and get up. there are about 30 uavcs in portugal, some of them still working under technical and human limitations. however, these units are but one of the links in the chain of care. at least in some areas, much is still to be done in this regard. | introductionwe discuss current status and developments in the care of stroke patients in portugal.theory and methodsstroke is the main cause of death in portugal and the main cause of disability on elderly people. this situation has raised great concern and several initiatives have been put in place to deal with the problem. the unidades de acidentes vasculares cerebrais (uavc) (stroke unit) were launched in 2001, aiming at reducing length of stay in acute care hospitals, functional incapacity and post - stroke complications, the number of patients in need of home nursing or specialized inpatient long - term care, as well as facilitating reintegration in family and society, namely return to workplace. the study is based on deep literature review and clinical experience in specialized care for stroke patients in portugal.resultsuntil the launch of uavcs, a patient suffering from stroke would be admitted to a servio de neurologia (neurology service) or to a servio de medicina interna (internal medicine service), probably in the hospital closest to the occurrence and care would be oriented to the acute phase. nowadays, patients with a diagnosis of stroke are preferably directed to a uavc. these units integrate health professionals especially trained for these situations and apply diagnostic and therapeutic procedures according to protocols that follow the most recent international recommendations. the multidisciplinary teams execute integrated care and rehabilitation plans based on the individual needs. complementary exams are done systematically aiming at more complete understanding. after discharge, patients are followed up in outpatient specific consultation. one of the most relevant practices adopted is physiotherapy care that promotes early mobilization and get up.conclusions and discussionthere are about 30 uavcs in portugal, some of them still working under technical and human limitations. however, these units are but one of the links in the chain of care. many attempts have been made to integrate care in portugal. at least in some areas, much is still to be done in this regard. |
frozen shoulder is one of the prevalent and painful joint disorders which is being found in 2 - 5% of the people of the society in the age range of 40 - 60. that is to say that this disease is scarce among children, also women suffer less than men, but there is no known race or genetic intention about this disorder. this disease is prevalent among patients suffering from diabetes, both dependant and independent of insulin ; and also among pre - diabetes patients (1). this disease forms up with activation of inflation mechanisms in the glenohumeral joint accompanied by synovial tissue inflation and will cause symptoms such as pain, movement limitations of joint, as well as muscle weakness without tangible disorders such as structural defects in the anatomy of the joint (fracture and dislocation) (1, 2). the restriction in the movement can take place in flexion, extension, and external rotation, but it is less in abduction and internal rotation (3 - 5). generally, the frozen shoulder can be divided into two groups, primary (idiopathic) and secondary (caused from a secondary disease) (6). the starting process of this disease is unknown and usually is accompanied by a report of some type(s) of traumas of shoulder. however mainly the disorder is self - limiting, but the length of the disease and the intensity of the disorder might be severely annoying (3, 7). about some patients the symptoms of the disease might be with the patient up to some years and they will influence the quality of the person`s life (sleeping disorders and disability in doing daily stuffs) (3, 7). the information about the frozen shoulder is insufficient and most of the information is obtained from interfered studies, as still there is no unanimous about a specific therapy method. some of the common therapies of this disorder consist of analgesic and anti - inflammatory medicines, massage therapy, heat therapy, ultrasound wave therapy, chiropractic techniques, and using stretch and isometric exercises and physiotherapy (8). also, surgery methods and anesthesia manipulation, intra - articular injection of corticosteroids and neural blocking of suprascapular nerve, are other approaches to heal the frozen shoulder. review and meta - analysis articles have not reported any privilege about any of these approaches. therefore, still in the new studies, the influence of different therapies on healing frozen shoulder is being surveyed, and one of them is acupuncture (9). acupuncture is an approach used from 5000 years ago to heal disorders ; and since it is safer than medical treatments and has less side effects, is being used nowadays to cure many types of diseases in the modern medicine, specifically to remedy of chronic joint pains. acupuncture is a method in which piercing certain spots of the body using very thin needles causes a pain relief or treating a certain problem of the patient (10). these needles are disposable and the material is stainless steel ; and are slightly thicker than human`s hair. there are reports from who, national center for complementary and integrative health (nccih), and american medical association (ama) that all declare effectiveness of acupuncture for treating many types of diseases (11). many studies had been implemented in order to evaluation of effectiveness and pain - relief strength of acupuncture, such as a study by ko sun in 2001 in hong kong, in which they concluded that acupuncture accompanied by shoulder exercises is more effective to cure frozen shoulder (9). in another study in 2008 by amanda tiffany, she declared that acupuncture has effective results to cure chronic pains of frozen shoulder (12). therefore, researchers unanimously decided to implement a study entitled determination of effectiveness of acupuncture to remedy the patients suffering from frozen shoulder who referred to baghiatallah hospital in 2013. in this controlled clinical trial, all the patients who referred to baghiatallah hospital in 2013 with a pain in the shoulder who were approved to have frozen shoulder according to clinical examinations were brought into the study. the standards which the participants were supposed to have in order to be brought in the study were restrictions in active and inactive movements of shoulder in flexion, extension, external rotation, and also night pains. also all the patients should had the symptoms for 4 - 6 weeks. in this study patients with background disorders such as renal, hepatic, and hematic disorders, patients under shoulder surgeries, patients with painful arc syndrome, patients with a fracture background, patients with neurologic and pathologic joint symptoms in the graphs of upper limb on the same side of painful shoulder, were dismissed. evaluation indexes in this study were intensity of pain in the suffering joint, movement domain of the joint, and life quality of the patient. in this study, patients with equal demographic properties were divided randomly into 2 groups with 20 members in each group (according to the formulation of calculating the volume of the sample) ; in the control group, physiotherapy and in the case group, acupuncture was used in addition to physiotherapy. notably, physiotherapy continued until the last session of acupuncture for all groups daily. in order to eliminate all interfering and confounding effects of intervention agents, we adopted two studied groups. about the