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birth defects are an important contributor to infant mortality among all racial / ethnic groups. major congenital anomalies are defined as those that threaten life, require major surgery, or lead to a significant disability. between 2% and 3% of all infants have a major congenital anomaly identified at birth and approximately 6% to 10% of such infants die within the first year of life (1). in more than 60% of such cases, the etiology of the congenital birth defect is unknown and primary prevention is impossible. in approximately 20% of congenital cases, the causes are monogeneous defects, 50% are caused by chromosome aberrations and 20% by virus infections, such as rubella, cytomegalovirus, and herpes virus (2, 3). many environmental factors have been suspected to play an etiologic role in the formation of congenital anomalies. chemical pollutants, dietary imbalances, ionizing radiation, pharmaceutical substances, and infections provide examples of known or suspected agents (4). unlike animal studies, molecular and biochemical studies in pregnant women are impossible. therefore, epidemiological data about congenital malformations is of vital importance to scientific research on pathomorphogenesis, aimed at prevention and public health education (3). only a small number of reports on birth defect monitoring are available from general hospitals (5 - 7). thus, our aim is to establish a multi - center birth defects monitoring system for the evaluation of the prevalence and serial occurrence of birth defects in korea. the study materials were all recorded deliveries at 10 medical centers in korea, between may 1999 and october 2002. the ten medical center were : the samsung cheil hospital, samsung medical center, asan medical center, cheil women 's clinic, ilsan cheil hospital, bombit women 's hospital, ewha women 's university mokdong hospital, kangnam st. records were obtained monthly, from delivery units and pediatric clinics by a visiting nurse. trained staff from each center provided the medical records that included delivery files, stillborn files and newborn files. the entire medical record of each case was reviewed for followings : hospital stay, prenatal diagnostic test results, birth certificate work sheet, labor and delivery records, progress notes, pathology / autopsy findings, physical examination findings, and a discharge summary. all live births, stillbirths, and spontaneous abortions after the 16th week of gestation were included. all neonates born at one of the 10 centers were examined by a pediatrician within the first week of life. in addition to the clinical examination, information from the prenatal and postnatal ultrasound examinations of the heart, brain and other organs were recorded. chromosome analysis was available for a number of birth defects that were coded according to the eurocat (european registration of congenital anomalies and twins, an european union registry) (4) and the international clearing - house for birth defects monitoring system (icbdms) (8). the nineteen groups of malformations as described by the icbdms (8) were 1) anencephaly, 2) spina bifida, 3) encephalocele, 4) hydrocephaly, 5) microtia, 6) cleft palate, 7) total cleft lip, 8) esophageal atresia or stenosis, 9) anorectal atresia or stenosis, 10) hypospadias, 11) renal agenesis / dysgenesis, 12) limb reduction defects, 13) omphalocele, 14) gastroschisis, 15) abdominal wall defects, 16) diaphragmatic hernia, 17) transposition of the great vessels, 18) hypoplastic left heart syndrome, and 19) down syndrome. the study materials were all recorded deliveries at 10 medical centers in korea, between may 1999 and october 2002. the ten medical center were : the samsung cheil hospital, samsung medical center, asan medical center, cheil women 's clinic, ilsan cheil hospital, bombit women 's hospital, ewha women 's university mokdong hospital, kangnam st. records were obtained monthly, from delivery units and pediatric clinics by a visiting nurse. trained staff from each center provided the medical records that included delivery files, stillborn files and newborn files. the entire medical record of each case was reviewed for followings : hospital stay, prenatal diagnostic test results, birth certificate work sheet, labor and delivery records, progress notes, pathology / autopsy findings, physical examination findings, and a discharge summary. all live births, stillbirths, and spontaneous abortions after the 16th week of gestation were included. all neonates born at one of the 10 centers were examined by a pediatrician within the first week of life. in addition to the clinical examination, information from the prenatal and postnatal ultrasound examinations of the heart, brain and other organs were recorded. chromosome analysis was available for a number of birth defects that were coded according to the eurocat (european registration of congenital anomalies and twins, an european union registry) (4) and the international clearing - house for birth defects monitoring system (icbdms) (8). the nineteen groups of malformations as described by the icbdms (8) were 1) anencephaly, 2) spina bifida, 3) encephalocele, 4) hydrocephaly, 5) microtia, 6) cleft palate, 7) total cleft lip, 8) esophageal atresia or stenosis, 9) anorectal atresia or stenosis, 10) hypospadias, 11) renal agenesis / dysgenesis, 12) limb reduction defects, 13) omphalocele, 14) gastroschisis, 15) abdominal wall defects, 16) diaphragmatic hernia, 17) transposition of the great vessels, 18) hypoplastic left heart syndrome, and 19) down syndrome. during the 4-yr period (may 1999-october 2002), we observed 1,537 cases of birth defects among 86,622 births, including live births and stillbirths, the prevalence rate being 1.8%. the mean maternal age was 29.9 (4.1, 95% ci) yr, with a range 19 - 45 yr. nineteen percent (291 cases) of the birth defects were associated with an elderly mother (35 yr). the mean gestational age at delivery was 32.58.6 weeks, ranging from 11 weeks to 43 weeks. the mean birth weight was 2,1981,319.5 g, with a range from 5 g to 5,060 g. non - living births (termination or intra - uterine fetal death) accounted for 31.5% of deliveries, and 438 babies (28.5%) were born via cesarean section. five cases had (0.3%) ambiguous genitalia, and in 45 cases (2.9%), the gender of the fetus could not be determined. table 3 shows the comparison of incidences of 19 congenital anomalies in korea as defined by icbdms (5) with those in japan and china and with atlanta, u.s.a. as determined by the metropolitan atlanta congenital defects program (macdp) (9). among 1,537 birth defect cases, 260 cases (16.9%) down syndrome was most common (137 cases), followed by edwards syndrome (57 cases). table 5 shows the birth defects according to the involved organs. the highest proportion of birth defects (17.5%) involved the cardiovascular system, followed by the genitourinary system (15.6%). thirty - four cases had polydactyly of the hand (4.0 per 10,000 fetuses) and 20 cases had polydactyly of the foot (2.4 per 10,000 fetuses). syndactyly of the hand and foot occurred in 9 cases (1.2 per 10,000 fetuses) and 7 cases (0.8 per 10,000 fetuses), respectively. the concept of congenital malformation is not strictly defined, and includes functional and metabolic disorders that, although present, may not necessarily be recognizable at birth (10, 11). the two most commonly used classification systems are : (1) the international classification of diseases system (12, 13), and (2) the international clearinghouse for birth defects monitoring system (icbdms) (14). in our study, the classification of birth defects was performed according to the european registration of congenital anomalies and twins (eurocat) and the icbdms (1994) system. the eurocat program was initiated in 1979. this surveillance system details 900,000 births per annum in 17 countries. the international clearing - house for birth defects monitoring system (icbdms) is a who - related non - governmental organization, and more than 25 countries including the united states, england, france, australia, japan, and china participate in the program. this system is a nationwide hospital - based monitoring system that covers about 10% of all births in japan. in our study, the 10 medical centers (six tertiary centers, one secondary center, and three large local obstetric clinics) covered only about 3% of births in korea. during the 4-yr study period, the overall incidence of birth defects from the 10 medical centers in korea was 1.8%. this is lower than the incidence of 2.4% provided by the eurocat registration system, which tends to produce lower figures than other passive registries (15). moreover, it has been reported that active monitoring systems detect 50% more congenital malformations than passive monitoring systems (16). therefore, the prevalence of birth defects in this study would be lower than the actual incidence. the incidence of total chromosomal abnormalities was 0.3% and the incidence of the trisomy 21 was 9.2 per 10,000 births, which is slightly lower than the incidence in glasgow (12.4 per 10,000 births) (17), in atlanta, u.s.a. (11.3 per 10,000 births), and in japan (10.4 per 10,000 births) (15). in the present study, the most frequent abnormality was a cleft lip with or without a cleft palate with a prevalence of 10.3 per 10,000 births, which is higher than in the atlanta (u.s.a.) study (9.9 per 10,000 births), but lower than in japan (15.9 per 10,000 births) or china (13.6 per 10,000 births) (15). the incidence of cleft palate without cleft lip in korea was found to be 1.4 per 10,000 births, which is lower than in other countries ; japan : 4.8 per 10,000 births, china : 2.4 per 10,000 births, and u.s.a. and the incidence of renal agenesis / dysgenesis in korea was 7.6 per 10,000 births, which is higher than in japan (4.3 per 10,000 births) and in the u.s.a. the incidence of anencephaly and microtia in korea (3.3 per 10,000 births and 2.7 per 10,000 births) were also higher than in japan (1.9 per 10,000 births and 1.6 per 10,000 births) and in the u.s.a. (1.8 per 10,000 births and 0.9 per 10,000 births). the incidence of hypospadia (1.2 per 10,000 births) and spina bifida (0.2 per 10,000) in korea were lower than in japan (3.5/3.2 per 10,000 births) and in the u.s.a. the incidences of hydrocephalus (3.6 per 10,000 births) and limb reduction defect (1.3 per 10,000 births) in korea were lower than in japan (7.5/3.8 per 10,000 births) and in the u.s.a. the incidence of gastroschisis (1.6 per 10,000 births) and abdominal wall defect (1.4 per 10,000 births) were lower than in japan (2.3/7.1 per 10,000 births) and in the u.s.a. the incidence of omphalocele (3.0 per 10,000 births) was lower than in japan (4.6 per 10,000 births) but higher than in the u.s.a. the frequency of birth defects varies markedly between countries and depends on the observation time after birth, the types of malformations included, and on differences in the reporting and statistical procedures used (18). on the other hand, this difference in the frequency of birth defects within countries would be affected by ethnicity, eating habits, and environmental factors, or combined. the highest proportion of birth defects involved the cardiovascular system (17.5%) followed by the genitourinary system (15.6%). recently, it was proposed that poor semen quality, cryptorchidism, hypospadias and testicular cancer are symptoms of one underlying entity, testicular dysgenesis syndrome (tds) (19). tds may be caused by genetic or environmental factors, or both. even though the clinical symptoms appear postnatally in addition, there are many recent reports that environmental factors, especially endocrine disrupting chemicals (edcs), can be the cause of congenital malformation (20). these edcs may cause a variety of defects in the endocrine and reproductive systems (21, 22). however, little is known about the underlying biochemical and molecular mechanisms, or the determinants of teratologic susceptibility, particularly in humans (20). possible explanations for the lower incidence of birth defects in the present study than in the eurocat program are : (1) we included neonates only within the first week of life, so that the defects recognizable thereafter might have been lost ; (2) in stillbirth cases, autopsy is carried out less frequently than in the eurocat program. the lower frequency of autopsy in our study might have resulted in a lower derection rate of defects in the internal organs. our aim is to establish a multi - center birth defects monitoring system to evaluate the prevalence and serial occurrence of birth defects in korea. birth defect registries that acquire data through active rather than passive reporting can provide additional important data on birth defects (21). such a multicenter birth defects monitoring system can provide high - quality information on birth defects in korea. to check the serial occurrence of birth defects, it is necessary to increase the number of participating hospitals and to launch a nation - wide multi - center study. | the aim of this study was to establish a multi - center birth defects monitoring system to evaluate the prevalence and the serial occurrence of birth defects in korea. ten medical centers participated in this program. a trained nurse collected relevant records from delivery units and pediatric clinics in participating hospitals on a monthly basis. we observed 1,537 cases of birth defects among 86,622 deliveries, which included live births and stillbirths. the prevalence of birth defects was 1.8%, and the sex distribution of the birth defect cases was 55.2% male and 41.6% female. the highest proportion of birth defects was in the cardiovascular system (17.5%), followed by birth defects involving in the genitourinary system (15.6%). chromosomal anomalies were detected 30.0 per 10,000 births. of these chromosomal anomalies, down syndrome was most frequently observed. this study led to an establishment of a multi - center active monitoring system for birth defects. to better understand the serial occurrence of birth defects in korea, it is necessary to increase the number of participating hospitals and to launch on a nation - wide multi - center study. |
paragangliomas in the mediastinum are rare tumors originated from the neuroendocrine cell. about 90% of paragangliomas exist in adrenal medulla (commonly called pheochromocytoma). the other 10% of tumors are extra - adrenal (paraganglioma), and 90% of these are intra - abdominal, most commonly arising from chromaffin cells near the aortic bifurcation or near the kidney. other sites include the paravertebral sympathethic ganglia, the urinary bladder, other automatic ganglia (celiac, mesenteric), the thorax (mediastinum, the heart, and paracardic regions) and the neck (in sympathetic ganglia, carotid body, cranial nerve or glomus jugulare). as far as we reviewed, there are no reports of ectopic acth syndrome with mediastinal paraganglioma in korea. we experienced a case with acth - secreting paraganglioma in the anterior mediastinum who was admitted because of cushing s syndrome. a 51-year - old - female was admitted to our hospital with 5 months duration of generalized edema and weakness. two years later, hypertension was detected and she had been taken antihypertensive medications intermittently. the patient had not any symptoms such as cough, sputum, fever and chest pain, except mild exertional dyspnea. her blood pressure was 170/100 mmhg, but other vital signs were stable. on physical examination, she was obese and had a moon face, buffalo hump and purplish abdominal striae. a 22 cm - sized hard and fixed the serum ast, alt, alp and ldh level was 50, 140, 131 and 1,065 u / l, respectively. the renal function test, including serum electrolytes, were within normal limits, except that potassium level was 2.5 fasting glucose was 472 mg / dl, hemoglobin a1c was 11.8% and spot urine glucose was above 1,000 mg / dl, but proteinuria was not found. chest x - ray (figure 1) and chest ct scan (figure 2) revealed a 1210 cm - sized well - defined and partially capsulated and lobulated homogeneous attenuated mass in the anterior mediastinum. there was no suppression of the plasma cortisol level (over 50 g / dl) after a high - dose dexamethasone suppression test. the plasma acth level was also elevated to 278 pg / ml, but pituitary mri scan did not reveal any evidence of abnormality, therefore ectopic acth syndrome was suspected. to find out the other focus of ectopic acth secretion, except mediastinal mass, abdominopelvic ct was performed, but there was no evidence of abnormalities, including adrenal gland, pancreas and ovary. the 24h - urine metanephrine, vanillymandelic acid (vma) and homovanilic acid (hva) levels were slightly elevated to 3.4, 11.4 and 13.9 mg / day, respectively, which are not typical ranges of pheochromocytoma. however, epinephrine and norepinephrine levels were within normal ranges. the 24h - urine 5-hiaa was elevated to 11.3 mg / day but serum calcitonin and parathyroid hormone levels were within normal ranges. the gross appearance was a soft, nodular and well - encapsulated 13106 cm - sized mass. light microscopic sections showed a predominantly diffuse pattern of monotonous neoplastic cells arranged in organoid cell nests (classic zellballen pattern) that are characteristics of paraganglioma (figure 3). tumor cells were polygonal with a clear or finely granular eosinphilic cytoplasm and a round or ovoid nucleus. immunohistochemical stains revealed a diffuse and strong cytoplasmic immunoreactivity for chromogranin in about 90% of the tumor cells. immunoreactivity for s-100 protein, confirming the presence of sustentacular cells which are characteristics of paraganglioma, was detected in scattered supporting cells, mostly at the periphery of the tumor cell nests(figure 4). she was confirmed as rarely occurring mediastinal paraganglioma secreting acth, cushing s syndrome and secondary diabetes. unfortunately, she died on the 22th postoperative day because of septic shock and respiratory failure due to uncontrolled mediastinitis with combined pneumonia. paragangliomas are unusual neuroendocrine cell tumors arising from paraganglia tissue which are widely dispersed groups of specialized neural crest cells. it has been known historically by a variety of names, including glomus tumor, chemodectoma, non - chromaffin paraganglioma, carotid body and tympanic body tumor, and receptoma. the frequent location of paraganglioma in the mediastinum is controversial. in a report of cesar., twelve tumors were located in the posterior mediastinum, and only three were anterior. in addition, tumors arising in an anterior mediastinum are claimed to be more often associated with older age, larger size and lower incidence of functionality, and are less amenable to surgical resection. mediastinal paragangliomas usually cause no symptoms. occasionally, however, they do present with varying degrees of hypertension, diabetes and hypermetabolism. vma and hva are the chief urinary excretion products but epinephrine and norepinephrine may also be secreted in the urine. the word however, because of potentially serious consequences of a catecholamine crisis during manipulation of a functioning tumor, evaluation of patients should include screening for symptoms and signs and the measurment of appropriate blood and urine product. the tumors may produce functioning peptides that can cause cushing s syndrome like this case, secretory diarrhea and polycythemia vera. in the thorax the 24h - urine metanephrine, vma, hva and serum serotonin levels may also be incerased, although slightly in this case. paraganglioma in association with other neoplasms (part of multiple endocrine neoplasia syndrome, men) and reports of multicentricity have been well documented. therefore, care should be exercised in differentiating between multicentric neoplasms and metastasis from a malignant tumor. malignancy is rare and typically defined by the existence of metastasis, rather than cellular characteristics. lung and bone were the usual sites of metastasis, whereas other sites included lymph nodes, liver, spleen, heart or kidneys. because paraganglial tissue is not usually found there, its presence in any of these organs constitutes metastasis. some patients with known lung and bone metastses can live as long as 25 years after the lesions are discovered. there was no evidence of men, but supraclavicular lymph node metastasis was found in this case. large masses may be visible on the chest radiograph but, in most patients, ct scans are necessary to visualize the tumors. the technetium - methoxyisobutylisonitrile (mibi) scan is also helpful to detect ectopic acth - producing tumors undiagnosed with ct and mri. when the tumors are located in the anterior mediastinum, the most important differential diagnosis is with thymic carcinoid. however, the presence of bands of connective tissue, nuclear pleomorphism with bizarre forms and absence of festfoons or rosettes are histological features more in favor of paraganglioma. in addition, the positive immunohistochemical reaction for keratin in the tumor cells is a feature that has been more often documented in carcinoid tumor. paraganglioma located in the posterior mediastinum should be differentiated from metastatic tumors, such as melanoma, renal cell carcinoma and alveolar soft part sarcoma. however, the patient should first undergo alpha blockade with penoxybenzamine for 1 week and then beta blockade with metoprolol or propranolol. the blood supply of paragangioma is usually rich. when surgery is planned, preoperative embolization of the main arterial supply and the tumor bed is helpful for safe and less morbid removal of large tumors the decision to operate or irradiate should be based on a formula that considers tumor size and location, patient age and health, symptoms or signs, potential morbidity and the expertise and availability of those involved in treatment. mediastinal paraganglioma, because of their unusual anatomic site, may pose problems in diagnosis. a detailed history, hormone study and immunohistochemical studies play an important role in separating these tumors from other neoplasms. | paragangliomas are unusual neuroendocrine cell tumors arising from paraganglia, of which acth - secreting cases in the mediastinum are exremely rare. a 51-year - old woman was admitted for generalized edema and weakness which began 5 months ago. chest x - ray and ct scan revealed a tumor mass in the anterior mediastinum. the plasma cortisol and acth levels were very high. other sources secreting acth, except mediastinal mass, were not found. surgical excision of mediastinal mass and left supraclavicular lymph node was performed. the postoperative microscopic finding and immunohistochemical staining revealed organoid tumor cell nests (zellballen) and s-100 protein positive sustentacular cells which are characteristics of paraganglioma. this was thus a case of cushing s syndrome resulting from ectopic acth production in anterior mediastinal paraganglioma. |
the growth of elderly populations is a global phenomenon, and these changes have been radical and accelerated in brazil. conservative projections indicate that brazil will have the sixth largest elderly population in the world by 2020, with over 30 million people classified as elderly.1,2 recent studies have shown that chronic disease and incapacity are not inevitable consequences of the aging process. prevention is effective in all age groups, even in the latest stages of life.1 therefore, an emphasis on prevention is important in terms of changing the current framework of incapacity and comorbidity. loss of muscle quality and quantity and reduced muscle strength with advancing age contributes significantly to a decline in the performance of activities of daily living (adl), resulting in reduced mobility, slower gait speed, and changes in balance as well as an increased risk of falls. thus, there is an increase in incapacity.2,3 the benefits of exercise in the elderly are well established, and include reduced fragility, increased gait speed, and increased performance of adl. exercise also provides independence and improves quality of life.3 elderly people who undertake a regular exercise program show better balance and have a reduced risk of falling.4,5 adherence is defined as the number of sessions attended divided by the number of sessions offered, and according to the literature, this relationship is frequently very low.6 the evidence suggests that 50% of people who begin a program of exercise discontinue within 6 months.79 an adherence level of at least 80%85% is recommended if the results of an intervention are to be satisfactory and if the intervention is to have therapeutic value.10 another important concept for exercise intervention is retention. this involves maintenance of the individual bond to a program of exercises as an experimental or clinical model. an individual may be linked to an exercise program throughout the period and without proper frequency, ie, without satisfactory adherence.7 kruger evaluated 6,000 elderly subjects and found that only 11% reported regular participation in a muscle strength training program.11 this study, which included a significant number of participants, showed the difficulties of adherence with exercise programs among the elderly. further, in a study investigating elderly subjects with chronic pain, sluijs showed that 70% of participants failed to adhere to a program of therapeutic exercise in the long - term. in addition, taylor found that rates of adherence with exercise among elderly individuals were approximately 14% to 17%. health professionals have made considerable effort to identify factors related to adherence with therapeutic exercise by elderly people, in view of the fact that it could allow for greater success of proposed treatments, maintain patient independence, increase muscle capacity and function, and minimize ongoing use of health care services.14 however, there is no consensus in the literature as to the factors that contribute to adherence with exercise programs in the elderly. in addition, there is a lack of standardized instruments for measurement of compliance.4,6,15 social, economic, and cultural factors may influence adherence, so surveys of adherence should be performed locally. we can not extrapolate the results of international studies to the brazilian population, which exists in a completely different context. further, there have been few studies in which adherence has been a primary outcome and in brazil there is no similar research. the objectives of the present study were to identify rates of adherence and retention in elderly women participating in aerobic exercise or muscle strength training programs and to identify the motivators, barriers, and clinical / functional factors associated with adherence of elderly women with therapeutic exercise programs. this study was approved by the ethical and research committee of the federal university of minas gerais (etic 38/2010, with approval of an addendum). details of the original project are in the registry of brazilian clinical trials (rebec rbr9v9cwf). our study population comprised a convenience sample of women aged 65 years or older who lived in the community and were recruited by advertising among third - age groups and associations as well as in newspapers. the sample was not randomized, and the study design involved development of two exercise programs at different points in time. the first volunteers were allocated to a muscle strengthening group, and when enough participants had been enrolled in this group, subsequent volunteers were allocated to an aerobic exercise group. we excluded anyone who showed cognitive changes detectable on the brazilian version16 of the mini - mental state examination. we also excluded subjects who had undergone orthopedic leg surgery, those with a history of fracture in the previous 6 months, those with a neurological disorder, and those with clinical or sensory conditions that precluded their participation in an exercise program. we calculated the sample size using the following regression model formula : 10 (k + 1), in which k represents the number of explanatory variables in the model.16,17 for each intervention group, three models of regression were used to explain adherence or failure to adhere. the ten explanatory variables used for the model, chosen from the theoretical framework, were : the timed up and go test,18 the sit - to - stand test,19 habitual gait speed,20,21 history of falls, presence of pain, number of comorbidities, self - rated health,22,23 depressive symptoms,24 self - rated stress,25 and cognitive capability.16 the other two models were constructed to include 13 motivators and 12 barriers according to the adherence questionnaire developed by researchers. thus, 10 (13 + 1) = 140 elderly women were necessary for each group. for sample characterization, we obtained sociodemographic data and information relevant to the clinical status of our elderly female study participants using a questionnaire developed by the researchers. folstein recommended the mini - mental state examination as a useful screening tool for detection of cognitive impairment in both the clinical and research setting. in 1994, bertolucci published a version of the mini - mental state examination adapted for the brazilian population.16 in the present study, we used this tool as a reference for cutoff points, with a score of 13 for illiterate elderly and a score of 24 for educated elderly, given that previous studies have demonstrated this scale to have good reliability.27,28 in the present study, the mini - mental state examination was used for subject exclusion and test scores were used as an explanatory variable in the regression model to evaluate the association between adherence and cognitive capacity in the study subjects.29,30 the geriatric depression scale (gds) was used to screen for the presence of depressive symptoms, and was administered by structured interview. the 15-item gds scale (gds-15) is a short version of the original scale developed by sheikh and yesavage24 and contains items that are most strongly matched with a diagnosis of depression. previous studies have demonstrated that symptoms of depression can interfere with the undertaking of physical exercise and that elderly people experiencing mood changes and general depressive symptoms are less committed to the intervention and therefore the results are worse. a 14-item scale of perceived stress was used to evaluate the level of stress in our study participants. this scale evaluated three factors considered to be the main components in the experience of stress, ie, the extent to which individuals rate their lives as unpredictable, uncontrollable, and overloaded.31 henry found that patients who are asked to perform many exercises do not perform as well in terms of adherence. in addition, a prescription for excessive exercise can negatively affect adherence in elderly women.10,25 self - evaluation of health status was determined by a simple question, in a general way, how do you consider your health ?. the answer to this question is one of the most commonly used indices in gerontological research and is an important indicator of functional decline and consequent restriction of activity.22,23 the literature demonstrates that elderly women with greater adherence are the most likely to get involved in other programs for promoting health, and that this is directly linked to their behavior, perception of their health, motivation, and self - efficacy.30 we selected three tests to evaluate functional capacity, all of which can be carried out simply and rapidly in clinic practice ; these tests are also valid and reliable in the elderly population. we used these tests because elderly people with lower functional performance find it more difficult to develop regular exercise habits.14 we measured walking speed over 10 minutes to evaluate habitual gait speed. pace was registered only in the middle 6 m of a lane, identified by tape markings, to avoid acceleration and deceleration bias.20,21,33 we used the timed up and go test to evaluate mobility. this test records the time taken for a subject to stand up without using their arms from a sitting position in a standardized chair with a seat height of 45 cm, walk 3 m, turn around, and return to the seat and sit down again. this test has high interrater reliability (intraclass correlation coefficient 0.99) and intrarater reliability (intraclass correlation coefficient 0.99).18,34 to evaluate functional performance in terms of sitting and standing, which is an indirect measure of strength in the legs and is associated with decline of function and capability, we used the sit - to - stand test with a standardized chair of 45 cm and floor height as the reference.19,20 there are no previous examples of adherence questionnaires validated and adapted for an elderly population in brazil.35 therefore, we developed our own adherence questionnaire based on the literature and on the cultural, economic, and social context of brazil. this questionnaire contained questions on the following three components : reasons for lack of attendance at sessions, motivating factors, and barriers to adherence with the different therapeutic exercise programs. as part of the development of this instrument, we trialed the questionnaire in ten elderly subjects participating in a physical exercise program. the necessary adjustments were made until the final version of the questionnaire was ready for use. intraobserver and interobserver variability were assessed by the kappa coefficient and this resulted in excellent intraobserver reliability (kappa coefficient 0.846) and interobserver reliability (kappa coefficient 0.822). first, the brazilian version26 of the mini - mental state examination was used to exclude elderly women with cognitive impairment. this was followed by a clinical sociodemographic questionnaire in the form of a structured interview for characterization of the sample.16 for the functional tests, all participants were given the same verbal instructions and the investigators demonstrated tests before the participants carried them out. after the initial evaluation, the elderly women were allocated to a therapeutic exercise group, ie, either muscle strength training or aerobic exercise. each program lasted 10 weeks and comprised three sessions weekly, each lasting approximately 50 minutes and guided by physiotherapists and members of the research team. the muscular strength training group carried out leg exercises involving concentric and eccentric contraction with resistance, with the load tailored to meet individual requirements and ability. the load appropriate for each participant was defined by calculation of maximum resistance.36 the aerobic exercise program was based on a protocol recommended by the american college of sport medicine (2009) for the elderly, and comprised 5 minutes of warming up, 40 minutes of aerobic activity, including exercises for the arms and legs while walking, orthostatic exercise, and a 5-minute recovery period. before and after each exercise session, blood pressure, and heart rate were measured. in addition, heart rate monitoring was undertaken in participants allocated to aerobic exercise to ensure that the program was safe for them and that their training range was appropriate.37 after completion of the 10-week training program, the volunteers were re - evaluated using the adherence questionnaire administered by an investigator who had not been involved in the intervention (to avoid information bias in collection of the data). elderly women who had abandoned their allocated program and were otherwise lost to follow - up were also contacted by the team ; a number of these women subsequently visited the federal university of minas gerais to answer the adherence questionnaire. successful intervention programs require an adherence rate of more than 85% to have satisfactory results and ensure internal validity. therefore, in the present study, elderly women with a participation rate of 85% or more in the exercise program were considered to have been adherent.31 thus, for a total of 30 sessions, five absences were allowed. those lost to the program comprised elderly women who did not finish the program or those who missed six or more exercise sessions during the 10-week intervention. three logistic regression models were built for each of the groups to identify factors accounting for adherence of these elderly women with an exercise program. the statistical analysis was blinded, performed without input from the researchers who developed the questionnaire, and without involvement of the study volunteers. the first regression model aimed to identify the contribution of the following explanatory variables : presence of pain, a history of falls, number of comorbidities, self - perception of health, level of stress, cognitive capacity, depressive symptoms, and functional capacity (measured by gait speed, timed up and go test, and sit - to - stand test). the other two regression models assessed the association between 13 motivational items and 12 items that acted as barriers to participation of elderly people in different therapeutic exercise programs. in the three models, the explanatory variables entered into the model were determined by theoretical reference. to define the variables in the model, the enter38 method was used and to define the model as significant was defined as p<0.20 and alpha 5%. interpretation of the logistic regression analysis was defined, by the odds ratio (or), as a relative risk of nonadherence. the level of statistical significance was set at 5% for all analyses, which were done using statistical package for the social sciences for windows version 17.0 software (spss inc, chicago, il, usa). our study population comprised a convenience sample of women aged 65 years or older who lived in the community and were recruited by advertising among third - age groups and associations as well as in newspapers. the sample was not randomized, and the study design involved development of two exercise programs at different points in time. the first volunteers were allocated to a muscle strengthening group, and when enough participants had been enrolled in this group, subsequent volunteers were allocated to an aerobic exercise group. we excluded anyone who showed cognitive changes detectable on the brazilian version16 of the mini - mental state examination. we also excluded subjects who had undergone orthopedic leg surgery, those with a history of fracture in the previous 6 months, those with a neurological disorder, and those with clinical or sensory conditions that precluded their participation in an exercise program. we calculated the sample size using the following regression model formula : 10 (k + 1), in which k represents the number of explanatory variables in the model.16,17 for each intervention group, three models of regression were used to explain adherence or failure to adhere. the ten explanatory variables used for the model, chosen from the theoretical framework, were : the timed up and go test,18 the sit - to - stand test,19 habitual gait speed,20,21 history of falls, presence of pain, number of comorbidities, self - rated health,22,23 depressive symptoms,24 self - rated stress,25 and cognitive capability.16 the other two models were constructed to include 13 motivators and 12 barriers according to the adherence questionnaire developed by researchers. thus, 10 (13 + 1) = 140 elderly women were necessary for each group. for sample characterization, we obtained sociodemographic data and information relevant to the clinical status of our elderly female study participants using a questionnaire developed by the researchers. folstein recommended the mini - mental state examination as a useful screening tool for detection of cognitive impairment in both the clinical and research setting. in 1994, bertolucci published a version of the mini - mental state examination adapted for the brazilian population.16 in the present study, we used this tool as a reference for cutoff points, with a score of 13 for illiterate elderly and a score of 24 for educated elderly, given that previous studies have demonstrated this scale to have good reliability.27,28 in the present study, the mini - mental state examination was used for subject exclusion and test scores were used as an explanatory variable in the regression model to evaluate the association between adherence and cognitive capacity in the study subjects.29,30 the geriatric depression scale (gds) was used to screen for the presence of depressive symptoms, and was administered by structured interview. the 15-item gds scale (gds-15) is a short version of the original scale developed by sheikh and yesavage24 and contains items that are most strongly matched with a diagnosis of depression. previous studies have demonstrated that symptoms of depression can interfere with the undertaking of physical exercise and that elderly people experiencing mood changes and general depressive symptoms are less committed to the intervention and therefore the results are worse. a 14-item scale of perceived stress was used to evaluate the level of stress in our study participants. this scale evaluated three factors considered to be the main components in the experience of stress, ie, the extent to which individuals rate their lives as unpredictable, uncontrollable, and overloaded.31 henry found that patients who are asked to perform many exercises do not perform as well in terms of adherence. in addition, a prescription for excessive exercise can negatively affect adherence in elderly women.10,25 self - evaluation of health status was determined by a simple question, in a general way, how do you consider your health ?. the answer to this question is one of the most commonly used indices in gerontological research and is an important indicator of functional decline and consequent restriction of activity.22,23 the literature demonstrates that elderly women with greater adherence are the most likely to get involved in other programs for promoting health, and that this is directly linked to their behavior, perception of their health, motivation, and self - efficacy.30 we selected three tests to evaluate functional capacity, all of which can be carried out simply and rapidly in clinic practice ; these tests are also valid and reliable in the elderly population. we used these tests because elderly people with lower functional performance find it more difficult to develop regular exercise habits.14 we measured walking speed over 10 minutes to evaluate habitual gait speed. pace was registered only in the middle 6 m of a lane, identified by tape markings, to avoid acceleration and deceleration bias.20,21,33 we used the timed up and go test to evaluate mobility. this test records the time taken for a subject to stand up without using their arms from a sitting position in a standardized chair with a seat height of 45 cm, walk 3 m, turn around, and return to the seat and sit down again. this test has high interrater reliability (intraclass correlation coefficient 0.99) and intrarater reliability (intraclass correlation coefficient 0.99).18,34 to evaluate functional performance in terms of sitting and standing, which is an indirect measure of strength in the legs and is associated with decline of function and capability, we used the sit - to - stand test with a standardized chair of 45 cm and floor height as the reference.19,20 there are no previous examples of adherence questionnaires validated and adapted for an elderly population in brazil.35 therefore, we developed our own adherence questionnaire based on the literature and on the cultural, economic, and social context of brazil. this questionnaire contained questions on the following three components : reasons for lack of attendance at sessions, motivating factors, and barriers to adherence with the different therapeutic exercise programs. as part of the development of this instrument, we trialed the questionnaire in ten elderly subjects participating in a physical exercise program. the necessary adjustments were made until the final version of the questionnaire was ready for use. intraobserver and interobserver variability were assessed by the kappa coefficient and this resulted in excellent intraobserver reliability (kappa coefficient 0.846) and interobserver reliability (kappa coefficient 0.822). first, the brazilian version26 of the mini - mental state examination was used to exclude elderly women with cognitive impairment. this was followed by a clinical sociodemographic questionnaire in the form of a structured interview for characterization of the sample.16 for the functional tests, all participants were given the same verbal instructions and the investigators demonstrated tests before the participants carried them out. after the initial evaluation, the elderly women were allocated to a therapeutic exercise group, ie, either muscle strength training or aerobic exercise. each program lasted 10 weeks and comprised three sessions weekly, each lasting approximately 50 minutes and guided by physiotherapists and members of the research team. the muscular strength training group carried out leg exercises involving concentric and eccentric contraction with resistance, with the load tailored to meet individual requirements and ability. the load appropriate for each participant was defined by calculation of maximum resistance.36 the aerobic exercise program was based on a protocol recommended by the american college of sport medicine (2009) for the elderly, and comprised 5 minutes of warming up, 40 minutes of aerobic activity, including exercises for the arms and legs while walking, orthostatic exercise, and a 5-minute recovery period. before and after each exercise session, blood pressure, and heart rate were measured. in addition, heart rate monitoring was undertaken in participants allocated to aerobic exercise to ensure that the program was safe for them and that their training range was appropriate.37 after completion of the 10-week training program, the volunteers were re - evaluated using the adherence questionnaire administered by an investigator who had not been involved in the intervention (to avoid information bias in collection of the data). elderly women who had abandoned their allocated program and were otherwise lost to follow - up were also contacted by the team ; a number of these women subsequently visited the federal university of minas gerais to answer the adherence questionnaire. successful intervention programs require an adherence rate of more than 85% to have satisfactory results and ensure internal validity. therefore, in the present study, elderly women with a participation rate of 85% or more in the exercise program were considered to have been adherent.31 thus, for a total of 30 sessions, five absences were allowed. those lost to the program comprised elderly women who did not finish the program or those who missed six or more exercise sessions during the 10-week intervention. three logistic regression models were built for each of the groups to identify factors accounting for adherence of these elderly women with an exercise program. the statistical analysis was blinded, performed without input from the researchers who developed the questionnaire, and without involvement of the study volunteers. the first regression model aimed to identify the contribution of the following explanatory variables : presence of pain, a history of falls, number of comorbidities, self - perception of health, level of stress, cognitive capacity, depressive symptoms, and functional capacity (measured by gait speed, timed up and go test, and sit - to - stand test). the other two regression models assessed the association between 13 motivational items and 12 items that acted as barriers to participation of elderly people in different therapeutic exercise programs. in the three models, the explanatory variables entered into the model were determined by theoretical reference. to define the variables in the model, the enter38 method was used and to define the model as significant was defined as p<0.20 and alpha 5%. interpretation of the logistic regression analysis was defined, by the odds ratio (or), as a relative risk of nonadherence. the level of statistical significance was set at 5% for all analyses, which were done using statistical package for the social sciences for windows version 17.0 software (spss inc, chicago, il, usa). a total of 382 elderly community - dwelling women participated in this study, comprising 231 in the aerobic exercise group and 151 in the muscle strength training group. there were no significant differences between the two groups in terms of clinical or demographic profile. the rate of adherence was 49.70% in the aerobic exercise group and 56.20% in the muscle strength training group. the rate of retention in the program was 71.40% for the aerobic exercise group and 66.80% for the muscle strength training group. the regression model comprising the ten explanatory variables selected from the theoretical framework did not find any statistically significant differences for the aerobic exercise group (p=0.947) or the muscle strength training group (p=0.104). none of the variables included in the regression model were predictors of adherence in either of the exercise programs. the multiple logistic regression model comprising motivators for exercise was significant (p=0.003) for the muscle strength training group (r=0.310). (p=0.037 ; or 0.234, 95% confidence interval [ci ] 0.0124.523) ; i practice exercises even when i do nt feel like it (p=0.004 ; or 0.062, 95% ci 0.0090.416) ; and companions in the group help me to deal with my problems this multiple logistic regression model comprising motivators for exercise was also significant for the aerobic group (p=0.008 ; r=0.154). variables that predicted adherence were exercises increase my concentration (p=0.003 ; or 0.598, 95% ci 0.0645.622) and i practice exercise even when i do nt feel like it (p=0.05 ; or 0.471, 95% ci 0.1671.329). the third regression model analyzed variables related to barriers to exercise reported by the elderly women. the result was significant only for the muscle strength training group (p=0.003 ; r=0.236). the variable that predicted adherence was the bad weather disrupts the accomplishment of the exercises although the models did not canvass many variables that were predictors of adherence, the results suggest that there are many barriers to adherence with exercise in the elderly. this was seen in the statement if my health was better i would be more active. the second most common barrier was difficulty performing exercise due to pain ; more than half of the elderly who did not adhere to their program agreed with this statement. the least important items for participants who did not adhere to their exercise program were the opportunity to have group companions and consideration of physical exercise as a leisure activity. the low rate of adherence compared with the rate of retention in the present study indicates that the number of absences among participants was very high. although the participants in our study did not abandon their exercise program, they found it difficult to attend often enough to reap the benefits of the proposed intervention. on investigating the reasons for nonattendance in each group, we noticed that changes in state of health was the most frequent reason given by adherent and nonadherent participants in both exercise groups. it is also important to highlight that family problems, lack of motivation, and pain were cited markedly more often by elderly women who did not adhere to the program than by those who did adhere. participants in the present study showed lower adherence rates than those previously recommended to ensure the effectiveness of therapeutic exercise programs. thus, completion of an exercise program, or rate of retention in the program, does not mean good adherence or good results. health services, community centers, and sports centers that implement exercise programs for the elderly should develop strategies to encourage these people to exercise and increase their frequency of exercise. brittle reported a high 82% retention rate in exercise programs for the elderly ; however, study participants attended an average of only 43% of the sessions offered, and only 18% of the sample participated in all of the sessions prescribed. thus, it is clear that while the elderly seem to enjoy this kind of physical activity, attendance at sessions offered is not guaranteed. in our study, the adherence rate was higher in the muscle strength group than in the aerobic exercise group. this finding is consistent with the results of a meta - analysis of 3,389 elderly subjects by hong showing that resistance exercise programs had higher rates of adherence than aerobic exercise programs.7 in elderly populations, the high prevalence of joint diseases, such as osteoarthritis, frequently hampers successful performance of aerobic exercises because many activities cause joint impact. muscle strength training exercises without weight - bearing provide more joint stability.7,10 elderly people tend to participate more effectively at the beginning of exercise programs ; however, adherence diminishes as time passes.10 if health professionals can identify patients at high risk of nonadherence with a proposed treatment, special attention can be given to them during the intervention, and may help to increase their adherence rate.6,10,40 in the present study, none of the explanatory variables selected from the theoretical framework were predictors of adherence in our regression models. heuvelen,6 a higher number of comorbidities and chronic diseases was correlated to lower adherence of subjects (n=118) to a program of physical exercise. in that study, the participants were divided in two groups, ie, one that carried out physical exercises and another that received a psychological approach. those in the psychological group who recorded only moderate results for adl and habitual gait speed before intervention had the lowest program adherence. in the physical activity group, adherence was low for subjects with low adl scores and modest habitual walking gait scores. worse functional performance is therefore an impediment to adherence with physical exercise programs in the elderly.6 forkan also found that changes in health state were a frequent reason for low adherence with continuing exercise regimes among the elderly.5 in their study, one of the factors associated with lack of participation (p<0.05) in an exercise program by the elderly was changes in the weather, especially the presence of cold weather. there is no severe winter in brazil ; however, the rainy season brings disruption and we found that this was an important barrier to exercise for participants in the muscle strength training group. this can be explained by the fact that the program was held in the first half of the year when there is a lot of rain, while the aerobic group program was conducted in the second half of the year. with the rain, use of public transport becomes even harder, there is a greater risk of falls, and elderly people struggle with the difficulty of having to walk while holding umbrellas and bags. understanding the benefits of an exercise program was an important motivator in both the muscle strength training group and the aerobic exercise group in this study. these elderly women mentioned that they practiced their exercises even when they were not in the mood, and realized that exercise was necessary regardless of their mood, indicating their belief in the benefits stemming from the practice. therefore, educational initiatives highlighting the importance of exercise need to be promoted.8,41 one important study found that improved concentration acted as a motivator and predicted adherence with exercise programs, and this could be due to increased circulation in the brain. concentration can be explained as intense brain activity and research suggests that physical exercise can increase cognitive control and the capacity to pay attention, resulting in better cognitive function.42 socialization, or the presence of other elderly people, was also a predictor of adherence in the muscle strength training group. exchange of experiences and encountering people with similar problems and similar difficulties, support, listening, and all the other components associated with a group activity are all very important to the benefit of physical exercise in groups. although individual exercise regimes may bring more directed physical benefits, the benefits of group exercise in terms of social and emotional health are critical and should be encouraged.30,43 our results reflect the difficulties associated with adherence. the present study did not show a direct correlation between low self - rated health and low adherence with exercise programs ; therefore, the influencing factors are multifactorial and should be investigated in terms of physical, emotional, and social health. although adherence to both programs was suboptimal in this study, some factors may have contributed to the increased adherence rate in relation to the population in general. the elderly women who participated in this study chose to do so and were active and healthy ; in other words, they were available for and interested in undertaking physical exercise. another limiting factor was the short duration of the study ; this is because it is difficult to maintain an ideal exercise frequency in long - term exercise programs and adherence rates in such programs are even smaller. in addition, a factor that could have contributed negatively to the programs developed for this study is the fact that they constituted general exercises and there was no specific individual objective. preventive exercises are less stimulating than exercises that aim to provide relief for a specific health problem. the scale developed to assess adherence may not have been sensitive enough to identify factors associated with adherence as dichotomous. the volunteer may have answered yes or no without thinking of any particular situation. although adherence to both programs was suboptimal in this study, some factors may have contributed to the increased adherence rate in relation to the population in general. the elderly women who participated in this study chose to do so and were active and healthy ; in other words, they were available for and interested in undertaking physical exercise. another limiting factor was the short duration of the study ; this is because it is difficult to maintain an ideal exercise frequency in long - term exercise programs and adherence rates in such programs are even smaller. in addition, a factor that could have contributed negatively to the programs developed for this study is the fact that they constituted general exercises and there was no specific individual objective. preventive exercises are less stimulating than exercises that aim to provide relief for a specific health problem. the scale developed to assess adherence may not have been sensitive enough to identify factors associated with adherence as dichotomous. future research on adherence with exercise programs for the elderly should incorporate specific strategies ; this will increase recruitment rates and boost adherence and retention of people of different cultures and ethnicities in exercise programs.30 future work in this area should focus on behavioral motivation. it is also important to look more broadly at social and environmental contexts and take into account direct variables, such as those related to commitment to exercise among older women, sedentary people, and patients with multiple diseases and functional deficits.44 we recommend that older people be asked more directly about what influences them, and qualitative studies should be encouraged in this regard. | backgroundparticipation of older people in a program of regular exercise is an effective strategy to minimize the physical decline associated with age. the purpose of this study was to assess adherence rates in older women enrolled in two different exercise programs (one aerobic exercise and one strength training) and identify any associated clinical or functional factors.methodsthis was an exploratory observational study in a sample of 231 elderly women of mean age 70.5 years. we used a structured questionnaire with standardized tests to evaluate the relevant clinical and functional measures. a specific adherence questionnaire was developed by the researchers to determine motivators and barriers to exercise adherence.resultsthe adherence rate was 49.70% in the aerobic exercise group and 56.20% in the strength training group. multiple logistic regression models for motivation were significant (p=0.003) for the muscle strengthening group (r2=0.310) and also significant (p=0.008) for the aerobic exercise group (r2=0.154). a third regression model for barriers to exercise was significant (p=0.003) only for the muscle strengthening group (r2=0.236). the present study shows no direct relationship between worsening health status and poor adherence.conclusionfactors related to adherence with exercise in the elderly are multifactorial. |
we report the successful treatment of a case of cystoid macular edema (cme) associated with topical tafluprost, which was accompanied by serous retinal detachment (srd). a 78-year - old woman underwent intraocular lens suture surgery, including anterior vitreous cutting, for crystalline lens dislocation in the right eye. however, the patient complained of blurred vision (20/200) 9 months after surgery. cme accompanied by srd was identified by optical coherence tomography (oct) and treated with subtenon triamcinolone injection. visual acuity rapidly increased to 20/50, and the volume of srd decreased in a few days. discontinuation of tafluprost and initiation of diclofenac eye drops improved visual acuity to 20/40 and resulted in improved oct findings within a few weeks. no recurrence of cme occurred over the following 3 months, and iop was controlled at 1015 mmhg. srd is considered to be a symptom of treatment - resistant cme, which may lead to poor visual acuity after recovery. in such cases, subtenon triamcinolone injection should be strongly considered at an early stage. cystoid macular edema (cme) has been reported in association with the use of latanoprost, travoprost, and bimatoprost.1,2 these drugs are ocular hypotensive lipids, and their side effects can be expected to be somewhat similar. in this report, we describe a case of cme associated with tafluprost. optical coherence tomography (oct) revealed typical cme appearance with serous retinal detachment (srd). the patient was successfully treated with subtenon triamcinolone injection. to our knowledge, this is the first report of cme associated with tafluprost. a 78-year - old woman, who had a history of chronic bronchitis, was under observation for glaucoma with use of tafluprost. intraocular pressure (iop) was controlled at 1316 mmhg without any tafluprost side effects. the patient underwent intraocular lens (iol) suture surgery, including anterior vitreous cutting, for crystalline lens dislocation in the right eye. however, 9 months after the surgery, the patient complained of blurred vision (20/200), and cme accompanied by srd was identified with oct (figure 1a). after injection of subtenon triamcinolone, visual acuity rapidly recovered to 20/50, and the srd lost its volume in a few days (figure 1b). discontinuation of tafluprost and initiation of diclofenac eye drops improved visual acuity to 20/40 and oct showed improvements within a few weeks (figure 1c and d). an iop spike after injection of subtenon triamcinolone was observed. because iop was above 20 mmhg, we resumed treatment with diclofenac eye drops and tafluprost 3 months after injection, as we considered that the damage to the blood no recurrence of cme occurred during the following 3 months, and iop was controlled at 1015 mmhg (figure 1e). tafluprost, like other prostaglandin analogs, exerts its effects on prostaglandin f receptors to reduce iop. several clinical studies have assessed the efficacy of tafluprost and reported that it was equivalent to the efficacy of latanoprost.3 furthermore, some recent studies have suggested that tafluprost has direct neuroprotective effects. mayama found that topical tafluprost enhanced optic nerve head blood flow in nave eyes without significant iop reduction.4 side effects of tafluprost, such as deepening of the eyelid sulcus and increased eyelid pigmentation, have been reported to be similar to those of other prostaglandin eye drops.5 the incidence of deepening of the eyelid sulcus has been shown to be lower with tafluprost than with latanoprost, travoprost, or bimatoprost.5 liang indicated that the benzalkonium chloride (bak) preservative is the primary cause of cytotoxicity due to prostaglandin analogs, given that bak - free products had minimal impact on the corneoconjunctival surface.6 uusitalo evaluated the pharmacokinetics and safety profiles of preservative - free and preservative - containing tafluprost ; although the incidence of ocular hyperemia was similar for both forms of tafluprost, it was less severe with preservative - free tafluprost.7 prostaglandin analogs have been shown to cause cme after cataract surgery. miyake reported that the preservatives in prostaglandin eye drops have a greater influence on the development of cme than the base compound.8 there are many reports of cme associated with latanoprost,919 which contains a relatively high bak concentration (0.02%), but there are few reports of cme associated with bak - free travoprost. bak - free tafluprost became available in western countries in 2012 ; the japanese product contains a very low concentration of bak (0.001%). to our knowledge, this is the first report of cme associated with tafluprost. risk factors for cme due to prostaglandin eye drops include a history of glaucoma surgeries, aphakia, complicated cataract surgery, scleral buckling, and vitrectomy, as well as a history of uveitis and other retinal diseases. it is rare for patients without risk factors to develop cme.915 wand reported that two of 38 high - risk cases (5.3%) developed clinical cme. fukuichi found no cases of clinical cme among 68 patients without any of the reported risk factors. in cases without known risk factors, cme can appear within 23 months after surgery,17,18 while in high - risk cases, cme can develop several years after surgery.9,1113,15,20 in the former, cme may develop immediately after surgery because of temporary damage to the blood aqueous barrier, while in cases with known risk factors, the damage to the blood aqueous barrier is protracted, resulting in a continuation of conditions that can lead to cme in the long term. in the case reported here, anterior vitreous cutting and the iol suture surgery were performed for crystalline lens dislocation ; thus, cme accompanied with srd occurred 9 months after surgery. as leakage from retinal capillaries increases, traction on the inner and outer segments of the fovea produces a small, pointed retinal detachment with a small base. when the barrier function of external limiting membrane breaks down, the serous rd enlarges and changes into a dome - shaped retinal detachment with a large base.21 tsujikawa reported that the outer retinal discontinuity on the external surface of the retina does not necessarily lead to poor vision.21 however, even after complete resolution of the macular edema and srd, diffuse disorganization of the outer photoreceptor layer beneath the fovea often results in poor visual acuity. in addition, a dome - shaped retinal detachment sometimes accompanies a focal defect of the outer segment of the photoreceptors above srd.21 several treatment options are available for prostaglandin - induced cme, including non - steroidal anti - inflammatory drugs (nsaids), steroids, dorzolamide hydrochloride eye drops, and oral acetazolamide ; however, in many cases, recovery can take a month or more.1113,17,19,22 we previously reported two cases in which travoprost- and latanoprost - induced cme was successfully treated in a few days with subtenon injection of triamcinolone.23 subtenon triamcinolone injection is considered to be effective and is the standard treatment for cme due to diabetic retinopathy, retinal vein occlusion, uveitis, and other conditions. therefore, in this case, we employed subtenon triamcinolone injection immediately after diagnosis, anticipating a quick recovery from cme and a subsequently minimized irreversible disruption in the sensory retina. we reported two cases in which cme did not recur for more than 3 years after resumption of prostaglandin eye drops.23 the first case was cme associated with travoprost following uneventful cataract surgery. the second case was cme associated with latanoprost after anterior vitreous cutting and iol suture. wand and gaudio20 reported two cases in which cme treated with nsaids did not recur after unoprostone and bimatoprost treatments were resumed. furthermore, wand reported one case in which cme did not recur after latanoprost treatment was resumed.12 callanan reported one case in which cme that was not treated with nsaids recurred after resuming treatment with latanoprost. prostaglandin has no systemic side effects and is currently the most effective drug in the form of eye drops. beta - blockers were not chosen as treatment in this case because of the patient s history of bronchitis, nor brimonidine, which was not commercially available until may 2012 in japan. thus, tafluprost use was resumed postoperatively, considering that the patient had been well - controlled and without tafluprost side effects before surgery. although we believe the risk of cme would be reduced by using tafluprost along with nsaids, the patient was informed of the necessity of careful observation. when possible, the use of prostaglandin eye drops can be resumed after a certain period of time after recovery, under careful observation. in certain cases, the concomitant use of nsaids and prostaglandin eye drops can be greatly beneficial to patients. srd was considered to be a symptom of treatment - resistant cme, which can lead to poor visual acuity after recovery. in such cases, subtenon triamcinolone injection should be considered at an early stage. | purposewe report the successful treatment of a case of cystoid macular edema (cme) associated with topical tafluprost, which was accompanied by serous retinal detachment (srd).casea 78-year - old woman underwent intraocular lens suture surgery, including anterior vitreous cutting, for crystalline lens dislocation in the right eye. tafluprost was initiated 12 weeks after surgery. intraocular pressure (iop) was controlled at 1014 mmhg. visual acuity remained at 20/4030/40. however, the patient complained of blurred vision (20/200) 9 months after surgery. cme accompanied by srd was identified by optical coherence tomography (oct) and treated with subtenon triamcinolone injection. visual acuity rapidly increased to 20/50, and the volume of srd decreased in a few days. discontinuation of tafluprost and initiation of diclofenac eye drops improved visual acuity to 20/40 and resulted in improved oct findings within a few weeks. three months after injection, tafluprost was resumed along with diclofenac. no recurrence of cme occurred over the following 3 months, and iop was controlled at 1015 mmhg.conclusionsrd is considered to be a symptom of treatment - resistant cme, which may lead to poor visual acuity after recovery. in such cases, subtenon triamcinolone injection should be strongly considered at an early stage. |
the prevalence of hypovitaminosis d has been reported to be high in various regions around the middle east. despite ample sunshine throughout the year, there are a large number of studies in the past decade suggesting that one - third of individuals living in sub - saharan africa and the middle east have serum 25-hydroxyvitamin d (25[oh]d) levels 25 kg / m), history of liver, renal, gestational, or endocrine disorder, medications that influence bone metabolism and current vitamin d and calcium intake, history of iron, folate, and vitamin b12 deficiencies, aplastic anemia, hemolytic anemia, sickle cell anemia, and thalassemia. therefore, 208 women (aged 1858 years) and 213 men (aged 1860 years) were included in the final study. all participants gave written consent after being fully informed of the study objectives and procedures and their right to withdraw from the study. the research protocol was approved by the research and ethics committees of the college of medicine and medical sciences, arabian gulf university, and research and ethic committee of bahrain defense force hospital. fasting serum calcium and phosphorus, creatinine, and bone specific alkaline phosphatase were analyzed immediately using the cobas 6000 analyzer system (roche diagnostics, basel, switzerland). hemoglobin levels were determined by cell - dyn 3700 (abbott laboratories, abbott park, il, usa). fasting plasma intact parathyroid hormone (pth) was determined using commercially available enzyme - linked immunosorbent assay kits (creative diagnostics, shirley, ny, usa). the intra - assay and inter - assay coefficients of variation for determination of intact pth in plasma were less than 4.5% and 7.1% for low control and 3.2% and 5.2% for high control, respectively. fasting serum total 25(oh)d (25[oh]d3 and 25[oh]d2) concentrations were determined by ultra performance liquid chromatography tandem mass spectrometry (waters limited, elstree, uk) using commercially available kits (chromsystems instruments and chemicals gmbh, grafelfing, germany). the intra - assay and inter - assay coefficients of variation for determination of 25(oh)d3 in serum were less than 2.7% and 3.9% for low control and 4.2% and 4.0% for high control, respectively. the intra - assay and inter - assay coefficients of variation for determination of 25(oh)d2 in serum were less than 3.9% and 5.7% for low control and less than 4.3% and 4.7% for high control, respectively. 30 nmol / l, insufficiency at levels between 3050 nmol / l, and optimal at levels 50 nmol / l.14 anemia was defined according to world health organization criteria with hemoglobin 65.0 pg / ml and hypocalcemia was defined as serum calcium these cutoff values are based on the reference range determination of these parameters determined in the authors clinical laboratory in the kingdom of bahrain. the 2.5th, 5th, 50th, 95th, and 97.5th percentiles were determined to observe the distributions of biometric and biochemical parameters in the participants of this study. the normality of serum total 25(oh)d and plasma pth distributions was assessed using the kolmogorov as they were negatively skewed, they were logarithmically transformed to reduce kurtosis before geometric means were calculated. student s t - test was used to compare the biometric and biochemical parameters between males and females. analysis of variance and scheffe s post hoc test were used to compare the hemoglobin and other biometric and biochemical parameters between participants with optimal, insufficiency, and deficiency of vitamin d. pearson s correlation coefficients were used to examine the correlation between hemoglobin levels with serum total 25(oh)d, pth, and other variables. stepwise multiple regression analysis was used to determine the predictors of blood hemoglobin using age, alkaline phosphatase, total 25(oh)d, creatinine, phosphorus, calcium, and pth as independent variables. univariate and multivariate logistic regression analysis were used to determine the association of anemia with vitamin d insufficiency and deficiency adjusted for pth as well as the association of anemia with hyperparathyroidism adjusted for total 25(oh)d. all statistical inferences were made based on a two - sided significance level of p < 0.05 and were performed using ibm spss statistics version 19.0 (ibm corporation, armonk, ny, usa). the 2.5th, 5th, 50th, 95th, and 97.5th percentiles were determined to observe the distributions of biometric and biochemical parameters in the participants of this study. the normality of serum total 25(oh)d and plasma pth distributions was assessed using the kolmogorov as they were negatively skewed, they were logarithmically transformed to reduce kurtosis before geometric means were calculated. student s t - test was used to compare the biometric and biochemical parameters between males and females. analysis of variance and scheffe s post hoc test were used to compare the hemoglobin and other biometric and biochemical parameters between participants with optimal, insufficiency, and deficiency of vitamin d. pearson s correlation coefficients were used to examine the correlation between hemoglobin levels with serum total 25(oh)d, pth, and other variables. stepwise multiple regression analysis was used to determine the predictors of blood hemoglobin using age, alkaline phosphatase, total 25(oh)d, creatinine, phosphorus, calcium, and pth as independent variables. univariate and multivariate logistic regression analysis were used to determine the association of anemia with vitamin d insufficiency and deficiency adjusted for pth as well as the association of anemia with hyperparathyroidism adjusted for total 25(oh)d. all statistical inferences were made based on a two - sided significance level of p < 0.05 and were performed using ibm spss statistics version 19.0 (ibm corporation, armonk, ny, usa). the distributions of biochemical and biometric parameters in males and females are shown in table 1. serum total 25(oh)d was significantly higher in males than females, whereas plasma pth was significantly higher in females than males. in table 2, the biochemical and biometric characteristics of participants of this study according to the vitamin d status are shown for females and males. in females, analysis of variance shows that age, calcium, and hemoglobin were significantly decreased and pth was significantly increased in vitamin deficient individuals than in those with optimal and insufficient vitamin d levels. scheffe s post hoc test for multiple comparison analysis shows that among all the parameters, hemoglobin was significantly lower in females with vitamin d deficiency (p 0.001) and insufficiency (p 0.001) compared with those with optimal levels of vitamin d. however, pth was significantly higher in females with vitamin d deficiency compared with those with optimal levels of vitamin d (p 0.03). in males, there were no significant differences between vitamin d groups in any parameters listed in table 2. further statistical analysis indicates that in females hemoglobin was significantly and negatively correlated with pth (r = 0.254, p = 0.0001) and significantly and positively correlated with total 25(oh)d (r = 0.338, p = 0.001). stepwise multiple regression analysis showed that in females the predictors of hemoglobin were total 25(oh)d (= 0.319, p 0.001) and pth (= 0.227, p = 0.01), contributing to 41.8% and 18.9% variance in hemoglobin, respectively. in table 3, univariate and multivariate logistic regression analysis shows that in females there was a significant 2.1-fold increased risk of anemia with hyperparathyroidism. however, when adjusted for vitamin d, this association disappeared and was no longer significant. in contrast, there was a significant 3.3-fold increased risk of anemia with vitamin d deficiency and this remained significant (2.9-fold) after adjustment for hyperparathyroidism. in males this is the first population - based study to provide evidence that vitamin d deficiency is associated with anemia in healthy female bahrainis. the association of vitamin d deficiency with an increased risk of anemia has been reported in some cross - sectional studies and also in patients with chronic kidney disease.15,16 the exact mechanism of association of vitamin d deficiency with anemia is still not known. it is suggested that vitamin d deficiency could lead to increased risk of reticulocytosis and iron deficiency anemia.8 in bone marrow, there are enormous vitamin d receptors and vitamin d is reported to stimulate erythroid precursors. high local concentrations of 1,25 di hydroxyvitamin d in hematopoietic tissues is suggested to activate erythroid precursor cells in a paracrine fashion.17,18 in addition, high doses of calcitriol the active form of vitamin d has been widely used to increase hemoglobin levels and reticulocyte count in hematological disorders.19,20 in vivo and in vitro studies have also demonstrated that calcitriol reduces cytokine production leading to the reduction of inflammatory milieu and anemia.3 an inverse relationship between plasma 25(oh)d and pth levels in females was also observed in this study. although this relationship was not statistically significant following multivariate regression analysis, it is consistent with a large number of studies in different populations and suggests that hypovitaminosis d could lead to secondary hyperparathyroidism, which is harmful to bone health.21,22 it is now well established that vitamin d causes the suppression of pth synthesis by increasing plasma calcium and by acting on parathyroid cells.23,24 in addition, there was a positive correlation between 25(oh)d and hemoglobin and a negative correlation between hemoglobin and pth. moreover, stepwise multiple regression analysis showed that total 25(oh)d and pth were the main and independent predictors of hemoglobin, but total 25(oh)d was a better predictor than pth (41.90% versus 18.0% variance in hemoglobin). in addition, although the results from logistic regression analysis shows that there was a significant association of vitamin d deficiency and hyperparathyroidism with anemia, this significant association of hyperparathyroidism with anemia disappeared when adjusted for 25(oh)d. this study demonstrates that vitamin d - deficient females are prone to hyperparathyroidism and anemia. over the past 30 years, in vitro and in vivo studies suggest that pth plays a significant inhibitory role in erythropoiesis and survival. secondary hyperparathyroidism is known to induce bone marrow fibrosis, impair erythropoiesis, and inhibit the endogenous production of erythropoietin. pth is reported to be a resistance factor in recombinant human erythropoietin therapy.5 a marked gender difference in vitamin d status has been reported by the authors group and the prevalence of vitamin d deficiency is significantly higher in females than in males in the kingdom of bahrain.25 the lower vitamin d status in females compared to males observed in the present study could be explained by females spending more time indoors than males and/or the type of clothing that females wear and sun protection and sun avoidance attitudes seen in bahraini women.26 the association of vitamin d deficiency with anemia observed in this study is consistent with some recent studies in different populations. perlstein reported that vitamin d deficiency is associated with specific subtypes of anemia in the elderly, especially in those with anemia of inflammation.15 sim reported an increased prevalence of vitamin d deficiency associated with anemia in a cross - sectional study.8 in addition, observational studies have shown that african americans have lower circulating levels of 25(oh)d and are more likely to be vitamin d deficient than other ethnic groups, and it is suggested that increased prevalence of anemia in non - hispanic blacks is correlated with vitamin d deficiency.27 there are some limitations in this study including the lack of data of other confounding factors related to erythropoiesis, lack of 1,25 dihydroxyvitamin d measurement, lack of information about other causes of anemia including iron, b12, and folate deficiency as well as sickle cell and thalassemia carriers among the participants (although individuals with history of anemia were excluded from this study). although this study demonstrates an association of vitamin d deficiency with anemia, a causal relationship can not be established and further studies are warranted to investigate the relationship of vitamin d deficiency and hyperparathyroidism with causes of anemia in anemic patients in the kingdom of bahrain. the results of this study indicate that vitamin d deficiency is associated with anemia in healthy bahraini females. these findings could have potentially broad public health implications given the high prevalence of vitamin d deficiency in bahrainis. these findings suggest that further investigations are warranted particularly those examining specific erythropoiesis and inflammatory pathways or host genetic changes that contribute to the pathogenesis of anemia before vitamin d deficiency can be implicated in the causal development of anemia. ultimately, if these results can be replicated by others and extended, they could lead to randomized clinical trials to evaluate vitamin d supplementation as therapy for patients with anemia. | vitamin d deficiency and anemia are common in the middle east, and vitamin d deficiency and hyperparathyroidism have been reported to be associated with an increased prevalence of anemia. in this study, the hypothesis that vitamin d deficiency and hyperparathyroidism may be associated with anemia in a bahraini population was tested. association of hyperparathyroidism and vitamin d levels (deficiency and insufficiency) with anemia was investigated in 421 bahrainis (213 males and 208 females). in females, the prevalence of anemia was significantly associated with vitamin d deficiency independent of parathyroid hormone levels (odds ratio : 2.9 ; 95% confidence interval : 2.310.5 ; p = 0.001). in females, the prevalence of anemia appeared to be significantly associated with hyperparathyroidism (odds ratio : 2.1 ; 95% confidence interval : 1.23.7 ; p = 0.01) ; however, this significant association disappeared when adjusted for vitamin d deficiency (odds ratio : 1.6 ; 95% confidence interval : 0.756.5 ; p = 0.154). results from this study suggest that vitamin d deficiency is independently associated with anemia in females but not males. further studies to determine whether vitamin d supplementation could be used to treat anemia are warranted. |
reports indicate that 25 to 80% of thoracic traumas are associated with pulmonary contusion (1, 2). various techniques have been proposed for detection of this lesion including clinical assessment, chest radiography (cxr), arterial blood gas, and computed tomography (ct) scan (3, 4). cxr is the most common diagnostic tool in detection of pulmonary contusion but presence of hemothorax or pneumothorax might complicate the diagnosis (5 - 7). moreover, identification of this lesion in cxr is not possible in the first 6 hours after injury (8, 9). ct scan is the most accurate diagnostic tool for pulmonary contusion and can detect the lesion right after the injury (10, 11). ultrasonography reported to have acceptable sensitivity and specificity in detection of pulmonary contusion (11 - 13). in the last 10 years many studies have evaluated the diagnostic values of ultrasonography and radiography in detection of traumatic thoracic injuries including pulmonary contusion (14 - 16), but reaching a consensus has been hindered by the vast disagreements on this subject. one of the ways to overcome this problem is conducting a systematic review and meta - analysis (17, 18). in this regard, we aimed to compare the diagnostic values of these two modalities in detection of pulmonary contusion through a meta - analysis of the available literature. search strategy and selection criteria search strategy was based on the keywords related to ultrasonography and chest radiography including ultrasonography or sonography or ultrasound or chest film or chest radiograph combined with pulmonary contusion - related terms including contusions or pulmonary contusion or lung contusion. the systematic search was carried out in databases of medline (via pubmed), embase, isi web of knowledge, scopus, cochrane library, and proquest directed at finding retrospective and prospective original articles. bibliographies of the related and review articles were scanned in order to find relevant undiscovered studies in our systematic search. the search keywords were extracted from medical subject heading (mesh) terms and emtree. review and editorial articles, case reports, letters to editors, poster presentations, and meeting abstracts were excluded from this survey. application of a reference test other than ct scan and conducting the study on animal samples were also considered as exclusion criteria. two reviewers (m.y, p.g) extracted data in true positive (tp), true negative (tn), false positive (fp), and false negative (fn). in cases where these values could not be obtained neither from the article nor by contacting the authors, the survey were excluded from the study. charecteristics of included studies 1, (+ / -) : (number of patient with contusion / number of patient without contusion) ; 2, number are presented as mean standard deviation or (range). ct : computed tomography ; cxr : chest radiography ; ep : emergency physician ; na : not applicable ; nr : not reported ; us : ultrasonography. two reviewers (m.y, p.g) independently assessed the titles and abstracts of the articles found in the systematic search. then the full texts of the potentially relevant articles were evaluated and the data from the studies that met the inclusion criteria were precisely summarized in details. no time or language limitations were established. quality assessment of the articles was performed according to the guidelines suggested by 14-item quality assessment of diagnostic accuracy studies (quadas-2) tool (28). based on this criteria, all included studies were screened for presence of selection, performance, recording, and reporting biases. demographic characteristics of the patients including age, gender, the number of patients with / without pulmonary contusion according to the results of ct scans, characteristics of ultrasound device (transducer, frequency) and its operator, blinding status, and sampling method (consecutive, convenience). finally, the number of tp, tn, tn, and fn cases were recorded. the method proposed by sistrom and mergo (29) was used to extract the data presented as charts. web - based programs were utilized to calculate the number of tp, tn, tn, and fn cases from the articles in which only the sensitivity and specificity were presented. summary receiver operative curves (sroc), sensitivity, specificity, positive likelihood ratio and negative likelihood ratio of ultrasonography and radiography in detection of pulmonary contusion with 95% confidence interval (95% ci) were estimated. in cases where data were presented separately for each hemi - thorax the information were included separately as presented in the original article. due to the significant heterogeneity between the included studies, mixed effects binary regression model was applied. heterogeneity was evaluated through calculation of i and tests and a p value of less than 0.1 along with an i greater than 50% were considered as presence of considerable heterogeneity (30). in order to recognize the sources of heterogeneity, subgroup analysis was performed considering the sampling method (consecutive/ convenience), operator (emergency physician/ other specialists) or the interpreting physician, the ultrasound device s frequency of the transducer (1 - 5 mhz/ 5 - 10 mhz), and sample size (less than 100 patients/ more than 100 patients). in all the analyses, p value of less than 0.05 study characteristics search in the mentioned databases yielded 15 studies that met the inclusion criteria. after summarization and quality assessment, 12 studies were included (11 - 13, 19 - 27) (figure 1). a total of 716 patients with pulmonary contusion and 965 subjects without were evaluated. their age ranged from 4 to 90 years old and male patients comprised 76% of the study population. diagnostic accuracy of ultrasonography and radiography in detection of pulmonary contusion were assessed simultaneously in eight studies (11 - 13, 19, 22, 23, 26, 27) and the accuracy of radiography was evaluated individually in four surveys (20, 21, 24, 25). considerable heterogeneity was observed between the studies (p<0.001). no publication bias was observed in evaluation of the diagnostic accuracy of ultrasonography (p = 0.97) and chest radiography (p = 0.15) (figure 2). area under the curve of sroc for ultrasonography in pulmonary contusion diagnosis was found to be 0.93 (95% ci : 0.91 - 0.95) (figure 3-a). pooled sensitivity of ultrasonography in this regard was 0.92 (95% ci : 0.81 - 0.96 ; i = 95.81, p < 0.001) and its pooled specificity was estimated to be 0.89 (95% ci : 0.85 - 0.93 ; i = 67.29, p < 0.001). ultrasonography had pooled positive and negative likelihood ratios of 8.94 (95% ci : 5.95 - 93.36 ; i = 67.92, p < 0.001) and 0.09 (95% ci : 0.04 - 0.22 ; i = 06.36, p < 0.001), respectively (figure 4). the sensitivity of this modality was lower when consecutive sampling method was used (0.87 vs. 0.97), procedure was performed via an emergency specialist (0.77 vs. 0.95), sample sizes of higher than 100 patients, the sensitivity (0.86 vs. 0.96), and frequencies of ultrasonography probe was higher than 5 mhz (0.86 vs. 0.93). - chest radiography data from 12 surveys were included in this part of meta - analysis (11 - 13, 19 - 27). area under the sroc for radiography in detection of pulmonary contusion was 0.72 (95% ci : 0.67 - 0.75) (figure 3-b). pooled sensitivity and specificity of this diagnostic tool were 0.44 (95% ci : 0.32 - 0.58 ; i= 87.52, p < 0.001) and 0.98 (95% ci : 0.88 - 1.0 ; i = 95.22, p < 0.001), respectively. pooled positive and negative likelihood ratios were also calculated to be 19.69 (95% ci : 3.59 - 108.07 ; i = 88.75, p < 0.001) and 0.57 (95% ci : 0.45 - 0.72 ; i = 93.13, p < 0.001), respectively (figure 5). subgroup analysis showed that the sensitivity of radiography is affected by the interpreting physician of the plain film (emergency physician/ other specialists) and sample size (table 2). according to the results of this analysis, the sensitivity of this imaging modality is higher when the radiographs were interpreted by a radiologist or intensivist (0.49 ; 95% ci : 0.30 - 0.68) compared to an emergency specialist (0.40 ; 95% ci : 0.21 - 0.58). subgroup analysis of diagnostic accuracy for chest radiography and ultrasonography in detection of pulmonary contusion p value < 0.1 was considered as significant for heterogeneity ; ci : confidence interval summary receiver operative curves (sroc) with prediction and confidence contours of ultrasonography (a) and chest radiography (b) in detection of pulmonary contusion. forest plot of screening performance characteristics of chest ultrasonography in detection of pulmonary contusion. sensitivity and specificity (a) ; diagnostic likelihood ratio (dlr) (b). sensitivity and specificity (a) ; diagnostic likelihood ratio (dlr) (b). the present meta - analysis is the first to assess the diagnostic accuracy of ultrasonography and radiography in detection of pulmonary contusion. the results illustrate a higher sensitivity of ultrasonography compared to radiography (0.92 vs. 0.44) in this regard, whereas the specificity of radiography was slightly higher (0.97 vs. 0.89). since these two imaging modalities are the first diagnostic tools for assessment of traumatic thoracic injuries, their screening accuracy is of utmost importance. accordingly, ultrasonography has better screening performance characteristics in detection of pulmonary contusion compared to radiography. various studies have pointed out the fact that diagnostic accuracy of ultrasonography is directly dependent on the skills of the operator (14, 15, 31, 32). the results demonstrated a higher sensitivity of ultrasonography in detection of pulmonary contusion when performed by a radiologist or an intensivist compared to emergency specialists. this might be due to the nature of pulmonary contusion whose diagnostic signs are very challenging to detect. the most important signs of pulmonary contusion identified by ultrasonography include multiple b - lines and an irregularly delineated tissue image which might be a moderately hypo - echoic blurred lesion (16). furthermore, after observation of these signs, the operator should rule out pneumothorax as well application of transducers with frequencies lower than 5mhz yield greater diagnostic values compared to higher. contusion in characterized by parenchymal injuries and accumulation of fluid and blood in the lung tissues (16). these tissues lie in the deepest layers of chest cavity and so the penetrating power of ultrasound wave is more important than the image resolution (which is directly related to the wave s frequency). since ultrasound waves with lower frequencies have greater penetrating powers, sample size was also found to have an effect on diagnostic values of ultrasonography and radiography in detection of pulmonary contusion. the sensitivity of both these modalities was found to be higher in the studies with sample sizes of less than 100 patients. this might be due to possible selection bias in these studies (33). selection of patients with severe traumas and so the higher chances of injury identification via imaging would be prominent in these studies. moreover in some of these surveys, pneumothorax patients had been excluded which might have made the diagnosis easier (27). firstly, comprehensive search in databases to include the maximum number of related surveys and secondly, elimination of publication bias. thirdly, the effects of heterogeneity between the studies were controlled by subgroup analysis. on the other hand, simultaneous inclusion of retrospective and prospective studies however, evaluation of outliers on the scatterplot based on standardized predictive random effects revealed that retrospective surveys are not the cause of diversity between the studies. moreover, due to the observational nature of included studies, precise assessment of causal relationships was impossible. the results of present meta - analysis revealed the better screening performance characteristics of chest ultrasonography compared to radiography in detection of pulmonary contusion. it should be mentioned that these characteristics where dependent on operator and characteristics of device. this research has been supported by tehran university of medical sciences & health services grant number : 93 - 02 - 38 - 25618. all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors. | introduction : ultrasonography is currently being used as one of the diagnostic modalities in various medical emergencies for screening of trauma patients. the diagnostic value of this modality in detection of traumatic chest injuries has been evaluated by several studies but its diagnostic accuracy in diagnosis of pulmonary contusion is a matter of discussion. therefore, the present study aimed to determine the diagnostic accuracy of ultrasonography and radiography in detection of pulmonary contusion through a systematic review and meta - analysis. methods : an extended systematic search was performed by two reviewers in databases of medline, embase, isi web of knowledge, scopus, cochrane library, and proquest. they extracted the data and assessed the quality of the studies. after summarization of data into true positive, false positive, true negative, and false negative meta - analysis was carried out via a mixed - effects binary regression model. further subgroup analysis was performed due to a significant heterogeneity between the studies. results:12 studies were included in this meta - analysis (1681 chest trauma patients, 76% male). pooled sensitivity of ultrasonography in detection of pulmonary contusion was 0.92 (95% ci : 0.81 - 0.96 ; i2= 95.81, p<0.001) and its pooled specificity was calculated to be 0.89 (95% ci : 0.85 - 0.93 ; i2 = 67.29, p<0.001) while these figures for chest radiography were 0.44 (95% ci : 0.32 - 0.58 ; i2= 87.52, p<0.001) and 0.98 (95% ci : 0.88 - 1.0 ; i2= 95.22, p<0.001), respectively. subgroup analysis showed that the sources of heterogeneity between the studies were sampling method, operator, frequency of the transducer, and sample size. conclusion : ultrasonography was found to be a better screening tool in detection of pulmonary contusion. moreover, an ultrasonography performed by a radiologist / intensivist with 1 - 5mhz probe has a higher diagnostic value in identifying pulmonary contusions. |