confounder variable of using analgesics during the research, for each group one type of analgesic was prescribed with the same dosage (ibuprofen 400 mg per day), in the case of intolerable pain. the patients were examined at the beginning of the study, 1.5 months after that (end of the sessions) and 3 months later, and the information was registered in the form of predetermined questionnaires, and eventually the obtained data were analyzed by spss 17 software. the qualitative variables were used by chi square test, and quantitative variables were used by student t - test. at first, the variables were introduced and then the raw data were evaluated by proper statistic tests. in this study that was implemented on 40 patients suffering from frozen shoulder, the participants were divided into two test groups of acupuncture (20 persons) and control group (20 persons). the age range in this study reported 44 - 71 and the average age in test group was 54.658.25 and in the control group was 54.458.04. in the test group, there was 40% male (8 persons) and 60% female (12 persons) and in the control group there was 45% male (9 persons) and 55% female (11 persons). results demonstrate that 8 persons (3 persons in the test group and 5 persons in the control group) were also suffering from diabetes, 3 persons (2 persons in the test group and 1 person in the control group) were suffering from hypothyroidism, 5 persons (3 persons in the test group and 2 persons in the control group) had a heart surgery and 3 persons (1 persons in the test group and 2 persons in the control group) were suffering from arthritis rheumatoid. about evaluating the location of the disorder, it was revealed that 56.3% of patients of the test group, had disorders in the right shoulder and 45.8% of patients in the control group had disorders in the right shoulder. the average of the length of frozen shoulder disorder in the test group was observed as 4.052.06 months, and for the control group it was 4.102.17 months. statistic tests demonstrate no meaningful difference about the variable of age among the mentioned two groups (p - value=0.930). the correlation of the location of the disorder (left or right shoulder) was evaluated using a proper statistic test. the results demonstrate no meaningful difference between these two variables (p - value=0.510). in this study, the domain of active and inactive movements (flexion, extension, abduction, adduction, and internal and external rotation) were evaluated for two groups of test and control group chronologically after 1.5 and 3 months after starting the therapy using independent samples t - test statistic test. the results showed that about both follow up times, there was a meaningful difference between the average of the movement domain in both active and inactive flexion and abduction in the control and test groups (p - value 0.05). also using the repeated measures anova test, it revealed that there is meaningful difference between the intensity of the pain, disability score and spadi in those three chronological times of before the therapy, 1.5, and 4 months of follow up (p - value 0.05), but after 3 months, the vas index reduced significantly in the test group comparing to the control group (p - value < 0.05). also using the repeated measures anova test revealed that there is a meaningful difference between vas amounts for the times of before, 1.5, and 3 months after the therapy (table 4). comparing the vas between the control and test groups and also chronologically for before the therapy, and follow up times of 1.5, and 3 months. in the case of treating the frozen shoulder using the acupuncture, we realized that totally the implementation of acupuncture causes an improvement in movements of the shoulder in the patients suffering from frozen shoulder. also the vas index had a tangible improvement after three month of starting the treatment comparing to the control group. regarding the obtained results, we found out that in the case of improvement of movements of the shoulder in the patients, after 1.5 months of starting the treatment and also after 3 months, active and inactive movements in flexion and abduction directions were significantly improved in the case of mentioned movements comparing to the past. in the general case, implementation of acupuncture causes a tremendous improvement of shoulder movements, but the improvement was more significant about flexion and abduction movements. therefore, the implementation of acupuncture can be offered as an approach to improve movements of the shoulder in the patients suffering from frozen shoulder. about the item of pain, generally about both groups of control and acupuncture, they had a reduced pain after treatment but in the acupunctured group the amount of this reduction was higher, but there was no meaningful difference between acupunctured and control group about this item. about the comparison of the disability score, generally about both groups of control and acupuncture, they had an improvement after treatment but in the case of acupunctured group the amount of this reduction was higher, but there was no meaningful difference between acupunctured and control group about this item. about the spadi index, generally about both groups of control and acupuncture, they had an improvement after treatment but in the acupunctured group the amount of this reduction was higher, but there was no meaningful difference between acupunctured and control group about this item. about the comparison of vas, generally both control and acupunctured groups had improvements after treatment and in the acupuncture group this improvement was observed as higher, but after 3 months after starting the treatment they had no significant difference. in 2005, during the systematic surveys of cochrane database, the influence of acupuncturing on shoulder pain disorders such as osteoarthritis, adhesive capsulitis of shoulder, and rotator cuff disorder was analyzed. among these researches, 9 trial clinical studies which had good enough quality, one of these trials had concluded that a compound treatment of acupuncture and physical treatment makes better results for increasing the movement domain comparing to the only physical treatment. notably, the patients were studied who had shoulder pain for more than 3 weeks. among the studies, those who reported the effectiveness of acupuncture declared that because of few numbers of the participants in these studies, methodological problems and few numbers of these researches, we can not judge precisely about effectiveness or inefficacy of acupuncture (13). in our study it was concluded that acupuncture causes an improvement in movements of frozen shoulder. in 2005, about physical treatment, johnson started a research from the rct type, entitled the effect of acupuncture versus ultrasound in the patients suffering from shoulder impingement syndrome. 85 patients with this syndrome diagnosis were treated with acupuncture and ultrasound randomly ; also both groups were under sport therapy plans. though there were symptoms of improvement in both groups, but the acupunctured group experienced improvement over 12 months (13). in our study, considering sport therapy which was performed during the acupuncture for both groups daily, it was shown that acupunctured group had more improvement in shoulder joint movements. gladys lv in 2008 in china studied electric acupuncture and interferential electrotherapy in order to healing the frozen shoulder in a double blind study. the third group, as the control group, was not under any type of therapy. this study surveyed therapy approaches for 4 weeks on target groups. in the case of vas, in certain intervals a significant improvement was observed in target groups (p - value=0.001). after 6 months of starting the therapy, the obtained results were stable ; the result showed that electric acupuncture and interferential therapy companied by shoulder exercises is effective to heal the frozen shoulder (14). the result of our study was confirming the same results of the mentioned study. in a study of dong he in 2004 in norway, they concluded that acupuncture has significant effects on reducing the intensity and frequency of the pain (15). but in our study we realized that however the reduction of pain is a real phenomenon, but had no meaningful difference with the control group. regarding the obtained results and safety and low side effects of acupuncture comparing to other methods of therapies of frozen shoulder, it is suggested that with determination of the effectiveness of acupuncture to cure diseases and make this matter acceptable and believable for the public, we can achieve a reduction of spent money for ineffective therapies and increasing the speed of the recovery and going back to work process and increasing the quality of the work provided by a proper therapy. generally, acupuncture causes improvement of all movements of the shoulder, but about flexion and abduction the improvement was evaluated to be more. also the vas index had improved after 3 months after remedy comparing to the control group, and eventually the acupuncture can be offered as a method to improve the movement of the shoulder in patients suffering from frozen shoulder. | background : adhesive capsulitis is a common disease that causes pain and reduced range of motion, but vague on the shoulder. woman are affected fewer than men, but there is no known racial or genetic tendency. most patients with adhesive capsulitis will improve with nonsurgical treatment. acetaminophen and nonsteroidal anti - inflammatory drugs for pain relief in patients without contraindication are first - line options. acupuncture considered being safe and effective in reducing pain. the aim of this study was to investigation of the effectiveness of acupuncture in the treatment of frozen shoulder.materials and methods : in a controlled clinical trial, patients referred to the baqiatallah clinic in 91 years with shoulder pain, frozen shoulder diagnosed based on history and physical exam, they have been enrolled. indicators measured in the study was included the involved joint pain, range of motion and quality of life. patients, first at baseline, one and a half months later (end of session) and then 3 months after the examination information about each individual entered in the from of questionnaires were pre - determined and data were analyzed by spss 17 software.results:in this clinical trial study total 40 patients with frozen shoulder (20 interference with the acupuncture and 20 people control) study that patients average age 55/54. age maximum 71 years and minimum 44 years. acupuncture in the treatment of frozen shoulder with the results achieved in the general case acupuncture may improve shoulder motion in patients. vas index at three months after treatment compared with the control group had a greater improvement.conclusion:in the case of acupuncture and ultimately improve the overall look of all the movement of flexion and adduction of the shoulder, but the movement has been further improved, vas index at three months after treatment compared with the control group had a greater improvement and finally, we perform acupuncture as a way to improve shoulder motion in patients with frozen shoulder offered. |
the structure types consist of the overall and the local models. as an effective tool to analyze gene expression data, the clustering analysis is comprehensively applied to many fields, such as gene expression spectrum analysis (1), genome study, biological regulatory networks (2), medicine filtering, new medicine development, clinical disease diagnosis 3., 4., and so on. the basic hypothesis included in the clustering analysis is that, the genes that have the same expression mode may have similar functions (5). however, traditional clustering methods have a series of problems in reducing noise, mining local information, and synthesizing the heterogeneous data, etc. the data dimension is becoming higher and higher due to the use of new biological microarray chips. different objects of the same cluster in the data of a high dimensional space could show the similarity only in a certain subspace. when this principle is applied to gene expression data, mutual - controlling genes could show similar expression patterns in some conditions for test samples. in fact, in the whole input space, the gene expression pattern is different. therefore, difficulties appear when we use traditional clustering methods to determine the object similarity by using the value approximation in high dimensional data. the biclustering algorithm presented by cheng and church (6) is different from traditional clustering algorithms, in which the similarity is not treated as a function of pairs of genes or pairs of conditions. instead, it is a measure of the coherence of the genes and conditions in the biclustering. this measure can be a symmetric function of genes and conditions involved and thus the finding of biclusters is a process that groups genes and conditions simultaneously. the most important innovation of cheng and church algorithm is that they put forward a definition called residue score 6., 7.. the algorithm divides an expression model into three parts : attribute residue, object residue, and -cluster residue (or background residue). the mathematical definitions are as follows:(1)eij=iieij|i|eij=jjeij|j|eij=ii, jjeij|i||j| where i and j are the row and column vector sets of the submatrix, respectively ; |i| and |j| are the number of rows and columns, respectively ; eij is the element of the submatrix ; eij, eij, and eij are the attribute residue, object residue, and -cluster residue, respectively. the definition of the residue score is as follows:(2)rsij(i, j)=eijeijeij+eij let x be the set of genes and y the set of conditions. let eij be the element of the gene - condition expression matrix representing the logarithm of the relative abundance of the mrna of the i gene under the j condition. let i x and j y be the subsets of genes and conditions. the pair (i, j) specifies a submatrix aij with the following mean squared residue score:(3)h(i, j)=ii, jjrsij2|i||j| the lowest score h(i, j) = 0 indicates that the gene expression levels fluctuate in unison. these trivial biclusters may not be very interesting but need to be discovered and masked so that more interesting ones can be found. the row variance may be an accompanying score to reject trivial biclusters:(4)v(i, j)=1|j|jj(eijeij)2 the higher the value of h is, the more disordered the data is. in cheng and church algorithm, a greedy method is used to select submatrix with a low h score. firstly, the method is to remove the row or column to achieve the largest decrease of the score. for the current submatrix, they calculate the average residue score of each row using d(i)=1|j|jjrsij(i, j) and the average residue score of each column using e(j)=1|i|iirsij(i, j), then choose the row or column with the maximal score and delete it from the current submatrix, until h(i, j) <. also they use a parameter, so that they can delete a set of nodes each time before the score is recalculated. without updating the score after the removal of each node, the matrix may shrink too much and one may miss some large -clusters. one may also choose an adaptive based on the score and the size during the iteration. secondly, they add rows and columns so that the matrix with the maximal size can be obtained. the most