in this article, we will review four critical issues related to the research and development of topical formulations containing analgesics such as amitriptyline, phenytoin, ketamine, and baclofen in neuropathic pain. they are as follows : the selection of the analgesic proper, formulation issues, dose - finding aspects, and phase iii, especially related to the selected clinical end points. there are a number of advantages of using topical analgesics over oral analgesics, such as the following : local application only on the pain area where relief is neededfast onset of actionhigher concentration of the analgesic in the pain arealow or no systemic drug levelsabsence of systemic side effectsabsence of drug drug interactionsease of implementing in multimodal therapiesease of combining more analgesics in one vehicle to obtain synergiesimprovement of complianceno risk of dependency or abuse local application only on the pain area where relief is needed higher concentration of the analgesic in the pain area low or no systemic drug levels absence of systemic side effects absence of drug drug interactions ease of implementing in multimodal therapies ease of combining more analgesics in one vehicle to obtain synergies improvement of compliance no risk of dependency or abuse the fundamentals enabling the development of topical analgesic formulations were identified at the end of the last century. the first hints of the efficacy of such topical formulations can be found in an article from 1981, where topical alcoholic solutions containing 5% amitriptyline or 5% doxepin were found to be effective against experimentally induced pruritus.1 somewhat later, there were also some indications that tricyclic antidepressants (tcas) could exert an analgesic effect via a peripheral mechanism of action, as tcas could inhibit inflammatory pain in arthritis models.2 subsequent experiments to determine the analgesic mechanism of action of clomipramine in a rat model for inflammation (inflamed carrageenan paw model) excluded peripheral mechanisms.3 though, further experiments from different groups supported a peripheral mechanism of action of tcas, and thus a neurophysiological foundation for the use of topical creams containing tcas emerged.4,5 in 1999, on the basis of these experiments, it was suggested that such findings supported the development of cream formulations against pain due to the fact that such creams could increase the local concentration of amitriptyline without increasing the plasma levels, leading to systemic side effects.6,7 the peripheral mechanisms of action of tcas could be understood while observing the receptor affinities of the tcas. for example, amitriptyline acts on many targets in the skin ; it blocks na, k, and ca voltage - gated ion channels as well as the muscarinic, cholinergic, nicotinic, histaminergic, -adrenergic, opioid, adenosine, and n - methyl - d - aspartate receptors.8 the fact that peripheral mechanisms contribute to neuropathic pain and that locally applied active pharmaceutical ingredient (api) could reduce such pain led to clinical trials to support the development of topical analgesics for the treatment of neuropathic pain.9 the documented therapeutic effects of capsaicin and lidocaine in neuropathic pain further supported the case for exploring the targeting of peripheral pathomechanisms in neuropathic pain.10,11 the complex interactions between nociceptors, immunocompetent cells, and epithelial cells, creating a pathogenetic network in neuropathic pain, support the use and evaluation of combining different apis in topical formulations to target these three components.12,13 as there are again a multitude of pharmacological targets related to these components, topical formulations containing a rational selection of a number of apis are in need of in - depth evaluation. here, we need to also clarify regulatory hurdles, as the competent the authorities european medicines agency (ema) and the us food and drug administration (fda), in general, recommend proving efficacy and safety for one api at a time. this suggests that when developing a topical formulation with two or more apis, the combination should be better compared to the single topical apis. such a philosophy is not optimal because even combinations between one established active drug together with another drug that on its own accord would not lead to efficacy can result in a superior therapeutic principle owing to synergistic mechanisms. single compound approaches might have worked in the past for a number of disorders, although for the treatment of complex disorders based on interactive networks of factors, we definitely need combinations of drugs to enhance the chances of finding a good therapeutic response. many studies examining oral medication for neuropathic pain support combination of analgesics of different classes.1417 nevertheless, some case studies and clinical trials have already supported the efficacy and safety of creams or gels containing one api only ; we will discuss, for the sake of simplicity, in this article only topical formulations containing phenytoin, ketamine, amitriptyline, and/or baclofen alone or as combinations. these drugs act on many different targets (eg, nmda receptors, ion channels, and gabab receptors) located on dermal cells such as nerve endings, keratinocytes, and immunocompetent cells, and this may enhance the chances of synergism.8,18,19 however, since some conflicting results have been reported in this field, we will raise four critical issues that are not often considered in clinical trials on the efficacy and safety of topical analgesics published so far. patents are interesting and important sources to study issues related to drug development, as formulations selected are specified and, mostly, a rationale is given for the key aspects of the invention. the first patent in the field of topical formulations of tcas and of ketamine, priority date of september 22, 1995, is the preparation of topical regional compositions for the relief of pain a number of vehicles were claimed, such as lecithin composition, aloe vera gel, cocoa butter, aquafor, petroleum jelly, and/or a standard cold cream. the selection was based on its ability to enhance transport of the api across the dermis and into the tissues below to exert the effects of these agents on the nerves in the region below the site of application. a clinical trial was performed in 34 patients using three different apis, alone or in combination, in preparations of lecithin matrix gels. the contents of these compositions were placebo (with no apis), a composition containing ketamine 0.5%, a composition containing amitriptyline 0.05%, a composition containing guanethidine 0.05%, a composition containing a combination of ketamine 0.5% and amitriptyline 0.05%, and a composition containing a combination of ketamine 0.5% and guanethidine 0.05%. a total of 97% of the patients achieved > 50% reduction in their rest pain 23 hours following application of lecithin matrix gel containing the combination of 0.5% ketamine plus 0.05% amitriptyline. in the placebo arm, none of the patients reported more than a 20% pain reduction. the ketamine 0.5% composition produced significant pain relief in 82% of patients at 2 hours and 62% at 3 hours following application., we can identify two key issues that we will discuss in more detail in the sections that follow : the absence of dose finding : only one dose was defined for each compound (amitriptyline 0.05% and ketamine 0.5%)no testing for the suitability of various vehicles : lecithin matrix gel was chosen without justification the absence of dose finding : only one dose was defined for each compound (amitriptyline 0.05% and ketamine 0.5%) no testing for the suitability of various vehicles : lecithin matrix gel was chosen without justification the selection of the api is one of the key issues leading to success or failure of any topical drug formulation. analgesics and co - analgesics differ greatly in their mechanism of action and their physicochemical properties, and it is mandatory to find the most optimal fit between such aspects and the pathogenesis of the targeted neuropathic pain. furthermore, the physicochemical properties of any api selected for a topical formulation (eg, whether the molecule selected is hydrophilic, lipophilic, or amphoteric) will strongly influence the choice of a proper vehicle. the concentration of an api can influence the stability of the formulation.20 remarkably, a specific analysis of the pathogenesis of a certain type of neuropathic pain and the mechanisms of action of a selected api is often missing in the literature. the pathogenesis of pain in hereditary motor and sensory neuropathies, and in diabetic neuropathy, and pain due to small fiber neuropathy (sfn) will perhaps have some similarities, but most probably more differences.21 for instance, we have found that sfn pain patients, in which pathology resides mainly in the skin, are good responders to phenytoin cream. elsewhere, we have outlined the pathogenesis of neuropathic pain syndromes related to the skin, pointing out the pathological triad of keratinocytes, immune - competent cells, and nociceptors targeted by topical creams containing sodium channel blockers such as phenytoin.22,23 many more pathophysiological mechanisms can be involved in the development and maintenance of a specific neuropathic pain syndrome. for example, for both capsaicin and lidocaine plasters, the approved indication is postherpetic neuralgia. the two apis have entirely different mechanisms of action and thus influence different parts of the multifactorial pathophysiological mechanism. however, both in the preclinical models for neuropathic pain and in clinical trials, a rational approach to the analysis of optimal combinations is missing. there are a multitude of neuropathic pain states, which most certainly differ in pathogenesis. sfn in sarcoidosis will have many different pathogenetic characteristics compared to chemotherapy - induced peripheral neuropathy (cipn), and diabetic neuropathy will differ from postherpetic neuropathy and chronic inflammatory demyelinating polyradiculoneuropathy. for instance, many inflammatory markers are upregulated in the skin of chronic inflammatory demyelinating polyradiculoneuropathy patients, just as in lyme disease, but not in the skin of diabetic patients.24 the same holds true for complex regional pain syndrome (crps), where keratinocyte and mast cell activation and proliferation as well as inflammatory mediator release can be found in the skin.25 in diabetes patients, the epidermal neuropilin-1 receptor expression is high in the epidermal layer of diabetic subjects suffering from polyneuropathy, compared to controls. neuropilin-1 receptor trafficking toward the membranes of all epidermal cells in diabetes might play an important role in the development of sfn.26 an understanding of the exact pathogenesis in the skin in a variety of neuropathic pain syndromes is still preliminary, but, clearly, it will differ considerably in various syndromes. topical ketamine and/or clonidine, for instance, might be more suitable and more directly linked to the pathogenesis in crps compared to topical baclofen.2730 second, the selected end points can make or break the study. in the amitriptyline - ketamine study, the primary analysis as defined in the protocol was an analysis of covariance (ancova) to assess changes in pain, numbness, and tingling from baseline to week 6.31 to expect analgesics to influence numbness, without any indications of such an effect from past studies, seems unrealistic. the selection of in the literature, pharmaceutical aspects related to the selected topical formulation with apis, such as ketamine, amitriptyline, and baclofen, are, with only a few exceptions, not described in detail. we will discuss two of those examples to illustrate that even seemingly comparable formulations, based on pluronic organogels, can differ owing to different ways of preparation., the composition of amitriptyline 7.5% gel was applied to a depressed patient who could not tolerate orally administered amitriptyline.32 a transdermal preparation was compounded with a pluronic lecithin organogel (plo) base. the recipe was given in the study : a solution was prepared by dissolving soy lecithin granules in isopropyl palmitate. a 20% water - based gel was prepared by dissolving pluronic f-127 (sigma aldrich, st louis, mo, usa) in distilled water. amitriptyline was then dissolved in the liquid pluronic f-127 gel, and the resulting mixture was added to the lecithin - isopropyl palmitate. apparently, this study might have set the standard for compounding topical amitriptyline formulations on the base of plo. however, the goal of this gel was to create clinically relevant plasma levels, and with success : serum amitriptyline and nortriptyline concentrations were reported as total tricyclic concentrations were within the therapeutic range : 50250 ng / ml. a second recipe of a plo gel can be found in the description of a ketamine 5% gel : soybean lecithin granules were mixed with 150 ml isopropyl palmitate. the mixture was stirred for at least 12 hours until a uniformly dark, amber - colored solution was obtained. ketamine (10 ml ; ketalar [parke - davis (india) ltd., mumbai, india ], 50 mg / ml) was added to reach a final concentration of 5 mg ketamine / ml gel.33 it remains quite unclear ; however, why this gel formulation was selected as a base vehicle for ketamine. lecithin organogel has certain physicochemical properties enabling the dissolution of lipophilic, hydrophilic, and amphoteric molecules, and these gels are regarded as suitable for trans - dermal transport of apis.34 the fact that plo described in the first case noted earlier did indeed lead to high amitriptyline plasma concentrations indicates its use for transdermal absorption for apis. furthermore, no information was given on aspects such as the stability or ph of the selected plo or on the convenience after application on the skin. if we compare the recipes of both plos, we can see that although both approaches lead to a plo, the procedures are quite different, and thus the pharmaceutical and physicochemical properties of the gels might also be different, possibly leading to different clinical effects. in a placebo - controlled study, 17 patients were randomized into either the treatment ketamine 5% cream or placebo cream.35 the selected vehicle was based on aquaphor gel, and subsequently the gel was compounded into a cream. also here, no further details related to ph or stability were given. the effect of the placebo was as robust as the effect of the ketamine 5% cream. in a recent pivotal trial with 462 patients, evaluating the efficacy and safety of a combination of 2% ketamine and 4% amitriptyline cream for reducing cipn symptoms, no details were given on the selected cream base (nor a rationale for the selected dose).31 the study was negative, and without discussing formulation issues, the authors came to the conclusion that topical formulations containing amitriptyline together with ketamine are not recommended for reducing cipn symptoms. this is an example of jumping to conclusions in the absence of arguments related to key drug development issues such as the pharmaceutical properties and the requirements of the selected formulation. a comparable randomized placebo - controlled trial in cipn patients (n=203) for the efficacy and safety of baclofen 0.75%, amitriptyline 3%, and ketamine 1.5% in plo was performed. the gel had 1-year stability data, but no rationale for the choice of this specific gel was given, nor a detailed composition.36 the authors, however, indicated that the selected gel was not optimal : patients had difficulty in applying the gel owing to suboptimal smearability and poor absorption into their skin. the authors recommended using a different liposomal transdermal base in the future, making it easier for participants to rub the formulation into the skin. whether transdermal absorption is indeed preferable compared to purely topical application clearly, in none of the studies a thorough line of arguments was given to support the choice of the vehicle. in most cases, a plo base was selected, although such a gel seems to induce patient compliance issues owing to lack of convenience in applying. the selection of the api is one of the key issues leading to success or failure of any topical drug formulation. analgesics and co - analgesics differ greatly in their mechanism of action and their physicochemical properties, and it is mandatory to find the most optimal fit between such aspects and the pathogenesis of the targeted neuropathic pain. furthermore, the physicochemical properties of any api selected for a topical formulation (eg, whether the molecule selected is hydrophilic, lipophilic, or amphoteric) will strongly influence the choice of a proper vehicle. the concentration of an api can influence the stability of the formulation.20 remarkably, a specific analysis of the pathogenesis of a certain type of neuropathic pain and the mechanisms of action of a selected api is often missing in the literature. the pathogenesis of pain in hereditary motor and sensory neuropathies, and in diabetic neuropathy, and pain due to small fiber neuropathy (sfn) will perhaps have some similarities, but most probably more differences.21 for instance, we have found that sfn pain patients, in which pathology resides mainly in the skin, are good responders to phenytoin cream. elsewhere, we have outlined the pathogenesis of neuropathic pain syndromes related to the skin, pointing out the pathological triad of keratinocytes, immune - competent cells, and nociceptors targeted by topical creams containing sodium channel blockers such as phenytoin.22,23 many more pathophysiological mechanisms can be involved in the development and maintenance of a specific neuropathic pain syndrome. for example, for both capsaicin and lidocaine plasters, the approved indication is postherpetic neuralgia. the two apis have entirely different mechanisms of action and thus influence different parts of the multifactorial pathophysiological mechanism. however, both in the preclinical models for neuropathic pain and in clinical trials, a rational approach to the analysis of optimal combinations is missing. there are a multitude of neuropathic pain states, which most certainly differ in pathogenesis. sfn in sarcoidosis will have many different pathogenetic characteristics compared to chemotherapy - induced peripheral neuropathy (cipn), and diabetic neuropathy will differ from postherpetic neuropathy and chronic inflammatory demyelinating polyradiculoneuropathy. for instance, many inflammatory markers are upregulated in the skin of chronic inflammatory demyelinating polyradiculoneuropathy patients, just as in lyme disease, but not in the skin of diabetic patients.24 the same holds true for complex regional pain syndrome (crps), where keratinocyte and mast cell activation and proliferation as well as inflammatory mediator release can be found in the skin.25 in diabetes patients, the epidermal neuropilin-1 receptor expression is high in the epidermal layer of diabetic subjects suffering from polyneuropathy, compared to controls. neuropilin-1 receptor trafficking toward the membranes of all epidermal cells in diabetes might play an important role in the development of sfn.26 an understanding of the exact pathogenesis in the skin in a variety of neuropathic pain syndromes is still preliminary, but, clearly, it will differ considerably in various syndromes. topical ketamine and/or clonidine, for instance, might be more suitable and more directly linked to the pathogenesis in crps compared to topical baclofen.2730 second, the selected end points can make or break the study. in the amitriptyline - ketamine study, the primary analysis as defined in the protocol was an analysis of covariance (ancova) to assess changes in pain, numbness, and tingling from baseline to week 6.31 to expect analgesics to influence numbness, without any indications of such an effect from past studies, seems unrealistic. in the literature, pharmaceutical aspects related to the selected topical formulation with apis, such as ketamine, amitriptyline, and baclofen, are, with only a few exceptions, not described in detail. we will discuss two of those examples to illustrate that even seemingly comparable formulations, based on pluronic organogels, can differ owing to different ways of preparation., the composition of amitriptyline 7.5% gel was applied to a depressed patient who could not tolerate orally administered amitriptyline.32 a transdermal preparation was compounded with a pluronic lecithin organogel (plo) base. the recipe was given in the study : a solution was prepared by dissolving soy lecithin granules in isopropyl palmitate. a 20% water - based gel was prepared by dissolving pluronic f-127 (sigma aldrich, st louis, mo, usa) in distilled water. amitriptyline was then dissolved in the liquid pluronic f-127 gel, and the resulting mixture was added to the lecithin - isopropyl palmitate. apparently, this study might have set the standard for compounding topical amitriptyline formulations on the base of plo. however, the goal of this gel was to create clinically relevant plasma levels, and with success : serum amitriptyline and nortriptyline concentrations were reported as total tricyclic concentrations were within the therapeutic range : 50250 ng / ml. a second recipe of a plo gel can be found in the description of a ketamine 5% gel : soybean lecithin granules were mixed with 150 ml isopropyl palmitate. the mixture was stirred for at least 12 hours until a uniformly dark, amber - colored solution was obtained. ketamine (10 ml ; ketalar [parke - davis (india) ltd., mumbai, india ], 50 mg / ml) was added to reach a final concentration of 5 mg ketamine / ml gel.33 it remains quite unclear ; however, why this gel formulation was selected as a base vehicle for ketamine. lecithin organogel has certain physicochemical properties enabling the dissolution of lipophilic, hydrophilic, and amphoteric molecules, and these gels are regarded as suitable for trans - dermal transport of apis.34 the fact that plo described in the first case noted earlier did indeed lead to high amitriptyline plasma concentrations indicates its use for transdermal absorption for apis. furthermore, no information was given on aspects such as the stability or ph of the selected plo or on the convenience after application on the skin. if we compare the recipes of both plos, we can see that although both approaches lead to a plo, the procedures are quite different, and thus the pharmaceutical and physicochemical properties of the gels might also be different, possibly leading to different clinical effects. in a placebo - controlled study, 17 patients were randomized into either the treatment ketamine 5% cream or placebo cream.35 the selected vehicle was based on aquaphor gel, and subsequently the gel was compounded into a cream. also here, no further details related to ph or stability were given. the effect of the placebo was as robust as the effect of the ketamine 5% cream. in a recent pivotal trial with 462 patients, evaluating the efficacy and safety of a combination of 2% ketamine and 4% amitriptyline cream for reducing cipn symptoms, no details were given on the selected cream base (nor a rationale for the selected dose).31 the study was negative, and without discussing formulation issues, the authors came to the conclusion that topical formulations containing amitriptyline together with ketamine are not recommended for reducing cipn symptoms. this is an example of jumping to conclusions in the absence of arguments related to key drug development issues such as the pharmaceutical properties and the requirements of the selected formulation. a comparable randomized placebo - controlled trial in cipn patients (n=203) for the efficacy and safety of baclofen 0.75%, amitriptyline 3%, and ketamine 1.5% in plo was performed. the gel had 1-year stability data, but no rationale for the choice of this specific gel was given, nor a detailed composition.36 the authors, however, indicated that the selected gel was not optimal : patients had difficulty in applying the gel owing to suboptimal smearability and poor absorption into their skin. the authors recommended using a different liposomal transdermal base in the future, making it easier for participants to rub the formulation into the skin. whether transdermal absorption is indeed preferable compared to purely topical application was not further discussed. clearly, in none of the studies a thorough line of arguments was given to support the choice of the vehicle. in most cases, a plo base was selected, although such a gel seems to induce patient compliance issues owing to lack of convenience in applying. it seems a simple question, but it is not, and one can not throw a dice. in drug development, there is a generalized tendency to shortcut development lines and avoid full - powered dose - finding phase ii trials. such a flawed strategy, sadly enough, seems to be quite popular in the development of topical formulations. such shortcuts, however, are always counterproductive, because the results of small trials are inconclusive, or because of false - positive findings one enters phase iii without sufficient proof of principle. in one of the first studies on topical formulations against pruritus, an alcoholic solution containing 5% amitriptyline or 5% doxepin was tested.1 no reasons were given for the choice of concentration. interestingly, doxepin 5% cream was registered by xepin - bioglan, malm, sweden, and is included in the british national formulary for the relief of itching associated with eczematous dermatitis interesting, because we can not find any published data to support the choice of concentration. the analysis of the aforementioned development of the ketamine / amitriptyline and the baclofen / amitriptyline / ketamine formulations demonstrates a number of development flaws and shortcuts. the selection of a gel containing a maximum of 2% ketamine and 4% amitriptyline, with or without 0.75% baclofen, has not been backed up by sufficient dose - finding data. or, to put it related to the gel containing baclofen 0.75%, amitriptyline 3%, and ketamine 1.5%, the fda apparently prohibited higher doses than initially proposed by the investigators owing to the lack of data on systemic absorption of this triple combination. instead of starting a small study to evaluate the systemic absorption of the three components of the api combination gel, the authors followed the fda specification and tested much lower concentrations of the apis than initially proposed, although they had to know that most probably there was insufficient clinical data available to back up that specific low dose selection. logically, by selecting a low dose for all active components, the study runs a great risk of ending as a negative study owing to underdosing. this was quite an expensive experiment leading to a new working hypothesis proposed by the authors : next time, select a higher concentration of apis. a higher concentration might indeed have led to a better clinical effect, as documented earlier in a study on the effects of topical ketamine 10% in allodynia in crps patients in a double - blind crossover placebo - controlled trial in 20 patients.29 ketamine reduced the allodynia significantly. plasma levels of ketamine and its active metabolite norketamine were below the limits of detection after application. apart from the issues discussed above, improvements in selecting the target indication, the end points, and other aspects of clinical trial design will contribute to the development of topical analgesics. owing to the fast onset of action of topical analgesics, responders can be identified quickly with a test application.37 meanwhile, we gathered experience using this responder identification method in our clinic. we test patients in a single - blind fashion, for instance patients suffering from sfn pain, by applying a finger - tip unit of placebo cream or the same amount of phenytoin 10% cream on burning and painful areas. patients mostly indicate within 10 minutes a 50% reduction of baseline pain on phenytoin cream, while placebo cream does not lead to relevant pain reduction. with this responder identification method, the most optimal api, the optimal concentration for a specific api, and various other formulations, can subsequently be tested in an enrichment design phase ii study.38 also, combinations of apis formulated in different bases can be tested by using a simple test design (blinded placebo or comparator - controlled crossover testing). this procedure can also be followed for a phase iii study, an enrichment design strategy.39 before entering the study, patients will receive a test application with the analgesic cream. when the patient responds in a clinically meaningful way and reports relevant pain reduction, ie, a decrease of two points on the nrs or 33% pain reduction,40 responders will be randomly assigned to the active arm or placebo arm, after baseline assessment. after applying the cream, patients will be assessed for a certain period again. to enhance the sensitivity of the clinical trial, a crossover design could be selected. after 1 week washout, patients receive the other cream. in this design also, an intrapatient evaluation could be made. this type of enrichment randomized double - blind crossover trial is now being executed in the netherlands for ketamine 10% cream and amitriptyline 10% cream. in evaluating the efficacy and safety of topical analgesics, many issues related to drug development are neglected. we pointed out the relevance of selecting the correct api, based on the mechanisms of action of the api related to what we know about the pathogenesis of the neuropathic pain state. we also highlighted how details related to the pharmaceutical form are missing, dose - finding studies are missing, and the selection of end points and other aspects of clinical trial design are suboptimal. in order to test the efficacy and safety of an api such as phenytoin, amitriptyline, ketamine, or baclofen, one needs to compare the suitability of the selected vehicle in well - designed pilot trials. furthermore, one can not skip well - designed phase ii studies in order to specifically define the active concentration range of the selected topical and the lowest effect dose. most pilot studies are only suited for feasibility testing, whether one can find patients for inclusion, and whether the selected vehicle is acceptable and convenient for patients to smear. we presented a simple phase ii test design in a limited group of patients (blinded placebo or comparator - controlled crossover n=1 testing) to quickly test the pain relief of various formulations and concentrations and compare and select the best. in addition to this explorative phase, we presented an enrichment design for a phase iii study to decrease placebo responses and increase the chances of finding and including responders. within this context, we developed a quick single - blinded responder identification method as a key element for such an enrichment study. topical analgesics are promising inroads for the treatment of neuropathic pain, once we learn to avoid development mistakes and shortcuts from the past. | topical analgesics can be defined as topical formulations containing analgesics or co - analgesics. since 2000, interest in such formulations has been on the rise. there are, however, four critical issues in the research and development phases of topical analgesics : 1) the selection of the active pharmaceutical ingredient. analgesics and co - analgesics differ greatly in their mechanism of action, and it is required to find the most optimal fit between such mechanisms of action and the pathogenesis of the targeted (neuropathic) pain. 2) issues concerning the optimized formulation. for relevant clinical efficacy, specific characteristics for the selected vehicle (eg, cream base or gel base) are required, depending on the physicochemical characteristics of the active pharmaceutical ingredient(s) to be delivered. 3) well - designed phase ii dose - finding studies are required, and, unfortunately, such trials are missing. in fact, we will demonstrate that underdosing is one of the major hurdles to detect meaningful and statistically relevant clinical effects of topical analgesics. 4) selection of clinical end points and innovatively designed phase iii trials. end point selection can make or break a trial. for instance, to include numbness together with tingling as a composite end point for neuropathic pain seems stretching the therapeutic impact of an analgesic too far. given the fast onset of action of topical analgesics (usually within 30 minutes), enrichment designs might enhance the chances for success, as the placebo response might decrease. topical analgesics may become promising inroads for the treatment of neuropathic pain, once sufficient attention is given to these four key aspects. |
the physics involved ranges from subnanosecond submillimeter electron avalanches governed by atomic and molecular ionization and attachment cross sections to centimeterscale propagating space charge waves (streamers) to microsecondscale meterlong channels of ionized gas described by nonequilibrium plasma and electrodynamic behavior. on a larger scale, spark behavior in nature ranges from 100 km lightning channels that last seconds and span conditions from the upper reaches of a thundercloud to the microsecondscale processes by which such a channel attaches to the ground. in such a diverse range of behavior one can find many puzzling phenomena. the process by which a discharge initiates in the cloud is not understood nor is the process by which lightning channels (leaders) extend and branch. the extension process sometimes proceeds as a series of microsecondscale steps separated by 10 s of microseconds of relative quiescence. more puzzling, the steps seem to be immediately preceded by formation of a hot conductive channel displaced from the end of the existing channel that rapidly connects with the main channel, though the mechanism by which this space leader the transition from streamer to leader is the focus of much research as are the dynamics of the streamer itself and the role of energetic particles in the process. this role of energetic particles is especially interesting in the light of observations of xray production by lightning and the hypothesis that energetic particles produced by lightning contribute to the production of terrestrial gamma ray flashes. xrays as produced by lightning seem to occur coincident with stepwise extensions of the channel [howard., 2010 ; dwyer., 2011 ] and have energies around a few 100 kev [moore., 2001 ; dwyer., 2003 ; these xrays imply the existence of a much larger population of higherenergy electrons. while lowenergy electrons encounter high dynamic friction, relativistic electrons encounter much lower friction and can run away to very high energies when driven by electric fields, a fact first recognized by wilson and modeled in the context of electric fields near lightning channels in carlson [2009, chapters 5 and 6 ]. seed energetic electrons necessary to initiate such a process can come from energetic background radiation (i.e., cosmic rays [carlson., 2008 ]), lowenergy electrons in local electric fields strong enough to overcome the maximum friction force [e.g., moss., 2006 ; li., 2009 ; chanrion and neubert, 2010 ], or by feedback from prior generations of energetic electrons [dwyer, 2003, 2007 ]. regardless, electric fields may drive avalanche growth of populations of such runaway electrons[gurevich. unfortunately, the relative importance of these processes for phenomena like xray production by lightning or terrestrial gamma ray flashes is not well understood. in the context of these puzzles, any additional information about the processes involved, laboratory studies can shed light on the detailed dynamics of leader extension, and while the energy scales are necessarily smaller (of order 1 mv compared to the 10 s of megavolts of natural lightning), lab sparks even produce xrays. dwyer [2005a ] report the first detection of xrays associated with such sparks in a study of 14 discharges of 1.5 mv, with total energy deposited in a detector up to mev scale. variation of signals among xray detectors with different attenuators suggest xray energies from 30 kev to 150 kev piling up to produce the mevscale observations. report similar results with total energy deposited up to several mev with 1 mv discharge. (elaborated in nguyen) also report similar results for 0.881 mv sparks with a much larger data set and with a variety of detectors, attenuators, and positions of detector. nguyen. show intensity variations with highvoltage (hv) polarity and with distance between detector and spark gap, suggesting that such intensity variations could result from xray production by positive streamers from metal structures near the detectors. report a further study of 241 discharges of 1 mv with various polarities and gap distances and push the maximum energy deposited in a single detector up to 50 mev with varied attenuators providing evidence for individual photon energies exceeding 300 kev and statistical evidence for an average photon energy at most 230 kev. dwyer. also report collimator experiments supporting the production of xrays by a diffuse source in the gap between the electrodes and that bursts of xrays produced late in the discharge come from elsewhere. march and montany report that hv pulses with rapid risetime tend to produce more xrays, and march and montany examines the effect of electrode geometry. kochkin., kochkin., and kochkin. add nanosecondresolution photography as a useful tool, mapping the streamer clouds produced by positive [kochkin., 2012 ] and negative [kochkin., 2015 ] hv discharges. in such experiments the hv risetime is much longer than the time for streamers to propagate across the gap, and in experiments with negative high voltage the negative streamers tend to appear and grow in bursts [see, e.g., kochkin., 2014, figure 6 ]. demonstrate for negative hv that the xrays tend to be emitted during these bursts of streamer development but are emitted on a much shorter timescale than the burst, supporting the suggestion that the large and transient electric fields that result from interaction of negative and positive streamer fronts may play a role in energetic electron and thus xray production. [2012, 2015 ] also describe that attenuator experiments they claim are consistent with 200 kev characteristic xray energy. these estimates are based simply on comparison of registration rate (the fraction of sparks for which xrays are observed) for various attenuators with the expected attenuation in number of photons incident on the detector, assuming all sparks emit the same number of photons and that pileup does not affect their observations, though they acknowledge that pileup has a significant effect. together, these studies provide a reasonably complete picture of xray emissions from sparks : free electrons are pushed to overcome friction in the highfield region ahead of a negative streamer (possibly briefly enhanced by a nearby positive streamer), gain at least enough energy to run away in the lower fields surrounding the streamer, then undergo bremsstrahlung in the surrounding air to emit xrays. these intense streamer fields exist only as the discharge develops and are unlikely to exist during the discharge itself, though the intense electrodynamic environment during discharge may drive discharges elsewhere in the lab that also produce detectable xrays. average energy, the properties of the photon spectrum are unknown and difficult to judge due to pileup, and the fact that some sparks produce copious xrays while others produce no measurable xrays is not addressed. the present paper attempts to shed light on these topics by examining the energies measured in xray detectors placed near a 1 m spark gap at the technical university of eindhoven during 921 shots as described in section 2. these shots give us the opportunity to statistically analyze the distributions of spark xray fluence, number of photons detected by multiple detectors, and photon energy. these distributions are described in section 3 and constrained by comparison to observations in section 4. the experiment was conducted in the highvoltage lab at the technical university of eindhoven with a haefly 2 mv marx generator configured to produce 1 mv pulses with 1 s risetime. all tests were carried out with a 1 m pointpoint gap with negative hv polarity. the spark voltage, ground electrode current, and highvoltage electrode current were recorded by lecroy fourchannel storage oscilloscopes configured to record 2 s of data at 10 ghz sample frequency when triggered by the marx generator, as were signals direct from the output of photomultiplier tubes monitoring a variety of energetic radiation detectors. the radiation detectors used in the experiment included scintillating plastic optical fibers placed at a variety of locations around the spark to monitor energetic electrons. analysis of these data has been presented [ostgaard., 2014 ] and will be published separately. due to the need to run many shots with the scintillating fiber detectors at various locations, a total of 950 shots were carried out from which we recorded usable data for 921. during all shots, two additional xray detectors were running, providing a wealth of data about the photon population. these two detectors are each composed of a 1.5 inch long 1.5 inch diameter labr3(ce) scintillator monitored by a photomultiplier tube (pmt). these detectors are placed next to each other (separation 10 cm) in an electromagnetic compatibility (emc) cabinet to shield the detectors from the electromagnetic noise produced by the spark. the location of the detectors relative to the spark corresponds to location h as shown in figure 1 of kochkin.. the scintillator material is separated from the spark by roughly 0.5 mm of aluminum in the scintillator housing and emc cabinet wall and approximately 2 m of air. the signals as recorded by the oscilloscopes are in volts and here have been converted to mev by use of cs137 and co60 gamma ray emitters as sources of known energy. the lowest energy detectable by this setup is 20 kev, comparable to the minimum photon energies transmitted through the 0.5 mm aluminum shielding, while the maximum depends on the settings of the oscilloscope. signal from detector 1 clips at just above 5 mev, while detector 2 clips at just above 3.5 mev. the highvoltage trace shown is very consistent from one spark to the next, as is the highvoltage electrode current. the pulselike features on the highvoltage electrode current occur when bursts of corona and streamer activity carry charge away from the electrode. see kochkin. for a detailed discussion of such processes as seen in highspeed camera imagery. (top left) the voltage of the highvoltage electrode with respect to the ground electrode. the pulses seen in the oscilloscope traces indicate deposition of energy in the scintillator, but a single pulse from the pmts may be produced by multiple xray photons entering the scintillator. these photons may arrive at slightly different times and produce a visibly altered pulse shape, but typically no time structure is visible. the photon spectrum is therefore not directly measurable, and pulse pileup must be treated statistically. to compile data for such statistics, we search through the data for pulses. to search, we first smooth the data by lowpass filtering once pulses are identified, we collect a variety of data including pulse time, height, integral, and duration. pulse integral is a more robust measure of deposited energy in case multiple pulses arrive near simultaneously, so our analysis here reports energy as determined by pulse integral. while pulse integral is somewhat resistant to saturation when oscilloscope signals clip, the energy of a saturated pulse can not reliably be determined, so we enforce a maximum energy for each detector corresponding to the energy of the largest unclipped pulse. in cases when multiple pulses are observed in a single detector, we add their energies together to better capture variation from one spark to the next, though note that the majority of sparks have zero or only one burst so this combination of bursts only affects a small fraction of our data. the data set we consider here is therefore a set of pairs of numbers, each pair associated with a single spark and each number with the pair giving the total energy deposited in a detector during the given spark. a scatterplot showing the energies deposited in each detector in each shot is shown in figure 2. most sparks (57%) produce no detectable signals in either detector, indicating less than 20 kev deposited, while approximately 3% of sparks saturate both detectors, indicating at least 35 mev of energy deposited. each point represents a spark, where e 1 was deposited in detector 1, while e 2 was deposited in detector 2. the dotted line shows e 1 = e 2. at e 1 = e 2 = 0, 523 points overlap, and 20 points the points clearly cluster in the lower left, but a significant number of points appear in the middle and upper regions of the plot. attempting to explain this distribution most simply, one might assume all sparks are identical and that all photon energies are equal and treat the observed distribution as solely due to poisson statistical fluctuations in the number of photons observed. in this case, the high number of points in the lower left (57% of events undetected) implies a poisson mean of 0.562. with this mean, observation of events that saturated both detectors is then incredibly unlikely as deposition of more than 8 mev (3 mev in one and 5 mev in the other) requires at least 40 photons for the mean energy 200 kev consistent with the results quoted above. observation of at least 40 photons from a poisson distribution with mean 0.562 has a probability around 10, much less than the 3% of observed sparks that saturate both detectors. adding the complication of a photon energy distribution is logical, but does not help enough, since the maximum photon energy must be less than 1 mev as the photons will be much less energetic than the 1 mv maximum spark voltage. assuming the same poisson mean as before and attempting to explain the events that saturated both detectors as due to an extreme fluctuation in photon energy such that all photons carried 1 mev of energy means that we must have at least eight photons instead of at least 40, but the poisson probability of observing at least eight photons given a mean of 0.562 is roughly 10, still much smaller than the 3% observed. clearly, there must be a significant variability in spark xray fluence at the location of the detectors from one spark to the next. this variability could come from intrinsic variability in spark xray luminosity or it could come from some variability in the geometry if xray emissions are not isotropic. regardless, a full explanation of the distribution of points in figure 2 must include the effect of the distribution of spark xray fluence, the distribution of numbers of photons detected by each detector (given the xray fluence), and the distribution of photon energies. these distributions and their properties are the focus of this paper. in attempting to build a statistical model of the distribution of points in figure 2, we need to know the form of the distributions of physical properties relevant to the point locations. there are three main distributions at work : the distribution of spark xray fluence, the distribution of numbers of photons incident on each detector (given spark fluence), and the distribution of photon energies. the distribution of spark fluence is determined in principle by the processes at work in electron acceleration. one spark has an intrinsically higher luminosity than another by having more energetic electrons, perhaps because random streamer branching events happened to lead to more negative streamers or perhaps fewer but more intense or more rapidly growing negative streamers or perhaps more interactions between negative streamers and positive streamers. fluence variability may also result from anisotropy in the directionality of xray emissions : perhaps all sparks emit many xrays, but the emissions are not always beamed toward the detectors. these processes may be chaotic or perhaps intrinsically random, but either way there will be some variability in xray fluence. to represent this variability, we treat the xray fluence as the expected value of the number of photons hitting a detector,, and assume some probability distribution for the occurrence of various values of. as there is yet no theoretical expectation for the form of this distribution, we must make some assumption. examining the clustering of points in the lower left of figure 2, we expect lower fluences to be more common than higher fluences, but the existence of points in the upper right suggests that the tail of the distribution to high fluences is quite strong. to represent distributions that follow this general trend of decreasing with a potentially long tail, we assume the distribution for is a power law with index : (1)p()=(+1)max+1min+1 where min and max are lower and upper limits on necessary to ensure that the distribution is normalizable for 1 and the leading constant ensures normalization. by examining the clustering in the lower left in figure 2, we expect 0, and n 2=0, n 1>0 terms in the probability distribution contain a single delta function. only the terms for which both n 1 and n 2 are greater than zero lack delta function divergence behavior. these delta functions make the probability distribution difficult to work with directly but are capable of representing the clustering of points at the origin and along the axes in figure 2. these probability distributions form the basis of our model, and we seek to infer the parameters,, min, and max by comparison of our model to data. comparison of a probability distribution to data can be done in many ways, for example, the kolmogorovsmirnov test or the andersondarling test. such tests work on the basis of the cumulative distribution function (cdf) f, which for a probability distribution p(x) in one dimension is defined as f(x)=xp()d. since here we work in two dimensions (e 1,e 2), we need a twodimensional analog of the cumulative distribution function. here we follow fasano and franceschini in taking our set of data points { p i } = { (e 1i, e 2i) } and for each point, assigning four cdflike values : fdati < <, fdati<, fdati <, and fdati, each giving the fraction of the observed data set in the corresponding quadrant relative to the data point in question. for example, fdati < < gives the fraction of data { p j } for which e 1j < e 1i and e 2j < e 2i. these { fdati } can be plotted if an order is assigned to the data points for use as an abscissa. here it is convenient to order each set of fdati separately such that fdati is monotonically increasing, i.e., sort each set of fdati from smallest to largest and use its place in the resulting list as its abscissa. for example, the grey, curve starts at 0.03 since 3% of sparks saturate both detectors : the data point that has the fewest data points with energies greater than or equal to its energy must be one of those doubly saturated events, and thus, 3% of the data satisfies the, condition. moving to the right along the, curve, larger cdf values correspond to data points that are below and/or to the left of the upper right corner, i.e., data points for which larger fractions of the data satisfy the, condition. the large jump from about 0.35 to 1.0 at cdf rank 400 occurs when incrementally increasing the fraction of the data set that satisfies the, condition requires looking at a point where e 1 = e 2 = 0, which thus includes all other such points (57% of all data) as well as points for which e 1 = 0 or e 2 = 0. the cdf therefore jumps up to 1 since all of the data has e 10 and e 20. the direction of the inequality is related to the shape of the distribution, with and < < related to the lower left / upper right balance and width while the < and < versions related to upper left / lower right balance and width. one can imagine other ways of constructing such cdfs, but this is a straightforward technique that as we will see shortly is quite effective. each horizontal point represents a point in the (2d) data set, for which the vertical coordinate represents the fraction of the data set with energies related to the energies of the given data point by the inequalities associated with the given curve (i.e., the fdati described in the text). each curve represents a different inequality, and the data have been reordered for each curve to make the curve in question monotonically increasing. two sets of curves are shown, thick curves from the data and thin curves predicted given the best fit parameters discussed in the text, but the curves overlap so much any deviations are difficult to judge. such curves as in figure 3 can be predicted on the basis of the probability density (equation (6)), assigning a predicted fpredi to each data point by integrating the expected distributions over the region relevant to the inequalities in question. for example, (7)fpredi<<=e1ide1e2ide2p,(e1,e2) similar calculations can be made for predicted fpredi <, fpredi<, fpredi by changing the limits on the integrals to go from the point in question to + as appropriate. regardless, the exponentials and powers in the integrand can be manipulated to convert the integrals here and in equation (6) into upper and lower incomplete gamma function evaluations (e.g., (k, x)=xxk1exdx), and the summations in equation (6) can be computed numerically and truncated without significant loss of accuracy. for a given,, min, and max, the result of this exercise is four sets of numbers ({ fpredi < < }, { fpredi < }, { fpredi< }, and { fpredi }) that can be compared to the corresponding sets associated with the data ({ fdati < < }, { fdati < }, { fdati< }, and { fdati }). treating these numbers as analogous to the cumulative distribution function, the kolmogorovsmirnov test statistic would be the maximum deviation between any two corresponding f i. the kolmogorovsmirnov test is sometimes criticized as not very powerful [e.g., razali and wah, 2011 ], so instead, we calculate an analog of the andersondarling test statistic : (8)s=i(fdati<<fpredi<<)2fpredi<<(1fpredi<<)+(fdati<fpredi<)2fpredi<(1fpredi<)+(fdati<fpredi<)2fpredi<(1fpredi<)+(fdatifpredi)2fpredi(1fpredi) i.e., the squared deviation between the predicted cdf and the observed cdf, weighted as in the andersondarling statistic, summed over all four cdf types (inequalities), and summed over all points in the observed data set. some numerical problems arise when both numerator and denominator are exactly zero, i.e., one of the fpredi is exactly 0 or 1, as occurs, for example, for fpredi < < for a point, where e 1i = e 2i = 0. since such points involve exact match between predicted and observed cdf and therefore should not contribute to the test statistic, we add a small factor (0.001) to the denominator of each term to ensure the division of zero by zero results in zero. this does not significantly affect the other terms in the test statistic or the overall results. we then fit our predicted distribution to the observed distribution minimizing s by varying the parameters of our calculated cdf with the downhill simplex algorithm (nelder and mead as implemented in johnson). the optimal values are = 1.29, = 86 kev, min = 0.022, and max = 113. since our multidimensional cdf and modified andersondarling test statistic are so far removed from their original application, we only use the statistic in judging the quality of the fit in the optimization process described above and make no attempt to apply the distributions typically associated with the andersondarling test statistic to assess the uncertainty in our fit results. instead, we judge uncertainty by bootstrap, repeating our fit process many times with alternative data sets produced from our original data by sampling with replacement. the fit results are each approximately normally distributed, with mean and standard deviation given as follows : = 1.29 0.04, = 86 7 kev, min = 0.022 0.006, and max = 110 25. plotting these bootstrapped fit results, one parameter versus another, shows very little correlation between results, with the exception of and min which are negatively correlated : more negative is associated with higher min. this makes sense given that more negative is associated with more events with low xray fluence, so raising the minimum fluence is necessary to retain the balance between events with and without detectable signal. the cdfs computed from the best fit parameters are also shown in figure 3 as thin curves, but they overlap with the data so much that no systematic deviations are evident. a residuals plot showing the differences between the two sets of curves is shown in figure 4. some deviations can be seen but are well within the typical size of the fluctuations due to random distribution of points in the data set. a positive deviation indicates a larger data cdf than computed from the best fit parameters. monte carlo simulations of data sets drawn from the distributions described above show no obvious deviations from the distribution of the data when plotted as in figure 2. it is also worth noting that since our fitting procedure captures the joint distribution, it also captures the distribution within each detector separately. in this paper, we have not attempted to compensate for saturation to construct a true energy distribution, but kochkin., using essentially the same experimental setup as used here, do present such a spectrum [kochkin., 2015, figure 12 ] constructed by a sophisticated procedure of fitting pulse shapes to observed oscilloscope records. though the analysis in kochkin. includes the data set used here, the analysis procedures are completely different, and kochkin. results are presented for energy deposition per burst not per spark, but the fact that relatively few sparks have multiple bursts means that it is still useful to directly compare the results from kochkin. to equation (5). evaluation of equation (5) with our fit result values of,, min, and max, normalized and overlaid with the data from kochkin., is shown in figure 5, demonstrating much better agreement than the single exponential distribution used in kochkin.. inferred a 200 kev characteristic burst energy (which is asserted to be roughly equal to characteristic photon energy) but showed an extremely poor fit at high burst energies that they attribute to pileup. properly accounting for pileup as we do here with the poisson distribution of observed counts (p (n)) and for fluence variability with the assumption of a power law (p ()) gives us a much better fit with a much lower mean photon energy (86 kev), suggesting that pileup and fluence variation is essential to consider in interpretation of such data. comparison of the predicted total energy deposition spectrum from this paper with observations from figure 12 of kochkin.. the red squares show the observed spectrum, while the black curves show the predictions based on our distributions. the thick curve is the prediction, while the thin curves represent the n standard deviation expected for the spread in the data. as a final comparison to data, while we ran no attenuator experiments in the data set described above, kochkin. they report results for lead attenuators of varying thickness, determining the fraction of sparks that produce observable signals in a single detector by running 50 sparks with each attenuator. the distributions determined above can easily be used to construct results for attenuated scenarios for comparison with the observations in kochkin. by monte carlo simulation. first, we draw a random spark fluence, then draw a random number of photons from such a spark, then draw that many random photon energies from the photon energy spectrum, then attenuate those photons probabilistically by use of the mass attenuation coefficients from hubbell and seltzer, and then finally classify the event as containing detected xrays or not based on whether or not any photons made it through the lead shield. we repeat this procedure 10 times, compute the overall fraction of events with detected xrays, and repeat for each attenuator thickness. comparison with data requires an estimate of the uncertainty in the data, here estimated by calculating a 68% confidence interval based on the binomial distribution. sixtyeight percent confidence corresponds to a 1 error bar, and all but one of our predictions are consistent with the observations at this confidence level. the distributions determined in this paper are thus also consistent with attenuator experiments, though further attenuator observations would be useful to narrow the uncertainties in the observations. predictions based on the distributions determined in this papera the 68% confidence intervals correspond to 1 error bars and are calculated based on binomial statistics. to summarize, we have studied observations of xray emission by sparks and attempted to understand those observations by modeling the process as a combination of a distribution of spark fluence, poisson statistics, and an xray energy spectrum. these distributions successfully reproduce not only the data used here but also independent analysis of similar experiments on overall energy distribution and signal attenuation in kochkin.. the two main results are a welldefined photon mean energy and the distribution of overall spark fluence. the 86 7 kev mean photon energy determined above is consistent with most earlier estimates but somewhat lower than some. we feel this mean energy is quite reasonable given that electrons accelerated in the environment of a 1 mv spark realistically must have at most a few hundred kev of energy and bremsstrahlung photons produced must have a lower energy than that of the source electrons. as a crude estimate of electron energy, assume bremsstrahlung produced by the relatively low energy electrons here follows approximately the dn / de 1/e distribution seen for bremsstrahlung produced by highenergy electrons. this 1/e distribution spans the range from the energy of the electron as a maximum to a minimum energy at most 20 kev. using 20 kev as the minimum energy and requiring that the average photon energy is 86 kev require the electron energy to be roughly 200 kev, while using 5 kev as the minimum energy gives nearly 400 kev as the electron energy. this 200400 kev energy range is not inconsistent with the potential available for electron acceleration at the time of xray production as seen in figure 1, but this is a crude estimate in need of refinement by modeling of electron energy distributions and the resulting bremsstrahlung before it can be taken very seriously. the exponential xray spectrum motivated by the expectation of an at least approximately exponential distribution in electron energies very closely reproduces the observed distributions, though is likely not the only such distribution to do so, and as noted above we have assumed that fluence () and mean energy are independent which may or may not be true. while the average energy should be close to correct, the other distributions at work in this system complicate the analysis. the more important factor at work is the very broad distribution of observed spark xray fluence (), which, as mentioned earlier, we assume captures both intrinsic variability in spark luminosity and variability in the spatial distribution of xray emissions. the results obtained above, which the distribution of is very hard (power law index 1.29) and covers a broad range, from 0.02 photons to over 100 photons for the experiment conducted here, pose a challenge to the idea that the strong field near the head of a negative streamer is all that is required to accelerate electrons into the energy regime needed for 100 kev xray production. all sparks involve extensive streamer production [see, e.g., kochkin., 2014, figure 2 ], so the number or distribution of streamer production can not directly explain the broad distribution of spark xray fluence. as discussed in kochkin., the very short duration and appearance of multiple bursts of xray emissions suggests that xray emission occurs during some fast transient process like streamer collision, but it is not clear how streamer collision frequency would be distributed and whether such a distribution could reproduce the characteristics observed here. while we have not proved that xray fluence truly follows a power law, only that our assumption of a power law is consistent with our data, it is perhaps not unreasonable that a power law distribution might arise here in the context of dielectric breakdown since power laws arise in studies of systems that similarly approach breakdown and then collapse [bak., regardless of the true form of the distribution, we hope our analysis here is useful for evaluation of xray production mechanisms, which must explain the frequency of both dim and bright sparks in roughly the balance described here. as for the relative contributions of spark luminosity variation and nonuniform xray spatial distribution to the observed fluence variability the observed fluence variability should correspond roughly to the degree of spatial variability in xray emissions. one can set an upper limit on the spatial variability present in xray emissions in an experiment such as this by examining the directional distribution of bremsstrahlung from a unidirectional beam of 500 kev electrons. [1979, figure 5 ] and khn and ebert [2014, figure 7b ]. both figures present plots of the intensity of emissions versus angle relative to the direction of the electron beam, with tseng. showing a factor of around 100 difference from highest to lowest fluence, while khn and ebert show a factor of around 300. that the best fit distribution constructed here requires a factor of max/ min = 100/0.02 = 5000 from highest to lowest fluence suggests that even this extremely aggressive assumption of unidirectional 500 kev electrons falls short of explaining the observed fluence variability, but this analysis is admittedly crude. a slightly less crude assessment can be made by monte carlo : first, draw a random beam direction uniformly from the highvoltage electrode in a hemisphere facing the ground electrode, then determine the angle from the center of that beam to the location of the detector and evaluate the relative fluence by extracting the distributions shown in tseng. and khn and ebert for a relatively high energy photon. though this ignores the xray energy dependence of the bremsstrahlung angular distribution, it should reasonably account for the degree of variability. repeat this procedure many times, compiling a histogram estimating the fluence distribution, then scale these histograms such that they are normalized and adjust the relative fluence to correspond to by shifting the relative fluence until the histograms correctly predict the fraction of events that would fall at intensities too low to be detected. histograms constructed in this manner are compared to the best fit power law in figure 6. predicted fluence distributions assuming only spatial variability due to beaming of bremsstrahlung from unidirectional 500 kev electrons. the curve marked tseng is derived from figure 5 of tseng., the similarity of slopes is striking, especially for the khn and ebert case ; but it is clear that fluence variability due to beaming can not fully account for the observations. in the context of the 86 kev average photon energy determined here, the maximum fluence of the bremsstrahlungderived cases, 10, would result in 800 kev deposited on average, far from the 35 mev needed to saturate the detectors. put another way, if we attempt to account for the large fraction of lowfluence events simply as emissions beamed away from the detectors, the maximum fluence would then be too low to account for events that saturate the detectors. the scale of variability in the distributions derived from bremsstrahlung, even for the extreme case of unidirectional 500 kev electrons, is therefore too low to fully account for the variability observed, providing clear evidence for strong sparktospark variability. crudely comparing the spread of the khn and ebert curve to our power law in figure 6, there is at least another order of magnitude variability in fluence that can not be explained as spatial variability and that is a very conservative estimate since in a more realistic scenario there will be directional dispersion of electrons, lower energy electrons, and possibly multiple beams, all of which lead to reductions in the spatial variability due to beaming alone, thus requiring even more sparktospark variation. we hope the results described above will provide a basis for comparison to results of other spark experiments to determine if the distributions of mean photon energy and spark xray fluence depend on the experimental setup. we further hope that theoretical work can find a basis for such distributions to confirm or refute our assumptions and that such theories can shed light on the process of runaway electron acceleration in sparks as suggested to be relevant to other electrical phenomena such as lightning or terrestrial gamma ray flashes. | abstractxray emission by sparks implies bremsstrahlung from a population of energetic electrons, but the details of this process remain a mystery. we present detailed statistical analysis of xray spectra detected by multiple detectors during sparks produced by 1 mv negative highvoltage pulses with 1 s risetime. with over 900 shots, we statistically analyze the signals, assuming that the distribution of spark xray fluence behaves as a power law and that the energy spectrum of xrays detectable after traversing 2 m of air and a thin aluminum shield is exponential. we then determine the parameters of those distributions by fitting cumulative distribution functions to the observations. the fit results match the observations very well if the mean of the exponential xray energy distribution is 86 7 kev and the spark xray fluence power law distribution has index 1.29 0.04 and spans at least 3 orders of magnitude in fluence. |
long duration space flights expose human body to microgravity which affects the physiological health drastically. some of the physiological effects that result from human space flights are bone loss, muscle atrophy, cardiovascular deconditioning, altered sensory motor reactions, fluid redistribution / orthostatic intolerance, etc. in order to counteract these changes and increase astronaut performance in orbit, cardio vascular exercises as well as resistive exercises has been made a part of the international space station missions. it has been evident that extravehicular activity (eva) exposes the crew member to substantial physical stress. use of eva suits during mars analogue work, particularly those with helmets exhibiting poor ventilation, has been reported to cause increased exertion, expressed as heart rate and stress levels response, during short period of physical work. it is of utmost importance that the crew of aircraft that is to land on mars should possess adequate physical fitness in order to handle their work tasks and to be prepared for possible emergency situations. hence, the astronauts need to be physically fit and be capable of performing intense activities. it is known that heart rate and cortisol are one of the parameters that can be measured to know the stress levels.[57 ] in the present study we characterized the physiological cost of mars analogue eva work by outfitting crew members with a portable expired gas analysis system during training eva sessions. also, to find out the correlation between heart rates, stress and cortisol levels, the heart rates, cortisol and workload during eva. the six - crew members were selected from 100b ilewg euromoonmars during mdrs (mars desert research station), utah, usa. the ages for the crew members (n = 6) aged 20 - 26 [23.6 (2.4) ] years. the average and calcium intake of the crew members during mission was 2400 kcal / day (range 2090 - 3200 kcal / day) and 1267 mg / day (1130 - 1400 mg / day), respectively. dietary sodium and potassium intake were maintained at 98 (80 - 103) and 86 (75 - 120) mmol / day, respectively. water intake was ad libitum 1236 (68) ml / day (940 - 1590) ml / day. each crew members was outfitted for gas analysis during two eva excursions and two graded hill runs, each performed on a separate day. the eva excursions were for the purpose of training the crew for geological surveying, were between one and two hours in duration, and were performed while wearing the mars eva suit provided for at the mars desert research station (mars society, usa). the hill runs consisted of continually running up and down a 200 m hill path (10% grade). peak was increased every two minutes at the encouragement of a principal investigator (br) and the participant was instructed to run until exhaustion. the peak 10 second mean value recorded over the two hill sessions was taken to represent vo2 peak (peak oxygen uptake or maximal aerobic capacity). aerobic energy expenditure determined by measured oxygen consumption (vo2) during eva sessions was compared relative to peak oxygen consumption (vo2 peak) recorded during graded hill runs performed on 14 separate days. the heart rates, cortisol, vo2 of the crew are measured before, during and after an eva. the workload of the crew is measured using nasa task load index (tlx) before and after the eva. expired oxygen and carbon dioxide were measured in a breath by breath manner by a cos med portable gas analysis system (cosmed, italy). saliva samples were collected (versi - sal, oasis, usa) immediately before eva and after eva. the average vo2 peak recorded during the hill runs was 56.6 (2.67) ml / kg / min and peak vo2 reached during a given eva was 35.87 (0.78) ml / kg / min. data for heart rate showed the same trend as vo2, with a maximal increase to 85% of peak [figure 1 ]. the rating of subscale showed a significant increase in eva as compared to run [figure 2 ]. also rating of subscale showed a significant increase during eva as compared to before and after. a good correlation between nasa tlx and salivary cortisol levels (r = 0.68, p < 0.001). salivary cortisol levels were increased in both groups [figure 3 ], although significant increased of cortisol levels (p < 0.05) more in eva as compared to hill running crew members. heart rates increased in both groups [figure 4 ], although significant increased of heart rates (p < 0.01) more in eva as compared to hill running crew members. there was positive relation between the rise in salivary cortisol concentration and max vo2 (r = 0.78, r = 0.75 ; eva and hill running, respectively). there were good correlation between heart rate and cortisol levels (r = 0.69, r = 0.72 ; eva and hill running, respectively). peak heart rate achieved during hill runs and eav (figure 1 showing that heart rate was significantly increased during eva task as compared to hill runs) average task load index (tlx) during hill runs and eva (figure 2 showing that average was significantly increased during eva task as compared to hill runs) salivary cortisol levels during hill runs and eva (figure 3 showing that salivary cortisol levels were significantly increased during eva task as compared to hill runs) heart rate during hill runs and eva. (showing that heart rate was significantly increased during eva task as compared to hill runs) this pilot study showed that a typical mars analogue eva requires a sustained challenging physical effort. the rating of subscale showed a significant increase in eva as compared to hill run. it might be due to higher stress situation during eva task as compared to hill run which is supported by significantly increased of salivary cortisol levels and heart rates during eva task as compared to hill run. during exercise, when the max o2 consumption exceeds 60% an increase in the epinephrine and cortisol concentrations occurs. during stress condition vasopressin stimulates the release of corticotropin - relasing factor, which in turn leads to the release of acth (adrenocorticotropic hormone). the highest value of cortisol was reported in aerobic capacity exercises. whereas anaerobic exercise for a brief period led to no changes whatsoever in the sporting subjects, with aerobic exercise an increase in the cortisol and acth hormone secretion was observed. a good correlation between nasa tlx and salivary cortisol levels the positive relation between the rise in salivary cortisol concentration and max vo2 was found. in the light of these findings a lack of accurate methods of vo2 max estimation by treadmill or cycle ergometer testing protocols and less number of subjects were main limitations of this study. notwithstanding of these limitations this investigation was able to observe authentic physiological phenomena in the unique environment of the mars analogue environment. continuation of aerobic fitness during the long trip to mars will be considerably more difficult. so, new training methods and medicinal therapy will have to be explored that can not only maintain aerobic fitness to the levels specified, but also address muscle atrophy and bone demineralization due to microgravity. these results suggest that salivary cortisol rhythms may be successfully employed for estimating circadian rhythms and related stress decrements in astronauts during space missions. we can use these salivary markers for mentoring of stress and workload parameters during selection of astronauts, during mission and after mission because being a noninvasive, cost - effective, less time consuming, easy to use and non - infectious. further study is required on large scale taken into account of limitations such as large sample size, no applicable of statistical analysis method due to small sample size, using standardized measuring methods, taking into account of more serum and salivary stress biomarkers, with and without helmet of this study and including other physiological and psychological parameters in mars analog environment. | background : extravehicular activity (eva), such as exercise performed under unique environmental conditions, is essential for supporting daily living in weightlessness and for further space exploration like long mars mission.aim:the study was planned stress, workload, and physiological demands of simulated mars exploration.materials and methods : in this study, the six - person crew lived (24 hours) for 14 days during a short - term stay at the mars desert research station. the heart rates, salivary cortisol, workload, peak oxygen uptake or maximal aerobic capacity of the crew are measured before, during and after an eva.results:data for heart rate showed the same trend as peak oxygen uptake or maximal aerobic capacity, with a maximal increase to 85% of peak. the rating of subscale showed a significant increase in eva as compared to run. salivary cortisol levels and heart rates were increased in both groups, although significant increased of cortisol levels and heart rates more in eva as compared to hill running crew members.conclusion:further study is required on large scale taken into account of limitations of this study and including other physiological and psychological parameters in mars analog environment. |
myelofibrosis is a myeloproliferative disease (mpd) in which bone marrow is replaced with collagen connective tissue fibres due to the proliferation of an abnormal clone of haematopoietic progenitor cells. splenectomy is sometimes performed before bone marrow transplant (bmt), especially if the patient has splenomegaly with hypersplenism. we describe a case of portal vein thrombosis (pvt) after splenectomy for mpd with an epidural catheter in situ which posed a therapeutic challenge. we wish to highlight the need for anaesthesiologists to be aware of the high risk of pvt after splenectomy for mpd. a 51-year - old male patient with myelofibrosis presented with gross splenomegaly with left - sided obstructive uropathy and no co - morbidities. other than anaemia and slightly deranged renal parameters (blood urea : 57 mg / dl, serum creatinine : 1.6 mg / dl), other investigations were normal. prior to bmt, he underwent splenectomy under a combination of epidural and general anaesthesia. (pod) 1, the patient developed fever, disproportionately severe abdominal pain in spite of adequate epidural analgesia and rise in liver enzymes (serum glutamic oxaloacetic transaminase : 1947 iu / l, serum glutamic pyruvic transaminase : 2798 iu / l). a contrast - enhanced computed tomography scan revealed right and main pvt extending up to proximal superior mesenteric vein, infarction of right lobe of liver (segment v, vi, vii), and oedematous wall of gastric and small bowel loops, moderate ascites and splenic bed collection [figure 1 ]. systemic heparinisation was immediately started to target an activated partial thromboplastin time of 23 times the control. computed tomography scan of patient showing portal vein thrombosis and infarction of liver the intensivist, anaesthesiologist, surgical oncologist, intervention radiologist and medical oncologist jointly discussed the pros and cons of removing the epidural catheter and waiting before starting local thrombolysis in an already anticoagulated patient. removing the catheter before thrombolysis would have significantly delayed thrombolysis with the risk of extension of liver infarction. hence, arterial thrombolysis keeping the catheter in situ with close neurological monitoring was planned. epidural local anaesthetic infusion was stopped, and intravenous patient - controlled analgesia with morphine was started. indirect thrombolysis of mesenteric and portal vein was started with injection alteplase (6 mg bolus + 1 mg / h) through the micro - catheter placed in the superior mesenteric artery. patient was clinically assessed for lower limb neuromotor deficit at close intervals and haemoglobin and lactate levels were monitored. on pod 4, there was reduction in haemoglobin levels with significant bleeding from epidural site with soakage of bed linen (approximately 100200 ml) and without any neurodeficit. repeat angiography demonstrated partial recanalisation of portal vein and hence the micro - catheter was removed, alteplase was stopped and systemic heparinisation continued. on pod 7, heparin was stopped, and the epidural catheter was removed under fresh frozen plasma cover. patient was subsequently discharged without any neurological deficit on a therapeutic dose of low molecular weight heparin. on one hand, there was an urgent need for thrombolysis for the pvt, while on the other hand there was a risk of neurological complications due to recent epidural catheter insertion. thrombolysis was considered to be more effective, despite the increased risk of bleeding, and inability to monitor, titrate or reverse its effect, as compared to anticoagulation alone. the american society of regional anesthesia and pain medicine (asra) suggests avoidance of thrombolytic therapy for 10 days after neuraxial puncture (grade 1a). asra recommends neurological monitoring with interval 2 h and avoidance of epidural infusion causing sensory and motor block to facilitate neurological assessment. there is no definitive recommendation for removal of neuraxial catheters in patients who unexpectedly receive thrombolytic therapy during a neuraxial catheter infusion. they suggest measurement of fibrinogen levels to evaluate the presence of residual thrombolytic effect and to time catheter removal accordingly. patients undergoing splenectomy have an increased risk of splenic / pvt (spvt). with improved quality and frequency of radiological imaging, it is clear that this occurs more frequently than was earlier thought. in a systematic review the incidence of pvt varies depending on the indication for splenectomy, being high in mpds and hereditary haemolytic anaemias and low in trauma and autoimmune thrombocytopaenia. in a prospective study of 101 patients who underwent splenectomy, 40% patients with mpd and 25% patients with haemolytic anaemia developed spvt. three of 4 patients (75%) with mpd and splenic weight > 3000 g developed pvt. our patient had the splenic weight of 3270 g. unfortunately, the weight of the spleen can not be accurately determined before surgery, but the presence of massive splenomegaly must lead to a presumption of increased risk of pvt. thrombosis of the splenoportal axis is being increasingly recognised even after laparoscopic splenectomy, being reported as high as 55% in one study. this high probability of spvt should make us reconsider the use of epidural anaesthesia and analgesia in such cases. though the median interval between splenectomy and symptomatic spvt is 812 days, it may present as early as 2 days as in our case, when an epidural catheter is likely to be in place. this case highlights the fact that despite the well - known benefits of epidural analgesia in upper abdominal surgery, the high risk of pvt requiring anticoagulation or thrombolysis in patients undergoing splenectomy for mpd may well preclude the use of central neuraxial blockade. anaesthesiologists should be aware that the risk of pvt after splenectomy varies with the indication for splenectomy and is extremely high in patients with mpd, especially with massive splenomegaly. considering this high likelihood of pvt requiring anticoagulation or thrombolysis, the central neuraxial blockade should be avoided. if post - operative thrombolysis is required in a patient already having an epidural catheter in situ, it should be done under close neurological monitoring, weighing the risks and benefits. fibrinogen levels should be monitored to evaluate the presence of residual thrombolytic effect to determine the appropriate time for catheter removal. | we describe management of portal vein thrombosis (pvt) in a patient with myeloproliferative disease after splenectomy. this case posed a unique therapeutic challenge in maintaining a fine balance between life - saving thrombolysis and the risk of neuraxial complications due to bleeding. the incidence of pvt after splenectomy in patients with myeloproliferative disorders is high (40%). anaesthesiologists should be aware of this and avoid central neuraxial blockade in such cases. if post - operative emergency thrombolysis is required in a patient having an epidural catheter in situ, it should be done under close monitoring, weighing the risks and benefits. fibrinogen levels should be monitored to evaluate the presence of residual thrombolytic effects and to time the catheter removal. |
it is characterized by progressive acquired loss of retinal ganglion cells and their axons, leading to optic nerve atrophy and appearance of characteristic cupping of the optic disc associated with corresponding visual deficit. this damage can lead to permanent vision loss if it is not treated.1,2 population studies indicate that one in 40 adults older than 40 years has glaucoma, with loss of visual function. the disease affects approximately 60 million people worldwide, with 8.4 million being bilaterally blind. even in developed countries, glaucoma is mostly asymptomatic until late in the disease when visual problems arise.3 elevated iop is an important and modifiable risk factor for the development and progression of glaucoma.1 it has been demonstrated that lowering iop effectively delays development of glaucoma in patients with ocular hypertension, as well as progression of established glaucoma.4 topical hypotensive medication is considered as first - line treatment in the initial management of increased iop. nevertheless, target iop levels are not always achieved with a single medication, and patients frequently require multiple medications, which can lead to unsatisfactory adherence with treatment.3,57 according to the ocular hypertension treatment study, after 5 years of iop - lowering treatment, 40% of patients need at least two drugs.8 fixed combinations for glaucoma and ocular hypertension treatment are increasing. these fixed combinations contain two medications in a single bottle, and offers several advantages, including enhanced convenience, improved adherence, and reduced exposure to preservatives.6 the efficacy and safety of fixed combinations has been evaluated previously in different studies.7 the purpose of this study was to compare the efficacy and safety of fixed combinations of timolol 0.5% + brimonidine 0.2% + dorzolamide 2% (tbd) versus timolol 0.5% + brimonidine 0.2% (tb) for iop reduction. we conducted a prospective 3-month, randomized, double - blind, multicenter clinical trial to compare the efficacy and safety of tbd versus tb on iop reduction. the protocol was reviewed and approved by our ethics committee, and was conducted in compliance with the declaration of helsinki and in accordance with good clinical practice standards. the study is registered on the international standard randomized controlled trial number register (isrctn48477221). adult patients with primary open - angle glaucoma, ocular hypertension, pseudoexfoliative glaucoma, or pigmentary glaucoma, and a mean iop of 2130 mmhg were enrolled. primary exclusion criteria included blindness in one eye, visual field loss indicative of end - stage glaucoma, normal - tension glaucoma, optic nerve cupping 0.8, any active ocular disease other than glaucoma or ocular hypertension that would interfere with study interpretation, history of cataract surgery within 3 months prior to baseline, contraindication of any medication used in the protocol, and pregnancy, risk of pregnancy, or breastfeeding. the primary efficacy endpoint was the iop - lowering effect after 3 months of treatment. patients were assessed for eligibility at a screening visit 6 weeks before baseline when a medical and ocular history was taken, iop was measured in both eyes (using a calibrated goldmann applanation tonometer), as well as snellen visual acuity measurement and visual field test using automated perimetry (humphrey s 24 - 2 sita standard). abnormal findings on slit - lamp examination (biomicroscopy) and fluorescein dye were graded as mild, moderate, or severe. all iop - lowering medications were suspended in eligible patients, who underwent a 6-week washout period prior to baseline and, if their iops were considered detrimental, they were excluded from the study. patients were evaluated at five study visits, ie, at baseline, and at 15, 30, 60, and 90 days after enrollment. at each study visit, iop was measured in each eye at 8 am and 4 pm, slit - lamp examination using biomicroscopy and snellen visual acuity measurement tests were performed, and use of concomitant medications were recorded. bottles of the study drugs were overlabeled so that they had an identical appearance when provided to patients. patients were instructed to instill one drop of either tbd (krytantek ofteno ; laboratorios sophia, guadalajara, mexico) or tb (combigan d ; allergan inc, irvine, ca) in each eye twice a day, ie, in the morning at 8 am (15 minutes) and in the evening at 8 pm (15 minutes) for 3 months. the trial medication was discontinued if either the investigator or patient thought that it was not in the patient s best interests to continue, or if the patient became pregnant. investigators recorded observed adverse events, as well as those reported by patients or elicited by questioning. adverse events were classified as mild, moderate, or severe. only randomized patients who concluded the study without major protocol violation and provided iop measurements at all visits were included in the per - protocol analysis. baseline and final differences in iop between the treatment groups were analyzed using the mann, it was determined that at least 42 patients were needed per group to detect a difference of at least 2 mmhg in mean iop reduction between treatments using a significance level of 0.05, with a power of 0.80. baseline and final differences in iop between the treatment groups were analyzed using the mann whitney test. before study initiation, it was determined that at least 42 patients were needed per group to detect a difference of at least 2 mmhg in mean iop reduction between treatments using a significance level of 0.05, with a power of 0.80. fifty - six patients per group were recruited for this study from 11 centers in mexico. the mean age was 62.5 10.2 years in the tbd group and 60.6 12.7 years in the tb group. the primary efficacy endpoint was the iop - lowering effect at both 8 am and 4 pm after 3 months of treatment. the mean baseline iop was similar between the groups at 8 am (tbd 22.3 0.9 mmhg, tb 22.4 1.8 mmhg, p = 0.558) and at 4 pm (tbd 19.02 1.3 mmhg, tb 19.08 1.2 mmhg, p = 0.536, table 2). after baseline, and at all study visits, the mean 8 am iop at 15, 30, and 60 days after enrolment was significantly lower in the tbd group (figure 1). the same pattern was observed when iop was assessed at 4 pm (figure 2). furthermore, at the conclusion of the study, the mean iop was significantly lower in the tbd group at 8 am (16.19 2.0 mmhg versus 18.35 1.4 mmhg, p = 0.000) and at 4 pm (14.74 2.4 mmhg versus 16.77 1.4 mmhg, p = 0.000, table 3). adverse events were documented in eight patients, of whom six were excluded from the study. these events included ocular burning (mild in one patient and moderate in another), itching (severe in one patient), foreign body sensation (mild in one patient), redness (severe in one patient), dizziness (severe in one patient), headache (mild in one patient), and bradycardia (mild in one patient), the latter not being related to the study drug. the aim of treating glaucoma and ocular hypertension is to reduce iop.5 elevated iop is an important risk factor for progression of glaucoma, so achieving and maintaining a low iop minimizes the risk of glaucomatous progression and vision loss.9,10 different classes of drugs are available for iop reduction, the most commonly used being the prostaglandin analogs, beta - blockers, alpha agonists, and carbonic anhydrase inhibitors. single medication is sometimes insufficient to reduce iop, and patients may require different combinations to achieve their target iop.6,10 fixed combinations, ie, two drugs contained in a single bottle, have emerged as a treatment option, offering several advantages and fewer adverse events.6 a number of studies have evaluated the efficacy and safety of these combinations, and it has been demonstrated that combinations are superior to monotherapy with their constituent parts.1,47,10,11 garca - snchez compared administration of a fixed combination of latanoprost + timolol with an unfixed combination of brimonidine and timolol in patients with elevated iop for 6 months. they observed that the fixed combination reduced iop more effectively at all assessment times and was better tolerated than the unfixed combination. in our study, we compared a triple fixed combination of tbd and a double fixed combination of tb. although our study is different because we used fixed combinations and did not use a prostaglandin analog, we can compare our results for iop reduction taking into account the use of fixed combinations, and we observed that the tbd combination was more effective than tb. in a previous study, baiza - durn compared two fixed combinations, ie, timolol + dorzolamide + brimonidine versus timolol + dorzolamide. the conclusion of their study was that the triple combination appeared to be safer and more effective for reducing iop than the double fixed combination. similarly, our investigation demonstrated that the tbd combination was more effective than tb in reducing iop in the population studied. finally, a study by fechtner compared triple therapy (a fixed combination of brimonidine - timolol plus latanoprost) with double therapy (timolol plus latanoprost) ; in this study, patients were on latanoprost treatment and required additional iop - lowering. adjunctive therapy comprised a fixed combination of brimonidine + timolol or timolol only plus latanoprost ; thus, a triple combination was used. this study concluded that triple therapy (fixed combination of brimonidine + timolol plus latanoprost) reduced iop significantly more effectively than double therapy (timolol plus latanoprost). our study results are similar in that we also demonstrated that a triple combination is more effective than a double combination when reducing iop. in the study by fechtner, triple therapy was used, but was not a fixed combination and involved two different bottles of medication. the ultimate goal of treating glaucoma is to preserve the remaining visual field. only treatment to reduce iop monotherapy remains the preferred initial choice of treatment in glaucoma, using prostaglandin analogs and -blockers as initial treatment for lowering iop. prostaglandin analog eye drops are preferred due to their strong iop - reducing action and the convenience of requiring only once - daily administration, except in the event of side effects, intolerance, or patient refusal. nevertheless, target iop levels are not always achieved with a single medication, and patients frequently require multiple medications, which can lead to unsatisfactory adherence with treatment.3,57,13 adherence with treatment for glaucoma is better when regimens are simple rather than complex.14 use of two or more bottles of iop - lowering medication may be associated with an increase in noncompliance, and the advantage of fixed combinations is that a single bottle can contain up to three medications, thus minimizing the number of bottles and drops that need to be used by the patients, and facilitating adherence to treatment. fixed combinations are important adjuncts for the treatment of glaucoma but should generally be used only when monotherapy has not provided adequate iop reduction. one possible disadvantage is that tbd must be administered twice a day as opposed to prostaglandins, which only need to be administered once a day. another possible disadvantage is that the incidence of treatment - related adverse events could be higher on tbd than on any other combination. another drawback could be that it is not possible to change the drug concentration or dosing schedule for one component medication independently of the other when using a fixed combination like tbd. further studies will be needed to determine the long - term safety and efficacy of a fixed combination of tbd, as well as its effectiveness in providing additional iop - lowering over 24 hours. this can be achieved using different drugs with different pharmacodynamics. using this fixed combination, low doses of different drugs can be administered, and in this way, the onset of adverse effects could be delayed or diminished. the use of fixed combinations is preferred over separate use of their components to improve adherence and persistence with treatment. a fixed combination of tbd administered twice a day is more effective than a fixed combination of tb twice a day for reducing iop. | backgroundthe purpose of this study was to evaluate the efficacy and safety of two fixed combinations, ie, timolol 0.5% + brimonidine 0.2% + dorzolamide 2% (tbd) versus timolol 0.5% + brimonidine 0.2% (tb) in patients with primary open - angle glaucoma or ocular hypertension.methodswe performed a 3-month, randomized, double - blind study in patients with primary open - angle glaucoma or ocular hypertension and an intraocular pressure of 2130 mmhg. patients were randomly assigned to receive one drop of tbd or tb twice a day. the primary efficacy endpoint was change in intraocular pressure after 3 months of treatment. safety measures were assessed by the presence of adverse events.resultsmean baseline intraocular pressure was similar at 8 am and 4 pm in the treatment groups (tbd 22.3 0.9 mmhg, tb 22.4 1.8 mmhg, p = 0.558 ; tbd 19.02 1.3, tb 19.08 1.2, p = 0.536, respectively). at the end of the study, the mean intraocular pressure was significantly lower in the tbd group at both 8 am (16.19 2.0 mmhg versus 18.35 1.4 mmhg, p = 0.000) and 4 pm (14.74 2.4 mmhg versus 16.77 1.4 mmhg, p = 0.000).conclusionfixed - combination tbd was more effective than fixed - combination tb for reducing iop in patients with primary open - angle glaucoma. |
achieving a balanced and suitable nutrition is one of the most important health objectives in the early years of the child 's life. the role of nutrition in healthiness, effectiveness, learning and its relationship with economic development has been confirmed. several factors are involved in providing nutritional health of the household, known as the household food security. a household is a place where the food security and nutrition develops in the daily life. teaching good eating habits and proper nutritional behaviors result in the persistence of these habits into adulthood. the parents should receive necessary education about the proper child nutritional behavior and provide a suitable environment for nutrition of the child. therefore, the childhood is a critical period in the term of establishing suitable patterns of nutritional behaviors. in this period, the nutritional behaviors of the child are directly affected by the family and interactions with the environment. the child is directly affected by the family 's beliefs, attitudes, culture, and traditions, in the early years of life. thus, investigation of the proper patterns of the parents nutrition behaviors, especially mothers, has a significant role in children 's nutrition. mothers awareness of the proper developing nutritional behaviors has a direct effect on the nutritional quality of the children. these measures have reduced the children death and increased the longevity in most countries. however, protein - energy malnutrition remains a major nutritional problem in most developing countries including iran. nowadays, although the knowledge and experience in the prevention of malnutrition is available, about 150 million of children less than five years old suffer from malnutrition in the world. in our country, an average of 30% of children between one to three years old suffer of mild to moderate degrees of malnutrition. national survey results (anis) in 1,377 shows that 15.4% of children below five years of age suffer from nutritional stunting, 10.9% have moderate and severe underweighting, and 4.9% suffer from impotence if the mild cases also be added, the extent of the problem will be more visible. improving the mothers nutritional knowledge, in addition to help reducing the malnutrition in children, can cause changes in their nutritional behavior. the aim of this study is to determine the level of knowledge and attitude of the mothers covered by the urban health centers of birjand about nutritional behaviors. this descriptive - analytical study was conducted on 294 of mothers with children less than five years old referring the health centers of birjand, using a simple non - probability sampling method. a questionnaire included two sections were used to conduct the research. the section i consists of 21 questions about the knowledge and attitudes. the questionnaire was filled out after interviewing with the mothers by the experienced staff of nine health centers of birjand. content validity of the questionnaire was confirmed by a number of expert faculty members. the questionnaire contained personal characteristics (age, occupation, mother 's education stand) and attitudinal information about the nutritional behaviors. a score of 0 and one was given to the wrong and correct answers, respectively. a score of 10 to 15 and 15 to 21 was considered as moderate and good knowledge level, respectively. the knowledge questions were (the main food groups, nutritional value of each food group, who are at risk of anemia, the iron content of the food, what food is causing iron absorption, the prevention of iron deficiency among mothers and children, and the effects of iodine, vitamins and minerals deficiency). a score of one to five was given to the answers based on the likert scale. the attitude level was classified based on the attained score in three levels, i.e. poor (less than 50% of total score), moderate (a score between 50 to 75% of total score) and good attitude (scores higher than 75% of total score). data were analyzed using spss software and chi - square, anova, tukey 's post hoc and pearson correlation coefficient statistical tests with a accuracy level of p < 0.05. a total of 294 subjects (mothers referring the health centers of birjand) were studied. the average age of the mothers was 27.7 5.8. seven point one percent of these people were illiterate or poorly educated, 65.6% were diploma and below, 12.9% were technicians and 14.4% had higher education stand (b.sc and higher). fifty nine point nine percent and 80.6% of the mothers had moderate knowledge and good attitude level, respectively [table 1 ]. pearson correlation test showed a meaningful correlation between the knowledge score (r = 0.2) (p = 0.000) and the mothers age. however, no significant correlation was observed between the attitude and the mothers age (r = 0.05) (p = 0.3). frequency distribution of the mothers based on the knowledge and attitude level sixty five point six percent, 47%, 82.7%, 64.6%, 91.5%, 38.4% and 29.3% of the mothers were aware of the onset and the amount of iron drop, the food groups that are a good source of iron, the iodine - containing foods, minerals and vitamins beneficial to the body, the effects of deficiency of vitamin a, the anemia caused by iron deficiency, the role of iron in the body and the best way to prevent iodine deficiency, respectively [table 2 ]. frequency distribution of the mothers in terms of answers to the attitude questions anova statistical test showed a significant difference between the knowledge scores of the mothers in terms of education stand (p = 0.00) [table 3 ] ; the tukey 's post hoc test indicated a difference between the knowledge scores of the mothers with diploma and associate education stand (p = 0.003), diploma and bachelor (p = 0.00), associate and illiterate (p = 0.001), bachelor and illiterate (p = 0.00). comparison of the knowledge and attitude scores in terms of the education stand of mothers the one - way anova also showed a meaningful difference between the attitude scores of the mothers in terms of the education stand (p = 0.002) [table 3 ]. furthermore, tukey 's post hoc test showed this difference between the attitude scores of the mothers with below diploma and associate education stands (p = 0.002). the chi - square statistical test showed a meaningful relationship between the knowledge level and the age group of the mothers (p = 0.01) [table 4 ]. in addition, a significant correlation between the attitude level and the age group was observed. the mothers of the 25 to 35 years of age group had a good attitude level about nutritional behaviors. recent studies (and studies conducted in other universities) indicate that people (especially women) have low awareness of the principles of nutrition. nutrition principles and nutritional behaviors consist of fine and accurate aspects which should be noted. the results showed that the knowledge of the most mothers about micronutrients (iron, iodine, vitamin a and d), their role in the body, food sources and deficiency symptoms is at desirable level. this finding is consistent with the results of a study on 160 mothers referring the urban centers of babol. the present study showed that the knowledge level of the mothers increases by increasing the age. an investigation was conducted on the knowledge level and the performance of 120 mothers about the child nutrition during diarrhea at the ardebil university of medical sciences. the obtained results showed a meaningful relationship between the mother 's education stand and the nutritional information with increasing the age and the knowledge of the mothers. assessment of the nutritional knowledge of 247 of the pregnant mothers in babol, showed that the nutritional knowledge among pregnant women was at a moderate level of 65.6%. the most important sources of information were through health care staff (32%) and radio and television (29%), which has a significant correlation with current study. in the present study, the nutritional knowledge level of the mothers was 59.9% and the most important source of information was through the health care staff (41.2%)., at the ardebil university of medical sciences revealed that only 2.1% and 15% of the mothers were aware of the onset of iron drop and the number of iron drops, respectively. but in the present study, 65.6%, 61.2 and 82.7 of the mothers were aware of the onset and amount of iron drops, the dietary sources of iron and the useful minerals and vitamins for the body, respectively. also, 38.4, 49.7 and 29.3% of the mothers knew the role of iron in the body, iodine deficiency symptom and the best way to prevent iodine deficiency, respectively. employing effective methods of intervention and training in health centers can be effective in improving knowledge level of the mothers. the result of a study conducted in gorgan indicated that from 248 of mothers, 62% were aware of the correct time of iron drop which is consistent with the results of present study. in the present study, the nutritional behavior knowledge level of 22.1%, 59.9% and 18% of the mothers was poor, moderate and good, respectively. so, improving the education level of the mothers is one of the most effective methods to improve the nutritional status of the children. in the present study, therefore, the role of health centers staff in raising the knowledge level of the mothers in various fields, particularly children 's nutrition is very important. based on the results of the present study, in addition to proper informing of the mothers, continuous re - training of the family physicians and health centers staff about the importance of nutritional behavior, the following items are recommended : periodic assessments of the mothers about proper nutrition of children (considering the importance of nutrients and supplements, the role of nutrients in the body) in health centers and health housesperiodic and regular assessment of the children 's growth can depicts an outlook of the nutritional and health status of the children in the region.conduct a comprehensive functional plan to prevention of malnutrition in the province, with special attention to the high - risk regions.using effective nutritional methods, especially in the case of iron and vitamin nutritional supplement that fits the demands and needs of the target groups. periodic assessments of the mothers about proper nutrition of children (considering the importance of nutrients and supplements, the role of nutrients in the body) in health centers and health houses periodic and regular assessment of the children 's growth can depicts an outlook of the nutritional and health status of the children in the region. conduct a comprehensive functional plan to prevention of malnutrition in the province, with special attention to the high - risk regions. using effective nutritional methods, especially in the case of iron and vitamin nutritional supplement that fits the demands and needs of the target groups. | background : achieving a balanced and proper nutrition is one of the most important health objectives in the early years of the child 's life. the aim of this study is to determine the level of knowledge and attitude of the mothers covered by the urban health centers of birjand about nutritional behaviors.materials and methods : this descriptive - analytical study was conducted on the mothers with children less than five years referring the health centers of birjand, in 1387. a questionnaire was prepared for data collection. data were analyzed using spss software, chi square, anova, tukey 's post hoc and pearson correlation coefficient statistical tests with a accuracy level of p < 0.05.results:two hundred ninety four patients were studied. a meaningful difference was observed between the knowledge and attitude scores in terms of the mothers education stand (p = 0.002). eighty three point seven percent, 65.6%, 82.7% and 64.6% of mothers were aware about the importance of iron absorption, the onset of iron supplement drop, the minerals and vitamins in the body, and the effects of vitamin a deficiency, respectively. the mothers knowledge and attitude about the nutritional behavior was evaluated at the average and good level, respectively.conclusions:considering the average level of the mothers knowledge and attitude about children 's nutritional behaviors, the retraining of family physicians and health centers staff about the importance of nutritional behaviors is recommended. |