important innovation of cheng and church algorithm is that they put forward a definition called residue score 6., 7.. the algorithm divides an expression model into three parts : attribute residue, object residue, and -cluster residue (or background residue). the mathematical definitions are as follows:(1)eij=iieij|i|eij=jjeij|j|eij=ii, jjeij|i||j| where i and j are the row and column vector sets of the submatrix, respectively ; |i| and |j| are the number of rows and columns, respectively ; eij is the element of the submatrix ; eij, eij, and eij are the attribute residue, object residue, and -cluster residue, respectively. the definition of the residue score is as follows:(2)rsij(i, j)=eijeijeij+eij let x be the set of genes and y the set of conditions. let eij be the element of the gene - condition expression matrix representing the logarithm of the relative abundance of the mrna of the i gene under the j condition. let i x and j y be the subsets of genes and conditions. the pair (i, j) specifies a submatrix aij with the following mean squared residue score:(3)h(i, j)=ii, jjrsij2|i||j| the lowest score h(i, j) = 0 indicates that the gene expression levels fluctuate in unison. these trivial biclusters may not be very interesting but need to be discovered and masked so that more interesting ones can be found. the row variance may be an accompanying score to reject trivial biclusters:(4)v(i, j)=1|j|jj(eijeij)2 the higher the value of h is, the more disordered the data is. in cheng and church algorithm, firstly, the method is to remove the row or column to achieve the largest decrease of the score. for the current submatrix, they calculate the average residue score of each row using d(i)=1|j|jjrsij(i, j) and the average residue score of each column using e(j)=1|i|iirsij(i, j), then choose the row or column with the maximal score and delete it from the current submatrix, until h(i, j) <. also they use a parameter, so that they can delete a set of nodes each time before the score is recalculated. without updating the score after the removal of each node, one may also choose an adaptive based on the score and the size during the iteration. secondly, they add rows and columns so that the matrix with the maximal size can be obtained. because the greedy method may not always lead to correct results, we use an additional course to avoid deleting the steps for the node addition in the original algorithm are as follows:1.compute eij (for all i), eij (for all j), eij, and h(i, j).2.add the columns j j with1|i|ii(eijeijeij+eij)2<h(i, j).3.recompute eij, eij, and h(i, j).4.add the rows i i with1|j|jj(eijeijeij+eij)2<h(i, j).5.for the i row that is still not in i, add its inverse if1|j|jj(eijeijeij+eij)2<h(i, j).6.if no node needs to add in the current iteration, return the final i and j. compute eij (for all i), eij (for all j), eij, and h(i, j). recompute eij, eij, and h(i, j). add the rows i i with 1|j|jj(eijeijeij+eij)2<h(i, j). for the i row that is still not in i, add its inverse if 1|j|jj(eijeijeij+eij)2<h(i, j). if no node needs to add in the current iteration, return the final i and j. considering that the search space of cheng and church algorithm is only a subspace of the result set, we make some improvements as follows. in order to maximize the size of the result submatrix, we amend the decision condition in the original algorithm by changing the original constraint(5)1|i|ii(eijeijeij+eij)2<h(i, j)into the following form(6)1|i|ii(eijeijeij+eij)2<kh(i, j) and when it is added into the former matrix, the value of h in the new matrix is less than the original value. the improved algorithm extends the searching scope and increases the number of the nodes that can be added into the cluster. our improved algorithm introduces a parameter k in equation (6) to ensure that the blind search is reduced. cross validation shows that the improved algorithm performs better when k is taken as 3.2. to speed up the improved algorithm, we first express the matrix by using the idea of chromosome used in evolutionary computation, and then change the chromosomes in the matrix into two - dimensional link lists, in which we calculate the value of h and save the values of the chromosomes in the field h. to examine the efficiency of the improved algorithm, we tested it using the yeast gene expression spectrum from the gene expression data set (2). for the ten clusters obtained in the simulation, we calculated the average computational time of the results and found that the original algorithm cost 65 s, while the improved algorithm cost 94 s. the comparison results are shown in table 1. the quality of result sets of the improved algorithm is enhanced obviously, on the tolerable condition that the time cost is increased by less than 0.5 times. the comparison of the submatrix rows and columns from the improved algorithm and the result sets of the original algorithm are also shown in table 1. it is obvious that from the results in the same condition, the improved algorithm can obtain better result sets and more information. in cheng and church algorithm, there are two important parameters and that need to be set before the algorithm running, where is a threshold of score function h and measures the extent of data consistency. the parameter influences the quality of matrix clustering and in general it is better if the value is smaller. but if is too small, the scale of the submatrix will be over small and easy to lose information. hence, a balance point should be found for this parameter before running the algorithm. the parameter is used in the deletion course of the first phase in the original algorithm, which is also an important threshold. we determined the value ranges through experiments to provide referable information for realizing adaptive setting for the parameters. firstly, we chose real data sets for testing. through a series of numerical experiments, we obtained the relation between the values of parameter and submatrix size, as shown in figure 1. the arithmetic average of space size is used to estimate the quality of the clustering. in the experiments, it was found that the size of the submatrix decreases monotonously with the descent of the value of. when is taken as around 120, the trend of the descent is gentle. even the value of goes down again, this trend does not change essentially. therefore, we suggest that for these data sets, it is better to take the value of in the range of [120, 180 ]. for the same data sets, we took the difference of the two systems clocks before and after the experiment as the time consumption of clustering, and only calculated the consuming time during the course of deletion. in this way we obtained the relation between the value of and the time consumption (figure 2). the value of was taken in the range of [2.8, 3.2 ]. we also need to make practical clustering test at = 2.8 to avoid any misvalue. if the clustering results are satisfied, we could keep the range, otherwise we have to increase the lower limit. for different data sets, we can obtain a series of results. through linear regression and suitable adjustment to these calculation results let [a, b ] be the value range of data sets, m the number of genes, and n the number of conditions. the simulating experiments showed that it is better to take the value of in the range of [3j, 4j ]. the results of simulations showed that it is better to take the value of in the range of [7l, 8l ]. the open human lymphoma b cell data set (8) was used to examine the proposed improved algorithm. compared with the original algorithm, the quality of clustering results using the improved algorithm is enhanced obviously, the mining expression models are better, and the data possess stronger consistency with fluctuation on the condition that the time cost is increased a bit. in addition, in spite that the noise level of data sets is very high of having the loss rate of 12.3%, simulation results showed that the improved algorithm could still keep a good clustering effect even if the noise interference is very strong. because the greedy method may not always lead to correct results, we use an additional course to avoid deleting the steps for the node addition in the original algorithm are as follows:1.compute eij (for all i), eij (for all j), eij, and h(i, j).2.add the columns j j with1|i|ii(eijeijeij+eij)2<h(i, j).3.recompute eij, eij, and h(i, j).4.add the rows i i with1|j|jj(eijeijeij+eij)2<h(i, j).5.for the i row that is still not in i, add its inverse if1|j|jj(eijeijeij+eij)2<h(i, j).6.if no node needs to add in the current iteration, return the final i and j. compute eij (for all i), eij (for all j), eij, and h(i, j). recompute eij, eij, and h(i, j). add the rows i i with 1|j|jj(eijeijeij+eij)2<h(i, j). for the i row that is still not in i, add its inverse if 1|j|jj(eijeijeij+eij)2<h(i, j). if no node needs to add in the current iteration, return the final i and j. considering that the search space of cheng and church algorithm is only a subspace of the result set, we make some improvements as follows. in order to maximize the size of the result submatrix, we amend the decision condition in the original algorithm by changing the original constraint(5)1|i|ii(eijeijeij+eij)2<h(i, j)into the following form(6)1|i|ii(eijeijeij+eij)2<kh(i, j) and when it is added into the former matrix, the value of h in the new matrix is less than the original value. the improved algorithm extends the searching scope and increases the number of the nodes that can be added into the cluster. our improved algorithm introduces a parameter k in equation (6) to ensure that the blind search is reduced. cross validation shows that the improved algorithm performs better when k is taken as 3.2. to speed up the improved algorithm, we first express the matrix by using the idea of chromosome used in evolutionary computation, and then change the chromosomes in the matrix into two - dimensional link lists, in which we calculate the value of h and save the values of the chromosomes in the field h. to examine the efficiency of the improved algorithm, we tested it using the yeast gene expression spectrum from the gene expression data set (2). for the ten clusters obtained in the simulation, we calculated the average computational time of the results and found that the original algorithm cost 65 s, while the improved algorithm cost 94 s. the comparison results are shown in table 1. the quality of result sets of the improved algorithm is enhanced obviously, on the tolerable condition that the time cost is increased by less than 0.5 times. the comparison of the submatrix rows and columns from the improved algorithm and the result sets of the original algorithm are also shown in table 1. it is obvious that from the results in the same condition, the improved algorithm can obtain better result sets and more information. in cheng and church algorithm, there are two important parameters and that need to be set before the algorithm running, where is a threshold of score function h and measures the extent of data consistency. the parameter influences the quality of matrix clustering and in general it is better if the value is smaller. but if is too small, the scale of the submatrix will be over small and easy to lose information. hence, a balance point should be found for this parameter before running the algorithm. the parameter is used in the deletion course of the first phase in the original algorithm, which is also an important threshold. we determined the value ranges through experiments to provide referable information for realizing adaptive setting for the parameters. firstly, we chose real data sets for testing. through a series of numerical experiments, we obtained the relation between the values of parameter and submatrix size, as shown in figure 1. the arithmetic average of space size is used to estimate the quality of the clustering. in the experiments, it was found that the size of the submatrix decreases monotonously with the descent of the value of. when is taken as around 120, the trend of the descent is gentle. even the value of goes down again, this trend does not change essentially. therefore, we suggest that for these data sets, it is better to take the value of in the range of [120, 180 ]. for the same data sets, we took the difference of the two systems clocks before and after the experiment as the time consumption of clustering, and only calculated the consuming time during the course of deletion. in this way we obtained the relation between the value of and the time consumption (figure 2). the value of was taken in the range of [2.8, 3.2 ]. we also need to make practical clustering test at = 2.8 to avoid any misvalue. if the clustering results are satisfied, we could keep the range, otherwise we have to increase the lower limit. for different data sets, we can obtain a series of results. through linear regression and suitable adjustment to these calculation results let [a, b ] be the value range of data sets, m the number of genes, and n the number of conditions. the simulating experiments showed that it is better to take the value of in the range of [3j, 4j ]. the results of simulations showed that it is better to take the value of in the range of [7l, 8l ]. the open human lymphoma b cell data set (8) was used to examine the proposed improved algorithm. compared with the original algorithm, the quality of clustering results using the improved algorithm is enhanced obviously, the mining expression models are better, and the data possess stronger consistency with fluctuation on the condition that the time cost is increased a bit. in addition, in spite that the noise level of data sets is very high of having the loss rate of 12.3%, simulation results showed that the improved algorithm could still keep a good clustering effect even if the noise interference is very strong. the process of the extended space in the second stage of cheng and church algorithm is improved. the numbers of rows and columns are increased about 20% by using the improved algorithm. the effects of the two important parameters on the speed of the algorithm and the clustering quality are discussed. on the basis of simulated experiments, the experienced values for selecting parameter ranges | cheng and church algorithm is an important approach in biclustering algorithms. in this paper, the process of the extended space in the second stage of cheng and church algorithm is improved and the selections of two important parameters are discussed. the results of the improved algorithm used in the gene expression spectrum analysis show that, compared with cheng and church algorithm, the quality of clustering results is enhanced obviously, the mining expression models are better, and the data possess a strong consistency with fluctuation on the condition while the computational time does not increase significantly. |