research into the relationship between on the one hand brain injury [be it vascular or traumatic (tbi) ] and on the other hand various cognitive processes remains a crucial part of cognitive neuroscience. some of the consciousness - related phenomena studied in brain injured patients are hemispatial neglect and blindsight. there is a constantly growing body of clinical as well as experimental data on various types of neglect and blindsight in focally lesioned patients (e.g., natsoulas, 1997 ; marcel, 1998 ; rossetti., 1998 ; kentridge., 1999 ; danckert and goodale, 2000 ; schindler., 2006 ; bartolomeo, 2007 ; silvanto., 2008 ; funk., 2010). even within other areas than visual perception lesion - induced dissociations between conscious and non - conscious processes are being addressed in attempts to yield information regarding the neural processes mediating subjective consciousness (e.g., lane., 1997). studies focusing on the manifestations of consciousness in brain injured patients fall within the framework of research addressing other cognitive domains exhibit a constantly growing body of such studies, e.g., language (e.g., thomas., 1997 ; thulborn., 1999 ; ansaldo., 2002 ; ansaldo and arguin, 2003 ; perani., 2003 ; baumgaertner., 2005 ; meinzer. associating various functions with specific brain regions is one of the main research traditions of cognitive neuroscience. but it is also an endeavor facing both methodological and theoretical challenges (e.g., mogensen and mal, 2009) not the least the apparent contradiction between the localization and post - traumatic recovery, respectively, of various functions. focusing on the recently developed reorganization of elementary functions (ref)-model (mogensen and mal, 2009) the present communication discusses these issues with respect to the understanding of the neural substrate and post - traumatic recovery of cognitive functions in general. a subsequent paper (overgaard and mogensen, 2011) will deal more specifically with the issue of what is required in terms of theoretical conceptualization as well as experimental documentation if post - traumatic functional recovery is to be taken as documentation of multiple realizations of neural substrates of consciousness. various brain structures and substructures perform apparently unique types of information processing and consequently participate differentially in the mediation of various types of behavior and cognition. in other and more commonly used words : the functions of the brain are regionally localized. one of these is the study of post - traumatic impairments in brain injured individuals patients or experimental animals. in such studies, the profile of post - injury symptoms the logics of these lesion experiments (see e.g., coltheart, 2001 ; selnes, 2001 ; controlled experiments in animal models and clinical studies in brain injured patients) is that in the absence of a brain structure, the symptoms must reflect the absence of functional contributions from the affected neural machinery and that with the proper analysis and comparisons across symptoms, conclusions can be drawn regarding the information processing of the missing brain circuitry. a more recent contribution to this type of study is the use of transcranial magnetic stimulation (e.g., pascual - leone., 1999, 2000 ; walsh and cowey, 2000) in which the functional integrity of a part of the brain is temporarily disturbed allowing an analysis of the consequences of what can be seen as a temporary lesion. the other main source of support for functional localization within the brain is the studies utilizing various types of neuroimaging techniques. by studying the regional pattern of brain activation during the performance of various tests, one can provided adequate baseline measures are utilized for the subtraction from the test condition provide information about whether or not a particular brain structure changes its level of activity (often reflected directly or indirectly as a change in metabolism and blood supply) in association with the performance of a particular task or stimulation. numerous contradictions exist within these branches of the neuroscientific literature, but there is an overall agreement that although often poorly understood, there is a regional functional specialization within the brain a functional localization (e.g., monakow, 1914 ; coltheart, 2001 ; selnes, 2001 ; kringelbach and rolls, 2004). as mentioned above, a crucial aspect of the interpretation of results from various types of lesion experiments is that in the absence of a brain structure and consequently the functional contributions from that circuitry the behavior and conscious manifestations of the individual must reflect the lack of whatever functional contributions were provided by the now missing part of the brain. but if it is assumed that the lost circuitry is post - traumatically never regained (an issue to which i will return later), one should expect the impairments after brain injury to be chronic. nevertheless, it is a well - established fact that a post - traumatic functional recovery does occur. in patients as well as in animal models of brain injury, most lesion - associated impairments post - traumatically undergo some level of recovery at least within cognitive domains not closely linked to the direct in- and output pathways. but for almost all higher cognitive functions, trauma - related impairments are followed by an apparent return toward the proficiency seen pre - traumatically (e.g., ramachandran and blakeslee, 1998 ; carney., 1999 ; buller and hardcastle, 2000 ; panksepp and panksepp, 2000 ; len - carrin and machuca - murga, 2001 ; mogensen., 2004, 2007 ; mogensen and mal, 2009 ; rohling., 2009). in most cases, such a functional recovery is associated with more or less formalized and institutionalized rehabilitative training, but in the absence of such training, spontaneous recovery is also seen (e.g., len - carrin and machuca - murga, 2001). it must, however, be remembered that even in the absence of a formalized post - traumatic training program, practically all brain injured organisms (patients and experimental animals alike) are subjected to the informal training of daily life activities. even the most basic daily activities and communicative efforts constitute challenges and tasks which the brain injured individual must attempt to meet and master. consequently, the absence of an externally imposed training regime does not allow a claim that the potential occurrence of a functional recovery is independent of interactions with the environment. some instances in which a spontaneous recovery may be of a more automatic nature can, however, be seen in cases where a lesion - induced phenomenon can briefly be described as a situation in which injury within one part of the brain causes other brain areas to receive a reduced level of blood supply. while being sufficient for the survival of neurons within that penumbra region, the reduced blood supply does not allow a normal level of functionality. consequently, the observable symptoms are not only associated with the trauma per se but also with the impaired neural activities within the penumbra. penumbras, however, mostly disappear spontaneously and allow a return to normal levels of functional performance within that region of the brain (e.g., choi., 2007). although sometimes incomplete, this post - traumatic functional recovery may both clinically and in animal models turn out to be complete defined as the acquisition of a post - traumatic proficiency equal to that seen in the absence of any brain injury (e.g., mogensen., 2004). in case of animal models, this is even seen under circumstances ensuring the complete removal of the brain structure in question as well as a well - established pre - traumatic functional baseline. if the relatively few instances in which post - traumatic symptoms can be associated with penumbras or similar phenomena are excluded, what remains is the apparent contradiction between the two phenomena of functional localization and recovery of function. a radical and maybe tempting way to utilize lesion experiments without having to deal with the contradiction between localization and recovery of functions is to accept only the post - traumatic symptoms as relevant to arguments regarding functional specialization, in case those symptoms turn out to be chronic never to demonstrate any functional recovery. it has to be realized, however, that if only those instances in which post - traumatic impairments persist chronically are to be considered when conclusions are drawn from lesion experiments, the vast majority of such clinical and animal model derived data would have to be discarded. but the arguments against dismissing all but the chronic post - traumatic symptoms for consideration when the principles of localization are considered, are not only of such a practical nature (although disregarding almost the entire mass of post - traumatic data is in itself not an insignificant obstacle !). the fact that a functional recovery can actually take place in spite of the continued presence of a lesion, which originally had such an impact on the information processing of the brain that significant symptoms occurred, is in itself a highly relevant phenomenon. it indicates dynamic changes, which must be an essential part of the functional organization of the brain. if these phenomena are not considered in neuroscientific studies and the construction of various models, the result will be an incomplete and lacking understanding of the functionally dynamic brain. but in which ways, then, can one imagine the apparent return (potentially to a normal level of proficiency) of the behavioral and cognitive abilities of a brain injured organism ? one possibility is that post - traumatically the brain is able to reconstruct within the region of injury or elsewhere a circuitry, which can accomplish an information processing similar to what has been lost to injury. although it is well known that the actual site of injury mostly turn into scar tissue, this does not in itself preclude the possibility of a recreation somewhere in the brain of circuitry fulfilling the same information processing demands as the lost structure. in order to evaluate the likeliness that a reconstruction of the lost circuitry can occur in the injured brain, it is relevant to compare the types of plasticity available in the injured adult brain to the plastic processes, which contribute to the original construction of such networks during development. when the adult brain is injured, the trauma itself induces a range of changes in gene expression both in the tissue immediately surrounding the site of injury and in more remote parts of the brain. some such changes are likely primarily to contribute to the detrimental effects of the injury for instance by promoting metabolic dysfunction, inflammatory responses, etc., 1995 ; hermann., 1999 ; harris., 2001 ; other changes in gene expression are likely to be supportive of rehabilitation promoting plasticity and reorganization (e.g., witte, 1998 ; frost., 2003 ; one such process may be the increased occurrence of long - term potentiation (ltp) like synaptic plasticity after brain injury (e.g., hagemann., 1998). relative to the issue of recreation of circuitry similar to what has been lost to injury, it might be especially relevant that injury to the brain potentiates the ongoing neurogenesis (e.g., magavi., 2000 ; scharff., 2000 ; arvidsson., 2002 ; nakatomi., 2002 ; chen., a reason for this to be important is that during maturation neurons undergo a number of changes reducing their similarity to the developing neurons, which originally formed various circuits (e.g., fawcett., 1989 ; in contrast, the newly formed neurons produced by adult neurogenesis are unlikely to have similar limitations. if the newly created neurons in the injured brain are to reach a specific destination and contribute to recreation of a circuit, they will have to migrate in an appropriate manner. after injury, mature astrocytes are able to transform themselves into radial glial cells similar to those guiding neural migration during development (e.g., rakic, 1971, 1985). such radial glial cells are able to guide the migration of immature neurons even in the adult brain (e.g., leavitt., 1999). further optimism regarding the potentials of the injured adult brain may come from the fact that most substances, which played a guiding role during the original outgrowth of dendrites and axons (e.g., keynes and cook, 1992 ; brose., 1999 ; chen., 2000 ; hiramoto., 2000 are also present in the adult brain (e.g., koeberle and bahr, 2004). this optimism may, however, be tempered by the observation that the distribution of these substances undergoes major changes during the maturation of the brain making it questionable whether they in an injured adult brain can play similar roles to those of development (e.g., harel and strittmatter, 2006). the most important factors preventing immature neurons in the adult nervous system from recreating the injured circuitry, may, however, be associated with glial cells and myelin (e.g., berry, 1982 ; schwab and thoenen, 1985 ; schfer., 2008). especially important may be the astrocyte - produced chondroitin sulfate proteoglycans (cspgs), which play an important role in terminating the developmentally critical periods (e.g., pizzorusso., 2002 ; berardi., 2004 ; mcgee., 2005). while consolidating the plastic processes occurring during critical periods, these substances may also play a role, which prevents an adult recreation of the circuit lost to injury (e.g., del rio and soriano, 2007 ; schfer., 2008). an improved functional recovery (potentially associated with recreation of lost circuitry) has been found when the cspgs are pharmacologically inhibited locally (e.g., del rio and soriano, 2007). while such a local inhibition may have therapeutic potentials in the future, the results also demonstrate that without an external intervention, the cspgs are likely to prevent or at least reduce the possibility of a post - traumatic re - establishment of the circuitry and thereby information processing lost to trauma. while it is important in these ways to establish whether the post - traumatically available neuroplasticity seems capable of processes, which can create a copy of what has been lost another and at least as important approach is to scrutinize the neural and cognitive processes accompanying the actual functional recovery. an extensive animal model - based research program (e.g., mogensen., 2002, 2003, 2004, 2005, 2007) has performed such an analysis and some of the results have been reviewed by mogensen and mal (2009). what has emerged is a pattern of principles regarding the mechanisms mediating post - traumatic recovery. three general principles are especially important describing the situation after a successful post - traumatic rehabilitation : modification of degree of contribution to task mediation by individual brain structures some structures exhibit an increased or decreased level of contribution to task mediation. task dependent and dissimilar neural substrates after a given lesion, the functional recovery of various cognitive tasks is mediated by unique and dissimilar neural substrates. application of new cognitive strategies the fully post - traumatically recovered individuals solve the task by applying new strategies that are dissimilar to those applied pre - traumatically. these three principles like the above consideration of the types of plasticity available in the developing and injured, mature brain, respectively indicate that a recreation of the lost circuitry is unlikely. both the second and third of the above principles show that the neural mechanisms mediating post - traumatic functional recovery do not include a copy of what has been lost to trauma. if any part of the injured brain at the end of rehabilitation training contained a circuitry similar to what was pre - traumatically available, it would be expected that all cognitive domains affected by the lesion would post - traumatically receive equal contribution to functional recovery from the brain region within which the circuitry had been (re)created. this possibility, however, is contradicted by principle 2. additionally, principle 3 contradicts the post - traumatic re - establishment of information processing identical to what was available pre - traumatically : if post - traumatic processes had re - established the information processing of the injured structure, one would expect not only task solution of a proficiency similar to that seen preoperatively, but also that such a task solution would employ similar strategies to those of the pre - traumatic situation. but if the post - traumatic functional recovery is not mediated via mechanisms recreating what has been lost to injury, how can a sometimes even complete level of proficiency be re - established within traumatically impaired cognitive domains ? the recently proposed ref model of mogensen and mal (2009) is an attempt to describe neural and cognitive mechanisms, which in spite of the absence of a recreation of the lost circuitry can account for a potentially full proficiency of post - traumatic cognitive recovery. at the most basic level of the ref - model the efs are truly localized in the sense that they are mediated by local circuitry within a structure of the brain. each traditionally defined neural structure (e.g., the hippocampus or the dorsolateral prefrontal cortex) contains the neural substrates of a huge number of efs. when a region of the brain is lost to injury, all efs mediated by the lost tissue are according to the ref - model irreversibly lost, too. the information processing of an individual ef is of a highly basic and modular type. an ef does not in itself have any of the functions traditionally described by psychology. psychologically defined functions such as object discrimination, explicit memory, or allocentric spatial orientation belong at a different level of analysis (to be described shortly). the function of an ef may more easily be described in mathematical terms rather than in the vocabulary of for instance cognitive psychology. in contrast, the functions and cognitive domains normally described by psychology are in the ref - model represented at the third and highest level the level of surface phenomena. the surface phenomena of the ref - model consist of observable behavior (for instance the performance of a task by a patient or an experimental animal) as well as conscious manifestations such as the subjective experience of recognizing a familiar face or planning a course of action in order to solve a problem. it is at the level of these surface phenomena that post - traumatic functional recovery is normally defined and evaluated. tests conducted in order to determine what is impaired by brain injury address the observable behavior as well as whatever representations can be obtained of the subjective experiences of the patient. and it is at the same level that the more or less complete recovery of these impaired dimensions of cognition and consciousness are determined using similar methods to those utilized during diagnosis. an essential component of the ref - model is the layer of analysis introduced between the basic layer of the efs and the uppermost layer of the surface phenomena. most if not all ass are established as the result of experience and learning. most efs are simultaneously part of several or many ass. while the individual efs are strictly localized within a subregion of the brain, the neural substrate of an as consists of the neural substrates of all its constituent efs as well as the interconnections between the neural substrates of these efs. this makes the neural substrate of an as highly distributed and in most cases components of the neural substrate of an as will be found within a number of brain structures. for instance, a specific type of solution of a task is obtained by activation of a particular as. unless special analytical techniques are employed, it may at the surface level not be possible to discriminate between behavioral or conscious phenomena reflecting two related but different ass. whenever brain injury destroys the neural substrate of one or more of the constituent efs within an as, that as is lost. consequently, the surface phenomena associated with the activity of the lost as are also lost and post - traumatically an impairment is registered. according to the ref - model, the mechanisms enabling a post - traumatic functional recovery are special cases of a more general mechanism, which in the intact brain has evolved as a crucial aspect of learning and problem solving. when an individual encounters a situation calling for a task solution for which there is no established procedure available, a mechanism is initiated during which various existing ass are tested out. a selector / evaluator mechanism which resembles (without being identical to) the supervisory attentional system (sas) of norman and shallice (1986) sequentially activates existing ass. when activated, an as results in the associated surface phenomenon and, in turn, the quality of the resultant behavior or mental manifestation is evaluated. in case the desired result is obtained, activation of that as will in the future be associated with the situation in question. this mechanism resembles without being identical to the mechanism of hypotheses evaluation described by krechevsky (1932, 1933). in case activation of an existing as can obtain the desired result, the neural plasticity associated with this entire process is restricted to modifications within the selector / evaluator mechanism plasticity ensuring a future association between the situation in question and activation of the successfully selected as. if, however, a situation requiring the solution of a problem can not be solved by activation of any existing as, a novel as will have to be established. the creation of a novel as involves a reorganization of the functional interaction between efs. such a reorganization (see mogensen and mal, 2009) utilizes a type of process resembling the backpropagation algorithm (e.g., rumelhart and mcclelland, 1986 ; werbos, 1994). such mechanisms constantly utilizing the feedback of the environment form a novel as by combining a set of efs, which previously did not constitute an interacting entity. whenever such a ref - process is required in order to successfully obtain a task solution, the required neuroplasticity includes modified connections between the neural substrates of the constituent efs. additionally, the complete process of eventual activation of the novel as and its association with the situation in question is also associated with neuroplasticity within the evaluator / selector mechanism. flow diagram depicting the sequence of events, which according to the ref - model leads to a successful development of a task solution potentially a successful functional recovery after brain injury. additional plasticity modifying the connections between the neural substrates of efs is only expected in case an actual reorganization of elementary functions (ref) process is required. for further details : see the present text as well as figures 3, 4, and 5 in mogensen and mal (2009). these processes of selection and potentially even de novo establishment of ass when a novel situation is encountered are according to the ref - model essential mechanisms in the mediation of normal problem solving. what is special about brain injury is that many situations, for which there used to be an established mechanism of task solution, will after the traumatic event have a status similar to novel situations. the as, which would normally be activated and ensure an efficient task solution, is no longer available since some of its constituent efs have been lost to injury. consequently, there is no immediately available mechanism of task solution. which leads to the above - described mechanisms of initial search for an appropriate as. since multiple ass might pre - traumatically have been able to allow an efficient task solution, some of these might post - traumatically still be available. if this is the case, the search for an available and appropriate as will be successful. alternatively, the ref - process (including backpropagation - based reorganization of interconnectivity between the neural substrates of efs) is required in order to obtain a satisfactory solution to the task. viewed in this way, the processes allowing a post - traumatic functionally recovery to take place have mostly if not exclusively evolved as mechanisms mediating problem solving in the intact brain. in one study addressing the types of neural and cognitive reorganizational processes described by the ref - model (mogensen., 2004) the mechanisms of post - traumatic functional recovery of a water maze based allocentric place learning task of the mapping type was addressed in rats subjected to various types of focal brain injury. lesions of the hippocampus provoked a major functional impairment, which, however, disappeared completely during an approximately 1 month long post - traumatic training period leaving the animals capable of a task proficiency indistinguishable from that of intact rats. relying on partly prefrontal cortical mechanisms, the animals could thus achieve a fully proficient surface phenomenon task performance. it turned out, however, that even rats subjected to hippocampal lesions as well as removal of the prefrontal cortex were able to achieve an equal that is : normal proficiency of task performance within a similar period of training. in the absence of both hippocampal and prefrontal contributions to the mediation of task performance, the neural substrate of task solution appeared to depend upon neural mechanisms within the parietal association cortex. in terms of behavioral parameters such as the time and swim distance required to reach the hidden target location, functional recovery mediated by mechanisms within the prefrontal cortex and parietal association cortex, respectively, were of equal proficiency. in the terminology of the ref - model this means that equally proficient ass can be constructed on the basis of populations of efs including either prefrontally based efs or efs mediated by the parietal association cortex, respectively. although equally proficient, the ass relying on prefrontal and parietal mechanisms, respectively, differed with respect to the cognitive mechanisms (as opposed to proficiency) of task solution (as would be expected from the ref - model) : while the recovered task solution in animals relying on prefrontal task mediation included cognitive representations of the goal position, such a knowledge of the spatial location of the goal appeared to be absent in even fully recovered animals relying on mechanisms within the parietal association cortex (mogensen., is encountered as described at the level of surface phenomena, the actual situation is that the post - traumatically selected and potentially established as is able to allow such a proficiency of task solution that using standard methods of analysis although each as is, in fact, associated with a particular way of solving the task, the observable behavior or subjective experience at the level of surface phenomena may in all of these cases be similar enough to be (in a sense wrongly) identified as the same. dissimilar surface phenomena that is a crucial aspect of the apparent contradiction between localization and post - traumatic recovery of functions. what is truly localized are the efs, and when the neural substrate of these basic information processing entities is lost, there is no recovery of the function associated with that information processing. in contrast, functional recovery is identified at the level of surface phenomena where highly detailed and special analytical techniques are required in order to discriminate between the phenomena associated with activation of various ass. the dynamic reorganizations associated with the ref - process only affect the input / output relationships of the efs. it does, however, contribute this information processing within a novel context within the newly established as. the situation in which an information processing module continues to perform its previous operations but on a novel input, bears a certain resemblance to some of the plastic processes found in uninjured brains. the somatosensory cortex, which after the amputation of a hand has become vacant, continues its functional activities but now operating on information regarding the face or arm (e.g., yang., 1994 ; weiss., 2000 such relative shifts within the somatosensory representations can also be seen after intensive training restricted to part of the body (e.g., merzenich and jenkins, 1993 ; elbert., 1995 ; tonotopic representations undergo plastic changes due to changes in input or experience (e.g., robertson and irvine, 1989 ; scheich, 1991 ; recanzone., 1993 ; irvine, 2007 ; thai - van., 2007). another obvious parallel is the situation in which a cortical area specialized in analysis of figure orientation within the visual domain can become engaged in apparently similar or at least related information processing on somatosensory information in the blind (ptito., 2005). given the highly specialized information processing units of the ref - model the efs this model falls within what is called massive modularity by for instance barrett and kurzban (2006). it should, however, be noted that the modularity of the ref - model is far from identical to the kind of modularity described by fodor (1983). that radical type of modularity has, however, subsequently been denounced by fodor (2000) himself. as emphasized above and by mogensen and mal (2009), according to the ref - model apparently the same surface phenomenon may be achieved by activation of a variety of ass. in the terminology of price and friston (price and friston, 2002 ; friston and price, 2003) such a situation represents a degeneracy relative to the manifestation of the surface phenomena. in cases where multiple ass give rise to surface phenomena that can not be distinguished from each other and that surface phenomenon is then characterized as one function, such a function is degenerate. it should, however, be stressed that according to the ref - model, such a degeneracy is the result of considering multiple surface phenomena, which might by a more or less superficial examination be indistinguishable from each other, the same in spite of the fact that these surface phenomena are in reality different (although perhaps only marginally so). this analysis seems to be in agreement with a number of the examples given by price and friston (2002). degeneracy is by price and friston (2002) illustrated by examples in which successful solution of a task (in the terminology of the ref - model : surface phenomenon) can be achieved via activity in separate and potentially not overlapping neural systems. it is recognized that for instance in case of a linguistic task the separate systems which are individually able to achieve a successful task solution do perform dissimilar types of information processing and thereby mediate dissimilar types of cognitive analysis. such systems would in the ref - model be identified as separate ass. while degeneracy might, thus, apply to the neural and cognitive mechanisms of a particular surface phenomenon, there is according to the ref - model no degeneracy with respect to the substrate of efs. an ef and its neural substrate are unique and if lost due to injury not replaced. when studying brain injured individuals it is important to realize that the result of the recovery process is not at the neural and more basic cognitive levels a return to the pre - traumatic situation. instead it constitutes a novel state of affairs, which has been constructed in an interaction with the environment during the period of rehabilitation. the ass which form the basis of the post - traumatically observable surface phenomena have been selected and potentially constructed via the interactions between the injured individual and the broadly defined environment. this means that a post - traumatic recovery process can be very situational specific. while the symptoms within a cognitive domain can appear to have disappeared completely when tested in one situation and under certain circumstances, symptoms within the same cognitive domain in the same individual may be evident and for that matter show no signs of recovery when tested under different circumstances. as has been emphasized elsewhere (e.g., mogensen, 2003, 2011 ; mogensen, in preparation ; overgaard and mogensen, 2011 ; wilms and mogensen, in preparation) animal models have frequently demonstrated that while one variant (/setup) of a cognitive test is able to reveal a striking level of post - traumatic impairment, another test (/setup), which characterized according to the formal demands of that cognitive test must be considered identical, shows no sign of post - traumatic impairment. across all studied species, animals subjected to lesions within the prefrontal cortex or the associated structures such as the prefrontal part of the neostriatum show an impaired performance of the task known as spatial delayed alternation (e.g., mogensen, 2003 ; mogensen., 2007, 2008). (1987) tested rats subjected to lesions of the prefrontal part of the neostriatum (a lesion which also renders the prefrontal cortex inoperable due to undercutting) in two variants of the spatial delayed alternation task. while the variant administered in a t - maze clearly revealed the expected symptoms, an operant chamber - based version of the task (in spite of fulfilling all the procedural / cognitive demands normally made on such a test setup) showed no sign of post - traumatic impairment. a somewhat related demonstration of the importance of the procedures employed in animal models can be found in a study by lepore. cats were tested for their ability to transfer visual discrimination - relevant information from one hemisphere to the other in the absence of the corpus callosum the main pathway between the two hemispheres. after having acquired the visual discrimination task based on one hemisphere exclusively (information was provided via one eye only in animals with a split optic chiasm), cats subjected to lesions of the corpus callosum were tested for their ability to perform the task with only the contralateral eye open a situation in which the task performance had to be based on the hemisphere contralateral to the one originally trained. the animals were tested for this ability in two experimental setups : a lashley - type jumping - stand and a traditional (maze - like) two - choice discrimination box. when tested in the discrimination box, the cats did not demonstrate any ability to transfer information between the hemispheres subcallosally, while such an ability was clearly revealed when the test was performed in the jumping - stand. while animal model - based studies are often able to more clearly demonstrate the procedure and setup - related differences in the degree or even presence of post - traumatic symptoms and post - traumatic recovery, clinical data are frequently of a more anecdotal nature. there are, however, studies in which such phenomena have been scrutinized in patients under controlled circumstances. the background of this study is a fascinating method of rehabilitative training of brain injured patients suffering hemispatial neglect (e.g., rossetti., 1998) : the prism adaptation therapy (pat ; e.g., rossetti., 1998 ; frassinetti., 2002). in this method, the patients are trained in a task requiring them to point (without being able visually to follow their arm during the pointing movement) to targets defined by the therapist and doing so when wearing prism goggles which diverts the visual field 10 to the right (the patients are exhibiting a hemispatial neglect of the left hemispace). normally, the feedback provided to the patient is the sight of the pointing finger at the moment when the pointing movement has been terminated. in most cases, the patient will gradually adapt to the perceptual shift and eventually show an after - effect in the form of a relative shift of the pointing movement even after the removal of the goggles. in other words, the procedure constitutes an at least partial therapeutic intervention regarding the neglect of the left hemispace. it has been demonstrated that an essential element of the procedure is the feedback regarding the precision of the pointing movements during the training period (e.g., frassinetti., 2002 ; serino., 2006, 2007 ; in the study by wilms and mal (2010) this traditional version of the pat - procedure was included and compared directly to a procedure in which the patients pointed to a touch - sensitive computer screen and feedback was provided graphically on the screen rather than via the direct sight of the pointing finger. surprisingly, in both patients and normal subjects the two procedures differed significantly the version in which an icon on the computer screen provided the feedback did not lead to any demonstrable after - effect. clinically, this specificity of the post - traumatic recovery poses a significant problem with respect to obtaining a therapeutic outcome, which generalizes to for instance the every - day - situations of the patient at home or at the work place. but with respect to studies addressing neural organization and reorganization and for that matter the neural substrate of consciousness the problems caused by the rather specific reorganizations provoked by a particular post - traumatic training process are primarily related to the (lack of) generality of the conclusions, which can be drawn from studies of a functionally recovered patient or animal. the pattern of neural mechanisms (e.g., regional activations seen in neuroimaging studies) and cognitive mechanisms allowing a more or less successful task solution can not be seen as a more global indication of which parts of the brain are able to take over from those lost to injury. instead, the observable pattern is the result of a specific process, which in principle is only designed to solve the manifestation of the task, which has been trained. but exactly this specificity may also become a window through which much more detailed information can be obtained. if certain demands are fulfilled, a novel more difficult and refined but also more promising type of localization research may be conducted. scrutinizing the post - traumatic situation in both patients and experimental animals, such research may elucidate central aspects of the organization and post - traumatic reorganization of the brain thereby providing a better insight into the neural mechanisms of cognition and consciousness. and which demands are then to be met by such studies of post - traumatic symptomatology and recovery ? an essential aspect will be a more refined conceptualization of what constitutes a function and what is actually obtained during post - traumatic functional recovery. replacing the old contradiction between localization and recovery of function with the more detailed concepts of for instance the efs and ass of the ref - model can provide a better framework for conceptualization of the results obtained. also the methodology of such studies needs refinement. realizing (like what is indicated by the ref - model) that rehabilitative training obtains a relatively task - specific reorganization rather than a recreation of what has been lost to injury, will have to provoke a different type of research strategy. a recovery process must be systematically addressed across various manifestations of what appear to be the same cognitive task as well as across cognitive domains. for each task it should also (as far as possible) be attempted to include studies utilizing various types of organic as well as behavioral / cognitive challenges as has been suggested by mogensen and mal (2009) and mogensen (2011). such challenge procedures can provide more detailed insights into the pattern of neural activities mediating a recovered task performance as well as the cognitive strategies allowing a particular surface phenomenon to be achieved. the plastic nature of the brain and especially the regionally injured brain can provide many surprises and frustrations to the neuroscientist trying to understand this most complicated product of nature. apparently similar tasks provide dissimilar results post - traumatically, and patients who apparently just need a precise feedback regarding their pointing errors during rehabilitative training of hemispatial neglect respond differentially in case of a graphic representation on a screen or the actual sight of their finger. these and many more frustrations can in the short term become obstacles to clinical utilization of methods as well as the possibility to reach more global conclusions. but like the apparent contradiction between localization and recovery of function they are also the types of data, which we should cherish and on which we should focus our attention. because here the dynamic brain is offering us windows through which we may eventually understand the neural mechanisms of cognition and maybe consciousness. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | in the search for a neural substrate of cognitive processes, a frequently utilized method is the scrutiny of post - traumatic symptoms exhibited by individuals suffering focal injury to the brain. for instance, the presence or absence of conscious awareness within a particular domain may, combined with knowledge of which regions of the brain have been injured, provide important data in the search for neural correlates of consciousness. like all studies addressing the consequences of brain injury, however, such research has to face the fact that in most cases, post - traumatic impairments are accompanied by a functional recovery during which symptoms are reduced or eliminated. the apparent contradiction between localization and recovery, respectively, of functions constitutes a problem to almost all aspects of cognitive neuroscience. several lines of investigation indicate that although the brain remains highly plastic throughout life, the post - traumatic plasticity does not recreate a copy of the neural mechanisms lost to injury. instead, the uninjured parts of the brain are functionally reorganized in a manner which in spite of not recreating the basic information processing lost to injury is able to allow a more or less complete return of the surface phenomena (including manifestations of consciousness) originally impaired by the trauma. a novel model [the reorganization of elementary functions - model ] of these processes is presented and some of its implications discussed relative to studies of the neural substrates of cognition and consciousness. |
in the previous issue of critical care, bruel and colleagues report findings from a small, prospective, observational study in which they investigate the feasibility, efficacy and safety of intra - arrest therapeutic hypothermia (th) for victims of out - of - hospital cardiac arrest (ohca). from an initial pool of 412 cardiac arrest victims, the study enrolled 33 patients with a variety of presenting rhythms. this represents the first study of its kind to investigate the feasibility of intra - arrest cooling in the clinical setting, an approach that has shown significant promise in animal models of cardiac arrest and brain injury [2 - 4 ]. sudden cardiac arrest, defined as the abrupt loss of mechanical cardiac activity and concomitant global loss of blood flow, is a leading cause of death in the united states and europe. approximately 200,000 people suffer ohca in the united states each year, and over 90% will succumb during resuscitation efforts or during subsequent hospitalization. survival to hospital discharge depends on a number of factors, including prompt delivery of cardiopulmonary resuscitation and defibrillation when indicated, the initial cardiac rhythm of arrest, and the quality of post - resuscitation care including provision of th. despite the significant effort that has been invested in this field, few therapeutic or pharmacologic interventions have yielded meaningful increases in overall survival from ohca over the past 20 years. the relatively new and evolving treatment modality of th, however, has been associated with markedly decreased mortality and neurologic injury among patients who initially survive ohca. th reduces both the cerebral metabolic rate and oxygen demand, and it is thought to attenuate reperfusion injury, global inflammation and endothelial dysfunction all consequences of cerebral and other organ ischemia. through such mechanisms, two landmark multicenter randomized controlled trials of th demonstrated over 20% absolute mortality reduction for initial survivors of ventricular fibrillation / ventricular tachycardia ohca. although both investigations documented strikingly improved survival in patients who had th implemented after blood flow was restored, provocative animal data suggest that initiation of cooling during cardiac arrest itself may yield further considerable improvements in survival and neurologic outcome when compared with the current standard of delayed th. the feasibility of such an approach in humans remains an active question with a paucity of data. the study by bruel and colleagues examines the feasibility of conducting intra - arrest cooling in the prehospital setting. although other investigators have evaluated prehospital implementation of post - resuscitation hypothermia, the current study represents the first implementation of this novel therapeutic approach in humans during the intra - arrest period. the current work establishes that th induction during the intra - arrest period, using chilled medical saline during advanced life support prehospital care, was feasible and without overt safety concerns. the technical and training hurdles in conducting such an investigation are formidable, and the authors should be applauded for their efforts. this study lays the foundation for future work on a larger scale that might incorporate randomization of patients to intra - arrest hypothermia, post - resuscitation hypothermia or normothermic resuscitation. furthermore, bruel and colleagues ' study demonstrates the feasibility of initiating intra - arrest hypothermia despite the significant, inherent challenges of the prehospital environment. the authors accomplished cooling with only peripheral venous access, although this may be technically more difficult during absence of flow (or limited flow during cardiopulmonary resuscitation), and may be very dependent on the quality of cardiopulmonary resuscitation and induced blood flow. finally, the study suggests that intra - arrest cooling in the prehospital environment may not only be feasible but also efficacious. more specifically, the time to reach mild hypothermia (34c) was an impressive 16 minutes, substantially faster than other studies of post - resuscitation th. although the sample size was small and the patient population was varied, 20/33 (60.6%) of patients who were successfully cooled had circulation restored, a trend that suggests utility of cooling in the intra - arrest period. despite successes in early cooling, there were surprisingly modest temperature differences at the time the patients were admitted to the intensive care setting. this should not be an insurmountable problem in future studies, and adjuncts to intravenous cooling could easily be used to maintain the hypothermic state. one advantage of the european system that employed this protocol was the presence of an emergency medical services physician and the ability to host the requisite refrigeration unit and associated equipment. this calls into question the ability to generalize this process to emergency medical services systems without these resources, although it is likely that the tasks could be accomplished by skilled prehospital personnel without direct physician oversight. finally, it is difficult to draw definitive conclusions about safety from this fairly limited study population. the overall number of enrolled patients was too small to detect differences in the appreciably rare complications, and it is not entirely clear over what time period the authors monitored for complications and what objective criteria were used to diagnose any observed adverse effects. in summary, bruel and colleagues ' innovative study is the first to implement the use of th prior to resuscitation from ohca. the authors demonstrate that prehospital, intra - arrest cooling is possible and may be efficacious. this notion is supported by our growing understanding of the pathophysiology of the ensuing injury associated with low or no - flow states. mechanistically, early cooling may reduce reperfusion - related injury by attenuating the oxidant burst seen within minutes of normothermic reperfusion or by the inhibition of reperfusion - activated apoptotic pathways. future studies should include objective measurements of patient pathophysiology to understand better the kinetics of injury and the beneficial effects of th. | over the past several years, the implementation of therapeutic hypothermia has provided an exciting opportunity toward improving survival from out - of - hospital cardiac arrest. there are compelling data to support the prompt use of therapeutic hypothermia for initial survivors from out - of - hospital cardiac arrest, but animal data have suggested that initiation of therapeutic hypothermia during the intra - arrest period may significantly improve outcomes even further. in the first feasibility study in humans, bruel and colleagues report on the implementation of this intra - arrest approach among patients suffering out - of - hospital cardiac arrest, an exciting prospect that is discussed in the present commentary. |
xylopia sericea (annonaceae) is a fast - growing native tree of the brazilian atlantic forest and cerrado ecosystems (lorenzi 1992), known locally as pimenteiro. during a collection of entomopathogenic fungi associated with armoured scale insects in the canopy of x. sericea (fig. 1a), a high proportion of the pruned branches showed abnormal development of lenticels : the latter appearing as prominent eruptions along the branches (fig. a discomycete fungus was observed consistently colonising the lenticular tissues, which was identified provisionally as similar to claviradulomyces dabeicola (evans., morphological differences were found that distinguished the fungus on x. sericea from c. dabeicola. the fungus was isolated into pure culture, from the sexual morph and from the purported asexual morph, allowing for molecular data to be generated to confirm the sexual relationship. a detailed description of the new species and a discussion of claviradulomyces phylogeny and its placement within ostropales are presented. stems bearing pronouncedly developed lenticels were collected from the canopy of xylopia sericea with the aid of a pruning pole (fig. 1) from two sites in the municipality of viosa (state of minas gerais, brazil) : at the edge of a well preserved stretch of atlantic rainforest, and a roadside stand adjacent to farmland. samples were air - dried and specimens were deposited in the collections of the universidade federal de viosa (vic) and of the cbs - knaw fungal biodiversity centre herbarium (cbs). isolations were performed either by transferring sporocarps to sterile distilled water agar (dwa), breaking them open with fine forceps, and streaking the spores across the agar surface to await germination, when germinating spores (ascospores, conidia) were selected with the aid of a sterile fine - pointed needle under a stereo - microscope with transmitted light and placed on potato carrot agar (pca), or by direct transfer of ascomata or pycnidia onto plates containing vegetable broth agar (vba), as described in pereira. colony characters were noted on malt extract agar (mea) and pca, either in the dark or with a 12 h light/ 12 h dark regime, at 25 c. morphological observations were made in lactic acid or lacto - fuchsin from hand sections of sporocarps or those teased from the lenticels and macerated. genomic dna was isolated from fungal mycelium grown on mea, using the ultracleantm microbial dna isolation kit (mobio laboratories, solana beach, ca) according to the manufacturer s protocols. the primers v9 g (de hoog & gerrits van den ende 1998) and lr5 (vilgalys & hester 1990) were used to amplify part of the nuclear rdna operon spanning the 3 end of the 18s rrna (ssu), its1, 5.8s rrna gene, its2 and the first 900 bases at the 5 end of the 28s rrna (lsu) genes. 2009a) were used as internal sequence primers to ensure good quality sequences over the entire length of the amplicon. lsu and mtssu sequences from the two claviradulomyces spp. were concatenated and incorporated into the alignments of baloch. data were analysed with bayesian phylogenetic methods using mrbayes v. 3.1.2 (huelsenbeck & ronquist 2001 ; ronquist & huelsenbeck 2003), with gaps treated as missing data, applying the gtr+i+g model for both genes, the models selected using the aic method in mrmodeltest v. 2.3 (nylander 2004). the data set was run with two chains for 10 m generations, and trees sampled every 1000 generations. convergence of all parameters was checked using the internal diagnostics of the standard deviation of split frequencies and performance scale reduction factors (psrf), and then externally with tracer v. 1.5 (rambaut & drummond 2007). on this basis, stems bearing pronouncedly developed lenticels were collected from the canopy of xylopia sericea with the aid of a pruning pole (fig. 1) from two sites in the municipality of viosa (state of minas gerais, brazil) : at the edge of a well preserved stretch of atlantic rainforest, and a roadside stand adjacent to farmland. samples were air - dried and specimens were deposited in the collections of the universidade federal de viosa (vic) and of the cbs - knaw fungal biodiversity centre herbarium (cbs). isolations were performed either by transferring sporocarps to sterile distilled water agar (dwa), breaking them open with fine forceps, and streaking the spores across the agar surface to await germination, when germinating spores (ascospores, conidia) were selected with the aid of a sterile fine - pointed needle under a stereo - microscope with transmitted light and placed on potato carrot agar (pca), or by direct transfer of ascomata or pycnidia onto plates containing vegetable broth agar (vba), as described in pereira. colony characters were noted on malt extract agar (mea) and pca, either in the dark or with a 12 h light/ 12 h dark regime, at 25 c. morphological observations were made in lactic acid or lacto - fuchsin from hand sections of sporocarps or those teased from the lenticels and macerated. genomic dna was isolated from fungal mycelium grown on mea, using the ultracleantm microbial dna isolation kit (mobio laboratories, solana beach, ca) according to the manufacturer s protocols. the primers v9 g (de hoog & gerrits van den ende 1998) and lr5 (vilgalys & hester 1990) were used to amplify part of the nuclear rdna operon spanning the 3 end of the 18s rrna (ssu), its1, 5.8s rrna gene, its2 and the first 900 bases at the 5 end of the 28s rrna (lsu) genes. 2009a) were used as internal sequence primers to ensure good quality sequences over the entire length of the amplicon. lsu and mtssu sequences from the two claviradulomyces spp. were concatenated and incorporated into the alignments of baloch. data were analysed with bayesian phylogenetic methods using mrbayes v. 3.1.2 (huelsenbeck & ronquist 2001 ; ronquist & huelsenbeck 2003), with gaps treated as missing data, applying the gtr+i+g model for both genes, the models selected using the aic method in mrmodeltest v. 2.3 (nylander 2004). the data set was run with two chains for 10 m generations, and trees sampled every 1000 generations. convergence of all parameters was checked using the internal diagnostics of the standard deviation of split frequencies and performance scale reduction factors (psrf), and then externally with tracer v. 1.5 (rambaut & drummond 2007). on this basis, diagnosis : similar to clavariadulomyces dabeicola, from which it is distinguished by the longer periphysoids (1639 m, shorter asci (3550 m), a longer ostiolar neck in the asexual state, shorter conidia (1434.5 m) with a discrete heel region, and molecular sequence data. type : brazil : minas gerais : viosa, pina, on living branches of xylopia sericea (annonaceae), 11 june 2010, r.w. barreto (vic31417 holotype ; cbs 133260 and cbs 133261 ex - holotype cultures). mata do seu nico, fazenda bonsucesso, on living branches of xylopia sericea, 20 may 2010, h.c. description : internal mycelium intra- and intercellular, 12 m diam, septate, branched, hyaline. ascomata erumpent from spongy tissues of lenticels on bark of living branches ; apothecial when mature and turgid, but perithecium - like and opening by a large, round pore when dry ; sessile, urceolate, 0.120.25 mm diam, wall black, extending above the surface of the hymenium, partially covering the hymenium when dry, opening pore lined with a whitish fringe of periphysoids. in vertical section, lower part of ascomatal wall often ill - formed and restricted to a loose hyphal layer from which asci and paraphyses arise, 515 m thick, composed of tangled hyphae 12 m diam ; upper part of wall dark brown to 37 m thick, narrowing and becoming paler towards the base and there 1012 m wide. periphysoids lining the upper wall above the level of the hymenium, cylindrical or club - shaped, sinuose or curved, 1639 m long and 34 m wide along the axis, often slightly swollen in the upper part up to 6 m, wall hyaline along most of the length, bearing abundant blunt denticles, arising from short brown smooth basal stalks. paraphyses 12 m wide, to 60 m long, apex swollen and bulbous, to 57 m wide and bearing abundant blunt denticles, imparting a mace - like or muricate appearance, extending beyond the asci but usually prostrate over the hymenial surface. asci parallel, clavate with a broadly rounded to somewhat flattened apex, becoming ellipsoidal when free of the hymenium, without a basal stalk, 3550 510 m, apex non - amyloid, 8-spored. ascospores in single fascicles extending to the base of the ascus, parallel to spirally or partly spirally arranged in the upper half, cylindrical to vermiform, attenuating towards the sub - acute ends, straight to slightly curved or sigmoid, sometimes strongly curved at the apices, (21) 2843 23 m, aseptate, hyaline, smooth, strongly guttulate. asexual morph : formed separately or in combination with the sexual morph in the same lenticel. conidiomata pycnidial, semi - immersed, globose, 76137 m diam ; thin walled, walls 415 m thick, with long cylindrical ostiolate necks, 110360 2446 m, composed of parallel hyphae 13 m wide, often reduced to a narrower cylinder of bristle - like hyphal tips at the apex, 1521 15 m. conidiophores usually reduced to conidiogenous cells, occasionally consisting of a small stalk of one or two cells. conidiogenous cells lining the pycnidial wall, holoblastic, seemingly monoblastic, subcylindrical, lageniform or oblong, straight or curved, solitary, occasionally branched, 4.512.5 1.52 m, hyaline, smooth. conidia probably mucilaginous, acerose to narrowly cymbiform, mostly straight or slightly curved or sigmoid, attenuated towards a basal subtle heel continuing as a short cylindrical penducle and ending in a rounded base, aseptate, guttulate, hyaline, smooth, 1434.5 23.5 m. cultures : slow growing, to 76 mm diam after 23 d, either totally immersed or flat to slightly raised centrally, with radiating grooves of compressed medium, immersed at periphery ; felt - like or entirely slimy centrally, comprising a dense, rosy vinaceous mat of dark brown monilioid hyphae within a pale hyphal matrix ; embedded, black setose pycnidia densely formed on pca in light, producing a white - creamy ooze of cylindrical to oval hyaline conidia (24 1.52 m) and oblong hyaline spermatia (1.5 1 m) ; pycnidia fewer and sterile in the dark. the sequences generated from both collections were identical (genbank accession numbers its jx843524 ; lsu jx843525 ; ssu jx843526). the two claviradulomyces spp. formed a strongly supported clade within the ostropales, but the family level relationship within the order was not resolved (fig. diagnosis : similar to clavariadulomyces dabeicola, from which it is distinguished by the longer periphysoids (1639 m, shorter asci (3550 m), a longer ostiolar neck in the asexual state, shorter conidia (1434.5 m) with a discrete heel region, and molecular sequence data. type : brazil : minas gerais : viosa, pina, on living branches of xylopia sericea (annonaceae), 11 june 2010, r.w. barreto (vic31417 holotype ; cbs 133260 and cbs 133261 ex - holotype cultures). mata do seu nico, fazenda bonsucesso, on living branches of xylopia sericea, 20 may 2010, h.c. description : internal mycelium intra- and intercellular, 12 m diam, septate, branched, hyaline. ascomata erumpent from spongy tissues of lenticels on bark of living branches ; apothecial when mature and turgid, but perithecium - like and opening by a large, round pore when dry ; sessile, urceolate, 0.120.25 mm diam, wall black, extending above the surface of the hymenium, partially covering the hymenium when dry, opening pore lined with a whitish fringe of periphysoids. in vertical section, lower part of ascomatal wall often ill - formed and restricted to a loose hyphal layer from which asci and paraphyses arise, 515 m thick, composed of tangled hyphae 12 m diam ; upper part of wall dark brown to 37 m thick, narrowing and becoming paler towards the base and there 1012 m wide. periphysoids lining the upper wall above the level of the hymenium, cylindrical or club - shaped, sinuose or curved, 1639 m long and 34 m wide along the axis, often slightly swollen in the upper part up to 6 m, wall hyaline along most of the length, bearing abundant blunt denticles, arising from short brown smooth basal stalks. paraphyses 12 m wide, to 60 m long, apex swollen and bulbous, to 57 m wide and bearing abundant blunt denticles, imparting a mace - like or muricate appearance, extending beyond the asci but usually prostrate over the hymenial surface. asci parallel, clavate with a broadly rounded to somewhat flattened apex, becoming ellipsoidal when free of the hymenium, without a basal stalk, 3550 510 m, apex non - amyloid, 8-spored. ascospores in single fascicles extending to the base of the ascus, parallel to spirally or partly spirally arranged in the upper half, cylindrical to vermiform, attenuating towards the sub - acute ends, straight to slightly curved or sigmoid, sometimes strongly curved at the apices, (21) 2843 23 m, aseptate, hyaline, smooth, strongly guttulate. asexual morph : formed separately or in combination with the sexual morph in the same lenticel. conidiomata pycnidial, semi - immersed, globose, 76137 m diam ; thin walled, walls 415 m thick, with long cylindrical ostiolate necks, 110360 2446 m, composed of parallel hyphae 13 m wide, often reduced to a narrower cylinder of bristle - like hyphal tips at the apex, 1521 15 m. conidiophores usually reduced to conidiogenous cells, occasionally consisting of a small stalk of one or two cells. conidiogenous cells lining the pycnidial wall, holoblastic, seemingly monoblastic, subcylindrical, lageniform or oblong, straight or curved, solitary, occasionally branched, 4.512.5 1.52 m, hyaline, smooth. conidia probably mucilaginous, acerose to narrowly cymbiform, mostly straight or slightly curved or sigmoid, attenuated towards a basal subtle heel continuing as a short cylindrical penducle and ending in a rounded base, aseptate, guttulate, hyaline, smooth, 1434.5 23.5 m. cultures : slow growing, to 76 mm diam after 23 d, either totally immersed or flat to slightly raised centrally, with radiating grooves of compressed medium, immersed at periphery ; felt - like or entirely slimy centrally, comprising a dense, rosy vinaceous mat of dark brown monilioid hyphae within a pale hyphal matrix ; embedded, black setose pycnidia densely formed on pca in light, producing a white - creamy ooze of cylindrical to oval hyaline conidia (24 1.52 m) and oblong hyaline spermatia (1.5 1 m) ; pycnidia fewer and sterile in the dark. the sequences generated from both collections were identical (genbank accession numbers its jx843524 ; lsu jx843525 ; ssu jx843526). the two claviradulomyces spp. formed a strongly supported clade within the ostropales, but the family level relationship within the order was not resolved (fig. claviradulomyces xylopiae represents a novel species on an indigenous brazilian plant distantly related to erythroxylum mannii, the host of the type species, c. dabeicola, in west africa (evans. this suggests that fungi in this genus could have a pantropical distribution, perhaps as endophytic colonisers of tropical woody plants, with the ability to sporulate on the lenticular tissues of living plants. this somewhat cryptic niche has not traditionally been explored by mycologists, and this could explain the absence of previous records of this genus. however, it remains to be proven whether or not these fungi are benign endophytes, or acting as systemic pathogens that promote abnormal lenticel growth to facilitate sporulation, or opportunistic invaders of trees with bark disorders. claviradulomyces xylopiae has considerable similarity to c. dabeicola, and has the same muricate periphysoids, which characterise the genus (evans. it can, however, be separated from the type species by the longer periphysoids, 1639 m compared to 6.514 m, and shorter asci, 3550 m compared with 6075 m. nevertheless, the most significant morphological divergences are to be found in the asexual state of the two species. the ostiolar neck in the new species is long, sometimes reaching up to three times the length of the pycnidial body, whereas in c. dabeicola the neck is reduced to a short protrusion. conidia in c. xylopiae are also shorter, 1434.5 m in length, than in c. dabeicola where they measure 3342 m, and also have a discrete heel region not seen in c. dabeicola. (2010) referred claviradulomyces to odontotremataceae based on morphology. at that time, no dna sequences of odontotrematcaeae had been published. 2010) allowed a re - evaluation of the phylogenetic position of claviradulomyces, which we now regard as incertae sedis within ostropales. the morphological similarity with the odontotremataceae is not reflected in the phylogenetic position of these purported woody plant endophytes. | claviradulomyces xylopiae sp. nov. is introduced for a fungus occurring in association with abnormal (enlarged, spongy) lenticels of xylopia sericea (annonaceae), a common tree of the atlantic forest and cerrado ecosystems in brazil. this is the second species described in the genus and, although it is morphologically distinct from the type species, c. dabeicola from west africa, it possesses the same characteristics. apothecial ascomata have periphysoids and paraphyses that are inflated apically (clavate), and ornamented with denticles (raduliform). furthermore, similar to the type species, it also has long - cylindric or acerose, aseptate ascospores and conidia. an additional asexual morph was produced in culture and is described. molecular studies of c. dabeicola and the new species confirmed a placement in ostropales, although a relationship to odontotremataceae was not supported. both species were consistently in association with abnormal lenticular development on their woody hosts. it remains to be ascertained, however, if these are the causal agents of the bark disorders, or, simply, opportunistic colonisers. the finding of the second species in the genus claviradulomyces on a plant from a distantly related family to that of the host of c. dabeicola (erythroxylaceae) for the genus on a different continent suggests that fungi in this genus may be common on lenticels of other woody plants, and could even have a pantropical distribution. it is possible that fungi in the genus have remained unreported until now because lenticels have remained neglected as a habitat surveyed by mycologists. |
we selected 101 patients with type 2 diabetes (diagnosed by 1999 world health organization criteria). the mean patient age was 63.45 8.38 years, and all patients were hospitalized in the department of endocrinology at fujian provincial hospital between january 2010 and january 2011. this study was approved by the hospital internal review board, and all subjects provided written informed consent. the exclusion criteria were as follows : presence of a cns disease that could cause mci, history of head trauma, history of inflammatory or infectious brain disease, depression, drug or alcohol abuse or dependence, and use of possible or known cognition - impairing drugs in the previous month. based on the criteria established by the 2006 european alzheimer 's disease consortium (7) and the montreal cognitive assessment (moca) scoring system, 58 patients (30 males and 28 females) had moca scores 0.05). compared with the control group, patients with mci had a significantly longer duration of diabetes ; elevated total cholesterol (chol), ldl cholesterol (ldl - c), triglyceride (tg), crp, intima - media thickness (imt), and ba - pwv ; and lower abi (p 0.05 for all). relationship of moca score with other clinical indicators in patients with type 2 diabetes and mci finally, we performed spearman rank correlation analysis of esrage (dependent variable) with other clinical indicators (independent variables) in all 101 patients. the results indicated that esrage was positively correlated with moca score and abi (r = 0.803 and r = 0.214, respectively), but negatively correlated with ba - pwv, chol, tg, and crp (r = 0.371, r = 0.303, r = 0.274, and r = 0.308, respectively) (table 4). there was no significant correlation of esrage with age, duration of diabetes, hba1c, bmi, waist circumference, hdl, ldl, or imt (p > 0.05 for all). relationship of esrage with other clinical indicators in type 2 diabetic patients with and without mci (n = 101) table 1 shows the demographic and clinical characteristics of enrolled type 2 diabetic patients with and without mci. there were no significant differences between the groups in age, sex, bmi, waist circumference, morning urinary microalbumin, hdl cholesterol (hdl - c), duration of hypertension, and history of smoking (p > 0.05). compared with the control group, patients with mci had a significantly longer duration of diabetes ; elevated total cholesterol (chol), ldl cholesterol (ldl - c), triglyceride (tg), crp, intima - media thickness (imt), and ba - pwv ; and lower abi (p 0.05 for all). relationship of moca score with other clinical indicators in patients with type 2 diabetes and mci finally, we performed spearman rank correlation analysis of esrage (dependent variable) with other clinical indicators (independent variables) in all 101 patients. the results indicated that esrage was positively correlated with moca score and abi (r = 0.803 and r = 0.214, respectively), but negatively correlated with ba - pwv, chol, tg, and crp (r = 0.371, r = 0.303, r = 0.274, and r = 0.308, respectively) (table 4). there was no significant correlation of esrage with age, duration of diabetes, hba1c, bmi, waist circumference, hdl, ldl, or imt (p > 0.05 for all). relationship of esrage with other clinical indicators in type 2 diabetic patients with and without mci (n = 101) our study of patients with type 2 diabetes indicated that mci was associated with duration of diabetes ; elevated levels of hba1c, chol, ldl, tg, imt, crp, abi, ba - pwv, and esrage ; and low levels of serum ages. in agreement with our results, numerous other studies have also reported associations of diabetes with vascular brain damage (8), degenerative nerve disease (9), cognitive decline (10,11), and dementia or mci (12). in addition, mci is associated with the onset, longer duration, and greater severity of diabetes (13). diabetic patients have increased levels of ages, inflammation, and oxidative stress, all of which can affect blood supply to the brain. we observed an association between hyperlipidemia and mci perhaps because hyperlipidemia itself causes denaturation of neurons responsible for cognitive function, or because hyperlipidemia accelerates the progression of atherosclerosis, which leads to mci (14). in agreement with our results, other studies (15) reported that sustained hyperglycemia, as indicated by elevated hba1c, is an independent risk factor for impairment of cognitive function. there may be several pathogenic mechanisms underlying this process, including accumulation of sorbitol and development of a hyperosmotic state in nerve cells that leads to edema and impaired brain function (16), insulin resistance syndrome (17), impaired insulin homeostasis in the brain (18), and/or hyperinsulinemia (19). our results indicate that type 2 diabetes patients with mci had significantly elevated imt and ba - pwv but lower abi than the control group, indicating a correlation between atherosclerosis and type 2 diabetes (20), who reported that individuals with peripheral arterial disease had worse cognitive function than healthy controls, and with the research of hanon. (21), who reported a significant relationship of reduced cognitive function and arteriosclerosis (based on pwv) in 308 elderly patients. our results also indicate that type 2 diabetic patients with mci had significantly higher crp levels than controls, suggesting an association of inflammation and mci. in agreement with this result, xu. (22) reported that patients with higher serum crp had lower mini - mental state examination scores and higher risk for development of ad. other studies have also shown that ad patients have increased brain inflammation and that this leads to deposition of amyloid -protein (23), formation of senile plaques, and neurofibrillary formation, resulting in damage or death of neurons. inflammation also increases apolipoprotein e synthesis in stellate cells, which is associated with increased risk of ad (24). rage appears to promote ad through a positive feedback mechanism with amyloid -protein (25). another study reported that low expression of esrage in the hippocampus was associated with ad (26). thus, the mechanism by which ages induce mci in patients with type 2 diabetes may include one or more of the following : 1) ages induce oxidative stress directly or by binding to specific receptors such as rage, leading to upregulation of nuclear factor-b and its target genes, resulting in inflammation and neural damage ; 2) ages induce vascular endothelial dysfunction, and the increased permeability of blood vessels increases accumulation of ages in the vascular wall and causes hardening ; and 3) ages activate microglial cells and damage the microtubular structure, leading to neuron dysfunction. (28) reported that serum esrage levels were significantly decreased in mci patients and even lower in ad patients than in healthy controls. similar to these reports, our results indicated that esrage levels were lower in patients with an mci group than those with type 2 diabetes alone, suggesting that esrage may have a protective effect against diabetic mci. while the precise mechanism of this relationship remains unclear, one or more of the following may be responsible : 1) chronic hyperglycemia in type 2 diabetic patients with mci directly inhibits synthesis and secretion of esrage ; 2) accumulated ages bind to esrage, which leads to increased clearance of esrage ; and 3) inflammation, which has been implicated in mci, can also reduce esrage synthesis. our results also indicated that the moca score was positively correlated with serum esrage (r = 0.942) and negatively correlated with chol (r = 0.364) in type 2 diabetes patients with mci. thus, measurement of esrage and chol can be helpful in the prediction and/or diagnosis of mci in middle - aged and older populations. we also found that esrage was negatively correlated with chol and tg, consistent with previous studies (29). additionally, lindsey. (31) reported that esrage was independently and negatively correlated with coronary atherosclerosis. on the other hand, there is some evidence that elevated esrage is associated with increased cardiovascular disease (32,33). (34) reported that esrage was negatively correlated with high - sensitivity crp (hs - crp) and tumor necrosis factor- (tnf-), but that tnf- was positively correlated with hs - crp. this result suggests that the production of esrage may be regulated by tnf-related hs - crp. another study involving 245 type 2 diabetic patients without coronary artery disease shows negative correlation between esrage and hs - crp (35). consistent with these reports, we found that esrage was negatively associated with hs - crp (r = 0.308). in conclusion, our study indicates that a lower level of serum esrage and a higher level of serum age in patients with type 2 diabetes are associated with mci, dyslipidemia, and atherosclerosis. thus, we suggest that future investigators consider the hypothesis that therapeutic interventions that increase serum esrage may be a novel approach to prevent rage - mediated diseases such as mci and ad. | objectivedetermine the serum levels of endogenous secretory receptor for advanced glycation end products (esrages) in patients with type 2 diabetes and mild cognitive impairment (mci) and in control patients with type 2 diabetes but no mci, and examine the relationship of esrage and mci with other clinical factors.research design and methodsa total of 101 patients with type 2 diabetes who were hospitalized in the department of endocrinology at fujian provincial hospital between january 2010 and january 2011 were enrolled. there were 58 patients with mci and 43 patients without mci (control). serum levels of esrage were measured using an enzyme - linked immunosorbent assay (elisa). other clinical parameters were also measured.resultstype 2 diabetic patients with mci had a longer duration of diabetes ; elevated hba1c, total cholesterol (chol), ldl cholesterol (ldl - c), triglyceride (tg), intima - media thickness, c - reactive protein (crp), and brachial - ankle pulse wave velocity (ba - pwv) ; and lower ankle brachial index (abi) and esrage relative to the control group. among patients with mci, the montreal cognitive assessment (moca) score was positively correlated with serum esrage but negatively correlated with chol. spearman rank correlation analysis indicated that esrage was positively correlated with moca score and abi but negatively correlated with ba - pwv, chol, tg, and crp in all subjects.conclusionsour results suggest that esrage may be a potential protective factor for dyslipidemia, atherosclerosis, and mci in patients with type 2 diabetes. |
participants enrolled in this study included all patients aged 70 years or older in 3 large prospective cohort studies in japan. ucas japan (umin - ctr c000000418) was a project of the japan neurosurgical society and was designed as a multicenter, prospective cohort study of ucas in the japanese population. patients with newly diagnosed ucas from january 2001 to april 2004 were enrolled in ucas japan. in a total of 5,720 patients who were 20 years of age or older, 6,697 saccular aneurysms that were 3 mm or more at the largest diameter met the eligibility criteria. we excluded ucas less than 3 mm from this study because the diagnosis accuracy for computer images of ucas decreases when under 3 mm in diameter with low - tesla mri. follow - up data on the patients ' clinical status, a description of the aneurysms, and the treatment management plan were recorded at 3, 12, and 36 months and at 5 to 8 years. the therapeutic strategy was chosen by the patient or was determined at the physician 's discretion. when a patient underwent a surgical intervention, data up to the time of the intervention were included in the analysis of the risk of rupture. we extracted the data from patients who were 70 years of age or older from ucas japan and analyzed a total of 1,577 patients with 1,844 aneurysms in this study. subarachnoid hemorrhage (sah) was identified by means of ct imaging or lumbar puncture or was documented at autopsy. for 2 patients extracted from ucas japan, the diagnosis was made on the basis of sudden severe headache or loss of consciousness. ucas ii (umin - ctr c000000420) was conducted to clarify the influence of surgical intervention for ucas on periodic health - related quality of life and cognitive function as part of the analysis of uca surgical complications, and to assess the natural history of ucas. in ucas ii, patients with newly identified ucas from january 2006 to january 2007 at 31 institutions in japan were prospectively enrolled. the patients ' status and aneurysm characteristics were monitored at 3, 6, and 60 months after enrollment. a total of 953 patients with 1,106 aneurysms were enrolled in ucas ii, and the data from 234 patients who were aged 70 years or older with 268 aneurysms were extracted from ucas ii for this pooled analysis. the organization members of ucas japan and ucas ii are listed in supplemental data. from january 2003 to december 2006, a total of 419 patients with 529 ucas were referred to the jikei university school of medicine in japan and were prospectively observed without treatment at this single institution. the data from 85 patients aged 70 years or older with 115 ucas were integrated with those from ucas japan and ucas ii in this pooled analysis. the jikei university school of medicine has a role as referral center to surgically treat ucas. many patients enrolled in ucas japan and in ucas ii were registered from general hospitals that served as secondary care centers. the characteristics of the 3 prospective japanese studies that were used in this pooled analysis are summarized in table e-1 on the neurology web site at neurology.org. patient characteristics that are common to the 3 prospective studies include the following : age, sex, history of sah, family history of sah, former or current smoking, multiplicity, and hypertension. the aneurysm size was divided as follows : 34 mm, 56 mm, 79 mm, 1024 mm, and 25 mm. the aneurysm location was categorized as follows : middle cerebral artery, anterior communicating artery (acoma), internal carotid artery (ica), internal carotid posterior communicating artery (ic - pcoma), basilar artery (ba), vertebral artery (va), and other. the ica includes ica paraclinoid location, the ica dorsal curvature location, ica bifurcation, and ica anterior choroidal artery and excludes other ica aneurysms located at the posterior communicating artery and cavernous portion. other includes aneurysms at the anterior cerebral artery a1 portion, distal anterior cerebral artery, and other supratentorial or infratentorial locations that are not categorized above. data were censored at the time of a patient 's death, a surgical or endovascular intervention, or the last follow - up assessment. the aneurysm rupture hazard ratios were studied individually per patient using cox proportional hazard regression models. when a patient had multiple aneurysms, the largest of these aneurysms along with its location and with or without the daughter sac was used to categorize the patient. when variables associated with aneurysm rupture had a probability value less than 0.2 using a univariable analysis, they were selected for a multivariable analysis. the cumulative rates of sah were estimated per patient using the kaplan - meier product - limit method, and the curves between 2 groups were compared using the log - rank test. all statistical analyses were performed using stata software, version 13.1 (statacorp, college station, tx). statistical tests were 2-sided and differences were considered to be significant when p values were less than 0.05. participants enrolled in this study included all patients aged 70 years or older in 3 large prospective cohort studies in japan. ucas japan (umin - ctr c000000418) was a project of the japan neurosurgical society and was designed as a multicenter, prospective cohort study of ucas in the japanese population. patients with newly diagnosed ucas from january 2001 to april 2004 were enrolled in ucas japan. in a total of 5,720 patients who were 20 years of age or older, 6,697 saccular aneurysms that were 3 mm or more at the largest diameter met the eligibility criteria. we excluded ucas less than 3 mm from this study because the diagnosis accuracy for computer images of ucas decreases when under 3 mm in diameter with low - tesla mri. follow - up data on the patients ' clinical status, a description of the aneurysms, and the treatment management plan were recorded at 3, 12, and 36 months and at 5 to 8 years. the therapeutic strategy was chosen by the patient or was determined at the physician 's discretion. when a patient underwent a surgical intervention, data up to the time of the intervention were included in the analysis of the risk of rupture. we extracted the data from patients who were 70 years of age or older from ucas japan and analyzed a total of 1,577 patients with 1,844 aneurysms in this study. subarachnoid hemorrhage (sah) was identified by means of ct imaging or lumbar puncture or was documented at autopsy. for 2 patients extracted from ucas japan, the diagnosis was made on the basis of sudden severe headache or loss of consciousness. ucas ii (umin - ctr c000000420) was conducted to clarify the influence of surgical intervention for ucas on periodic health - related quality of life and cognitive function as part of the analysis of uca surgical complications, and to assess the natural history of ucas. in ucas ii, patients with newly identified ucas from january 2006 to january 2007 at 31 institutions in japan were prospectively enrolled. the patients ' status and aneurysm characteristics were monitored at 3, 6, and 60 months after enrollment. a total of 953 patients with 1,106 aneurysms were enrolled in ucas ii, and the data from 234 patients who were aged 70 years or older with 268 aneurysms were extracted from ucas ii for this pooled analysis. the organization members of ucas japan and ucas ii are listed in supplemental data. from january 2003 to december 2006, a total of 419 patients with 529 ucas were referred to the jikei university school of medicine in japan and were prospectively observed without treatment at this single institution. the data from 85 patients aged 70 years or older with 115 ucas were integrated with those from ucas japan and ucas ii in this pooled analysis. the jikei university school of medicine has a role as referral center to surgically treat ucas. many patients enrolled in ucas japan and in ucas ii were registered from general hospitals that served as secondary care centers. the characteristics of the 3 prospective japanese studies that were used in this pooled analysis are summarized in table e-1 on the neurology web site at neurology.org. patient characteristics that are common to the 3 prospective studies include the following : age, sex, history of sah, family history of sah, former or current smoking, multiplicity, and hypertension. the aneurysm size was divided as follows : 34 mm, 56 mm, 79 mm, 1024 mm, and 25 mm. the aneurysm location was categorized as follows : middle cerebral artery, anterior communicating artery (acoma), internal carotid artery (ica), internal carotid posterior communicating artery (ic - pcoma), basilar artery (ba), vertebral artery (va), and other. the ica includes ica paraclinoid location, the ica dorsal curvature location, ica bifurcation, and ica anterior choroidal artery and excludes other ica aneurysms located at the posterior communicating artery and cavernous portion. other includes aneurysms at the anterior cerebral artery a1 portion, distal anterior cerebral artery, and other supratentorial or infratentorial locations that are not categorized above. data were censored at the time of a patient 's death, a surgical or endovascular intervention, or the last follow - up assessment. the aneurysm rupture hazard ratios were studied individually per patient using cox proportional hazard regression models. when a patient had multiple aneurysms, the largest of these aneurysms along with its location and with or without the daughter sac was used to categorize the patient. when variables associated with aneurysm rupture had a probability value less than 0.2 using a univariable analysis, they were selected for a multivariable analysis. the cumulative rates of sah were estimated per patient using the kaplan - meier product - limit method, and the curves between 2 groups were compared using the log - rank test. all statistical analyses were performed using stata software, version 13.1 (statacorp, college station, tx). statistical tests were 2-sided and differences were considered to be significant when p values were less than 0.05. a total of 1,896 patients who were 70 years of age or older with 2,227 ucas from the 3 prospective cohort studies were investigated in this pooled analysis (figure e-1). two hundred six patients (10.9%) who were 80 years of age or older had 239 aneurysms. four hundred eighty - two (29%) of 1,690 patients aged 70 to 79 years and 8 (4%) of 206 patients 80 years or older underwent surgical repair of aneurysm during the follow - up period. patient characteristics aneurysm characteristics the median (interquartile range) and mean follow - up period were 990 (1031,115) and 802.7 days, respectively. sixty - eight patients (3.6%) experienced sah during the follow - up period and the overall annual rupture risk for these cohorts was 1.6% (68 sahs/4,167 patient - years) (95% confidence interval [ci ], 1.32.1). additional 2 cases with multiple aneurysms experienced sah not caused by the represented aneurysms, which were excluded from the patient - based analysis. the cumulative rate of sah for all patients was 3.8% (95% ci, 2.94.9) at 2 years after diagnosis, and 6.3% (95% ci, 4.68.5) at 5 years after diagnosis (figure 1a). in addition, the kaplan - meier curve analyzed according to age showed that the cumulative rates of sah in patients older than 80 years were higher than in those of patients aged 70 to 79 years (p < 0.001) (figure 1b). a total of 191 patients died during follow - up in this study : 46 patient deaths were related to sah and 145 patient deaths were not sah - related. (a) kaplan - meier curve showing the cumulative rates of sah for all patients. (b) kaplan - meier curve showing the cumulative rates of sah for patients in 2 groups according to age : 7079 years, and 80 years or older. results of a univariable and multivariable analysis of factors related to uca rupture per patient are summarized in table 3. patients 80 years or older (hazard ratio [hr ], 2.02 ; 95% ci, 1.163.49, p = 0.012), aneurysms 7 mm or larger (hr, 3.08 ; 95% ci, 1.357.03, p = 0.007 for 79 mm ; hr, 7.82 ; 95% ci, 3.6016.98, p < 0.001 for 1024 mm ; and hr, 43.31 ; 95% ci, 12.55149.42, p < 0.001 for 25 mm), and ic - pcoma aneurysms (hr, 2.45 ; 95% ci, 1.234.88, p = 0.011) were risk factors for uca rupture in a multivariable analysis. although female sex, multiplicity, and ba aneurysm were significant risk factors by the univariable analysis, they did not reach statistical significance by the multivariable analysis. the influence of difference among the 3 cohorts did not have statistical significance on rupture risk. risk factors associated with rupture : univariable and multivariable cox proportional - hazard analysis per patient the median (interquartile range) and mean follow - up period were 990 (1031,115) and 802.7 days, respectively. sixty - eight patients (3.6%) experienced sah during the follow - up period and the overall annual rupture risk for these cohorts was 1.6% (68 sahs/4,167 patient - years) (95% confidence interval [ci ], 1.32.1). additional 2 cases with multiple aneurysms experienced sah not caused by the represented aneurysms, which were excluded from the patient - based analysis. the cumulative rate of sah for all patients was 3.8% (95% ci, 2.94.9) at 2 years after diagnosis, and 6.3% (95% ci, 4.68.5) at 5 years after diagnosis (figure 1a). in addition, the kaplan - meier curve analyzed according to age showed that the cumulative rates of sah in patients older than 80 years were higher than in those of patients aged 70 to 79 years (p < 0.001) (figure 1b). a total of 191 patients died during follow - up in this study : 46 patient deaths were related to sah and 145 patient deaths were not sah - related. (a) kaplan - meier curve showing the cumulative rates of sah for all patients. (b) kaplan - meier curve showing the cumulative rates of sah for patients in 2 groups according to age : 7079 years, and 80 years or older. results of a univariable and multivariable analysis of factors related to uca rupture per patient are summarized in table 3. patients 80 years or older (hazard ratio [hr ], 2.02 ; 95% ci, 1.163.49, p = 0.012), aneurysms 7 mm or larger (hr, 3.08 ; 95% ci, 1.357.03, p = 0.007 for 79 mm ; hr, 7.82 ; 95% ci, 3.6016.98, p < 0.001 for 1024 mm ; and hr, 43.31 ; 95% ci, 12.55149.42, p < 0.001 for 25 mm), and ic - pcoma aneurysms (hr, 2.45 ; 95% ci, 1.234.88, p = 0.011) were risk factors for uca rupture in a multivariable analysis. although female sex, multiplicity, and ba aneurysm were significant risk factors by the univariable analysis, they did not reach statistical significance by the multivariable analysis. the influence of difference among the 3 cohorts did not have statistical significance on rupture risk. risk factors associated with rupture : univariable and multivariable cox proportional - hazard analysis per patient the prevalence of both unruptured and ruptured aneurysms increases with age, and this tendency is of peculiar note given the recent trends in population aging. it is important to stratify the risks of uca rupture exclusively in elderly patients and to effectively perform preventive surgical treatment for the patients who are at a high risk of rupture. all 3 studies that we used for this pooled analysis were representative of large - scale studies in japan ; we evaluated the risk factors for uca rupture exclusively in elderly patients in the largest prospective study. an aneurysm size of 7 mm or larger was an independent predictor for aneurysm rupture in elderly patients according to the multivariable analysis in this investigation. many reports also showed that aneurysm size was one of the most important factors in determining risk of subsequent aneurysm rupture. ucas japan reported that there was a significant positive correlation between the patient 's age and the aneurysm size : aneurysms 7 mm or larger were found in 32.6% of patients aged 70 to 79 years and in 39.7% of patients aged 80 years or older. the cumulative risk of aneurysm growth at 1, 3, 5, and 7 years was reported to be 2.8%, 7%, 15.3%, and 21.8%, respectively, in patients aged 70 years or older. in addition, it was demonstrated that the annual rupture risk after growth was 18.5% per person - year. these data indicate the dynamic nature of ucas in elderly patients, and thus careful observation is also warranted even in elderly patients. this pooled analysis demonstrated that, for the aneurysm site, only the ic - pcoma aneurysm was a significant risk factor for uca rupture in elderly patients. ba aneurysms had an increased risk of aneurysm rupture according to the univariable analysis. in ucas japan, the aneurysms most prone to rupture were located in the acoma and ic - pcoma. ucas japan also reported that the aneurysms located in ba, va, and ic - pcoma were significantly larger than those in middle cerebral artery, acoma, ica, and others and that elderly patients had significantly more posteriorly located aneurysms compared with nonelderly patients. the difference in the distribution of aneurysms regarding size and age may explain why only ic - pcoma aneurysms remained as a significant predictor of rupture in this pooled analysis, which was limited to elderly patients. the annual rupture rate of ucas in this study is higher than that in other reports for general japanese and non - japanese populations. in addition, our pooled analysis revealed that patient age of 80 years or older was an independent risk factor for aneurysm rupture in our study that exclusively comprised patients 70 years or older, and the cumulative rates of sah in patients 80 years or older were significantly higher than those in patients 70 to 79 years in this investigation. the prognosis of elderly patients with ruptured aneurysm is poor, but it has also been revealed that patients ' age was a strong predictor of surgical outcomes and major complication following endovascular uca treatment. in the analysis of endovascular treatment of ucas in elderly patients, the paradoxical relationship between the relatively high annual and cumulative risk of uca rupture and the risk for surgical treatment of ucas makes the management of elderly patients with ucas more difficult. when deciding therapeutic strategy for elderly patients with ucas, we have to consider various factors, such as the difference between chronological age and physiologic age, the difference in life expectancy between patients aged 70 to 79 years and those older than 80 years, and one 's view of life and death. it was reported that female sex was a significant risk factor for rupture of ucas. ucas japan also demonstrated that female sex was a predictor for rupture although the statistical value was marginal (p = 0.05). our pooled data did not indicate that female sex was a significant predictor for rupture in the multivariable analysis. in ucas japan, where the mean patient age was 62.5 10.3 years, the prevalence of aneurysms larger than 7 mm was higher in females than in males (p = 0.04, pearson test). this might partly explain the higher incidence of sah in nonelderly, female patients because larger aneurysms are more prone to rupture. however, there was no significant difference in the prevalence of aneurysms larger than 7 mm between women and men in this study, where the mean age of patients was 74.3 3.9 years (p = 0.40, pearson test). this is probably one of the reasons why female sex disappeared as a risk factor for aneurysm rupture in elderly patients with ucas in our pooled analysis. our analysis did not identify the shape of the aneurysm as a risk factor that influences rupture unlike ucas japan. first, the population in this pooled analysis was limited to japanese patients with ucas. the rate of uca rupture in the japanese population has been reported to be high, and the japanese population is an independent predictor of aneurysm rupture according to previously reported pooled data. therefore, the result in this report should be applied with caution to elderly patients with uca in other populations, because the geographical region is related to the risk of rupture. ideally, it is better to stratify the risk factors for uca rupture in elderly patients in each population. the baseline characteristics of patients with surgical / endovascular treatment and with observation are shown in table 4. the patients with smaller and ba aneurysms were prone to be treated conservatively, and ones with family history of sah and aneurysms with daughter sacs tended to be surgically treated. also, the difference in the frequency of surgical intervention between patients aged 70 to 79 years and patients older than 80 years could be a cause of the bias associated with age. it is a crucial perception that multivariable analysis in this study revealed that patient age 80 years or older was a significant risk for uca rupture regardless of the age - related bias. these types of biases are thought to be unavoidable because of comorbidity, physiologic age of the patients, and the wishes of the patients and their families. third, the number of variables investigated in this study is relatively small because we assessed the variables common to 3 prospective studies. to determine the influence of comorbid diseases on the risk of uca rupture in elderly patients, hyperlipidemia, diabetes mellitus, polycystic kidney disease, cerebral infarction, ischemic heart disease, and malignant tumor should be investigated. in addition, it is important to investigate the medications administered to elderly patients with ucas and evaluate the risks and benefits of these medications for ucas. ucas japan was funded by the ministry of health, labor and welfare of japan and others (ucas japan umin - ctr c000000418). ucas ii was funded by the ministry of health, labor and welfare of japan (ucas ii umin - ctr c000000420). t. hishikawa, i. date, k. tokunaga, s. tominari, k. nozaki, y. shiokawa, and k. houkin report no disclosures relevant to the manuscript. y. murayama reports grants from stryker, siemens, and ntt docomo and personal fees from stryker and asahi intecc, outside the submitted work. t. ishibashi reports grants from stryker, siemens, and ntt docomo and personal fees from stryker, outside the submitted work. h. takao reports grants from stryker, siemens, and ntt docomo, outside the submitted work. t. kimura, t. nakayama, and a. morita report no disclosures relevant to the manuscript. | objectives : the aim of this study was to identify risk factors for rupture of unruptured cerebral aneurysms (ucas) in elderly japanese patients aged 70 years or older.methods:the participants included all patients 70 years of age or older in 3 prospective studies in japan (the unruptured cerebral aneurysm study of japan [ucas japan ], ucas ii, and the prospective study at the jikei university school of medicine). a total of 1,896 patients aged 70 years or older with 2,227 ucas were investigated. the median and mean follow - up periods were 990 and 802.7 days, respectively.results:the mean aneurysm size was 6.2 3.9 mm. sixty - eight patients (3.6%) experienced subarachnoid hemorrhage during the follow - up period. multivariable analysis per patient revealed that in patients aged 80 years or older (hazard ratio [hr ], 2.02 ; 95% confidence interval [ci ], 1.163.49, p = 0.012), aneurysms 7 mm or larger (hr, 3.08 ; 95% ci, 1.357.03, p = 0.007 for 79 mm ; hr, 7.82 ; 95% ci, 3.6016.98, p < 0.001 for 1024 mm ; and hr, 43.31 ; 95% ci, 12.55149.42, p < 0.001 for 25 mm) and internal carotid posterior communicating artery aneurysms (hr, 2.45 ; 95% ci, 1.234.88, p = 0.011) were independent predictors for uca rupture in elderly patients.conclusions:in our pooled analysis of prospective cohorts in japan, patient age and aneurysm size and location were significant risk factors for uca rupture in elderly patients. |
there has been a progressive increase in the incidence of type 1 dm, and several advances in its treatment have been achieved. as a result, an increasing number of patients, who are older and have longer disease durations, are more severely affected by chronic complications [13 ]. chronic subclinical inflammation, impaired fibrinolytic system activity, and elevated procoagulant factor levels form the basis of atherosclerotic diseases. consequently, dm has been regarded as a major risk factor for cardiovascular diseases [4, 5 ]. insulin resistance is characterized by limited stimulation of glucose metabolism in muscle and the liver and has been described in patients with poorly controlled type 1 dm [68 ]. cytokines such as leptin, resistin, and adiponectin released from these tissues critically impact nutritional status, body fat distribution, metabolic parameters, inflammatory status, atherosclerotic alterations, and insulin resistance. the levels of adiponectin, which is regarded as an antidiabetic, anti - inflammatory, and antiatherogenic cytokine, are reported to be depressed in patients with type 2 dm [10, 11 ]. however, in some studies, increased adiponectin levels have been reported in patients with type 1 dm. resistin, on the other hand, impairs cellular glucose intake, because it is stimulated by insulin ; as a result, hepatic glucose production is increased, leading to impaired glucose tolerance and eventual development of insulin resistance. owing to its augmenting effect on the production of adhesion molecules, resistin is considered to be having proinflammatory effects in the vascular endothelium [13, 14 ]. leptin has been shown to have important effects on both body energy balance and fat distribution [15, 16 ]. glucose uptake rates by peripheral tissues, which can be stimulated by insulin in skeletal muscles, decrease over time in patients with type 1 dm and poor glycemic control. these patients have significant hepatic insulin resistance, and the effects of insulin are impaired because of plasma free fatty acids (ffas) [1719 ]. despite recent advances in the management of dm, it has been suggested that cardiovascular disease - related mortality rates increase as more intensive therapies are required to ensure tight blood glucose control. therefore, combination therapies are required to ensure tight glycemic control, minimize the risk of macrovascular disease, and reduce other cardiovascular risk factors. thiazolidinediones (tzds) act by binding to nuclear peroxisome proliferator activated receptor - gamma (ppar-), which is chiefly expressed in fatty tissue, and mediate their effects by activating the transcription of the genes that influence adipocyte differentiation as well as glucose and lipid metabolism [2123 ]. tzds, apart from their direct effect on fatty tissue, might influence the release of adipocyte - derived signal factors that determine the insulin sensitivity of muscles, such as ffas, adiponectin, leptin, and tumor necrosis factor - alpha (tnf-). in addition to their favorable effects on glycemic control, tzds directly influence vessel walls, decrease vasoconstriction, and inhibit inflammation. therefore, they inhibit insulin resistance and slow down the atherosclerotic process [2427 ]. in addition, it has been shown that tzds could inhibit hyperglycemia - induced reactive oxygen species production from mitochondria (mtros) by activating the ppar- coactivator-1 alpha (pgc-1) pathway which could contribute to the prevention of diabetic vascular complications [28, 29 ]. agonists of ppar - gamma and ppar - alpha have been shown to upregulate the heme - oxygenase- (ho-) system which has been shown to increase insulin sensitivity, improve glucose / lipid metabolism, suppress inflammation / oxidative stress, decrease immune response, and modulate cell - growth / differentiation. it has been also shown that there were beneficial effects of the ho - system in the pathogenesis of type 1 diabetes and related cardiometabolic complications. although, euglycemic - hyperinsulinemic clamp study is accepted standard for measurement of insulin sensitivity in patients with type 1 dm, it is not practical for use and is labor - intensive. estimated glucose disposal rate (egdr) is a derived measure of insulin resistance and can be calculated using routine clinical measures such as waist circumferences or waist - to - hip ratio, presence of hypertension, and hba1c levels. as an insulin sensitivity index, it is well correlated with results obtained from clamp studies and it should be emphasized that lower egdr levels indicate greater insulin resistance [32, 33 ]. the purpose of the present study was to investigate the efficacy of combined therapy of insulin and the oral antihyperglycemic agent rosiglitazone, a ppar- agonist, on blood glucose regulation, total administered daily insulin dose, metabolic parameters, egdr, ffas, inflammatory indicators, and adipocytokine levels in patients with type 1 dm and poor glycemic control despite intensive insulin therapy. the study was conducted between march 2007 and january 2008 at the clinic of endocrinology and metabolism diseases, following the approval of the uluda university medical school ethics committee. after providing written consent, the patients were considered eligible based on the following criteria : age 1865 years, diagnosis of type 1 dm, and a glycosylated hemoglobin (hba1c) level > 6.5% despite 40-unit (u) intensive insulin therapy, on average, for 6 months. exclusion criteria were renal failure (glomerular filtration rate 1.5 mg / dl), chronic hepatic disease or aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels 2.5 times the normal values, current antidiabetic therapy other than insulin therapy, known history of rosiglitazone allergy, stage ii iv heart failure according to the new york heart association (nyha) classification, inability of the patient to enforce strict lifestyle changes, medical nutrition therapy or self - monitoring of blood glucose levels, ongoing or planned pregnancy, and current lactation. this prospective, open - label, randomized trial investigated the effectiveness of 4 mg / day rosiglitazone for 18 weeks in patients undergoing insulin therapy and without acute metabolic complications in combination with strict lifestyle changes and effective medical nutrition therapy. the patients attended a screening visit (visit 1) two weeks before randomization, and they were classified into two open - label groups, such that clinical and demographical characteristics were similar in the two groups. the patients were evaluated for lifestyle changes, diet and exercise compliance, and insulin requirements. their therapies were modified, and the randomization visit (visit 2) occurred two weeks later. the patients in both groups underwent insulin titration at visit 2, and the patients in one group had 4 mg / day rosiglitazone added to their ongoing therapy (group 1), while the patients in the other group were monitored from that point with their most recent insulin titration (group 2). to reduce the risk of side effects, 4 mg rosiglitazone then, the patients attended a control visit (visit 3) four weeks after randomization and a final visit (visit 4) after 16 weeks. during each of the four visits, variables within six different categories were monitored, including a detailed physical examination ; self - monitoring of bg levels ; glycemic control ; therapy alterations and insulin requirements ; adverse events ; and biochemical, hematological, and inflammatory parameters, including adipocytokine levels. in order to measure the direct effect of rosiglitazone recurrent hypoglycemia, presence of symptomatic hyperglycemia, diabetic ketosis (dk), diabetic ketoacidosis (dka), and nonketotic hyperosmolar syndrome (nkhs) were considered metabolic complications. while patients with dk, dka, nkhs, or major hypoglycemia were planned to be excluded from the study and hospitalized if necessary, patients with symptomatic hyperglycemia and recurrent minor hypoglycemia underwent insulin titration. patients with symptoms of polydipsia, polyuria, weight loss, and nocturia were considered to be having symptomatic hyperglycemia if they also had mean bg levels > 276 mg / dl ; minor hypoglycemia if they were aware enough of their condition to administer self - therapy, were symptomatic, and had bg levels 1.1 u / kg were administered 8 mg / day rosiglitazone. the investigators reported that the change in hba1c levels was not significant when compared with the placebo group. for decades, type 1 diabetes has been traditionally known as insulin - dependent, while type 2 has been known as noninsulin - dependent diabetes. however, it is becoming increasingly clear that insulin deficiency and insulin resistance are manifested in both forms of diabetes at different stages. unlike other studies [39, 40 ] which investigate the effects of tzds on insulin resistance and consist of obese or overweight patients with type 1 dm, almost all of the patients were lean in present study. egdr is a validated clinical tool for estimating insulin sensitivity in patients with type 1 dm. it was near normal at baseline and did not change significantly during our study period in both groups. this result was important to show effects of rosiglitazone, except insulin - sensitizing characteristics. thus, it can be thought that the effects of rosiglitazone observed in present study were not associated with insulin sensitivity. one of the known major effects of these drugs is on oxidative stress and mitochondrial ros production. it has been shown that tzds could inhibit hyperglycemia - induced mtros and contribute to the prevention of diabetic vascular complications. although there were some studies investigating these effects in patients with type 2 dm, it is unclear in patients with type 1 dm. it is obvious that there is a need to investigate this effect in prospective cohort studies composed of patients with type 1 dm. in addition, agonists of ppar - gamma might increase insulin sensitivity via upregulating heme - oxygenase - system. the ho - system and related products have been shown to decrease inflammation and enhance insulin sensitivity. more importantly, in experimental models of type 1 dm, upregulating ho - system caused increase in pancreatic beta cell insulin production. beneficial effects of tzds on insulin resistance and inflammation resulting from ho - system have been studied in patients with both type 2 and type 1 dm [30, 31, 42 ]. these developments may offer new options, either prevention of disease or development of the complications. we observed significant bw and bmi changes in all patients at the final visit compared with baseline values. the most significant side effect of tzds, particularly when combined with sulfonylurea and insulin, is weight gain. tzds increase overall fat tissue, and the most affected area is subcutaneous fat tissue. another major cause of tzd - related weight gain is increased water and salt retention, which leads to increased plasma volume. edema is caused by depressed renal sodium excretion and free water retention [4345 ]. reported a 10% increase in left ventricular mass without significant alterations in cardiac structure and function in patients undergoing rosiglitazone therapy. although we observed weight gain in our patient groups in this present study, no patient had heart failure. moreover, peripheral edema or dyspnea was not among the adverse events reported by our patients. this could be attributed to the relatively young mean age in our sample or to the fact that heart failure at baseline was among the exclusion criteria. furthermore, no patient in this cohort had diabetic nephropathy that could lead to edema, which might have decreased the risk for edema. the hypoglycemic effects of tzds include increased insulin sensitivity that is mediated through tg and ffa metabolism and is associated with the agonistic effects of ppar-. increased ffa levels lead to insulin resistance and fasting plasma ffa levels in patients with type 2 diabetes administered tzd might decrease by 2030%. ppar- activation impairs tg and fatty acid synthesis, leading to decreased very - ldl - c and hdl - c synthesis as well as increased ldl - c and total - c levels [47, 48 ]. the patients in the present study with bmi and wc levels in the normal range had baseline tg and hdl - c levels within the desired range, and the ldl - c levels in all patients were close to 100 mg / dl. while slight decreases in total - c, ldl - c, tg, and ffa levels were noted in the patients receiving rosiglitazone at the end of the present study, hdl - c levels had increased slightly. patients receiving only insulin had similar values to those in the combined therapy group except for slightly higher tg levels, although this was not statistically significant. we suggest that the strict enforcement of lifestyle changes during the follow - up period might have contributed to these results. tzds have been demonstrated to have significant anti - inflammatory characteristics in studies conducted on patients with type 2 diabetes. reported that rosiglitazone reduces diabetes - related atherosclerosis and that this effect is possibly associated with oxidative stress and inflammation, independent of metabolic effects, unrelated to the insulin dose. in particular, the diabetes control and complications trial, as well as a number of other studies, revealed that hs - crp levels increased in patients undergoing intensive therapy. furthermore, hs - crp and increased fibrinogen levels are independent risk factors for coronary heart disease, and a number of studies have reported elevated fibrinogen levels in patients with diabetes [50, 51 ]. the esr levels in the present study were within the normal range at baseline, and they continued to be so until the end of study. while hs - crp levels decreased with respect to baseline values in the patients receiving rosiglitazone in the present study, they increased slightly in the patients receiving insulin alone. to the best of our knowledge, this is the first study to measure fibrinogen levels in patients with type 1 diabetes after rosiglitazone administration. the levels decreased significantly in these patients, while the patients undergoing therapy with insulin alone showed minimal decreases in fibrinogen levels. independent of the improvement in bg regulation, levels of hs - crp and fibrinogen, which are conventional inflammatory markers, decreased significantly following rosiglitazone therapy in the present study. plasma leptin levels are positively correlated with female sex, bmi, and age but not with diabetes duration, hba1c, or total insulin dose per kilogram. most patients with type 1 diabetes are either underweight or in the normal bw range. data concerning leptin levels in this group of patients vary, and exogenous insulin therapy leads to elevated leptin levels in patients with type 1 diabetes [53, 54 ]. resistin, which is released from adipose tissue, is directly associated with insulin resistance factors such as wc and whr. reported increased serum resistin levels in patients with type 1 diabetes and reported that levels returned to the normal range after pancreas transplant [55, 56 ]. while no significant differences were observed between the groups at the baseline or final visit or in the change between the visits, the within - group changes during the follow - up period were significant, despite the weight gain. resistin levels were low in both groups, and the change in the patients receiving rosiglitazone was significant, despite weight gain, and was associated with the favorable effects of rosiglitazone. adiponectin has been shown to have positive effects on cardiometabolic risk, and adiponectin levels are negatively correlated with insulin resistance and weight gain. moreover, good glycemic control increases adiponectin levels, whereas poor glycemic control decreases adiponectin levels [57, 58 ]. in the cacti trial, maahs. reported negative correlations between adiponectin levels and male sex, central adiposity, sbp, dbp, daily insulin dose, hba1c, fibrinogen, albumin excretion rate, and tg levels ; positive correlations were noted with type 1 diabetes, hdl - c, and homocysteine. in the present study, adiponectin levels increased parallel to glycemic improvement both in patients undergoing combined therapy and in those receiving insulin therapies alone. the diverse favorable effects of tzds previously reported in patients with type 2 diabetes were not fully experienced in patients with type 1 diabetes in the present study. the addition of 4 mg / day rosiglitazone to intensive insulin therapy did not significantly improve glycemic parameter, lipid parameter, ffa, esr, hs - crp, leptin, or resistin levels. patients receiving rosiglitazone showed weight gain, a major side effect of tzds, whereas insulin sensitivity was not significantly different and the incidence of hypoglycemia was not lower. hba1c, fpg, and total daily insulin doses decreased significantly after combination therapy with tzd and insulin in patients with type 2 diabetes mellitus, suggesting that the insulin - sensitizing characteristics of tzds are likely more pronounced in patients who are not totally devoid of endogenous insulin secretion. we acknowledge the limitations of the current study including single centre experience and small sample size. patients treated in our series did not appear to have driven any meaningful benefit from combination therapy. due to small number of patients included in study, prospective clinical trials are however required to define optimal treatment regimens. clinical prognostic factors evaluated in our series may be useful for stratification and eligibility considerations in future clinical trials. | aim. to investigate the efficacy of combined therapy of insulin and rosiglitazone on metabolic and inflammatory parameters, insulin sensitivity, and adipocytokine levels in patients with type 1 diabetes mellitus (type 1 dm). material and methods. a total of 61 adults with type 1 dm were randomly and prospectively assigned in open - label fashion to take insulin and rosiglitazone 4 mg / day (n = 30) or insulin alone (n = 31) for a period of 18 weeks while undergoing insulin therapy without acute metabolic complications. results. combination therapy did not significantly improve metabolic and inflammatory parameters, insulin sensitivity, and adiponectin levels. while leptin and resistin levels decreased in both groups (group 1 : resistin 6.96 3.06 to 4.99 2.64, p = 0.006 ; leptin 25.8 17.6 to 20.1 12.55, p = 0.006 ; group 2 : resistin 7.16 2.30 to 5.57 2.48, p = 0.031 ; leptin 16.72 16.1 to 14.0 13.4, p = 0.007) hgb and fibrinogen levels decreased only in group 1 (hgb 13.72 1.98 to 13.16 1.98, p = 0.015, and fibrinogen 4.00 1.08 to 3.46 0.90, p = 0.002). patients in both groups showed weight gain and the incidence of hypoglycemia was not lower. discussion. the diverse favorable effects of tzds were not fully experienced in patients with type 1 dm. these results are suggesting that insulin sensitizing and anti - inflammatory characteristics of tzds were likely to be more pronounced in patients who were not totally devoid of endogenous insulin secretion. |