evidence - based practice (ebp) conducted in the health and medical field refers to clinical decision - making based on the best available external clinical evidence, and on individual clinical expertise1. the health and medical profession is a practical field of scholarship ; hence practical technique, theory, and research should all be developed together. when the newest information from systematic research is applied in the clinical field, it can be referred to as client - centered medical service2. however, the use of research findings in the practical field is not very common. smith3 stated that only 15% of clinical services use objective evidence based on research. gwendolijne.4 stated that, although doctors practicing in hospitals have a positive attitude towards research, only 5% of them actually make use of scientific research findings when making clinical decisions. from these results, we can infer that there are barriers to applying scientific research findings in the clinical field. some difficulties present themselves regarding the application of research : for example, lack of time because of the overload of work ; environmental factors, such as lack of organizational support ; lack of understanding of evidence based on practice and research ; personal factors, such as the lack of ability to search for study articles ; research factors regarding the quality of published articles ; and how the results of the research are communicated5,6,7. however, because there are numerous professions within the health and medical field, it is very important to research the barriers according to the types of professions that have not been actively making use of research materials. research has been performed on occupational therapists and on nurses5, 8, but no research has been performed specifically on physical therapists. since practical physical therapy is performed on patients in the clinical environment, significant evidence - based practice also needs to be conducted in the clinical environment. the application of evidence - based practice is an important part of clinical decision making when providing evaluation of rehabilitation and therapeutic services9. therefore, the purpose of this research was to investigate the most effective way that evidence - based practice can be performed in conjunction with the actual activities of physical therapists. investigation also needed regarding the differences in barriers to the use of research findings by physical therapists in evidence - based practice. the subjects of this research were physical therapists employed by hospitals that agreed to cooperate with the research in b city. a questionnaire survey was conducted in the period from september 2014 to october 2014. two hundred copies of the questionnaire were distributed, of which 163 copies were completed and returned, giving a recovery rate of 81.5%. the purpose of the research was explained to the participants, and instructions were given on how to answer the questionnaire. this study complied with the ethical principles of the declaration of helsinki and all participants signed a written consent that explained that participation was voluntary, the withdrawal procedure, and confidentiality of the information. it was stipulated that the information collected by the questionnaire would not be used for purposes other than the research for this project. to examine the barriers to using research findings, the barriers to research utilization scale that was founded by funk.10 was used. the questionnaire was made up of 6 research items, 8 physical therapist items, 6 presentation items, and 8 setting items, a total of 28 items. responses ranged from 1 point indicating not at all to 4 points indicating very much. the content validity of the questions was tested using an expert panel (n=5). each expert panel member evaluated the items for content validity on a 5-point scale. so, the content validity of the barriers to research utilization scale used in this study was supported by the content validity index (cvi). 21.0) was used to conduct statistical analyses, and the significance level used in the analysis was 0.05. descriptive statistics were calculated for general and clinical characteristics, barriers to using research findings, research related activities, and recognition and performance of evidence - based practice. the differences in the barrier level to using research findings according to the general characteristics of the result were analyzed using the t - test, and anova, and scheffe s test was used as a post hoc test11. the results of the barriers to using research findings by the physical therapists in a hospital environment are shown in table 1table 1.barriers to using research findings (n=158)type of barrier(mean)barrier itemsmeansdn%rankorderresearch : qualities of the research (2.3)the literature reports conflicting results2.20.64025.319the research has methodological inadequacies2.20.74427.815the research has not been replicated2.40.76742.48the conclusions drawn from the research are not justified2.10.53320.923research reports / articles are not published fast enough2.20.74427.815the physical therapist is uncertain whether to believe the results of the research2.50.88352.56physical therapist : the physical therapist s research value, skills, and awareness (2.1)the physical therapist is unwilling to change / try new ideas2.10.84729.721the physical therapist feels the benefits of changing practice will be minimal2.20.74729.719the physical therapist does not see the value of research for practice1.90.6148.928the physical therapist sees little benefit for self2.00.72717.127there is not a documented need to change practice2.00.73421.526the physical therapist is unaware of the research2.30.86943.711the physical therapist is isolated from knowledgeable colleagues with whom to discuss the research2.30.86138.613the physical therapist does not feel capable of evaluating the quality of the research2.30.866138.613presentation : presentation and accessibility of the research (2.5)the research is not reported clearly and readably2.50.88453.25the relevant literature is not compiled in one place2.30.75635.412implications for practice are not made clear2.70.811069.61statistical analyses are not understandable2.40.77144.99the research is not relevant to the physical therapist s practice2.40.76943.79research reports / articles are not readily available2.40.87849.47setting : setting barriers and limitation (2.3)the physical therapist feels results are not generalizable to own setting2.70.810163.92the physical therapist does not feel she / he has enough authority to change patient care procedures2.10.74327.224the physical therapist does not have time to read research2.20.74729.715the facilities are inadequate for implementation2.60.89258.23there is insufficient time on the job to implement new ideas2.50.88755.14administration will not allow implementation2.10.73622.821physicians will not cooperate with implementation2.20.74327.215other staff are not supportive of implementation2.00.62113.325total (2.3)reporting item as a moderate or great barrier, barriers were classified as being characteristics of physical therapist, the setting, the research, or its presentation. the greatest barrier among the research, physical therapy, presentation and setting categories was presentation, 2.5. the physical therapist feels results are not able to be generalized to his / her own setting at 2.7. on the other hand, the average barrier scores that were 2 included the physical therapist does not see the value of research for practice at 1.9 ; the physical therapist sees little benefit for self was 2.0 ; and there is not a documented need to change practice was 2.0. the effects of physical therapist s general characteristics and clinical characteristics on the barriers to using research findings are shown in table 2table 2.differences in barriers to using research findings by characteristics of participants (n=158)variablesdivisionn (%) meansdgendermale103 (65.2)2.20.5female55 (34.8)2.30.3age (years)2419 (12.0)2.50.3252961 (38.6)2.30.5303452 (32.9)2.20.53526 (16.5)2.40.3educationcollege39 (24.7)2.50.4university72 (45.6)2.20.4graduate school or more47 (29.7)2.20.4hospital typeuniversity hospital51 (32.3)2.30.4general hospital81 (51.3)2.20.4rehabilitation