contraceptive implants have been licensed in over 60 countries in the world and used by millions of women for over four decades.1 they are highly effective and safe with evidence suggesting that women do not experience serious health events at rates higher than those who use nonhormonal methods or women in the general population.12 other benefits of the implants include their ease of use, long duration of action, noninterference with intercourse, little ongoing attention, and immediate return to fertility after removal.23456 disruption of the menstrual bleeding pattern, especially in the early months after insertion, is their major drawback, a problem they share with all other progestin - only contraceptives2 and account for the most number of early discontinuations.45678910 this unpredictability of the bleeding pattern causes immense nuisance and negatively affects women 's daily lives and restricts their social, sexual, community, and religious activities in many climes.10 in addition, initiation and discontinuation of implants including jadelle are provider dependent, a factor that can open users to abuse and coercive prescribing by providers.2310 jadelle contains levonorgestrel just like the 6-rod norplant but has the comparative advantage of containing only two rods, which makes both insertion and removal a lot easier.10 the literature demonstrates that jadelle and norplant are similar in many respects, except in the higher incidence of removal - related adverse events that occurred more with norplant.101112 the main contraceptive mechanism of these implants is the inhibition of ovulation through the suppression of the luteinizing hormone surge. other secondary mechanisms are the increase in the viscosity of the cervical mucus making it impenetrable to spermatozoa and the thinning out of the endometrial lining making it atrophic.410 jadelle was introduced into the contraceptive method mix of the jos university teaching hospital in october 2007. our facility has had experience with two different implants before this time : norplant which was introduced in our center in 1985 and implanon in may 2006. there was therefore the need to understand the use - dynamics of jadelle after the withdrawal of norplant. besides, there are generally limited data on the pattern of use, continuation rates, and reasons for discontinuation of jadelle in northern nigeria. the objective of the study therefore was to determine the sociodemographic profiles of acceptors of jadelle and the reasons for discontinuation in a region with a high fertility rate. this was a retrospective chart review carried out at the family planning clinic of the jos university teaching hospital between october 2007 and october 2013 among women who accepted jadelle a levonorgestrel - containing contraceptive implant. all women who opted for and had jadelle inserted after counseling were included in the study. high body weight, elevated blood pressure, or the presence of any chronic medical conditions did not constitute any reason to preclude the insertion of the implant. the only exclusion criteria were known or suspected pregnancy, hypersensitivity to levonorgestrel, or being < 6 weeks postpartum. all contraceptive methods including jadelle were provided free to clients in our unit although a surcharge of 1000 naira was collected for consumables. data on the sociodemographic characteristics, reproductive history, medical history, presence of any chronic condition, and previous experience with contraceptives were extracted from the charts. the dates of insertion and removal of the implant were noted as well as the presence of any complication and/or adverse effect from the use of the contraceptive method. for those who had their jadelle removed during the study period, the length of implant use in months was noted in addition to the reason for the discontinuation. a urine pregnancy test was usually carried out to rule out any existing pregnancy before insertion. this was also repeated once there was any suspicion of a pregnancy with the implant in situ. if the pregnancy test became positive while using the implant, an ultrasound scan was carried out to date the pregnancy. they were however advised to return to the clinic any time they experienced any untoward effect with the method or they wanted it removed. during each follow - up visit, the subjects were asked about the presence of any adverse effect from the use of the implant. those women who did not come for any of the yearly visits and the contraceptive efficacy of jadelle was determined by the number of in - treatment pregnancies that occurred. this was a chart review and therefore the sample size was based on clinical feasibility and not any formal statistical power calculation. every woman who initiated jadelle had her profile included in the analysis of the sociodemographic characteristics. descriptive and analytic statistics were carried out on all data where appropriate using epi info version 3.5.1 (cdc, atlanta, ga, usa). the descriptive statistics were frequency and percentage for categorical variables while mean, median, standard deviation, and interquartile range were used for noncategorical variables. this was a chart review and therefore the sample size was based on clinical feasibility and not any formal statistical power calculation. every woman who initiated jadelle had her profile included in the analysis of the sociodemographic characteristics. descriptive and analytic statistics were carried out on all data where appropriate using epi info version 3.5.1 (cdc, atlanta, ga, usa). the descriptive statistics were frequency and percentage for categorical variables while mean, median, standard deviation, and interquartile range were used for noncategorical variables. during the study period, 1401 women accepted jadelle as family planning methods in the family planning clinic of the jos university teaching hospital, jos nigeria. sociodemographic characteristics of jadelle acceptors women from all but four states (bayelsa, kebbi, lagos, rivers) of the federal republic of nigeria accepted jadelle during the study period with 4 states (plateau [66.4% ], kaduna [4.4% ], benue [3.7% ], and nasarawa [2.3% ]), constituting at least 2% of the acceptors. about 132 different ethnic groups across nigeria were represented with about a dozen ethnic groups accounting for over three - quarters (75.8%) of the acceptors : berom (26.8%), afizere (6.4%), hausa (5.8%), yoruba (5.5%), mwaghavul (5.1%), ron (4.5%), igbo (4.2%), irigwe (4.2%), ngas (3.8%), rukuba (2.7%), idoma (2.7%), taroh (2.1%), and fulani (2.0%). the mode of delivery in the last confinement before initiating jadelle is depicted in figure 1. figure 1 also shows the number of women whose delivery was attended by a complication. the mode of delivery / presence of complication in the most recent pregnancy the mean parity of the subjects who initiated the jadelle implant was 4.1 1.9. the parity and other components of the reproductive history of the women are summarized in table 2. reproductive history of jadelle acceptors about 90% of all the women who opted for jadelle as a contraceptive method had regular menstrual cycles at the time of accepting the method. percentage of women with regular and irregular menstruation at insertion of jadelle just under half of the women (49.5%) were breastfeeding and over half (55.9%) had future fertility desires at the time of commencing the use of jadelle. about 82% had some experience with other contraceptive methods with only 18% starting jadelle as the first - ever contraceptive method. figure 3 shows the proportion of the women breastfeeding at the time of initiating jadelle and their future reproductive intentions. it also presents the proportion of women with previous contraceptive experience, while figure 4 shows the percentages of the different types of contraceptive methods used by the women previously. proportion of subjects that were breastfeeding, had previously used contraceptives and their future fertility desires at insertion of jadelle types of contraceptives and percentages of jadelle acceptors that had used each previously about 42 women had jadelle reinserted after the expiration of the first one. further, during the study period, 337 women discontinued the implant for different reasons. most women discontinued the jadelle so as to fulfill a further fertility desire after using the implant for an average of 29.6 months. however, menstrual irregularity accounted for 34.6% of the discontinuation in the first 6 months of use. two women had their jadelle removed, following its failure to protect them against pregnancy ; the first after 26 months of use while she was 37 years and weighed 67 kg and the second after 55 months of use at 32 years and she weighed 88 kg at the time of the failure. the average period of use of the implant generally in this study was 28.8 months. during the study period, 1401 women accepted jadelle. the mean age of the women was 33.4 5.9 years. there were only five teenagers which accounted for < 0.5% of those who accepted the method. even worse is the initiation of long - acting reversible contraceptives (larcs) by this age group.1314 lack of access to larc method is one of the reasons advanced by adolescents for not using them.13 in nigeria, family planning clinics are not adolescent - friendly, especially for unmarried adolescents, since it is regarded as the exclusive preserve of the married.131516 this situation is much different from many climes in europe and america where teenagers constitute a significant proportion of users of family planning clinics and larc methods including implants.171819 over 73% of the jadelle initiators had at least secondary school education or more while 88.8% were christians. education and religion in nigeria are important predictors of the use of modern contraceptives.202122 in a representative sample of nigerian women, it was found out that women with tertiary education were 2.10 times more likely to have ever used modern contraceptives than women with no formal education. in addition, christians were 1.41 times more likely than their muslim counterparts to have used a modern contraceptive method.20 the means of parity, number of children born alive, and number of children still alive to women in this study at baseline were 4.1, 4.1, and 3.8, respectively. despite these figures, over half of the women (55.9%) still had desires to have more children in future. this is an attestation to the high total fertility rate (tfr) in nigeria put at 5.7. tfr is higher in northern nigeria than in the southern part of the country.1623 the contraceptive prevalence rate in nigeria is generally low.1624 however, majority (82%) of the women in this study had used at least one form of modern contraceptive in the past. figure 4 shows that there was an overrepresentation of the short - acting methods such as injectables, oral contraceptive pills, and condoms in the women 's previous contraceptive experience. previous research had shown that in sub - saharan africa, the contraceptive method mix practiced by women was mostly dominated by the less effective short - acting methods such as pills and injectables.2526 majority of women in our study discontinued the implant to fulfill a further desire to have more children. these women used jadelle for an average of 29.6 months before planning the next pregnancy. this was not surprising since 55.9% of the women had expressed a desire for more children at baseline. about 41% of the women at baseline accepted jadelle even though they felt that they had completed their family size. have shown that in some nigerian cultures, sterilization is associated with loss of vitality and related to sterility in the afterlife and therefore, larcs are a viable alternative to bilateral tubal ligation.27 menstrual irregularities account for most early discontinuation of implants in many series.45678910 our study has also reconfirmed this finding. over one - third (34.6%) of the discontinuation of jadelle within 6 months of use was as a result of menstrual pattern disruption. over the course of the entire study, however, menstrual disruption accounted for only 11.3% of the reasons women discontinued jadelle. disruption of vaginal bleeding pattern is almost inevitable with contraceptive implants, especially in the early months.10 while these bleeding disturbances are not known to threaten the lives of the women, it may however lead to an increase in investigations for cervical or endometrial pathologies.10 it also leads to disruption of the daily lives of the women and affects their social, sexual and religious practice (praying, fasting, attending funerals, going to places of worship).6 wearing a pad or tampon because of prolonged bleeding is not only uncomfortable but also comes with some cost.10 preinsertion counseling on menstrual pattern disruption is therefore a sine qua non for the provision of quality services for all progestin - only implants since this is the most likely reason to prompt early discontinuation.6 this is because women choosing and continuing a contraceptive method are actually making a trade - off between its perceived advantages and disadvantages.6 other nonmenstrual adverse effects that resulted in the removal of jadelle in this study were weight gain, elevated blood pressure, and headache. each of these resulted in the discontinuation of jadelle for < 5% of the entire reasons for the removal of jadelle. these nonmenstrual reasons for removal of jadelle are similar to other studies of discontinuation of contraceptive implants.611 two women had a method failure with jadelle in our series. these two failures were not in keeping with the well - documented efficacy of jadelle in clinical trials.1314 these women had used jadelle for 26 and 55 months, respectively. these are likely to be proper method failures because they happened quite remote from the time of insertion and are not likely to be preexisting pregnancies that predated the insertion. it is a retrospective chart audit which relied heavily on information already obtained within the setting of clinical consultation. some of the data entries were missing and this may have biased the outcome of the study. however, the results of this study have improved our understanding of the profile of those women who accepted jadelle as one of the larc methods and the reasons for its discontinuation. this information will be useful for further research that is aimed at targeting the underserved members of the community. jadelle is used by diverse ethnic groups in nigeria especially by women who were very educated. the main reason for discontinuation was to have more children although menstrual pattern disruptions accounted for earlier removals. | background : contraceptive implants (including jadelle) are highly effective, safe, and easy to use and have a long duration of action. they do not interfere with intercourse with immediate return to fertility after removal. however, disruption of the menstrual bleeding pattern is almost inevitable and coercive prescription may be a problem because insertion and removal of implants are provider dependent. the objective of this study was to determine the sociodemographic profiles of acceptors of jadelle and the reasons for discontinuation in jos, nigeria.materials and methods : this was a 6-year retrospective chart review carried out at the jos university teaching hospital.results:about 1401 women accepted jadelle with a mean (standard deviation) of 33.4 5.9 years. about 88% of the women were christians and almost three - quarters (73.5%) had at least secondary school education. the means of parity and number of children still alive at the time of accepting jadelle were 4.1 and 3.8, respectively. half of the women (49.5%) were breastfeeding and over half (55.9%) had future fertility desires at the time of commencing jadelle. about 82% had previously used other contraceptives (mostly short - acting methods such as injectables, pills, and condoms), with only 18% starting jadelle as the first - ever contraceptive method. about 90% of the women had regular menstrual cycles. the major reason for discontinuation of jadelle was desire for pregnancy although menstrual pattern disruption was the most common reason for removal in the first 6 months of use.conclusion:the main reason for discontinuation of jadelle was to have more children although menstrual pattern disruptions accounted for earlier discontinuation. |
worldwide, lung cancer remains the leading cause of cancer death in both men and women. spinal metastasis in patients with all types of cancer combined varies between 30 and 70%, and is most frequent in patients with lung cancer (60%). all these patients are at risk of developing symptomatic spinal cord compression with an incidence of 5 - 14%. spinal metastasis often causes severe pain and neurologic dysfunction, and may exacerbate the prognosis and quality of life of patients. thus, metastatic spinal disease is one of the important therapeutic targets in the management of patients with advanced cancer. metastatic spinal tumor is treated by surgery, radiotherapy or chemotherapy as well as more conservative palliative treatments such as opioids, non - steroid anti - inflammatory agents or corticosteroid. surgical procedures often contribute to pain relief, neurological recovery and mechanical stability of the site of metastasis. it can stop rapidly progressive paralysis and recover neurological symptoms in some cases in which such recovery is not attainable with other kinds of interventions., surgery is indicated only if a relatively long survival is expected and if the anticipated improvement in quality of life outweighs the risks. there have been some studies on the outcome of surgical treatment of metastatic spinal disease from various cancers. the prognosis of patients with spinal metastasis and surgical treatment is relatively good, when all kinds of cancers are considered together. moreover, it was reported that patients with lung cancer and melanoma had the poorest prognosis in a series of 76 patients with various cancers. chen. reported good symptom relief for metastatic spinal disease of lung cancer, and 18 patients (58.1%) had a relatively good eastern cooperative oncology group (ecog) performance status (ps) score of 0 or 1. in the present study, including patients with poor performance status, we studied the usefulness of surgery in symptomatic spinal metastasis of lung cancer. we retrospectively reviewed all records of patients with lung cancer in the department of respiratory medicine, kyoto university hospital, japan, from january 1999 to july 2007. information was obtained from patient records, including age, pathological diagnosis, findings of spinal mri and ct from the neck to the pelvis, ecog ps, symptoms (paralysis, pain and movement capacity), pre- and postoperative movement capability, operative method, location of operation, pre- and postoperative prescription, duration from symptom appearance to operation, and postoperative complications and prognosis. to evaluate neurological status activities of daily living (adl) were classified into 3 categories : can walk independently, can move with wheel chair, and can not move. pain was expressed as grade of prescription necessary to control pain according to the world health organization pain relief ladder (who ladder). a total of 772 lung cancer patients were diagnosed or referred to the department of respiratory medicine, kyoto university hospital, from january 1999 to july 2007, and 14 (1.7%) underwent surgery for metastatic spinal disease. surgery was not considered if the extent of the metastatic disease precluded adequate stabilization with segmental instrumentation and if the expected survival was <3 months. the most common site of involvement, in 9 patients, was the thoracic spine. eleven patients underwent spinal decompression by laminectomy and posterior spinal fusion, and 1 underwent spinal decompression and anterior spinal fusion. there was 1 postoperative complication and recovery without reoperation. 1 and table 2. more than 1 grade of improvement in frankel 's scale was noted in 10 of 14 cases (71%). improvement in adl was noted in 9 of 14 cases (64%) (fig. 2). ecog ps before the symptoms caused by spinal metastasis was a mean of 2.29 (ps1, 2 ; ps2, 6 ; ps3, 6). ps immediately before surgery was a mean of 3.61 (ps3, 5 ; ps4, 8). after surgery, ps recovered to a mean of 1.29 (ps1, 5 ; ps2, 5 ; ps3, 2 ; ps4, 2). ps of 2 patients with ps4 did not change, and none of the cases worsened after surgery (table 2). symptoms of spinal pain were reported by all patients before surgery, and pain grade was 2 or 3 on the who ladder. after surgery, improvements of more than 1 grade were noted in 12 of 14 cases (86%), and 6 of these became grade 0, with complete relief of pain (fig. the group with a good postoperative ps (0 - 2) was shown to have better median postoperative survival than that with a poor postoperative ps (3 - 4) (table 3). prognosis of patients with extensive lung cancer is still poor even with the development of chemotherapeutic drugs. thus, symptom relief and maintenance of quality of life are important aims of medical intervention in patients with advanced - stage lung cancer, especially after failure of the initial therapy. lung cancer frequently develops metastasis to the spine, and it often leads to compression fracture or spinal dysfunction. once compression fracture or spinal dysfunction has occurred, the quality of life of the patient deteriorates severely. surgical procedures can immediately improve pain, neurological dysfunction, mechanical stability, and quality of life, although it is the most invasive treatment for patients with advanced lung cancer, and the criteria for surgical treatment of metastatic spinal tumor are still not clear. the life expectancy is one of the important factors to select the treatment modality, and the prognosis of the patients with metastatic spine tumors is related to the primary site of malignancy. studied 246 patients with metastatic spinal tumors and proposed scoring systems to evaluate prognosis and the suitability of the subsequent treatment strategy. in their series, lung cancer belonged to the poor prognosis groups, and they concluded that lung cancer is a negative factor for indication of surgery. weigel. also stated that survival of patients with lung cancer is the poorest (2.1 months) among all patients with solid cancer in their series. these reports may indicate that surgical indication of patients with lung cancer needs to be discussed separately from other solid cancers with a relatively longer life expectancy. according to the past few reports which studied patients with lung cancer, an expected survival time in a range of 3 to 6 months has been used as a criterion for choosing surgical treatment. in 1995,. showed that the median survival was 6 months after surgical treatment for spinal metastasis. in the study of chen. in 2007, median survival after surgery was 8.8 months, and 10 of 31 patients survived more than 1 year. the survival in other series ranged from 1.5 to 9.9 months [1, 10, 11, 12 ].. found that the prognostic factors of patients with spinal metastases from non - small cell lung cancer were ps, serum calcium, and serum albumin, and that ps was a significant factor for survival in the postoperative period among the operated patients. the ps of patients involved in the current study was 3 or 4, which is poorer than in previous reports. however, postoperative median survival in the current study was 5 months (1 - 25 months), and it was compatible with those of previous reports. tanaka. studied 100 cases of metastatic spinal tumors with various origins, and they found that preoperative pain and paralysis improved by 88.0 and 53%, respectively, and that quality of life score improved by 53%. bach. reported 102 cases of metastatic spinal cord compression secondary to lung cancer. chen. studied 31 patients with lung cancer and spinal metastasis and showed that 23 regained the ability to walk after surgery and that neurological improvement was noted in 25. in our study, after surgical treatment, neurological improvement, improvement of movement capacity and improvement in pain was 69, 62 and 84%, respectively. these results show that, in advanced lung cancer with spinal metastases, improvement of symptoms by spinal surgery can be expected in a substantially high percentage of patients. in the current study, the group with a good postoperative ps (1 or 2) was shown to have a better median postoperative survival of 13 months, compared with 3 months in the group with a poor ps (3 or 4). commonly, it takes about 1 month for the patient to recover to the best postoperative condition and the patient suffers from degradation of the general condition and limitation of adl for 1 month or so until death. median postoperative survival was 5 months (1 - 24 months) in this series. it is suspected that the beneficial period to the patients was less than 3 months in this series. some authors have suggested that symptoms have no impact on survival in patients with spinal metastases, and hosono. reported that walking ability is not a prognostic factor in patients with spinal metastases. however, other authors [12, 15, 16 ] have suggested that postoperative ambulation ability is associated with longer survival after surgery, even in patients with lung cancer. in the present study, postoperative but not preoperative postoperative complications are another important factor in selecting treatment modality. in our study, 1 (7.7%) patient had postoperative infection. previous reports showed that complications occurred in a range from 13 to 25.8% [1, 3, 7 ]. reported that operation - related death occurred in 1 (3.2%) and 2 (2.3%) patients, respectively. other minor complications reported were wound infection, wound dehiscence, hoarseness and respiratory insufficiency. in recent studies the limitations of our study are a relatively small number of patients studied and lack of a control group. however, lung cancer is often thought to be one of the poorest prognosis groups among solid cancers. surgical treatment of metastatic spinal disease might be considered to improve quality of life and recovery of independent ambulation. further prospective studies are needed on metastatic spinal tumor of lung cancer to confirm the utility of the surgical treatment of metastatic spinal disease especially compared with radiation therapy. surgical treatment for symptomatic metastatic spinal tumor of lung cancer can improve quality of life in a substantially high percentage of patients. | backgroundspinal metastases of patients with advanced stage lung cancer are an important target for palliative therapy, because their incidence is high, and they often cause severe symptoms and worsen the quality of life. surgery is one of the most effective treatment options, but the indication of surgery is unclear as the procedure is invasive and patients with spinal metastasis have a rather short life expectancy. furthermore, there have been few studies that have focused on lung cancer with poor prognosis.methodswe reviewed all of the cases of lung cancer from january 1999 to july 2007 in the department of respiratory medicine, kyoto university hospital, japan. thirteen patients with metastatic spinal tumor of lung cancer underwent surgery, and all of them had a poor performance status score (3 or 4).resultsneurological improvement by at least 1 frankel grade was seen in 10 of 14 cases (71%). improvement of the movement capacity was noted in 9 of 14 cases (64%), and pain improvement was noted in 12 of 14 (86%). median postoperative survival was 5 months (125 months). in particular, the group with a good postoperative performance status score (02) was shown to have a better median postoperative survival of 13 months.conclusionssurgical treatment for symptomatic metastatic spinal tumor of lung cancer can improve quality of life in a substantially high percentage of patients. surgery should be considered even if preoperative performance status is poor. |
loss of lean body mass, and muscle tissue in particular, may in critically ill patients be detrimental, and it increases complications and impairs the clinical outcome. therefore, nutritional therapies together with therapies reducing the catabolic burden and improving lean body mass are clinically important. growth hormone has major effects on metabolism and affects the utilization of substrates and changes the tissue specific metabolism. several earlier studies have shown its potential benefits with regard to protein metabolism, which also is reflected in clinically positive effects following surgery and in burn injuries [39 ]. however, a large multicenter study involving critically ill patients revealed an increased mortality in the gh - treated group indicating that its effects in situations with uncontrolled ongoing inflammation are associated with major negative consequences. the present study was planned and completed before the result from the multinational study parts of the action of growth hormone are mediated through the insulin - like growth factor-1 (igf-1). administration of growth hormone induces a rise in circulating igf-1 that has important metabolic effects in stimulating glucose and amino acid uptake in muscle and improving muscle protein synthesis [12, 13 ]. in catabolic situations the levels of igf-1 decrease while its binding proteins increases leading to a lower local igf-1 activity, contributing to the decreased insulin sensitivity seen in catabolism [1416 ]. the metabolic effects of gh are at least in part mediated through the action of igf-1 produced in the liver and in the peripheral tissues by the influence of growth hormone [1719 ]. in skeletal muscle a reduced gene - expression of the gh - receptor is seen following surgical trauma. this reduces the local igf-1 synthesis, an effect that may be counteracted by gh supplementation. the interrelation between gh, igf-1 and igf - binding proteins (gh / igf-1/igfbp - axis) is thus affected mirrored by a lower gh sensitivity, leading to lower igf-1 and higher igfbp levels that have been described in icu patients leading to a lower igf-1 bioavailability [14, 22 ]. in septic patients the change in the gh / igf-1/igfbp - axis is possible to counteract by amino acid supplementation. a similar effect on the gh / igf-1/igfbp - axis is seen in cardiac surgery when a glucose - insulin - potassium (gik) infusion is given peroperatively. igf-1 levels have also been shown to be possible to increase in catabolic patients, either by gh administration or by giving igf-1 [11, 26 ]. a combination of growth hormone and igf-1 has a theoretical beneficial potential since the decreased insulin sensitivity induced by growth hormone may be outbalanced by an addition of igf-1. gh increases the binding protein for igf and concomitant administration may therefore increase the bioavailability of igf-1 and increase its effects on the peripheral tissues. combination of growth hormone and igf-1 with glutamine containing tpn has been shown to improve protein balance in critically ill patients [27, 28 ], but the combined effect on nitrogen, amino acid, and protein metabolism has so far not been explored following surgical trauma. this study was designed to elucidate the effects on muscle amino acid, glutathione, and protein metabolism of growth hormone alone or in combination with igf-1 given together with continuous nutritional supplementation following colonic resection as a human model of trauma. the present study was discontinued when the report of an increased mortality of icu patients given gh was published, but since the results may be of general interest we hereby communicate this almost 10 years later. as a model of elective trauma metabolically healthy individuals under the age of 80, undergoing elective colonic resection because of nonspread colonic cancer or earlier diverticulitis, were included in this randomized and blinded study (n = 26). colonic resection was chosen as a surgical trauma model leading to muscle catabolism, used in earlier studies on muscle and nitrogen metabolism [29, 30 ]. the operation is standardized, and metabolic changes indicating a catabolic situation are seen during the early postoperatve period [29, 30 ]. the characteristics of the patients and of the operative procedures are shown in table 1. they were preoperatively weight stable, not under medication affecting the metabolism, and none of them had signs of ongoing inflammation. the patients were postoperatively given isocaloric (28 kcal / kg/24 h) and isonitrogenous (0.15 g n / kg/24 h) tpn (total parenteral nutrition), not containing glutamine, for 3 days as a continuous infusion. one group served as a control group (n = 10), another group was given gh postoperatively (gh ; n = 7) twice a day subcutaneously (0.15 iu / kg body weight / injection) and a third group (gh - igf-1 ; n = 9) was given the same amount of gh and igf-1 (40 g / kg body weight / injection) twice a day. this dosages has in earlier studies been shown to influence protein and substrate metabolism [5, 11, 3135 ]. the study was designed to include 10 patients in each group, but when the use of growth hormone in catabolic patients was stopped by the results from the multicenter study on icu patients inclusion of patients in the present study was stopped. preoperatively, after induction of anaesthesia, a muscle biopsy was taken with percutaneous technique from the vastus lateralis femoris muscle. the muscle tissue of about 200 mg wet weight was divided into portions for the analyses of the amino acid and glutathione concentration and the content and size distribution of ribosomes, reflecting protein synthesis. all visible fat and connective tissue were removed from the tissue sample and the muscle tissue was weighed on an electrobalance and then plunged into liquid nitrogen and thereafter stored at 80c, until analysis. a second biopsy was taken on the third postoperative day in local anaesthesia confined to the skin and fascia only. urine was collected in 24 hour portions for the determination of the cumulated nitrogen losses. the study protocol has been approved by the swedish medical products agency (lkemedelsverket) and by the ethical committee of the karolinska institute, stockholm, sweden. the patients were informed about the study procedure and of the possible risks involved before consent was obtained. energy expenditure and the respiratory quotient were measured by indirect calorimetry preoperatively and on the second postoperative day (deltatrac, datex oi, helsinki, finland). the first measurement was made after an overnight fast before premedication. although the substrate availability may be different comparing these two situations the patients were measured in a standardized way that allows comparisons between the three groups. glucose was determined by an enzymatic colorimetric method, urea by a kinetic absorbance method and crp by a quantitative immunological method. the whole - body nitrogen balance was determined by subtracting the measured nitrogen excretion in urine every 24-hour from the amino acid nitrogen content in the tpn. the extrarenal losses were approximated to be 1.5 g/24 hours that was included in the calculations. the volume was measured and aliquots were stored at 20c prior to analysis of the daily losses of urea (urease enzymatic 's uv test, boeringer, mannheim, germany) ammonia (gld, kinetic uv test merk, darmstadt, germany), and creatinine (jaff reaction, beckman astra, palo alto, usa). the total nitrogen excretion was determined using chemoluminescence (771 c pyroreactor, 720 c nitrogen detector ; antek, houston, texas, usa). since treatment of growth factors significantly decreased the urea levels postoperatively, the contribution of the whole body urea shift was also taken into account expressing a urea related cumulated nitrogen balance. changes both in total body water and urea were taken into account in calculating the urea - related nitrogen balance. in order to assess total body water body impedance analysis (bia) the urea related nitrogen balance was calculated as follows : urea related n balance = n intake n urine n extrarenal (approximated to be 1.5 g n/24 h) diff urea. diff urea = (urea end urea start) total body water (start) 0.028 + (body water end body water (start)) urea end 0.028. the frozen muscle biopsy specimen was homogenized in 4% sulphosalicylic acid (ssa), containing norleucine as internal standard. the amino acids were separated on an ultropae 8 lithium form ion exchange resin (biochrom) using lithium citrate buffers. the amino acids were detected and quantified by postcolumn derivatization with o - phthaldialdehyde (opa) and fluorescent detection at ex 350 nm em 420 nm. the concentrations of the amino acids were expressed as mmol / kg wet weight in muscle. muscle tissue with a weight between 20 and 40 mg was used for analysis. the frozen muscle tissue was homogenized and deproteinized in 6.5% sulphosalicylic acid (ssa) in a glass homogenizer on ice. the protein precipitate was later used for the determination of protein - bound gsh and cysh. samples of gsh and cysh standards or ssa - soluble fraction from muscle biopsies (100 l) were mixed with monobrombimane (mbbr) (8 m in sodium n - ethylmorpholine ph 8.0, 100 l), allowed to react for 5 minutes in the dark. thereafter the reaction was stopped by the addition of 100% ssa (10 l). total glutathione (gsh + gssg), total cysteine and precipitated protein were also evaluated by the present method by performing a reduction step of gssg and cyss with dithiothreitol (dtt) after protein precipitation. the hplc separation of thiol - bimane adducts was achieved on a column (150 4.5 mm) packed with 3 m octadodecylsilica reversed - phase resin, followed by fluorescent detection at ex 394 nm, em 480 nm. the protein synthesis in skeletal muscle tissue the samples were homogenized in a medium containing a ribonuclease inhibitor and then centrifuged for 10 minutes at 1.500 g. the pellet was saved for dna determination by a fluorescence method. the supernatant was ultracentrifuged for 2 hours at 102.000 g. the pellet containing the ribosomal particles was resuspended in 200 l of a medium containing a ribonuclease inhibitor. the ribosome concentration was determined spectrophotometrically at 260 nm and was expressed as optical density (od) units per milligram of dna. the remainder of the ribosomal suspension, 150 l, was layered onto a gradient of sucrose between 0.4 m and 1.5 m. the tubes were ultracentrifuged for 60 minutes in a swing - out rotor. afterwards the gradient was displaced through a continuous - flow cuvette and the absorbance at 260 nm was recorded to show the distribution of ribosomes and polyribosomes in the sucrose density gradient. the area under the curve was measured and the percentage of polyribosomes in the total ribosome area was calculated. the values are expressed as medians with lower and upper quartiles. a nonparametric anova (kruskal - wallis anova) was used followed by wilcoxon test for post hoc comparisons within the groups and mann - withney u - test for comparisons between the controls and the groups given growth factors. this was a pilot study in which the effects on the primary effect variables were not known and therefore a power analysis was not perforned. since the variation coefficient was < 5% for the amino acid analyses, < 10% for the ribosome analyses, and < 8% for the analyses of glutathione, groups including 10 individuals each was considered sufficient to detect a difference of a larger magnitude as the coefficient of variation in paired samples. the energy expenditure increased postoperatively in the gh - group by 15% (p =.024) but was unaltered in the other groups. no statistically significant changes were seen in respiratory quotient within or between the groups, reflecting that the relation between substrates used for oxidation was not influenced by the surgical trauma or administration of growth factors. the controls were normalized on the second postoperative day, while levels in the gh and igf-1/gh groups remained elevated throughout the study period. plasma urea decreased in both the gh- and gh - igf - i groups on the postoperative day 2 by 43% and 39% and on day 3 by 50% and 54%, respectively, compared to the control group (p <.05) in which no change was seen. the cumulated postoperative nitrogen balance was positive in all groups without any differences between the groups. the urea corrected whole body cumulated nitrogen balance (expressed as g n) was numerically more positive in the gh group (11.43 (7.4512.87)) compared to the control group (6.84 (3.198.96)) and compared to the gh - igf-1 group ((8.92 (5.8218.27)) but without reaching a significant level, p =.063 and p =.22, respectively. muscle glutamine decreased in the control group by 28 %, by 24% in the gh - group, and by 28% in the gh - igf-1-group (p <.05). in parallel muscle bcaa increased by 44% in the control group, by 39% (p <.05) in the gh - igf-1 group while no change was seen in the gh - group. a similar increase was also seen in the aromatic amino acids in muscle by 61% in the control group and by 46% (p plasma glutamine concentration decreased by 17% (p <.05) in the gh - igf-1 group while the level was unchanged in the other groups. total glutathione (tgsh) and reduced glutathione (gsh) decreased in the gh group by 16% and 12%, respectively, (p <.05) and by 15% and 19%, respectively, in the gh - igf-1 group (p <.05). in the controls a numerical decrease by 13% was seen for tgsh, that did not attain statistical significance (p =.06) while gsh was unaltered. the polyribosome concentrations decreased by 19 % in the control group and by 28 % in the gh group (p <.05) while the concentration was unchanged in the gh - igf-1 group. in a human model of elective trauma the metabolic effects of gh either given alone or in combination with igf-1, together with total parenteral nutrition during three days postoperatively were evaluated. growth factors increased the energy expenditure, increased the glucose and lowered the urea levels. gh preserved the levels of bcaa and aaa in skeletal muscle indicating that protein breakdown was unaltered compared to increased postoperative levels in the other groups. a combination of gh and igf-1 preserved the level of polyribosomes postoperatively indicating an unaltered protein synthesis compared to decreases levels in the other groups. to combine growth hormone with either insulin or igf-1 may be advantageous since gh increases peripheral insulin resistance and secondly since surgical trauma furthermore increases gh resistance with lower levels of circulating igf-1. the effect of igf-1 given alone in catabolic conditions is not as clear - cut as during growth hormone treatment. in some studies igf-1 given alone postoperatively showed no effect on nitrogen [26, 36 ]. igf-1 treatment during 5 days postoperatively reduced plasma insulin and glucose levels, indicating an improved insulin sensitivity following surgery. in the present study a daily supply of gh (0.3 iu / kg / day) and igf-1 (80 g / kg / day) was chosen since these amounts have shown effects on protein and substrate metabolism in earlier studies during the postoperative period [5, 11, 3135 ]. plasma glucose levels increased in both groups given growth factors as compared to the control group. although not statistically significant, towards higher levels in the gh - group compared to the gh - igf-1-group was observed. a sufficient energy and protein supply is a prerequisite for an optimal igf - i effect and response to growth hormone. when compared to the resting energy consumption by indirect calorimetry an excess of energy in the range between 400 to 600 kcal per day was given in the present study every day. treatment with growth factors increased the resting energy expenditure postoperatively compared to the control group without changing the rq. this may be explained by the action of growth factors, increasing the metabolism of substrates. both growth hormone and igf - i have in earlier studies been shown to increase the resting energy metabolism following surgery and in healthy volunteers [4143 ]. hyperglycemia has in mitochondrial studies been shown to affect mitochondrial metabolism and substrate and energy metabolism and increase the action of the uncoupling proteins. this may also be a contributing factor to the increased energy metabolism seen in the groups given growth factors. urea levels decreased during the study period in both the gh- and gh - igf-1 groups mirroring a lower urea - formation, an indirect sign of a decreased protein breakdown. since the shift in whole body urea was significant in the gh- and gh - igf-1 groups this was taken into account in a calculation determining the cumulated nitrogen balance related to the shift in urea. a tendency of an improved whole body nitrogen balance during the study period was seen in the groups given growth factors, the effect being close to significant in the gh - group. in all groups a positive nitrogen balance was seen which may be explained by continuous administered parenteral nutrition. this effect on nitrogen balance has earlier been reported when parenteral nutrition is administered continuously. in all three groups a similar decline in muscle glutamine was seen postoperatively. this decline in glutamine was not that extensive as has been shown in control groups in earlier glutamine studies receiving a similar amino acid composition as in the present study [30, 46 ]. there is no clear explanation to this, but it is plausible that the continuous administration of parenteral nutrition optimizes the metabolic conditions to an extent that reduces the changes in catabolic parameters. in the present study conventional tpn was given without glutamine since that was not a clinical routine at the time of the study. a decline in muscle free glutamine has earlier been reported to be reduced by gh - treatment but this finding was not reproduced. changes in the total sum of bcaa, aromatic and essential amino acid concentrations differed between the groups. an increase in bcaa and aaa indicating an increased proteolysis was observed in the control group and in the group given gh together with igf-1. these changes indicate that protein degradation was reduced by gh treatment but not by the combined therapy. the total amount of glutathione and its reduced form decreased postoperatively in all groups with the exception of maintained levels of reduced glutathione in the control group. this observation is in line with results from earlier studies in which muscle glutathione levels are affected by surgical trauma. muscle protein synthesis was assessed by determination of the total amount and size distribution of ribosomes in muscle tissue. the concentration of polyribosomes reflects the protein synthesis activity in the tissue studied [32, 47 ]. the polyribosome concentrations decreased in the control and gh - group but were statistically unchanged in the gh - igf-1 group. however using a nonparametric anova no differences were seen between the three groups, making this observation not statistically robust enough. there are historically studies with clinical beneficial effects with the use of gh [3, 6, 7, 9 ], but when used in a heterogeneous group of icu patients with ongoing acute general inflammation an increased mortality is seen. the mechanism behind that adverse effect may be increased insulin resistance, uncontrolled hyperglycemia, uncontrolled nutritional supply, a triggering of the inflammatory response, and a shortage of substrates for the immune system induced by a decreased peripheral release of glutamine. it is important to consider a proper timing with regard to the inflammatory response, and a proper glucose control and to pay attention to the nutritional treatment. this has been elucidated in a recent study presenting metabolically positive effects when gh was administered in a pulsative way together with glutamine in icu patients. despite the negative effect of gh treatment used in unselected icu patients earlier there the present study showed effects on muscle amino acids indicating a lower protein degradation when gh was used, an prevention of a drop in postoperative protein synthesis when the combination of gh- and igf-1 was given. even when the change in urea was taken into consideration a significant difference was not seen between the control and groups given growth factors. the patients were all in positive nitrogen balance, a situation that was not further pronounced by treatment with growth factors. | this study explored if a combined supplementation of gh and igf-1 had an additive effect on whole body nitrogen economy, energy, substrate and skeletal muscle metabolism following surgical trauma. patients were randomized to controls (c ; n = 10), to gh (0.15 iu / kg / injection) (gh ; n = 7) or gh combined with igf-1 (40 g / kg / injection) subcutaneously twice a day (gh - igf-1 ; n = 9) together with standardized parenteral nutrition. muscle amino acids, glutathione and the ribosomal pattern reflecting protein synthesis, and nitrogen balance were measured. gh- and gh - igf-1 groups showed lower urea and higher plasma glucose concentrations. energy expenditure increased in the gh - group. gh - igf-1 prevented a decrease in muscle polyribosomes indicating a preserved muscle protein synthesis. in the gh group unaltered bcaa and aaa levels were seen in muscle indicating an unchanged protein breakdown, while the other groups showed increased muscle concentrations postoperatively. without statistically difference gh marginally improved the nitrogen balance, in terms of higher values, and growth factors improved the nitrogen balance when the shift in urea was taken into account. to conclude, growth factors influences urea metabolism, protein degradation and protein synthesis. there was no clearcut additional effect when combining gh and igf-1 but the study was probably underpowered to outrule this and effects on nitrogen balance. |
hashimoto 's thyroiditis (ht) is an autoimmune disease and the most frequent cause of hypothyroidism. though most patients with ht have no pain, rare cases presenting with serious neck pain leading pathologies that can present with neck pain include intranodular bleeding, subacute thyroiditis (sat), and infectious pathologies of the neck and thyroid gland.[24 ] here we present a ht case that developed sat during her clinical follow - up. a 69-year - old female patient with ht and multinodular goiter has been followed up in the outpatient clinic of an endocrinology department since 2003. her first ultrasonography revealed a heterogeneous parenchyma compatible with chronic thyroiditis and coincidental multiple isoechoic thyroid nodules in the left lobe. the largest nodule was measured as 15 9 mm and was located in the left lobe. a fine needle aspiration biopsy was performed from the largest nodule and was reported as being compatible with ht. the patient presented to our department with a symptom of neck pain radiating to the right ear and neck persisting for 2 months. she was prescribed analgesics and antibiotics by other physicians during the 2 months period, but the pain persisted. she described some symptoms of a possible upper respiratory tract infection including fever which began prior to the presenting symptom. her tsh level was 3.94 miu / ml (0.35 - -4.94), esr 23 mm / h, crp 3.2 mg / l, wbc 4900/l. thyroid ultrasonography revealed that the right lobe volume was 28.5 ml, the left lobe volume was 12.0 ml. the thyroid parenchyma was heterogeneous and a hypoechoic area was noticed over the tender lobe [figure 1 ]. power doppler imaging (pdi) revealed almost no blood flow in that area [figure 2 ]. she was started on methylprednisolone 32 mg / day. at day 10 of therapy, repeated ultrasonography showed that the hypoechoic area had disappeared and the right lobe volume had decreased to 9.5 ml [figures 3 and 4 ]. gray - scale sonography of the patient 's thyroid gland at presentation (right lobe, transverse view) doppler sonography of the patient 's thyroid gland at presentation (power doppler mode, right lobe, transverse view) gray - scale sonography of the patient 's thyroid gland during follow - up (right lobe, transverse view) gray - scale sonography of the patient 's thyroid gland during follow - up (right lobe, longitudinal view) the differential diagnosis of painful neck conditions includes a wide spectrum from benign and malign proliferative pathologies to inflammatory and infectious states of neck structures including the thyroid gland. painful ht is a concept first brought into the literature by doniach. in 1960. new reports since the definition of the concept are very limited in number.[34815 ] debate seems to be continuing whether it is a different clinical entity or is simply a variant of sat. it may be challenging to differentiate the two entities in a patient with a previous diagnosis of ht. a viral prodrome is common, though not a rule during the course of sat. fever, neck pain, increased esr, and crp can be seen in both entities which are acute inflammatory conditions.[24 ] sat is usually characterized by thyrotoxicosis in the acute phase, but this may not be a discriminatory feature from painful ht in cases with preexisting ht such as ours. reported in 2009 that ultrasonographically focal thyroiditis areas of two patients with painful ht showed increased blood flow with pdi. in sat, ht presenting as painful swelling is a rare entity and pain usually starts at presentation and not during follow - up. the cases of painful ht defined to date had generally persistant pain refractory to glucocorticoid therapy, but not invariably. some patients have even undergone thyroidectomy.[1214 ] the clinical course and the laboratory findings of our case with a viral prodrome, decreased local blood flow, glucocorticoid responsiveness probably coincide with sat. the laboratory findings were compatible with the recovery phase of sat and the toxic phase was likely missed. more research is needed on the topic to be able to reach a consensus for a clear - cut differentiation of both entities. the presenting clinical and laboratory findings of the patient is compatible with sat, which is an unusual, but a much more common presentation than a painful attack of ht to be considered in differential diagnosis. it should be kept in mind that such painful attacks some of which may require intervention may develop during follow - up of ht cases. | hashimoto 's thyroiditis (ht) is an autoimmune disease and the most frequent cause of hypothyroidism. subacute thyroiditis (sat) overlapping ht is a rare entity. a 69-year - old female patient with ht and multinodular goiter has been followed on levothyroxine replacement therapy for 7 years. she presented with neck pain radiating to the right ear persisting for 2 months. she was prescribed analgesics and antibiotics by other physicians during that period, which did not work. her vital signs were stable with no tachycardia or fever. the right lobe of the thyroid gland was tender on palpation. her tsh level was 3.94 miu / ml, esr 23 mm / h, crp 3.2 mg / l, wbc 4900/l at presentation. thyroid ultrasonography revealed a hypoechoic area over the tender lobe. power doppler imaging revealed almost no blood flow in that area. she was started on methylprednisolone 32 mg / day. at day 10 of therapy, her symptoms had completely resolved. ultrasonography repeated showed that the hypoechoic area had disappeared. glucocorticoid dosage was tapered and stopped. emergence of subacute thyroiditis in a case with preexisting hashimoto 's thyroiditis is a quite rare condition, but should be kept in mind along with a painful attack of ht in the differential diagnosis. |
microsporidia are a group of obligate intracellular eukaryotic parasites first identified as the cause of pbrine disease of silkworms in 1857. over the past two decades several genera and species of microsporidia were found in humans, and diagnosis and clinical management of microsporidiosis cases have improved significantly. despite these scientific advances, the epidemiology of human microsporidiosis is still unclear. most of what is now known about human microsporidiosis can be attributed to experience with patients infected with human immunodeficiency virus (hiv). since 1985 and the first recognition of enterocytozoon bieneusi as an acquired immunodeficiency syndrome (aids)-associated opportunistic pathogen, several hundred patients with chronic diarrhea attributed to this organism have been reported. cases of intestinal microsporidiosis due to e. bieneusi have been increasingly reported in other immunocompromised individuals such as organ transplant recipients, as well as in travelers, children, and elderly [312 ]. some reports in humans suggested that this species can produce asymptomatic infections in immunocompromised and immunocompetent individuals [1620 ]. in addition, e. bieneusi has been commonly identified in animals, especially mammals [2132 ], and water, raising public health concerns about zoonotic and waterborne transmission of microsporidia [3335 ]. enterocytozoon bieneusi infections in humans are mainly detected by light microscopy of stained fecal smears, electron microscopy, and pcr - based methods. therefore, infection rates are difficult to compare because of the considerable differences in the diagnostics methods employed, the specimens analyzed, the geographical locations, the patient groups, and patients characteristics (sex, age, socioeconomic conditions, immune status, and clinical features). in developed countries in north america, europe, and australia, studies involving hiv - seropositive persons with diarrhea reported rates between 2% and 78% [17, 3645 ]. lower infection rates (between 1.4% and 4.3%) were reported in hiv - seropositive persons without diarrhea [17, 39, 43, 45 ]. in one study of hiv - seronegative patients with diarrhea or pneumonia, spores of e. bieneusi were detected in 2.5% of urine, 11.5% of fecal, and 16.2% of sputum samples examined. in majority of the studies performed in developed countries e. bieneusi was the species detected most often, followed by e. intestinalis ; nevertheless, these findings are not in accordance with results of a study performed recently in czech republic, involving immunocompetent humans. in this study, e. bieneusi was detected four times less frequently than e. cuniculi, and e. intestinalis was identified only sporadically. also the majority of microsporidial infections (reaching 94%) were reported for nondiarrheal specimens. it spans a period between 1999 and 2009, involving 856 patients (561 hiv - seropositive and 295-seronegative) and respective clinical data. the authors observed that e. bieneusi was more common in the hiv - seropositive group of patients (7.0%39) than in the hiv - seronegative group (5.1%15). in developing countries, e. bieneusi prevalence rates were reported between 2.5% and 51% in hiv - seropositive adult patients with diarrhea [4760 ], and in 4.6% of patients without diarrhea. also in developing countries, in hiv - seronegative persons with and without diarrhea, e. bieneusi was detected in 5.35% to 58.1% of the fecal samples examined [19, 51, 53, 54, 61, 62 ]. in studies conducted in children, e. bieneusi prevalence ranged from 17.4% to 76.9% in hiv - seropositive children with diarrhea, and from 0.8% to 22.5% in the immunocompetent or apparently immunocompetent groups of children with or without diarrhea [18, 19, 30, 49, 6368 ]. the recovery of spores of e. bieneusi in 0.8% of african children considered hiv seronegative, and in 5.9% of healthy orphans and 1.9% of child - care workers in asia, indicated the presence of enteric carriage of infection in immunocompetent persons in tropical countries [49, 69 ]. table 1 gives an overview of the studies conducted on the prevalence of e. bieneusi infection in humans, in various areas. in industrialized nations, some studies reported e. bieneusi infection rates between 6.1 and 10% in travelers who suffered from self - limited diarrhea and returned from tropical destinations [6, 8 ]. in addition, a prevalence of 17.0% of e. bieneusi infection in elderly persons was observed, leading to the speculation that age - related diminishment of the immunity might predispose these persons to microsporidial infections. a few prospective studies conducted in developed countries indicate that the prevalence of e. bieneusi in hiv - seropositive patients is progressively decreasing, probably due to the use of highly active antiretroviral therapy (haart) [70, 71 ]. similar observations are also made recently in some developing countries. in a study conducted in brazil, comparing enteric parasitic infections in hiv / aids patients before and after the haart, a statistically significant reduction in the prevalence of these infections was observed (63.9% versus 24%). also in a 1-year longitudinal study of e. bieneusi infection in the same orphanage in bangkok, thailand, a decreasing pattern of prevalence, similar to the one observed for the incidence, was detected. microsporidia are ubiquitous in nature, and so it should come as no surprise that they can infect humans and cause clinical disease. e. bieneusi was the first microsporidia species observed by transmission electron microscopy in 1982 in villus epithelial cells in small intestinal biopsies from aids patients with diarrhea. this species is closely linked with persistent diarrhea and wasting in adults who are hiv / aids positive ; however, the outcome of follow - up studies involving children who are also hiv / aids - positive and severely malnourished children may be entirely different and warrants further study. in the last decade, several studies reported the recovery of e. bieneusi spores from children ' stools associated to different clinical spectra (symptomatic and asymptomatic infections in hiv - seropositive and seronegative children), in different settings (table 1). in immunocompetent or apparently immunocompetent children, several cases of e. bieneusi infection have been described. in a study conducted in children considered hiv seronegative, attending two primary care centers in niamey, niger, e. bieneusi spores were recovered in 8 of 990 stool samples from these children (six males and two females, with ages ranging between 3 and 26 months). three of the symptomatic ones presented other intestinal coinfections with giardia intestinalis (one), entamoeba histolytica (another), and g. intestinalis and trichomonas intestinalis (the third child) and complaint of diarrhea. due to these associations, the pathogenic role of microsporidia, in these children, is difficult to ascertain. although, for the other four symptomatic children, whose stools were positive for e. bieneusi, this was the only enteropathogen identified ; two had diarrhea only, one had diarrhea and vomiting, and the last one complaint of vomiting, dehydration, and fever without diarrhea. also a case of dual infection with e. bieneusi and cryptosporidium was reported in a healthy three - year - old turkish girl (hiv seronegative) living in germany that accompanied her family on a vacation trip to turkey, where they resided in a rural area. two months later the child developed profuse watery, nonbloody diarrhea, and nausea with recurrent vomiting, weight loss, and serious dehydration. as it happens in the above study the pathogenic role of microsporidia is difficult to ascertain in this child, due to the concomitant presence of cryptosporidium in stools. another investigation was carried out to search for microsporidian spores in the stool specimens of 344 toddlers, essentially hiv seronegative, aged 1 to 24 months, and hospitalized at a pediatric institution in tucumn, argentina. they were classified in two groups : i, made up of 222 children suffering from severe diarrheas, and ii by 122 affected by different pathologies, except gastroenteritis. the detection of microsporidia was done by light microscopy in smears of stained stools although the determination of the species involved was not done. coproparasitological and coprobacteriological studies were also carried out and the nutritional status of each child was determined. in group i, microsporidia were found in 7.2% (16/222) of the children, 4/68 (5.9%) belonged to eutrophic children, and 12/137 (8.8%) to undernourished children ; 8/16 positives were found to be related with other enteropathogenics. in group ii, microsporidia were detected in 8.2% (10/122), 4/47 (8.5%) in eutrophic children, 4/54 (7.4%) in undernourished children, and without data in two cases ; five out of 10 positives were related with other enteropatogenics. in this study, the occurrence of intestinal microsporidia was important and did not show significant differences between toddlers with or without diarrhea, eutrophic, or undernourished children. an unusual e. bieneusi genotype (peru 16), transmitted from guinea pigs, was found in a 2-year - old immunocompetent child complaining of diarrhea, in peru. this child suffered weight loss that started almost concurrently with the episode of diarrhea at the beginning of this infection episode. this finding suggests that this species may cause short - lived gastrointestinal manifestation in immunocompetent persons. also, in a study conducted in portugal, involving 295 hiv - seronegative patients with gastrointestinal complaints, of whom 18.6% (55) were children, it was observed that microsporidians were more common in children 18.2% (10/55), than in adults 6.3% (15/240), and this difference was statistically significant (p = 0.012). the age of the hiv - seronegative children infected ranged from 20 months to 8 years, with a median of 3 years. the percentage of infection by e. bieneusi was similar in both age groups, 5.5% for children against 5.0 % for the adults. in the same study, the overall percentage of hiv - seronegative patients with diarrhea (4.5% ; 7), due to e. bieneusi, was slightly smaller than that of nondiarrheal (5.7% ; 8). studies have been conducted also in immunocompromised children, especially hiv - seropositive ones. in a prospective study conducted in madrid, spain, to determine the prevalence rates of microsporidiosis and other enteroparasites in 83 hiv - seropositive children treated in three hospitals of the city, e. bieneusi the infected child was 10 years old and presented nonchronic diarrhea and a cd4 count of 298/mm. in another investigation carried out in a day care treatment center and the national hospital of niamey, niger, in hiv - seropositive patients, before the introduction of highly active antiretroviral therapy (haart), one child out of a group of 24 patients, all adults, was positive for e. bieneusi, by real - time pcr. a recent study conducted in, lisbon, portugal, involved 126 hiv - seropositive children, in a total of 561 hiv - seropositive patients with gastrointestinal complaints of whom 18.6% (55) were children, it was observed that microsporidians were more common in children 19.0% (24), than in adults 12.4% (54). the percentage of infection by e. bieneusi was higher in children (10.3 % ; 13) than in adults (6.0% ; 26), but this difference was not statistically significant. in the same study, the overall percentage of hiv - seropositive patients with diarrhea (8.6% ; 29/339), due to e. bieneusi, was lower than that of nondiarrheal (4.5% ; 10/222). also here also studies have been carrying out in institutionalized and inpatient children, independent of the hiv status. a study was undertaken in dar es salaam, tanzania, to relate the protozoan infections by cryptosporidium, microsporidia, and cyclospora with the clinical features and hiv status of children, aged 1560 months, and adults hospitalized with diarrhea. included in the study there were two groups of inpatient symptomatic children : children with chronic diarrhea (of whom 23 of 59 were hiv - seropositive) and children with acute diarrhea (of whom 15 of 55 were hiv seropositive). microsporidia spores were identified by staining methods and the species confirmed by transmission electron microscopy. enterocytozoon was identified in specimens from 2/59 children with chronic diarrhea (one hiv seropositive) and in 0/55 children with acute diarrhea. another study designed in bangkok, thailand, investigated the prevalence and clinical features of intestinal microsporidiosis in hospitalized hiv - seropositive and seronegative children with diarrhea. of the 95 hiv - seropositive and 87 seronegative children, 25.3% (24) and 14.9% (13), respectively, were diagnosed with intestinal microsporidiosis. species identification of microsporidia spores by transmission electron microscopy demonstrated e. bieneusi in five cases. malnutrition was commoner in the hiv - seropositive group (79.2% versus 23.1% ; p = 0.003). this study indicated that intestinal microsporidiosis was an important disease in both hiv - seropositive and -seronegative thai children with diarrhea. in a cross - sectional, case - control study, involving children aged 14 days), admitted to the same hospital in kampala, uganda, using microscopy and pcr methods, screened for the presence of e. bieneusi and cryptosporidium in the children ' stools. they found that 32.9% (80) of them were excreting e. bieneusi, and 31.3% (76) were excreting cryptosporidium. ninety - one of the 243 children were hiv seropositive, of whom 76.9% (70) had e. bieneusi, versus 6.6% (10) of the 152 hiv - seronegative (odds ratio = 47.33 ; 95% ci = 19.88 to 115.97), while 73.6% (67) had cryptosporidium, versus 5.9% (9) without hiv (odds ratio = 44.36 ; 95% ci = 18.39 to 110.40). children with counts 10% of the baseline (63% ; 19/30). in a 10-year study, performed in portugal, involved 856 (675 adults and 181 children) hiv - seropositive and -seronegative patients with gastrointestinal complaints, an immunosuppressive condition and youth (children) were the risk factors observed for microsporidian infection. other risk factors associated with intestinal microsporidiosis, in general, or particularly to e. bieneusi infection have been reported. a us study of hiv - seropositive patients with diarrhea showed significant association of intestinal microsporidiosis with contact with horses, having been stung by a bee, hornet, or a wasp, and having used injection drugs. the use of hot tub or spa and occupational contact with water also reached statistical significance in this study. also person - to - person transmission has been suggested in an orphanage, where a multivariate analysis showed that orphans who were 1223 months old, girls, and living in one particular house were independently associated with e. bieneusi infection. all infected children presented the same e. bieneusi genotype in stools [5, 19 ]. another study in zimbabwe showed association of e. bieneusi infection in hiv - seropositive patients with living in rural areas, consumption of nonpiped water, contact with cow dung, and contact with a person with diarrhea. these authors postulated the reasons for the unclear association between injection drug use and enteric microsporidiosis stating that injection drug users were as likely or more likely to contact potential infectious sources, such as contaminated water. contact with water is an important risk factor consistently associated with e. bieneusi infection in epidemiologic studies, and e. bieneusi spores have been detected in ground, surface, ditch, and crop irrigation water sources [33, 105107, 132 ]. however, no significant seasonal variation has been detected in the prevalence of intestinal microsporidiosis in hiv - seropositive patients. in a study of hiv / aids patients performed in peru, risk factors for e. bieneusi infection differed by genotype. infection with genotype peru-1, one of the more common genotypes found, was associated with contact with duck or chicken droppings and lack of running water, flush toilet, or garbage collection. e. bieneusi has been able to cause clinical disease in immunocompromised and immunocompetent persons with different patterns, and the risk factors associated with these pathologies are partially known as a result of improvement in genetic typing and molecular epidemiology. the combination of epidemiologic studies with genotyping techniques has contributed to a better understanding of the characteristics of microsporidia that infect humans and of its reservoirs and transmission patterns. before the introduction of the molecular tools, the characterization of this important group of parasites relied exclusively on electron microscopic observations of the morphological differences in spores and/or endogenous developmental stages and host occurrence. the recent application of pcr - based molecular methods for species - specific identification and genotype differentiation of microsporidia has increased research interests in this group of microorganisms. the enhanced diagnostic capacity obtained with the molecular methods has improved the identification of human cases of microsporidiosis due to e. bieneusi, not only in developed countries [6, 7, 17, 20, 30, 38, 45, 62, 66, 81, 87, 92, 108, 112, 115 ], but also, and especially, in developing countries where e. bieneusi may be a public health problem due to the magnitude of the hiv pandemic and poor sanitary conditions [18, 19, 30, 47, 48, 5052, 5456, 62, 64, 68, 88, 89, 99, 102 ]. enterocytozoon bieneusi is a complex species with multiple genotypes and diverse hosts range and pathogenicity. since different strains can not be discriminated morphologically, typing of this species relies on molecular methods. pcr analysis of the internal transcribed spacer (its) of the rrna gene, a hypervariable sequence with about 243 bp long, followed by sequencing of the pcr products and comparison of obtained its sequences with those in the databases, is the standard method for genotyping e. bieneusi isolates from humans and animals. for several years these were the only genetic markers available [22, 56, 104, 133136 ]. a few researchers genotyped e. bieneusi based on the analysis of the its by pcr - restriction fragment length polymorphism (rflp) [92, 109 ]. and recently, two groups of researchers reported the analysis of the genbank e. bieneusi its sequence collection, using statistical methods, with the aim to obtain information about diversity, transmission, and evolution of this species [135, 136 ]. these studies have identified the presence of host - adapted e. bieneusi genotypes in a variety of domestic animals and wild mammals, as well as a large group of e. bieneusi genotypes that do not appear to have host specificity [24, 25, 135, 136 ]. e. bieneusi genotypes in humans have been shown to differ from each other in virulence and geographic distribution [48, 136 ]. other authors, taking advantage of the current genome sequence surveys of e. bieneusi, identified four polymorphic microsatellite and minisatellite markers. together with the its, they became part of a multilocus sequence typing (mlst) technique developed for genotyping e. bieneusi that could be useful in epidemiologic investigations of e. bieneusi transmission, especially those concerning the public health significance of parasites of animal origin. the observations, obtained with the study of the its locus, were also corroborated with the phylogenetic analysis of the polymorphic microsatellite and minisatellite markers, identified by feng and collaborators in the e. bieneusi genome. these authors observed the formation of two large groups of e. bieneusi : zoonotic genotypes and host - adapted genotypes. actually, more than 90 different genotypes of e. bieneusi, identified by its sequence, have been described in humans and animals, and some genotypes have multiple names that have been reviewed in satn and fayer, 2011 who tabulated all the existing synonyms for the first time and proposed a standardized nomenclature for e.bieneusi genotypes based on the its sequences. about 34 of these genotypes have been found only in humans [5, 18, 24, 48, 75, 76, 92, 94, 99, 109, 112, 114, 115 ] (table 3), and 11 genotypes have been reported both in humans and animals [18, 21, 2429, 48, 55, 56, 75, 76, 92, 95, 99, 102, 109, 111114, 116, 118127, 129, 139 ] (table 4). all these observations were made in populations from different geographic regions, involving all continents. in studies conducted in france involving hiv seropositive and seronegative patients infected with e. bieneusi, liguory and collaborators found five genotypes (types i to v) [109, 112 ]. type i, synonym of genotype b, was the most prevalent genotype (75%). genotypes b (type i) and c (type ii) were also found in other populations [94, 95, 99, 111, 112, 115 ]. genotype b (type i) even showed predisposition toward hiv / aids patients and was the only genotype found in a study performed in hiv - seropositive patients in australia. type iv, also named genotype k / peru2/ptebiii / beb5/beb5-var / cmits1, was rare (only in one human isolate). in contrast, type iv (genotype k) was the only genotype found in a population in cameroon. moreover, this genotype was also found in humans in other regions [18, 48, 56, 75, 76, 99, 111, 112 ] and in various animals [2527, 122125 ]. peru, in a population of 2,672 hiv / aids patients, 11 genotypes were identified (peru-1 to peru-11). the most prevalent genotypes were peru-1 (39%), synonym of genotype a, peru-2 (19%), synonym of type iv, and peru-9 (10%), also named genotype d ; the remaining eight genotypes were found in the population at much lower frequencies (19%) [48, 56, 75 ]. some of these genotypes were also found in animals, including genotype a (peru-1), type iv (peru-2), peru-4, also named genotype ebpc / e / wl13/wl17, to peru-6, also named pteb1/ptebvii, peru-7, d (peru-9), peru-10 and peru-11, also named peru-12 [21, 2428, 32, 55, 75, 95, 114, 116, 117, 121126 ]. three genotypes (a / peru-1, peru-7, and peru-11), found until recently only in humans, were also identified in animals [32, 117 ]. both genotypes d and type iv (reported as genotype k) were the most prevalent among patient groups from malawi and the netherlands. genotype d that was commonly detected in hiv - seropositive patients was also found in hiv - seronegative individuals, confirming its widespread nature [56, 99, 101, 102, 111, 113 ]. in a study conducted in renal transplant recipients in spain, also in peru, in a prospective pediatric - cohort study of enteric parasites, e. bieneusi infection was identified in 31 of 388 (8%) children. thirty of these children had infections with genotypes peru-1 (synonym of a) through peru-15 and peru-17, and one child was infected with genotype peru-16. fecal samples of all animals in the household of child infected with peru-16 were also analyzed including guinea pigs, chickens, dogs, and cats. peru-16 was identified in seven of the eight guinea pigs studied, and all the other animals were negative for microsporidia. this genotype was genetically very different from other known e. bieneusi genotypes in humans and was placed, in an independent clade, outside the cluster of genotypes considered to have broad host specificity in a phylogenetic analysis of the its sequences. the finding of genotype peru-16 in guinea pigs from unrelated households, and the close contacts between the study child and infected guinea pigs, all strongly suggested that the child probably acquired the infection from the guinea pigs in the household. this finding indicated that even some e. bieneusi genotypes that are apparently unique in some animals have zoonotic potential. one of the genotypes, peru-14, also named genotype wl15/wl16, was also found in animals. genotype ug2145, identified for the first time in uganda, was the only e. bieneusi detected in a population of 1,779 children with diarrhea. some genotypes like genotypes b and c are considered host specific because they were exclusively found in humans. however, it must be kept in mind that they differ in very few base pairs from other sequences commonly found in other hosts. for instance, genotype type iv differs from b in 3 bp, and from a in just 1 bp. in contrast, some other genotypes have a zoonotic potential (such as genotypes d and type iv, and more recently genotype a) because they have been seen in various animals [18, 2427, 29, 48, 75, 76, 95, 111, 112, 116, 118, 119, 122124, 126, 127, 129, 139 ]. breton and collaborators revealed for the first time the presence of genotypes a and b in africa. these genotypes were also reported from hiv - seropositive and hiv - seronegative populations in europe [92, 94, 109, 111, 112, 121 ], peru [56, 75 ], thailand [5, 114 ], and niger. in breton and collaborators study, genotype d was found in one hiv - seropositive patient in gabon, and it was identified for the first time in isolates from three hiv - seronegative individuals in cameroon. it is considered a genotype with a broad host and geographic range, having been found in several countries in europe [26, 111, 112, 127 ], usa [24, 119 ], peru [48, 56, 75 ], malawi, thailand [5, 102 ], vietnam, and korea [124, 129 ]. three new genotypes caf1, caf2, and caf3 that are close relatives of genotypes e, type iv, and d, respectively, were found for the first time in the hiv - seropositive patients from gabon, with 10 out of 15 isolates. another new genotype, caf4, a highly divergent genotype reported from humans, was equally present in hiv - seropositive patients in gabon and hiv - seronegative individuals in cameroon. its high frequency (25%) in both countries may indicate that this genotype is common in central africa. ten hove and collaborators found nine new genotypes : six (s1-s6) were from malawi, and three (s7-s9) were from the netherlands. genotype s7 showed no linkage to any of the described genotypes and seems to be more related to the genotypes isolated from cattle. the patient (hiv infection was considered unlikely) infected with this e. bieneusi genotype was a middle - age man presenting severe diarrhea and rectal bleeding. two other new unnamed genotypes were detected in this study, af502396 and ay371283, actually named as ue2145 and peru-8, respectively. in a study conducted in changchun city, china, involving 40 fecal samples from diarrheal children and 180 fecal samples from animals (61 from pigs, 26 from dogs and 93 from cows), all collected in the same area around the city, zhang and collaborators found two previously reported genotypes (genotypes i and j), and 10 new genotypes (chn1 - 10). genotypes i (also named beb2/cebe) and j (also named beb1/cebb, pteb x), originally detected in cattle, were identified in both cow and human samples in this study. both genotypes coinfected with other genotypes in all positive samples. of the novel genotypes, chn1, which was detected in five children, nine cows, and four pigs, was the most common genotype found in this study. genotypes chn3 and chn4 were also found in both human and cow samples, and genotypes chn5 - 10 were only detected in animals. the its sequences of the 10 new genotypes were highly homologous to those of genotypes i, j, type iv, and g published earlier [25, 75, 101, 109, 129 ]. genotypes chn1, chn2, and chn3 differed from j by one to four positions, while genotype chn4 is 2 bp shorter than type iv. due to the high level of similarity of the sequences, no obvious clusters related to host preference were observed. in a study that investigated the occurrence and prevalences of the microsporidial species enterocytozoon bieneusi and encephalitozoon spp. in 382 immunocompetent humans in the czech republic the sequence analyses of e. bieneusi - positive samples revealed seven different genotypes. for the first time, after so many studies performed in humans, in different geographic areas, genotypes that were previously reported only from cattle (beb4 and ebpa) and pigs (pigebits5, ebpa, and bfrmr2) were identified in humans, and three new genotypes were detected (e. bieneusi cz1 to cz3). the most prevalent e. bieneusi genotypes were ebpa (in 10 individuals), cz3 (in 4 individuals), and pigebits5 (in three individuals), followed by bfrmr2 and beb4 (each in two individuals). the cz1 and cz2 genotypes were identified only in single individuals. in the same study e. cuniculi genotype ii these data suggest that isolates of this group are able to switch hosts from animals to humans and should be regarded as isolates with high zoonotic potential. the high prevalences of microsporidia infection surprisingly found in different populations of immunocompetent humans in this study raise the question of whether these findings represent true infection resulting in shedding of parasites or ingested parasites that just passed through the gastrointestinal tract without establishing an infection. however, the chance of detecting the temporary passage of spores is quite low in the number of individuals sampled analyzed in this study. several studies indicate that the distribution of genotypes of e. bieneusi can vary by geographical locations, and it has been recently proposed that predominant genotypes in different geographical sites could be related to distinct sources of transmission. in a comparative study of the molecular epidemiology of microsporidiosis among hiv - seropositive patients in two separate geographical areas, niamey, niger and hanoi, vietnam, interesting results were obtained. in hanoi, two zoonotic genotypes d and e were identified. both genotypes have been previously recovered in both humans and animals [21, 24, 26, 56 ]. these genotypes were also predominant in a study conducted in thailand in hiv - seropositive patients. thus, there is a potential zoonotic transmission of e. bieneusi in thailand and vietnam. zoonotic transmission can be from direct exposure to animals or by contamination of surface water by discharged domestic wastewater or animals. on the contrary, the niamey data highlighted a higher frequency of genotypes known to be human specific (genotype a), similar to results obtained in children from an orphanage in thailand. these results suggest that transmission of microsporidiosis due to some e. bieneusi genotypes could occur through person - to - person contact. this mode of transmission could be facilitated by chronic carriage of e. bieneusi, as described in previous studies [49, 140 ]. all these findings suggest different transmission modes of e. bieneusi in diverse geographic regions and in special groups in the same region. however, further studies with larger number of e. bieneusi samples are needed to confirm these findings. despite recent advances in the understanding and diagnosis of e. bieneusi, emerging microsporidian pathogens, more research is necessary to elucidate their complex epidemiology. in fact, studies that reflect true human infecting e. bieneusi prevalence are still insufficient. a few prospective studies conducted in developed countries indicate that the prevalence of e. bieneusi in hiv - seropositive patients is progressively decreasing, probably due to the use of haart. the recovery of spores of e. bieneusi in fecal samples of children considered hiv - seronegative, and in healthy ones, indicated the presence of enteric carriage of infection in immunocompetent persons in tropical countries, but also in other regions of the globe. also, the finding of asymptomatic chronic carriers of e. bieneusi emphasizes their importance in the transmission cycle, warning for the potential risk of reactivation of latent infection in cases of immunosuppression causing life - threatening disease in these individuals. the pathogenicity of microsporidia is still not clearly defined, and the mechanism by which they induce diarrhea has not been determined. even though some genotypes of e. bieneusi can cause chronic diarrhea and wasting syndrome in aids patients once the cd4 + t - lymphocyte count drops below 100 cells / mm. the recent application of pcr - based molecular methods to e. bieneusi identification and characterization has led to more reliable results compared with prevalence rates determined by light microscopy of stained biological smears, to better study the risk factors associated with these pathologies, to study the distribution of genotypes of e. bieneusi by geographical location, to improve source tracking, and to calculate the host range and pathogenic potential of an isolate. all typing studies performed until now were based on the analysis of the its sequences, the only molecular marker existent until recently. these studies identified the presence of host - adapted e. bieneusi genotypes in several animals, and e. bieneusi genotypes with no host specificity, which are considered zoonotic. some studies suggest that transmission of microsporidiosis due to some e. bieneusi genotypes could occur through person - to - person contact. this mode of transmission could be facilitated by chronic carriage of e. bieneusi, as described in some studies. besides, several microsporidian species that infect humans have been identified in animals, especially mammals, raising public health concerns about zoonotic and waterborne transmission of microsporidia. in addition, some studies indicate that even some e. bieneusi genotypes, which are apparently unique in some animals, have zoonotic potential. other studies indicate that the distribution of genotypes of e. bieneusi can vary by geographical locations and virulence. despite of the great genetic variation of e. bieneusi observed worldwide with the analyses of the its region of the rrna gene, studies with additional independent markers for e. bieneusi are highly desirable in order to clarify the genetic structure of the parasite 's populations. the discrimination among strains of e. bieneusi is very important, not only for the clarification of the reservoirs and of the transmission modes of this pathogen but also to help to explain its variations in pathogenicity that can not be answered with the study of a single locus. | a review was conducted to examine published works that focus on the complex epidemiology of enterocytozoon bieneusi infection in humans. studies on the prevalence of these emerging microsporidian pathogens in humans, in developed and developing countries, the different clinical spectra of e. bieneusi intestinal infection in children, in different settings, and the risk factors associated with e. bieneusi infection have been reviewed. this paper also analyses the impact of the recent application of pcr - based molecular methods for species - specific identification and genotype differentiation has had in increasing the knowledge of the molecular epidemiology of e. bieneusi in humans. the advances in the epidemiology of e. bieneusi, in the last two decades, emphasize the importance of epidemiological control and prevention of e. bieneusi infections, from both the veterinary and human medical perspectives. |
traumatic brain injury (tbi) is a significant public health problem leading to mortality, morbidity, and socioeconomic losses in india. the majority (60%) of tbi cases are a result of road traffic accidents (rta). among rta, pedestrian injuries are most common followed by two - wheelers and bicyclists. the roads of different types, widths, with varying topographic and climatic conditions, influence the travel pattern in rural and urban areas. walking and cycling the pedal bicyclist constitutes a significant share of total traffic on indian roads accounting for 15 - 35% of total trips. the incidence of head injuries in cyclists ranges from 14 - 36% of the total injuries. their demographic details, risk, cause, severity, helmet benefit, mortality rate and preventive measure studies have been well studied in developed countries. in our country, cycling forms a major mode of non - motorized transportation in villages and small cities, but literature on head injuries sustained in bicycle crashes is lacking. the present study aimed to explore the knowledge regarding the demographic, clinical and imaging findings, and outcome of bicycle - related injuries at our institute. this study was done at a tertiary level referral centre for treatment of neurological, neurosurgical, and psychiatric patients. patients with tbi, who have a different registration process, are evaluated by neurosurgery residents and data is entered in a structured head injury proforma, which consists of comprehensive demographic, clinical and computed tomography (ct) scan findings. data of all patients during a period from 15 may 2011 to 15 november 2011 were retrospectively reviewed. the ct scan findings were confirmed independently from images archived in picture archival communication system (pacs). from these records the data of all patients who sustained head injuries while riding a bicycle were identified. the outcome assessment tools used were glasgow outcome scale (gos), rivermead post - concussion symptom questionnaire (rpcsq), and rivermead head injury follow - up questionnaire (rhfuq). the assessment was done at three months to six months after injury. to assess these scales we strictly followed the duration with minimum three months to maximally six months after injury because during the initial three months after head injury the majority of patients (70 - 80%) with mild to moderate severity manifests with concussion symptoms, but after three months these symptoms decrease to 15 - 20% and may be persistent for long time. data were expressed using descriptive statistics such as mean and standard deviation for continuous variables, and frequency and percentage for categorical variables. logistic regression was used to compare moderate to severe injury with mild injury on crash characteristics. the independent variables considered were victim 's occupation, ear, nose and throat bleed (ent bleed) (present or absent), loss of consciousness (present or absent), ct findings (normal or abnormal), skull fracture (present or absent). the significance was calculated by chi square for categorical groups and independent t test for continuous data. data were expressed using descriptive statistics such as mean and standard deviation for continuous variables, and frequency and percentage for categorical variables. logistic regression was used to compare moderate to severe injury with mild injury on crash characteristics. the independent variables considered were victim 's occupation, ear, nose and throat bleed (ent bleed) (present or absent), loss of consciousness (present or absent), ct findings (normal or abnormal), skull fracture (present or absent). the significance was calculated by chi square for categorical groups and independent t test for continuous data. rta accounted for 3388 (50%) cases. among them, bicyclists accounted for 108/3388 (3.18%) cases. most of these patients were young males, either student or laborers, from poor socioeconomic status, and sustained injuries during daytime due to collision with other vehicles.. demographic profile of patients the clinical and imaging findings are given in table 2. the head ct scan was done for 98 (90.7%) cases, and was normal in nearly half. clinical and imaging findings of all 108 cases out of 108 patients, 74 were available for outcome assessment at mean 4.5 months after injury by telephonic interview. the outcome of these patients is given in tables 3, 4a and 4b. the follow - up details of functional outcome of 74 patients post - concussion symptoms follow - up details of 74 patients head injury follow - up details of 74 patients most of the survivors had a good recovery. the post - concussion symptoms were more frequent among middle - aged patients presenting with moderate tbi. six patients died due to vehicle collision and two patients due to fall. in five cases the mortality was attributed to severe tbi, and in three cases to extra - cranial injuries. only one fatal case was from an urban area, the rest were from rural areas. in the present study model the dependent variable, moderate and severe traumatic brain injury, was significantly affected by the occupation of the patient, and history of loss of consciousness (loc). the analysis results showed that labor workers were more severely injured (p = 0.033) with five times more risk of sustaining moderate to severe injuries as compared to students. patients presenting with loss of consciousness were more severely injured (p = 0.014) with four times more risk of sustaining moderate to severe injuries when compared to patients manifesting with no loss of consciousness [table 5 ]. logistic regression analysis for severity of injury there was no significant difference between rural and urban cycle injuries as compared with patient 's occupation, crash type, primary care, associated injuries, injury severity and ct findings [table 6 ]. it is an easy way to get exercise that builds up muscle strength and tone, and keeps the heart healthy. the expenses for using and maintaining it are minimal. it is the commonest mode of transportation among the rural population and low - income urban population in a developing country. the cyclists are vulnerable to injuries in rural areas due to bad, narrow roads, and sometimes domestic animals may either stand or sit on the roads, thereby obstructing the passage. the present study showed that 50% of injuries occurred in the middle age group. in the middle - aged group most were males of the labor class who sustained injuries during daytime while going to work. studies from developed countries have described the injury patterns and possible preventive measures among preschool and school children cyclists. a prospective study by davidson., showed that the majority of injured cyclists were men on roads without separate lanes during daylight hours. our data also showed that bicycle collision with moving vehicles, especially motorcycles, auto - rickshaws, and cars was the commonest crash type in all age groups., have shown that the common cause of injury was fall from moving bicycle followed by collision with other moving vehicle. on logistic regression our results showed that the menial workers and patients manifesting with loc after injury are at risk of developing moderate to severe injury. it could be that this group of workers has to use busy arterial roads where they are exposed to high - speed vehicular traffic., reported that the risk of serious injuries among cyclists was increased by vehicle collision, self - reported speed > 15 mph, and age over 39 years. the present study data showed that eight patients (7.4%) died, all were males. six patients (5.5%) died due to vehicle collision and two patients (1.8%) due to fall. probably, village roads have unpaved paths or improper surfaces with partial concrete and craters which explains the increased accidents in rural areas as observed from our study. also, a fall on these roads could result in head injury and/or polytrauma which could be more severe. in the literature the mortality rate among pedal cyclist ranges from 0.18 - 1.5%, and tbi is the leading cause of death. none of the patients in our study used a helmet while cycling due to lack of information and laws to enforce the rules. outcomes were measured after head injury that accessed improvement in functioning and quality of life, measures of activity and participation, measure of psychological and psychosocial adjustment. the outcome was favorable in most of the patients but a significant number of cases had post - concussion symptoms like headache, giddiness, tiredness, and fatigue and decreased interactions with others, which were more frequent among middle - aged patients presenting with moderate tbi. we could not explain the exact details of one - third of the patients who did not respond to telephonic interview for outcome assessment. separation of cyclists from motor vehicles by dedicated bicycle tracks. pre - event and event phase separate signals and low speeds at intersection. event and post - event phase wear proper bicycle helmets. post - event medical management and rehabilitation for persistent concussion symptoms. we could not explain the exact details of one - third of the patients who did not respond to telephonic interview for outcome assessment. separation of cyclists from motor vehicles by dedicated bicycle tracks. pre - event and event phase bicycle injuries are common among middle - aged males from a rural background due to vehicular collision. the laborers and patients presenting with loss of consciousness were at high risk of developing moderate to severe injuries. | objectives : to describe the epidemiology of head injuries sustained due to bicycle accidents in india.materials and methods : data were retrospectively collected over a period of six months (15 may 2011 to 15 november 2011). demography of patients, glasgow coma scale (gcs), clinical and imaging findings, and mortality and outcome using glasgow outcome scale (gos), rivermead post - concussion symptom questionnaire (rpcsq) and rivermead head injury follow - up questionnaire (rhfuq), were analyzed. outcome was assessed by telephonic interview.results:there were 108 patients (100 males) with mean age of 27.7 years. seventy - four (68.5%) were from rural areas. accidents due to vehicular collision accounted for 60 (55.6%) cases. none wore a helmet. the admission gcs was 14 - 15 in 68.5% cases, 13 - 3 in 31.5%. the risk of moderate to severe injuries was increased among working laborers (or = 5), and patients with loss of consciousness (or = 4). sixty - three (49%) patients had abnormal computed tomography (ct) findings ; most common finding was skull fracture 25 (23.1%). four patients needed surgery. the gos assessment at three to six months revealed favorable outcome in 66 patients (61.1%) and death in 8 (7.4%). the common post - concussion symptoms were headache, fatigue, and poor concentration.conclusion:the majority of hospitalized cyclists were from a rural background and of the lower income group. after three months the majority of patients had good recovery with few persistent concussion symptoms. |
on august 2007, a 29-year - old man from tucuru in northern brazil, who worked in power grid maintenance in a forested area, came to his ophthalmologist with an intraocular larva in the left eye. there was no familial history of ophthalmologic disorders, and ophthalmologic examinations showed that the patient had visual acuity and corrected vision of 20/25 in both eyes. biomicroscopy showed a transparent cornea in the right eye without lesions or edema, an anterior cavity without an inflammatory reaction, and an anterior subcapsular cataract of + /4 +. the cornea in the left eye was transparent and did not have lesions or edema, and the anterior chamber did not show an inflammatory reaction. no funduscopic alterations were found in either eye by direct and indirect ophthalmoscopic examination. an 4-mm worm with undulating movements was observed between the muscular fibers of the iris (figure 1, panel a). the patient underwent surgery 1 day after the consultation, and he consented to the publication of this clinical case. after peribulbar anesthesia, a 2-mm corneal incision was made at the 11 oclock position. the nematode was extracted by aspiration (video) and placed in saline solution. no surgical complications occurred (figure 1, panel b), and the patient did not have ocular symptoms during the 6 months after surgery. b) iris after surgery, showing a mild residual scar (cr) in the region where the nematode had been located. surgical removal of apelecitus sp. a portion of the material in this video was previously published in the journal parasites and vectors (http://www.parasitesandvectors.com/content/pdf/1756-3305-4-41.pdf). direct link : http://streaming.cdc.gov/vod.php?id=bec4b66c4804f440dce5072b04eeba0c20110426172737138 the worm was fixed in 2% acetic acid, 3% formaldehyde, and 95% ethanol ; mounted in glycerine jelly ; and later transferred into lactophenol. the specimen was preserved in absolute alcohol at the national museum of natural history (paris, france) (accession no. 138 yu). this male nematode (length 4.5 mm, width 300 m at mid - body) had a coiled and twisted body that tapered at both extremities (figure 2, panel a). the cuticle (thickness 6 m) showed 2 rounded, lateral, cuticular alae (thickness 20 m) along the body and postdeirids 530 m from the posterior extremity (figure 2, panel b). the head was bluntly rounded and contained 4 externolabial papillae, 4 cephalic papillae, 2 amphids, and a buccal cavity (length 5 m, width 4.5 m) with a tiny cuticular ring. the esophagus was 765 m long, increased slightly in diameter in the posterior half, and did not have a distinct glandular part. five pairs of caudal papillae (2 pedunculated, precloacal, lateral ; 1 small subventral closely posterior to the cloacal opening ; and 2 pedunculated lateral pairs on posterior half of the tail) were observed, and the 2 phasmids were subterminal. the 2 spicules (length 66 m and 82 m) (figure 2, panels c, d) were dissimilar. parasitic nematode isolated from the eye of the patient, a 29-year - old man from brazil. a) nematode that was removed from the iris, showing anterior (ae) and posterior (pe) extremities. b) caudal region, subdorsal view, showing lateral alae, spicules, and the 2 postdeirids. many morphologic characteristics of the filarial worm resembled those of pelecitus spp (6). (coiled and twisted body that was attenuated at both extremities ; lateral alae from the cervical region to distal tip of body ; postdierids within alae in the posterior half of body ; and a delicate, preesophageal, cuticular ring). pelecitus spp. include mainly parasites of birds and a few mammals, some of which have been identified as loaina spp (6,7). the specimen from the patient was compared with the 16 known species of pelecitus described (6,8,9), but the specimen did not match any of them. the worm differed from the only 2 species found in lagomorphs (p. scapiceps and p. meridionaleporinus) (9) in north america and mexico, which had a beveled extremity on the right spicule (8,9) instead of the left spicule. a small male filaria was recovered from the anterior chamber of a human eye in colombia (5). this filaria was originally assigned to the genus loaina but was later identified as a species of pelecitus (6). like the nematode specimen we assigned the worm found in the anterior chamber of the eye of the patient to the genus pelecitus (6). the species of pelecitus that infected the eyes of 2 humans (reported here and in colombia) (5) remains unidentified. these cases were found in the tropical amazon region (par, brazil) and the department of antioquia (northwestern colombia). the male specimen of pelecitus sp. described here and the species that infected a human in colombia are similar but distinct. however, in both cases, a mammalian origin of these zoonotic agents seems unlikely because of differences identified by comparing these worms with parasitic species infecting lagomorphs (8,9). vectors of pelecitus spp. are mosquitoes, chewing lice, and tabanids, as shown with the 3 cycles elucidated (1012). although infection of birds in south america by pelecitus spp many nematode species have not been identified because of lack of basic information on filarial fauna of animals. this dearth of information is particularly true for regions, such as the amazon rain forest in brazil, where wide biodiversity and many unidentified animal and plant species are found (15). consequently, species identification of filarioid nematodes that infect human eyes is difficult if not impossible. however, our identification of this filarid should help clarify the zoonotic role of filarioid infections in humans in tropical regions and increase awareness of physicians and ophthalmologists of the variety of nematodes that may be found in the human eye. | a male nematode was extracted from iris fibers of a man from the brazilian amazon region. this nematode belonged to the genus pelecitus but was distinct from the 16 known species in this genus. similarities with pelecitus spp. from neotropical birds suggested an avian origin for this species. |