hospital21 (13.3)2.50.3clinic5 (3.2)2.30.5working placemodality room27 (17.1)2.20.5exercise room116 (73.4)2.30.4manual therapy room15 (9.5)2.30.5assigned patientmusculoskeletal58 (36.7)2.30.5adult neurology86 (54.4)2.30.4pediatric neurology14 (8.9)2.20.2career (months)1223 (14.6)2.20.6133634 (21.5)2.40.4376028 (17.7)2.30.56173 (46.2)2.20.4professional satisfactionvery dissatisfied4 (2.5)2.20.1dissatisfied34 (21.5)2.50.3satisfied107 (67.7)2.20.4very satisfied13 (8.2)2.00.5p b, c by post hoc test, : b > d by post hoc test. age (p e by post hoc test. there was a significant relationship between the barrier level and research participation in clinical research (p d by post hoc test, : b > c, d, c > d by post hoc test. the recognition level of evidence - based practice (p b, c by post hoc test, : b > d by post hoc test p e by post hoc test p d by post hoc test, : b > c, d, c > d by post hoc test among the barriers to using research findings, the item that had the highest barrier score was implications for practice are not made clear followed by the physical therapist feels the results are not generalizable to his / her own setting. these results are very similar to those of a study in which nurses were subjects of the research5. generally, scores of 2 points and below are not considered barriers no barriers to using research findings. the items scored at 2 points and below included, the physical therapist sees little benefit for self, and there is no documented need to change practice. the low barrier score of the physical therapist category (which includes the value and recognition of the physical therapist regarding the research) has a very positive effect on the use of this research. because physical therapists are the subject of this research, changes in value and recognition on the part of the therapists is not easy. in contrast, items in the research, presentation and setting categories could be corrected in a short period if efforts were made. analysis of the results of barriers to using research findings regarding the general characteristics of the physical therapists revealed that participants aged 24 years old and younger had the highest barrier scores. it is probable that this is because therapists who are 24 years old and younger usually depend heavily on guidelines in their practice, so they are less inclined to use research findings. physical therapists who are 34 years old and older also had a high barrier score because their greater clinical experience reduces their inclination to use research findings in the decision making process on behalf of their patients. this result parallels the results of the research of cameron.12 and dysart & tomlin13. melnyk.14 also stated that as the therapists have more experience when their clinical careers, they tend to rely more upon their own existing knowledge or experience in treating their patients. as regards educational level, the college graduates had higher barrier scores than those who had graduated from university or postgraduate school. compared to the curriculum of junior colleges, it is understood that the curriculum of universities and other higher education centers weights the importance of reading study articles, understanding research methodologies, and understanding the relationship between practice and research. hence, to improve the quality of physical theory, and to apply evidence - based physical therapy, there needs to be arrangements to educate physical therapists to read, study, and evaluate study articles. in addition, the physical therapists who reported satisfaction in their job had lower barrier scores than those who were not satisfied with their jobs. therefore, it is desirable to improve the job satisfaction of physical therapists, rather than simply emphasizing the application of study results. the results demonstrate that the more experience physical therapists had in clinical research participation, the greater their frequency of reading research articles, and the higher the level of support they received from managers to use the research, the lower the barrier score becomes. however, the physical therapists who participated in clinical research amounted to only 34.8% of the sample, significantly lower than physical therapists in the netherlands, 61.7%4. the percentage of physical therapists participating in clinical research was also lower than the percentage of occupational therapists at 40.2%8. this demonstrates the need for active participation of therapists in the research. among the physical therapists who were the subjects of this research, 52.5% answered that they were aware of evidence - based practice, and 61.4% of them were putting it into practice. those physical therapists who had high recognition and performance of evidence - based practice demonstrated a lower barrier level to using research findings, indicating that more emphasis should be placed on education to improve the recognition of evidence - based practice for the application of research results. from the results described above, it can be concluded that the age, educational level, and professional satisfaction of physical therapists are major factors influencing barriers to using research findings. among the items that were related to research, participation in clinical research, frequency of reading research articles, and support of managers to use research, affected the barrier scores. hence, we conclude that to improve the utilization of research findings, there is a need to provide continuous education, and more opportunities should be created to allow participation in research and environments where research results can be applied. | [purpose ] this research study was performed to investigate the barriers to using the research findings of physical therapists on evidence - based practice. [subjects ] the subjects of this research were physical therapists employed by hospitals that agreed to cooperate with the research in b city. [methods ] a questionnaire made up of 6 research items, 8 physical therapist items, 6 presentation items, and 8 setting items, for a total of 28 items, was distributed. the responses were scored so the higher result scores indicate a higher barrier level to using research findings. differences in barrier levels related to the likelihood of therapists using research findings in their practice varied according to the general characteristics of the result as according to the t - test and anova. scheffe s test was used as a post hoc test. [results ] the analysis of 158 returned questionnaires revealed that there were significant relationships between the age, educational level, and professional satisfaction of the therapists and the barriers to using research finding. significant relationships were also found between the items of research participation in clinical research, frequency of reading research articles, and support of manager to use research and the barrier level. no relationship was demonstrated between the recognition level of evidence - based practice and the performance level with the barrier score to using research findings. [conclusion ] this study demonstrated that to improve the utilization of research findings, there is a need to provide therapists with continual education and opportunities to participate in research, and environments and ways in which the research results can be given practical applications. |