nearly 20 years ago, a unique cd4 t cell type lacking the expression of costimulatory cd28 surface receptors was first described in rheumatoid arthritis (ra) patients [1, 2 ]. t cell receptor (tcr) stimulation as primary and cd28 costimulation as secondary signal are necessary for t cell activation. cd28 is further involved in survival, il-2 production, metabolic activity, and clonal expansion of t cells [13 ]. as these cells also lack cd28 mrna, it became clear that cd28 expression is blocked at the transcriptional level. downstream of the tata box in the promoter region of the cd28 gene, there are two regulatory motifs, called sites and, as described in two transformed t cell lines (cd28 jurkat and cd28 hut78), two primary cell lines (h28p and h28n), and two t cell clones (pl65 and k2) [5, 6 ]. the cd28 -initiator is activated by the binding of a protein complex consisting of the site specific proteins nucleolin and the a isoform of heterogeneous nuclear ribonucleoprotein - d0 (hnrnp - d0a). both proteins were separately found in the cd28 jurkat and the cd28 hut78 t cell lines, but as a complex they were only detectable in cd28 t cells, suggesting that complex formation is disturbed by posttranscriptional changes, such as phosphorylation of serine or threonine residues. the exact mechanism causing the inhibition of the complex formation and further the loss of cd28 is still unclear. nevertheless, the provided data support the hypothesis of a cell type distinguishable on transcriptional level, due to the lack of cd28 mrna. a notable factor involved in cd28 downregulation is tnf-, an inflammatory cytokine also produced in cd4cd28 t cells itself [8, 9 ], leading to downregulation of surface cd28 and affecting mrna expression by long term exposure to jurkat cells. also peripheral blood mononuclear cells cocultured with tnf- showed downregulation of the cd28 costimulatory receptor, and high levels of tnf- are associated with higher levels of cd4cd28 t cells in patients with unstable angina, supporting the findings of the in vitro studies. in contrast to tnf-, exposure to il-12 leads to reexpression of cd28 on cd4cd28 t cells isolated from peripheral blood mononuclear cells but has no effect on cd4cd28 t cells. in these experiments, however, il-12/tcr costimulation resulted in reexpression of cd28 in about 50% of the cd28 t cells, suggesting heterogeneity in this cell population. the reexpressed cd28 receptors are functional, meaning the cells regain their complete costimulatory signalling capacity, including the following expression of responsive genes, like the cd40-ligand (cd40l). it is still a question to solve how il-12 can reverse transcriptional inhibition of the cd28 gene by modifying the complex proteins nucleolin and hnrnp - d0a. taken together, loss of cd28 transcription is a typical feature of a specific cd4 t cell subset but is still reversible in some cells. this review focuses on the characteristics of cd4cd28 t cells and their physiological and pathogenetical role in humans. usually both cd4cd28 and cd8cd28 t cells are present in the peripheral blood, but cd4cd28 t cells occur less frequently than their cd8 counterparts. in healthy individuals older than 65 years cd4cd28 t cells accumulate to up to 50% of total cd4 t lymphocytes [3, 5, 10 ], whereas healthy, young people have only few cd4cd28 t cells accounting for 0,1 to 2,5% of all cd4 t cells [1, 14 ]. the enormous accumulation of cd4cd28 t cells with age indicates an involvement of the immune system in aging and represents cd28 as a marker of t cell aging. at first, researchers detected elevated levels of cd4cd28 t cells in patients with ra [15, 16 ] followed by a broad range of other immune - mediated diseases, (like ankylosing spondylitis (as) [9, 17 ], dermatomyositis and polymyositis, polymyalgia rheumatica and giant cell arteritis, systemic lupus erythematosus, granulomatous polyangiitis, multiple sclerosis, and active crohn 's disease), in acute coronary syndrome [2426 ], abdominal aortic aneurysms, and chronic kidney graft rejection and even in infectious diseases, like infections with cytomegalovirus (cmv) [29, 30 ], human immunodeficiency virus (hiv) [31, 32 ], trypanosoma cruzi, and chronic hepatitis b. in summary, prevalence of cd4cd28 t cells is high in chronic inflammatory diseases [25, 35 ], immunodeficiency [28, 32 ], and specific infectious diseases. based on these findings, presence of cd4cd28 t cells has been proposed as a biomarker not only for normal aging, but also for early aging of the immune system under pathological conditions. however, phenotypic and functional characteristics of cd4cd28 t cells may vary depending on the underlying disease, disease activity, and concurrent treatment and were not performed at the same extent in all of the mentioned diseases. interestingly, cd4cd28 t cells show limited tcr diversity in the peripheral blood and form oligoclonal populations [1, 37 ], most likely as the result of repeated exposure to the same antigen. these clonally expanded cd4cd28 t cells were also found in the synovia of ra patients even though some of the detected clonotypes were cd28, suggesting that the special environment of the synovia may support reexpression of cd28 on cd4 t cells. the permanent activation resulting in oligoclonality could be triggered by either endogenous or exogenous antigens including viral antigens expressed during chronic viral infections. one of the analysed viruses in this context, the human cytomegalovirus (cmv), which occurs in more than 90% of the elderly [38, 39 ], is still discussed as a candidate for ongoing stimulation of the immune system. however, the high infection rate of cmv results in a virtually absolute cooccurrence with cd4cd28 t cells. a good argument for cmv involvement is the finding that 30-fold more cd4cd28 t cells respond to cmv compared to their cd28 counterparts. cmv alters production of diverse cytokines in host cells possibly supporting the induction of the cd4cd28 t cell phenotype. for example, in dendritic cells cmv infection may lead to cellular activation via the viral toll - like receptors (tlr) 7 and 9 and secretion of high amounts of ifn-, causing downregulation of the costimulatory molecule cd28 [40, 41 ]. proliferation and cytokine production of cd4cd28 t cells are induced by cmv stimulation but not by stimulation with other antigens like tetanus toxin or varicella - zoster virus. viral dna of cmv was also found in the synovial tissue and fluid of ra patients and is associated with the development of cardiovascular diseases, suggesting a common pathogenetic background of these diseases with cmv and cd28 t cell expansion [29, 30 ]. this hypothesis is supported by a study, which shows that cd4cd28cd57 t cells were only expanded in cmv seropositive ra patients, indicating a previous cmv infection. moreover, some as patients were also tested positive for cmv and epstein - barr virus (ebv), but only titres of immunoglobulin (ig) g for cmv but not for ebv correlated with prevalence of cd4cd28 t cells. these results together with the finding that cd4cd28 t cells could be stimulated by cmv antigens strongly suggest an involvement of cmv in cd4cd28 t cell activation. besides, it was shown that more than 50% of cultured cd4cd28 t cells from patients suffering from acute coronary syndrome recognize the 60 kd human heat - shock protein (hhsp-60), an ubiquitously expressed intracellular chaperone protein. however, hhsp-60 could only be recognized by cd4cd28 t cells from coronary artery disease patients during the acute phase and not from patients with chronic stable angina during the stable phase or healthy individuals. heat - shock proteins can be expressed by all cells under specific stress conditions, including increased temperature, an inflammatory environment or oxidative stress. the detection of hsp - reactive t cells suggests that these proteins represent self - antigens triggering immunoregulatory pathways in inflammatory diseases. the production of ifn- and perforin after activation of cd4cd28 t cells with hhsp-60 confirms its stimulatory feature and indicate, especially due to its ubiquitous expression that hhsp-60 is the factor responsible for permanent t cell activation leading to oligoclonality in acute coronary syndromes. hhsp-60-tcr interaction alone could not induce the cytotoxic phenotype of these special t cells in contrast to the interaction with kir2ds2, suggesting a t cell activation via nk receptors. at the current state there are only data available regarding the role of hhsp-60 in activation of cd4cd28 t cells in acute coronary syndrome patients. it would be of interest if this interaction, hhsp-60-kir2ds2, is also observable in other diseases with kir2ds2 expressing cd4cd28 t cells. the telomere length becomes shorter after each cell division, and at the point, which is also known as the hayflick limit when the telomeres reach a critically short length, the cells are senescent and undergo apoptosis. the cd4cd28 t cells found during ageing and in immune - mediated diseases also show significantly shortened telomeres indicating a replicative history [36, 48, 49 ]. as telomerase activity depends on the expression of cd28, it decreases with cd28 reduction and could explain the shortened telomere length in cd4cd28 t cells. this phenomena have been studied in aged cd4 t cells both in ra and axial spondyloarthritis (including as) so far [51, 52 ]. one could expect that loss of the most important costimulatory signal cd28 and a shortened telomere length result in anergy and apoptosis [5, 53 ]. however, cd4cd28 t cells show the opposite ; they undergo less apoptosis than their cd28 counterparts [16, 54 ] and are able to survive in the periphery over years [36, 55 ]. one cause of apoptosis is activation - induced cell death (aicd), eradicating activated t cells by generation of a death signal after fas - fasl interaction, leading to phosphorylation of the fas receptor death domain and finally to cell death by activation of several cascades, including caspases 1, 3, 8, and 10 [56, 57 ]. the inhibitor of this pathway, the fas - associated death domain - like il-1-converting enzyme inhibitory protein (flip), however, is increased in cd4cd28 t cells and interacts with caspases 8 and 10 or with the fas - associated death domain and thus inhibits cell death by interrupting fas signalling. besides, analysis of bcl-2 family members revealed an antiapoptotic effect with increased bcl-2 levels in cd4cd28 t cells after withdrawal of il-2 as apoptotic stimulus. as cd4cd28 t cells produce large amounts of ifn-, il-2, so as the cytotoxic molecules perforin and granzyme b, they can be classified as cytotoxic t - helper 1-type cells [46, 54, 58, 59 ] (see figure 1). usually, th1 cells are characterized by the secretion of cytokines such as ifn- and mediate in this way inflammatory reactions but have no cytotoxic features. activation of these cells results in a reduction of intracellular granzyme b and perforin levels, suggesting that activation leads to degranulation, release, and further cytotoxicity. in vitro studies showed that the cytotoxicity of these cells causes cell death of endothelial cells. the secretion of the proinflammatory cytokines tnf- and ifn- promotes the inflammatory environment whereas local tnf- contributes to the downregulation of cd28. particularly the proinflammatory cytokine ifn- is highly produced and involved in the development of rheumatoid synovitis but also in atherosclerosis development. it is suggested that the secretion of ifn- mediates disease by causing the perturbation of inflammation. as a possible consequence of this th1 profile, activation of macrophages by ifn- leads to secretion of matrix metalloproteinases, which are implicated in tissue damaging processes. the cytotoxic properties of granzyme b and perforin may result in destabilization of unstable plaques and culprit lesions as well as damage of the vascular smooth muscle cells and endothelial layer by these cells in vitro, amplified by high sensitivity - c - reactive protein [62, 66 ]. the c - reactive protein has direct proinflammatory effects on t cells and is also able to increase the sensitivity of huvecs (human umbilical vein endothelial cells) to the cytotoxicity of these special t cells in culture. increased production of ifn- and perforin by cd4cd28 t cells has been shown not only in ra but also in as [9, 17 ], polymyalgia rheumatica / giant cell arteritis [15, 19 ], granulomatous polyangiitis, and abdominal aortic aneurysms. classification of cd4cd28 as th1-type t cells has also been supported by the chemokine receptor expression profile, which is similar to that of th1 cells, meaning upregulation of cxcr3 as well as the cxcr3/ccr4 ratio as identified in patients with as. particularly expression of th1 receptor cxcr3 strongly indicates that these t cells derive from cd28 th1 cells. to further examine the relationship between ifn- producing cd4cd28 t cells and th1 cells the dna methylation status of the ifng locus was analysed. hypomethylation of the ifng locus is induced during the differentiation to th1 cells and leading further to the expression of ifn-. this hypomethylation of the ifng locus was also detected in cd4cd28 t cells. the similarity of their dna methylation patterns further supports the th1-type of cd4cd28 t cells. beside the loss of cd28 these proinflammatory cd4 t cells show even more typical phenotypes different from their cd28 counterpart, at least in part with the potential of alternative costimulation of the cd28 t cells (see figure 1). for example, expressions of cd7 and cd40l [58, 69 ] are reduced and the receptor cx3cr1, the natural killer (nk) cell receptors nkg2d [19, 70 ], cd11b, cd57, kir2ds2, cd161, and toll - like receptor (tlr) 2 and tlr4 upregulated on cd4cd28 t cells. icam-1 is a surface adhesion molecule densely expressed on cd4cd28 t cells in ra and multiple sclerosis (ms) patients, assuming a high migratory potential. cytotoxic cd4cd28 t cells in ms patients additionally express the vla-4 and lfa-1 receptor, whereby the lfa-1 interaction with icam-1 promotes the secretion of proinflammatory cytokines by inducing various transmembrane signals [22, 74 ]. interaction and communication of classical cd4cd28 t cells with b cells require cd40l (= cd154), which is important by binding cd40 on the b cell surface. reduction of cd28 on cd4cd28 t cells coincides with a decreased expression of cd40l [58, 69 ], resulting in deficiency of the cd40-cd40l interaction. in vitro, four different cd4cd28 t cell clones failed to provide signals obligatory for b - cell differentiation and immunoglobulin secretion. furthermore, it has to be studied if this downregulation also influences b cell activation, recruitment of germinal centre precursors, and induction of memory b cells as it was observed in studies with normal cd4 t cells. contrary to these findings a newer study showed an increase of cd40l in cd4cd28 t cells in bronchiolitis obliterans syndrome. future studies will address the question if expression of cd40l is unique for cd4cd28 t cells in bronchiolitis obliterans syndrome or not. amongst the chemokine receptors, cx3cr1 on the surface of cd4cd28 t cells is best characterised in the peripheral blood of ra, ms, and primary sclerosing cholangitis patients. these studies showed cd4cd28cx3cr1 t cells in the synovial fluid of ra and in the brain of ms patients. more than 60% of cd4cd28 t cells in ra patients expressed the cx3cr1 receptor, while their cd28 counterparts were constantly negative for cx3cr1. this receptor interacts with the chemokine fractalkine, which is presented on the surface of cultured fibroblast - like synoviocytes in the synovial tissue from ra patients as well as increasing in the serum of ra and in the cerebrospinal fluid (csf) of ms patients compared to healthy controls. furthermore, fibroblast - like synoviocytes stimulated with either tnf-, ifn-, or both could induce the fractalkine mrna expression in vitro, suggesting a similar role in vivo. binding of fractalkine to cx3cr1cd28 t cells results again in enhanced inflammation with tissue damage after secretion of ifn- and tnf- by the effector cells as well as angiogenesis [74, 79 ], supporting its pathogenic effects in patients with ra. in ms patients it was observed that the chemokine gradient of soluble fractalkine is responsible for migration of these cells to the inflamed brain lesions. in ra, the expressed chemokine receptors include cxcr4 and ccr5, which are responsible for tissue invasion, and ccr7, the lymphoid homing chemokine receptor. expression of the ccr5 and ccr7 receptors gives the t cells the abilities of short - lived effector and long - lived memory t cells by allowing them either to home into the lymphoid organs or to invade into tissues like the synovial fluid and membrane. invasion into tissues and production of inflammatory cytokines then lead to an enhancement of local inflammation and autoreactivity [72, 8082 ]. in contrast, ccr5 expression was extremely low in as patients not only in cd28 but also in cd28 t cells, while cxcr3 was highly expressed in cd4cd28 t cells. these data may explain why cd4cd28 t cells in as have a reduced capacity to invade into synovial tissues compared to their ccr5-rich counterparts in ra. cd4cd28 t cells produce natural killer cell receptors, like cd11b and cd57 but lack their specific cd16 molecule. despite nk receptor expression they are not classified as typical natural killer t cells due to the fact that their characteristic expression of an invariant tcr -chain could not be detected. in diseases like ra, granulomatous polyangiitis, sjgren 's syndrome, and even in insulin - dependent diabetes mellitus cd4cd28 t cells were detected, which are able to express nkg2d receptors together with other killer cell immunoregulatory inhibitory receptors (kir), mediating cellular cytotoxicity [71, 72, 83 ]. nkg2d is usually expressed on nk cells, cd8 t cells but not on cd4 t cells and interacts with its stress - inducible, major histocompatibility complex class i - chain related ligand (mic) a and micb, which are present in the intestinal epithelium and the synovia of ra patients. the activation by mic causes survival of the autoreactive cd4cd28 t cells and leads to endocytosis and degradation of nkg2d [69, 85 ]. this supports upregulation of nkg2d, reversing its downmodulation by mic and promoting the proinflammatory status as the tissue damaging effect. furthermore, nkg2d was also expressed on cd4cd28 t cells in giant cell arthritis and polymyalgia rheumatica, accumulating around the vasa vasorum in the temporal arteries suggesting a ligand interaction in this region. the functional role and mechanism of kirs are still elusive but a subtype of this family, kir2ds2, was detected on the surface of cd4cd28 t cells from ra and acute coronary syndrome patients. it was confirmed that this receptor has active costimulatory functions and is considered to be a risk factor for the development of vasculitis but not synovitis. kir2ds2s expressed on cd4cd28 t cells are supposed to recognize hla - c molecules [46, 86 ] and human hsp-60 on major histocompatibility complex class 1 molecules, inducing the expression of perforin. additionally, it was shown that cytokine expression of ifn- was independent of kir2ds2 indicating separate pathways for proinflammatory and cytotoxic functions [43, 45, 87 ]. whether expression of nk receptor cd161 on cd4cd28 t cells in ra patients is relevant remains open. the ligand of cd161 is not known, but it is suggested that it differs from kir and nkg2d receptors. it is most likely that cd161 is involved in tissue invasion and in t cell adhesion. interestingly, not only nk receptors but also tlrs are expressed on cd4cd28 t cells. tlrs represent one of the four classes of the pattern recognition receptor families [88, 89 ], which are expressed on cells of the innate immune system. each of the 10 human tlrs recognizes specific pathogen - associated molecular patterns of either bacterial or viral origin [88, 90, 91 ]. for patients with ra, as, and psoriatic arthritis (psa) it was confirmed that tlrs, sensitive to lipopolysaccharides (lps), are expressed on cd4cd28 t cells. tlr4 was highly expressed on t cells in all three disease groups, while tlr2 was only detectable on cells from as patients, but in a lower frequency. activation via lps / tlrs increased the cytotoxic capacity of cd28 t cells, which was shown by the upregulated intracellular release of perforin. the induction of the perforin expression resulted from a tcr independent activation of tlr4 on cd4cd28 t cells without a concomitant tcr cross - linking. the high affinity of tlr4 on cd4cd28 t cells to lps indicates a possible involvement of components of gram - negative bacteria, especially in as. cd28 costimulation is characterized amongst others by an upregulation of the il-2 production and thus preventing efficiently apoptosis [80, 92 ]. searching for an appropriate costimulatory pathway instead of cd28 with these same effects led to several different pathways as independent stimulators. here we discuss the most important ones, but it can be expected that even more pathways can be involved to achieve production of il-2 and prevent apoptosis without the cd28-cd80/86 pathway. as the first alternative pathway vallejo cd47 is constitutively and highly expressed on the surface of peripheral t cells and bound by its ligand thrombospondin-1 (tsp1), an extracellular matrix protein, which is in turn bound on cd36. analysis of the cd47-tsp1-cd36 pathway in cd4cd28 t cells from ra patients resulted in a comparable production of il-2 and proliferation rates as observed for the cd28 pathway. tsp1 occurs mostly on damaged and repairing tissues raising the question whether this costimulus is involved in t cell recruitment and tissue inflammation : indeed tsp1 is highly expressed in the synovia of ra patients and may support the migration of cd4cd28 t cells [80, 93, 94 ]. an upregulation of cytotoxic t - lymphocyte - associated protein 4 (ctla-4 = cd152) was detected on cd4cd28 t cells from healthy subjects and patients with bronchiolitis obliterans syndrome. ctla-4 is a homolog of cd28 expressed on the surface of t cells, also activated by the cd28-ligands cd80 and cd86. ctla-4 acts as immune downmodulator and inhibits il-2 production as well as cell cycle progression. the antiapoptotic properties of the ctla-4-cd80/86 pathway are mediated by disturbing the fas - fasl interaction (see section 2.3) through the inhibition of bad. furthermore, activation of ctla-4 leads to an increase of bcl-2 with its antiapoptotic effects. both ox40 (= cd134) and 4 - 1bb (= cd137) are members of the tumour necrosis factor receptor family and are expressed on the surface of different cell types including activated t cells. they recognize their ligands ox40l and 4 - 1bbl and use the tumour necrosis factor receptor associated factor adaptor molecules to activate pi-3 kinase, akt / pkb, c - jun n - terminal kinase, and the nf-b pathway [96, 97 ]. ox40 and 4 - 1bb are expressed and activated independent of cd28 on t cells and enhance cytokine secretion, proliferation, and survival (by upregulating bcl-2 family members) [96, 97 ]. cd4cd28 t cells detected in acute coronary syndrome patients express enhanced levels of ox40 and 4 - 1bb compared to their cd28 counterparts. inhibition of these costimuli resulted in decreased production of the proinflammatory cytokines ifn- and tnf- and altered degranulation and release of perforin from cd4cd28 t cells. despite the marginally changed intracellular level of granzyme b, blocking ox40 and 4 - 1bb could be a possible treatment option for acute coronary syndrome patients by inhibiting inflammation and cytotoxicity. these and other important results helped to characterize cd4cd28 t cells with typical effector features, characterized by the expression of cd69 as indicator of activation, production of inflammatory cytokines, cytotoxic molecules, and expression of natural killer (nk) cell receptors, although it was found that they are still able to express the nave t cell marker cd45ra [15, 27, 63, 98 ]. because of their cytotoxic and proinflammatory features it is hypothesized that they might have an important role in the pathogenesis and maintenance of chronic immune - mediated diseases. the majority of the above - mentioned studies to characterize cd4cd28 t cells were performed with t cells from ra and as patients. interestingly, cd4cd28 t cells are found in one - third of all ra patients and occur with a higher frequency in the peripheral blood than in the synovial fluid [68, 99 ]. recently, it was discovered that cd4cd28 t cells from the synovial fluid, but not from the peripheral blood were able to produce the proinflammatory cytokine il-17. it is speculated that higher concentrations of tnf- in the synovial fluid compared to the peripheral blood may induce additional cellular functions like production of il-17. the amount of cd4cd28 t cells is directly associated with disease severity and the development of extra - articular manifestations with the highest levels of cd4cd28 t cells in patients with subcutaneous nodule formation and rheumatoid organ disease [2, 37, 100 ]. the main cause of acute coronary syndromes including unstable angina, myocardial infarction, and subsequent sudden cardiac death is rupture of atherosclerotic plaques characterized by the inflammatory infiltration of macrophages and t cells. cd4cd28 t cells occur frequently in patients with unstable angina but are rare in stable angina and healthy controls, leading to the speculation that these cells are involved in the development of cardiovascular events and apoptotic effects on vascular wall cells [24, 25 ]. the presence of this type of t cells in unstable but not in stable plaques indicates an important role in plaque disruption. the high level of produced ifn- induces the recruitment and activation of macrophages and their secretion of metalloproteinases. the tissue damaging effect of metalloproteinases is well known and may result in the destabilization of the plaque, rupture, and superimposed thrombosis and thus raises the risk of acute ischemia [24, 62, 102 ]. cd4cd28 t cells in patients with acute coronary disease additionally produce enhanced levels of granulysin compared to cd28 t cells, which is known to be involved in the immune defense against bacteria and fungi. this result together with the fact that myocardium infarction is more common after an infection strengthens the hypothesis that cd4cd28 t cells expressing tlrs might be activated by microbial agents. for further confirmation it will be interesting if tlrs, as already found on the surface of cd4cd28 t cells in as, are also present on cells in acute coronary disease and if cells from as patients are also able to produce granulysin. an increased risk for angina pectoris, myocardial infarction, and cardiac death has also been described for patients with rheumatoid vasculitis [64, 101 ] and patients with chronic kidney disease depending on their level of cd4cd28 t cells. also cd4cd28 t cells occur in diabetes mellitus, with higher levels in case of the first cardiovascular event and acute coronary syndrome compared to diabetes mellitus patients without a cardiovascular syndrome and patients with acute coronary syndrome but without diabetes mellitus. these cardiovascular complications could be in conjunction with the accumulation of glycosylated end - products and the poor glycaemic control, maybe associated with cd4cd28 t cells. in patients with granulomatous polyangiitis cd4cd28 t cells were studied in the peripheral blood, the granulomatous lesions, and the bronchoalveolar lavage fluid. it was suggested that cd4cd28 t cells are recruited to granulomatous lesions by cd18-icam1 interaction and support in this manner granuloma formation by secreting inflammatory cytokines. however, it is not clear if these cells are recruited already in the cd28 negative status or still as cd28 cells losing their receptor in the inflammatory environment of the granulomatous lesion after repeated activation [21, 98, 105 ]. only recently the analysis of cd4cd28 t cells in patients with primary sclerosing cholangitis revealed that these cells occur with a frequency of 3,3% of all cd4 t cells in the peripheral blood and 30,3% in the liver tissue, where they accumulate around the bile ducts. cx3cr1 was higher expressed on cells in the blood than in the liver tissue, but tissue infiltration was additionally promoted by cxcr6 and ccr10, which give the t cells the ability to migrate toward biliary epithelial cells. as a consequence the concentrations of tnf- and ifn- increase with the accumulation of cd28 t cells in the liver and induce in turn the expression of adhesion molecules and secretion of chemokines in biliary epithelial cells. bronchiolitis obliterans syndrome is also associated with increased levels of cd4cd28 t cells compared to stable lung transplant patients. downregulation of cd28 was correlated with an increased need for steroids (steroid resistance) and an upregulation of specific costimulators ox40 (or cd134), 4 - 1bb (or cd137), ctla-4 (cd152), and cd40l (cd154). the molecules 4 - 1bb and ctla-4 are responsible for the production of cytotoxic t cells before or during graft rejection and ox40 and cd40l are important for proliferation and cytokine production of cd4cd28 t cells in bronchiolitis obliterans syndrome. this finding is contrary to those of another study showing a downregulation of cd40l together with cd28, indicating an alteration of cd4cd28 t cells in bronchiolitis obliterans syndrome. indeed there is ongoing interest in the evaluation of the functional role of cd4cd28 t cells and their involvement in disease development, progression, or maintenance, and targeting cd4cd28 t cells as treatment option appears to be an intriguing topic for further studies. several studies concluded that the number of cd4cd28 t cells correlates with severity and extra - articular involvement in ra [82, 100 ] ; additionally high levels of proinflammatory cytokine tnf- were found in both the peripheral blood and synovia. occurrence of cd4cd28 t cells together with tnf- in ra patients raises the assumption that targeting tnf- also reduce the level of cytotoxic t cells [10, 106 ]. indeed, ra patients treated with disease modifying antirheumatic drugs like methotrexate showed no changes of cytotoxic t cells [28, 64, 100 ] but showed downregulation of cd4cd28 t cells and even reexpression of cd28 under treatment with the tnf- inhibitor infliximab [11, 65 ]. besides, tnf- inhibition with infliximab led to downregulation of tlr expression on cd4cd28 t cells in as patients however, cd4cd28 t cells could not be fully eradicated using infliximab, which results in a lowering of 3336% of the cd28 t cell level in ra and unstable angina patients [12, 64, 65 ]. treatment of unstable angina patients with statins led to an even less pronounced reduction of cd4cd28 t cells. statins are 3-hydroxy-3-methylglutaryl - coenzyme a reductase inhibitors, resulted in reduction of soluble cd40l in patients with hypercholesterolemia and coronary artery disease. independent trials to test the effect of statins on cd4cd28 t cells in patients with either stable coronary artery disease / unstable angina / acute coronary syndrome or diabetes mellitus show conflicting data with downregulation of cd4cd28 t cells in unstable angina and acute coronary syndrome, but lack of influence in the other two diseases [66, 103, 107, 109 ]. reduction of cd4cd28 t cells was also observed in ra patients treated with abatacept. abatacept is a fusion protein consisting of the extracellular domain of ctla-4 and the modified fc portion of human igg1. it binds the cd28/ctla-4 ligands cd80 and cd86 and consequently inhibits their signalling pathway. also the combination of abatacept with methotrexate showed a beneficial effect in ra patients, even after failure of other medications, like tnf- blockers [110112 ]. so far we are only aware of one approach for full eradication of cd4cd28 t cells, namely, that achieved by polyclonal antilymphocyte globulins (atg) in vitro and in transplant patients. atgs are suppressors of the immune system used since decades as prophylaxis against graft versus host disease in transplant patients. the high diversity of their antigens allows them to interact with all cell types of the peripheral blood mononuclear cell compartment and subsequently induces apoptosis in these cells. analysis of t cells from transplant recipients treated with atg revealed preferred eradication of cd4cd28 t cells in comparison with their cd28 counterpart, suggesting a specific effect of atgs against cd4cd28 t cells. ageing coincides with the development of cd4cd28 t cells showing typical phenotypes and costimulatory pathways. thus properties from nk cells and monocytic lineages have been described as well as a functional role of these cells as th1 cells. many aspects of cd4cd28 t cells have been extensively described, although not in all diseases, but exemplary. to our understanding some important questions are still open : (1) why are not all cells restored by il-12 ? (2) can the observed phenomena be generalized for all diseases ? (3) how to define the prognostic effect of cd4cd28 t cells, and could prognosis be improved by eradication of these cytotoxic cells ? finally we assume that the potential role of cd4cd28 t cells as biomarkers and prognostic factor is still underestimated for clinical practice, and it appears that there is still an ongoing strong interest of the scientific community in these cells. | cd4+cd28 t cells are a unique type of proinflammatory t cells characterised by blockade of costimulatory cd28 receptor expression at the transcriptional level, which is still reversible by il-12. in healthy individuals older than 65 years, these cells may accumulate to up to 50% of total cd4 + t lymphocytes as in many immune - mediated diseases, immunodeficiency, and specific infectious diseases. here we focus on cd4+cd28 t cells in chronic immune - mediated diseases, summarizing various phenotypic and functional characteristics, which vary depending on the underlying disease, disease activity, and concurrent treatment. cd4+cd28 t cells present as effector / memory cells with increased replicative history and oligoclonality but reduced apoptosis. as an alternative costimulatory signal instead of cd28, not only natural killer cell receptors and toll - like receptors, but also cd47, ctla-4, ox40, and 4 - 1bb have to be considered. the proinflammatory and cytotoxic capacities of these cells indicate an involvement in progression and maintenance of chronic immune - mediated disease. so far it has been shown that treatment with tnf- blockers, abatacept, statins, and polyclonal antilymphocyte globulins (atg) mediates reduction of the cd4+cd28 t cell level. the clinical relevance of targeting cd4+cd28 t cells as a therapeutic option has not been examined so far. |
all along in millions of years, the interaction between immune cells and the micro environment intertwined each other in an inseparable relationship. human immune cells build up their recognition database of microbe 's antigens as well as how to cease pathogenic microorganism invasion then pass it on to the next generations. similarly microbes at times changes their antigenic structure to evade recognition and destruction from immune cells, and those that survived the screening process also pass on their genomic structure as a new strain of species. this relationship goes on and on for as long as the human history and, like a scale, it skewed each time changes happened in either side of the hand. however, not all microbes are harmful to the body ; some microbes are classified as commensal or harmless residents, this population even offers protection from other pathogenic microbes.interestingly, the commensal microorganism sometimes shares the same general antigenic features like any other pathogenic microbes but was not attacked by the immune cells. the mechanism of how the immune cells differentiate between pathogen and harmless microbes need more in depth exploration, but the cause behind the phenomena is that the pathogenic microbes express a structure called pathogen - associated molecular patterns (pamps) which are easily recognized by the immune cells. innate immune response works as a first liner in confronting pathogen invasion, these cells responds very fast and kill anything that is marked as threat to the body ; adaptive immune response is more antigen - specific, however, works slower and takes time to be developed. pattern recognition receptor (prr), a compartment that binds to pamps, from the innate immune system responded to antigens then triggered adaptive immune response through multiple pathways[579 ]. in general, adaptive immune response will be halted if innate pathway is sufficient to cover the damage caused by pathogens immediately and usually the response is not a systemic one. if the damage can not be compensated by these cells, then these immune cells will eventually send signals and release cytokines to recruit more cells to the site ; meanwhile antigen presenting cells (apcs) which also express prr will be responsible to carry on the antigenic information to t lymphocytes and start the adaptive immune response. once those t cells receive the antigenic information it can be activated and proliferate with an antigenic - specific site expressed on their surface. some of them will induce b lymphocytes activation to produce antigenic - specific antibodies, and some will migrate out to the infected sites for pathogen eradication. mostly activated cells will become apoptotic soon after they finish their task, only a small amount of these cells will become memory cells and keep all the antigenic information for later use. so when there is a second exposure with the same antigen in the future, rapid response will follow and more cells will be released to the site. immune protection in the gut is considered to be more tolerable compared to other places. there are billions of microbes ingested into the intestinal lumen daily. to protect the intestinal mucosa from harmful pathogen, a tight junction is built in the surface of the lumen by the intestinal epithelial cells (iecs). these cells also produce mucus to lubricate and trap the pathogen on the surface preventing it to cross the barrier. apart from physical barrier, an ample amount of soluble immunoglobulin a (iga) is secreted to the lumen daily to keep control of the microorganism population. iecs also express prr and have the ability to recruit immune cells when bind to pamps from the pathogen inside the lumen. however the professional apcs in the gut, dendritic cells (dcs) and macrophage, seemingly more tolerant to luminal microbes in sending alert and phagocyting them compared to apcs from other sites. it is even thought that when the immune cells are no longer tolerant to commensal microbes then unwanted inflammation occurs, such as that in colitis. immune over - reactivity is a condition where immune cells over - react toward innocuous agents, whether self cells or harmless microbes. it happens when an incompletely digested protein breaks through a leak in the intestinal barrier, captured by an apc and flagged as an antigen then presented to the t cells, henceforth that certain proteins will be classified as pathogens and thus at the second exposure antigen - specific antibodies flood the intestinal lumen to cause unwanted protection and inflammation. in 2001, mcintire found a gene family related to a mendelian trait known as t cell and airway phenotype regulator (tapr) which has a restricted immunoglobulin variable domain and mucin domain. the gene family is found expressed on t cells so hence called t cell immunoglobulin and mucin domain (tim). up until now there are 3 tim subtype discovered in human (tim-1, tim-3, and tim-4), and 8 in mice (tim1 to tim8). in this review, we will discuss mainly about tim-4 and another member related to it. in the association with natural selection over thousands of years, tim-4 molecule has been through positive natural selection pressure along with other immunoglobulin superfamily gene like cd3, cd4, cd48 and several others, indicating that tim-4 plays a role in either innate or adaptive immune response. tim-4, previously known as smuckler, gene is conserved in human and mouse apcs unlike other tim gene family which is expressed mainly in t cells. tim-4 expression in apcs is restricted only to certain subtypes that express cd11c+ and cd11b+. tim-4 has the ability to engage tim-1 and co - stimulate t cell proliferation in activated t cells[2426 ]. found that disruption of tim-1 and tim-4 binding with anti - tim-1 antibody and with tim-4 interfered gene expression can deplete th2 and allergic response in peanut allergy mice model. however in another study, meyer at al discovered that tim-4 fusion protein induce an increase of t cells proliferation but in lower concentration it inhibited t cells response. other findings show that in vivo administration of tim-4 antibody apparently induced th1 cells proliferation and that tim-4 fusion protein is able to induce an increase of interferon gamma (ifn - g) and tumor necrosis factor - alpha (tnf - a) rate in vitro. albacker found that over - expressed tim-4 in apcs decreased antigenic specific t cell activity, while mizui found that blocking tim-4 will increase antigenic specific t cell activity but decrease inflammation and t cell proliferation rate. apart from that, like other members of the tim gene, tim-4 is also a phosphatidylserine (ps) receptor that enhances phagocytosing activity of apoptotic cells by macrophages to maintain the homeostasis. for instance, blocking of the ps receptor in apcs will consequently produce negative effect in peritoneal macrophage phagocytosing activity. ps expressed on the surface of an apoptotic cell is a signal that attracts apcs for immediate clearance or else too much linger will eventually cause auto - immunity reaction and chronic inflammation. after phagocytosing, apcs will release transforming growth factor beta (tgf - b) that plays a part in inducing regulatory t (treg) cells proliferation. in the gut, treg cells are responsible to maintain immune tolerance to commensal flora and acts as a negative feedback for hyperactive pro - inflammatory response hence keeping the immune response in balance[3234 ]. xiao discovered that a fusion protein using tim-4 monoclonal antibody with the immunoglobulin region only could increase t cell proliferation but another fusion protein which has both the immunoglobulin and mucin domain in contrast inhibit t cell proliferation. whereas park. discovered that without its cytoplasmic tail and the transmembrane region, tim-4 is still capable of enhancing phagocyting activity in the apcs the immunoglobulin region of tim-4 is responsible in recognizing apoptotic cells through ps receptor binding and this binding site is also the same site for anti - tim-4 monoclonal antibodies binding. has discovered that the leucocyte mono - immunoglobulin - like receptor 5 (lmir5) can bind to tim-1 at the ps binding site and the binding does not interfere with the phagocyting ability of tim-4. found that tim-4 knock - out resident peritoneal macrophages are able to phagocytose necrotic and other opsonised targets but incompetent in phagocytosing apoptotic cells either in vitro or in vivo. regarding tim-1 - tim-4 binding site, it requires both tims intact glycosylated mucin stalk but mucin stalk alone is not yet sufficient for binding. found that tim-1 - tim-4 binding may be a kind of intercellular signalling via exosome that exposed ps. however, tim-4 may also bind with ligand other than tim-1 and causing inhibition of naive t cells activation. several findings indicated that tim-4 plays a significant role in intestinal allergic response and interaction with tim-1 is related to t cell antigen - specific proliferation not only in the gut but also elsewhere, like the liver and kidney[29314143 ]. when an iec encounters a commensal flora, it will release cytokines such as il-10 and tgf - b to induce tolerance. however in the case of inflammation, iecs in the mucosa layer are damaged and the continuity of the structure is broken hence giving pathogens more chances to break into the lamina propria. in colitis, apoptotic iecs may probably relate to the pathogenesis of the disease leading to a th1 immune response. from our own unpublished data, we found that tim-4 expression is significantly higher in colitis patients compared to healthy adults imposing that through ps receptor signalling pathway tim-4 expressing apcs will direct proliferation toward th1 response. it is known that t helper type 2 (th2) cells are responsible in the development of allergic response. in several in vivo and in vitro experiments, exposure to resident flora toxin markedly increases antigenic - specific response to allergen through increase of th2 polarization and mast cells degranulation. after administration of anti - tim-4 and/or anti - tim-1 antibodies, the allergic response decreases with a lesser level of th2 polarization. the antibody blocking works at the ps binding site at the immunoglobulin region, then apoptotic cells phagocytosing activity of tim-4 expressing apcs should have been diminished. if tim-4 expressing apcs are still able to phagocytose necrotic cells and pathogens without ps signalling through other glycosylated ligands, then the inhibition of naive t cells proliferation seen by mizui probably caused by a pathway other than tim-1 binding. found that tim-4 phagocyting activity of apoptotic cells markedly increase in the antigen - specific t cell population in inflammation but then help control the number of the remain antigen - specific t cells after the clearance. interestingly, a group of apoptosis resistant dcs have the ability to skew the immune response to th2 polarization. this population can be isolated from antigen - primed mice then cultured with antigen - specific t cells ; those that survived are able to induce th2 differentiation from naive t cells either in vivo or in vitro. tim-4 ability to recognize apoptotic cells is dependent to the ps receptor on its immunoglobulin region and possibly with the help of tim-1 that also has the same receptor. however without tim-1 interaction, tim-4 is able to bind with other glycosylated ligands. this versatile feature may be responsible for the bimodal character that somewhat contradicting each other. a future study is needed to see whether tim-4 is present in mature apcs that are apoptotic resistant population and the phagocytosing activity in apoptotic t cell antigen - specific cells are aided by tim-1 through ps receptor which favours the th2 response. regarding the induction of th1 response such as that in colitis, tim-4 may also play a role in the process either through other ligands or signalling pathways. | human being living in constant contact with microbes and pathogen and in the process has developed a recognition pattern of pathogenic structure in the immune cells. the gut lumen has high density of microbes thus the immune response is slightly tolerable to certain microbes, known as commensal flora. these microbes along with other innocuous agents do not cause any inflammation response normally, and are considered as harmless by the immune cells. in immune hypersensitivity condition, such as colitis or food allergy, this mechanism is disturbed. t cell immunoglobulin and mucin domain (tim)-4 is a phosphatidylserine receptor expressed in mature antigen presenting cells. it is shown that tim-4 and its ligand tim-1 are associated in intestinal immune response. however the characteristic of tim-4 sometimes seems to be two - faced and there is a possibility that tim-4 also bind to other ligands. |
adverse reactions to systemic drug administration can have different clinical patterns such as erythema multiforme minor, major, steven johnsons syndrome, anaphylactic stomatitis, intraoral fixed drug eruptions, lichenoid drug reactions, and pemphigoid - like drug reactions. based on the severity and the number of mucosal sites involved, the disease has been subclassified into em minor and major. steven johnson syndrome is a more severe condition characterized by wide spread small blisters on torso and mucosal ulcerations with atypical skin target lesions triggered by drug intake. typical target skin lesions are necessary along with mucosal ulcerations to consider diagnosing them as em minor and major. many investigators have reported cases of oral mucosal ulcerations and lip lesions typical of em without any skin lesions. it has been reported that even if the primary attacks of oral em are confined to the oral mucosa the subsequent attacks can produce more severe forms of em involving the skin and hence it is important to identify and distinguish them from other ulcerative disorders involving oral cavity for early management and proper follow up. this article reports two cases of oral em highlighting the importance of distinguishing this disorder. a 21-year - old female patient visited the dental opd with the complaint of extensive ulceration of oral cavity and pain and inability to eat for the past 4 days. she gave a history of leg sprain for which she took diclofenac sodium subsequent to which she developed multiple small ulcerations that later transformed into extensive, irregular ulcerations of the oral mucosa. on extra oral examination, both upper and lower lips showed extensive irregular ulcerations, showing cracking and fissuring with blood encrustation. intraoral examination showed extensive, irregular ulcerations with yellow base and erythematous borders on buccal mucosa, palate, dorsal and ventral surfaces of the tongue [figures 1 and 2 ]. case 1 irregular lip ulcerations with blood encrustations case 1 irregular buccal mucosal and tongue ulcerations with lip lesions the sudden onset, positive drug history, extensive ulcerations of the oral cavity, cracking, and fissuring of lips with bloody crusting lead to the diagnosis of oral erythema multiforme. the patient was advised to stop the diclofenac sodium medication and was treated with topical corticosteroids, mild analgesics, and local application of lignocaine gel to facilitate oral fluid intake. healing was noticed on the third day and the lesions were completely cleared without scarring in 10 days time [figure 3 ]. patient was advised not to take any drug from the diclofenac group. case 1 after 10 days of treatment the oral mucosal and lip ulcerations are healed a 23-year - old female patient presented to the dental op with the complaint of painful ulcerations of the oral cavity for the past 5 days. she gave a history of bronchial asthma for which she took homeopathy medicine few weeks back, within days she developed oral ulcerations. she gave a history of multiple vesicles of the oral mucosa, buccal, and labial mucosa, which ruptured to form painful ulcerations. patient was unable to eat any hot and spicy food and was on liquid diet for the last 2 days. intraorally multiple ulcerations of the buccal and labial mucosa and palate were seen [figure 4 ]. tongue showed white coating on the dorsal surface with irregular ulcerations of the right lateral border. case 2 palatal ulcerations with irregular blood encrusted lip lesions patient was advised to discontinue the homeopathic medicine and treated with cortico steroids (prednisolone 10 mg) twice a day for 3 days followed by tapering dose for 10 days and local application of topical anesthetic gel for pain relief. patient responded well to the treatment and healing of the lesions occurred within a week [figure 5 ]. positive association between the drug intake and incidence of the lesions and the clinical appearance of the lesions lead to the diagnosis of oral erythema multiforme. a 21-year - old female patient visited the dental opd with the complaint of extensive ulceration of oral cavity and pain and inability to eat for the past 4 days. she gave a history of leg sprain for which she took diclofenac sodium subsequent to which she developed multiple small ulcerations that later transformed into extensive, irregular ulcerations of the oral mucosa. on extra oral examination, both upper and lower lips showed extensive irregular ulcerations, showing cracking and fissuring with blood encrustation. intraoral examination showed extensive, irregular ulcerations with yellow base and erythematous borders on buccal mucosa, palate, dorsal and ventral surfaces of the tongue [figures 1 and 2 ]. case 1 irregular lip ulcerations with blood encrustations case 1 irregular buccal mucosal and tongue ulcerations with lip lesions the sudden onset, positive drug history, extensive ulcerations of the oral cavity, cracking, and fissuring of lips with bloody crusting lead to the diagnosis of oral erythema multiforme. the patient was advised to stop the diclofenac sodium medication and was treated with topical corticosteroids, mild analgesics, and local application of lignocaine gel to facilitate oral fluid intake. healing was noticed on the third day and the lesions were completely cleared without scarring in 10 days time [figure 3 ]. patient was advised not to take any drug from the diclofenac group. a 23-year - old female patient presented to the dental op with the complaint of painful ulcerations of the oral cavity for the past 5 days. she gave a history of bronchial asthma for which she took homeopathy medicine few weeks back, within days she developed oral ulcerations. she gave a history of multiple vesicles of the oral mucosa, buccal, and labial mucosa, which ruptured to form painful ulcerations. patient was unable to eat any hot and spicy food and was on liquid diet for the last 2 days. intraorally multiple ulcerations of the buccal and labial mucosa and palate were seen [figure 4 ]. tongue showed white coating on the dorsal surface with irregular ulcerations of the right lateral border. case 2 palatal ulcerations with irregular blood encrusted lip lesions patient was advised to discontinue the homeopathic medicine and treated with cortico steroids (prednisolone 10 mg) twice a day for 3 days followed by tapering dose for 10 days and local application of topical anesthetic gel for pain relief. patient responded well to the treatment and healing of the lesions occurred within a week [figure 5 ]. positive association between the drug intake and incidence of the lesions and the clinical appearance of the lesions lead to the diagnosis of oral erythema multiforme. erythema multiforme (em) is an inflammatory disorder that affects the skin or mucous membrane or both. according to von hebra, who first described the disease in 1866, the patients with erythema multiforme should have acrally distributed typical target lesions or raised edematous skin papules with or without mucosal involvement. in 1968, kenneth described an inflammatory oral disorder with oral lesions typical of em but without any skin involvement. when lips are involved the typical blood encrusted lesions were seen. in this series of cases, the typical target skin lesions were seen during the recurrences not in their initial attacks. many investigators have suggested this as a third category of em known as oral em that are characterized by typical oral lesions of em but no target skin lesions. our two cases showed extensive irregular erythematous ulcerations in the buccal mucosa, labial mucosa, tongue, and palate along with blood encrusted lip ulcerations. biopsies are advised only in early vesicular lesions of erythema multiforme not in ulcerated ones since histopathologic appearances are nonspecific and nondiagnostic. our patients reported to us with advanced ulcerated lesions and hence the diagnosis had to be established based on the positive drug history, clinical appearance, and distribution of the lesion and exclusion of other ulcerative lesions. we were able to establish a temporal relationship between the drug intake and occurrence of the oral mucosal lesions. the oral ulcerations in our cases started within a few days of the drug intake and were resolved upon cessation of the drug. erythema multiforme is usually triggered by herpes simplex infections, but rarely by drug intake. when the lesions are confined only to the oral cavity the different differential diagnosis that has to be considered are herpes, autoimmune vescicullobullous lesions such as pemphigus vulgaris or bullous pemphigoid and other patterns of drug reactions. herpetic ulcers are smaller with regular borders than ulcers associated with em. extensive irregular ulcerations in the lining nonkeratinized mucosa as seen in our patients were typical of em and are not a feature of herpes infection. the presence of a temporal relationship between the drug intake and onset of the disease excludes the possibility of any infectious aetiologies. the positive drug histories associated with onset of ulcerations in our cases ruled out the possibility of other autoimmune vescicullobullous lesions like pemphigus vulgaris. unlike pemphigus vulgaris oral em have an acute onset and does not show any desquamative gingivitis. bullous lichen planus lesions that may have similar ulcerations should have wickham 's striae, which were absent in our cases excluding it as the diagnosis. other patterns of drug reactions like lichenoid drug reactions, pemphigoid - like drug reactions that resemble their namesake can be easily differentiated based on the clinical patterns as above mentioned. anaphylactic stomatitis often shows urticarial skin reactions with other signs and symptoms of anaphylaxis which were absent in our cases. in mucosal fixed drug eruptions the lesions are confined to localized areas of oral mucosa but in our cases there were wide spread lesions affecting labial, buccal, palatal, and tongue mucosa along with lip involvement. lesions of em minor are characterized by single mucosal ulcerations and typical target lesions of skin. erythema multiforme major is considered to be a more aggressive form characterized by involvement of multiple mucosa accompanied by typical target skin lesions.[57 ] the third category of em, also described by many investigators as oral em has the lesions confined to the oral mucosa and lips with no skin involvement. since our cases were evidently triggered by drug intake and they had typical lesions of em in the oral mucosa and lips with no skin involvement we came to a diagnosis of oral em. the most common drugs that trigger em lesions are long acting sulfa drugs especially sulphonamides, co - trimoxazole, phenytoin, carbamazepine and nonsteroidal antiinflammatory drugs such as diclofenac, ibuprofen, and salicylates. management of oral em involves identification of triggering agent. if it is found to be hsv infection patients have to be put on antiviral medications. if hsv is ruled out as a triggering agent and the culprit is an adverse drug reaction, the drug is immediately stopped. usually lesions of oral em can be treated palliatively with analgesics for oral pain, viscous lidocaine rinses, soothening mouth rinses, bland soft diet, avoidance of acidic and spicy food, systemic and topical antibiotics to prevent secondary infection. lesions of em usually respond to topical steroids, for more severe cases systemic corticosteroids are recommended. even though primary attacks of oral em are confined to the oral mucosa the subsequent attacks can produce more severe forms of em (em minor and major) involving the skin. hence, it is important to distinguish oral em for their early diagnosis, prompt management, and proper follow up. | oral erythema multiforme (em) is considered as a third category of em other than em minor and major. patients present with oral and lip ulcerations typical of em but without any skin target lesions. it has been reported that primary attacks of oral em is confined to the oral mucosa but the subsequent attacks can produce more severe forms of em involving the skin. hence, it is important to identify and distinguish them from other ulcerative disorders involving oral cavity for early management. this article reports two cases of oral em that presented with oral and lip ulcerations typical of em without any skin lesions and highlights the importance of early diagnosis and proper management. |
acinar cell carcinomas (accs) of the pancreas are a rare tumor accounting for only about 2% of all pancreas tumors. because accs morphologically resemble neuroendocrine tumors (nets) and solid pseudopapillary tumors (spts), differential diagnosis of acc based on the cytological findings we recently encountered a case of acc, which was difficult to distinguish morphologically from an net and spt on the basis of the pancreatic endoscopic ultrasound - guided and fine - needle aspiration biopsy findings. we report herein on this case and discuss how to distinguish accs from nets and spts morphologically and immunohistochemically. at another hospital, a 41-year - old woman was found to have multiple tumors in the liver on ultrasound (us) and computed tomography (ct) scan images, and a pancreatic head tumor was detected on a contrast - enhanced ct scan. she was thus referred to our hospital with the suspected diagnosis of pancreatic cancer and multiple liver metastases. a low echoic 20 mm mass at the pancreatic head was punctured with a 22 gauge needle. an acc was suggested from the findings, but the possibility of net or spt was not ruled out. subsequent cell block immunostaining strongly suggest a diagnosis of acc. a definitive diagnosis of acc was confirmed with a liver biopsy. the clusters were solid and assumed an acinar structure, with numerous loosely bound atypical cells found around the clusters. the nuclear - cytoplasmic ratio was high, and the cytoplasm was granular, but zymogen granules were not evident. chromatin was fine granular in shape and distributed nonuniformly, accompanied by evident nucleoli [figure 1a d ]. cell block staining was positive for -catenin (cell membrane and part of nuclei) [figure 1e ], synaptophysin (focal), chromogranin a (focal) and chymotrypsin [figure 1f ] were all positive thus allowing a diagnosis of acc. (a) the tumor cells forming clusters or showing sporadic distribution (pap, 100). (b) acinar structures (pap, 200). (c, and d) the nuclei are small round in shape with fine granular chromatin (c : pap, 400, d : diff -quik, 400). (e) -catenin shows cell membrane and nuclear positivity (ihc, 400). (f) chymotrypsin shows cytoplasmic positivity (ihc, 400) tumor cells with evident nucleoli, densely stained nuclei and slightly eosinophilic scant cytoplasm had proliferated, assuming a fused gland - like and cribriform structure [figure 2a and b ]. immunostaining was positive for -catenin (cell membrane and part of nuclei), synaptophysin (focal), chromogranin a (focal), chymotrypsin and amylase [figure 2c f ], thus confirming a definitive diagnosis of acc. (a and b) the tumor cells showed growth into fused ductal or cribriform structures (a : h and e, 100 ; b : h and e, 200). (c) -catenin shows cell membrane and nuclear positivity (ihc, 100). the clusters were solid and assumed an acinar structure, with numerous loosely bound atypical cells found around the clusters. the nuclear - cytoplasmic ratio was high, and the cytoplasm was granular, but zymogen granules were not evident. chromatin was fine granular in shape and distributed nonuniformly, accompanied by evident nucleoli [figure 1a d ]. cell block staining was positive for -catenin (cell membrane and part of nuclei) [figure 1e ], synaptophysin (focal), chromogranin a (focal) and chymotrypsin [figure 1f ] were all positive thus allowing a diagnosis of acc. (a) the tumor cells forming clusters or showing sporadic distribution (pap, 100). (b) acinar structures (pap, 200). (c, and d) the nuclei are small round in shape with fine granular chromatin (c : pap, 400, d : diff -quik, 400). (e) -catenin shows cell membrane and nuclear positivity (ihc, 400). tumor cells with evident nucleoli, densely stained nuclei and slightly eosinophilic scant cytoplasm had proliferated, assuming a fused gland - like and cribriform structure [figure 2a and b ]. immunostaining was positive for -catenin (cell membrane and part of nuclei), synaptophysin (focal), chromogranin a (focal), chymotrypsin and amylase [figure 2c f ], thus confirming a definitive diagnosis of acc. (a and b) the tumor cells showed growth into fused ductal or cribriform structures (a : h and e, 100 ; b : h and e, 200). (c) -catenin shows cell membrane and nuclear positivity (ihc, 100). acinar cell carcinomas morphologically resemble tumors of a neuroendocrine feature such as pdas, nets, and spts, and for this reason, the accurate diagnosis rate of acc based on cytological findings is low. sigel and klimstra have reported a discrepancy between the cytological and histological diagnoses in 14 of the 29 cases of malignant acinar tumors of the pancreas, stating that many cases of acc were cytologically rated as nets or pdas. also, in the present case, a definitive diagnosis of acc was not possible on the basis of the cytological findings alone, and its distinction from a net and spt was difficult. the prognosis of accs is poor as compared with nets and spts and different chemotherapeutic regimens may be indicated for cases not indicated for surgery. cytological features allowing distinction between accs and nets include structural and cytoplasmic features and the nuclear chromatin pattern. accs assume an acinar structure and possess zymogen granules in the cytoplasm. however, the cytological distinction of an acinar structure from rosette formation associated with nets is difficult, and the zymogen granules in the cytoplasm are also difficult to detect with diff - quik or pap staining. for detection of zymogen granules, electron microscopy or the postperiodic acid - schiff staining diastase digestive test is used, but these techniques are not specific to accs because zymogen granules are small in number or may have been replaced with mitochondria in the case of accs. additionally, in the present case, distinction from rosette formations and the detection of zymogen granules were difficult based on the cytodiagnosis although acinar structures and granular cytoplasms were noted. the nuclear chromatin in this case was fine granular and distributed nonuniformly, unlike the salt and pepper - like form associated with nets. labate. also pointed out a similar nuclear chromatin pattern, reporting that it would be useful in distinction from nets. thus, differences in the nuclear chromatin pattern would appear to be useful in differential diagnosis. furthermore, considering that an inclusion body in the nucleus was present in cases of nets, but absent in the present case, the nuclear inclusion bodies may also be useful as a feature toward differential diagnosis. distinction from spts is also difficult because the cytological features of an acc resemble an spt and because the cytological findings from nets are partially in common with accs. additionally, in the present case, distinction from an spt was necessitated by the finding of clusters appearing pattern, a scattered distribution of numerous cells with biased nucleus location and myxomatous stroma. spts can be characterized by the pseudopapillary arrangement and the presence of hyaline globules, which are usually absent in accs. these features were absent also in the present case, suggesting that the pseudopapillary arrangement and hyaline globules are useful in distinguishing acc from an spt. immunohistologically, chymotrypsin and trypsin are positive in accs, but negative in nets and spts. however, there is a report that enzymatic markers were positive in 5 - 66% of all cases of nets, so care is needed when differential diagnosis is attempted on the basis of enzymatic markers alone. in net cases, neuroendocrine markers show a strong positive reaction diffusely, but these markers are often positive locally in accs and spts. furthermore, in the present case, neuroendocrine markers were locally positive and enzymatic markers were diffusely positive. for distinguishing acc from net by means of immunostaining, the expression pattern and intensity of neuroendocrine markers and enzymatic markers seem to be important. -catenin is positive in the nucleus and cell membrane of accs while it is positive only in the cell membrane of nets. distinguishing accs from spts on the basis of -catenin seems to be difficult because -catenin is positive in the nucleus and cytoplasm of spts, resembling its positive sites in acc cases. however, since vimentin is strongly positive in spts, the presence / absence of expression of enzymatic markers and vimentin would appear to be useful in distinguishing accs from spts. although the cytological distinction of accs from nets and spts is difficult, the nuclear chromatin pattern and nuclear inclusion bodies, pseudopapillary arrangement and hyaline globules seem to play an important role in the differential diagnosis. for precise distinction from nets and spts, immunocytochemical staining and immunohistochemical staining are needed, and not only enzymatic and neuroendocrine markers, but also antibodies to -catenin, vimentin and so on seem to be useful in the differential diagnosis. | acinar cell carcinomas (accs) of the pancreas are a rare tumor accounting for only about 2% of all pancreas tumors. we report herein on this case and discuss how to distinguish accs from neuroendocrine tumors (nets) and solid pseudo - papillary tumor (spts) morphologically and immunohistochemically. in cytological findings, the nuclear - cytoplasmic ratio was high, and the cytoplasm was granular, but zymogen granules were not evident. the nucleus was biased in location, assuming small circular and irregular forms. chromatin was fine granular in shape and distributed nonuniformly, accompanied by evident nucleoli. immunohistochemically was positive for -catenin (cell membrane and part of nuclei), synaptophysin (focal), chromogranin a (focal) and chymotrypsin were all positive. although the cytological distinction of accs from nets and spts is difficult, the nuclear chromatin pattern and nuclear inclusion bodies, pseudopapillary arrangement and hyaline globules seem to play an important role in the cytological differential diagnosis. furthermore, not only enzymatic and neuroendocrine markers, but also antibodies to -catenin, vimentin and so on seem to be useful in the differential diagnosis. |
the study was advertised and participants were examined at one of the following five locations : 1) department of research, southern college of optometry, 2) the ohio state university college of optometry, 3) the glaucoma and diabetes eye institute, 4) indiana university school of optometry, and 5) state university of new york, college of optometry. the institutional review board approved the study at individual sites, and the tenets of the declaration of helsinki were observed. the investigators of the study were not aware of the participants measurements prior to the study. each study participant underwent ophthalmic examination, including external eye evaluation with a slit lamp biomicroscope and retinal evaluation. individuals with corneal diseases or with a history of intraocular surgery were not included in the study. only one eye of an individual was included in the data analysis for the study. a total of 120 eyes of 120 individuals were included in the study. of these, 81 were women and 39 were men ; 88 individuals were caucasian and 32 were african american ancestries. subjects underwent iop measurement with a gat and a pascal dct, which were mounted on a slit lamp. a drop of fluress (benoxinate 0.4% with fluorescein sodium 0.25%) was instilled prior to the measurement of iop with the gat. a drop of proparacaine 0.5% was instilled before the measurement of iop with the pascal dct. the pascal dct measures pressure continuously as long as it is in adequate contact to the eye. the tonometer gives out a whistling sound, the rhythm of which coincides with the ocular pulse and the cardiac cycle. it is recommended that the iop be measured for a period of 68 cardiac cycles. the instrument s software gives a quality score that ranges from 1 to 5, with 1 being the best reading and 5 being the worst reading. all measurements performed in the study had a quality reading of 13. because reliable and accurate measurement of cct was of paramount importance for the present study, all centers used a pachymeter that was obtained from the same manufacturer. the ultrasonic pachymeter that was used to measure cct was corneo - gage plus from sonogage, cleveland, ohio. this instrument uses a 50-mhz ultrasonic probe, which gives a 2.5 times better resolution when compared with other commercially available pachymeters, which uses a 10- to 20-mhz probe. subjects were instructed to keep the eye closed for 30 seconds to 1 minute to ensure good anesthesia. measurements were performed in rapid succession (up to 10 readings), and the lowest was taken as the cct. patients were instructed to blink after each measurement to avoid desiccation of the corneal epithelium. subjects underwent iop measurement with a gat and a pascal dct, which were mounted on a slit lamp. a drop of fluress (benoxinate 0.4% with fluorescein sodium 0.25%) was instilled prior to the measurement of iop with the gat. a drop of proparacaine 0.5% was instilled before the measurement of iop with the pascal dct. the pascal dct measures pressure continuously as long as it is in adequate contact to the eye. the tonometer gives out a whistling sound, the rhythm of which coincides with the ocular pulse and the cardiac cycle. it is recommended that the iop be measured for a period of 68 cardiac cycles. the instrument s software gives a quality score that ranges from 1 to 5, with 1 being the best reading and 5 being the worst reading. because reliable and accurate measurement of cct was of paramount importance for the present study, all centers used a pachymeter that was obtained from the same manufacturer. the ultrasonic pachymeter that was used to measure cct was corneo - gage plus from sonogage, cleveland, ohio. this instrument uses a 50-mhz ultrasonic probe, which gives a 2.5 times better resolution when compared with other commercially available pachymeters, which uses a 10- to 20-mhz probe. subjects were instructed to keep the eye closed for 30 seconds to 1 minute to ensure good anesthesia. measurements were performed in rapid succession (up to 10 readings), and the lowest was taken as the cct. patients were instructed to blink after each measurement to avoid desiccation of the corneal epithelium. ehlers iop was calculated from the goldmann iop using the nomogram published by ehlers.1 paired - samples t - test was used to evaluate the difference in iop measured using the gat and the pascal dct. pearson correlation coefficient (r) was used to evaluate the associations between cct and the goldmann and pascal iops, and the difference between goldmann iop and the pascal iop. linear regression was used to analyze the residual association between the cct and the difference in ehlers iop and pascal iop. both the goldmann iop and the ehlers iop were compared with the pascal iop using bland patients were stratified on the basis of self - reported race, and the agreement between iop values was examined. as known previously, cct has a weak yet statistically significant positive correlation with the iop measured by the gat (r = 0.20, p = 0.03 ; figure 1) ; that is, the goldmann iop increases with increase in cct. however, there is no association between the cct and the pascal iop (r = 0.02, p = 0.84 ; figure 2). the difference in the goldmann iop and the pascal iop was related to the measured cct, indicating that part of the difference in iop can be accounted by the physiological variation in cct (r = 0.18, p = 0.04 ; figure 3). the difference in the goldmann iop and the pascal iop was significant (p < 0.0001). examining the limits of agreement between the goldmann iop and the pascal iop by using bland altman plots, we find that the limits of agreement are wide, with the lower limit of agreement being 6.9 mmhg and the upper limit of agreement being + 4.1 mmhg (figure 4). there is no systematic error in measurement, and the difference in iop measured using the two devices did not vary as a function of the iop. examining the limits of agreement between the ehlers iop and the pascal iop, we find that the limits of agreement are wider than that of the goldmann iop and pascal iop with the lower limit of agreement being 8.25 mmhg and upper limit of agreement being + 4.15 mmhg (figure 5). linear regression analysis indicates that there is a negative trend when comparing ehlers iop and the pascal iop, which indicate an overcorrection of iop (figure 6). the trends were similar even when groups were segregated by race, with limits of agreement between the ehlers iop and the pascal iop being wider, in both the caucasian and african american ancestries when compared with limits of agreement between the goldmann iop and the pascal iop. examining the limits of agreement between the ehlers iop and the pascal iop, we find that the limits of agreement are wider than that of the goldmann iop and pascal iop with the lower limit of agreement being 8.25 mmhg and upper limit of agreement being + 4.15 mmhg (figure 5). linear regression analysis indicates that there is a negative trend when comparing ehlers iop and the pascal iop, which indicate an overcorrection of iop (figure 6). the trends were similar even when groups were segregated by race, with limits of agreement between the ehlers iop and the pascal iop being wider, in both the caucasian and african american ancestries when compared with limits of agreement between the goldmann iop and the pascal iop. the iop measured by the gat is affected by cct as shown by ehlers and numerous other studies subsequently.2026 however, studies have shown that the iop estimates obtained using the pascal dct is independent of the effect of cct.613 furthermore, the iop estimates obtained by the pascal dct closely matches the manometry values,14,15 making it a closer representation to the true iop when compared with the goldmann iop. a portion of the difference in the measured values and agreement between the goldmann iop and the pascal iop is attributed to the errors in tonometry induced by the physiological variations in biomechanical properties of the cornea.14,15 if the ehlers correction nomogram was indeed correct and used on the goldmann iop to correct for cct, it is expected that the agreement with the pascal iop should improve. this study shows that the limits of agreement between the ehlers iop and the pascal iop are wider than that of the agreement between goldmann iop and pascal iop, indicating that correcting the goldmann iop for the errors in cct may in fact worsen the estimate of iop and widen the difference from the true iop in eye. further, it was apparent from the negative trend observed in the regression analysis (figure 6) that the ehlers nomogram1 overcorrects and overestimates the effect of the cct on iop. furthermore, the corneal thickness may be only a part of the error in tonometry and parameters like corneal rigidity and hydration may affect corneal biomechanics and are other sources of errors in tonometry. this study is additional evidence to a finding that was reported in a prior study,27 which showed the effect of cct on iop after correcting it with the ehlers nomogram. this study shows that the ehlers nomogram did not improve the agreement even when the participants were stratified by race. this is interesting because it was expected that the ehlers nomogram would be more accurate for the individuals with caucasian origin as the participants in ehlers study were primarily of caucasian origin, and there would be an increased similarity of ocular parameters like the corneal thickness, corneal curvature, and axial length. there could be a few reasons why the ehlers iop was not in agreement with the pascal iop. the ehlers correction factor was derived from manometry experiments, and although manometry is the best direct measure of iop, it is not free from errors. manometry results can be significantly erroneous if the needle that is used for cannulation of the eye is blocked by air bubbles or particles.28 further, the manometry experiments performed by ehlers also included patients with acute angle closure glaucoma who were undergoing surgery for glaucoma. this could have a significant impact because there are changes in biomechanical properties of the cornea that has edema secondary to angle closure. also, there could be other factors that may account for the differences in the goldmann iop and the pascal iop, such as the age - related and physiological variation in the hydration of cornea, which can not be measured in vivo at present time. other studies that have examined the effect of cct on goldmann iop have also discounted the benefit of using the correction algorithms that are solely based on corneal thickness because the error in tonometry due to corneal parameters is multidimensional, of which corneal thickness is a small part of the equation and may not aid in better clinical care or outcome.10,29,30 there are other correction algorithms that are present as an alternative to ehlers correction factor,1 notably, the orssengo pye algorithm,31 which is based on finite element analysis and does not assume linearity as done by the ehlers correction factor. pye algorithm31 to calculate iop correction requires the need of having corneal curvature details, which were not available in the present study, and thus, we were not able to evaluate its efficacy. this study is in agreement with prior studies and showed that there was a systematic error in the gat - measured iop, with physiological variation of cct.2026 this study also confirms the finding that the iop measured by the pascal dct is less affected by the variation in cct when compared with the goldmann gat.12,13,32,33 thus, it may, in fact, be a better tonometer when compared with the goldmann gat in measuring iop. there is a definite need to estimate the iop accurately because the current treatment and management of glaucoma, to an extent, are based on the level of iop. however, the results from this study indicate that the use of ehlers nomogram1 to correct for the error induced due to cct is not advisable, as it may be increasing the error rather than decreasing it. | purpose : to evaluate if using the ehlers correction factor on the intraocular pressure (iop) measured using the goldmann applanation tonometer (gat) improves its agreement with the pascal dynamic contour tonometer (dct).patients and methods : a total of 120 eyes of 120 individuals were examined. participants underwent iop measurement with both the dct and the gat and central corneal thickness measurement. the ehlers correction factor was applied on the gat iop measurements to calculate ehlers - corrected gat iop. the agreement between the dct and gat, and dct and ehlers - corrected gat iop was analyzed. the analyses were repeated by stratifying the data by race.results:the mean iop of the gat, dct, and the ehlers - corrected gat was 15.30, 16.78, and 14.68 mmhg, respectively. the agreement as assessed by bland altman plot for the gat with the dct and dct and ehlers - corrected gat iop was + 4.1 to 6.9 and + 4.15 to 8.25 mmhg, respectively. the results were similar even when stratifying the data by race.conclusion:using ehlers correction factor to account for the effect of corneal parameters on the iop measured by the gat worsens the agreement with the dct. this effect remains even when stratifying the data by race. |
photodynamic therapy (pdt) is a successful and minimally invasive therapeutic approach used in the treatment of various solid tumors as well as non - malignant diseases. photodynamic therapy has been clinically approved for the treatment of lung, esophageal, bladder, skin and head and neck cancers. photodynamic therapy action is dependent on three essential components : photosensitizer that is applied topically or administered systemically ; visible light, usually generated by laser sources ; and molecular oxygen. none of these elements is toxic, only when combined together they exert a cytotoxic effect to tumor cells. in a clinical setting pdt is a two - step procedure involving administration of a photosensitizing agent that preferentially accumulates in the tumor tissue, followed by local irradiation of the lesion with the light of appropriate wavelength able to activate the sensitizer. the light activated photosensitizer can either transfer its energy directly to molecular oxygen, leading to production of highly reactive singlet oxygen, or can react with macromolecules and generate radicals. further reaction with molecular oxygen results in formation of superoxide ion, hydroxyl radical or hydrogen peroxide. all these reactive oxygen species (ros) are responsible for oxidative damage of proteins, lipids and other intracellular molecules, causing pdt - mediated direct tumor cell death through apoptosis, necrosis or autophagy. antitumor effects of pdt result from its unique mechanism involving not only direct cytotoxicity to tumor cells, but also indirect actions such as disruption of tumor vasculature and induction of acute local inflammatory response (fig. 1). the latter may facilitate the development of antitumor and antigen - specific immune response. it was widely reported that pdt treatment leads to oxidative stress in tumor tissue associated with massive photooxidative damage in cancer cells, tumor vasculature and stroma. this pdt - mediated injury is a local trauma and threat for tissue integrity and homeostasis. therefore, host inflammatory response is elicited in order to remove dead and damaged cells, heal injured tissue and restore its function as well as the disrupted homeostasis. oxidative stress caused by pdt may induce various signals transduction in the cell which are responsible for production of stress - induced proteins, activation of genes regulating apoptosis and cytokine gene expression. photooxidative damage of cell membranes results from peroxidation of lipids that leads to extensive release of arachidonic acid metabolites which are potent inflammatory mediators. pdt - mediated damage of endothelial cells causes disruption of their barrier function, loss of junctions, and exposure of the vascular basement membrane. exposure of sites enabling clot formation initiates clotting cascade, activation of platelets and the release of proaggregatory and vasoactive agents. this is followed by vessel contraction which facilitates leucocytes and thrombocytes adhesion, whereas released mediators increase vessel permeability. the process of inflammation is one of the most essential mechanisms in antitumor action of pdt. this inflammatory reaction is mediated by various factors such as vasoactive substances, components of the complement and clotting cascades, acute phase proteins, proteinases, peroxidases, ros, leukocyte chemoattractants, cytokines, growth factors and other immunoregulators. pdt - induced damage of tumor and stroma cells leads directly to an increased level of cytokines and other mediators of inflammation. further release of cytokines is a consequence of activation of various signaling pathways and transcription factors such as activator protein 1 (ap-1) or nuclear factor b (nf-b). after pdt treatment in animal and human studies, elevated levels of numerous cytokines such as interleukin (il)-1, il-2, il-6, il-10, tumor necrosis factor (tnf-) were confirmed. however, it seems that the most important cytokine in pdt outcome is il-1. there are studies showing that il-1 activity is critical for the therapeutic efficacy, since its neutralization reduces the cure rates of pdt - treated tumors in a mouse model. interestingly, blocking of anti - inflammatory cytokines such as il-10 and tgf- improved the cure rates of pdt - treated tumors. release of immunomodulating factors together with acute phase response from pdt - injured tissue is followed by rapid and massive accumulation of various immune cells into the tumor site. the infiltrating cells mainly comprise neutrophils, but also mast cells and monocytes / macrophages. shortly after pdt, neutrophils massively migrate to the tumor site and orchestrate other immune cells and further development of immune response. there is increasing evidence that neutrophils are indispensable for pdt efficacy. depletion of these cells in tumor bearing mice and rats led to attenuation of pdt - mediated destruction of tumor cells. furthermore, macrophages / monocytes are also considered as effector cells in the elimination of pdt - treated tumor tissue. a number of studies indicate that low dose pdt can stimulate macrophages, leading to enhanced phagocytic activity and release of tnf- and nitric oxide (no). moreover, tumoricidal activity of these cells was confirmed by in vivo studies showing that stimulation of macrophages potentiates antitumor effects of pdt, whereas inactivation of macrophages causes decreases in cure rates in mice. another class of non - specific immune cells contributing to the antitumor effect of pdt is nk cells. it was reported that depletion of nk cells in a mouse model leads to abrogated antitumor response towards distant lesions, indicating that nk cells play a pivotal role in pdt outcome. one of the earliest events in pdt - treated cells is massive release of various natural and oxidatively modified tumour antigens together with cell stress factors known as damage - associated molecular patterns (damps). damps are intracellular molecules, which act as immunostimulators when exposed on the surface or released by damaged and/or dying cells. damps are molecules recognized by the innate immune system through pattern recognition receptors (prr). it has been shown that pdt enhances the immunogenicity of dead tumor cells by inducing release of damps and tumor antigens. altogether, tumor antigens and damps with other pro - inflammatory signals can activate both innate and adaptive immune response. dendritic cells (dcs) are the most potent antigen presenting cells (apcs) that link innate and adaptive immune response. dendritic cells capture and recognize antigens, become activated and home to local lymph nodes, where they mature and present antigen - derived peptides in association with major histocompatibility complex (mhc) molecules to t lymphocytes. this results in activation of cd4 + t helper cells and cd8 + cytotoxic t cells, b cells and initiation of the adaptive immune response. the pro - inflammatory pdt effect, together with released tumor antigens and danger signals, enhances maturation and activation of dcs and further stimulation of t cell effector functions. it was reported that immature dcs injected into pdt - treated tumors can capture tumor antigens, become mature and migrate into draining lymph nodes, where they stimulate specific t cells. during an effective immune response, activated t lymphocytes become effector t cells and migrate to the lesion and kill the tumor cells. the role of the immune system in pdt outcome has been broadly investigated for the last decades indicating that antitumor effects of pdt depend on the presence and activity of adaptive immunity. reported long - term cures in balb / c mice inoculated with emt6 mammary carcinoma cells, whereas the same pdt regimen was not able to cure scid mice. notably, adoptive transfer of splenocytes obtained from previously cured mice fully restored the curative effect of pdt. interestingly, there are also human studies reporting that pdt is effective in achieving local and distant tumor control. the main cluster of tumors on the right upper limb was treated with fotolon - pdt. unexpectedly, 2 months after therapy, the untreated tumors on the same limb underwent spontaneous remission. similarly, 4 months after treatment spontaneous remission of the untreated tumors on the other limb was observed. moreover, biopsy of the irradiated site revealed strong infiltration of cd4+t lymphocytes and subsequent massive accumulation of cd8 + t lymphocytes. activation of antitumor immune response by pdt is of great interest, whereas little attention is paid to the immunosuppressive side of pdt. pdt - mediated tissue damage causes release of various agents leading to activation of immune response. every strong immune activation induces counteraction such as immunosuppressive mechanisms in order to restore homeostasis and prevent from dangerous over - active immune responses. therefore, it is not surprising that there are studies reporting induction of immunosuppression by pdt. treatment of mice with hematoporphyrin derivative (hpd) pdt resulted in 50% suppression of contact hypersensitivity (chs) to 2,4-di - nitrofluorobenzene (dnfb). subsequently, it was shown that pdt can lead to systemic immunosuppression that can be adoptively transferred by macrophages. pdt - activated dcs transport tumor antigens to the lns not only to stimulate but also to inhibit immune response. the function of activated t cells might be prevented by regulatory t cells (tregs) or immunosuppressive cytokines such as il-10 or transforming growth factor (tgf-) that are elevated in response to pdt treatment. those cytokines stimulate cd4 + t cells to differentiate into tregs, leading to generation of anergic or tolerogenic cd8 + t cells. recently, it has been reported that the level of tregs is significantly elevated in spleens and lymph nodes (lns) in a tumor bearing mouse after pdt treatment. additionally, pdt - induced ros production possibly may cause inactivation of immunogenic molecules, such as damps, released after pdt. moreover, enhanced production of vascular endothelial factor c, vegfc, after pdt treatment might affect the maturation process of dcs that results in development of tolerogenic and immunosuppressive environment. mechanisms of pdt - induced immunosuppression is still poorly understood and mostly tested in the chs model. ideal anticancer therapy would lead to eradication of not only the primary tumor, but also induce specific antitumor immune response allowing for the control of distant metastases and protection from tumor relapse. it seems that pdt may meet these expectations as it induces acute inflammation, attracts immune cells to distant tumors and is able to develop systemic antitumor immune response. under certain experimental settings, pdt itself can lead to complete regression of tumor and to development of long - lasting tumor - specific immunity. however, such situations are unusual ; in most cases pdt alone is insufficient in inducing immune response that would lead to complete tumor rejection. nevertheless, antitumor pdt creates a unique microenvironment for further development of effective antitumor immune response. therefore, pdt with its unique features gives new possibilities for combination treatments, especially with drugs stimulating immune response. there are several studies reporting enhanced anti - tumor immune response when pdt is combined with immune - stimulating agents such as recombinant cytokines [granulocyte - colony stimulating factor (g - csf), granulocyte - macrophage colony - stimulating factor (gm - csf) and tnf ] ; epigenetic drug, 5-aza-2-deoxycytidine or intratumoral administration of dcs [8, 16, 2729 ]. on the other hand, pdt can induce immunosuppression leading to activation of anti - inflammatory cytokines and suppressing immune cells. it was widely reported that neutralization of anti - inflammatory cytokines such as il-10 or tgf- improved pdt outcome. moreover, application of pdt combined with cyclophosphamide led to depletion of tregs and enhanced pdt - mediated immunity as well as to long - term survival and development of memory immunity in mice. to summarize, all those events involved in immune response against tumors induced by pdt should be further studied. understanding of these mechanisms will allow for rational design of combination therapies that could be applied in clinical settings. | photodynamic therapy (pdt) of cancer is an efficient and promising therapeutic modality approved for the treatment of several types of tumors and non - malignant diseases. it involves administration of a non - toxic photosensitizer followed by illumination of the tumor site with a harmless visible light. a light activated photosensitizer can transfer its energy directly to molecular oxygen, leading to production of highly toxic reactive oxygen species (ros). antitumor effects of pdt result from the combination of three independent mechanisms involving direct cytotoxicity to tumor cells, destruction of tumor vasculature and induction of the acute local inflammatory response. pdt - mediated inflammatory reaction is accompanied by tumor infiltration of the leukocytes, enhanced production of pro - inflammatory factors and cytokines. photodynamic therapy is able to effectively stimulate both the innate and the adaptive arm of the immune system. in consequence, this regimen can lead to development of systemic and specific antitumor immune response. however, there are limited studies suggesting that under some specific circumstances, pdt on its own may exert some immunosuppressive effects leading to activation of immunosuppressive cells or cytokines production. in this report we briefly review all immunological aspects of pdt treatment. |
it appears that the potentially fatal pathogen burkholderia cepacia has taken hold of another reservoir for transmission in the form of moisturizing body milk. b. cepacia, a gram - negative rod bacterium known for its affinity towards and its virulence in moist environments, presents a clinical challenge in the treatment and management of susceptible populations and in the subsequent identification of causative sources during nosocomial outbreaks. capable of person - to - person transmission and transmission through contact with surfaces such as medical devices and medicines, b. cepacia is an opportunistic pathogen highly resistant to most antimicrobial agents and it possesses a 42% mortality rate. the need for all clinicians to examine more closely their environments in an effort to halt the spread of b. cepacia is therefore critical. alvarez - lerma and colleagues observed over an 18-day period that five critically ill patients admitted to a multidisciplinary 18-bed intensive care unit contracted the nosocomial infection b. cepacia. microbiologic analysis was performed on the collected oropharyngeal mucosa, urine, and bronchial aspirates from all patients in the intensive care unit and on samples of antiseptics, eau de cologne, and moisturizing body milk used in routine nursing care. it was determined that the strains of b. cepacia isolated from the patients and from the moisturizing body milk were of the same clone, indicating the lotion was the source of the outbreak. upon this discovery, the hospital immediately withdrew the product from routine nursing care hospital wide and notified the manufacturing company about the b. cepacia contamination. alvarez - lerma and colleagues strongly recommend cosmetic products that do not guarantee sterilization during the manufacturing process should not be used in critical care settings. from an all or nothing perspective and within the context that b. cepacia only survived in moisturizing body milk, this approach would appear practical and the right thing to do in an effort to promote quality and safe patient care. removing nonsterile cosmetic products such as lotion from the critical care setting, however, would not completely eradicate or protect patients from b. cepacia infecting humans in the hospital setting. the fact remains that b. cepacia has the potential to find its way into reservoirs that have undergone sterile manufacture because of its affinity for moist environments. several recent studies illustrate that strains of the b. cepacia complex were found in antiseptics, ultrasound gel, enteral feedings, and albuterol and nasal sprays products manufactured in sterile environments [3 - 6 ]. in an effort to first do no harm, the clinician must carefully weigh the risks and benefits of all interventions for every patient. the critically ill patient in a state of imbalance is more susceptible and vulnerable to pathogens in their environment ; therefore, a greater need for enhanced analysis of the benefits and risks inherently exists. it is feasible to ban the use of lotions in critically ill patients if it is known that b. cepacia clinically demonstrates higher colonization rates within moisturizing body milks than any other products, sterilized or nonsterilized, used in the hospital. without such evidence that lotions possess a higher colonization rate for b. cepacia, from a nursing perspective the potential benefits of the use of nonsterilized lotion for the patient might be overlooked. lotion, commonly applied to patients with dry, itchy skin, helps to decrease scratching, which can lead to skin excoriation and can leave the body open to infection. lotion may also be utilized in patients who are bed - ridden and who present with red areas on bony prominences. here, the lotion helps to create a gentle friction on the reddened areas of the body, which helps to stimulate blood flow to the surrounding tissue in an effort to ward off potential development of pressure ulcers. additionally, the use of lotions can assist in the donning of latex - free compression stockings commonly used in a multitude of medical conditions such as lymphedema and venous insufficiency. from a holistic perspective the use of aids in the bonding process of parents of babies in the neonatal intensive care unit by helping to facilitate infant massages, and helps facilitate sensory integration therapies in children with autism and sensory processing disorders. there is no doubt of the tragic magnitude that would ensue if a widespread deadly outbreak of b. cepacia was to occur from the use of moisturizing body lotions, especially if those greatly affected were infants, children, and adults concurrently battling critical or chronic illnesses. halting the spread of b. cepacia infection can not occur only through the discontinuation of nonsterilized cosmetic products, and neither can it be guaranteed only with the use of sterilized products. a tried and true way to take hold of b. cepacia therefore appears to be through the continued and regimented practice of adequate hand washing. | alvarez - lerma and colleagues observed over an 18-day period that five critically ill patients admitted to a multidisciplinary 18-bed intensive care unit contracted burkholderia cepacia from unopened containers of moisturizing body milk, calling into question the use in critical care settings of cosmetic products that do not guarantee sterilization during the manufacturing process. is this the answer to the problem, however, or should the use of lotions in such settings be re - examined ? |
raoultella planticola is a gram - negative, aerobic, non - motile, encapsulated rod - shaped bacterium belonging to the family enterobacteriaceae. it is closely related to klebsiella bacteria species and thus is easily misidentified as klebsiella pneumoniae or klebsiella oxytoca. it is an uncommon pathogen and has rarely been reported to infect humans. to the best of our knowledge, a 62-year - old male patient with a history of diabetes mellitus type 2, hypertension and benign prostatic hypertrophy presented with complaints of fatigue, increased urinary frequency, mild epigastric tenderness, and nausea and vomiting for 5 days. on admission, physical examination revealed a mildly ill - appearing white male, alert and oriented, and in moderate distress. vital signs revealed a temperature of 37 c, pulse of 127 beats per minute, and a blood pressure of 117/76 mmhg. his physical exam was unremarkable except for tenderness to palpation in the right upper quadrant (fig. laboratory data on admission were notable for the following (reference ranges provided parenthetically) : creatinine 3.1 mg / dl (0.81.3 mg / dl) with a baseline of 1.4 mg / dl, glucose 500 mg / dl, wbc 12 k / mm with 93% neutrophils. liver function results were : alkaline phosphatase 351 unit / l (50100 u / l), and bilirubin 2.1 mg / dl (0.31.9 mg / dl). there was an anion gap of 26 with lactic acid of 1.74 mmol / l (0.51 urinalysis revealed 2 + protein, large bacteria, negative nitrites, positive leukocyte esterase and 3 white blood cells / hpf. treatment was initiated for diabetic ketoacidosis secondary to underlying sepsis with intravenous fluid resuscitation, insulin drip and empiric antimicrobials therapy with piperacillin - tazobactam. initial blood and urine cultures grew gram - negative bacilli later identified as r. planticola. an abdominal ct scan revealed a complex multicystic mass in the medial left hepatic lobe suggestive of a hepatic abscess. based on these findings, antimicrobials were changed to ceftriaxone to provide better biliary and hepatic penetration and the patient underwent a percutaneous drainage of the hepatic abscess, during which 80 ml of purulent fluid was removed. the patient 's drainage tube was removed after 8 days and the patient discharged home to complete a 2-week course of iv ceftriaxone 2 g daily followed by ciprofloxacin 500 mg bid for an additional 28 days. follow up of the patient at 2 months post treatment revealed resolution of his symptoms and improvement of his liver abscess on a ct scan. raoultella species are gram - negative, non - motile, aerobic bacilli that are primarily considered as environmental bacteria closely related to the genus klebsiella. the organism has been isolated from a variety of human tissues and biological fluids, and constitutes a potential, although rare, cause of severe infections in hospitalized and immunocompromised patients. raoultella planticola and italicize r. ornithinolytica also have the ability to produce histidine decarboxylase and have been associated with scrombroid fish poisoning by metabolizing the histadine in the fish tissues to histamine raoultella planticola has been found to cause urinary tract infections, cholangitis, cholecystitis, pneumonia, and soft tissue infection, but has not previously been reported as a cause of liver abscess. raoultella planticola was originally considered a member of environmental klebsiella, which consisted of klebsiella terrigena, klebsiella ornithinolytica, klebsiella planticola, and klebsiella trevisanii. in 1986, the last two species were combined under the name k. planticola because of indistinguishable phenotypic characteristics and high levels of dna homology in 2001, k. terrigena, k. ornithinolytica, and k. planticola were transferred to the new genus raoultella on the basis of 16 s rrna and rpob sequences. this case describes a liver abscess in a patient who presented with diabetic ketoacidosis, urinary tract infection and bacteremia. the putative primary causative organism r. planticola was isolated from urine, blood, and hepatic drainage. the most frequently isolated pathogens responsible for hepatic abscesses are escherichia coli, k. pneumonia and streptococcus anginosus group such as s. intermedius. other causative organisms reported in the literature include actinomyces species, entamoeba histolytica and staphylococcus aureus. cases involving aspergillus sp. and candida albicans have also been reported. as a result, definitive bacteriologic diagnosis based on blood or tissue specimen cultures is necessary to guide antimicrobial treatment. chun and yun provided a retrospective study of 20 patients with r. planticola bacteremia characterizing clinical features. the majority of the patients had underlying malignant conditions, most commonly adenocarcinoma involving the gallbladder or bile duct. in fact, one possible scenario suggested for r. planticola s natural course of infection is that it occurs when systemic impairment of the host immune system enables dormant colonizers to become invasive,,,. in the present report, r. planticola was the etiologic agent responsible for bacteremia, urinary tract infection, and liver abscess. the patient was successfully treated with surgical drainage and a 6-week course of antibiotics, including ceftriaxone and ciprofloxacin. this case represents the first reported case of a liver abscess caused by r. planticola and illustrates the importance of recognizing that uncommon organisms can cause significant disease. | raoultella species are a group of gram - negative, non - motile bacilli commonly isolated from the environment. the group was considered a member of the genus klebsiella until the late 1990s. raoultella planticola is a rare cause of human infections. we report the first case of liver abscess caused by this organism. the patient was successfully treated with appropriate antimicrobials combined with operative drainage. |