mullerian duct anomalies (mdas) are rare and to witness a successful pregnancy outcome after surgical correction of the same is a delightful experience for the treating clinician. mda results from nondevelopment (agenesis or hypoplasia), defective vertical or lateral fusion, or resorption failure of the mullerian ducts. in the general population, the prevalence of these anomalies ranges from 0.001% to 10% while in women with bad obstetric history it ranges from 8% to 10%. didelphic uterus is seen in about 11% of mda cases and are associated with unilateral anomalies, i.e., obstructed hemivagina and ipsilateral renal agenesis in 1530% of cases. this association of didelphic uterus with obstructed hemivagina and ipsilateral renal agenesis has been referred to in the literature as the herlyn werner the term obstructed hemivagina and ipsilateral renal anomaly (ohvira) syndrome is used to denote the triad of ohvira and uterine anomaly other than didelphys. both the syndromes are rare and are discussed as same entities in many case reports and their incidence is estimated to be between 0.1% and 3.5% of all mullerian anomalies. the cause is attributed to embryologic arrest affecting both the mullerian and metanephric ducts concurrently at about 8 weeks gestation. the most common presenting symptoms are dysmenorrhea within first few years of menarche and increasing pelvic pain. a palpable mass can be encountered due to the associated hematocolpos or hematometra, which result from retained menstrual blood in the obstructed vagina. due to their extreme, narrated here is an interesting case of a 26-year - old woman with hwws who had an eventful journey from her menarche to successful motherhood. a 26-year - old primigravida was admitted from our antenatal clinic at 19 weeks gestation with threatened abortion. she had a history of having undergone abdominal metroplasty at the age of 15 years. she was a familiar face as she had been a frequent visitor at our gynecological outpatient clinic with complaint of severe dysmenorrhea since her menarcheal age of 13 years. her early symptoms included irregular menses, progressive pelvic pain, constipation, and rectal pain during defecation. her clinical workup showed an ultrasonography report suggestive of bilateral cystic ovarian mass with absent left kidney. diagnostic laparscopy was performed which revealed two hemiuteri out of which the left hemiuterus was markedly distended. since the laparascopic findings were suggestive of gross mullerian anomaly and the magnitude of symptoms was deteriorating the patient 's quality of life, laparatomy was carried out subsequently after a week. the findings of diagnostic laparascopy were confirmed on laparatomy, showing a didelphic uterus with obstructed hemivagina and absent left kidney. as the hemiuteri were hypoplastic, strassman 's metroplasty was performed to drain the left noncanalized hemiuterus and to create a single uterine cavity with a bigger caliber, aiming to provide better prospects for her future obstetric career. postoperatively, the patient was greatly relieved of her symptoms and in due course of time, she got married in 2007. following this, she had tedious 7 years of primary infertility and was under regular followup. her husband 's semen analysis was found normal. the hormone profile (luteinizing hormone, follicle stimulating hormone, and estradiol), serum prolactin, and thyroid profile of the patient were normal. hysterosalpingography was performed which revealed a deformed uterine cavity resembling an arcuate uterus with bilateral spillage of dye. finally, she conceived spontaneously and was admitted at 19 weeks with threatened abortion. on examination, her uterus corresponded to 20 weeks, and a gentle speculum examination revealed an old clot with no active bleeding and her cervical os was closed. an ultrasonography was done immediately to ascertain fetal wellbeing and cervical length which was found normal. she was planned for institutional observation and strict surveillance throughout her pregnancy till safe confinement. her surveillance program included maternal and fetal monitoring with regular ultrasonography scans to estimate cervical length, rule out intrauterine growth retardation, and doppler studies to detect placental invasion. all her antenatal investigations were reported normal, and we had planned her delivery at 37 weeks gestation by elective cesarean section. in anticipation of preterm labor, she was administered corticosteroids. she was carrying her pregnancy well until at 36 weeks 6 days gestation, she developed a premature rupture of membranes and was taken for emergency cesarean. her intraoperative findings revealed a unified arcuateshaped uterus having a deep midline scar [figures 1 and 2 ]. the posterior surface of the unified uterus revealed the remnant rectovesical ligament which was transected during previous procedure of strassman 's metroplasty [figure 3 ]. clinical photograph showing the unified uterus with deep midline scar of strassman 's metroplasty the two cavities with midline scar indicating former didelphic uterus unified to form present arcuate shaped uterus remnant of rectovesical ligament which was transected during strassman 's metroplasty, now seen adhered to the posterior surface of the unified uterus the placenta was delivered with ease, and there were no intraoperative complications. cesarean was successfully conducted followed by an uneventful postpartum period, and our patient went home on 7 day postoperatively with a healthy baby. mda is an infrequent finding in routine clinical practice and often their accurate diagnosis and complete assessment are missed. most of the patients with mda present during their reproductive years with infertility, recurrent pregnancy loss, prematurity, and other obstetric complications, whereas in pubertal age group, the symptoms include amenorrhea, cryptomenorrhea, or dysmenorrhea. a symptom such as dysmenorrhea is often treated conservatively, and the question arises as to what extent we can go while evaluating a case of primary dysmenorrhea. this was the salient feature of our case where a young teenage girl presenting with agonizing dysmenorrhea was evaluated to the extent of diagnostic laparoscopy followed by laparotomy and surgical correction. our patient had a rare variant of mda known as the hwws with a didelphic uterus, obstructed left hemivagina, and left renal agenesis. this was a striking feature contrary to the evidence provided in the literature of predominance of rightsided renal involvement. while investigating such rare cases, the imaging modalities are of variable utility as the hematometra and the hematocolpos frequently distorts the pelvic anatomy leading to inconclusive results. laparoscopy and laparotomy prove to be appropriate in defining the abnormality and treating it as seen in our case. it was noteworthy to see the compliance of our patient for nearly a decade and how we pursued her to accomplish the goal of successful motherhood after corrective strassman 's metroplasty. in an era where laparoscopic surgeries are more rampant, to witness a fruitful obstetric outcome in a patient who underwent abdominal strassman 's metroplasty provides relevance to this lesser practiced technique. moreover, it also finds its place in lowresource set up like ours, where facilities for laparoscopic surgeries are unavailable. our case was a great learning experience and a boost for us because our endeavors were not only merely providing symptomatic relief to our patient but also giving her a promising obstetric career ahead. | herlyn werner wunderlich syndrome is an uncommon variant of mullerian duct anomaly and the approach to its diagnosis requires a high index of suspicion and vigilant work up. presented here is a case of a 26-year - old woman who had the aforementioned anomaly and was pursued for nearly 10 years to provide her with a fruitful obstetric outcome after having undergone strassman 's metroplasty at a young age of 15 years. |
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