human adenovirus (ad) was first isolated from adenoid tissue in the 1950s as novel viral agents associated with respiratory infections (1,2). over 100 ad family members have been identified and characterized in a wide range of host organisms, from a variety of mammals and birds, to reptiles and amphibians (3). in the early 1960s, researchers showed that some human ads can cause tumours in rodents (4,5), which led to a surge in studies of the molecular biology, genetics and physiology of ads which continues to this day. since ads must manipulate the host cell to promote a microenvironment conducive to virus replication, studies of basic ad biology have contributed a great deal of novel insight into all fields of cellular biology, including dna replication, tumourigenesis and control of gene expression in the host cell. while the pool of knowledge regarding the ad lifecycle is immense, few studies have investigated the structure and protein association of ad dna within the infected cell nucleus. considering the fundamental importance of chromatin in regulating gene expression in host cells, it is surprising that, until recently, it remained unclear whether ad dna interacted with cellular histones or assembled into chromatin. this review summarizes our current knowledge of the nucleoprotein structure of the ad genome within the infected cell. the virion is a non - enveloped icosahedral capsid with a diameter of 8090 nm, containing a linear double stranded dna genome of 3040 kb (figure 1) (3). of the human ads, serotype 2 (ad2) and 5 (ad5), both of subclass c, are the most extensively characterized. the ad5 genome is 36 kb in size and encodes 39 genes, which are classified as either early or late depending on whether they are expressed before or after dna replication (figure 1a) (6). four early transcription units (e1a, e1b, e3 and e4) encode proteins that are required for transactivating other viral regions, modifying the host cellular environment or altering the immune response. all major late proteins, organized in the transcription units l1 to l5, are expressed from a common major late promoter and are generated by alternative splicing of a single transcript. however, recent work has shown that the l4 - 22k and l4 - 33k proteins, which are themselves involved in regulation of the major late promoter, are initially expressed from a novel promoter (7). four other small late transcripts are also produced : protein ix (pix, encoding a minor structural protein), iva2 (encodes a protein involved in encapsidating the viral dna into the immature virion) and va rna i and ii (the rna itself blocks activation of the interferon response). inverted terminal repeats (itr) of 100 bp flank both ends of the viral dna and contain the origins of replication. directly adjacent to the left itr is the viral packaging sequence (150 bp). (a) a simplified map of the ad5 genome showing the early genes (e1e4) and the region from which the major late transcript is produced (the extensively spliced l1l5 transcripts produced from alternative splicing of the major late transcript are not shown). the relative position of pix, va rna i and ii and iva2 are indicated. also shown are the viral inverted terminal repeats (itr) located at each end of the genome, the viral packaging element () located adjacent to the left itr, and the position of the major late promoter (mlp). (b) model of the ad5 virion, adapted from (9), with modifications based on additional data provided by (8,10,11). (a) a simplified map of the ad5 genome showing the early genes (e1e4) and the region from which the major late transcript is produced (the extensively spliced l1l5 transcripts produced from alternative splicing of the major late transcript are not shown). the relative position of pix, va rna i and ii and iva2 are indicated. also shown are the viral inverted terminal repeats (itr) located at each end of the genome, the viral packaging element () located adjacent to the left itr, and the position of the major late promoter (mlp). (b) model of the ad5 virion, adapted from (9), with modifications based on additional data provided by (8,10,11). the ad5 capsid is composed of three major (ii, iii and iv) and five minor (iiia, iva2, vi, viii and ix) polypeptides (figure 1b) (811). the facets are composed primarily of hexons (trimers of protein ii) with pentons (five molecules of protein iii) capping each vertex. the latter is the base from which extends fibre (trimer of protein iv), the distinctive projections at the ad capsid vertices. within the capsid, the viral dna is associated with three highly basic proteins, core proteins vii, v and mu () (1214). protein vii is a protamine - like protein and is responsible for wrapping and condensing the viral dna (15). the protein vii - dna nucleoprotein complex is organized into a central dense core with 12 large spherical nucleoprotein projections, termed adenosomes, which extend into each vertex (16,17). a shell of protein v is thought to coat the protein vii - dna complex (16,18). protein v is believed to make contact with the outer capsid in several different ways ; protein v interacts directly with penton, and indirectly with peripentonal hexon and the remainder of hexon bridged through iiia and protein vi, respectively (10,11,1922). mu is synthesized as a 79 amino acid precursor protein, pre - mu, which is cleaved by the ad - encoded proteinase to its final 19 amino acid, highly basic mature form (23). pre - mu is speculated to interact with and aid in tightly condensing the viral dna within the capsid, and cleavage of pre - mu may serve to partially relax this structure prior to its entry into the nucleus (24). although the viral dna does not interact directly with the major capsid proteins (10,25,26), the dna still appears to contribute to the physical stability to the virion ; packaging of subgenomic sized dna [< 90% of the wild - type (wt) genome length ] results in virions that are less stable than wtad (27,28). initially, the ad fibre protein binds to the coxsackie - adenovirus receptor (car), which is the primary receptor for both ad5 and coxsackie b virus (29,30). this binding is followed by a secondary interaction between ad penton and v3 or v5 integrins (31). recent studies have shown that ad5 can enter cells using heparin sulfate proteoglycans as an alternative receptor, either through direct binding to the ad fibre shaft (32), or bridged through interaction of ad with blood factors such as factor ix, factor x or complement component c4-binding protein (3335). ad is internalized by receptor - mediated endocytosis and evades degradation by escaping from the early endosome (36). the virion is transported through the cytoplasm to the nucleus along the microtubule network (36), and the capsid is slowly disassembled en route (37). upon reaching the nuclear pore complex, the protein vii - wrapped ad dna enters the nucleus (14,37,38), while the rest of the capsid remains at the nuclear membrane and is subsequently degraded. viral dna replication and assembly of progeny virions the life cycle takes 2436 h, although the time for completion of the lifecycle is slightly extended in primary cells. an overview of our current understanding of ad dna chromatin state in the infected cell is shown in figure 2. although a number of ad capsid proteins reach the nucleus, it is only the protein vii - wrapped dna that enters the nucleus (14). histone h1 (h1) escorts the ad dna protein complex through the nuclear pore (39) ; however, this function appears to be independent of any structural role for h1 on the viral dna. during this phase of infection, protein vii protects the viral dna from activating the dna damage response (40). the ultimate fate of protein vii after entry into the nucleus is currently a topic of debate. some studies suggest that protein vii stably associates with ad dna throughout the early phase of infection (14,41), while other groups suggest that vii is removed gradually from at least certain regions of the genome during this same time period (42,43). other reports have shown that the overall level of vii within the infected cell declines rapidly within the first few hours of infection with a concomitant decline in vii association with viral genomes (44). whether the eviction of protein vii requires active transcription of the ad genome is also in dispute (41,44,45). dna in the ad capsid is highly condensed with core protein vii (1), along with protein v and (not shown). the protein vii - dna complex transits to the nucleus (2), and undergoes remodeling to decondense the core before transcription of early genes can begin (3). remodeling may involve loss of at least some vii. at the time when viral gene expression is first detected histone variant h3.3 is preferentially deposited on the viral dna, through the action of hira and chd1 (4). onset of viral dna replication may be accompanied by a shift to deposition of h3.1 by the caf1 complex on the dna, as is observed for hsv1 (5). as the histone pool is depleted and the intracellular levels of newly synthesized pre - protein vii increase, there is a transition of ad dna association from nucleosomes to pre - protein vii, possibly mediated by taf - iii (6). the viral dna condensed with pre - protein vii is packaged into the ad capsid (1). dna in the ad capsid is highly condensed with core protein vii (1), along with protein v and (not shown). the protein vii - dna complex transits to the nucleus (2), and undergoes remodeling to decondense the core before transcription of early genes can begin (3). remodeling may involve loss of at least some vii. at the time when viral gene expression is first detected histone variant h3.3 is preferentially deposited on the viral dna, through the action of hira and chd1 (4). onset of viral dna replication may be accompanied by a shift to deposition of h3.1 by the caf1 complex on the dna, as is observed for hsv1 (5). as the histone pool is depleted and the intracellular levels of newly synthesized pre - protein vii increase, there is a transition of ad dna association from nucleosomes to pre - protein vii, possibly mediated by taf - iii (6). the viral dna condensed with pre - protein vii is packaged into the ad capsid (1). cell - free systems developed to study ad dna replication have shown that the compacted nature of the vii - wrapped viral dna allows for only limited transcription and dna replication (46,47). this observation suggests that the core / dna structure must be remodeled to allow these processes to proceed with greater efficiency. three cellular proteins have been identified that can remodel the ad core in these cell - free systems : template activating factor i (taf - i) [also known as set (46) ], taf - ii [nap-1 (48) ] and taf - iii [b23/nucleophosmin (49) ]. using the cell - free system, all three taf 's were shown to stimulate replication from the ad core, while taf - i and taf - ii were also shown to enhance transcription. taf-1, the best characterized taf in the context of ad core remodeling, forms a tertiary complex with the vii - wrapped dna (41,50,51), which results in increased accessibility of the viral dna to nucleases and restriction enzymes and, presumably, transcriptional activators (47). it is not clear if increased accessibility was due to actual removal or only shifting of vii on the dna template. sirna - mediated knockdown of taf - i in infected cells delayed virus gene expression, dna replication and virus yield (42), although the effect was relatively modest. knockdown of taf - i did not affect the binding level of protein vii on viral dna as assessed by chromatin immunoprecipitation (chip), at least at 4 hpi (43). thus, additional proteins are likely involved in preparing the ad core for efficient gene expression and dna replication within the infected cell nucleus. several lines of evidence suggest that vii remains associated with ad dna during the early stage of infection. first, vii can be cross - linked to the viral dna at virtually all stages of infection (52). second, based on immunofluorescence analysis, foci of vii can be observed in the nucleus of infected cells, which represent the vii - wrapped viral dna (40,41,45,53). third, chip studies have shown directly that vii is bound to the viral dna up to at least 10 hpi (41,43,44,51,54). there is some disagreement in these studies regarding the level of vii association over time ; some studies indicate that vii association is constant and does not change throughout the early stage of infection (41,45,52), while others suggest a gradual (or more rapid) decline in vii association with the viral dna during this time period (40,43,44,51). based on chip experiments by komatsu. (43), it appears that the degree and timing of vii association with the viral genome during the early phase of infection can vary depending on the region of the genome that is analysed. for example, between 1 and 10 hpi, vii remains stably associated with the late - gene hexon coding region, but shows declining association over time with the major late promoter (43). plasmid - based in vitro assays, addition of small amounts of protein vii with the dna actually enhanced transcription over naked dna, suggesting that small quantities of protein vii may function in part to keep repressive histone / chromatin features from forming on certain promoter regulatory elements (43). taken together, these results suggest that dynamic regulation of protein vii is necessary for optimal viral growth ; sufficient protein vii must be removed or remodeled to decondense the viral dna nucleoprotein complex to allow access to the transcription machinery, but some protein vii must remain to stimulate transcription. it is unclear whether transcription through the ad dna template is required for removal or remodeling of vii. inhibition of transcription has been correlated with prolonged retention of vii on the ad genome (40,45), although in other studies inhibition of transcription or transcription elongation did not affect loss of vii (43,44). although it has been suggested that de novo expressed e1a (the first viral gene product expressed within the infected cell) associates with protein vii and is involved in stimulating transcription on the viral genome which subsequently strips vii from the viral dna (45,54), this function is likely not completely necessary since vii is still removed in the absence of e1a or active transcription (43,44). it is possible that other proteins within the cell can perform this function in the absence of e1a ; indeed, e1-deleted ad can complete a full replication cycle in certain cell types, although the time required to complete the replication cycle is extended (55), suggesting that compensating proteins may exist. in eukaryotic cells, the basic unit of chromatin is the nucleosome, with 147 bp of dna wrapped around a histone octamer, composed of two sets of h2a h2b and h3h4 dimers. the notion that nucleosomes are simply beads on a string has been challenged by the realization that histones and nucleosomes play key roles in gene regulation (56). the post - translationally modified n - terminal tails of histones serve as docking / recognition sites for other regulatory proteins (57), providing the epigenetic information governing gene expression, as dictated by the histone code (58). conflicting data from the 1980s suggested that ad dna is or is not associated with cellular histones within the infected cell (5963). with the development of more sensitive techniques, the subject of the ad nucleoprotein structure within the infected cell has been revisited recently. based on chip analysis, ad and its derivative vectors (either e1-deleted, replication defective ad or helper - dependent ad [hdad devoid of all ad protein coding sequences (64) ]) do interact with cellular histones within a few hours of infection (43,44,65). histones can be detected on the ad dna as early as 1-h post - infection, and chip / re - chip experiments show that both protein vii and histones can be found associated with the same dna molecule in the cell (43). since the histones almost certainly bind directly to the ad dna, at least some vii must be removed from the viral dna at these time points to allow for binding of histones. the mechanism by which vii is removed, and the cellular protein(s) involved in this process, have yet to be determined. within the cell, deposition of cellular histones can occur either through a replication - coupled or replication - independent mechanism, and there are specific histone variants and chaperones associated with each mechanism (66). histone variant h3.1 is expressed exclusively during s - phase and is deposited on de novo synthesized dna by the chromatin assembly factor i (caf1) complex in what is considered a replication - coupled mechanism (67). in contrast, the replacement histone variant h3.3, which differs from h3.1 by only five amino acids, is expressed at all phases of the cell cycle, and is deposited through a replication - independent mechanism (66). h3.3 is deposited on actively transcribed genes by the histone chaperone hira, or on specific regions of the chromosome [such as pericentric dna repeats and on telomeres (6870) ] by the h3.3 chaperone daxx (68). the h3.3 variant is also deposited on incoming male pro - nuclear dna shortly after fertilization utilizing the histone chaperone hira (71). although taf - i can act as a chaperone to transfer histones to dna templates (47), it does not appear to perform this function during ad infection (43). as ad can infect both dividing and non - dividing cells (and only induces cell cycle progression after viral gene expression has initiated), it suggests that ad dna is likely to be chromatinized by exploiting a mechanism independent of dna replication. chromatin immunoprecipitation (chip) experiments have recently demonstrated hdad and e1-deleted ad (44), and wtad (our unpublished data) dna preferentially associates with h3 variant h3.3 as early as 4 hpi, suggesting that chromatinization does indeed occur by a replication - independent mechanism. a preferential association with h3.3 was also found with herpes simplex virus 1 (hsv1) dna during the early phase of infection (72). sirna - mediated knockdown of hira reduced the total association of h3 with the hdad and hsv1 dna, as well as reducing expression of virally encoded genes for both viruses, suggesting that chromatinization was necessary for efficient expression. the involvement of the h3.3 chaperone hira, and not daxx, is consistent with the observation that daxx is actively degraded during normal ad infection (73). as deposition of h3.3 on ad (44) and on hsv1 (72) was dependent on hira, it suggests a common mechanism for deposition of histones on the genomes of invading dsdna nuclear viruses. moreover, the similarity between the chromatinization of sperm dna and nuclear virus dna suggests that both use a similar pathway to achieve chromatinization in the absence of cellular dna replication. in vitro observations suggest that histone chaperones, such as hira, either do not assemble nucleosomes or assemble them at a greatly reduced rate in the absence of atp - utilizing factors (74). in the male pronucleus, hira is necessary for delivery of h3.3 to the site of nucleosome formation (71,75), but it is the atp - dependent chromatin remodeling complex chd1 that is required for h3.3 deposition (74). in hela extracts, hira interacts directly with chd1 (74). thus, it is likely chd1 that is directly involved in deposition of histones on the ad dna, although this has yet to be formally proven. (43) showed that wtad can be found associated with all members of the nucleosome, h2a h2b and h3h4, as early as 1 hpi which, together with studies showing ad dna in the nucleus is wrapped in a repeating 200 bp structure, suggests that the dna may be wrapped in complete, physiologically spaced nucleosomes (44,5962,76). it has been estimated that up to 40% of infecting wtad dna is contained in nucleosomes at 3 hpi, and all regions of the genome are represented in micrococcal nuclease - protected fractions (59). the observation that both protein vii and histones can be found bound to the same viral dna molecule at the same time suggests that the viral chromatin may not completely resemble that of the host cell (43). interestingly, hsv1 genomic dna associates with regularly spaced nucleosomes in a latent infection, but the spacing becomes unstable during a lytic infection and generates heterogeneously sized fragments upon mnase digestion (77). whether wtad assembles into stable or unstable chromatin during a productive infection remains to be determined. electron microscopy analysis of viral genomes isolated during late time points of infection (1618 hpi) showed irregularly spaced nucleosome - like particles at approximately one - tenth the density of cellular chromatin in hela cells [3 versus 26 nucleosomes per m of dna, respectively (61) ]. however, it is not clear whether this is due to unstable chromatin or the limited quantities of histones that are available late in ad infection. during the replicative phase of the hsv1 lifecycle, there is a switch from early association with h3.3 to deposition of h3.1 (72). whether a similar phenomenon occurs with wtad has yet to be determined. the observation that ad - induced shut - off of host protein synthesis results in a reduction in histone gene expression (78,79) suggests that the virus may simply switch from association of nucleosomes containing h3.3 to re - deposition of pre - protein vii in preparation for dna packaging into the viral capsid at the final stage of the virus lifecycle (discussed below). since ad dna is chromatinized, this suggests that epigenetic regulation may be as important for expression of ad - encoded genes as is it for expression of genes encoded by the host cell. chip analysis showed that there was an increase over time in the level of association of acetylated h3 at all ad promoters tested (43). since acetylated histones are commonly associated with actively transcribed genes, it suggests that as these promoters become active, they adopt an epigenetic status similar to cellular genes, which may aid in recruiting appropriate co - factors for optimal gene expression. interestingly, the cell also uses an epigenetic approach in an attempt to down - regulate expression from some foreign, invading dnas, including ad. indeed, daxx can act as an anti - ad defense factor and down - regulate gene expression during wtad infection (73,80). thus, ad - induced degradation of daxx at late times during infection may be a mechanism that the virus uses to evade down - regulation of its expressed genes (73). a similar phenomenon occurs for vectors based on ad (65), and this can affect vector function in vitro and in vivo (81). in these latter studies, the vector chromatin was preferentially associated with deacetylated histones, which is a marker of transcriptionally inactive chromatin (65) ; thus the dna was these observations clearly illustrate the ongoing battle between host and pathogen, and the importance of epigenetic regulation of viral dna at the chromatin level. during the final stage of virus replication, the viral dna must be condensed once again into the compact structure required for packaging within the viral capsid. the histones must therefore be displaced from the ad dna and replaced with pre - protein vii, the precursor of the mature protein vii [the n - terminus of pre - protein vii is cleaved by the viral - encoded protease during virion maturation (3) ]. little is known about how this switch occurs. in eukaryotic cells, expression and synthesis of new histones interestingly, however, there is a dramatic decline in histone synthesis at late times during ad infection (78,79). this puts forth the hypothesis that at the late stage of infection, the decline of available histones relative to the increased levels of pre - protein vii leads, by default, to the deposition of pre - vii on the newly synthesized viral dna (60,62,83). experiments in cell free systems have shown that simply mixing ad dna and purified pre - protein vii leads to the formation of an insoluble complex, suggesting that a specific cellular chaperone(s) mediates the placement of pre - protein vii on ad dna (84). based on co - immunoprecipitation studies using extracts from infected cells, taf - iii / nucleophosmin was shown to have a greater affinity for pre - protein vii than the mature protein vii, suggesting that taf - iii may be involved in placing pre - protein vii on the viral dna (85). in a cell free system, taf - iii was able to transfer pre - vii onto dna, suggesting that taf - iii is indeed a pre - vii chaperone. however, additional studies are required to further support the role for taf - iii as a chaperone during normal ad infection of a cell. insight into the mechanism of ad chromatinization has the potential to impact three specific areas of research. first, ad is widely used as a gene delivery system for basic studies and gene therapy applications (64), and improved understanding of the parameters that aid in establishing gene expression within the host cell will improve vector efficacy and safety. second, ad is a significant and often overlooked human pathogen (86), and understanding early events in the cell that permit expression of viral genes may lead to the identification of new therapeutic targets to limit or prevent wtad - induced morbidity and mortality. finally, numerous studies of basic aspects of ad biology have contributed significantly to our understanding of how the host cell works (3). undoubtedly, delineation of the proteins and pathways involved in ad dna chromatinization will also improve our understanding of this process within the host cell. canadian institutes of health research and cancer research society (cihr) (canada) (grants for research in the parks laboratory) and ontario graduate scholarships in science and technology (ogsst) from the ontario government (to a.n.g. and a.r.d.). funding for open access charge : cihr. | for more than half a century, researchers have studied the basic biology of adenovirus (ad), unraveling the subtle, yet profound, interactions between the virus and the host. these studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. in the infecting virion, the viral dna is tightly condensed in a virally encoded protamine - like protein which must be remodeled within the first few hours of infection to allow for efficient expression of virus - encoded genes and subsequent viral dna replication. this review discusses our current knowledge of ad dna protein complex within the infected cell nucleus, the cellular proteins the virus utilizes to achieve chromatinization, and how this event contributes to efficient gene expression and progression of the virus life cycle. |
depression constitutes the second most common chronic condition in clinical practice and will become the second leading cause of premature death or disability worldwide by the year 2020. reduced monoamine signaling and monoamine metabolite levels have been found in cerebrospinal fluid of depressed individuals ; likewise serotonin, norepinephrine or dopamine depletion exerts pro - depressive effects. in case of depression, the level of monoamine oxidase enzyme in brain is increased, which in turn reduces levels of monoamines. flowers of h. rosa - sinensis linn (malvaceae) are reported to possess cardio- protective, hypotensive, antidiabetic, anticonvulsant and antioxidant activity. therefore, the present research was aimed to evaluate the effect of methanol extract containing anthocyanins (mhr) and anthocyanidins (ahr) in tail suspension test (tst) and forced swim test fst. adult male swiss albino mice (22 2 g) were used for this study. the animals were housed at 24 2c and relative humidity 55 5 with 12:12 h light and dark cycle. the experimental protocol was approved by the institutional animals ethics committee (iaec) of mgv 's pharmacy college, nasik. prazosin (sigma - aldrich, usa), p - cpa (sigma - aldrich, usa), imipramine (diamin, reliance formulation pvt. ltd., india), haloperidol (serenec, searl, india) were used for this study. flowers of h. rosa - sinensis were collected from aushadhi bhavan, ayurved seva sangh, nasik and authenticated by dr. the methanolic extract (mhr) was obtained by maceration of fresh sepal - less flowers of h. rosa - sinensis for 72 h, followed by filtration and concentrated to remove methanol. the anthocyanidins (ahr) fresh sepal - less flowers (200 g) of h. rosa - sinensis were macerated in 2 l methanol : 2 m hcl (85:15 v / v) solution for 72 h. the extract was then concentrated to 500 ml and filtered. mixture was heated in round bottom flask under reflux for 2 h. the mixture was then refrigerated until crystals of anthocyanidins (ahr) were separated out. uv - visible spectra- ahr revealed peak at 286.50 nm and 537 nm while mhr revealed peak at 287.50 nm and 576 nm when spectra was run using shimadzu-2450. ftir- ahr depicted presence of functional groups like phenolic oh (1205.55 cm), aromatic c - c stretching (1510.10 cm), oh - bend and c = o stretching (1332.086 to 1446.66 cm), 6-member ring with carbonyl group (1612.54 cm). mhr exhibited presence of aromatic c - c stretching (1618.33 cm), oh - bend and c = o stretching (1278.85 cm) with shimadzu - ftir 8400s. on the day of experiment, the animals (n imipramine (10 mg / kg, i.p) was administered to animals 30 min before experiment. ahr (30 and 100 mg / kg) was suspended in 0.1% cmc and mhr (30 and 100 mg / kg) was dissolved in water and administered orally to animals 1 h before the test. the antidepressant - like activity was evaluated using tail suspension test (tst) and forced swim test (fst). mice were suspended on the edge of a table 58 cm above the floor by the adhesive tape placed approximately 2 - 3 cm from the tip of the tail. animal was considered to be immobile when it does not show any movement of body and remain hanging passively. mice were forced to swim individually in a glass jar (25 12 25 cm) containing fresh water of 15 cm height and maintained at 25 c. after an initial period of vigorous activity, each animal assume a typical immobile posture. a mouse was considered to be immobile when it remains floating in the water without struggling, making only minimum movements of its limbs necessary to keep its head above water. the change in immobility duration was studied after administering drugs in separate groups of animals. probable involvement of dopamine, adrenaline and serotonin in antidepressant - like activity of mhr and ahr was evaluated using animals pre - treated with haloperidol (50g/ kg, i.p.), prazosin (62.5 g / kg, i.p) and p - cpa (100 mg / kg, i.p.). the study was performed according to irwin schedule and doses were selected from pilot studies performed in our lab. haloperidol, prazosin, p - cpa were administered intraperitoneally in a fixed volume of 1 ml/100 g body weight. p - cpa was administered for three consecutive days before treatment with mhr and ahr. on third day, animals received mhr and ahr 30 min after treatment with p - cpa. duration of immobility was measured 30 min after treatment with mhr (30 and 100 mg / kg, p.o.) and ahr (30 and 100 mg / kg, p.o.). statistical analysis was carried out by one - way anova followed by dunnett 's test. adult male swiss albino mice (22 2 g) were used for this study. the animals were housed at 24 2c and relative humidity 55 5 with 12:12 h light and dark cycle. the experimental protocol was approved by the institutional animals ethics committee (iaec) of mgv 's pharmacy college, nasik. prazosin (sigma - aldrich, usa), p - cpa (sigma - aldrich, usa), imipramine (diamin, reliance formulation pvt. ltd., india), haloperidol (serenec, searl, india) were used for this study. flowers of h. rosa - sinensis were collected from aushadhi bhavan, ayurved seva sangh, nasik and authenticated by dr. the methanolic extract (mhr) was obtained by maceration of fresh sepal - less flowers of h. rosa - sinensis for 72 h, followed by filtration and concentrated to remove methanol. the anthocyanidins (ahr) fresh sepal - less flowers (200 g) of h. rosa - sinensis were macerated in 2 l methanol : 2 m hcl (85:15 v / v) solution for 72 h. the extract was then concentrated to 500 ml and filtered. mixture was heated in round bottom flask under reflux for 2 h. the mixture was then refrigerated until crystals of anthocyanidins (ahr) were separated out. uv - visible spectra- ahr revealed peak at 286.50 nm and 537 nm while mhr revealed peak at 287.50 nm and 576 nm when spectra was run using shimadzu-2450. ftir- ahr depicted presence of functional groups like phenolic oh (1205.55 cm), aromatic c - c stretching (1510.10 cm), oh - bend and c = o stretching (1332.086 to 1446.66 cm), 6-member ring with carbonyl group (1612.54 cm). mhr exhibited presence of aromatic c - c stretching (1618.33 cm), oh - bend and c = o stretching (1278.85 cm) with shimadzu - ftir 8400s. on the day of experiment, the animals (n imipramine (10 mg / kg, i.p) was administered to animals 30 min before experiment. ahr (30 and 100 mg / kg) was suspended in 0.1% cmc and mhr (30 and 100 mg / kg) was dissolved in water and administered orally to animals 1 h before the test. the antidepressant - like activity was evaluated using tail suspension test (tst) and forced swim test (fst). mice were suspended on the edge of a table 58 cm above the floor by the adhesive tape placed approximately 2 - 3 cm from the tip of the tail. animal was considered to be immobile when it does not show any movement of body and remain hanging passively. mice were forced to swim individually in a glass jar (25 12 25 cm) containing fresh water of 15 cm height and maintained at 25 c. after an initial period of vigorous activity, each animal assume a typical immobile posture. a mouse was considered to be immobile when it remains floating in the water without struggling, making only minimum movements of its limbs necessary to keep its head above water. the change in immobility duration was studied after administering drugs in separate groups of animals. probable involvement of dopamine, adrenaline and serotonin in antidepressant - like activity of mhr and ahr was evaluated using animals pre - treated with haloperidol (50g/ kg, i.p.), prazosin (62.5 g / kg, i.p) and p - cpa (100 mg / kg, i.p.). the study was performed according to irwin schedule and doses were selected from pilot studies performed in our lab. haloperidol, prazosin, p - cpa were administered intraperitoneally in a fixed volume of 1 ml/100 g body weight. p - cpa was administered for three consecutive days before treatment with mhr and ahr. on third day, animals received mhr and ahr 30 min after treatment with p - cpa. duration of immobility was measured 30 min after treatment with mhr (30 and 100 mg / kg, p.o.) and ahr (30 and 100 mg / kg, p.o.). statistical analysis was carried out by one - way anova followed by dunnett 's test. adult male swiss albino mice (22 2 g) were used for this study. the animals were housed at 24 2c and relative humidity 55 5 with 12:12 h light and dark cycle. the experimental protocol was approved by the institutional animals ethics committee (iaec) of mgv 's pharmacy college, nasik. prazosin (sigma - aldrich, usa), p - cpa (sigma - aldrich, usa), imipramine (diamin, reliance formulation pvt. ltd., india), haloperidol (serenec, searl, india) were used for this study. flowers of h. rosa - sinensis were collected from aushadhi bhavan, ayurved seva sangh, nasik and authenticated by dr. a voucher specimen has been retained there (pashir-1). the methanolic extract (mhr) was obtained by maceration of fresh sepal - less flowers of h. rosa - sinensis for 72 h, followed by filtration and concentrated to remove methanol. the anthocyanidins (ahr) fresh sepal - less flowers (200 g) of h. rosa - sinensis were macerated in 2 l methanol : 2 m hcl (85:15 v / v) solution for 72 h. the extract was then concentrated to 500 ml and filtered. to the filtrate, mixture was heated in round bottom flask under reflux for 2 h. the mixture was then refrigerated until crystals of anthocyanidins (ahr) were separated out. uv - visible spectra- ahr revealed peak at 286.50 nm and 537 nm while mhr revealed peak at 287.50 nm and 576 nm when spectra was run using shimadzu-2450. ftir- ahr depicted presence of functional groups like phenolic oh (1205.55 cm), aromatic c - c stretching (1510.10 cm), oh - bend and c = o stretching (1332.086 to 1446.66 cm), 6-member ring with carbonyl group (1612.54 cm). mhr exhibited presence of aromatic c - c stretching (1618.33 cm), oh - bend and c = o stretching (1278.85 cm) with shimadzu - ftir 8400s. on the day of experiment, the animals (n = 6) were divided into control and experimental groups. imipramine (10 mg / kg, i.p) was administered to animals 30 min before experiment. ahr (30 and 100 mg / kg) was suspended in 0.1% cmc and mhr (30 and 100 mg / kg) was dissolved in water and administered orally to animals 1 h before the test. the antidepressant - like activity was evaluated using tail suspension test (tst) and forced swim test (fst). mice were suspended on the edge of a table 58 cm above the floor by the adhesive tape placed approximately 2 - 3 cm from the tip of the tail. animal was considered to be immobile when it does not show any movement of body and remain hanging passively. mice were forced to swim individually in a glass jar (25 12 25 cm) containing fresh water of 15 cm height and maintained at 25 c. after an initial period of vigorous activity, each animal assume a typical immobile posture. a mouse was considered to be immobile when it remains floating in the water without struggling, making only minimum movements of its limbs necessary to keep its head above water. the change in immobility duration was studied after administering drugs in separate groups of animals. probable involvement of dopamine, adrenaline and serotonin in antidepressant - like activity of mhr and ahr was evaluated using animals pre - treated with haloperidol (50g/ kg, i.p.), prazosin (62.5 g / kg, i.p) and p - cpa (100 mg / kg, i.p.). the study was performed according to irwin schedule and doses were selected from pilot studies performed in our lab. haloperidol, prazosin, p - cpa were administered intraperitoneally in a fixed volume of 1 ml/100 g body weight. p - cpa was administered for three consecutive days before treatment with mhr and ahr. on third day, animals received mhr and ahr 30 min after treatment with p - cpa. duration of immobility was measured 30 min after treatment with mhr (30 and 100 mg / kg, p.o.) and ahr (30 and 100 mg / kg, p.o.). all data were expressed as mean sem. statistical analysis was carried out by one - way anova followed by dunnett 's test. from phytochemical analysis, mhr and ahr revealed presence of flavonoids, saponins, cardiac glycosides and phenols. spectroscopic data confirmed presence of anthocyanins and anthocyanidins like quercetin. in tst and fst, mhr and ahr (30 and 100 mg / kg, p.o.) decreased the immobility periods significantly (p < 0.05) compared to vehicle treated group [table 1 ]. effect of h.rosa-sinensis extract on duration of immobility haloperidol (50 mcg / kg, i.p.) alone significantly (p < 0.05) increased the immobility period when compared to the vehicle treated group. treatment of animals with mhr and ahr (30 and 100 mg / kg, p.o.) significantly reversed the increased immobility time observed after treatment with haloperidol [table 2 ]. effect of low dose haloperidol pre - treatment on antidepressant activity of h. rosa - sinensis prazosin (62.5 mcg / kg, i.p.) alone significantly (p < 0.05) increased the immobility period when compared to the vehicle treated group. treatment of animals with mhr and ahr (30 and 100 mg / kg, p.o.) significantly reversed the increased immobility time observed after treatment with prazosin in tst and fst [table 3 ]. effect of low dose prazosin pre - treatment on anti - depressant activity of h. rosa - sinensis p - cpa (100 mg / kg, i.p.) alone significantly (p < 0.05) increased the immobility period when compared to the vehicle treated group, whereas, after treatment with mhr and ahr (30 and 100 mg / kg, p.o.) significantly reversed the increased immobility time observed after treatment with p - cpa in tst and fst [table 4 ]. effect of p - cpa pre - treatment on anti - depressant activity of h. rosa - sinensis tail suspension test represents the behavioral despair model, claimed to reproduce a condition similar to human depression. remarkably, tst detects the anti - immobility effects of a wide array of antidepressants, including tricyclic antidepressants (tca), selective serotonin reuptake inhibitors (ssri), monoamine oxidase inhibitors (maoi), electro - convulsive shock (ecs) and even atypical antidepressants. the immobility displayed by rodents when subjected to unavoidable stress such as forced swimming is thought to reflect a state of despair or lowered mood, which are thought to reflect depressive disorders in humans which is reduced by treatment with antidepressant drugs. the monoamine theory of depression proposes that depression is due to a deficiency in one or another of three monoamines, namely serotonin, noradrenaline and or / dopamine. in support to monoamine hypothesis, haloperidol (classical d2-dopamine receptor antagonist) pre - treated group exhibited significant increase in duration of immobility which was reduced by treatment with mhr and ahr, suggesting involvement of dopamine in antidepressant - like activity of mhr and ahr. proposed behavioral model of fst for the screening of new antidepressant compounds. in agreement with this information, in the present study, effect of mhr and ahr prazosin pre - treatment exhibited significant increase in duration of immobility in fst and tst which was reduced by treatment with mhr and ahr, suggesting involvement of adrenaline in antidepressant - like activity of mhr and ahr. p - cpa inhibits synthesis of serotonin by inhibiting selectively and irreversibly enzyme tryptophan hydroxylase. p - cpa (100 mg / kg, i.p., 3 consecutive days) treatment exhibited significant increase in duration of immobility which was blocked by treatment with mhr and ahr. recently, several studies have suggested the antidepressant effect of quercetin glycosides such as hyperoside, isoquercitrin and rutin using the positive results of fst. previous studies on h. rosa - sinensis revealed presence of quercetin and cyanidin flavonoids which was confirmed by uv, ir studies. it was reported that, anthocyanin and their aglycones (anthocyanidins) are characterized by two absorption bands, one in the uv region (275 - 280 nm) and the other in the visible region (475 - 560 nm) and cyanidin group shows absorbance maxima at 535 nm. results from previous workers also showed that cyanidin absorbs at 280 nm and 530 nm which is in accordance with the spectroscopic analysis in this study. the ir spectrum of h. rosa sinensis flowers showed characteristic absorption bands at 3421 (oh) and 1710 (c = o) cm. thus, the activity of h.rosa-sinensis extract may probably involve one of the mechanisms as described above. the finding of the present investigation suggests that antidepressant - like effect of h. rosa - sinensis is mediated through dopaminergic, adrenergic and serotonergic mechanisms. in our further study, we will try to explore selective mechanism of action of h. rosa - sinensis flowers responsible for antidepressant - like activity. | aim : flowers of hibiscus rosa - sinensis linn (malvaceae) popularly known as china - rose flowers contain flavonoids. flavonoids have been found to have antidepressant activity. the aim of the present study is to evaluate the antidepressant activity of flavonoids in h. rosa - sinensis flowers with possible involvement of monoamines.materials and methods : anti - depressant activity of methanol extract containing anthocyanins (mhr) (30 and 100 mg / kg) and anthocyanidins (ahr) (30 and 100 mg/ kg) of h. rosa - sinensis flowers were evaluated in mice using behavioral tests such as tail suspension test (tst) and forced swim test (fst). the mechanism of action involved in antidepressant activity was investigated by observing the effect of extract after pre - treatment with low dose haloperidol, prazosin and para - chlorophenylalanine (p - cpa).results : present study exhibited significant decrease in immobility time in tst and fst, similar to that of imipramine (10 mg / kg, i.p.) which served as a positive control. the extract significantly attenuated the duration of immobility induced by haloperidol (50 g/ kg, i.p., a classical d2-like dopamine receptor antagonist), prazosin (62.5 g / kg, i.p., an 1-adrenoceptor antagonist) and p - chlorophenylalanine (100 mg / kg, i.p., 3 days ; an inhibitor of serotonin synthesis) in both tst and fst.conclusion:it can be concluded that mhr and ahr possess potential antidepressant activity (through dopaminergic, noradrenergic and serotonergic mechanisms) and has therapeutic potential in the treatment of cns disorders and provides evidence at least at preclinical levels. |
campylobacter is the most common bacterial cause of gastroenteritis in industrialized and non - industrialized countries. between 40 000 and 60 000 laboratory - confirmed cases in england and wales annually are estimated to represent over 300 000 infections, similar to other industrialized countries. about 93% of laboratory - confirmed cases of campylobacteriosis in england and wales are due to campylobacter jejuni. phenotypic subtyping has made limited impact in understanding this infection with weak prediction of genetic relatedness by assayed phenotypes. multilocus sequence typing (mlst) has been applied to human c. jejuni isolate collections from many countries [612 ] and to non - human animal isolates [6, 9, 1319 ]. this has allowed genetic attribution of human infections to source [13, 14, 18 ], which indicates that mlst may also support investigation of basic patterns of epidemiology such as temporal and spatial distribution. a summer peak in campylobacter is consistently reported from temperate countries [1, 2022 ] often doubling the weekly incidence at the same time each year with later peaks in colder countries [21, 22 ]. exceptions include estonia and germany with less distinct or repeatable summer peaks [21, 22 ]. seasonality may be driven by exposure to differing sources or varying campylobacter prevalence in sources of infection with a lack of empirical evidence to differentiate between these. prevalence of different multilocus sequence types varies between reservoirs [13, 14, 18 ]. a 1-year uk human study noted a peak of st45 complex in c. jejuni isolates during that summer. the temporal pattern of c. jejuni subtypes infecting humans may thus help to explain seasonality. these differences could represent geographical structuring, seasonal patterns, or transient expansions of particular clones [8, 12 ]. a common - source local outbreak is suggested as the source of a sequence type (st) common in a new zealand collection. the same main clonal complexes contribute to human disease in much of the world, which finding is less affected by sampling concerns than observed differences. substantial published multilocus sequence - typed human isolate collections from australia, new zealand and finland allow direct comparison of infecting subtypes. variation in subtype with age and gender has not been studied apart from one study reporting increased serotype diversity with older age. the integration of mlst in epidemiological analysis of structured longitudinal samples and joint analysis of data from separate studies may address some of these questions. this study applied mlst to a two - site longitudinal sample of c. jejuni in humans and extended this by joint analysis with published international datasets. the uk sample was a 3-year population - based isolate collection with accompanying descriptive epidemiological information. the main questions addressed were whether population structure changed systematically over a 3-year period ; varied seasonally ; correlated with demography or symptoms ; was distinguishable in geographically separate uk populations ; and varied among countries. the campylobacter sentinel surveillance scheme (csss) ran between may 2000 and april 2003. two populations in the csss, nottingham and southampton and parts of their surrounding counties, were selected because they were geographically separated, 168 miles apart, and recruitment had been successful and relatively stable. isolates linked to a questionnaire with complete postcode and no recent international travel were included. travel - related cases were excluded to maximize the data available to study endemic disease within available resources. post - code completeness exceeded 96% apart from the nottingham area during the first year when it was 72%. amplification and sequencing used methods and primers from published protocols [16, 29 ] and the mlst website (http://pubmlst.org/). sequence extension reactions used bigdye reaction mix at 1/32 of the manufacturer 's recommended quantities. traces were assembled using sequence typing analysis and retrieval system software developed by dr man - suen chan and dr nicki ventress [(http://sara.molbiol.ox.ac.uk/userweb/mchan/stars/adminguide.htm) university of oxford, 2001 ]. consensus were queried against the pubmlst campylobacter database (http://pubmlst.org/campylobacter/) to give an allele number and sequence types using standard campylobacter mlst nomenclature. published structured human disease datasets from defined time and place were from populations in australia, new zealand and finland (table 1). the australian dataset was from a population - based case - control study of sporadic cases of campylobacter in the catchment area of two laboratories in new south wales. the new zealand isolates were consecutive isolates form the hospital laboratories serving eight district health board populations in new zealand excluding repeat isolates from individuals or members of the same family. the finnish isolates were from domestically acquired human sporadic cases of gastroenteritis collected at helsinki university central hospital laboratory during separate years. jejuni isolates by country and year assembled from published literatureyearcountryaustraliafinlandnew zealand1999200115319969220021092003972006107 c. jejuni isolates by country and year assembled from published literature the two english populations were compared and then combined for comparison with the datasets from other countries. comparison was by test for clonal complex distribution, fst fixation indices calculated by arlequin at the nucleotide and allele level for gene pools and simpson 's index for diversity. pairwise fst measures the extent to which isolates differ between populations compared to within populations. a value of 0 indicates identical population (i.e. no genetic differentiation) and a value of 1 complete genetic separation. a peak day of year was identified by poisson regression of daily counts modelling seasonal and secular variation using date in sine and cosine terms and in a linear term. the 50% of isolates closest to this day of year were considered peak and others non - peak. a test on clonal complex and fst tests having confirmed significant variation, a harmonic logistic regression model was used to explore seasonal patterns by clonal complex in more detail and without the assumption that seasonality was limited to the presence of absence of a summer peak. this modelled season using sine and cosine coefficients for day of year and with year modelled as both a continuous and categorical variable to assess nonlinear effects of year. for example, to assess seasonal st45 complex distribution compared to other isolates the model was:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ $ \eqalign { \tab { \rm logit}\lpar { \rm st}45\sol { \rm notst}45\rpar \equals \beta _ { \setnum{0 } } \plus \beta _ { \setnum{1 } } { \rm sin e}\lpar 2\pi \lpar { \rm day}\rpar \sol 365.25\rpar \cr \tab \quad \plus \beta _ { \setnum{2 } } { \rm cosine}\lpar 2\pi \ast \lpar { \rm day}\rpar \sol 365.25\rpar \plus \beta _ { \setnum{3 } } \lpar { \rm year}\rpar. \cr } $ $ \end{document } subtype distribution was compared by gender, between older (aged 60 years) and younger (aged < 30 years) people, and between children (aged < 18 years) and adults using the same analyses as for geographical analyses. the association of genotype with presence of particular symptoms and with illness duration was tested using logistic and linear regression models, respectively. a phylogeny was estimated using clonalframe a model - based algorithm inferring phylogeny from sequence data assuming a clonal genealogy with recombination. the model assumption that distant sequence blocks are not affected by single recombination events holds for c. jejuni. the assumption that recombination is from outside the dataset studied can lead to incorrect inference of relatedness in deeper branches. analysis used 50 000 burn - in and 50 000 subsequent iterations, sampling each 100th iteration. of 1596 cases, the laboratory sample was missing or contaminated for 36 (23%) cases. six isolates proved to be c. coli on mlst and seven mixed cultures (with at least two alleles for mlst one locus) that could not be resolved. of the remaining 1547 c. jejuni isolates 1505 were typed by mlst (table 2). allele distributions and demographic characteristics of cases with complete profiles and partial profiles were similar. table 2.typed isolates by location and study yearyearnottinghamhampshiretotalmay 2000april 2001316276592may 2001april 2002292262554may 2002april 2003188112300total7966501446cases reporting date of onset of illness. of the 59 cases not recording onset date an estimated date based on laboratory data indicated onset of 17, 30 and 12 cases in years 1, 2 and 3, respectively. of the 59 cases not recording onset date an estimated date based on laboratory data indicated onset of 17, 30 and 12 cases in years 1, 2 and 3, respectively. 1) with no statistical support for variation between them at clonal complex (, p = 079) nucleotide (fst = 0, p = 04), or allele (fst = 0, p = 06) levels. the two sentinel site populations are combined as english c. jejuni isolates in comparisons below with other countries. clonal complex distribution of 1505 isolates in two geographically distinct uk populations, may 2000april 2003 (likelihood ratio test, p = 079). clonal complex distribution of 1505 isolates in two geographically distinct uk populations, may 2000april 2003 (likelihood ratio test, p = 079). the fst values are highest between finland and each of the other three countries and show that the finnish c. jejuni population is distinct from the others (table 3). there was also substantial variation of the c. jejuni clonal complex distribution among countries (fig. some clonal complexes were widely distributed with st21 complex the most common in england, second most common in finland and new zealand and fourth in australia. st48 complex was the most abundant in australia and new zealand, fourth in england and fifth in finland. however, in finland 46% of isolates were st45 complex, ranging between 40% and 53% in the 3 years studied, which was fivefold higher than england, and tenfold higher than australia or new zealand. within the st45 complex, st45, the central genotype, comprised 62% of isolates and st137 12%, with no marked differences among countries in these proportions. the st677 complex comprising 16/92 isolates in 1996 and 21/206 in 20022003 in finland was otherwise rare with eight (05%) isolates in england and none from australia or new zealand. by contrast, st257 complex was rare in finland (one isolate) but comprised 14%, 13% and 5% of isolates in australia, england, and new zealand, respectively. there were 50 st574 isolates in england with this sequence type absent from other countries, and just one st574 complex isolate seen elsewhere, in finland. 2[colour online ]. international comparisons of clonal complexes of c. jejuni in population - based samples. ua, unassigned. table 3.fst values for c. jejuni isolates from human infections in four countries, allele - based fst values above diagonal and sequence - based fst values below. all differences are statistically significant with p < 00001country (sample dates)englandfinlandaustralianew zealandengland (20012003)011600530028finland (1996, 2002, 2003) 015401730177australia (19992001) 003502140073new zealand (2006) 003202390033 [colour online ] fst values for c. jejuni isolates from human infections in four countries, allele - based fst values above diagonal and sequence - based fst values below. all differences are statistically significant with p < 00001 there were differences in clonal complex distribution between isolates from peak and non - peak periods (supplementary fig. ;, p < 00001). fst differences were small but also highly significant at nucleotide (0011, p < 00001) or allele (0007, p < 00001) levels. secular and seasonal analysis for each clonal complex using harmonic logistic regression showed that the abundant st21 complex decreased, while st257, st353 and st574 complexes increased. strong seasonal effects were evident for st45 and st283 complexes, both peaking during the overall seasonal peak, and for st257 complex which did not rise with the seasonal peak but comprised a greater proportion of isolates at other times of the year. other clonal complexes with evidence for seasonal patterns were st354 complex peaking in the first half of the year and st49 and st52 complexes peaking in the latter part of the year (table 4). table 4.statistical evidence for secular and seasonal variation of clonal complexes in human c. jejuni infections in hampshire and nottinghamshire, may 2000april 2003 (n = 1505)clonal complex (cc)number (in cc)seasonalyearlinear effect (year)st21557056000108 (070092)st2572010020004129 (106155)st45142<00001040st48125098002080 (063102)st20656007099st35352052011148 (104210)st574500290003173 (120247)st35444001012st44336092078st283230002069st6123055045st5222004 011st4221061033st66121056011st2215023031st4912004 023st57310087002 238 (105542)unassigned45083097minor50078012bold indicates p values < 005.likelihood ratio test of a model to predict being a member of the clonal complex under study using season modelled by sine and cosine terms and year as a categorical variable.likelihood ratio test of a model to predict being a member of the clonal complex under study using year as a categorical variable and including season modelled by sine and cosine terms.likelihood ratio test of a model using year as a continuous variable and season modelled by sine and cosine terms used to perform statistical testing. values in parentheses are 95% confidence intervals.no isolates in these clonal complexes present in 2003 sample reducing sample size to 1446 for analyses with year as a categorical variable. analysis of seasonal effect with year as a continuous variable to include the full dataset gave similar statistical support for the seasonal variation of st52 and st49 complexes. statistical evidence for secular and seasonal variation of clonal complexes in human c. jejuni infections in hampshire and nottinghamshire, may 2000april 2003 (n = 1505) bold indicates p values < 005. likelihood ratio test of a model to predict being a member of the clonal complex under study using season modelled by sine and cosine terms and year as a categorical variable. likelihood ratio test of a model to predict being a member of the clonal complex under study using year as a categorical variable and including season modelled by sine and cosine terms. likelihood ratio test of a model using year as a continuous variable and season modelled by sine and cosine terms used to perform statistical testing. no isolates in these clonal complexes present in 2003 sample reducing sample size to 1446 for analyses with year as a categorical variable. analysis of seasonal effect with year as a continuous variable to include the full dataset gave similar statistical support for the seasonal variation of st52 and st49 complexes. subtypes varied with age. comparing individuals aged 60 years with those aged < 30 years (test on clonal complexes, p < 00001 ; fst = 0012, p < 00001 for alleles and 0017, p < 00001 for nucleotides). st257 complex (p < 0001) and st574 complex (p = 0002) were more and less frequent among older cases, respectively. there was no difference between simpson 's index (094 for each), or proportion of infections due to uncommon clonal complexes. children and adults also differed (test on clonal complexes p = 0007 ; fst = 0003, p = 005 for alleles and 0004, p < 004 for nucleotides). isolates from children comprised 11% of the dataset overall, 30% for st283 complex and 5% for st257 complex. subtypes infecting men and women were similar (, p = 041). reported bloody stool (34% of cases) varied with clonal complex but vomiting, abdominal pain and fever did not. analysis adjusting for age (using 10-year age groups) reduced the estimated association between clonal complex and bloody diarrhoea and rendered it non - significant (p = 009), with bloody diarrhoea more common in younger cases. st353 (48%) and st52 (67%) complexes were those most associated with bloody diarrhoea and st206 complex (20%) least. st574 comprised 51 isolates, of which 50 were st574, all from the english sample. st257 complex, common in australia, england and new zealand was dominated by the st257 sequence type (89% of isolates). this study integrated population genetic analysis in the descriptive epidemiology of c. jejuni infection using a 3-year sample from two areas in england and published international datasets. the dominant clones were relatively stable in the english dataset over the 3-year study mirroring consistent patterns over time in finland. within this stability a decrease in the abundant st21 complex and rises in st257 and st574 complexes were the most substantial changes. the narrow clonal structure of the increasing st574 and st257 complexes, with a single sequence type dominating in each case contrasts with the prevalent, diverse, but decreasing st21 complex. these findings suggest st257 and st574 as recently expanding clones and st21 as more longstanding. while the two english populations showed nearly identical subtype distributions of c. jejuni the international comparison showed clear differences. these large effects compared to change over time in the english and finnish datasets indicate true geographical differences. differentiation was not proportional to distance with the english sample being more similar to the samples from australia and new zealand than to the finnish sample, which was from the helsinki area. similarities in subtypes between england, australia and new zealand may therefore represent similarity of food sources of c. jejuni infection in these countries and rather different sources in finland. one finnish study identified that subtypes present in finnish cases have a smaller overlap with poultry types than reported in uk studies [14, 18 ]. the current study further supports substantial differences in the sources of c. jejuni infection in england and finland. seasonally, two clonal complexes (st45 and st283 complexes) were particularly concentrated during the summer peak, some (e.g. st21 and st48 complexes) maintain a similar proportion over the year, while others (e.g. st257 complex) contribute a greater proportion outside the seasonal peak. more complex seasonal patterns are also suggested, e.g. for st354 and st52 complexes which have their peaks away from the overall one. varying seasonal patterns by subtype provides a framework for work to study whether these represent exposure of humans to different sources across the year, different dominant types of infection in these sources over the year, or seasonal variation of survival in the food chain by type. complexes with lower relative prevalence during the seasonal peak were either absent (e.g. st257, st354, st49) or uncommon (e.g. st52 complex) in finland but quite common in england, australia and new zealand. in contrast the st45 complex, which contributed substantially to the summer peak in the english study, was very common in the finland, which has a marked seasonal peak [10, 22 ] but less common in new zealand which has less seasonal variation. a 1-year study in northern england also reported st45 complex as being more common during summer in humans and that this type was isolated from diverse animal species and surface water samples. environmentally adapted. a uk study evaluating campylobacter carriage in broilers at depopulation over 2 years reported a summer peak in st45 complex infection at first depopulation. laboratory studies suggest that st45 complex has different stress responses compared to st21 complex isolates with better survival in response to oxidative and freezing stress but poorer survival in response to heat or chilling. the st45 complex seasonal and geographical patterns in the current study and ecological and biological findings in other work suggest that identifiable biological characteristics of clonal groupings affect the dynamics of subtype distribution. alongside st45 the less abundant st283 complex showed a marked summer peak in the current study. st283 complex isolates were present in the english and finnish samples but not the australian or new zealand collections. in the clonalframe analysis of population structure st283 and st45 complexes formed a single clonal group. these findings in the dimensions of time, space and genotype show that st45 and st283 complexes form a single clade with shared biological and ecological features. this study identified little evidence for variation in virulence, as measured by type of symptoms and duration of illness, between clonal groups ; no evidence of an association of gender and infecting subtype ; limited, although clearly present, variation with age group ; and marked similarity between the c. jejuni populations isolated from the two geographically separate uk study populations. the essentially identical subtype distributions across these areas sampled at the same time period confirm and extend the similar patterns of clonal complex distribution described in oxfordshire compared to a slightly earlier study in northern england. a consequence of the finding that similar types infect such separated populations is that sentinel site surveillance may be an efficient way to monitor this infection for public health purposes. given the large number of cases annually a sentinel approach would be particularly attractive. these findings are also consistent with widely distributed foods being the main sources of infection. a caveat is the english populations in the current study comprised a city and the surrounding areas. the lack of effect does not therefore rule out geographical differences between, for example, urban and rural populations, which may have different patterns of c. jejuni epidemiology. this study has identified different ecologies for identifiable subtypes of c. jejuni affecting humans and the power of integrating population genetic analytical approaches in epidemiological analyses to the explanation of unresolved epidemiological features such as the seasonality of this infection. the findings of a coherent clade comprising st45 complex and st283 complex isolates with particular temporal, spatial, and population genetic characteristics is an area for further exploration in understanding the drivers of the summer peak in this infection. these findings also define a population structure and biological questions to guide experimental work on c. jejuni in general and this clade in particular. | summarywe sought to explain seasonality and other aspects of campylobacter jejuni epidemiology by integrating population genetic and epidemiological analysis in a large 3-year longitudinal, two - centre, population - based study. epidemiological information was collected for 1505 isolates, which were multilocus sequence - typed. analyses compared pathogen population structure between areas, over time, and between clinical presentations. pooled analysis was performed with published international datasets. subtype association with virulence was not observed. uk sites had nearly identical c. jejuni populations. a clade formed by st45 and st283 clonal complexes showed a summer peak. this clade was common in a finnish dataset but not in new zealand and australian collections, countries with less marked seasonality. the uk, new zealand and australian collections were otherwise similar. these findings map to known in - vitro differences of this clade. this identifies a target for studies to elucidate the drivers of the summer peak in human c. jejuni infection. |
tumors of the gastrointestinal (gi) system constitute a major part of the cancer incidence and mortality statistics. worldwide, colorectal cancer is the most frequent type of gi cancer : it is the third most common cancer in men and the second most common in women. moreover, colorectal cancer accounts for the largest share of gi cancer - related deaths in women, while liver cancer is the most common cause of death among gi tumors of men. despite the recent advances in diagnosis and therapy, outcomes for patients with gi tumors remain very poor. often, gi tumors are diagnosed only at advanced stages due to the lack of specific symptoms and screening methods. as a result, adoptive cell immunotherapy might be used in combination with standard therapies as adjuvant postsurgical treatment and as palliative treatment to improve survival and quality of life of gi cancer patients. cytokine - induced killer (cik) cells have the best credentials to be effective in this therapeutic approach. compared to lymphokine - activated killer (lak) cells, cik cells can be obtained more easily and reveal a higher tumor - specific cytotoxic activity [610 ]. similarly, there are several factors hampering the adoptive cell therapy with tumor - infiltrating lymphocytes (tils), for example, the difficulty to recover sufficient quantities of these cells and their poor migration to the tumor side [11, 12 ]. cik cells can be easily developed from peripheral blood lymphocytes (pbls) and stimulated with interferon- (ifn-), monoclonal antibody against cd3 and interleukin (il)-2. the heterogeneous cell population gains its potent, nonmajor histocompatibility complex (mhc)-restricted cytotoxicity mainly through expansion of cd3cd8cd56 cells to cd56-positive natural killer (nk) t cells [10, 13, 14 ]. cik cell cytotoxicity is mediated by perforin release and dependant on nkg2d recognition and signaling [15, 16 ]. the addition of high doses of il-2 during generation of cik cells is critical for the expression of the nkg2d adapter protein dap10 which in turn is essential for cytolysis. nkg2d ligands (e.g., mica, micb, and ulbp 14) are expressed on both solid and hematologic tumors [1719 ]. in the following sections, clinical studies applying cik cells for the treatment of gi tumors gastric cancer is the third leading cancer - related cause of death among men and the fifth most common among women. apart from dietary aspects, helicobacter pylori, a bacterium colonizing the stomach, is the most prominent known risk factor for gastric cancer [1, 20 ]. today, combinations of surgical resection, different platin, fluoropyrimidine, and taxane - derived chemotherapies and radiotherapy are the standard treatment options for patients with stomach cancer. at the time of diagnosis these patients generally present with advanced or even metastatic disease. provide a study applying cik cells in combination with chemotherapy in patients with advanced gastric cancer who had all undergone palliative gastrectomy. after gastrectomy, twenty - five patients were treated with three cycles of folinic acid, 5-fluorouracil (5-fu), and oxaliplatin (folfox) chemotherapy. thirty - two patients were treated with folfox chemotherapy plus autologous cik cell therapy consisting of five transfusions of cik cells (1 10) after each chemotherapy. in both treatment groups, serum levels of tumor markers were significantly (p 19 months and an improved quality of life. this case report gives a promising view on an individual course of cik cell therapy in a patient with pancreatic cancer. the patient was at poor health and not able to receive chemo- or radiotherapy. here hepatocellular carcinoma (hcc) is the most common histological type of liver cancer and the fifth most common cause of malignancy worldwide. liver cancer rates are more than twice as high in men as in women and the second most common cause of cancer - related death in men. the majority of cases occur in developing countries, where liver cancer is strongly related to infections with hepatitis b and c viruses (hbv and hcv). in industrial countries, where the incidence of hcc is increasing, the most common risk factors are alcohol abuse and obesity resulting in fatty - liver disease, liver fibrosis, and finally liver cirrhosis. in 80% of the cases, hcc is developing on top of a liver fibrosis / cirrhosis. in most of the cases therefore, the majority of patients can only be treated with palliative therapies. despite the evolving new medical treatments such as the tyrosine kinase inhibitor sorafenib, therapy of hcc is still challenging. in 2000 a study investigating postsurgical recurrence rates in hcc patients hcc patients who had all undergone hepatic resection were randomly divided into two groups : one group received adjuvant cytokine - stimulated lymphocyte immunotherapy ; the other group received no additional treatment. peripheral blood was drawn from the patients one day before surgery and lymphocytes were cultured with il-2 and anti - cd3 for two weeks. patients in the immunotherapy group received autologous lymphocytes at weeks two, three, four, twelve, and 24 after surgery. in the end, 72 patients received all five cik cell transfers, two received only four courses of cik therapy because of detection of extrahepatic metastases, and two received only one cycle and refused subsequent cell infusions. adverse events occurred in 45 patients and were all who grade 1 or 2 and self - limiting. the recurrence rate of hcc was significantly lower in the immunotherapy group (59%, 45 patients) than in the control group (77%, 57 patients ; p = 0.01). also the time to first recurrence was significantly longer in the immunotherapy group (p = 0.008). hence, adjuvant cell therapy was able to lower the frequency of recurrence and to extend the recurrence - free time after hepatic resection. four years later a small clinical trial about cik cell therapy in hcc patients was published by shi.. at days ten, thirteen, and fifteen of cik cell culture. before treatment and ten days after cik cell therapy, the patients ' pbmcs were analyzed using a flow cytometer : percentages of cd3cd8, cd25, and particularly of cd3cd56 cells were significantly increased (p < 0.05). patients were followedup for up to 108 days after cik cell therapy. at that time, the composition of lymphocyte subpopulations was still similar to the levels determined ten days after therapy. this indicates that the induced changes in the subpopulations can last for at least 108 days. as all patients had a background of chronic hbv infection, the influence of cik therapy on the hbv viral load was of great interest : on average, the hbv content decreased from 1.85 10 to 1.41 10 copies of dna / ml three months after therapy. it is well established that dc function is suppressed in patients with hcc and chronic hbv or hcv infections. therefore, the frequencies of dc1, which induce th1 cell differentiation and immunity, and dc2, which induce th2 differentiation and immunogenic tolerance, were analyzed during this study. the percentages of both cell types were significantly increased in blood after cik cell therapy (p < 0.01). these results suggest that cik cells are able to play a major role not only in tumor treatment but also in restriction of viral infections. in a study by zhang. cik therapy is reported as being safe, effective, and without side effects. without giving reasons, after three months a tumor specimen of this patient showed large lymphocyte infiltration much more than before treatment. the cells were stained for t cell, t memory cell, and mono / macrophage markers and all cell types were increased after cik therapy. unfortunately, no clinical parameters or results are stated and therefore no conclusions on the clinical effect of the cik cell transfer can be drawn from this study. all patients had undergone transarterial chemoembolization (tace) and additional radiofrequency ablation (rfa). each of the 33 patients in the study group received eight autologous cik infusions every three to four weeks either via the peripheral vein or the hepatic artery. after a relatively short followup of one year, 29 patients in the study group and 23 patients in the control group were recurrence - free. as in the study discussed above, the hbv dna content was determined before and after treatment. in the study group, the number of patients with a viral dna content lower than 1 10 increased from 19 patients before treatment to 29 afterwards. in the control group, again, this study gives an idea of what adoptive cik cell therapy is capable of ; it may reduce recurrence, prolong recurrence - free time, and fight hbv. eighty - five hcc patients were treated by tace and rfa and were divided into two groups. the 45 patients in the study group received additional cik cell therapy via the hepatic artery. cik transfusions were given fortnightly after sequential tace / rfa, four times as a course of treatment. patients in the control group (n = 40) received no additional treatment. following the transfusions, eleven patients developed a light fever and shiver ; all adverse effects were grade 1 or 2. the proportions of several t cell subsets in the patients ' peripheral blood were measured by flow cytometry two weeks after cik transfusions. cd4, cd3, cd56, and cd3cd56 t cell populations and the cd4/cd8 ratio were significantly increased (p < 0.05). remarkably, the percentages of these cells were lower in patients who experienced recurrence than in patients who were recurrence - free (concerning all 85 patients ; p < 0.05). interestingly, the percentage of cd8 t cells, which was decreased after cik cell therapy, was increased again in recurrent patients. during eighteen months of followup fourteen patients (31.1%) of the immunotherapy group had hcc recurrence compared to 32 patients (80.0%) in the control group (p = 0.001). the median recurrence - free survival was significantly longer for patients who received cik cell transfer (p = 0.012). also the recurrence - free survival rates were significantly higher in the immunotherapy group than in the control group (31 patients = 68.9% versus 8 patients = 20.0% ; p = 0.01). this study shows that cik cell immunotherapy can be an effective adjuvant therapy to improve recurrence and survival prognosis for patients with hcc. thus, cik cell therapy might be particularly helpful to eradicate microscopic residual tumor lesions to prevent recurrence and improve survival. a similar study by pan. focuses on changes in serum alpha - fetoprotein (afp) levels in patients with hcc after tace / rfa and cik therapy. this protein is not only known to play a role in the inhibition of the immune system but also in the promotion of cancer cell growth. the aim of this study was to examine afp as a potential marker to predict the clinical outcome of the combinational therapy of tace / rfa and cik cells. six to eight weeks after tace / rfa therapy the possibility of residual tumor burden was excluded by imaging techniques. before randomization, two consecutive afp measurements assured a stable afp level. patients were then randomly divided into a control group (n = 39) and a study group (n = 42) ; patients within the study group received autologous cik cell infusions once every week via the common hepatic artery or peripheral veins. further afp levels were examined one and four weeks after tace / rfa and only in the study group before each cik cell transfer and once every four weeks within one year after cik cell therapy. comparing afp levels during followup to baseline levels before tace / rfa, no significant decrease in afp concentration the differences between the two patient groups were significant (p < 0.05). within the control group, nine patients experienced recurrence within one year ; six of them had afp concentrations slightly higher than the baseline level. in the study group three patients developed recurrence within one year, with two of them having afp levels slightly higher than the baseline level. in summary, the one - year recurrence rate was 7.14% in the study group, which was significantly lower than in the control group (23.1% ; p = 0.04). serum afp levels were also reduced in patients after cik cell transfusions indicating that a sustainable decrease of afp might be useful to predict the clinical efficacy of cik cell therapy. regarding these promising results, the researchers recently published another retrospective study including 174 hcc patients from january 1999 to april 2012. eighty - nine patients in the tace + rfa group were treated by tace / rfa alone and 85 patients in the tace + rfa + cik group received additional cik cell therapy. after sequential therapy with tace and rfa, blood was collected from patients in the cik group. two weeks later ; four to 25 (median = 9) successive cik cell infusions were given per patient. no major complications occurred during treatment and the overall frequency of adverse effects was similar in both groups. the evaluation of the clinical efficacy was based on modified response evaluation criteria in solid tumors (mrecist). differences in the short - term efficacy (three months after therapy) of the two treatment modalities were not significant. after a median followup of 78 months os and pfs were calculated for both groups. the median survival time (mst) and the median pfs were significantly longer in the cik group than in the control group (mst : 56 months versus 31 months, p = 0.023 ; pfs : 17 months versus 10 months, p < 0.001). the 3-, 5-, and 10-year os was also significantly higher in the cik group (p 0.005). the results of this study suggest that additional cik cell infusions can be beneficial for patients treated with sequential tace / rfa. cik cell therapy can prolong recurrence - free survival, which points to a safe and effective treatment option for patients with hcc. published a nonrandomized controlled clinical trial combining only tace with cik cell therapy. from 2005 to 2008, 146 patients with unresectable hcc were assigned by request either to the tace group or to the combination group. no significant differences were found in the baseline characteristics between the two groups. at first, all patients were treated by tace. the 72 patients in the combination group received four additional cik cell transfusions as one course of cik treatment with maximally four courses in one patient every year. according to recist criteria, short - term responses were similar in both groups : 18% (13 patients) in the combination group and 14.9% (11 patients) in the tace group had pr ; 81.9% (59 patients) in the combination group and 85.1% (63 patients) in the tace group had sd. the pfs in the combination group was significantly improved compared to the tace group ; the 6-month, 1-year, and 2-year pfs rates were 72.2%, 40.4%, and 25.3% in the combination group versus 34.8%, 7.7%, and 2.6% in the tace group (p < 0.001). among other factors, cik cell therapy and times of tace before disease progression were associated with pfs and os as identified by univariate analysis. the median os was significantly longer in the combination group (31 months) than in the tace group (10 months ; p < 0.001). in this study, cik immunotherapy was able to prolong pfs and os in patients with unresectable hcc. especially the times of tace combined with cik cell therapy were critical for the clinical outcome. this might be due to the low residual tumor burden after several courses of tace on which cik cells might be most effective. in a recently published trial, i.p. perfusions of autologous cik cells were combined with local radio frequency (rf) hyperthermia in 31 patients with advanced hcc. perfusions of 3.2 10 to 3.6 10 cik cells with local rf hyperthermia performed two hours later. levels of peripheral blood lymphocytes, the afp concentration, and the abdominal circumference were recorded every two weeks. the level of peripheral blood cd4, cd3cd8, and cd3cd56 cells increased significantly after treatment with cik cells (p < 0.05). the afp level (p = 0.001) and the abdominal circumference (p = 0.002) significantly decreased. after a median followup of 8.3 months (range 212 months), the median ttp was 6.1 months and the median os 8.5 months. the 3-, 6-, 9-months, and 1-year survival rates were 93.5%, 77.4%, 41.9%, and 17.4%, respectively. although a control group is missing in order to draw definite conclusions, the authors suggest a benefit of this treatment modality for patients with advanced hcc. they also emphasize the necessity of more clinical trials including higher numbers of patients to provide evidence and evaluate combinations with other therapeutic approaches. a relatively large randomized controlled study on postoperative cik immunotherapy was conducted from january 2000 to 2002. the aim of this study was to evaluate the clinical outcome of two different schedules of adjuvant cik cell therapy. for this purpose, 127 patients with hcc, who underwent radical hepatic resection, were randomly assigned to three groups. the first group (cik - i) comprised 41 patients who received three courses of cik cell transfer, while the 43 patients in group cik - ii were given six courses of cik cell transfer. the control group (n = 43) did not receive any postoperative adjuvant therapy. the patients were followedup for a relatively long period of five to seven years. in total, the dfs rates were significantly longer in the cik - treated groups than in the control group (p < 0.005), but there were no significant differences between the cik - i and the cik - ii groups (p = 0.345). comparing the 5-year dfs rates, the results of the cik - i and the cik - ii groups are relatively close together (23.3% and 19.4%), while the result of the control group is about half the percentage (11.2%). interestingly, there were no significant differences in the os rates (p = 0.884). apart from the treatment modality, liver cirrhosis, tumor size, tumor differentiation, and vascular invasion were identified as significant factors influencing the dfs (p < 0.05). this randomized study was conducted with a relatively high number of patients and a long followup. the authors demonstrate that adoptive cik cell therapy can prevent or at least delay recurrence of hcc after hepatic resection. however, adjuvant cik cell therapy does not seem to be able to improve the os. similar to the study about -gal - pulsed dcs and cik cells for the treatment of pancreatic cancer, the same authors tested this strategy on hcc. eighteen patients with clinical stage iii hcc were included in this study and treated with conventional therapies. nine patients were enrolled in the study group and nine patients in the control group. autologous tumor cells were synthesized with -gal epitopes, then bound by natural anti - gal antibody, and finally lysed. dcs were induced from pbmcs and co - cultured with this -gal epitope - expressing hcc tumor cell lysate. cik cell transfusions in the study group started three days after completion of the chemo- or radiotherapy and were then given every week for two to seven times. the median survival of the study group was 17.1 months versus 10.1 in the control group (p = 0.00121). increases in cd3, cd4, cd45ro, cd8, and cd56 cells could be detected in the patients ' peripheral blood after cik therapy. in summary. it would yet be interesting to compare the clinical effect of these tumor - specific cik cells to standard cik cells in order to elucidate whether the coincubation with the -gal - pulsed dcs gives a significant benefit. colorectal cancer belongs to the most common and deadliest types of cancer. on the one hand, the incidence of colorectal cancer is decreasing in some countries, for example, in the united states. this decrease is due to an increase in detection and removal of precancerous lesions. on the other hand, the incidence is increasing in some other countries, for example, in several asian and eastern european countries, probably due to several lifestyle factors such as increasing obesity and smoking. in general, nonlifestyle risk factors for colorectal cancer include age, genetic predisposition, and chronic inflammatory bowel disease. to our knowledge the only clinical trial with cik cells including colorectal cancer patients was published by schmidt - wolf. in 1999. here ten patients with metastatic disease were included in this study seven patients with colorectal cancer, two patients with lymphoma, and one patient with renal carcinoma. infusions of il-2-transfected cik cells and five infusions of untransfected cik cells ; the second treatment cycle followed three weeks after the first one. cik cell therapy was well tolerated ; only three patients developed who grade 2 fever. transfected cells could be detected in the patients ' blood for up to two weeks after immunotherapy. moreover, significantly increased serum levels of ifn-, gm - csf, and transforming growth factor- (tgf-) were measured (p < 0.05). the clinical evaluation in this study was based on comparison of ct scans before and after treatment and on the who handbook for reporting results of cancer treatment. at the beginning of this study, after treatment six patients remained in pd, three patients had sd, and one patient had a complete response (cr) with no signs of tumor for at least four weeks. in conclusion, this clinical trial proved cik cell administration to be safe and promising for patients with colorectal cancer. the twenty clinical studies discussed here prove cik cell therapy to be an effective treatment option after or along with conventional therapies for patients with gastrointestinal tumors. in addition, as it was shown to be safe and not to induce major adverse effects, cik cell therapy is a valuable alternative for patients not able or willing to tolerate side effects of conventional chemotherapy [24, 33 ]. within the different studies, cik cells were applied either via peripheral veins, i.p., or via the common hepatic artery and all approaches were well tolerated. most probably, the best application depends on the tumor side itself and on the prevailing circumstances ; for example, in case a catheter is introduced for tace, the application of cik cells through the same catheter is apparent. still, as cik cells are easily applicable i.v. several studies showed that after cik cell application the levels of cd3, cd4, cd3cd8, cd3cd45ro, and cd3cd56 cell populations and the cd4/cd8 ratio in the patients ' blood increased, which indicates a boosting cellular immunity induced by cik cell transfusions [22, 31, 35, 45 ]. the secretion of ifn- by pbmcs and the ifn- serum level, respectively, were also shown to increase after cik cell therapy. in the study of qui and colleagues the increased inf- secretion lasted for the followup period of two years and elevated cd3, cd4, cd3cd8, cd3cd45ro, and cd3cd56 levels returned to normal six to nine months after cik cell therapy. shi and colleagues detected significantly increased levels of cd3cd8, cd25 and cd3cd56 cells during the whole followup period of up to 108 days. remarkably, in the study of weng and colleagues the percentages of cd4, cd3, cd56, and cd3cd56 cells were lower in recurrent patients. therefore, the immunomonitoring of these t - cell subtypes seems to be critical to predict the clinical response to cik - cell therapy as increases in these t - cell subtypes suggest a prolonged ttp. some types of cancer such as hcc are closely associated with underlying viral infections (hbv, hcv) making the viral load an important prognosis factor. therefore, one therapeutic goal is to decrease the viral load in order to improve the clinical outcome. in some clinical studies on cik cell therapy for hcc, the viral load of hbv has been determined before and after cik transfusions in order to evaluate the effect of cik cells on viral replication. this indicates a positive effect of cik cells on viral infections and thus a clear benefit for patients with tumors and chronic viral disease [35, 39 ]. in summary cik cells have a favorable biology with non - mhc - restricted tumor targeting and uncomplicated isolation and cultivation. in all studies, there are several questions yet to be elucidated, for example, the optimal application schedule and the best therapeutic combination with conventional treatment modalities. a few of the abovementioned studies are retrospective studies and many authors stated that more large - scale randomized controlled studies are needed. indeed, these retrospective studies only give an idea of the effectivity of cik cell therapy while the improved data quality of prospective clinical studies results in a higher significance. the great advantage of cik cell therapy is that it is a personalized therapeutic approach unfortunately, this makes cik cell therapy very cost- and time - intensive, hampering the conduction of large prospective randomized trials. there are many variations in the methodology and in the clinical evaluation between the research groups, which impede the comparison of the trials. generate cik cells by addition of ifn- and il-2 on day 0, anti - cd3 antibody and il-1 on day 1, and then il-2 every third day. add anti - cd3 antibody, il-1, and ifn- at day 0, il-2 after 24 hours, and then ifn- and il-2 every five days. shi and colleagues do not use any il-1 at all but add only ifn- at day 0 and anti - cd3 antibody and il-2 at day 1. the same applies for the concentration of cytokines and the time of incubation till harvesting. often not even information about the resulting phenotype composition in the cik cell cultures is given, which makes the drawing of definite conclusions even more difficult. the international registry on cik cells (ircc) was established to collect data about cik cell therapy and to set new standards on the report of results from cik cell application. making the generation of cik cells, the treatment, and the reporting of results more uniform will definitely push the application of cik cell therapy forward and result in advantageous treatment options for cancer patients. | tumors of the gastrointestinal system represent a significant share of solid tumors worldwide. despite the advances in diagnosis and treatment, the prognosis of gastrointestinal tumors is still very poor and improved therapies are indispensable. cytokine - induced killer (cik) cells are feasible for an immunotherapeutic approach as they are easily available and have an advantageous biologic profile ; they are rapidly proliferating and their high cytotoxicity is non - mhc - restricted. we summarize and discuss twenty recent clinical studies applying cik cells for the treatment of gastric, pancreatic, hepatocellular, and colorectal cancer. autologous cik cells were transfused intravenously, intraperitoneally, or via the common hepatic artery. in all studies side effects and toxicity of cik cell therapy were mild and easily controllable. the combination of cik cell therapy with conventional adjuvant or palliative therapies was superior to the standard therapy alone, indicating the benefit of cik cell therapy for cancer patients. thus, cik cells represent a promising immunotherapy for the treatment of gastrointestinal tumors. the optimal treatment schedule and ideal combination with conventional therapies should be evaluated in further clinical studies. |
nucleoli are non - membrane - bound structures within the nucleus where ribosomal rna (rrna) genes are transcribed. these atypical cellular organelles consist of three major compartments with specific functions : (1) fibrillar centers (fc, pre - rrna synthesis), (2) dense fibrillar components (dfc, pre - rna processing), and (3) granular components (gc, ribosome assembly). their organization allows hosting a number of proteins and rnas with an important role in various cellular processes. thus the perturbation of nucleolar dynamic assembly exerts profound effects on several cellular functions. a detailed description of nucleolar morphology and organization has been provided elsewhere [1, 2 ]. here we focus on the mechanisms by which nucleolar activity may regulate neuronal function and survival and contribute especially to neurodegenerative diseases. one intriguing mechanism to control embryonic development, differentiation, and survival is the nucleolar shuttling of cell cycle regulators and transcription factors dictating cell lineage to the nucleoplasm. for example, during the embryonic development, transcription of rrna genes is repressed by lineage - commitment transcription factors turning off the pluripotency genes. during neural lineage commitment there is no direct evidence that rrna transcription is inhibited ; it is likely, however, that mechanisms involving nucleolar dynamics could be in play. for example in brain and retina the levels of the nucleolar protein nucleostemin, a controller of pre - rrna processing, are rapidly reduced before cell cycle exit and neural differentiation [4, 5 ], suggesting that rrna biosynthesis may have a regulatory effect on neurogenesis. however, the major regulatory function associated with altered dynamics of nucleolar proteins is connected to the stress response and involves among others the release of ribosomal proteins (rps) to the nucleoplasm. in response to adverse growth conditions, metabolic deficits, and oxidative stress, rrna synthesis disruption of ribosome biogenesis releases rps such as l5, l11, l23, and s7 into the nucleoplasm where they interfere with the activity of the e3 ubiquitin ligase mdm2. normally, this enzyme promotes proteasomal degradation of the transcription factor p53, but this function is impaired by rps leading to accumulation of p53, which in turn initiates transcriptional and non - transcriptional programs. by sensing cellular stress signals and transmitting them to the p53 stabilization system the nucleolar protein nucleophosmin is dowregulated upon excitotoxic stimuli in neurons and alters p53 levels. nevertheless, nucleophosmin - induced cell death appears to be p53 independent, suggesting that other yet unknown pathways control the stress response. more regulatory functions can be postulated based on the observation that the nucleoli are also composed of rnas, which are involved in the processing and maturation of cellular rnas. for example, small nuclear rnas are modified in the nucleolus, suggesting a possible link between nucleolar activity and splicing regulation. in mature neurons changes in protein synthesis during synaptic activity are linked to increased number of nucleoli, by the postsynaptic protein aida-1d that regulates the generation of functional nucleoli by enhancing the release of small nuclear ribonucleoproteins (snrnps) to the nucleoli. more recently, a significant number of small non - coding rnas (ncrnas) regulating mrna translation have been located in the gc of the nucleolus. although the role of this nucleolar transit is not understood, it is tempting to speculate that the regulation of micrornas (mirnas) processing and location is an additional mechanism linking nucleolar activity to protein synthesis regulation. recently, it has been demonstrated that also specific stress stimuli such as acidosis and heat shock induce immobilization of proteins by long ncrnas in the nucleolus. in turn, reduced levels of ribosomal proteins may alleviate mirna - mediated repression of translation initiation, highlighting the finely tuned cross - talk of the translational components that have their epicenter in the nucleolus. these premises and the increasing evidence of the role of ncrnas in neuronal development and plasticity support the unexplored functional link between nucleolus and ncrnas in neuronal survival and activity. in summary, by sequestering regulatory proteins and rnas, and by influencing their dynamics upon specific stimuli, the nucleolus can finely tune distinct cellular functions beyond protein synthesis under physiological conditions. it is well known that nucleolar malfunction contributes to the pathology of several rare human genetic disorders, such as werner syndrome, dyskeratosis congenita, treacher collins syndrome and predisposes to certain forms of cancer [17, 18 ]. moreover, decline in rrna synthesis and nucleolar size occur during aging, which is the principal risk factor for neurodegenerative diseases [19, 20 ]. one of the major problems in neurodegenerative diseases is that the majority of cases are sporadic and, even when an inherited basis is ascertained, there is a high inter - individual variability. current treatments only help to ameliorate the symptoms but treatments that stop or reverse the pathology are still missing. the interest on nucleolar stress is gaining momentum and the number of studies is rapidly growing over the last years (fig. 1). a focus on the role of nucleolar stress for neuronal activity and survival offers a basic and yet transformative perspective to the field of research on neurodegeneration. for example, silencing of rdna may occur during early stages of alzheimers disease (ad) pathology and play a role in ad - related ribosomal deficits and, ultimately, in dementia. in line with this speculation, differential methylation activity of the human rdna has been proposed as a mechanism to decrease rrna gene expression in ad patients. thus, rdna specific methylation pattern could be used as a marker of the disease or of its progression.fig. 1diagram showing the increasing number of publications and citations related to nucleolar stress. a number of publications found in pubmed database searching for nucleolar + stress. only four out of 39 publications have been published before the year 2005. b number of times the publications in (a) have been cited ; asterisk, last updated on aug 31, 2012 diagram showing the increasing number of publications and citations related to nucleolar stress. a number of publications found in pubmed database searching for nucleolar + stress. only four out of 39 publications have been published before the year 2005. b number of times the publications in (a) have been cited ; asterisk, last updated on aug 31, 2012 nucleolar integrity is disrupted in dopaminergic neurons of parkinsons disease (pd) patients in human post - mortem brain samples. treatment of mice with the neurotoxin mptp (widely used in pharmacological models of pd to inhibit complex i activity in dopaminergic neurons), decreases rrna synthesis causing early nucleolar disruption. the findings demonstrating that impaired mitochondrial activity affects nucleolar function support the concept that nucleolar stress may occur at early disease stages and that contributes to the pathogenesis. although the molecular basis of this control is still not understood, regulators of rrna transcription might be involved. in fact, previous studies identified nucleolin as a protein - regulating rrna synthesis and ribosome biogenesis [24, 25 ] and interacting with alpha - synuclein and dj-1, two major proteins involved in familial pd pathogenesis. strikingly, the expression levels of nucleolin are dramatically reduced in the substantia nigra pars compacta of human pd patients. more recently, misfolded dj-1 protein caused by l166p mutation has been shown to alter rrna biogenesis in cellular models of pd, further supporting the association of impaired nucleolar activity with pd pathogenesis. these neurodegenerative diseases are associated with expansion of trinucleotide in repeats transcribed or untranscribed region of different genes. huntington s disease (hd), for instances, is associated with an expansion in exon 1 of the huntingtin gene, which leads to an aberrant polyglutamine tract in the huntingtin protein. rrs1 (regulator of ribosome synthesis), a protein inhibiting transcription of both rrna and ribosomal protein genes, is highly expressed in a presymptomatic hd mouse model. the levels of the basal rna polymerase i factor ubf1 are decreased in cellular and animal hd models. in post - mortem specimens of human hd cases, insoluble aggregates of huntingtin a hallmark of this disorder are found in the nucleolus. the presence of insoluble protein aggregates is not limited to hd and is a rather common feature of neurodegenerative diseases. for instances, aggregates are found in the nucleolus of spinocerebellar ataxia patients, an inherited disorder caused by cag or ctg expansion [30, 31 ]. in these neurodegenerative diseases nucleolin appears to play a role because cag rnas deprive rrna promoter of this histone chaperone, downregulating rrna transcription. abnormal interaction with nucleolin also accounts for the neurological disorder ataxia with oculomotor apraxia type 1, due to mutated aprataxin. in several human cancers the angiogenic ribonuclease angiogenin (ang) is upregulated and acts as a transcription factor, binding to rrna promoters and stimulating rrna transcription. moreover, nucleolar activity may be repressed by inhibition of the proteasomal machinery, another hallmark of neurodegenerative diseases. dysfunction of snrnp biogenesis involves protein relocalization to the nucleolus in type i spinal muscular atrophy, an autosomal recessive disorder leading to degeneration and death of motor neurons caused by loss or mutations of the survival motor neuron 1 gene. mutant rna molecules downregulating rrna transcription in polyglutamine diseases and mutant proteins altering the localization of nucleolar proteins in pd show an active role of mutant gene products to cause rrna transcription failure, supporting similar mechanistic studies in other neurodegenerative disorders. the association of nucleolar stress with neurodegenerative diseases raises the question of what cellular and molecular alterations depend on it. recently developed mouse models in which nucleolar function is specifically impaired have provided the first mechanistic insights into nucleolar stress in the nervous system. these models are based on the genetic ablation of tif - ia, an evolutionary conserved transcription factor essential for the recruitment of rna polymerase i to rrna promoters. since its identification, it was evident that tif - ia activity is strongly dependent on external signals. by now, it is known that tif - ia is regulated by a variety of protein kinases at distinct serine residues : erk and rsk in response to mitogenic signals, s6k in response to growth stimuli [39, 40 ], jnk2 in response to oxidative stress, ampk in response to cellular energy status, perk - dependent phosphorylation in response to endoplasmic reticulum stress (fig. 2). in addition, proteasome activity is required for pre - rrna synthesis and tif - ia may represent a potential link by which proteasomes are recruited to rrna genes.fig. 2schematic of the rna polymerase i machinery at the rdna promoters with the different signals and pathways regulating tif - ia activity and exerting positive or negative control on nucleolar activity and integrity schematic of the rna polymerase i machinery at the rdna promoters with the different signals and pathways regulating tif - ia activity and exerting positive or negative control on nucleolar activity and integrity tif - ia global knock - out in mice results in early embryonic lethality. specific loss of tif - ia in neural progenitors leads to rrna synthesis inhibition, nucleolar disruption, rapid apoptotic death of developing neurons, and consequent anencephalia. the conditional ablation of tif - ia in mice offers the major advantage of allowing focused investigation on the impact of nucleolar stress not only on cell cycle and growth regulation of neural progenitors, but also on quiescent postmitotic neurons in living organisms. inactivation of the tif - ia gene in adult dopaminergic neurons by a drug - inducible approach leads to a phenotype closely resembling pd, characterized by depletion of dopamine in the striatum, oxidative stress, mitochondrial impairment, progressive and differential loss of dopaminergic neurons in the substantia nigra pars compacta, as well as marked deficiencies in motor performance. at the mechanistic level, mtor activity is downregulated by tif - ia ablation in dopaminergic neurons, underscoring a negative feedback that co - regulate and coordinate the translational process. in addition, the p53-dependent apoptosis in proliferating cells appears to be conserved in dopaminergic neurons under nucleolar stress, as shown by increased neuronal survival in absence of p53. the importance of p53 for the pathogenesis of neurodegenerative diseases has been clearly shown for hd, and further confirmed also for ad and pd. it is suggested that p53 could also serve as a convergence point in the molecular pathways for different neurodegenerative diseases. p53 induces cell cycle arrest, senescence, and apoptosis in response to different stress signals such as dna damage, hypoxia, nutrient deprivation, and nucleolar stress. however, identifying the key elements that define a particular p53-mediated stress response outcome remains a central, yet unresolved, question. the identification of such downstream effectors is crucial to manipulate the deleterious consequences of cellular stress mediated by nucleolar disruption. nucleolar stress may start a progressive series of events affecting protein translation, which can be deregulated in consequence of altered distribution of nucleolar proteins and/or biogenesis of mirnas, as well as of other cellular mechanisms. because ribosomes seem to be specialized for the synthesis of specific mrna, nucleolar stress by regulating ribosomal composition could specifically affect the translation of particular sets of mrna in developing and adult neurons. the activation of p53 and the negative feedback on mtor call in play metabolic and survival pathways executing cell fate decision, by inhibiting mitochondrial function and protein synthesis (fig. non - traditional nucleolar roles are depicted as concentric circles with the early events closer to the nucleolus and intersecting with the traditional nucleolar role in protein synthesis summary of the cellular and molecular alterations derived from nucleolar stress. the non - traditional nucleolar roles are depicted as concentric circles with the early events closer to the nucleolus and intersecting with the traditional nucleolar role in protein synthesis the tif - ia - based models will allow exploring initial adaptive mechanisms to stress conditions and by indirectly interfering with p53 turnover in specific cells can be used to gain understanding of context - dependent p53 functions. some attempts have been reported to overexpress p53 in a constitutive and/or inducible way by the generation of transgenic mice, but engineered mice having high p53 activity in specific cell types are not available because of the difficulty to generate mutant mice which reproduce the natural levels of proteins. ultimately, tif - ia mutant mice can be used to develop and validate therapeutic intervention and identify biomarkers associated with a specific degenerative pattern (table 1). modulation of the pol i transcriptional machinery is already proposed for anticancer therapies either by targeting specific components or the upstream pathways. inhibition of pol i in cancer cells has been shown to induce their apoptotic death. intriguingly, the tif - ia mutant mice have been already useful for testing the neuroprotective role of the phosphatase pten in dopaminergic neurons, supporting their possible use for evaluating new therapeutic approaches.table 1potential applications of the different types of tif - ia mutant mice for the investigation of features linked to nucleolar stresstype of mutationphenotypeapplicationglobal knock out embryonic lethality (e8.5) ; growth deficitsstudy of early embryonic compensatory mechanismsembryonic neural progenitors anencephalia at birth ; impaired neurogenesis (e13.5)study of effects on early neuronal differentiationadult hippocampal neurons progressive but differential degeneration of adult hippocampal neuronsanalysis of changes of neuronal activity and protein synthesis before neuronal deathembryonic or adult dopaminergic neurons [23, 51]parkinsonism ; early mitochondrial dysfunction and mtor downregulationsearch for biomarkers ; neurorestorative / neuroprotective strategiesother neuronsunpublished observationsanalysis of context - specific responses to nucleolar stressoverexpression ; heterozygositynot determined yetrole of nucleolar stress as a disease modifier potential applications of the different types of tif - ia mutant mice for the investigation of features linked to nucleolar stress increasing evidence point to a multifactorial basis of neurodegenerative disease onset and progression. perhaps there is time to bridge the gap between the reductionist approach focused on particular molecules involved in the pathology of a particular disease. the research for identifying common molecular checkpoints as possible new targets for pharmacotherapy seems necessary. despite the total market of 2.2 billion usd estimated for anti - pd drugs major pharmaceutical companies discouraged by the relatively little progresses announced cuts to brain disorder research in favor of sequencing projects to identify genetic risk factors. investing more efforts in defining the toxic species causing rrna transcription failure and the molecular alterations of nucleolar stress in specific cellular contexts will shed new light onto the mechanisms underlying differential vulnerability to stress conditions and indicate potential targets to manipulate them. otherwise there will always be a missing piece in our understanding of neurological disorders and in our way to cure them. | nucleoli are the sites where synthesis of rrna and ribosomal assembly take place. along with these traditional roles, the nucleolus controls cellular physiology and homeostasis. the cellular and molecular alterations associated with impaired nucleolar activity (nucleolar stress) have just started to be systematically explored in the nervous system taking advantage of newly available animal models lacking rrna synthesis in specific neurons. these studies showed that nucleolar function is necessary for neuronal survival and that its modality of action differs between and within cell types. nucleolar function is also crucial in pathology as it controls mitochondrial activity and critical stress signaling pathways mimicking hallmarks of human neurodegenerative diseases. this mini - review will focus on the modes of action of nucleolar stress and discuss how the manipulation of nucleolar activity might underscore novel strategies to extend neuronal function and survival. |
bone is the second most transplanted tissue after blood and is used to provide support, fill voids, and enhance biologic repair of skeletal defects. autologous bone provides the optimal source, but the harvest has considerable limitations, including donor - site morbidity, inappropriate form, and lack of sufficient quantities in procedures requiring large amounts of graft [1, 12 ]. this is of special concern in revision surgery of failed total hip and knee prostheses, and in limb salvaging resection of musculoskeletal tumors ; cadaver allograft is the only alternative. generally, bone allografting is performed without tissue matching between donors and recipients, but in processing procedures, blood, bone marrow, and soft tissue are removed and freezing and thawing of the donor bone reduces the antigenicity. antibodies against human leukocyte antigen (hla) are induced by mismatched bone transplants [10, 30, 32 ], but it remains unknown whether donor - specific hla sensitization influences the incorporation of bone allografts and their subsequent biologic performance. for example, strong. reported an increase in hla antibody sensitization from 39% to 67% associated with bone allografts, but they were unsure if there was any associated effect on allograft incorporation. in general, the rate of failure with allograft reconstruction owing to complications has been reported to range between 15% and 25% [3, 4, 6, 7, 9, 14, 15, 22 ]. however, results vary to some extent with the type of graft, anatomic site, and stage of the lesion, ie, the complexity of the surgery and the relative necessity for chemotherapy. the most important factors influencing graft survival are recurrence, infection, nonunion, and fracture. reported long - term results of allograft replacement in the management of bone tumors and found an infection rate of 11%, nonunion rate of 17% and fracture rate of 19%. mankin and hornicek reviewed 144 patients with giant cell tumors who had resection and implantation of cadaveric allografts from 1971 to 2001. most procedures were done in the distal femur, proximal tibia, proximal femur, and proximal humerus. allograft fracture occurred in 30 (21%) of the 144 patients, nonunion occurred in 12 (8%), and infection occurred in 12 (8%). reported three infections, 12 fractures, and nine nonunion treatments in a 10-year followup of 35 grafts. ultimately, six grafts failed, with infection and length of resection as predisposing factors. reviewed 59 consecutive patients after segmental resection in 52 for malignant bone tumors and in seven for benign aggressive bone tumors. infection, nonunion, and fracture rates were 5%, 9%, and 7%, respectively. processed frozen bone allografts lack viable donor cells and may trigger an indirect pathway of alloantigen recognition in the recipient. the indirect pathway involves presentation of processed allogeneic hla shed from foreign cells through cell necrosis and apoptosis. recipient antigen presenting cells (apc) present the processed peptides in the context of self - hla class ii to mhc class ii restricted cd4 + t cells [19, 25 ]. this type of recognition may induce either a chronic type of rejection or an immunologic state of tolerance to grafted antigens that can not be measured with human leukocyte blood tests. a chronic type of host - antigraft response may result in late deterioration of graft function characterized by inflammation and interstitial fibrosis. clinical studies have some disadvantages when studying hla sensitization to bone allografts, such as blood transfusions during surgery and the variability of the grafts in terms of processing, implantation, fixation techniques, fixation quality, complications, soft tissue envelope, periosteal preservation, supplemental bone grafting at the junctions, anatomic distribution, and use of radiation or chemotherapy. also, tissue injury in major surgery leads to the release of endogenous damage - associated molecular patterns that activate the innate immune cells, and one study suggests antibodies to degraded bone soluble proteins may develop in patients with osteoarthritis. therefore, we asked whether isolated mismatch for mhc antigens of deep frozen bone allografts in the long - term causes (1) immune reactions, and whether these reactions have any effect on (2) morphologic features of the graft, (3) radiographic graft healing, and (4) graft strength. for the experiment we obtained 24 pvg rats that were operated on under general anesthesia (hypnorm / dormicum, vetapharma, sheburn, leeds, england) (fig. 1). using an anterior incision, we exposed the proximal part of either the right or left tibia and carefully elevated the muscles. we then made two osteotomies at the shaft of the bone, 8 mm and 16 mm from the knee, using a fine - toothed circular saw blade mounted on an electric drill, and removed the segment. by random selection, the rats received a transplant of either a deep frozen syngeneic segment (pvg bone) or with a deep frozen allogeneic segment (pvg.1u bone), from the bone bank. we inserted a 0.8-mm steel pin (cannula), press fit, for intramedullary stabilization. we achieved fixation without radiographic control, but confirmed proper pin placement using radiographs taken at the end of the experiment. the strength of deep frozen autogenous grafts in this model attained approximately 70% of intact bone at 16 weeks. we therefore assumed that with a followup of 6 months the grafts would have attained the strength of the intact bone. with a difference between the two experimental groups of 15% as clinically interesting, six rats in each group for biomechanical testing would have a power of 80% with a two - tailed alpha of 0.05. one year before the experiments, six pvg.1u (rt1) and six pvg (rt1) rats (harlan uk limited, shaw s farm, blackthorn, bicester, england) were sacrificed and both tibial bones were excised for the grafts. to make the grafts we made two osteotomies at the shaft of the bones, 8 mm and 16 mm from the tibial plateau, using a fine - toothed circular saw blade mounted on an electric drill. by careful steel burring to the endosteal cortex, we emptied the bone segments of cancellous bone and bone marrow. we then sealed and froze the 24 bone segments at 80c in our bone bank system. the pvg.1u and pvg rats are identical except for the major histocompatibility complex (mhc of u haplotype versus mhc of c haplotype), where pvg.1u rats have their mhc (rt1) complex derived from the ao strain. therefore, an isolated effect of mhc mismatches (class i, class ii, and other polymorphic antigens in the mhc) on graft survival and other biologic parameters can be monitored. the experiment conformed to the norwegian council of animal research code for the care and use for experimental purposes. we subcutaneously injected buprenorphine (temgesic ; reckitt & benckiser, slough, uk), 0.05 mg per kilo, twice daily for the first 3 postoperative days for analgesia, and observed the rats daily for limb function and signs of improper healing., we sacrificed the animals with pentobarbital (s produksjonslaboratorium, s, norway), 100 mg / kg (0.1 ml/100 g) intraperitoneally (ie, 0.35 ml). postoperatively at the time of grafting and at termination of the experiments after 6 months, we obtained blood via a cannula from the tail vein of five rats from each group, and blood serum was examined for antibodies against mhc antigens. as described previously, we measured the antibody response in individual rats. in short, we incubated rat lymphoid cells, yb2/0, and expressing mhc - i (rt1 class i) antigens of rtiu haplotype with serum dilutions from the transplanted rats at 4c for 30 minutes, washed, then incubated with a fitc - labeled secondary goat anti - rat igg heavy + light chain (jackson immunoresearch laboratories, west grove, pa, usa) for another 30 minutes before washing again. we measured the fluorescence intensity in a facscalibur (becton dickinson, san jose, ca, usa), with gates set to exclude dead cells. in this test system, substantial antibody synthesis can be detected only when the peak fluorescence intensity has shifted so much to the right that there is only partly or no overlap between the two sets of curves. to show how an antibody response manifests itself in this system, we also included a standard mab (nr5/10) against mhc class i of the rt1u haplotype (positive control). we confirmed the validity of this method in a clear shift of the histogram to the right as evaluated by two of us (hs and br). the transplanted bones of these five animals of each group then were harvested and fixed by immersion in 4% phosphate - buffered formaldehyde immediately after sacrifice, decalcified for 3 weeks in 7% ethylenediaminetetraacetic acid (edta), and embedded in paraffin according to a routine protocol. sections were coded to blind the observer regarding the experimental group from which the section emanated. we used three to five hematoxylin and eosin - stained sections, including the central marrow area, to evaluate the tissue. one of us (fpr) used semiquantitative scoring with a three - grade scale (0, +, + +) to grade cortical integrity, identifiable remnants of graft, marrow fibrosis, and inflammation. the parameters were scored as follows : (1) cortical integrity = + one cortex intact, + + both cortices intact ; (2) identifiable remnants of graft = + remnants observed in less than 50% of the graft area, + + remnants observed in greater than 50% of graft area ; (3) marrow fibrosis = + fibrous tissue / collagen fibers in greater than 5% of the marrow area, + + fibrous tissue / collagen fibers in greater than 20% of the marrow area ; (4) inflammation = + one focus with more than 20 leukocytes in a high power field of vision, + + two or more such foci (fig. 2). the grading was performed twice by the same pathologist (fpr) at in interval greater than 1 month, and the reproducibility of the semiquantitative scoring was 90% (kappa 0.825).fig. 2a c(a) a low - power photomicrograph is shown that includes the area of the graft, a bone with allogeneic graft on the left, and a syngeneic graft on the right. both bones show + + bone continuity (stain, hematoxylin & eosin). (b) a medium - power photomicrograph shows an area with remnant of the graft outlined with arrows. the micrograph is from a bone with + + remnants of implant (stain, hematoxylin & eosin). (c) another medium - power photomicrograph shows an area of bone marrow with dead bone (rounded structure in the lower middle of the photomicrograph) surrounded by inflammatory infiltrate and fibrosis. this is from a + inflammation bone (stain, hematoxylin & eosin). (a) a low - power photomicrograph is shown that includes the area of the graft, a bone with allogeneic graft on the left, and a syngeneic graft on the right. both bones show + + bone continuity (stain, hematoxylin & eosin). scale bar = 1.5 mm. (b) a medium - power photomicrograph shows an area with remnant of the graft outlined with arrows. the micrograph is from a bone with + + remnants of implant (stain, hematoxylin & eosin) (c) another medium - power photomicrograph shows an area of bone marrow with dead bone (rounded structure in the lower middle of the photomicrograph) surrounded by inflammatory infiltrate and fibrosis. this is from a + inflammation bone (stain, hematoxylin & eosin) we harvested their transplanted and their intact legs, and performed radiography of the transplanted leg with a standard clinical digital system (siemens axiom aristos ; siemens ag, mnchen, germany) after removal of the intramedullary pin. the xray tube settings were 46 kv, 1.0 ma per second, and focus - to - film - distance (source - to - image receptor distance) of 115 cm. two of us (or, hs) assessed all radiographs for healing using the following criteria : (1) complete healing, defined as bone bridging without gaps, (2) incomplete healing, defined as a radiolucent gap with some areas of bridging bone trabeculae, and (3) no healing, defined as a continuous radiolucent gap at the osteotomy site with no bridging bone trabeculae. after radiographic examinations, we examined the bone mineral content (bmc) and density (bmd) of the transplanted and contralateral intact bones using dual energy radiographic absorptiometry (dexa). dexa was performed on a ge lunar piximus (lunar corporation, madison, wi, usa) with a tube current of 400 a. the frequency of the scanning unit was 50/60 hz, and the xray tube had a focal spot size of 0.25 mm by 0.25 mm with a coefficient variation of 1%. after scanning the whole tibia, we centralized a region of interest at the graft. thereafter, we tested the composite (transplanted) and the contralateral bones for torsional strength (mts 858 ; mts systems corporation, eden prairie, mn, usa). we fixed the tibial ends with a clamp and ran the instrument at a constant rate of 0.08 radii per second. the load values were recorded and transformed to a load deformation curve (origin 7.5 ; originlab corporation, northampton, ma, usa). we defined the strength as the torsion necessary to produce fracture of the composite bone. we determined the torsion stiffness from the slope of the linear elastic part of the curve and defined the energy as the energy absorbed during loading to fracture. data for biomechanical parameters and bmc and bmd are presented as mean and standard deviation of the mean. statistical analysis was performed with spss 16.0 (spss inc, chicago, usa.) none of the five allotransplanted rats exhibited any detectable antibody response as the facs distribution curves were identical to the controls (fig. 3).fig. 3a cthe facs plots show no detectable antibody production in pvg rats transplanted with either frozen bone from pvg (syngeneic) or pvg.1u (allogeneic mhc mismatched) rats. the plots show (a) fitc labeled secondary ab alone (gray filled), mab ox6 against mhc - ii (thin line) and a standard antibody against mhc - i of the rt1u haplotype mab nr5/10 (thick line), (b) counts for three pretransplanted rats, and (c) from two representative rats of each group (syngeneic [gray filled and stapled line ] and allogeneic [thin and thick lines ]). the facs plots show no detectable antibody production in pvg rats transplanted with either frozen bone from pvg (syngeneic) or pvg.1u (allogeneic mhc mismatched) rats. the plots show (a) fitc labeled secondary ab alone (gray filled), mab ox6 against mhc - ii (thin line) and a standard antibody against mhc - i of the rt1u haplotype mab nr5/10 (thick line), (b) counts for three pretransplanted rats, and (c) from two representative rats of each group (syngeneic [gray filled and stapled line ] and allogeneic [thin and thick lines ]). by histologic examinations, all samples showed intact cortical bone in the transplanted segment and adjacent bone, except for one focal defect in one of the animals in the allogeneic group (fig. we observed remnants of the graft in greater than 50% of the transplanted segment in four of five bones in each group (fig. one animal in each group showed focal inflammation surrounding remnants of devitalized bone along with focal fibrosis (fig. 2c).table 1semiquantitative assessment of histologic features in 10 bonesanimal type / numberintact cortexrest of implantmarrow fibrosisinflammationallogeneic 1++++00allogeneic 2++++00allogeneic 3+++00allogeneic 4++++00allogeneic 5+++++syngeneic 1++++00syngeneic 2++++00syngeneic 3++++00syngeneic 4++++00syngeneic 5+++++ intact cortex = + one cortex intact, + + both cortices intact ; rest of implant = + remnants observed in less than 50% of the implant area, + + remnants observed in greater than 50% of implant area ; marrow fibrosis = + fibrous tissue / collagen fibers in greater than 5% of the marrow area, + + fibrous tissue / collagen fibers in greater than 20% of the marrow area ; inflammation = + one focus with greater than 20 leukocytes in a high power field of vision, + + two or more such foci. semiquantitative assessment of histologic features in 10 bones intact cortex = + one cortex intact, + + both cortices intact ; rest of implant = + remnants observed in less than 50% of the implant area, + + remnants observed in greater than 50% of implant area ; marrow fibrosis = + fibrous tissue / collagen fibers in greater than 5% of the marrow area, + + fibrous tissue / collagen fibers in greater than 20% of the marrow area ; inflammation = + one focus with greater than 20 leukocytes in a high power field of vision, + + two or more such foci. complete radiographic healing of the grafted segment occurred in all transplanted bones (fig. 4a bthe radiographs show fully incorporated (a) frozen allogeneic and (b) syngeneic grafts (between arrows) after 6 months. the radiographs show fully incorporated (a) frozen allogeneic and (b) syngeneic grafts (between arrows) after 6 months. we observed no differences in either bmc or bmd between the two groups (table 2), and bone mineralization was similar to that in the intact bones (table 2). there were no differences in torsion strength between the groups, nor were there any differences in either stiffness or fracture energy (table 3). furthermore, the biomechanical parameters of the transplanted bones were similar to those of the intact bones (table 3).table 2bmc and bmd of the transplanted and intact bonesmineralizationsyngeneicallogeneicintactp valuebmc (10 g)41.8 2.1 44.6 12.235.6 3.40.364bmd (10 g / cm)19.9 2.219.5 4.217.4 0.70.473bmc = bone mineral content ; bmd = bone mineral density ; mean standard deviation.table 3biomechanics of the transplanted and intact bonesbiomechanicssyngeneicallogenicintactp valuemoment (nm 10)13.6 2.60 12.0 2.5414.0 1.780.295stiffness (nm / degree 10)3.06 0.642.55 0.603.06 0.500.229energy (nm degree 10)66.7 25.849.3 13.650.5 10.70.185 mean standard deviation. bmc and bmd of the transplanted and intact bones bmc = bone mineral content ; bmd = bone mineral density ; mean standard deviation. biomechanics of the transplanted and intact bones mean standard deviation. bone allografting is being used increasingly in patients for bone reconstruction. however, on implantation of allogeneic bone, the host is expected to experience an intricate immune response. deep freezing may reduce the immunity, but also may alter the properties of the graft. therefore, we asked whether isolated mismatch for mhc antigens of deep frozen bone allografts in the long - term causes (1) immune reactions, and whether these reactions have any effect on (2) morphologic features of the graft, (3) radiographic graft healing, and (4) graft strength. first, the results of experimental studies generally must be transferred to human clinical practice with caution, especially in this case, based on the magnitude of the difference in graft size and the inherent differences in the immune systems. however, the rats in this study were genetically identical except for the mhc region, and rats, in general, are biologically similar to humans when it comes to the biology of bone healing. second, we used intercalary allografts, and our findings may be restricted to these kinds of allografts. third, the reproducibility of semiquantitative histologic scoring systems is reportedly low, but we believe semiquantitative scoring may be reasonable with a high degree of reproducibility if the parameters are kept basic and the grades are limited and well defined.. however, our data were normally distributed with a power for the null hypothesis of 83% with a two - tailed alpha of 0.05. fifth, a torsion test is not osteotomy site - specific, as it determines the weakest point of the bone rather than measuring the strength at the osteotomy sites. however, this is also why torsion tests usually are applied to bone segments and grafts. in rats, the rt1 gene region on chromosome 20 encodes mhc classes i and ii molecules that are highly polymorphic and provoke strong alloimmune responses to organ transplantation, leading to their rejection. based on differences solely in the mhc region, strong antibody responses to allogeneic mhc - i molecules after mhc mismatched transplantation of fresh bone in this rat model have been observed. direct recognition occurs when recipient th lymphocytes recognize mhc - ii antigens on intact apc in the allograft. animal and human studies show that the cellular components of bone express sufficient mhc molecules to alert the host immune system with activation of cytotoxic t cells, leading to destruction of cells in the graft [8, 18, 26 ]. indirect recognition occurs when recipient apc ingests donor mhc antigens from broken down cells and present peptides of donor mhc in the groove of the recipient s own mhc molecules. this latter scenario may be predominant when donor tissue is damaged by freezing, and releases peptides that can lead to a more chronic state of inflammation in the graft. to obtain a sufficient igg response against mhc - i or ii molecules in the current study, none of the five allotransplanted rats exhibited any detectable antibody response ; the facs - distribution curves at 6 months were identical to the controls. this implies that mhc - derived antigens from the donor were not present in quantities that provoked a measurable antibody response. however, we can not formally exclude that low concentrations of donor antigens were taken up by host macrophages or antigen presenting cells, and could provoke an indirect antigen presentation to host t cells. some experimental studies reported an antibody response after cryopreserved bone grafts [2, 19, 27, 29 ], but such reactions usually are weak. bos. performed iliac crest grafting of frozen bone with minor and major histocompatibility differences. grafting in the face of a minor histocompatibility difference failed to generate second - set skin - graft reactivity rejection, whereas grafting across a major histocompatibility difference provided in vivo evidence of accelerated skin - graft rejection and in vitro evidence of cytolytic cells and antibodies. when grafting of frozen bone with mhc mismatch was performed without second - set skin grafting, antibodies were seen between days 30 and 50 with a peak at day 40, but never achieved high activity levels. there are some factors in experimental design that can explain this difference to our observations. first, bos. performed iliac crest grafting with a high bone marrow concentration. it is well recognized that bone marrow cells are strongly immunogenic. in our experiments on intercalary bone, we removed bone marrow content as much as possible in line with clinical practice. second, in the experiments of bos. the grafts were frozen to 20 for a minimum of 10 days, whereas our grafts were deep frozen to 80 for a year. had an observation period of 50 days. at this time the antibody response declined, and they stated that longer followup studies would be highly desirable. although bos. found immune activity, histologic evaluation at 50 days showed that frozen bone fared the same regardless of the genetic relationship, and neither was there any discernible difference in the scores of frozen grafts that were mismatched or syngeneic. our histologic results are in agreement with these observations, and these results may explain clinical difficulties in correlating hla mismatches of frozen bone allografts with performance and failures. it is assumed that t cells primed by the indirect pathway may play the dominant role in the chronic state of inflammation and rejection of allografts. histologic features of graft inflammation are well established as infiltration with large numbers of lymphocytes and macrophages, and a fibroproliferative process. in a study of osteochondral allografts in dogs, the authors found that the ingrowths of fibrous connective tissue were directly proportional to the immunogenicity of the bone grafts. it also was reported that revascularization was slow in mismatched grafts. in our study, the frozen allogeneic grafts did as well as the syngeneic grafts by histologic evaluations. the grafts consisted of a mixture of living and dead bone tissue, but complete healing at the osteotomy sites occurred in all cases. this reflects that the process of creeping substitution of grafts by the ingrowths of vessels is slow, and incorporation may be limited. one rat in each group showed localized inflammation and marrow fibrosis related to small dead bone fragments, most likely emanating from the grafts. one may speculate that these fragments represent a sign of early fatigue microdamage, heralding a later graft fracture. but along with the antibody tests, our histologic data indicate that there was no indirect antigen presentation to the host t cells that might provoke any inflammation or affect the healing of the graft. bone graft incorporation is a complicated process with multiple variables influencing rate, pattern, and completeness ; one method of evaluation would not adequately show the various aspects. our radiographic examinations were in agreement with the histologic findings and showed complete healing by complete bone bridging without gaps in all grafts. however, successful incorporation of bone grafts should result in a composite that can bear physiologic loads, and neither radiographic nor histologic methods can measure the biomechanical competence of grafted bones. measurements of bone mineralization provide a valuable estimation of bone quantity and quality that correlates to biomechanical properties, but the technique can not accurately predict biomechanical competence. however, our data on bone mineralization and biomechanical testing showed that, at 6 months, the biomechanical constructions were equivalent to intact bone ; either we used deep frozen grafts matched or mismatched for mhc antigens. few studies on bone grafting report biomechanical data, but our findings agree with observations 11 months after fresh and frozen tissue antigen matched and mismatched osteochondral allografts in dogs. our observations suggest mismatches for mhc antigens are not serious obstacles to transplantation of long - term frozen - bone intercalary allografts. if these results can be translated to the human setting, hla matching of the bone donor with the recipient does not, in itself, improve the outcome of the transplantation. | backgroundthe use of bone grafting in orthopaedic surgery has increased dramatically in recent years. however, the degree to which immune responses are important for the survival of the allograft is not fully understood. in particular it remains unclear whether differences in the major histocompatibility complex (mhc) influence incorporation of bone allografts and their subsequent biologic performance.questions/purposestherefore, we asked whether isolated mismatch for mhc antigens of deep frozen bone allografts in the long - term causes (1) immune reactions, and whether these reactions have any effect on (2) morphologic features of the graft, (3) radiographic graft healing, and (4) graft strength.methodswe used an established orthotopic tibial segment transplantation technique that allows determination of mechanical strength, histologic evaluation, and immune responses. tibial segments that had been deep - frozen at 80c for 1 year were transplanted into 24 pvg (rt1c) rats from either 12 syngeneic donors or 12 mhc congenic donors pvg.1u (rt1u). we determined immune responses using an indirect coombs reaction and determined graft healing radiographically and mechanically after 6 months.resultswe detected no alloantibody production to graft mhc - i antigens, and found no differences between syngeneic and mhc mismatched grafts in terms of remodeling with host bone, graft healing, and mechanical strength.conclusionsmismatches for mhc antigens do not seem to play a decisive role in healing of long - term, deep - frozen bone allografts. |
the primary cohort of animals whose data are the basis for this study has been described in detail recently. it consists of 51 rhesus macaque monkeys (macaca mulatta), 40 female and 11 male, ranging in age from 1.2 to 22.6 years (median, 9.6 years). data from a secondary group of n = 4 adult rhesus macaques with unilateral orbital optic nerve transection also were included for analytical comparison to experimental glaucoma (eg). all procedures were performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih ; bethesda, md, usa) and were approved and monitored by the institutional animal care and use committee (iacuc) at legacy health (usda license 92-r-0002 and olaw assurance a3234 - 01). all experimental methods and animal care procedures also adhered to the association for research in vision and ophthalmology 's (arvo) statement for the use of animals in ophthalmic and vision research. each animal had a minimum of three weekly baseline spectral - domain optical coherence tomography (sdoct) imaging sessions, which are described below. argon laser photocoagulation then was applied to the trabecular meshwork of one eye of each animal to induce chronic elevation of iop. initially, 180 of the trabecular meshwork was treated in one session, then the remaining 180 was treated in a second session approximately 2 weeks later. if necessary, laser treatments were repeated in subsequent weeks (limited to a 90 sector) until an iop elevation was first noted or if the initial postlaser iop had returned to normal levels. after initiation of laser photocoagulation, sdoct imaging was repeated approximately every 2 weeks until euthanasia. the stage of eg at euthanasia was predetermined for each animal based on the primary study to which it was assigned. because there were four such primary studies with differing targets for postmortem histopathology, the combined cohort provided a relatively wide range of damage for the cross - sectional analysis of this study. all experimental procedures began with induction of general anesthesia using ketamine (1025 mg / kg intramuscularly [i m ]) in combination with either xylazine (0.81.5 mg / kg i m) or midazolam (0.2 mg / kg i m), along with a single injection of atropine sulfate (0.05 mg / kg i m). animals then were intubated with an endotracheal tube to breathe a mixture of 100% oxygen, air and 1% to 2% isoflurane gas to maintain anesthesia to effect and oxyhemoglobin saturation as close to 100% as possible. intravenous fluids (lactated ringer 's solution, 1020 ml / kg / h) were administered via the saphenous vein. vital signs were monitored throughout and recorded every 10 to 15 minutes, including heart rate, blood pressure, arterial oxyhemoglobin saturation, end tidal co2, and body temperature. body temperature was maintained at 37c, heart rate above 75 beats per minute, and oxygen saturation above 95%. intraocular pressure was measured in both eyes at the start of every session using a tonopen xl (reichert technologies, inc., depew, ny, usa). the value recorded for each eye was the average of three successive measurements. all sdoct scans were acquired using a spectralis instrument (heidelberg engineering, gmbh, heidelberg, germany) 30 minutes after iop was manometrically stabilized to 10 mm hg. this is important to minimize elastic components of deformation that are known to exert a greater effect on the onh than on peripapillary rnfl thickness. a clear, rigid gas permeable contact lens filled with 0.5% carboxymethylcellulose solution was placed over the apex of each cornea. spectral - domain oct scans recorded at each session included an 80-radial b - scan pattern centered on the onh (30 wide, 1536 a - scans / b - scan, figs. 1a, 1b) and a peripapillary circular b - scan with 12 diameter (1536 a - scans, fig. nine to 16 individual sweeps were averaged in real time to form each b - scan. at the first baseline imaging session, all follow - up scans were acquired at the same location as baseline using the instrument 's automatic active eye tracking software. the left column shows baseline data, the right column shows final follow - up data. the inset shows the b - scan location indicated by the bold green line overlaid onto the infrared confocal scanning laser ophthalmoscopy (cslo) reflectance image. structures delineated in each radial b - scan include the ilm (yellow) and bmo points (red). the green segments connecting bmo points to the ilm represent the pair of mrw measurements made in each radial b - scan. (d) derivation of mra from the 160 radial trapezoidal sectors of each onh (see methods text for details). note deformation of the onh apparent in the right column for mrw / mra, including a deeper cup and thinner rim. in this eg eye, global average mra decreased from 1.16 mm at baseline to 0.63 mm at the final time point (45.6%). (e) segmentation of peripapillary circular b - scans to obtain the parameter rnfla are shown for the same eye and time points, ilm (red), and posterior rnfl boundary (green). retinal nerve fiber layer thickness decreased from 113.8 m at baseline to 96.1 m at the final time point ; rnfla decreased from 1.07 mm at baseline to 0.90 mm at the final time point (15.5%). (f) retinal nerve fiber layer area represented by the gold colored ribbon in projected 3d image from baseline (left) and final time point (right). the much larger decrease in mra than rnfla suggests substantial transverse compression of axons (and possibly also astrocytes) at the onh rim. this can be appreciated by comparison of the images inset at the bottom of each column, which are the infrared cslo reflectance image painted onto the ilm surface at each time point ; the rnfl appears stretched over the onh rim at the final time point. image segmentation was performed manually offline using custom software (atl 3d suite). for onh radial scans, two image features in particular required segmentation for the quantitative measurements used in this study : the inner limiting membrane (ilm ; yellow lines in figs. ac) and the pair of bruch 's membrane opening points (bmo ; red dots in figs. figures 1a and 1b, respectively, show a horizontally oriented b - scan and a vertically oriented b - scan through the onh at the first baseline session (left column) and at the final time point (right column) for a single representative eg eye (i.e., whose data are close to the average effect among the entire group of n = 51). each of the green line segments in figures 1a to 1c represents the minimum rim width (mrw) vector defined as the shortest distance from the bmo point to the ilm segmentation within the plane of the b - scan. the onh parameter minimum rim area (mra ; fig. 1d) was derived in a similar manner as mrw ; that is, two measurements for each radial b - scan, as described previously in detail. in brief, the mra for each onh at each imaging session was represented by the sum of the areas of 160 contiguous individual trapezoids whereby the base of each trapezoid is centered on the corresponding bmo point, and the height of each trapezoid is defined as the distance between the bmo point and the ilm segmentation that minimizes the area of the trapezoid (fig. two image features of each peripapillary circular b - scan required segmentation for this study : the ilm and the posterior boundary of the rnfl (fig. 1f) was derived from the circular b - scan by measuring the distance between the ilm and posterior rnfl and multiplying the average thickness (of 1536 a - line samples) by the scan circumference (9.37 mm, which assumes a visual angle for the macaque eye of 247.7 m / deg, the same transverse scaling applied for mra measurements). in total, there were 605 onh sdoct volumes analyzed in this study with a median scan quality score of 29.8 db and interquartile range of 27.6 to 32.0 db ; the lowest scan quality score was 17.6 db, which was the only value below 20 db. the peripapillary circle scans had a median scan quality score of 31.3 db and interquartile range of 27.9 to 34.3 db ; fewer than 1% had a score below 20 db, the lowest of which was 13.9 db. statistical analysis was performed using a commercial software package (prism 5 ; graphpad software, inc., la jolla, ca, usa). for each instance of reported results, the specific statistical test applied and the corresponding p value are included. the fundamental null hypothesis forming the basis for this study states that the cross - sectional area of peripapillary rnfl tissue will change by exactly the same amount as the cross - sectional area of onh rim tissue if the only structural change contributing to each is loss of retinal ganglion cell axons (since fewer than 1.5% of retinal ganglion cells are located proximal to the location of the peripapillary rnfl scan). the primary cohort of animals whose data are the basis for this study has been described in detail recently. it consists of 51 rhesus macaque monkeys (macaca mulatta), 40 female and 11 male, ranging in age from 1.2 to 22.6 years (median, 9.6 years). data from a secondary group of n = 4 adult rhesus macaques with unilateral orbital optic nerve transection also were included for analytical comparison to experimental glaucoma (eg). all procedures were performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih ; bethesda, md, usa) and were approved and monitored by the institutional animal care and use committee (iacuc) at legacy health (usda license 92-r-0002 and olaw assurance a3234 - 01). all experimental methods and animal care procedures also adhered to the association for research in vision and ophthalmology 's (arvo) statement for the use of animals in ophthalmic and vision research. each animal had a minimum of three weekly baseline spectral - domain optical coherence tomography (sdoct) imaging sessions, which are described below. argon laser photocoagulation then was applied to the trabecular meshwork of one eye of each animal to induce chronic elevation of iop. initially, 180 of the trabecular meshwork was treated in one session, then the remaining 180 was treated in a second session approximately 2 weeks later. if necessary, laser treatments were repeated in subsequent weeks (limited to a 90 sector) until an iop elevation was first noted or if the initial postlaser iop had returned to normal levels. after initiation of laser photocoagulation, sdoct imaging was repeated approximately every 2 weeks until euthanasia. the stage of eg at euthanasia was predetermined for each animal based on the primary study to which it was assigned. because there were four such primary studies with differing targets for postmortem histopathology, the combined cohort provided a relatively wide range of damage for the cross - sectional analysis of this study. all experimental procedures began with induction of general anesthesia using ketamine (1025 mg / kg intramuscularly [i m ]) in combination with either xylazine (0.81.5 mg / kg i m) or midazolam (0.2 mg / kg i m), along with a single injection of atropine sulfate (0.05 mg / kg i m). animals then were intubated with an endotracheal tube to breathe a mixture of 100% oxygen, air and 1% to 2% isoflurane gas to maintain anesthesia to effect and oxyhemoglobin saturation as close to 100% as possible. intravenous fluids (lactated ringer 's solution, 1020 ml / kg / h) were administered via the saphenous vein. vital signs were monitored throughout and recorded every 10 to 15 minutes, including heart rate, blood pressure, arterial oxyhemoglobin saturation, end tidal co2, and body temperature. body temperature was maintained at 37c, heart rate above 75 beats per minute, and oxygen saturation above 95%. intraocular pressure was measured in both eyes at the start of every session using a tonopen xl (reichert technologies, inc., depew all sdoct scans were acquired using a spectralis instrument (heidelberg engineering, gmbh, heidelberg, germany) 30 minutes after iop was manometrically stabilized to 10 mm hg. this is important to minimize elastic components of deformation that are known to exert a greater effect on the onh than on peripapillary rnfl thickness. a clear, rigid gas permeable contact lens filled with 0.5% carboxymethylcellulose solution was placed over the apex of each cornea. spectral - domain oct scans recorded at each session included an 80-radial b - scan pattern centered on the onh (30 wide, 1536 a - scans / b - scan, figs. 1a, 1b) and a peripapillary circular b - scan with 12 diameter (1536 a - scans, fig. nine to 16 individual sweeps were averaged in real time to form each b - scan. at the first baseline imaging session, all follow - up scans were acquired at the same location as baseline using the instrument 's automatic active eye tracking software. the left column shows baseline data, the right column shows final follow - up data. the inset shows the b - scan location indicated by the bold green line overlaid onto the infrared confocal scanning laser ophthalmoscopy (cslo) reflectance image. structures delineated in each radial b - scan include the ilm (yellow) and bmo points (red). the green segments connecting bmo points to the ilm represent the pair of mrw measurements made in each radial b - scan. (d) derivation of mra from the 160 radial trapezoidal sectors of each onh (see methods text for details). note deformation of the onh apparent in the right column for mrw / mra, including a deeper cup and thinner rim. in this eg eye, global average mra decreased from 1.16 mm at baseline to 0.63 mm at the final time point (45.6%). (e) segmentation of peripapillary circular b - scans to obtain the parameter rnfla are shown for the same eye and time points, ilm (red), and posterior rnfl boundary (green). retinal nerve fiber layer thickness decreased from 113.8 m at baseline to 96.1 m at the final time point ; rnfla decreased from 1.07 mm at baseline to 0.90 mm at the final time point (15.5%). (f) retinal nerve fiber layer area represented by the gold colored ribbon in projected 3d image from baseline (left) and final time point (right). the much larger decrease in mra than rnfla suggests substantial transverse compression of axons (and possibly also astrocytes) at the onh rim. this can be appreciated by comparison of the images inset at the bottom of each column, which are the infrared cslo reflectance image painted onto the ilm surface at each time point ; the rnfl appears stretched over the onh rim at the final time point. image segmentation was performed manually offline using custom software (atl 3d suite). for onh radial scans, two image features in particular required segmentation for the quantitative measurements used in this study : the inner limiting membrane (ilm ; yellow lines in figs. ac) and the pair of bruch 's membrane opening points (bmo ; red dots in figs. figures 1a and 1b, respectively, show a horizontally oriented b - scan and a vertically oriented b - scan through the onh at the first baseline session (left column) and at the final time point (right column) for a single representative eg eye (i.e., whose data are close to the average effect among the entire group of n = 51). each of the green line segments in figures 1a to 1c represents the minimum rim width (mrw) vector defined as the shortest distance from the bmo point to the ilm segmentation within the plane of the b - scan. 1d) was derived in a similar manner as mrw ; that is, two measurements for each radial b - scan, as described previously in detail. in brief, the mra for each onh at each imaging session was represented by the sum of the areas of 160 contiguous individual trapezoids whereby the base of each trapezoid is centered on the corresponding bmo point, and the height of each trapezoid is defined as the distance between the bmo point and the ilm segmentation that minimizes the area of the trapezoid (fig. two image features of each peripapillary circular b - scan required segmentation for this study : the ilm and the posterior boundary of the rnfl (fig. 1f) was derived from the circular b - scan by measuring the distance between the ilm and posterior rnfl and multiplying the average thickness (of 1536 a - line samples) by the scan circumference (9.37 mm, which assumes a visual angle for the macaque eye of 247.7 m / deg, the same transverse scaling applied for mra measurements). in total, there were 605 onh sdoct volumes analyzed in this study with a median scan quality score of 29.8 db and interquartile range of 27.6 to 32.0 db ; the lowest scan quality score was 17.6 db, which was the only value below 20 db. the peripapillary circle scans had a median scan quality score of 31.3 db and interquartile range of 27.9 to 34.3 db ; fewer than 1% had a score below 20 db, the lowest of which was 13.9 db. statistical analysis was performed using a commercial software package (prism 5 ; graphpad software, inc., la jolla, ca, usa). for each instance of reported results, the specific statistical test applied and the corresponding p value are included. the fundamental null hypothesis forming the basis for this study states that the cross - sectional area of peripapillary rnfl tissue will change by exactly the same amount as the cross - sectional area of onh rim tissue if the only structural change contributing to each is loss of retinal ganglion cell axons (since fewer than 1.5% of retinal ganglion cells are located proximal to the location of the peripapillary rnfl scan). figure 1 provides an individual example of results for a representative eg eye selected because its results are close to the average observed across the whole group. for each figure (fig. f), the left side shows results from the first baseline session and the right side shows results from the final session. figure 1a shows one of the 80 radial b - scans through the onh (a section close to the horizontal) and figure 1b shows another section close to the vertical. the pair of green line segments in each b - scan represent the mrw measurements. these b - scans clearly show posterior deformation of the onh surface at the final time point. figure 1c shows a projection of the complete 3d set based on the segmentations of all 80 b - scans. at the final time point, several aspects of glaucomatous cupping are apparent in figure 1c, including substantial posterior deformation of the onh surface (shown by the change in the yellow ilm segmentation lines), thinning of the onh rim tissue as represented by the shortened mrw vectors, axial bowing of the bmo into a more saddle - shaped opening and a decrease in the mrw angle, which had become more acute (i.e., the green lines are oriented more toward the plane of the bmo). figure 1d shows the mra results, which also show signs of glaucomatous cupping, such as a reduced rim area with a corresponding increase in area, as well as reduced mra angle (the red - colored trapezoids representing the 160 mra measurements are oriented more toward the bmo plane at the final time point). figure 1e shows the peripapillary circular b - scan at the baseline and final time points unwrapped and splayed out in typical fashion, along with the segmentations of rnfl tissue used to measure the total rnfla. figure 1f shows a projected 3d image with this ribbon of rnfl tissue area represented as a gold band surrounding the onh mra. in this eg eye, rnfla had decreased from 1.07 mm at baseline to 0.90 mm at the final time point (15.5%). minimum rim area had decreased from 1.16 mm at baseline to 0.63 mm at the final time point (45.6%). the degree of glaucomatous onh deformation in this eg eye also can be appreciated in the more familiar clinical view presented as insets adjacent to the 3d projections in figure 1f. figure 2 graphically depicts important experimental parameters for the entire group of 51 nonhuman primates (nhps). the number of baseline oct imaging sessions per animal ranged from 3 to 11 (median, 5 ; fig. mean iop over the span of postlaser follow - up ranged from 10.4 to 31.0 mm hg in eg eyes (median, 19.6 mm hg ; fig. mean iop over the same period in the fellow control eyes ranged from 8.4 to 23.3 mm hg (median, 11.4 mm hg). the peak iop observed during the postlaser follow - up period ranged from 15.3 to 60.3 mm hg in eg eyes (median, 43.0 mm hg ; fig. 2c) and from 10.0 to 31.3 mm hg in fellow control eyes (median, 15.3 mm hg). the duration of postlaser follow - up ranged from 3 to 37 months (median, 7.9 months ; fig. total study duration ranged from 7 to 46 months (median, 13 months). the median time between the final imaging session and euthanasia was 5 days (range, 0 to 14 days ; fig. age at the end of the experiment ranged from 2 to 26 years (median, 11.0 years ; fig. frequency histograms show (a) the number of baseline oct imaging sessions per animal, (b) the postlaser mean iop in eg eyes, (c) the postlaser peak iop in eg eyes, (d) duration of follow - up (number of months between first laser and final oct imaging session), (e) number of days between final oct imaging session and euthanasia, (f) age at the end of the experiment. figure 3 shows the distribution of raw parameter values found at baseline (hatched boxes) and at the final imaging session for the entire group of n = 51 nhps. as predicted by the hypothesis, the total cross - sectional area of onh rim tissue was very close to the total cross - sectional area of peripapillary rnfl tissue (1.0 mm), though the population variance was larger for mra than for rnfla. figure 3 also shows that mra and rnfla parameter values are repeatable from baseline to the final session in control eyes. in control eyes, mra was 1.00 0.19 mm at baseline and 1.00 0.19 mm at the final session (p = 0.77, paired t - test). minimum rim area decreased in eg eyes from 1.00 0.19 mm at baseline to 0.63 0.21 mm at the final session (p < 0.0001). retinal nerve fiber layer area in control eyes was 0.95 0.09 mm at baseline and 0.95 0.10 mm at the final session (p = 0.96). retinal nerve fiber layer area decreased in eg eyes from 0.95 0.09 mm at baseline to 0.74 0.19 mm at the final session (p < 0.0001). longitudinal change in minimum rim area (mra, left) versus rnfl area (rnfla, right). n = 51) of raw parameter values in control eyes (ctl) and eyes with eg at baseline (bl, hatched) and at the final imaging session (mra for eg eyes in solid red, rnfla for eg eyes in solid gold to match fig. 1 color scheme). in figure 4 the magnitude of longitudinal mra change minimum rim area decreased by 36.4 20.6% in eg eyes, which was significantly more than the rnfla decrease (21.7 19.4%, p < 0.0001, paired t - test). although these distributions of change passed a formal normality test in both cases (p = 0.36 for mra, p = 0.06 for rnfla, d'agostino and pearson omnibus test), the latter was borderline for rnfla, so the data also were compared nonparametrically, which also found that the mra change (median decrease of 31%) was significantly greater than the rnfla change (median decrease of 17%, p < 0.0001, wilcoxon matched - pairs signed rank test). notwithstanding that there was a significant difference between the magnitude of mra loss versus rnfla loss, longitudinal change for these two parameters was strongly correlated (pearson r = 0.84 ; 95% confidence interval, 0.740.91 ; p < 0.0001). longitudinal change in mra (left) versus rnfla (right) expressed relative to baseline values. box plots represent the distribution (median, interquartile range and extremes, n = 51) of changes in parameter values at the final imaging session expressed as a percentage of the baseline average value for each eye. control eyes (ctl) shown in gray and eyes with eg are shown in red for mra and in gold for rnfla. the group average (sd) and median values of change are listed for the eg eyes. in figure 5a the magnitude of longitudinal rnfla change at the final session is plotted against the magnitude of mra change for eg eyes (in red) and fellow control eyes (in blue). the vertical dashed gray line represents the lower limit of the 95% range of test retest repeatability for mra at baseline (12%) while the horizontal dashed gray line represents the same for rnfla (7.0%). none of the control eyes exhibit longitudinal change at the final session beyond the limit for either parameter, thus the specificity of mra and rnfla is 100%. among the group of n = 51 eg eyes, 45 (88%) had significantly decreased mra while only 38 (75%) had significantly decreased rnfla (p = 0.04, z - test to compare proportions). the dashed black diagonal line represents the 1:1 locus where all points would plot if the mra change equaled the rnfla change in each eye. the eg eyes, however, are shifted to the left of the 1:1 line indicating a greater degree of mra loss compared to rnfla loss throughout the wide range of damage studied in this group. the solid red line represents the result of deming regression applied to the eg eye data ; the slope is 1.01 (not significantly different from 1.0) and the x - intercept (where y = 0) is 15%, indicating that mra change is approximately 15% worse than rnfla change across nearly the entire dynamic range of these parameters. the differential between mra change and rnfla change was unrelated to age (r < 0.01, p = 0.72), sex (r = 0.01, p = 0.41), mean iop (r = 0.06, p = 0.08), or peak iop (r = 0.02, p = 0.31) when these variables were each considered alone or in combination in multiple linear regression models. collectively, the data analysis presented in figures 3 to 5 robustly support rejection of the null hypothesis of this study. longitudinal change in mra is greater than rnfla in eg, but the opposite pattern occurs after surgical retrobulbar optic nerve transection. (a) scatter plot showing the longitudinal change in rnfla versus the longitudinal change in mra for n = 51 eg eyes (red symbols) and their fellow control eyes (blue symbols). the dashed black diagonal line represents the 1:1 locus where all points would plot if the mra change equaled the rnfla change in each eye. the solid red line represents the result of deming regression (rnfla change = 1.01 mra change + 0.15). longitudinal change for these two parameters was strongly correlated (pearson r = 0.84, p < 0.0001), but the total cross - sectional area of the onh rim was 15% less than the total cross - sectional area of the peripapillary rnfl tissue throughout the range of eg severity studied. the vertical dashed gray line represents the lower limit of the 95% range of test retest repeatability for mra at baseline, while the horizontal dashed gray line represents the same for rnfla. (b) scatter plot showing results of the same analysis for n = 4 nhp 50.3 1.0 days after unilateral optic nerve transection ; in contrast to eg, this group of eyes shows greater loss of peripapillary rnfla than loss of onh mra (p = 0.0005). we performed the same analysis on data from four nhp obtained 50.3 1.0 days after unilateral optic nerve transection (ont ; see recent reports for detailed description of cohort and ont procedures). figure 5b shows that all four of the ont eyes plot to the right of the 1:1 line indicating that they exhibit greater loss of peripapillary rnfla (42.4% 2.4%) than they do loss of onh mra (24.3% 4.2%, p = 0.0005). finally, as described in figure 1 for the individual example eg eye, there were subtle changes for other onh parameters as well, such as a decrease of the mra angle (whereby it became significantly more acute at the final session in eg eyes, fig. 6a), a significant increase in the bmo nonplanarity (whereby the bmo became less planar and more bowed into a saddle shape, fig. 6c) and a significant decrease in the bmo aspect ratio (i.e., the aspect ratio of the best 3d - fit of an ellipse to the bmo, which became rounder, fig. however, there was no significant change in the area of the bmo projection (fig. n = 51) of raw parameter values in ctl eyes and eyes with eg at bl (hatched boxes) and at the final imaging session (filled boxes). the p values listed represent the results of a paired t - test in each case (comparing eg eyes at the final imaging session to their own bl and separately to their fellow control eyes at the final session ; p values in red font indicate statistical significance). the results of this study demonstrated that thinning of the onh neuroretinal rim is more extensive than thinning of the peripapillary rnfl tissue throughout a wide range of severity in a nhp model of eg. as predicted, the total cross - sectional area of the optical slice through these two adjacent tissue sites was similar at baseline in eg and control eyes and remained similar to each other at the final time point in control eyes. in contrast, longitudinal change in eg eyes exhibited substantially greater loss of onh rim tissue area than peripapillary rnfl tissue area. this finding provided evidence that axon bundles are compressed transversely within the onh rim as a specific manifestation of glaucomatous deformation. if the only structural change contributing to thinning at each site were loss of retinal ganglion cell axons alone, then their cross - sectional areas should have changed by exactly the same amount. several assumptions require consideration, perhaps most important among them is whether the tissue constituents at these two adjacent sites are similar at baseline and whether the nonaxonal constituents change at all or in a similar manner. first, the onh rim as measured by the mra parameter will contain all retinal ganglion cell axons, whereas the peripapillary rnfla measurement will contain approximately 1.5% fewer axons (since those arising from retinal ganglion cells located between the peripapillary circle scan and the bmo will not contribute). although it is possible these ganglion cells most proximal to the onh are at higher risk of degeneration, their number is far too few to account for the differential effect observed in this study between mra and rnfla. second, although the total content of nonaxonal tissue is similar, it is possible that longitudinal changes occur differently at these two sites. the radial peripapillary capillary plexus is the primary circulatory bed serving the rnfl at the site where it was assayed. these capillaries are fed by arterioles branching from the central retinal artery, as are the most anterior prelaminar capillaries of the onh rim tissue with which they are continuous. although onh capillary volume is thought to decrease following axon loss, capillary density does not change, maintaining a constant ratio of capillary area to neural tissue area. in fact, there is evidence that onh capillary blood flow actually increases in the earliest stages of nhp eg, which would be difficult to reconcile if capillary volume within the onh was reduced dramatically. nevertheless, onh capillary loss is unlikely to be so much greater within the onh rim as compared to the peripapillary rnfl tissue that it could account for the differential observed in this study. for example, recent studies using oct angiography in glaucoma suggest that loss of capillaries (patent to flow) appears to be continuous between the onh rim and peripapillary plexuses. in fact, the density of onh vessels patent to flow measured using commercial oct angiography is effectively a surrogate reflecting onh rim area, consistent with the earlier demonstration that loss of capillary volume is proportional to loss of neural rim tissue. indeed, onh vessel densities measured in glaucomatous eyes by oct angiography remained within the range of healthy eyes until rim area loss reached a moderate - to - severe stage. of course, it is possible that onh tissue compression also might reduce vessel caliber within the rim, but this alone does not seem sufficient to explain the substantial differential documented in this study without also inferring a differential effect on axon caliber within the rim versus the peripapillary rnfl. moreover, despite being plausible, reduced caliber of capillaries would be inconsistent with the aforementioned evidence from onh capillary blood flow studies in early - stage nhp eg. third, there is at least one potentially important difference between the onh rim and peripapillary rnfl in terms of glial content, in that the septa between axon bundles within the rnfl consist of both muller cell and astrocyte processes whereas the septa separating axon bundles of the onh rim contain only astrocyte processes. however, overall glial content does not vary significantly between the onh rim and peripapillary rnfl tissue, although it does increase slightly with increased depth into the onh. in moderate to severe glaucoma, both astrocytes and muller cells exhibit increased expression of glial fibrillar acidic protein (gfap) in the retina along with this well - known sign of astrocyte activation within the onh. however, there is little evidence to suggest that glial content decreases within the onh rim tissue to a greater extent that it does within the peripapillary rnfl ; on the contrary, both sites exhibit hypertrophy of the glial intermediate filament gfap. thus, our findings suggested that axon bundles (and possibly also astrocytes) are compressed (which may represent in part a consequence of longitudinal stretch) within the onh neuroretinal rim as a result of glaucomatous connective tissue deformation. this may represent a potentially important mechanism of pathophysiologic insult to axons in glaucoma. for decades the primary site of injury in glaucoma was thought to be at the level of the lamina cribrosa. strong evidence supporting this conclusion includes the accumulation of radioactive amino acid tracers and cellular organelles (chiefly swollen, distorted mitochondria but also vesicles) within the posterior aspect of the lamina cribrosa after acute or chronic iop elevation in nhps. these findings are thought to reflect interruption, or even severe blockade of fast axonal transport at the level of the posterior lamina cribrosa. however, this also is precisely the location where there is normally a very steep gradient of mitochondria between the unmyelinated portion of axons within the prelaminar onh and the start of myelination in the retrolaminar orbital optic nerve where action potentials abruptly switch to saltatory conduction. recent evidence from the mouse eye also demonstrates that astrocytes within the myelin transition zone (which extends approximately 1 to 2 mm behind the mouse sclera) are responsible for recycling damaged mitochondria from rgc axons by transcellular phagocytosis or the phenomenon of transmitophagy in mice suggests that the apparent accumulation of damaged mitochondria (and vesicles laden with membranous debris) within the immediate retrolaminar region of the primate onh may represent as much a back - up of this astrocyte - driven, normal clearance mechanism as it does a primary back - up of fast axonal transport. in fact, numerous examples exist within the classic papers on axonal transport interruption in nhp eg models where transport tracers accumulate within the onh rim tissue (see, e.g., fig. 2 in the report of quigley and anderson ; figs. the hypothesis that the onh rim can be a site of injury does not require the rim to be the only site of damage, nor does it undermine the classical evidence from human specimens stained with silver impregnation techniques showing that axons ultimately appear abruptly discontinuous at the posterior aspect of the lamina cribrosa, since this may be the site where injured axons are pruned. however, neither do these classic data rule out the possibility that local damage to the axonal cytoskeleton occurs as a direct result of axon deformation (compression and/or stretch) within the onh rim tissue, with a subsequent predictable failure of axonal transport there as well. in summary, the results of this study demonstrated that onh neural rim tissue thinning exceeds peripapillary rnfl thinning over a wide range of severity in nhp eg. these results support the hypothesis that axon bundles (and astrocytes) are compressed (which may include longitudinal stretch) within the onh rim by glaucomatous connective tissue deformation. | purposewe tested the hypothesis that experimental glaucoma (eg) results in greater thinning of the optic nerve head (onh) neural rim tissue than the peripapillary retinal nerve fiber layer (rnfl) tissue.methodslongitudinal spectral - domain optical coherence tomography (sdoct) imaging of the onh and peripapillary rnfl was performed every other week under manometric iop control (10 mm hg) in 51 nonhuman primates (nhp) during baseline and after induction of unilateral eg. the onh parameter minimum rim area (mra) was derived from 80 radial b - scans centered on the onh ; rnfl cross - sectional area (rnfla) from a peripapillary circular b - scan with 12 diameter.resultsin control eyes, mra was 1.00 0.19 mm2 at baseline and 1.00 0.19 mm2 at the final session (p = 0.77), while rnfla was 0.95 0.09 and 0.95 0.10 mm2, respectively (p = 0.96). in eg eyes, mra decreased from 1.00 0.19 mm2 at baseline to 0.63 0.21 mm2 at the final session (p < 0.0001), while rnfla decreased from 0.95 0.09 to 0.74 0.19 mm2, respectively (p < 0.0001). thus, mra decreased by 36.4 20.6% in eg eyes, significantly more than the decrease in rnfla (21.7 19.4%, p < 0.0001). other significant changes in eg eyes included increased bruch 's membrane opening (bmo) nonplanarity (p < 0.05), decreased bmo aspect ratio (p < 0.0001), and decreased mra angle (p < 0.001). bruch 's membrane opening area did not change from baseline in either control or eg eyes (p = 0.27, p = 0.15, respectively).conclusionsoptic nerve head neural rim tissue thinning exceeded peripapillary rnfl thinning in nhp eg. these results support the hypothesis that axon bundles are compressed transversely within the onh rim along with glaucomatous deformation of connective tissues. |
idiopathic ventricular tachycardia (vt) or frequent premature ventricular contractions (pvcs) from the right ventricular outflow tract (rvot) occur in patients without structural heart disease. although the patients with these arrhythmias can be highly symptomatic, these arrhythmias tend to follow a benign clinical course,. the aim of the treatment is therefore to reduce symptoms and improve quality of life. radiofrequency ablation (rfa) is a safe and effective treatment to reduce symptoms and eliminate vt and pvcs. as many of the patients are young and healthy individuals, a single intervention with rfa may be preferred over the lifelong use of antiarrhythmic drugs. in patients with frequent pvcs from rvot some patients experience recurrent tachycardia, which potentiates the development of cardiomyopathy and heart failure. rfa is generally more successful in patients with vt and pvcs originating from the rvot than from other heart structures. a high success rate of rfa treatment is well documented at mid- and short - term follow - up (fu), but knowledge about long - term fu is still limited. the aim of this retrospective study is to examine the effect of the rfa treatment at long - term fu, measured by the patient 's symptomatic complaints, use of antiarrhythmic medication, general health perception and their reported fitness to work. the study population consisted of patients recruited from a local quality register at our hospital. all patients suffered from idiopathic vt or frequent pvcs from rvot and had been treated with rfa in the time period from 2002 to 2005. the patients had been examined by echocardiography, magnetic resonance images / computer tomography and/or coronary angiography prior to the procedure to rule out structural heart disease, as such patients were excluded from the study. the study was approved by the regional ethical committee and informed consent was obtained from all patients. all patients were fasting and under mild sedation to avoid potential suppression of clinical vt or pvcs. if spontaneous vt or pvcs was not observed, they were induced by pace and/or isoproterenol infusion. this was registered on a 12-lead surface electrocardiography (ecg) and used as template for pace mapping (bard, electrophysiology, lowell, usa). activation mapping was used with (n = 18) or without (n = 16) a 3-dimensional mapping system (ensite array, endocardial solutions, sj medical, st. radiofrequency ablation was performed using a 4 mm - cool tip ablation catheter (biosense webster cordis, diamond bar, usa) in all procedures except one, where a 4 mm non - irrigated tip catheter was used. the ablation was performed at the best achievable pace map and/or the site of earliest local activation. the elimination of spontaneous clinical vt or pvcs via isoproterenol administration and non - inducibility of the clinical ventricular arrhythmias was the procedural endpoint. the patients were examined using an in - house questionnaire comprising a self - evaluation score. they also rated their overall general health perception as a consequence of their arrhythmia on a scale from 1 (poor) to 4 (excellent), as well as their fitness to work on a scale from 1 (incapacitated) to 5 (full time employment) prior to rfa, immediately after treatment with rfa and at long - term fu. all patients were contacted by telephone and given sufficient information to avoid any misinterpretation of the self - evaluation scores. a 12-lead ecg recording was performed in patients who were able to sustain an outpatient examination, either in the ambulatory clinic at our hospital or at their local hospital. all follow - ups and ecg were conducted at long - term fu, 10 years after the rfa procedure. statistical analyses were performed using spss version 22 (spss, chicago, il, usa). discrete variables were reported as percentages and continuous variables as mean sd, and compared using the paired student 's t - test and nonparametric wilcoxon rank test. the study population consisted of patients recruited from a local quality register at our hospital. all patients suffered from idiopathic vt or frequent pvcs from rvot and had been treated with rfa in the time period from 2002 to 2005. the patients had been examined by echocardiography, magnetic resonance images / computer tomography and/or coronary angiography prior to the procedure to rule out structural heart disease, as such patients were excluded from the study. the study was approved by the regional ethical committee and informed consent was obtained from all patients. all patients were fasting and under mild sedation to avoid potential suppression of clinical vt or pvcs. if spontaneous vt or pvcs was not observed, they were induced by pace and/or isoproterenol infusion. this was registered on a 12-lead surface electrocardiography (ecg) and used as template for pace mapping (bard, electrophysiology, lowell, usa). activation mapping was used with (n = 18) or without (n = 16) a 3-dimensional mapping system (ensite array, endocardial solutions, sj medical, st. radiofrequency ablation was performed using a 4 mm - cool tip ablation catheter (biosense webster cordis, diamond bar, usa) in all procedures except one, where a 4 mm non - irrigated tip catheter was used. the ablation was performed at the best achievable pace map and/or the site of earliest local activation. the elimination of spontaneous clinical vt or pvcs via isoproterenol administration and non - inducibility of the clinical ventricular arrhythmias was the procedural endpoint. the patients were examined using an in - house questionnaire comprising a self - evaluation score. they also rated their overall general health perception as a consequence of their arrhythmia on a scale from 1 (poor) to 4 (excellent), as well as their fitness to work on a scale from 1 (incapacitated) to 5 (full time employment) prior to rfa, immediately after treatment with rfa and at long - term fu. all patients were contacted by telephone and given sufficient information to avoid any misinterpretation of the self - evaluation scores. a 12-lead ecg recording was performed in patients who were able to sustain an outpatient examination, either in the ambulatory clinic at our hospital or at their local hospital. all follow - ups and ecg were conducted at long - term fu, 10 years after the rfa procedure. statistical analyses were performed using spss version 22 (spss, chicago, il, usa). discrete variables were reported as percentages and continuous variables as mean sd, and compared using the paired student 's t - test and nonparametric wilcoxon rank test. of the 36 patients originally included in the study, one patient pulled out of the study whilst one patient died during the rfa procedure, leaving 34 patients eligible for analysis. 11 participants suffered from idiopathic vt whilst 23 patients experienced frequent pvcs from the rvot. the final study population that participated in a long - term - follow - up program comprised 18 females and 16 males with an average age of 56.3 12.6 (range 3281) years. the rfa procedure was performed using activation mapping, with (n = 18) or with out additional 3d mapping system (n = 16), and pace mapping. x - ray duration in the 3d group was significantly higher compared to the pace mapping group (56.4 30.5 versus 24.3 18.6 min, p 10 years) after rfa treatment on patients with idiopathic ventricular arrhythmias originating from the rvot. this as extensive research focusing on rhythm surveillance and ecg morphology previously had been documented, and the clinical aim of rfa treatment was to eliminate symptoms and improve quality of life,. furthermore, patients reported an improvement in both their general health perception and fitness to work. reasons for unsuccessful rfa treatment were either that the arrhythmia foci were anatomically inaccessible, or that the arrhythmias were non - inducible prior to the procedure. three of the patients with unsuccessful rfa undertook a new rfa in the time period from 2002 to 2005 or later. the second attempt proved successful for all three patients. the last patient with an unsuccessful ablation chose antiarrhythmic drug therapy instead of a new rfa, and reported no symptomatic burden at ten years dizziness is a common medical symptom that can significantly affect the patient 's daily activities and can be caused by factors other than cardiac arrhythmias. it is thus difficult to determine whether the dizziness was caused by vt or pvcs, and not from other common causes. however, our study showed that 91.2% of patients reported a statistically significant improvement in ailments related to heart rhythm disorder immediately after rfa treatment and during long - term fu, supporting the suspicion that ailments actually was related to the heart rhythm disorder. although they still suffered from aliments related to their vt or pvc after rfa treatment, it did not affect their fitness to work and general health perception during long - term fu. our study showed a significant reduction in the use of antiarrhythmic medication after rfa at long - term fu. this is not unexpected, as 35.3% of patients experienced a decrease in symptomatic burden only, and 8.8% of patients reported no improvement in aliments overall. for patients who used antiarrhythmic medicine before and not after rfa, and got an improvement in aliment after rfa we can assume that the improvement is due to rfa. for patients using antiarrhythmic medication before and after rfa, but still got an improvement in aliments after rfa, we can not rule out that the improvement of the ailments are partially placebo effect. use of antiarrhythmic medication is still the preferred first choice of treatment of idiopathic vt or pvc,. however, undergoing rfa treatment may prevent many patients from the lifelong need of antiarrhythmic medication, with their potential side effects, as well as the risk of polypharmacy. the potential medical and financial implications could make rfa the preferred treatment among the patients. general health, symptoms and employability are all interlinked factors affecting a patient 's quality of life. although there was no statistically significant improvement in general health perception at long - term follow - up compared to immediately after rfa treatment, an improving trend was observed. approximately 21% of the patients reported that their general health was very good or good prior to rfa. this perception was due to limited or no symptomatic burden, which was consistent with previous studies. the percentage of patients in full time employment more than doubled at long - term follow up to 55.9% compared to 26.5% prior to rfa, demonstrating the medical and social benefits of rfa. whilst one patient (2.9%) was incapacitated due to arrhythmia, the remaining 41.2% of the patients are not in full time employment due to retirement, incapacitation or sick leave for other reasons than the heart rhythm disorder. this supports our finding that rfa positively affects fitness to work during long - term fu. none of the patients had clinically developed heart failure, malignant clinical arrhythmia or sudden arrhythmogenic death at long - term follow - up. however, we have no data available concerning the echocardiographic findings in our patient population. the average x - ray duration in the 3d mapping group was higher compared to the pace mapping group. this might be explained by the use of the 3d system in an early stage of development whereas pace mapping has been a part of clinical routine for several years. the results of this study can be influenced by the population size and the number of patients lost to follow - up. the small sample size may have reduced the power of the statistical analysis resulting in non - significant results. furthermore, if the patients lost to follow up had answered significantly different from the average population it would affect the results. according to isaac and michael a response rate of at least 80% is necessary to obtain good estimates. of all patients who were given the questionnaire only one patient chose not to participate in the study. the response rate in the patient population was thus 97%, and the results are unlikely to have been skewed by this absence. this is evident as previous studies on rfa treatment of vt or pvc (including non - idiopathic arrhythmia) use similar sample sizes,,,. this is a descriptive study of a relatively small number of patients without a control group. as there was no validated template questionnaire available for the main purpose of this study, an in - house questionnaire with disease specific questions was considered the most appropriate approach to identify patients ' overall burden of arrhythmia. to ensure that the answers were reliable an additional follow - up as it could be challenging to accurately recall details from events occurring ten years previously, the patient 's medical records were used as supporting documentation, allowing for possible recall bias. no long - term rhythm surveillance (holter) was obtained, as the patients were geographical spread across the country. however the procedural records do not include procedure time and radiofrequency time that might be of interest. this is a descriptive study of a relatively small number of patients without a control group. as there was no validated template questionnaire available for the main purpose of this study, an in - house questionnaire with disease specific questions was considered the most appropriate approach to identify patients ' overall burden of arrhythmia. to ensure that the answers were reliable an additional follow - up as it could be challenging to accurately recall details from events occurring ten years previously, the patient 's medical records were used as supporting documentation, allowing for possible recall bias. no long - term rhythm surveillance (holter) was obtained, as the patients were geographical spread across the country. however the procedural records do not include procedure time and radiofrequency time that might be of interest. in conclusion, a reduction in the use of antiarrhythmic medication and symptoms, as well as an improvement in general health perception and fitness to work, after rfa treatment of idiopathic vt or pvcs can be demonstrated after a long - term follow up of ten years. | backgroundthe aim of this study was to examine the effect of radiofrequency ablation (rfa) of ventricular arrhythmias from right ventricular outflow tract (rvot) during long - term follow-up.methodsa follow - up analysis was conducted using an in - house questionnaire, as well as a qualitative assessment of the patients ' medical records. the study population of 34 patients had a previous diagnosis of idiopathic vt or frequent pvcs from the rvot, and received rfa treatment between 2002 and 2005.resultsthe main symptoms prior to rfa were palpitations (82.4%) and dizziness (76.5%). a reduction in symptoms following rfa was reported by 91.2% of patients (p < 0.001). furthermore, there was a reduced use of antiarrhythmic medication after rfa (p < 0.001). general health perception classified on a scale of 1 (poor) to 4 (excellent), improved from median class 1 to 3 (p < 0.001) during long - term follow - up. the fitness to work increased from median class 3 to class 5 (1 = incapacitated, 5 = full time employment, p = 0.038), while the rate of patients in full time employment increased from 26.5% to 55.9% after rfa (p = 0.02).conclusionsa reduction of symptoms and use of antiarrhythmic medication, as well as an improvement in the general health perception and fitness to work after rfa of idiopathic ventricular arrhythmias can be demonstrated at ten - year follow - up. |
when present, it has been shown to be associated with a systemic autoimmune disease such as goodpasture 's syndrome, systemic lupus erythematosus (sle), and antineutrophil cytoplasmic antibodies (anca)-associated vasculitis or microscopic polyangiitis (1 - 6). less frequently, it has been reported to be associated with henoch - schonlein purpura (7), iga nephropathy (8) or idiopathic immune complex - associated glomerulonephritis (9). dah associated with acute post - streptococcal glomerulonephritis (psgn) is extremely rare ; there have been no cases reported in korea to date. a review of the literature has identified two cases worldwide (10, 11). in this report, we describe a patient who clinically presented with pulmonary - renal syndrome ; the renal histology revealed post - infectious glomerulonephritis of immune complex origin. a 59-yr - old woman, previously in good health, was admitted because of generalized edema, progressive oliguria, and blood - tinged sputum. two weeks prior to admission, the patient developed a sore throat and tonsillar swelling. on admission, the patient had facial swelling and generalized edema, with a blood pressure of 220/130 mmhg, a regular heart beat of 90 beats / min, a rapid breathing rate of 25/min, and a temperature of 37.2. on oral examination, left tonsillar swelling and throat injection were observed. chest radiography showed bilateral diffuse pulmonary infiltrates with blunting of both costophrenic angles (fig.. respiratory tract infection was suspected, and blood culture and sputum examination were performed. laboratory studies revealed an elevated white blood cell count (22,000/l) (segmented neutrophil 83%, lymphocyte 12.3%, eosinophil 0.1%, basophil 0.2%). the hemoglobin level was normal (13.4 g / dl), and the platelet count was normal (22810/l). c - reactive protein and erythrocyte sedimentation rate were slightly elevated (11.2 mg / l and 74 mm / hr, respectively). the plasma creatinine was 2.3 mg / dl, and the urea was 52.3 mg / dl. the concentrations of aspartate aminotransferase (ast), alanine aminotransferase (alt), total bilirubin, direct bilirubin, alkaline phosphatase, and creatine kinase (ck) were all normal (40 u / l, 45 u / l, 1.0 mg / dl, 0.3 mg / dl, 338 iu / l, and 147 iu / l, respectively). lactate acid dehydrogenase (ldh) was elevated to 1,103 urinalysis showed a large number of dysmorphic red blood cells and several wbcs on microscopic examination. the urine chemistry showed proteinuria at 1.2 g/24 hr at serum c3 was 10.5 mg / dl (normal 90 - 180 mg / dl), ch50 was below 5 u / ml (normal 30 - 45 u / ml) and, c4 was 19.4 mg / ml (10 - 40 mg / ml). the serum igg, iga, ige, and igm were all normal (795 mg / dl, 307mg / dl, 105 mg / dl, and 126 mg / dl, respectively). the anti - streptolysin - o titer (aso) was 275.4 (normal 0 - 166 iu / ml). autoantibodies including antineutrophil cytoplasmic autoantibody (anca), cryoglobulin, antinuclear antibody (ana), anti - dsdna antibody, and anti - phospholipid antibody were all negative. the indirect immunofluorescence assay for anti - glomerular basement membrane (gbm) igg and iga were negative. the sputum for acid - fast bacilli was negative. on the third hospital day, the patient developed red brick - colored hemoptysis and respiratory distress ; the clinical status deteriorated progressively with moderate respiratory failure. the chest radiography revealed a marked infiltrative haziness or edema over the whole lung field (fig. the hemoglobin level decreased to 9.8 g / dl and hematocrit to 27.8% after hemoptysis developed. the arterial blood gas analysis showed a ph 7.39, pco2 33.7 mmhg, po2 58 mmhg, hco3 20.9, and sao2 92.5%. the renal function progressively declined, with the creatinine values increasing to 2.8 mg / dl and the urea to 78.4 mg / dl. 2), which revealed bilateral pleural effusion and alveolar consolidation or alveolar hemorrhage in both middle and lower lung fields. macroscopically hemorrhagic fluid was obtained from a broncho - alveolar lavage. because of the absence of clinical findings suggestive of typical bacterial pneumonia and the presence of evidence for streptococcal infection such as medical history of upper respiratory infection, physical finding of tonsilar swelling, and laboratory finding of a high aso titer, we discontinued the ceftriaxone and started daily doses of injected methylprednisolone (1,000 mg) for three days. the bronchial fluid was negative for bacterial culture, gram stain, or afb smear. the cell block result from the bronchial washing fluid showed acute inflammatory cells and hemosiderin - laden macrophages. lung biopsy revealed eosinophils, polymorphonuclear cells and nuclear dust deposition in the alveolar interstitium as well as irregular septal thickening and fibrin clots attached to the interalveolar septa (fig. the serum creatinine level and serum aso titer were normalized (0.8 mg / dl and 165 iu / ml, respectively). the patient has been doing well without any additional symptoms ; she is currently treated as an outpatient with an ace inhibitor because of mild proteinuria. the renal biopsy was performed under ultrasound sonography guidance on the fourth hospital day. under light microscopy evaluation, the kidney biopsy specimen showed abundant glomeruli, all of which were diffusely and markedly hypercellular with various degrees of infiltration by polymorphonuclear neutrophils and fibrin / platelet aggregates. there were no crescent, vasculitis, or basement membrane duplication interstitium ; the tubules and blood vessel were normal (fig., there were dome - shaped or flame - shaped electron - dense granular subendothelial deposits (" hump ") (fig. the renal biopsy was performed under ultrasound sonography guidance on the fourth hospital day. under light microscopy evaluation, the kidney biopsy specimen showed abundant glomeruli, all of which were diffusely and markedly hypercellular with various degrees of infiltration by polymorphonuclear neutrophils and fibrin / platelet aggregates. there were no crescent, vasculitis, or basement membrane duplication interstitium ; the tubules and blood vessel were normal (fig., there were dome - shaped or flame - shaped electron - dense granular subendothelial deposits (" hump ") (fig. glomerulonephritis develops, on average, 10 days after pharyngitis or two weeks after skin infection (impetigo) with a nephritogenic strain of group a -hemolytic streptococcus (12). gross hematuria, oliguric acute renal failure, edema, and hypertension are the classic manifestations ; however, most patients present with a milder disease (13). it is a very unusual event for diffuse alveolar hemorrhage (dah) to develop in association with psgn. it is not clear why our patient developed such a rare and life - threatening condition, given the fairly typical course of the acute psgn. dah is usually associated with goodpasture 's syndrome with crescent glomerulonephritis and pulmonary hemorrhage as the typical characteristic features (1). goodpasture 's syndrome has been shown to have circulating anti - gbm antibody ; this antibody is observed as linear deposits on basement membrane (14, 15). in our case, there was no circulating anti - gbm antibodies detected. in addition, the granular deposition, of immune complexes on the subendothelium, excluded the possibility of the glomerulonephritis due to anti - gbm disease. dah has been identified in other causes of immune - mediated nephritis such as sle, wegener 's granulomatosis, microscopic polyarteritis, mixed cryoglobulinemia, behcet 's syndrome, henoch - schonlein purpura, and pauch - immune glomerulonephritis (3 - 7, 16, 17). in our case, lupus nephritis and cryoglobulinemia were excluded because of the absence of characteristic clinical features as well as negative anti - nuclear antibodies, anti - dsdna ab, and cryoglobulins. in addition, the absence of histological evidence of vasculitis in renal tissue also excluded the possibility of systemic vasculitis such as microscopic polyarteritis or anca - associated vasculitis. dah has been reported to be associated with anti - phospholipid antibody syndrome on rare occasions (18) ; this was excluded because of the absence of anti - phospholipid antibody. on the basis of clinical history, serologic tests, and the pathologic findings of the renal biopsy, the most reasonable diagnosis was psgn with dah. the pathogenic mechanism to explain the presence of dah with psgn is not well established. there are some reports that suggest that the clinical picture of goodpasture 's syndrome may not always be etiologically related to anti - gbm disease ; in some cases an immune complex mechanism may be operative. fukuda. reviewed an autopsy case of dah with acute nephritis (19). the lungs showed hemorrhagic interstitial pneumonia, with granular patterns of igg and c3 along the alveoli by immunofluorescence and electron - dense subepithelial deposits with electron microscopy. electron microscopy showed numerous subepithelial deposits, and immunoelectron microscopy revealed that igg was not present in the gbm itself but present in the subepithelial deposits. anti - gbm antibody activity was not detected in the serum or the kidney evaluated. these findings suggest that the renal and pulmonary lesions occurred by a similar mechanism in association with immune complex deposits (19). immune complex deposition on broncho - alveolar epithelium and capillary basement membrane may induce an acute inflammatory reaction with a neutrophil - mediated disruption of the alveolar bed and capillary basement membrane. previous uncontrolled observations suggest that high - dose intravenous pulse therapy with corticosteroids produces a prompt improvement in the nephritis associated dah (21). consistent with expectations, the hemoptysis in our patient as well as the respiratory distress resolved quickly after intravenous methylprednisolone treatment. a review of the literature has identified only two previous cases of psgn with pulmonary hemorrhage. one case was a 12-yr - old girl who developed a near - fatal pulmonary hemorrhage with acute non - crescent psgn (10) ; there was dramatic improvement of hemoptysis and chest radiography abnormalities within 48 to 72 hr after intravenous administration of methylprednisolone ; the renal function also improved. the other patient was a 38-yr - old male who developed pulmonary hemorrhage with acute crescent psgn. he also demonstrated dramatic improvement of the hemoptysis and chest radiography abnormalities within 72 hr after steroid treatment ; but the renal failure progressed to end - stage disease (11). reviewed nine patients with diffuse intrapulmonary hemorrhage and glomerulonephritis not due to anti - gbm antibody, which included systemic vasculitis of unspecified type in two patients with seropositive rheumatoid arthritis, idiopathic crescentic glomerulonephritis with negative immunofluorescence in two, wegener 's granulomatosis in two, and one each of polyarteritis nodosa, henoch - schonlein purpura, and mixed connective tissue disease. pulmonary hemorrhage was reported to improve markedly within 24 - 72 hr in eight of the nine patients ; however, there was no effect on renal function in most of these patients (22). these data suggest that early recognition and timely treatment with corticosteroids could be lifesaving in some patients with dah and glomerulonephritis. in conclusion, our case demonstrated that 1) post streptococcal noncrescent glomerulonephritis can be associated with pulmonary hemorrhage, 2) the clinical picture of goodpasture 's syndrome may not always be etiologically related to anti - gbm disease, and in some cases an immune complex mechanism may be implicated, and 3) intravenous administration of high - dose steroids, although not proven by controlled studies, may be beneficial for the patients with dah. | acute post - streptococcal glomerulonephritis (psgn) is characterized by an abrupt onset of edema, hypertension, and hematuria. life - threatening diffuse alveolar hemorrhage (dah) is rarely associated with acute psgn. there have been only two reported cases worldwide, and no case has been reported previously in korea. here, we present a patient who clinically presented with pulmonary - renal syndrome ; the renal histology revealed post - infectious glomerulonephritis of immune complex origin. a 59-yr - old woman was admitted with oliguria and hemoptysis two weeks after pharyngitis. renal insufficiency rapidly progressed, and respiratory distress developed. chest radiography showed acute progressive bilateral pulmonary infiltrates. the clinical presentation suggested dah with psgn. three days after treatment with high - dose steroids, the respiratory distress and pulmonary infiltrates resolved. electron microscopy of a renal biopsy specimen sample revealed diffuse proliferative glomerulonephritis with characteristic subendothelial deposits of immune complex (" hump "). the renal function of the patient was restored, and the serum creatinine level was normalized after treatment. |
hyperphosphatemia has been associated with poor outcomes and mortality in chronic kidney disease (ckd)stage 5d, and high normal serum phosphorus levels have been associated with mortality in non - ckd patients and in ckd stage 3 patients. therefore, in patients with ckd stage 5d, kidney disease : improving global outcomes (kdigo) suggest lowering elevated phosphorus levels toward the normal range. the rationale for controlling serum phosphorus is based on epidemiological evidence suggesting that hyperphosphatemia is an important risk factor not only for secondary hyperparathyroidism, but also for cardiovascular disease. dietary phosphorus restriction and removal of phosphorus by dialysis are usually inadequate for controlling serum phosphate. consequently, it becomes necessary to prescribe phosphate binders in order to reduce the amount of dietary phosphorus absorbed from the intestine. currently available phosphate binders, while effective, are not considered optimal for treating hyperphosphatemia because they may lead to unacceptable adverse effects such as hypercalcemia (e.g., calcium carbonate) or potential toxicities (e.g., aluminum toxicity). moreover, some binders are expensive (sevelamer and lanthanum carbonate). thus, there is a need for phosphate lowering agents that overcome some of the defects of current therapy. the major clinical use of niacin has been to increase high - density lipoprotein (hdl) cholesterol and reduce triglyceride levels. animal studies have shown that niacinamide prevents an increase in serum phosphate in animals with renal failure by reducing sodium - phosphate 2b transporter expression in the jejunum and inhibiting intestinal phosphorus absorption. its major side effects are vasodilation and flushing, which appear to be mediated through prostaglandin production, and thus can be attenuated by premedication with aspirin. however, recently the selective prostaglandin d2 receptor subtype 1 inhibitor laropiprant has been used in combination of niacin for reducing the flushing side effect of drug. niacinamide, which is a circulating form of niacin, inhibits phosphate uptake by brush border membrane vesicles isolated from the rat small intestine. recent human clinical trials studies have also shown that niacinamide and niacin accomplish clinically significant reductions in serum phosphate in patients undergoing dialysis.[812 ] niacin has potential advantages over current phosphate binders in that it does not need to be administered at the time of a meal. the aim of the present study was to examine whether niacin lowers serum levels of phosphorus and intact parathyroid hormone (ipth) in long - term hemodialysis patients. this study was a randomized, double - blind clinical trial to evaluate the effectiveness of niacin versus placebo in reducing plasma phosphorus levels in hd patients. this study was approved by the human research ethics committee of the arak university of medical sciences (irct registration number : irct138812153492n1). all 163 patients were screened for inclusion according to their most recent monthly plasma phosphorus value. inclusion criteria were age > 18 years, capable of giving informed consent, duration of hd > 3 months, serum phosphorus levels 5 - 7 mg / dl, unchanged treatment protocol (calcium components and vit d) during last 2 weeks and regular and steady dialysis program. exclusion criterias were pregnancy, known liver disease, active peptic ulcer disease, treatment with carbamazepine, drug intolerance and necessity of changing treatment protocol because of safety criteria (such as severe hyperphosphatemia or two consecutive serum phosphorus levels > 7 mg / dl). were packaged in identical 100-mg tablets, and dosages were titrated from 400 mg / d (taken as two tablets twice a day) to 1000 mg / d (five tablets twice a day) over 8 weeks. every 2 weeks the dose was increased to 600, 800 and 1000 mg / d, respectively. after 8 weeks, patients underwent a 2-week washout period, followed by 8 weeks of the alternative therapy (from niacin to placebo, and vice versa). concurrent binder therapy (calcium carbonate) and calcitriol were continued without any dosing adjustments during the study. patients were counseled to take 100 mg aspirin 1 hr before niacin in case of flush symptoms. calcium and phosphorus values were collected every 2 weeks ; complete blood counts were collected every 4 weeks ; total cholesterol, triglycerides, hdl cholesterol, low density lipoprotein (ldl) cholesterol and intact parathyroid hormone (ipth) were collected at study start and end in each arms. the week 0 was used as a baseline for comparison to the final study (week 8) value for all lab parameters. glucose and liver enzymes values were measured every 4 weeks to look for adverse drug effects. the primary end point was the change in serum phosphorus from the first to the last week of each arm. predefined secondary end points were the change in ipth, platelets and lipid profile (hdl, ldl, triglycerides). forty - seven patients were consented and randomized as placebo or niacin to participate in the study. five in the placebo arm were withdrawn prematurely because of the need for change and adjustment of phosphate binder treatment protocols (prescription of aluminum hydroxide) and five patients in the niacin arm were withdrawn due to intractable flushing and gastrointestinal upset. thirty - seven patients (18 men ; age, 5711 years) remained in study until the end. the mean dose of niacin at the end of the 8-week treatment period was 740204 mg / day. the minimum and maximum doses of niacin were 400 and 1000 mg / day respectively. the mean value of phosphorus and the mean dose of niacin at the end of the 2, 4, 6 and 8-week treatment period in both drug - treated and placebo groups have been shown in table 2. the consecutive values of phosphorus and niacin in the two groups there were statistically significant decrements in serum phosphorus levels throughout the first 2 weeks of study period in both niacin - treated and the placebo groupsth. after week 2 no significant changes occurred in phosphorus levels in every 2 weeks serial measured values in the niacin - treated group [table 3 ]. however, there was a significant increase in phosphorus value between w6 and w8 in the placebo arm, despite being relatively stable in the two previous measuring (p=0.003). this acute increase in phosphorus levels led to significant differences in the mean values of phosphorus at 8 weeks between drug - treated and placebo groups (p=0.039). also, as has been shown in the table 2, serum phosphorus decreased from (6.661.40) to (5.960.87) mg / dl (p=0.006) in the niacin - treated group at the 8-week treatment. the mean differences of serial serum phosphorus values in both groups changes in serum phosphate concentrations the mean values of phosphorus of week 8 have been compared in two groups of niacin - treated and placebo [table 4 ]. as has been shown, there was significant difference between phosphorus levels in w8 for patients able to be titrated to more than 800 mg / day niacin (57%) and placebo ; however, no meaningful difference was observed in the lower doses (p=0.006). comparison of phosphorus values base on niacin dose additionally, no statistically significant changes were demonstrated for serum calcium or ipth values [table 5 ]. summary of laboratory findings niacin increased hdl cholesterol from 35.37.26 to 40.610.1 mg / dl (p=0.018) in the high - dose group ; however, no significant changes occurred in the low - dose or placebo groups (p>0.05). there were no statistically significant changes in serum total cholesterol, ldl cholesterol and serum triglyceride levels throughout the study period [table 5 ]. there was no statistically significant changes in mean platelets levels among the niacin - treated participants (p=0.240). the most common adverse events which, possibly related to niacin treatment were flushing and gastrointestinal symptoms. severe flushing which led to discontinuing of the drug, were observed in three patients. however, 40% (15/37) of cases suffered from mild to moderate flushing, which were major barrier for drug titration. as pointed before, in spite of introduction of new phosphate - bindering drugs, improved membrane technology and quality improvements of dialysis, control of hyperphosphatemia remains a major challenge in hemodialysis patients care. in recent years, introduction of niacin and niacinamide derivatives as phosphate - binder agents has given birth to new hopes to solve the problem of hyperphosphatemia in advanced renal failure. some studies have evaluated niacinamide for demonstrating the phosphate - lowering effect of these derivatives. they started niacinamide at 500 mg / d in 65 hemodialysis patients, which was titrated to a maximum dose of 1750 (1080370) mg / day. during the 12-week treatment, phosphorus levels fell from 6.91.5 to 5.41.3 mg / dl (p<0.001) with no meaningful change in serum calcium levels, while intact parathormone decreased significantly. muller., used extended - release niacin (niaspan) with a mean dose of 1470110 mg / day in 20 dialysis individuals for the 12-week. the goal was to tolerate at least 1000 (1470110) mg / d of niaspan, for which 17 individuals qualified. serum phosphorus decreased from 7.20.5 to 5.90.6 mg / dl (p<0.015) and hdl cholesterol increased from 403.2 to 595.5 mg / dl (p=0.0005). cheng., used niacinamide in a randomized, double - blind, placebo - controlled crossover trial with 33 individuals on hemodialysis. serum phosphorus decreased from 6.261.28 to 5.471.49 mg / dl (p=0.02) with niacinamide ; while there was no significant change in the placebo group. hdl cholesterol increased from 5017 to 6121 mg / dl with niacinamide but not placebo. in this study there were no statistically significant changes to serum calcium or ipth levels. maccubbin., performed a post hoc data analysis of serum phosphorus concentrations among 1547 patients who had dyslipidemia and were randomly assigned to treatment with extended release niacin (ern ; 1 g / d for 4 weeks and dose advanced to 2 g / d for 20 weeks) combined with the selective prostaglandin d2 receptor subtype 1 inhibitor laropiprant (ern - l) (n=761), ern alone (n=518), or placebo (n=268). they demonstrated that ern - l and ern alone caused a sustained approximately 11% reduction in serum phosphorus [0.41 mg / dl (0.46 to 0.37 mg / dl) ], accompanied by lowering of the calcium - phosphorus product, without raising serum calcium concentrations, which is unaffected by estimated gfr ranging from 30 to 90 ml / min per 1.73 m (i.e., stages 1 through 3 chronic kidney disease). we recorded a sharp decrease in phosphate values in the first 2 weeks of the study in both groups, which have been justified as the initial reduction of phosphorus levels in control group was intriguing, possible effect of study participation. tracing phosphate values in consecutive weeks showed changes in phosphate levels at 2, 4, 6 and 8 weeks in the treatment group. however, significant increase was observed in phosphate values at w8 in patients on placebo. our impression was that this gap is due to inhibitory effect of niacin on rising serum phosphorus after meal consumption, because drug - treated patients were on average dose of 740204 mg / day of niacin at the end of week 8. additionally, by comparing the mean value of phosphate at week 8 and niacin dosage, these results were reproducible only in cases able to be titrated to more than 800 mg / day of niacin (n=21, 57%). on the other hand, it seemed doses lower than 800 mg / day are not effective in short term treatment., where they have shown significant phosphorus reduction on a 375 mg / day extended - release niacin in 30 out of 34 individuals. in all other studies, restrepo valencia., observed that niacin generated a significant decrease in serum phosphate levels at 8 months, but not at 4 months. they assumed that the required dose the patients should take inorder to achieve the desired effect may be 1,000 mg, since at that time 100% of them were already receiving that dose. also, we detected significant increase in hdl cholesterol level, which is consistent with previous reports.[813 ] in our study ldl levels decreased, but the changes were not statistically significant. also, there were no statistically significant changes to serum calcium. in our study, intact pth levels were not significantly changed despite decrease in phosphate levels. however, a longer follow - up period and a higher number of patients are required to really obtain data with higher statistical power. in this respect our data was consistent with finding from most of the previous studies. on a contrasting note, all previous investigations were implemented by extended - release form of the drug.[91113 ] niacin marketing in our country is limited to short - acting form of oral 25 mg and 100 mg tablets and therefore one of the main barriers for satisfactory compliance was a number of pills patients had to take. also, one of the other limitations in our study was the assessment of compliance by face - to - face interviews and relience upon patients remarks. the main troublesome constraint in our study was the erratic changes in phosphorus values caused by food variety. although, we had a dietician on hand to counsel patients almost throughout the study period, the influence of food variety effect is irrefutable especially because of the short duration of the study. our study suggests niacin may emerge as a safe, low - cost therapy in combination with other phosphate binders for phosphate control. however, larger and longer term controlled trials are needed to establish the optimal dosage and the clinical significance of niacin treatment. | hyperphosphatemia is common in patients with end - stage renal disease. recent studies have shown that niacinamide and niacin achieve clinically significant reductions in serum phosphate in patients undergoing dialysis. the aim of the present study was to evaluate the serum phosphorus lowering effect of niacin in long - term hemodialysis patients. in this 8-week randomized, double - blind clinical trial, 37 patients were assigned to niacin or placebo with titration from 400 to 1000 mg daily. a 2-week washout preceded the switch from niacin to placebo or vice versa. the mean dose of niacin at the end of the 8-week treatment period was 750200 mg / day. serum phosphorus decreased from 6.661.40 to 5.960.87 mg / dl (p = 0.006) in the niacin - treated group after 8-weeks. however, the main reduction occurred at the beginning of study and seems not to be related to the phosphate - lowering effect of drug. in spite of a sharp increase in phosphorus level between w6 and w8 in patients on placebo, phosphorus values in drug - treated group showed nearly steady trend, presumably due to the inhibitory effect of niacin on phosphate absorption from gut. niacin also increased the high density lipoprotein (hdl) cholesterol (p = 0.018). our study suggests that niacin should be considered as adjunctive therapy for patients with hyperphosphatemia despite management with phosphate binders. the modest increase in hdl values may be another beneficial effect of this treatment. |
the usual progression of liver disease in patients with hepatitis c (hcv) is a process of inflammation accompanied by periportal necrosis and fibrosis. the inflammation that results from the virus causes stimulation of stellate cells which ultimately leads to the deposition of collagen which leads to fibrosis progression within the liver. if this process is rapid and unhindered then the usual outcome is the development of cirrhosis which is the final irreversible stage characterized by parenchymal nodules with encircling fibrous septa. the hepatitis c virus is not considered to directly injure the liver but it rather triggers an hcv - specific lymphoproliferation. through profuse cytokine production and also a direct cytopathic effect, these t cells result in hepatocyte apoptosis. 1 many patients with chronic hcv are also noted to have a degree of steatosis present on their liver biopsies. hepatic steatosis is defined as excessive lipid accumulation within the hepatocyte cytoplasm and has been more recently recognized as a significant cause for cirrhosis in the united states. 2 there are two forms of steatosis present in patients with hepatitis c, specifically metabolic steatosis and hcv - induced steatosis. metabolic steatosis is a process which occurs in the setting of obesity, hyperlipidemia, and insulin resistance. this form of steatosis is also similar to the type of fatty infiltration which occurs from excessive alcohol consumption. metabolic steatosis is not triggered in anyway by the hepatitis c virus however the combination of this form of steatosis and the presence of hcv has been associated with a more rapid progression of fibrosis. the other type of steatosis found in patients with hcv is fatty infiltration that is directly elicited by the virus. though the precise mechanism is not well known, hcv - induced steatosis is recognized as the sole route for a direct cytopathic effect by the hepatitis c virus. this review will focus on the two different forms of steatosis and its implications on the natural history of hcv. the etiology for hepatic steatosis can be determined by the distribution and size of the lipid accumulation within the hepatocytes. microvesicular steatosis that is seen in reye 's syndrome and acute fatty liver disease of pregnancy occurs due to dysfunctions in -oxidation of free fatty acids and this can result in acute liver failure. 3 on the other hand, macrovesicular steatosis is the histologic finding in patients with non - alcoholic fatty liver disease (nafld). nafld is characterized by gross macrovesicular fatty change with lobular or portal inflammation in the absence of a significant alcohol history. this disease is frequently under - recognized and is now identified as the most common cause of cryptogenic cirrhosis. 4 nafld occurs in the setting of obesity, hyperlipidemia, and diabetes all of which is now accepted under one syndrome called the metabolic syndrome. the metabolic syndrome (also known as syndrome x, the deadly quartet, the insulin resistance syndrome, and the obesity dyslipidemia syndrome) consists of abdominal obesity leading to insulin resistance, hypertension, and hypertriglyceridemia and is now recognized as the major predisposition to hepatic steatosis. 5 - 6 the most widely supported theory recognizes insulin resistance as the major mechanism in the pathogenesis of hepatic steatosis. 7 - 11 in an autopsy report from 1990, investigators found that of 22 patients with histologically confirmed fatty liver disease, 20 were also obese and had diabetes mellitus. 12 the presumption that there is a causal relationship between the metabolic syndrome and disease progression in hcv makes sense given the connection between steatosis and necroinflammatory activity in patients with hcv. 13 - 14 previous studies have indicated that obesity is a risk factor independent of elevated alt levels which predict fibrosis progression. a study from spain found that there is a larger proportion of fast progressors and a lower proportion of slow progressors in patients with body mass index (bmi) levels of greater than 30 kg / m. 15 recognizing this risk factor may warrant weight loss in our obese patients just as much as alcohol abstinence in our patients with hcv. a small study of 19 patients showed that a weight loss of only 5.9 kg and a reduction in bmi by only 2 kg / m may account for a significant reduction in both steatosis and fibrosis progression in patients with hepatitis c. 16 thus, our more motivated patients with hepatitis c absolutely deserve the extra means available for weight loss, not only for cardiovascular health but also to reduce the risk of fibrosis and ultimately cirrhosis. diabetes was once thought to be an ailment secondary to the lack of insulin production but is now recognized as an illness of insulin resistance and resultant hyperinsulinemia. first line therapy now specifically targets insulin - mediated glucose utilization in the liver and peripheral tissue. 17 - 18 it has now been demonstrated that improvement occurs in both steatosis and the resultant inflammation by the use of insulin sensitizing agents. 19 therefore, utilization of these medications are becoming more and more necessary in our patients with non - alcoholic steatohepatitis and certainly in our patients with hcv and metabolic steatosis. the presence of steatosis on liver biopsy in patients with hepatitis c is more frequent when compared to other chronic liver diseases such as chronic hepatitis b and autoimmune hepatitis. 20 steatosis is also 2.5 times more prevalent in patients with hcv when compared to the general population. 21 the macrovesicular steatosis present in patients with hcv is also distributed in the periportal areas rather than the centrilobular region which is more commonly seen in nafld. 22 this all infers that the hepatitis c virus may be directly inducing steatosis in these patients rather than simply being an unrelated finding. it has been shown that hcv genotype 3 is independently associated with hepatocellular steatosis in patients with chronic hepatitis c. 23 furthermore, the severity of steatosis in these patients is directly related to the burden of the hcv rna load. this relationship between the hcv viral load and the magnitude of steatosis was not observed in other hcv genotypes. 24 it has also been noticed that the steatosis which was initially present in patients with genotype 3 infection resolves after a sustained virologic response is achieved through treatment with pegylated interferon- and ribavirin. 25 not only does the steatosis resolve with eradication of the virus but it also recurs if relapse transpires. all of these findings were only observed with the hcv genotype 3 virus and was not reproducible with other hcv genotypes. these findings all point to the ability of the hcv genotype 3 virus to directly induce steatosis, although the mechanism still remains elusive. thus, there seems to be two distinct forms of steatosis in patients with chronic hepatitis c. metabolic steatosis generally occurs in all genotypes of hcv infections and likely worsens the progression of hcv induced fibrosis. then there are those patients with genotype 3 infections who have a form of steatosis that is directly induced by the hepatitis c virus and which also resolves with successful treatment. these two forms of steatosis can certainly coexist in patients with genotype 3 infections with other underlying metabolic diseases. in these patients, we would expect that the steatosis would only partially resolve with successful eradication of the virus and that the remaining fatty infiltration is a result of nafld. most, if not all, studies to date have used hcv genotype 1 constructs in vitro, thus our current understanding of the process leading to lipid accumulation is limited. however, putative mechanisms exist where the presence of a particular viral component leads to lipid accumulation within the hepatocyte. hcv core protein has been studied at length in both cell cultures and in transgenic mice. 26 the core protein has been located at the surface of lipid droplets within the cytoplasm in cell cultures that are transfected with hcv while absent in control cells. 27 hcv core protein may be interacting with apolipoprotein aii which is a major component of high - density lipoproteins and this interaction may be causing hepatocellular steatosis. 28 other proposed mechanisms suggest an interaction between the core protein and retinoid x receptor (rxr) which is a transcriptional regulator that has many cellular functions, one of which is the metabolism of lipids. 29 other theories propose that the core protein induces oxidative stress within the mitochondria which leads to or contributes to lipid accumulation. 30 this notion was formulated due to the association between hepatic steatosis in transgenic mice and the presence of increased lipid peroxidation. 31 all of these premises point to a complex interaction between the hcv core protein and other components of the hepatocyte which ultimately contributes to the onset of steatosis. though the exact mechanism remains elusive, it seems firmly established that the hepatitis c virus, in and of itself, can directly induce cytoplasmic lipid accumulation. further studies examining the genotype 3 virus are warranted to further recognize the process involved in hcv - induced steatosis. it is difficult to ascertain whether hcv - induced steatosis and metabolic steatosis contribute equally to the overall disease progression in patients with hepatitis c. as discussed before, there have been many studies which have demonstrated a relationship between the severity of steatosis and the extent of fibrosis on liver biopsy specimens. however, the majority of studies to date have not separated the two types of steatosis when examining its affects on fibrosis progression in hcv patients. nonetheless, it has been generally accepted that either steatosis by itself aggravates fibrosis or the factors that are causing steatosis may be aggravating fibrosis. 32 there has also been some suggestion that the two forms of steatosis may act synergistically to trigger severe advancement of fibrosis in patients with genotype 3 infections. 32 overall, steatosis, whether metabolic or hcv induced, worsens the sequence of events leading to advanced fibrosis in patients with hcv and needs to be addressed when managing our patients with hcv. ultimately, further studies are warranted to distinguish viral steatosis between metabolic steatosis and both of their effects on fibrosis progression in patients with hcv. all in all, there is a clinically significant relationship between hcv infection and hepatic steatosis. the first type of steatosis occurs secondary to metabolic factors namely alcohol use or the metabolic syndrome. this form of steatosis is not initiated by the hepatitis c virus however it can very well increase the progression of fibrosis ultimately towards cirrhosis. the other form of steatosis occurs as a direct result of the hcv genotype 3 virus through complex interactions between the hcv core protein and the hepatocyte, the knowledge of which remains to be fully unravelled. it is important to recognize the category of steatosis present in our patients with hcv in order to properly treat them. those with metabolic steatosis warrant strict attention to weight loss and countering the effects of insulin resistance while focus on hcv eradication can be given to those with hcv - induced steatosis. it is well established that there is an association between hcv and steatosis and the mechanisms behind this relationship are currently being unravelled. in order to fully understand the intricate molecular processes involved in hcv genotype-3 induced hepatocellular steatosis, research should also continue to focus on therapies for insulin resistance and steatosis - induced fibrosis in chronic hepatitis c. in order to devise specific therapies for hcv - induced steatosis, further investigation needs to be done on the complex derangements in lipid metabolism with focus on the genotype 3 virus. | there are two discrete forms of steatosis that may be found in patients infected with hepatitis c virus (hcv). metabolic steatosis can coexist with hcv, regardless of genotype, in patients with risk factors such as obesity, hyperlipidemia, and insulin resistance. the second form of hepatic steatosis in hcv patients is a result of the direct cytopathic effect of genotype 3 viral infections. there have been proposed mechanisms for this process but it remains elusive. both categories of steatosis tend to hasten the progression of liver fibrosis and therefore prompt recognition and management should be initiated in patients with hcv and steatosis. the authors review the current understanding of the relationship between hepatitis c infection and hepatic steatosis and discuss future research directions. |
unless otherwise stated, all chemicals and solvents were purchased from commercial suppliers in reagent analytical grade quality and used directly as received, including dcm (emsure), acs, iso, reag. ph eur, dcm for analysis (m=84.93 g mol), water - free acn (aldrich), water - free toluene (aldrich), pvk (aldrich ; 3683505 g, secondary standard, mp 300 c, d=1.2, polydispersity2, average m=35 000). samples for pl quenching experiments were prepared either by drop - casting or spin - coating from solution onto glass or a glass / ito substrate. the film thicknesses were measured on a bruker dektakxt stylus profiling system, using a stylus - type radius of 2 m, a stylus force of 5 mg, and a lateral resolution of 0.33 m pt over 1000 m. the rhenium complex was prepared with slight modifications to literature methods.[21, 22 ] an equimolar amount of re(co)5cl (0.304 g, 0.84 mmol) and 2,2'bipyridine (0.131 g, 0.84 mmol) was dissolved in hot toluene (50 ml). the reaction mixture was stirred under reflux for 2 h. afterwards the solution was removed from the heat source and cooled down by an ice - salt mixture. the powder was further dried at a rotary evaporator (5 mbar for 1 h). h nmr (200 mhz, cd3cn) : =7.62 (t, 2 h, bipy - h 5 and 5 '), 8.18 (t, 2 h, bipy - h 4 and 4 '), 8.41 (d, 2 h, bipy - h 6 and 6 '), 9.00 ppm (d, 2 h, bipy - h 3 and 3 '). wavelength scans were taken on a perkinelmer lambda 1050 double monochromator spectrometer (source - doubling mirror) between 400700 nm in 2 nm steps, with a slit width of 2 nm and a detector response time of 0.2 s. signal - to - noise was optimized by attenuating the reference beam with internal attenuators (automatic 2 and 3 a attenuation). for all spectra, auto - zero (100 and 0 %) correction scans were taken (baseline correction). the spectrometer was equipped with a deuterium (d2) lamp as the uv source and a tungsten lamp as the visible and near - infrared source. photomultiplier r6872 and pmt were used for detection in the uv / vis, a peltier cooled ingaas detector for use in the 8002600 nm region, and a peltier cooled pbs detector for the range from 25003300 nm. pl spectra were recorded on a pti quantamaster 400 spectrofluorimeter with a continuous xenon arc lamp (75 w) light source in the emission range 185680 nm, a czerny turner type excitation monochromator (throughput 65 % at 300 nm) with a focal length of 300 mm, a excitation grating with 1200 lines per mm (300 nm blaze), an emission grating with 200 lines per mm (400 nm blaze), and a multimode pti pmt detector (model 914), with spectral response from 185 to 900 nm [quantum efficiency at 260 nm (peak) 25.4 % ]. for a schematic drawing of the experimental setup for pl quenching in acn solution, esr spectra were recorded on bruker emx x - band spectrometer (9.45 ghz) with a rectangular te102 cavity and oxford esr910 continuous flow he cryostat operating between 4 and 300 k. for solutions containing polar solvents, highly absorbing microwave radiation were soaked into quartz capillaries and placed in esr tubes, otherwise a small amount of the solutions was added directly into the esr tube. the samples were measured in the dark and under illumination with a halogen lamp to check for light - induced species. the samples were measured at various power levels from 20 w to 200 mw with a modulation amplitude of 1 gauss. unless otherwise stated, all chemicals and solvents were purchased from commercial suppliers in reagent analytical grade quality and used directly as received, including dcm (emsure), acs, iso, reag. ph eur, dcm for analysis (m=84.93 g mol), water - free acn (aldrich), water - free toluene (aldrich), pvk (aldrich ; 3683505 g, secondary standard, mp 300 c, d=1.2, polydispersity2, average m=35 000). samples for pl quenching experiments were prepared either by drop - casting or spin - coating from solution onto glass or a glass / ito substrate. the film thicknesses were measured on a bruker dektakxt stylus profiling system, using a stylus - type radius of 2 m, a stylus force of 5 mg, and a lateral resolution of 0.33 m pt over 1000 m. the rhenium complex was prepared with slight modifications to literature methods.[21, 22 ] an equimolar amount of re(co)5cl (0.304 g, 0.84 mmol) and 2,2'bipyridine (0.131 g, 0.84 mmol) was dissolved in hot toluene (50 ml). the reaction mixture was stirred under reflux for 2 h. afterwards the solution was removed from the heat source and cooled down by an ice - salt mixture. the powder was further dried at a rotary evaporator (5 mbar for 1 h). h nmr (200 mhz, cd3cn) : =7.62 (t, 2 h, bipy - h 5 and 5 '), 8.18 (t, 2 h, bipy - h 4 and 4 '), 8.41 (d, 2 h, bipy - h 6 and 6 '), 9.00 ppm (d, 2 h, bipy - h 3 and 3 '). wavelength scans were taken on a perkinelmer lambda 1050 double monochromator spectrometer (source - doubling mirror) between 400700 nm in 2 nm steps, with a slit width of 2 nm and a detector response time of 0.2 s. signal - to - noise was optimized by attenuating the reference beam with internal attenuators (automatic 2 and 3 a attenuation). for all spectra, auto - zero (100 and 0 %) correction scans were taken (baseline correction). the spectrometer was equipped with a deuterium (d2) lamp as the uv source and a tungsten lamp as the visible and near - infrared source. photomultiplier r6872 and pmt were used for detection in the uv / vis, a peltier cooled ingaas detector for use in the 8002600 nm region, and a peltier cooled pbs detector for the range from 25003300 nm. pl spectra were recorded on a pti quantamaster 400 spectrofluorimeter with a continuous xenon arc lamp (75 w) light source in the emission range 185680 nm, a czerny turner type excitation monochromator (throughput 65 % at 300 nm) with a focal length of 300 mm, a excitation grating with 1200 lines per mm (300 nm blaze), an emission grating with 200 lines per mm (400 nm blaze), and a multimode pti pmt detector (model 914), with spectral response from 185 to 900 nm [quantum efficiency at 260 nm (peak) 25.4 % ]. for a schematic drawing of the experimental setup for pl quenching in acn solution, esr spectra were recorded on bruker emx x - band spectrometer (9.45 ghz) with a rectangular te102 cavity and oxford esr910 continuous flow he cryostat operating between 4 and 300 k. for solutions containing polar solvents, highly absorbing microwave radiation were soaked into quartz capillaries and placed in esr tubes, otherwise a small amount of the solutions was added directly into the esr tube. the samples were measured in the dark and under illumination with a halogen lamp to check for light - induced species. the samples were measured at various power levels from 20 w to 200 mw with a modulation amplitude of 1 gauss. as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors | this work investigates the photoinduced energy transfer from poly(n - vinylcarbazole) (pvk), as a donor material, to fac-(2,2'-bipyridyl)re(co)3cl, as a catalyst acceptor, for its potential application towards co2 reduction. photoluminescence quenching experiments reveal dynamic quenching through resonance energy transfer in solid donor / acceptor mixtures and in solid / liquid systems. the bimolecular reaction rate constant at solution film interfaces for the elementary reaction of the excited state with the quencher material could be determined as 8.8(1.4)1011 l mol1 s1 by using stern volmer analysis. this work shows that pvk is an effective and cheap absorber material that can act efficiently as a redox photosensitizer in combination with fac-(2,2'-bipyridyl)re(co)3cl as a catalyst acceptor, which might lead to possible applications in photocatalytic co2 reduction. |
the eurodiab prospective complications study (pcs) is a follow - up of the eurodiab iddm complications study (17). full details of the design, methods, and recruitment to the eurodiab cohort have been published elsewhere (17,18). baseline investigations were performed on 3,250 men and women with type 1 diabetes aged between 15 and 60 years and recruited from 31 centers in 16 european countries. the sampling frame contained all type 1 diabetic patients attending at least once in the last year for each center. sample selection was stratified by age, sex, and duration of diabetes to ensure sufficient representation in all categories (17). type 1 diabetes was defined as a diagnosis made before the age of 36 years, with a need for continuous insulin therapy within a year of diagnosis. of those invited, 85% participated. the local ethics committees approved this study at each center, and all people provided written informed consent. definition of severe hypoglycemia was based on information obtained from questionnaires completed by subjects at both the baseline and the follow - up visit. all patients were asked, over the past year, how many hypoglycemic attacks have you had, serious enough to require the help of another person ? the questionnaire also provided information on physical activity, smoking habits, frequency of insulin injections, and number of daily insulin units injected per kilogram body weight (17,19). at the follow - up examination, information on numbers of nonsevere hypoglycemic episodes, defined as hypoglycemic episodes over the past year not requiring the help of another person, was also collected. all patients were recalled for follow - up assessment 68 years later. at the time of the follow - up study, data on mortality and morbidity forms were collected from available hospital case notes or other sources of clinical data in every participating center, detailing cause of death or the presence or absence of severe complications at their most recent visit. when death certificates could not be obtained, information considering the cause of death was reported by the physician or extracted from the hospital record. in the whole eurodiab cohort, cause of death could not be obtained for 35 of 102 deceased subjects because of legal restrictions. causes of death were coded according to the icd-9 classification and assigned to different categories, such as coronary heart disease, stroke, other cvd, non - cvd, and unknown. two observers (n.c. and j.h.f.) separately allocated cause of death with 100% agreement. cvd was defined as a positive medical history of a cv event, including myocardial infarction, angina pectoris, coronary artery bypass graft and/or stroke, and/or ischemic changes on a conventional 12-lead electrocardiogram (ecg) (18). ecg abnormalities suggestive of probable ischemia consist of major q / qs waves and complete left bundle branch block. possible ischemia consists of minor q waves, st segment abnormalities, and t wave abnormalities (18,20). hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg and/or the current use of blood pressure lowering drugs (21). retinopathy was assessed by centrally graded retinal photographs, and each patient s level of retinopathy (absent, nonproliferative, or proliferative) was defined by the level of the worst eye (20). nephropathy was assessed using the albumin excretion rate (aer) calculated centrally from a single, timed, 24-h urine collection (22). distal symmetrical polyneuropathy (dsp) was assessed on the basis of neuropathic symptoms and signs, including measurement of vibration perception threshold. the aer value was defined as normal (< 20 g / min), microalbuminuria (20 and < 200 g / min), or macroalbuminuria (200 g / min). glomerular filtration rate (gfr) was estimated using the four - component abbreviated equation from the modification of diet in renal disease study group (23). subjects with an estimated glomerular filtration rate (egfr) < 60 ml / min/1.73 m were defined as having chronic kidney disease. additional measurements included triglycerides, total cholesterol, hdl cholesterol, and hba1c. a cross - sectional, nested case - control study was designed at the follow - up examination (2430). case subjects were selected to have the greatest complication burden as possible to provide sufficient numbers for subgroup analyses. thus, case subjects were all those with cvd, proliferative retinopathy, or micro / macroalbuminuria at follow - up. blood samples from both case subjects and control subjects were collected at the follow - up examination, and markers of both endothelial damage and inflammation were measured. soluble vascular cell adhesion molecule 1 (svcam-1) (25), soluble e - selectin (s - e - selectin) (25), interleukin 6 (il-6) (25,27), tumor necrosis factor- (tnf-) (25,27), heat shock protein 27 (hsp27) (28), and anti - hsp60 and anti - hsp70 antibodies (29) were measured by commercially available elisa (r&d systems, oxon, u.k. ; hsp27, calbiochem san diego, ca ; anti - hsp60 and anti - hsp70 antibodies : anti eks-650 and eks-750, stressgen biotechnologies corporation). plasma levels of c - reactive protein (crp) were measured by an in - house elisa (26,27). variables with skewed distributions were logarithmically transformed for statistical analysis and expressed as geometric mean (25th75th percentile). follow - up duration for fatal and nonfatal cvd incidence was calculated as the time between the baseline examination and date of first event (myocardial infarction, angina pectoris, coronary artery bypass graft, or stroke), date of abnormal ecg finding suggestive of ischemia, date of loss to follow - up, or date of follow - up examination. as there was little variation in follow - up time among individuals, logistic regression was used to analyze the association of severe hypoglycemic episodes at baseline with incidence of fatal and nonfatal cvd. odds ratios (ors) were adjusted for age, sex, diabetes duration, and dsp at baseline. in addition, in models 2 and 3, adjustment was also performed for nonsevere and severe hypoglycemic episodes at the follow - up examination, respectively. in the nested case - control study, logistic regression analysis was used to estimate the ors of severe and nonsevere hypoglycemia for all complications and cvd, independently of age and sex. variables were retained in the final model if they added significantly to the likelihood of models or to the estimated coefficients of predictors. the 2 log likelihood ratio test was used to assess the overall significance of models. all reported p values are two sided, and a p value of < 0.05 was considered to indicate statistical significance. all analyses were performed with stata (stata release 10.0 ; stata corporation, college station, tx). definition of severe hypoglycemia was based on information obtained from questionnaires completed by subjects at both the baseline and the follow - up visit. all patients were asked, over the past year, how many hypoglycemic attacks have you had, serious enough to require the help of another person ? the questionnaire also provided information on physical activity, smoking habits, frequency of insulin injections, and number of daily insulin units injected per kilogram body weight (17,19). at the follow - up examination, information on numbers of nonsevere hypoglycemic episodes, defined as hypoglycemic episodes over the past year not requiring the help of another person, was also collected. all patients were recalled for follow - up assessment 68 years later. at the time of the follow - up study, data on mortality and morbidity forms were collected from available hospital case notes or other sources of clinical data in every participating center, detailing cause of death or the presence or absence of severe complications at their most recent visit. when death certificates could not be obtained, information considering the cause of death was reported by the physician or extracted from the hospital record. in the whole eurodiab cohort, cause of death could not be obtained for 35 of 102 deceased subjects because of legal restrictions. causes of death were coded according to the icd-9 classification and assigned to different categories, such as coronary heart disease, stroke, other cvd, non - cvd, and unknown. two observers (n.c. and j.h.f.) separately allocated cause of death with 100% agreement. cvd was defined as a positive medical history of a cv event, including myocardial infarction, angina pectoris, coronary artery bypass graft and/or stroke, and/or ischemic changes on a conventional 12-lead electrocardiogram (ecg) (18). ecg abnormalities suggestive of probable ischemia consist of major q / qs waves and complete left bundle branch block. possible ischemia consists of minor q waves, st segment abnormalities, and t wave abnormalities (18,20). hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg and/or the current use of blood pressure lowering drugs (21). retinopathy was assessed by centrally graded retinal photographs, and each patient s level of retinopathy (absent, nonproliferative, or proliferative) was defined by the level of the worst eye (20). nephropathy was assessed using the albumin excretion rate (aer) calculated centrally from a single, timed, 24-h urine collection (22). distal symmetrical polyneuropathy (dsp) was assessed on the basis of neuropathic symptoms and signs, including measurement of vibration perception threshold. the aer value was defined as normal (< 20 g / min), microalbuminuria (20 and < 200 g / min), or macroalbuminuria (200 g / min). glomerular filtration rate (gfr) was estimated using the four - component abbreviated equation from the modification of diet in renal disease study group (23). subjects with an estimated glomerular filtration rate (egfr) < 60 ml / min/1.73 m were defined as having chronic kidney disease. a cross - sectional, nested case - control study was designed at the follow - up examination (2430). case subjects were selected to have the greatest complication burden as possible to provide sufficient numbers for subgroup analyses. thus, case subjects were all those with cvd, proliferative retinopathy, or micro / macroalbuminuria at follow - up. blood samples from both case subjects and control subjects were collected at the follow - up examination, and markers of both endothelial damage and inflammation were measured. soluble vascular cell adhesion molecule 1 (svcam-1) (25), soluble e - selectin (s - e - selectin) (25), interleukin 6 (il-6) (25,27), tumor necrosis factor- (tnf-) (25,27), heat shock protein 27 (hsp27) (28), and anti - hsp60 and anti - hsp70 antibodies (29) were measured by commercially available elisa (r&d systems, oxon, u.k. ; hsp27, calbiochem san diego, ca ; anti - hsp60 and anti - hsp70 antibodies : anti eks-650 and eks-750, stressgen biotechnologies corporation). plasma levels of c - reactive protein (crp) were measured by an in - house elisa (26,27). variables with skewed distributions were logarithmically transformed for statistical analysis and expressed as geometric mean (25th75th percentile). follow - up duration for fatal and nonfatal cvd incidence was calculated as the time between the baseline examination and date of first event (myocardial infarction, angina pectoris, coronary artery bypass graft, or stroke), date of abnormal ecg finding suggestive of ischemia, date of loss to follow - up, or date of follow - up examination. as there was little variation in follow - up time among individuals, logistic regression was used to analyze the association of severe hypoglycemic episodes at baseline with incidence of fatal and nonfatal cvd. odds ratios (ors) were adjusted for age, sex, diabetes duration, and dsp at baseline. in addition, in models 2 and 3, adjustment was also performed for nonsevere and severe hypoglycemic episodes at the follow - up examination, respectively. in the nested case - control study, logistic regression analysis was used to estimate the ors of severe and nonsevere hypoglycemia for all complications and cvd, independently of age and sex. variables were retained in the final model if they added significantly to the likelihood of models or to the estimated coefficients of predictors. the 2 log likelihood ratio test was used to assess the overall significance of models. all reported p values are two sided, and a p value of < 0.05 was considered to indicate statistical significance. all analyses were performed with stata (stata release 10.0 ; stata corporation, college station, tx). of the 3,250 patients recruited at baseline, response data on severe hypoglycemic episodes were available for 3,248 (99.9%) subjects. out of these subjects, out of these 2,899 subjects, vital status at follow - up was not obtained for 383 people because of the following reasons : four local centers did not participate in the prospective study (n = 358), 7 patients did not fulfill the inclusion criteria, and 18 patients had an unknown vital status. we also excluded 335 subjects with missing morbidity and/or mortality (22 of 74) data, resulting in 2,181 individuals with complete data. baseline distribution of severe hypoglycemic episodes did not differ (p = 0.46) between patients remaining in the study (none, 1,495 [68.5% ] ; one to two, 418 [19.2% ] ; three or more, 268 [12.3% ] severe hypoglycemic episodes) and those who were lost at follow - up (none, 477 [66.5% ] ; one to two, 141 [19.6% ] ; three or more, 100 [13.9% ] severe hypoglycemic episodes), including subjects with unknown cause of death (none, 14 of 22 [63.6% ] ; one to two, 2 of 22 [9% ] ; three or more, 6 of 22 [27.2% ]). people who had experienced three or more recent severe hypoglycemic episodes had lower hba1c values but longer duration of diabetes with higher prevalence of both retinopathy and dsp. baseline characteristics and 7.3-year cvd incidence of type 1 diabetic patients according to hypoglycemic categories after a median follow - up of 7.3 years (interquartile range 6.97.9), 176 patients had incident cvd (either fatal or nonfatal). the percentages of case subjects with incident cvd were similar among patients with none (7.6% ; n = 114 ; 12 fatal and 102 nonfatal), one to two (7.9% ; n = 33 ; 3 fatal and 30 nonfatal), and three or more severe hypoglycemic episodes (10.8% ; n = 29 ; 0 fatal and 29 nonfatal). the proportion of patients who had fatal / nonfatal cv events was comparable (p = 0.22) in patients reporting (9.0%) and not reporting (7.5%) severe hypoglycemia at baseline as well as at follow - up (nonfatal only, 9.1 and 7.8% ; p = 0.40). numbers of severe hypoglycemic events at baseline and at follow - up showed a statistically significant correlation (r = 0.29 ; p = < 0.0001). moreover, a significant correlation between numbers of severe and total hypoglycemic episodes (r = 0.57 ; p < 0.0001) was found at the follow - up examination. logistic regression analysis showed that severe hypoglycemia at the baseline examination was not associated with incident fatal and nonfatal cvd (table 2). further adjustment for numbers of both severe and nonsevere hypoglycemic events at follow - up did not modify ors for nonfatal cvd. role of severe hypoglycemia in incidence of cvd in the prospective study ; results of logistic regression analyses as shown in table 3, almost one - third (33%) of the 531 subjects of the nested case - control study declared one or more severe hypoglycemic episodes over the past year. nineteen percent of the patients had one to two episodes and 14% had three or more episodes. those with vascular complications had a more adverse risk factor profile than control individuals, as we have previously reported (2931). there were no significant differences in serum crp, il-6, svcam, hsp27, anti - hsp60, and anti - hsp70 among groups. differences in e - selectin levels were clinically not relevant, though statistically significant. in case subjects, tnf- values were lower in patients who did not experience any severe hypoglycemic episode than in patients with severe hypoglycemic episodes. however, after adjustment for egfr, the difference was no longer significant (p = 0.84). adjustment for treatment with beta blockers and lipid - lowering therapy did not modify observed associations. as shown in table 4, in logistic regression analysis, people with severe hypoglycemic episodes at follow - up had a 53% (or 0.47 [95% ci 0.230.93 ]) lower risk of cvd after adjustment for age and sex, and independently of main confounders (diabetes duration, systolic blood pressure, log aer, ldl cholesterol, smoke, and log tnf-). however, after further adjustment for hba1c, this association was no longer significant (0.61 [0.281.30 ]). nonsevere hypoglycemic episodes in the past year were not associated with either all complications or cvd. finally, further adjustments for treatment with beta blockers (all complications or 0.62 [0.331.15 ] ; cvd 0.58 [0.271.26 ]) and lipid - lowering drugs (all complications or 0.62 [0.331.16 ] ; cvd 0.59 [0.271.27 ]) did not modify observed associations. variables associated with severe hypoglycemia in the nested case - control study of the eurodiab pcs result of logistic regression analysis of the case - control study within the eurodiab pcs after a median follow - up of 7.3 years (interquartile range 6.97.9), 176 patients had incident cvd (either fatal or nonfatal). the percentages of case subjects with incident cvd were similar among patients with none (7.6% ; n = 114 ; 12 fatal and 102 nonfatal), one to two (7.9% ; n = 33 ; 3 fatal and 30 nonfatal), and three or more severe hypoglycemic episodes (10.8% ; n = 29 ; 0 fatal and 29 nonfatal). the proportion of patients who had fatal / nonfatal cv events was comparable (p = 0.22) in patients reporting (9.0%) and not reporting (7.5%) severe hypoglycemia at baseline as well as at follow - up (nonfatal only, 9.1 and 7.8% ; p = 0.40). numbers of severe hypoglycemic events at baseline and at follow - up showed a statistically significant correlation (r = 0.29 ; p = < 0.0001). moreover, a significant correlation between numbers of severe and total hypoglycemic episodes (r = 0.57 ; p < 0.0001) was found at the follow - up examination. logistic regression analysis showed that severe hypoglycemia at the baseline examination was not associated with incident fatal and nonfatal cvd (table 2). further adjustment for numbers of both severe and nonsevere hypoglycemic events at follow - up did not modify ors for nonfatal cvd. role of severe hypoglycemia in incidence of cvd in the prospective study ; results of logistic regression analyses as shown in table 3, almost one - third (33%) of the 531 subjects of the nested case - control study declared one or more severe hypoglycemic episodes over the past year. nineteen percent of the patients had one to two episodes and 14% had three or more episodes. those with vascular complications had a more adverse risk factor profile than control individuals, as we have previously reported (2931). there were no significant differences in serum crp, il-6, svcam, hsp27, anti - hsp60, and anti - hsp70 among groups. differences in e - selectin levels were clinically not relevant, though statistically significant. in case subjects, tnf- values were lower in patients who did not experience any severe hypoglycemic episode than in patients with severe hypoglycemic episodes. however, after adjustment for egfr, the difference was no longer significant (p = 0.84). adjustment for treatment with beta blockers and lipid - lowering therapy did not modify observed associations. as shown in table 4, in logistic regression analysis, people with severe hypoglycemic episodes at follow - up had a 53% (or 0.47 [95% ci 0.230.93 ]) lower risk of cvd after adjustment for age and sex, and independently of main confounders (diabetes duration, systolic blood pressure, log aer, ldl cholesterol, smoke, and log tnf-). however, after further adjustment for hba1c, this association was no longer significant (0.61 [0.281.30 ]). nonsevere hypoglycemic episodes in the past year were not associated with either all complications or cvd. finally, further adjustments for treatment with beta blockers (all complications or 0.62 [0.331.15 ] ; cvd 0.58 [0.271.26 ]) and lipid - lowering drugs (all complications or 0.62 [0.331.16 ] ; cvd 0.59 [0.271.27 ]) did not modify observed associations. variables associated with severe hypoglycemia in the nested case - control study of the eurodiab pcs result of logistic regression analysis of the case - control study within the eurodiab pcs in this study, we found that frequency of severe hypoglycemia at baseline was not a predictor of subsequent cv events. in addition, hypoglycemic episodes, both severe and nonsevere, were not cross - sectionally associated with a rise in the circulating levels of markers of both inflammation and endothelial injury. therefore, our data do not support the hypothesis that in type 1 diabetic patients, severe hypoglycemia increases the risk of cvd. in the prospective study, furthermore, the proportion of patients who experienced cv events was comparable in patients reporting and not reporting severe hypoglycemic episodes at baseline. finally, results of logistic regression analysis showed that severe hypoglycemia at the baseline examination was not associated with incident fatal and nonfatal cvd. indeed, in the dcct study in type 1 diabetic patients, a glucose - lowering therapy that dramatically increased the risk of severe hypoglycemia did not cause long - term cv harm (911). furthermore, a recent meta - analysis of the four major intervention trials in type 2 diabetic patients (uk prospective diabetes study, action to control cardiovascular risk in diabetes, action in diabetes and vascular disease : preterax and diamicron modified - release controlled evaluation, and veterans affairs diabetes trial) has shown that intensive therapy, carrying a 2.5-fold increased risk of severe hypoglycemic events, reduced, though modestly, the overall risk of first occurrence of major cv events (32). however, intervention trials do not explore the direct role of hypoglycemia, but only if an intervention, increasing the risk of hypoglycemia, enhances the risk of cv outcomes. whether hypoglycemia is a risk factor for future cv events is best assessed in a prospective epidemiological study (33), and ours is the first large, clinically based, epidemiological study exploring the relationship between hypoglycemia and incident cvd in type 1 diabetic patients. the epidemiological analysis of the steno-2 trial has shown no association between hypoglycemic events and both total and cv mortality during the 13-year follow - up (34). in a recent epidemiological analysis of the action to control cardiovascular risk in diabetes trial, participants with severe hypoglycemic episodes had an increased risk of all - cause mortality (35). likewise, various prospective epidemiological studies have reported an increased all - cause mortality in diabetic patients with myocardial infarction who experienced in - hospital hypoglycemic episodes (6,8,36,37). however, data on cv events were either not available or not significant in these studies. this raises the hypothesis that people who are prone to hypoglycemia are at higher risk of death due to the coexistence of other risk factors, such as hepatic disease, renal disease, cognitive decline, cancer, and medications, rather than because of enhanced cv risk (33). in the nested case - control study, we found no association between frequencies of severe hypoglycemic episodes over the past year and markers of both inflammation / stress response (crp, il-6, tnf-, anti - hsp60, and anti - hsp70) and vascular injury (svcam and s - e - selectin). a significant increase in tnf- levels was observed in case subjects, but it was no longer present after adjustment for renal function. previous reports have shown that hypoglycemia induces an acute increase in markers of inflammation / endothelial injury (1316). our results are not in conflict with these findings, as hypoglycemia - induced changes are transient and our measurements were not performed immediately after a hypoglycemic episode. however, our data show that the cumulative and deleterious effects of repeated / severe hypoglycemic episodes over the previous year did not alter the circulating levels of markers, potentially linking hypoglycemia to diabetic vascular complications. therefore, our data indicate that hypoglycemia does not induce sustained changes in markers of potential relevance in cvd and argues against the hypothesis that hypoglycemia - induced changes in nontraditional cv risk factors play a long - term role in cvd. we acknowledge that in the case subjects, who were selected to have the highest burden of complications, small hypoglycemia - induced differences in markers of inflammation / endothelial injury might have been masked by major changes due to concurrent micro / macrovascular disease ; however, no differences were found in the control subjects, who were completely free of complications. furthermore, a role for hypoglycemia - induced inflammation / vascular damage in cvd has been proposed particularly for people with pre - existing vascular disease (1). an important strength of our work is the prospective clinically based design, evaluating a large, multicenter, european cohort of type 1 diabetic patients. type 2 diabetes is often associated with numerous other cv risk factors, so that a disentangled analysis may remain open to imprecision, and type 1 diabetes offers a better model. however, cv events are relatively rare among younger type 1 diabetic patients and a similar study could be carried out only in such a large population as the one studied prospectively by the eurodiab pcs. first, we can not exclude the possibility that in our work the consequences of hypoglycemia were underestimated because we relied on self - reports of spontaneous hypoglycemia and, thus, some patients were probably misclassified. validation of suspected hypoglycemic episodes through self - measurement of blood glucose levels would have improved the accuracy in identifying the patients with true severe hypoglycemia. however, patients may not experience hypoglycemic symptoms severe enough to prompt a measurement of finger stick glucose, even when blood glucose levels fall below 50 mg / dl because of hypoglycemia unawareness. we acknowledge that only studies based on continuous glucose monitoring would provide unbiased definitive data. however, the feasibility and/or practicality of recruiting large numbers of subjects to assess person - years using continuous glucose monitoring - based methods and/or observations for cv risk assessment is currently limited. second, the potential benefits for vascular complications of better glucose control, leading to increased risk of severe hypoglycemia, may have mitigated the deleterious consequences of hypoglycemia, thus obscuring any harm. indeed, in the dcct study, participants in the intensive therapy group had a 41% reduction in cv events at the end of the active treatment period despite a much higher risk of severe hypoglycemia. furthermore, analyses suggested that the difference in hba1c during the active treatment period accounted for the cv benefit, though the risk of severe hypoglycemia was strongly and inversely correlated to the achieved hba1c (10,11). consistently, in our case - control analysis, severe hypoglycemia was protective with respect to cvd, but this effect was merely due to hba1c. on the other hand, potential deleterious effects of severe hypoglycemia on cv outcomes may be overestimated because patients experiencing severe hypoglycemia had a longer duration of diabetes and greater burden of microvascular complications. however, our results were unaltered by adjustment for both diabetes duration and microvascular complications. third, both prospective analysis on incidence of cvd and case - control analysis at the follow - up examination were based on severe hypoglycemic episodes occurring in the previous year and no data were available throughout the follow - up period. however, we found a correlation between baseline and follow - up of severe hypoglycemic events in keeping with the dcct results, showing that prior episodes of severe hypoglycemia are the strongest predictor of risk of future episodes (9). fourth, repeated mild / modest hypoglycemic episodes may have a greater impact on cvd than severe ones, and we had no baseline information on nonsevere hypoglycemic episodes. however, at follow - up, severe episodes correlated with total hypoglycemic episodes, and inclusion of nonsevere hypoglycemic episodes in the logistic analyses did not alter the results. fifth, the release of the confidential cause of death data was difficult due to legal restrictions in a number of countries, which led to a relatively large number of unknown causes of death. however, based on the incidence of cv death in the eurodiab cohort, the few missing cases of cv death would have had a negligible effect on the final results. in addition, the baseline distribution of severe hypoglycemic episodes did not differ between patients remaining in the study and those who were lost to follow - up, including subjects with unknown causes of death. sixth, type 1 diabetes was defined based on age and insulin treatment ; however, only patients with continuous insulin therapy initiated < 1 year from diagnosis were selected, making misclassification of diabetes unlikely. finally, in the nested case - control study, the number of control subjects was lower than the overall number of case subjects, thus reducing the power of analyses. in conclusion, our data suggest that severe hypoglycemic episodes in type 1 diabetes are not associated with changes in either nontraditional cv risk factors or an increase in the incidence of cvd. therefore, our data do not support the hypothesis that severe hypoglycemia in patients with type 1 diabetes increases the risk of cvd. | objectivefrequent episodes of severe hypoglycemia may increase the risk of cardiovascular disease (cvd) in people with diabetes. our aim was to study the relationship between severe hypoglycemic episodes and cvd incidence in subjects with type 1 diabetes, and further, to assess if markers of inflammation / endothelial injury were enhanced in individuals who experienced hypoglycemic episodes.research design and methodsthe prospective study included 2,181 type 1 diabetic patients from the eurodiab prospective complications study. at baseline, frequency of self - reported severe hypoglycemia, defined as episodes serious enough to require the help of another person, was assessed based on responses to a patient questionnaire. both fatal / nonfatal cvd was assessed 7.3 years after baseline examination. at the follow - up visit, data on both severe and nonsevere hypoglycemic episodes in the previous year were collected through a questionnaire and markers of inflammation / stress response / endothelial injury measured by enzyme - linked immunosorbent assays in the 531 subjects of the nested case - control study, including 363 case subjects with one or more complications of diabetes and 168 control subjects with no evidence of any complication.resultsduring the follow - up period, 176 patients had incident cvd. logistic regression analysis showed that severe hypoglycemia at the baseline examination was not associated with incidence of cvd (adjusted odds ratios [95% ci ] : one to two episodes, 0.87 [0.551.37 ] ; three or more episodes, 1.09 [0.681.75 ]). furthermore, follow - up serum levels of markers of endothelial damage / inflammation were not cross - sectionally associated with the frequency of hypoglycemic episodes.conclusionstaken together our data do not support the hypothesis that in type 1 diabetes, severe hypoglycemia increases the risk of cvd. |
burnout among general practitioners (gps) appears to be common, with reported prevalence proportions varying from approximately 12% to 42%. the span in prevalence proportions may reflect actual differences in examined populations stemming from variations in demographic characteristics and in organization of general practice. methodological issues such as differences in applied cutoffs for classification of burnout cases and cultural variations concerning the attitude to disclose poor well being may of course also play a role. burnout is a psychological construct defined as a prolonged response to chronic emotional and interpersonal stressors on the job and is characterized by emotional exhaustion, depersonalization, and a subjective experience of decreased personal accomplishment. the negative effects of burnout on gps ' job performance are controversial. on the one hand, burnout has been associated with decreased empathy in medical students and with an increased number of self - reported medical errors in surgeons. on the other hand, two studies did not reveal significant associations between depersonalisation and patient - rated interpersonal skills and between burnout symptoms and the gps ' awareness of patients ' psychosocial problems. studies of burnout have often used a cross - sectional design and there is no good knowledge of the incidence and the risk of burnout. taken the supposedly adverse effects of burnout into consideration, it becomes highly important to establish knowledge about the risk of burnout and gain insight into possible preventive initiatives. various personal and occupational factors have been associated with burnout such as working hours, list size, and number of practice partners. being married and having children have been shown to be associated with less burnout in gps, but other studies have not supported such associations [2, 10, 11 ]. likewise, results concerning the role of age and seniority are inconsistent [6, 10 ]. women have fairly consistently been shown to report less depersonalization than men [6, 9, 12 ] but have also been shown to report higher levels of emotional exhaustion than their male colleagues. the results of one cross - sectional study have revealed an association between the intention to change medical specialty and low job satisfaction. while it seems likely that low job satisfaction can boost intentions to change specialty, the reverse causality would imply important perspectives. if wishing to change specialty is a precursor of burnout, this knowledge would be very important to, for example, colleagues and supervisors who guide gps. furthermore, a few studies have shown that lower job satisfaction is linked to intentions to withdraw from general practice [13, 15, 16 ], but whether job turnover rates are actually higher in gps with burnout compared to gps without burnout is unknown. on this background, the purpose of this study was to analyse (1) the risk of burnout among a sample of danish gps during a 7-year follow - up period, (2) whether thoughts about changing medical specialty increase the risk of burnout, and (3) whether the job turnover rates are higher for gps with burnout than for gps without burnout. all gps in denmark are independent contractors with the regional health authorities, and they are fully responsible for the organization of the work in their practice., the practice has to be open from 8 a.m. until 4 p.m., from monday to friday. the patient list size is on average 1550 patients per gp (including children) and 99% of citizens are registered with a particular general practice, which they have to consult. gps act as gatekeepers to the rest of the health care system except for emergencies. gps are remunerated on a mix of capitation and fee - for - service (25/75%). in may 2004, all 458 active gps in the county of aarhus, denmark, were invited to participate in a survey on job satisfaction, burnout, and working conditions (the gp profile 2004). the active gps were identified by the registry of health providers, which is managed by the national board of health. nonrespondents were sent a reminder with a new questionnaire after four weeks, and gps were remunerated with 16 for responding. in january 2012, the survey on job satisfaction, burnout, and working conditions was repeated (the gp profile 2012) and all active gps in the central denmark region (which since 2007 has included the former county of aarhus) were invited to participate. non - respondents were sent a reminder after four and thirteen weeks and gps were remunerated in the amount of 50 for responding. according to danish law, the study was not submitted to an ethical committee since questionnaire surveys do not require an ethical approval. the questionnaire included a burnout scale and items about practice organisation (solo or group practice), provision of walk - in open access, weekly working hours in practice, monthly working hours in out - of - hour primary care, time used per consultation, and marital status for the gp. we used the maslach burnout inventory human - services - survey (mbi - hss), which is considered to be a valid instrument for assessment of burnout symptoms. the mbi - hss consists of 22 items and each item is scored on a 7-point likert scale. the 22 items are divided into three subscales : (1) emotional exhaustion (9 items), (2) depersonalization (5 items), and (3) personal accomplishment (8 items). each subscale receives a score which is categorised as low or high based on normative population score. a high level of emotional exhaustion is defined as a score > 26, and a high level of depersonalization is defined as a score > 9. although it has been argued that the mbi subscale scores should be treated as continuous data, a categorical approach has often been applied [1, 2, 9, 11, 12 ]. in this study, a moderate degree of burnout was defined as a high score on the emotional exhaustion subscale and/or a high score on the depersonalization subscale. a high score on the emotional exhaustion and depersonalization subscales and a low score on the personal accomplishment subscale are defined as a high degree of burnout. thoughts about leaving practice were assessed with one question : would family medicine still be your choice if you were to select your medical specialty today ? the answers were yes, i definitely would, yes, i probably would, neutral, no, i would probably not, or no, i would definitely not. the answers yes, i definitely would and yes, i probably would were categorized as yes and the answers no, i would probably not or no, i would definitely not were categorized as no. from the registry of health providers, we received data on all the individual gps in relation to date when starting in a specific practice, changing to another practice or stopping as gp. data on age and gender was also included. from the national health insurance registry, we got data on the number of listed patients per gp at may 1, 2004, and at february 1, 2012, and number of consultations provided per gp in 2003 and in 2011. the incidence of burnout was estimated as the proportion of new cases of burnout in the 2012 survey to number of gps who were burnout - free in 2004. we calculated the association between new cases of burnout and possible risk factors assessed in 2004, including sex, age, marital status, practice organisation, open access, weekly working hours in practice, monthly working hours in out - of - hour primary care, estimated time uses per consultation, number of enrolled patients, number of provided consultations, and the gps ' answer to the hypothetical question whether general practice would still be their choice if they were to select medical specialty today. the association was calculated as odds ratio (or) in a logistic regression model. we calculated the crude or and an adjusted or controlling for the influence of sex and age. as some of the gps were working in the same practice, analyses were corrected for clusters of gps within the same practice using robust variance estimates. p values of 5% or less were considered statistically significant and data was analyzed using stata 11. figure 1 provides an overview of the response and dropout at the two measurement points. in this study, 105 (28%) the panel group, that is, gps who had completed both surveys, consisted of 216 gps. the 105 retired gps were older than the 216 gps in the panel group as well as the 59 nonrespondents who did not complete the 2012 survey but were still active (p < 0.001). characteristics of the 381 gps who participated in the 2004 survey and the 216 gps who participated in both surveys are shown in table 1 stratified for gender. the proportion of gps indicating high levels of emotional exhaustion, depersonalization, and low levels of personal accomplishment is shown in table 1. the proportion of female gps reporting high emotional exhaustion had increased from 8.7% in 2004 to 18.6% in 2012. among men the proportions of male and female gps reporting high levels of depersonalization and low levels of personal accomplishment had decreased from 2004 to 2012 as the proportion of male gps had reached the criteria for moderate burnout (from 25.0% in 2004 to 18.5% in 2012). among females, the proportion of gps with moderate burnout had increased from 20.8% to 23.7%. with respect to a severe degree of burnout, the proportion had increased slightly (2.6% in 2004 to 3.7% in 2012). a total of 152 gps were burnout - free in 2004 and in 2012 ; 20 new cases of moderate or severe degree of burnout were revealed, resulting in a risk of developing a moderate or high degree of burnout during the 7-year followup on 13.2% (95% ci : 8.219.6%). for male gps, the risk was 11.9% (95% ci : 5.920.8%) and for women was 14.7% (95% ci : 7.325.4% ; p = 0.637). of those with burnout in 2004, 26 (40.6%) were without burnout in 2012. associations between new cases of burnout and possible risk factors assessed in 2004 are depicted in table 2. gps who were inconclusive or reported no to the question about choosing family practice as medical specialty again had a 4.5 (95% ci : 1.216.5, p = 0.023) risk of being a new case of burnout in the 2012 survey compared to gps who answered that they would choose family practice as specialty again. twenty - two (25.0%) of the 88 gps with burnout in 2004 had withdrawn from general practice during followup. in comparison, 78 (28.8%) of the 271 gps who were burnout - free in 2004 had left general practice during followup. adjusting for age and sex, there was no association between burnout status in 2004 and retirement rates during followup (or = 0.99 ; 95% ci = 0.482.03 ; p = 0.975 ; data not shown). the risk of developing burnout in this sample of danish gps during the 7-year followup was 13.2%. a greater proportion of gps who had developed burnout during the followup reported in 2004 that they were inconclusive or would not choose family medicine if they were to select medical specialty again compared to the proportion of gps who were burnout - free in both surveys. age and sex were not associated with the risk of developing burnout as were neither of the other risk factors examined. there was not a higher job turnover rates during followup in gps who were burned out in 2004 than in gps who were burnout - free in 2004. this indicates that gps are able to or must stay in their practices until it is possible to retire. among the strengths of this study are the prospective design, use of an international validated scale for measuring burnout, and its high response rate. although the study enjoyed a high response rate, a small group of gps did not respond, which may have caused selection bias. it may be assumed that gps experiencing burnout would have less energy and enthusiasm to complete the questionnaire. insofar nonrespondents had a higher prevalence of burnout than respondents ; the risk of burnout would have been underestimated. however, the opposite scenario could perhaps have been the case as some gps suffering from high levels of burnout symptoms may have felt more inclined to participate in the study. one strength was the use of register based data which made it possible to make a nearly complete followup and to include data on the activity. the long followup strengthens this study since burnout is assumed to be a condition which takes time to develop. however, as many of the included variables are alterable, more assessment points during followup would have benefitted the study. for instance, it can not be excluded that we have missed some incident cases of burnout during followup if the afflicted gps had recovered before the 2012 survey. a study of first - year internal medicine residents from five different training programmes in us yielded a risk of burnout during a 1-year followup on 75%. the much higher burnout risk obtained in this study compared to the risk obtained in our study occurred despite fairly consistent definitions of burnout. thus, the difference could be an indication of the influence of experience and age, as the study populations consisted of trainees and specialist doctors, respectively. to our knowledge, the observed strong association between being reserved about choosing family practice as medical specialty again and increased risk of burnout has not been reported before. thoughts about switching specialty may indicate a poor match between the personality of the gp and the job demands. this falls in line with maslach and leiter 's model proposing that the greater the perceived mismatch between the person and the job, the greater the likelihood of burnout. the findings of this study suggest that the hypothetical question about whether one would select the same medical specialty again may be used as a screening tool to identify gps in risk of developing burnout. even though low levels of job satisfaction and high levels of perceived job - related pressure have been associated with intentions to leave general practice [13, 15, 16 ], we did not find a higher job turnover in gps with burnout compared to gps who were burnout - free in 2004. thus, when a burned out gp discloses an intention to leave general practice, it may reflect a state of despair, but in reality leaving practice or giving it up may act against the personality of the burned out gp often characterized by high levels of conscientiousness and a performance - based self - esteem. congruent with previous findings [6, 21 ], we found nonsignificant tendencies that the prevalence of burnout was higher in younger gps than in older gps and lower in gps in solo practices than in gps in other types of practices. it has been documented fairly consistently that female physicians report lower levels of depersonalization and higher levels of emotional exhaustion than male physicians [6, 9, 12 ], and our results supported this. the risk of developing a moderate or high degree of burnout in this sample of danish gps during the 7-year followup was 13.2%. gps who in 2004 reported that they would not choose family medicine if they were to select medical specialty again or were inconclusive about this issue more than four - doubled their risk of burnout in 2012 compared to gps whose choice of medical specialty would still be family medicine. we suggest more research into whether this particular item can be used as a screening tool among gps to identify gps at risk for developing burnout whereby an adjuvant intervention would be possible. burned out gps appear not to have higher job turnover rates than burnout - free gps. | background. we assessed risk of burnout in gps during a 7-year followup and examined whether (1) thoughts about changing medical specialty increased the risk of burnout and (2) burned out gps had higher job turnover rates than burnout - free gps. methods. in 2004 and 2012, all gps in the county of aarhus, denmark, were invited to participate in a survey. retirement status of physicians who participated in 2004 was obtained through the registry of health providers in 2012. results. 216 gps completed both surveys. the risk of developing burnout during the 7-year followup was 13.2% (8.219.6%). gps who in 2004 were burnout - free and reported that they would not select general practice as medical specialty again had a statistically significant increased risk of burnout in 2012 (or = 4.5 ; 95% ci = 1.216.5 ; p = 0.023). among gps with burnout in 2004, 25.0% had withdrawn from general practice during followup compared to 28.8% of burnout - free gps in 2004 (adj. or = 0.99 ; 95% ci = 0.482.02 ; p = 0.975). conclusion. the 7-year incidence of burnout was 13%. thoughts about changing medical specialty were an important predictor of burnout. burned out gps had not higher job turnover rates than burnout - free gps. |
tooth discoloration may be classified as intrinsic, extrinsic, and a combination of both.1 intrinsic discoloration occurs following a change to the structural composition or thickness of the dental hard tissues. intrinsic tooth discoloration is caused by aging, microcracks in the enamel, tetracycline medication, restoration, dental caries and excessive fluoride ingestion.2 excessive fluoride in drinking water, greater than 1 to 2 ppm, can cause metabolic alteration in the ameloblasts, resulting in a defective matrix and improper calsification of teeth. an affected tooth shows a hypomineralized, porous subsurface enamel and acid - resistant well - mineralized surface layer (figure 1).3 several authors investigated bond strength between composite materials and fluorosed enamel or dentin.47 nganga reported no significant differences in the bond strengths between fluorosed and normal enamel. on the other hand, weerasinghe found that severity of fluorosis affects the micro - shear bond strength of a self - etching bonding system to fluorosed enamel. some investigators have recommended extended enamel conditioning with phosphoric acid when bonding composite resin to the fluorosed enamel to increase bond strength.5,9 to date, only limited and contradictory data are available on shear bond strength of brackets to fluorosed human enamel. patients increased awareness of esthetic outcomes and their desire to look and feel better about themselves have led to enormous demands on dentists to perform esthetic procedures. fluorosed teeth can be treated with various restorative techniques, such as direct composite veneers, indirect porcelain veneers, ceramic crowns, microabrasion technique and bleaching.1013 effect of bleaching on the bond strength of adhesives to enamel is well documented. however, a consensus has not been reached on the effects of bleaching on bond strengths of brackets. several authors reported that tooth bleaching reduce the shear bond strength of resin composite materials to enamel or dentin surfaces.1418 in contrast, some authors claimed that bleaching procedures did not affect shear bond strengths.1921 however, to our knowledge, no study evaluated bond strengths of orthodontic brackets to bleached fluorosed teeth. therefore, the aim of this invitro study was to evaluate the effects of fluorosis and bleaching of fluorosed teeth on shear bond strengths of orthodontic brackets. our null hypothesis was that fluorosis and bleaching of fluorosed teeth do not affect shear bond strengths of orthodontic brackets bonded to enamel. a total of 45 (30 fluorosed and 15 non - fluorosed) non - carious freshly extracted human permanent premolar teeth, extracted for orthodontic reasons and without any caries or visible defects, were used in this study. fluorosed teeth were selected according to the modified thylstrup and fejerskov index (tfi), which is based on the clinical changes in fluorosed teeth.22 specific features of teeth with tfi scores of 4 are a marked opacity and a chalky white appearance on the entire surface (figure 1). each tooth was individually embedded in auto polymerizing acrylic resin (meliodent, herause kulzer, hanau, germany). the specimens were kept in distilled water except during the bleaching, bonding and testing procedures. a group of 15 non - fluorosed teeth (tfi score of 0) served as the control group. group ii (fluorosis+bleaching) : teeth were bleached with a 35% hydrogen peroxide (opalescence xtra, ultradent products inc, south jordan, utah), exposed to a fast halogen curing light (1000 mw / cm) (blue swan, dentanet, istanbul, turkey) for 20 seconds, and left standing for 15 minutes. the gel that had been applied to the tooth was washed away and a fresh gel was reapplied, light activated, left standing for another 15 minutes, and washed away. before bonding, the teeth were stored in distilled water for 2 days at room temperature., the facial surfaces of the teeth were cleaned with a mixture of water and pumice. the teeth were rinsed thoroughly with water and dried with oil and moisture - free compressed air. then, all teeth were rinsed with water / spray combination for 30 seconds and dried until characteristic frosty white etched area is observed. ormco mini 2000 (ormco corp, glendora, calif) bicuspid metal brackets with 9.63 mm surface area were used., a thin uniform layer of sealant was applied on the etched enamel and cured for 20 seconds. a thin coat of sealant was also painted on the metal bracket base and cured for 10 seconds before applying paste. using a syringe tip, the bracket was then positioned on the tooth and pressed lightly in the desired position. excess adhesive was removed with a sharp scaler and cured with a heliolux dlx (vivadent ets, schaan, liechtenstein) for 40 seconds (20 seconds on the mesial and 20 seconds on the distal surfaces of the brackets). all specimens were stored in distilled water at 37c for 24 hours and thermocycled for 500 cycles between 5c and 55c, using a dwell time of 30 seconds. each specimen was loaded into universal testing machine (lloyd ; fareham, hants, england) using nexjen software for testing, with the long axis of the specimen being perpendicular to the direction of the applied force. the standard knife edge was positioned to make contact with the bonded specimen (figure 2). bond strength was determined in the shear mode at a crosshead speed of 0.5 mm / min until fracture occurred. values of failure loads (n) were recorded and converted into megapascals (mpa) by dividing the failure load (n) by the surface area of the bracket base (9.63 mm). after debonding, all teeth and brackets in the test groups were examined under 10x magnification. any adhesive remained after debonding was assessed and scored according to the modified adhesive remnant index (ari).23 the scoring criteria of the index are as follows : 1= all of the composite, with an impression of the bracket base remained on the tooth ; 2= more than 90% of the composite remained on the tooth ; 3= more than 10% but less than 90% of the composite remained on the tooth ; 4= less than 10% of composite remained on the tooth ; 5= no composite remained on the tooth. descriptive statistics, including the mean, standard deviation, standard error, minimum and maximum values were calculated for each of the groups tested. the chi - square test was used to determine significant differences in the ari scores among test groups. descriptive statistics, including the mean, standard deviation, standard error, minimum and maximum values were calculated for each of the groups tested. one - way analysis of variance (anova) and tukey multiple comparison tests were used to compare shear bond strengths of the groups. the chi - square test was used to determine significant differences in the ari scores among test groups. all statistics were performed with spss version 11.0.0 (spss inc., chicago, il, usa). the descriptive statistics on the shear bond strengths (mpa) of the groups are presented as boxplots in figure 3. analysis of variance (anova) indicated a significant difference between groups (p.05). frequency distribution of the ari scores and the chi - square comparison of the test groups are presented in table 2. there was a greater frequency of ari scores of 3, 4 and 5 in group ii (fluorosis+bleaching), which indicated that failures in this group were mainly in the enamel - adhesive interface. this study was designed to evaluate the effects of fluorosis and bleaching of fluorosed teeth on shear bond strengths of orthodontic brackets. for this purpose, fluorosed teeth (tfi score 4) were collected and selected by two examiner s agreement (n.a, h.t). since fluoride content can vary between different teeth, only fluorosed human maxillary premolar teeth were used in this study. in our study, all specimens were etched with 37% phosphoric acid gel for 30 seconds. then, all teeth were rinsed with water / spray combination for 30 seconds and dried until characteristic frosty white etched area is observed. some investigators have recommended extended enamel conditioning with phosphoric acid when bonding composite resin to the fluorosed enamel.5,9 however, al - sugair and akpata24 demonstrated in an in vitro study that the mean depth of enamel dissolutions were not significantly different for the enamel specimens between non - fluorosed (tfi score 0) and fluorosed teeth (tfi score 4) etched for 30 seconds. therefore, we did not extend etching time as was done by ateyah and akpata5 and opinya and pameijer9. the findings of the present study demonstrate that fluorosis significantly reduced the shear bond strengths of the brackets to enamel. the mean shear bond strength of the fluorosed teeth group was 13.943.24 mpa, while the control group was 19.294.71 mpa. this result is consistent with weerasinghe who reported that severity of fluorosis affected the micro - shear bond strength of a self - etching bonding system to fluorosed enamel. their study also revealed that severe fluorosis decreased the shear bond strength even with the traditional acid etching using 37% phosphoric acid. fluorosed teeth have the highest concentration of fluoride in the outer 200 m of enamel surface.5 weerasinghe removed this hypermineralized, acid resistant enamel surface before the shear test. since this procedure is not suitable for orthodontic practice, we did not remove the enamel surface layer in our study. in contrast to the present study, nganga found no statistically significant difference between the mean values for bond strength of orthodontic brackets in fluorosed and non - fluorosed teeth. they bonded brackets with a composite resin after over - etching the enamel surface with 40% phosphoric acid for 60 seconds. the result of their study revealed that fluorosis decreased the bond strength compared with non - fluorosed teeth. however, the difference between the means for bond strength was not statistically significant. with the increasing number of adult patients seeking orthodontic treatment, orthodontists may face bonding brackets to previously bleached fluorosed teeth. up to date, no data was available concerning shear bond strength of brackets to bleached fluorosed enamel. therefore, we aimed to investigate not only the effect of fluorosis, but also bleaching procedure on shear bond strength of orthodontics brackets on fluorosed enamel surface. in the present study, mean shear bond strength for fluorosis+bleaching group was 12.572.34 mpa that was the lowest in all groups. these results are in agreement with nour el - din, titley and dishman who also reported reduced bond strengths after bleaching procedure. nour el - din investigated the shear bond strength, degree of resin infiltration and failure mode when organic solvent - based adhesives were used in immediate bonding to enamel bleached with 10% carbamide peroxide or 38% hydrogen peroxide systems. the shear bond strengths of 38% hydrogen peroxide and 10% carbamide peroxide were significantly lower compared to the non - bleached controls. moreover, scanning electron microscopy revealed few, thin and fragmented resin tags when 38% hydrogen peroxide and 10% carbamide peroxide were used. on the other hand, bishara,27 reported that in - office bleaching and at - home bleaching did not affect the shear bond strength of orthodontic brackets to enamel. uysal suggested that office bleaching with hydrogen peroxide did not adversely affect the bond strengths of brackets bonded immediately after bleaching or 30 days after bleaching. our results agree with nour el - din al16, however contradicting the results of uysal and bishara. the lowest values obtained in fluorosis+bleaching group may be explained with bleaching induced morphological alterations in the most superficial enamel crystallites. the bleaching agents significantly decrease the calcium and phosphate content of the enamel.28,29 in addition, residual oxygen in the enamel pores may interfere with resin infiltration into enamel30 or inhibit polymerization of the resin.31 it must be emphasized that this study was performed in vitro. therefore, shear bond strengths obtained in this study may not correspond well with clinical success. further in vivo studies are still needed to substantiate the results obtained in this study. enamel fluorosis significantly decreased the bond strength of orthodontic brackets. although bleaching of fluorosed enamel decreased the bond strengths more, the difference between fluorosis+bleaching and fluorosis groups was not statistically significant. though fluorosis and bleaching of fluorosed teeth reduce bracket bond strength to enamel, the bond strength with these still exceed the minimum 6 to 8 mpa required to expect adequate clinical performance. | objectivesto evaluate the effects of fluorosis and bleaching on shear bond strengths of orthodontic brackets.methodsa total of 45 (30 fluorosed and 15 non - fluorosed) non - carious freshly extracted human permanent premolar teeth which were extracted for orthodontic reasons and without any caries or visible defects were used in this study. fluorosed teeth were selected according to the modified thylstrup and fejerskov index (tfi), which is based on the clinical changes in fluorosed teeth. first group consisted of 15 fluorosed teeth. second group of fluorosed teeth were bleached with a 35% hydrogen peroxide office bleaching agent. third group served as control. no bleaching procedure was applied. orthodontic brackets were bonded with a light cure composite resin and cured with a halogen light. after bonding, shear bond strengths of the brackets were tested with universal testing machine.resultsthe results showed that fluorosis only and bleaching of fluorosed teeth significantly reduced the bond strengths of the orthodontic brackets (p.05).conclusionsfluorosis and bleaching of fluorosed teeth reduce bracket bond strength to enamel, but the bond strength with these still exceed the minimum 6 to 8 mpa required to expect adequate clinical performance. |
subjective tinnitus is characterized by the perception of sound or noise in the absence of an objective physical sound source (moller, 2003)., 2010 ; lanting., 2010) and neurophysiologic studies (weisz., 2007a, b) that tinnitus is related to abnormal functioning of the central auditory system (moller, 2003). based on these findings repetitive transcranial magnetic stimulation (rtms) of the temporal and temporoparietal cortex has been proposed as a potential treatment for chronic tinnitus (eichhammer., 2003). transcranial magnetic stimulation (tms) is a non - invasive tool for inducing electric currents in the brain (hallett, 2000). fast oscillating magnetic fields created by a strong electric current circulating within a coil, penetrate the skull and result in depolarization of superficial cortical neurons (ridding and rothwell, 2007). rtms can induce alterations of neuronal activity that outlast the actual stimulation period for a considerable amount of time (hallett, 2000). therefore, this technique has gained increasing attention as a potential clinical tool for the treatment of different neuropsychiatric disorders. although the direct effects of the magnetic field are limited to directly stimulated superficial brain areas (siebner., 2003), indirect effects can also occur in functionally connected remote areas (hallett, 2000 ; siebner. such remote stimulation effects have also been demonstrated in thalamic regions after temporal rtms by using voxel - based morphometry (may., 2007). several clinical studies consistently showed a reduction of tinnitus severity after repeated 1 hz rtms applied to the temporal cortex, whereas sham treatment had no effect (kleinjung., 2005 ; plewnia., 2007 ; rossi., 2007 ; however, treatment results are burdened by only moderate improvement and high inter - individual variability indicating the need for optimization strategies. as hypothesized already more than 20 years ago (jastreboff, 1990) and confirmed by recent neuroimaging findings, tinnitus is related to (i) abnormal activity in both auditory and non - auditory brain regions (lanting., 2009 ; leaver., 2011) and to (ii) abnormal functional connectivity between these regions (schlee., 2008, 2009a, b ; de ridder., 2011). in these studies the right dorsolateral prefrontal cortex has been identified as an important hub (schlee., 2008, 2009a, b ; vanneste., 2010a). it has been hypothesized that this area might especially be related to the affective components of tinnitus (vanneste., 2010a ; de ridder., 2011 ; langguth., 2011). it has even been speculated that based on the emotional relevance, involved limbic and paralimbic structures may effectively switch the perceived signal on and off (rauschecker., this is in line with electrophysiological studies that demonstrated the relevance of dysfunctional top - down inhibitory mechanisms originating in the prefrontal lobe for tinnitus generation (norena., 1999). the critical relevance of the dlpfc for tinnitus annoyance has been affirmed by recent studies that demonstrated symptom reduction after bifrontal tdcs (vanneste. furthermore, it has been shown that rtms over the dlpfc is apt to modulate the activity in functionally connected central limbic pathways such as the anterior cingulated cortex (paus., 2001). modulation of neuronal activity in the anterior cingulate, parahippocampus, and auditory cortex has also been reported in tinnitus patients after transcranial direct current stimulation of the prefrontal cortex (vanneste and de ridder, 2011). a further rationale for low - frequency stimulation of the right dlpfc derives from affective research. frontal asymmetry is known to influence emotion regulation and the emotional reaction to sensory stimuli (davidson, 1992 ; schmidt and hanslmayr, 2009). it has also been shown that low - frequency rtms of the right dlpfc exerts antidepressant effects of similar magnitude like high frequency rtms of the left dlpc, which is conventionally applied in depressive disorders (schutter, 2010). based on these data and the right lateralized alterations of frontal cortex activity in tinnitus patients (schlee., 2008) we hypothesized that low - frequency rtms of the right dlpfc might enhance treatment effects of low - frequency rtms in tinnitus patients and compared the combined prefrontal and temporal rtms therapy in tinnitus patients with the standard procedure of temporal rtms. fifty - six patients with chronic unilateral or bilateral tinnitus were enrolled in the study after having given written informed consent. the study has been registered with clinicaltrials.gov (http://clinicaltrials.gov/ct2/show/nct01261949), was approved by the local ethics committee and performed according to the declarations of helsinki. all patients suffered from disturbing tinnitus and had tried several standard treatment modalities such as cognitive behavioral therapy, hearing aids, white - noise generators, vasodilators, or antidepressants in the past. normal middle ear status was demonstrated by tympanometry, stapedius reflex tests, and otoscopy. patients with a history of seizures, a suspected diagnosis of organic brain damage, as well as patients with cardiac pacemakers, mobile metal implants, or implanted medication pumps were excluded. all data in the text and table is given as mean sd. rtms was applied with the use of a medtronic system with a figure-8 coil (cool b-65 butterfly ; medtronic, minneapolis, mn, usa). patients were enrolled in the study on a monday and received stimulation on 10 subsequent working days. one protocol (standard protocol) consisted of 2000 stimuli at a frequency of 1 hz and an intensity of 110% resting motor threshold (rmt) over the left auditory cortex. in the second treatment protocol (combined protocol), low - frequency stimulation (1000 stimuli, 1 hz, 110% motor threshold) applied to the right dlpfc preceded left temporal stimulation (1000 stimuli, 1 hz, 110% rmt). thus, the total number of applied stimuli per session was identical for both groups. stimulation was administered over the right dlpc and the left temporal cortex regardless of handedness or tinnitus laterality (kleinjung. thus, the induced current in the brain was directed approximately perpendicular to the location of the superior temporal gyrus. during treatment the tms coil was localized over the right dlpfc according to a standard algorithm by moving the coil from the optimal position for stimulation of the left abductor minimi 6 cm in the anterior direction and transferring this spot to the contralateral hemisphere in respect of the distance to the sagittal axis of the skull (george., 1995). the rmt was determined over the left motor cortex for the right abductor digiti minimi and defined as the lowest intensity at which at least four of eight consecutive meps were 50 mv in amplitude while the muscle being investigated was at rest. tinnitus severity was assessed before treatment (baseline), at the end of treatment (week 2), and during a certain follow - up period after rtms treatment (week 4 and week 12). tinnitus assessments included the german versions of the tinnitus questionnaire (tq ; goebel and hiller, 1994), the tinnitus handicap inventory (kleinjung., 2007a), the beck depression inventory (beck and steer, 1984), several tinnitus numeric rating scales (loudness, discomfort, annoyance, distractibility, unpleasantness ; landgrebe., 2010), and a quality of life scale (whoqol - bref ; murphy., 2000). data management was conducted according to the data handling plan (tri - dhp v07, may 9th, 2011). data analysis was conducted according to the standard operating procedure (tri - sa v01, may 9th, 2011), thereby following a study - specific statistical analysis plan (sap-003, may 18th, 2011) that was written according to the sap template (tri - sap v01, may 12th, 2011). all documents are to be found under http://database.tinnitusresearch.org/. the statistical analysis was performed on an intention - to - treat basis including all patients who participated in at least one measurement time point using a last observation carried forward or backward approach. for this purpose, we conducted an analysis of variance (anova) with the within - subjects factor time (baseline vs. week 2) and the between - subjects factor group (combined vs. temporal group). secondary outcome measures and exploratory analyses included chi - square tests for the variables group and treatment response which was defined as amelioration of at least 5 points in the tq. we pooled the group of responders and non - responders of both treatments and compared them in regard to demographic and clinical characteristics with chi - square and t - tests. furthermore, we compared baseline corrected tq scores (week 2 minus baseline) between the treatment groups with student s t - tests. in addition, we again conducted an anova with the factor group (between - subjects factor) and time (within - subjects factor), this time including five measurement time points (screening, baseline, week 2, week 4, and week 12). this anova was also computed for all other secondary outcome parameters (i.e., thi, bdi, and whoqol - bref). furthermore, we compared baseline corrected tq scores (week 2 minus baseline ; week 4 minus baseline) between the treatment groups. the analysis of secondary outcome parameters followed an exploratory approach, without corrections for multiple comparisons. both stimulation protocols were well tolerated, and all patients except one completed the treatment. this patient (combined stimulation group) refused further stimulation after day 3 because she feared a possible deterioration of the symptoms. a total of seven patients (including the one mentioned before) did not complete the course of the study (not shown up for follow - up - visit without giving further explanation). three of them were treated in the combined stimulation group, four in the conventional group. transient mild to moderate headache and feelings of twitching muscles at the stimulation site were reported as side effects. primary outcome analysis indicated a significant change over time for both groups as indicated by a significant main effect of time (f = 6.1 ; df = 1.54 ; p = 0.017), but no group differences (main effect of group : f = 0.8 ; df = 1.54 ; p = 0.375 ; interaction effect time by group f = 0.434 ; df = 1.54 ; p = 0.513). response rate was comparable between groups (combined : 40% ; temporal : 37% ; = 0.1 ; df = 1 ; p = 0.800). effect sizes were near zero for the non - responder groups (combined : d = 0.085 ; temporal : d = 0.104) and medium to high for the responder groups (combined : d = 0.700 ; temporal : d = 0.454). contrasts between these groups indicated no significant differences for age, gender, tinnitus laterality, duration, and hearing loss. we only found an effect for the change in tq and thi from screening to baseline for the non - responder group in contrast to the responder group of the combined treatment (tq : t = 2.156 ; df = 24 ; p = 0.041 ; thi : t = 3.675 ; df = 24 ; p = 0.001), i.e., there was a reduction of questionnaire scores from screening to baseline for the non - responder (tq : 5.2 11.7 ; thi : 5.6 7.7) and an increase for the responder group (tq : 3.5 6.1 ; thi : 5.8 8.0). comparable to the primary outcome analysis, anovas with five time points for tq, thi, and bdi indicated significant main effects of time (all fs > 2.3 ; df = 4.212 ; all ps 0.115) nor time by group (all fs 0.462). post hoc tests indicated an amelioration of symptoms after beginning of treatment and a return to baseline levels during the last follow - up (see figure 1), i.e., tinnitus scores were significantly bettered for week 2 and week 4 in contrast to screening, baseline and follow - up. (a) tinnitus questionnaire score (tq ; mean sem), (b) tinnitus handicap inventory (thi ; mean sem), and (c) beck depression inventory (bdi ; mean sem). baseline corrected group contrasts (week 2 minus baseline ; week 4 minus baseline) indicated no significant differences for week 2 (all ps > 0.265) and week 4 (all ps > 0.088) for tq, thi, and bdi. range of effect sizes were between 0.168 and 0.461 (0.176 for primary outcome analysis) indicating more pronounced improvement for the combined group in contrast to the temporal group for all variables. the main finding of this trial is that additional low - frequency stimulation of the right dlpfc failed to significantly improve the effects of low - frequency temporal stimulation in the treatment of tinnitus. however, on a descriptive level the combined treatment protocol yielded better results in all assessment instruments that have been applied. primary outcome analysis showed an effect size of 0.176 (group contrast in week 2) indicating a small effect according to cohen (1988). thus, the effect size was smaller than expected resulting in limited power and the failure to demonstrate significant effects. in this context it should be noted that significant improvement after rtms was observed in both stimulation groups. the effect size for responders from temporal stimulation was medium (d = 0.45) for the responders from the combined stimulation protocol high (d = 0.70) according to cohen (1988). interestingly, the responder group differed significantly from non - responders in the change of thi and tq scores from screening to baseline (non - responders : mean effect 4 tq/7 thi points ; responders : mean effect + 2 tq / thi points). this might be interpreted as a hint for the induction of homeostatic effects by tms (siebner., 2004). further analysis did not reveal any differences between responders and non - responders in respect to gender, age, tinnitus duration, laterality, number of previous treatments, hearing loss, numeric rating scales, and values (screening / baseline) for thi, bdi, and tq. a further important finding was the decrease of mean baseline values for tq, thi (for the 1/1-hz group), and bdi from screening to baseline. first screening scores are based on completion of the tq at home before the first consultation in our tinnitus clinic. the examination and consultation in the tinnitus clinic, which also involves counseling, may have resulted in the reduction of the tinnitus scores. alternatively the improvement can be interpreted as an anticipation effect. similar effects were observed in patients enrolled in waiting list control groups (hesser., 2011). facing the fact that the combined stimulation group has been compared to an actively treated control group [that has undergone an already established standard treatment protocol (kleinjung., 2005 ; plewnia., 2007 ; rossi., 2007 ; smith., 2007) ] it would be a bit too early to draw the conclusion that the small effect sizes might not possibly reflect clinically relevant changes especially taking into consideration the much higher effect sizes of the responder group. even if this pilot study might have been designed with limited power presumptions, the results suggest at least that the applied combined study protocol did prove to be non - inferior in comparison to the established stimulation pattern of 1 hz to temporal targets. in this study all patients received rtms over the left temporal cortex and in the combined group additionally over the right dlpfc. for both targets it remains a matter of debate, whether a more individualized strategy may not be more efficient. for the temporal cortex there are conflicting results whether stimulation ipsi- or contra - lateral to the perceived tinnitus laterality is more efficient (frank., 2010 ; we chose left temporal stimulation in all patients for better comparison with previous studies that investigated enhancement strategies for rtms (kleinjung., 2008, 2011 ; langguth., 2008). with respect to frontal stimulation individualized targeting based on function imaging data revealed conflicting results as well (kimbrell., 1999 ; herwig., 2003) and more consistent antidepressant efficacy has been reported for low - frequency rtms over the right dlpfc independent from imaging data (schutter, 2010). there is growing evidence from many recent neuroimaging studies that the influence of non - auditory brain structures may have been underestimated in the pathophysiology of chronic tinnitus in the past. a study investigating long - range connectivity of brain areas in patients suffering from chronic tinnitus by means of magnetoencephalography detected mainly the prefrontal cortex and the orbitofrontal region as hubs in tinnitus related networks (schlee., 2009b moreover with increasing tinnitus duration non - auditory areas seem to gain importance in tinnitus related networks in comparison to auditory areas (schlee., 2009b). but not only the dlpfc and neighboring regions seem to be of decisive relevance ; also the left hippocampus (landgrebe., 2009), parahippocampus (lockwood., 1998 ; schecklmann., 2011), the anterior (plewnia., 2007) and posterior cingulate cortex (vanneste., 2010a ; schecklmann., 2011), the temporoparietal junction (= auditory association area ; shulman, 1995 ; giraud., 1999 ; lockwood., 1999 ; gardner., 2002), the dorsolateral prefrontal cortex (mirz., 2000 ; voisin., 2006), and the cerebellum (lanting., possibly other rtms techniques with different target locations, frequencies, and stimulation protocols (e.g., burst protocols ; arfeller. very recently a new rtms coil, the so - called double - cone - coil with increased stimulation depth in the brain, has been introduced. based on the use of this device it has been shown that a direct modulating influence of rtms can be exerted to the limbic system, namely the anterior cingulate cortex (hayward., 2007). in first clinical trials this new technique has been proven to be safe and its application is feasible and well tolerated (personal communication s vanneste and d de ridder). even if the present study has not been placebo controlled, the results further support the efficacy of low - frequency rtms for the treatment of tinnitus as demonstrated in previous studies (kleinjung., 2005 ; plewnia., 2007 ; rossi., 2007 the inter - individual variability has been high in both treatment groups, highlighting the relevance of a more individualized treatment approach. the limited accuracy of the coil positioning procedure over the dlpfc together with the large anatomic inter - individual variability of the dlpfc in brodman area 9 and brodman area 46 (herbsman., 2009) may play a role in this context as well as potential genetic influences on neuromodulatory effects as proposed for the bdnf polymorphisms (cheeran., it has been shown that clinical characteristics have only limited value for predicting treatment outcome (frank., 2010). neuroimaging such as electro- or magnet - encephalography may be more promising for identifying patients who may respond well on specific stimulation protocols (lorenz. this may lead to the development of individualized multi - site - rtms - stimulation techniques for the treatment of tinnitus, but also in other indications such as depression or chronic pain. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | objectives : low - frequency repetitive transcranial magnetic stimulation (rtms) of the temporal cortex has been investigated as a new treatment tool for chronic tinnitus during the last years and has shown moderate efficacy. however, there is growing evidence that tinnitus is not a pathology of a specific brain region, but rather the result of network dysfunction involving both auditory and non - auditory brain regions. in functional imaging studies the right dorsolateral prefrontal cortex has been identified as an important hub in tinnitus related networks and has been shown to particularly reflect the affective components of tinnitus. based on these findings we aimed to investigate whether the effects of left low - frequency rtms can be enhanced by antecedent right prefrontal low - frequency rtms. study design : fifty - six patients were randomized to receive either low - frequency left temporal rtms or a combination of low - frequency right prefrontal followed by low - frequency left temporal rtms. the change of the tinnitus questionnaire (tq) score was the primary outcome, secondary outcome parameters included the tinnitus handicap inventory, numeric rating scales, and the beck depression inventory. the study is registered in clinicaltrials.gov (nct01261949). results : directly after therapy there was a significant improvement of the tq - score in both groups. comparison of both groups revealed a trend toward more pronounced effects for the combined group (effect size : cohen s d = 0.176), but this effect did not reach significance. a persistent trend toward better efficacy was also observed in all other outcome criteria. conclusion : additional stimulation of the right prefrontal cortex seems to be a promising strategy for enhancing tms effects over the temporal cortex. these results further support the involvement of the right dlpfc in the pathophysiology of tinnitus. the small effect size might be due to the study design comparing the protocol to an active control condition. |
periodontitis is an inflammatory disease of the dental supporting tissues that leads to bone destruction and tooth loss. its severe form affects ~11% of the adult population worldwide (reviewed by kassebaum and richards2). it is now known that the underlying tissue damage observed in this disease results from an excessive immune response to subgingival pathogens.3 different inflammatory components have been suggested to participate in this response, including cytokines and metalloproteinases (mmps).4,5 abnormal recognition of bacterial products leads to the activation of resident cells such as fibroblasts and dendritic cells, in addition to promoting cell (neutrophils and mononuclear cells) influx in to the gums, triggering an ongoing scenario of inflammation the tissue.4,5 innate immunity cytokines (tnf-, il-1, and il-6) are then produced, which amplify the inflammation by increasing cell migration via upregulation of adhesion molecules and inflammatory mediators, promoting bone resorption and contributing to tooth loss.4 also, as a result of cytokine generation, an increase in mmps is noted. mmps comprise a group of proteases that target the extracellular matrix and are associated with remodeling of the soft and mineralized periodontal tissues.5 as the disease progresses, another cytokine, il-10, is released into the gum by activated t - cells and macrophages.4 il-10 is produced in an attempt to counteract the inflammatory response and is known to trigger the expression of molecules that downregulate the transcription of proinflammatory cytokines and mmps.4,5 current treatment of periodontitis includes surgical and nonsurgical (systemic or local antibiotics) procedures.6 side effects, dysbiosis of gut microbiota, and increased bacterial resistance to antibiotic therapy have raised concerns regarding its use to treat periodontitis.6 on the other hand, the recent knowledge on the inflammatory pathways involved in this disease has unleashed novel venues for therapy, with increased attention to strategies that modulate the inflammatory response.7 recently, an anti - inflammatory effect was suggested for antihypertensive drugs (angiotensin ii receptor blockers, angiotensin - converting enzyme inhibitors, -blockers) in periodontitis.8,9 more recently, the cholesterol - lowering drug simvastatin, used as adjuvant in hypertensive patients with dyslipidemia, was suggested to have a protective effect against experimental10,11 and human periodontitis.9,12 this effect was attributed to its capacity to reduce bone loss and modulate the expression / release of inflammatory markers in gingival crevicular fluid samples. herein, this study evaluated the effect of simvastatin on the synthesis of cytokines tnf- and il-10 and the mmps 2 and 9 in a rat model of ligature - induced periodontitis. we found that this drug is able to increase the levels of il-10 while reducing mmp-9 expression in gingival tissue samples. rats were obtained from the centro multidisciplinar para investigao biolgica na rea da cincia em animais de laboratrio (campinas, brazil), and experiments were performed in the biological service unit of the university of campinas. animals were kept (n=5/cage) in a climatically controlled environment (room temperature of 22c2c and humidity of around 60%) under 12:12 hour light - dark cycle). this study and all procedures were approved by the ethics committee of the university of campinas and carried out in accordance with the guidelines of the brazilian society for animal welfare (sbcal). briefly, animals were anaesthetized with a single intramuscular injection of ketamine (90 mg / kg ; dopalen, ceva, brazil) and xylazine (10 mg / kg ; rompun, bayer, brazil). periodontitis was then induced as previously described by branco - de - almeida,13 with minor modifications. for this, a cotton ligature was placed in a subgingival position encircling the entire cervix of both sides of the first molar on the left (ipsilateral) side of the mandible. in order to immobilize the ligature, the right (contralateral) side of the mandible had no ligature placed and was used as the control. disease was allowed to develop for 14 days, and then the rats were euthanized by anesthetic overdose. gingival tissue samples were collected from the mandibular first molar regions, immediately frozen, and kept at 80c for further analysis. in order to confirm the establishment of periodontal disease, contralateral and ipsilateral mandibular alveolar bone specimens were collected at day 14 and submitted to macroscopic and histologic analysis. for macroscopic analysis, mandibles were immersed in sodium hypoclorite 1% solution for 4 hours and the whole remaining bone soft tissue was mechanically removed. the pieces were stained with methylene blue (1 g/100 ml) for 1 minute to delimit the cement enamel junction. for histological analysis, the mandibles were fixed in 10% neutral buffered formalin and then demineralized in 5% formic acid for 7 days. 7 m sections from the decalcified bone were stained with hematoxylin and eosin staining and evaluated to confirm bone loss. after ligature placement, animals were randomly assigned to two experimental groups (n=10 animals / group) : 1) rats with ligature + vehicle (saline ; 10 ml / kg ; orally) and 2) rats with ligature + simvastatin (25 mg / kg ; orally). ipsilateral mandible samples were immediately fixed in 10% neutral buffered formalin for 24 hours, washed 3 in phosphate - buffered saline, and then stained with 1% methylene blue.11 bone loss was evaluated in both contralateral and ipsilateral sides by comparison of digital images. gingival tissue samples were homogenized in 300 l of ripa lysis buffer (santa cruz biotechnology, dallas, tx, usa) and then centrifuged for 10 minutes at 10,000 rpm at 4c. the supernatants were collected and used for evaluation of cytokine (tnf and il-10) and mmp (mmp-2 and 9) levels. gingival tnf-, il-10, mmp-2, and mmp-9 levels were evaluated by using commercially available kits (r&d systems, minneapolis, mn, usa) according to the manufacturer s instructions. the percentage of inhibition is reported as the mean standard deviation for each individual experiment. statistical comparison was performed using unpaired student s t - test in graphpad prism 6.0 (graphpad software, inc., la jolla, ca, usa). rats were obtained from the centro multidisciplinar para investigao biolgica na rea da cincia em animais de laboratrio (campinas, brazil), and experiments were performed in the biological service unit of the university of campinas. animals were kept (n=5/cage) in a climatically controlled environment (room temperature of 22c2c and humidity of around 60%) under 12:12 hour light - dark cycle). this study and all procedures were approved by the ethics committee of the university of campinas and carried out in accordance with the guidelines of the brazilian society for animal welfare (sbcal). briefly, animals were anaesthetized with a single intramuscular injection of ketamine (90 mg / kg ; dopalen, ceva, brazil) and xylazine (10 mg / kg ; rompun, bayer, brazil). periodontitis was then induced as previously described by branco - de - almeida,13 with minor modifications. for this, a cotton ligature was placed in a subgingival position encircling the entire cervix of both sides of the first molar on the left (ipsilateral) side of the mandible. in order to immobilize the ligature, the right (contralateral) side of the mandible had no ligature placed and was used as the control. disease was allowed to develop for 14 days, and then the rats were euthanized by anesthetic overdose. gingival tissue samples were collected from the mandibular first molar regions, immediately frozen, and kept at 80c for further analysis. in order to confirm the establishment of periodontal disease, contralateral and ipsilateral mandibular alveolar bone specimens were collected at day 14 and submitted to macroscopic and histologic analysis. for macroscopic analysis, mandibles were immersed in sodium hypoclorite 1% solution for 4 hours and the whole remaining bone soft tissue was mechanically removed. the pieces were stained with methylene blue (1 g/100 ml) for 1 minute to delimit the cement enamel junction. for histological analysis, the mandibles were fixed in 10% neutral buffered formalin and then demineralized in 5% formic acid for 7 days. 7 m sections from the decalcified bone were stained with hematoxylin and eosin staining and evaluated to confirm bone loss. after ligature placement, animals were randomly assigned to two experimental groups (n=10 animals / group) : 1) rats with ligature + vehicle (saline ; 10 ml / kg ; orally) and 2) rats with ligature + simvastatin (25 mg / kg ; orally). simvastatin (sigma - aldrich, st. ipsilateral mandible samples were immediately fixed in 10% neutral buffered formalin for 24 hours, washed 3 in phosphate - buffered saline, and then stained with 1% methylene blue.11 bone loss was evaluated in both contralateral and ipsilateral sides by comparison of digital images. gingival tissue samples were homogenized in 300 l of ripa lysis buffer (santa cruz biotechnology, dallas, tx, usa) and then centrifuged for 10 minutes at 10,000 rpm at 4c. the supernatants were collected and used for evaluation of cytokine (tnf and il-10) and mmp (mmp-2 and 9) levels. gingival tnf-, il-10, mmp-2, and mmp-9 levels were evaluated by using commercially available kits (r&d systems, minneapolis, mn, usa) according to the manufacturer s instructions. the percentage of inhibition is reported as the mean standard deviation for each individual experiment. statistical comparison was performed using unpaired student s t - test in graphpad prism 6.0 (graphpad software, inc., la jolla, ca, usa). figure 1 shows the bone integrity of both the contralateral and ipsilateral sides of the mandible. the ipsilateral (ligature ; figure 1b), but not the contralateral (figure 1a) side, presented loss of the alveolar crest (black arrow), indicating the successful induction of periodontitis in the tested rats. in addition, the histological analysis demonstrated an inflammatory cell infiltration with cementum and alveolar process destruction confirming the periodontitis (ligature, figure 1d). the levels of tnf- and il-10 were evaluated in gingival tissue samples with established periodontitis obtained from vehicle- and simvastatin - treated rats. however, il-10 was upregulated in simvastatin - treated animals (1.8-fold increase) in comparison with those of the vehicle - treated group (figure 2b). gingival levels of mmp-2 and -9 were measured in samples obtained from vehicle- and simvastatin - treated rats. oral treatment with simvastatin reduced the gingival levels of mmp-9 (64.3%) in comparison with vehicle - treated samples (figure 3b). gingival inflammation triggered by bacteria is a hallmark of periodontitis and progressively leads to bone resorption and tooth loss.14 a plethora of cells and inflammatory mediators are involved in this process, leading to the higher production of proinflammatory molecules such as tnf and mmps, accounting for disease progression.15 nonsurgical treatment of periodontitis is currently used in to the clinics and is primarily based on administration of either systemic or local antibiotics.6 increased bacterial resistance to the therapy, in addition to its side effects,16 has lead to the search for novel therapeutic approaches. the increased knowledge on the inflammatory pathways involved in periodontitis has prompted the investigation of the effects of anti - inflammatory drugs as strategies to reduce gingival inflammation.16 recent studies have suggested that cholesterol - lowering drugs such as simvastatin are protective against experimental2,4,17 and human periodontitis.18,19 these evidences have suggested that simvastatin is able to reduce bone loss and modulate the expression / release of inflammatory markers in gingival crevicular fluid samples. however, little is known of simvastatin effects on the gingival tissue itself. herein, we provided data that this drug modulated inflammation in the gingival tissue of animals with experimental periodontitis induced by ligature placement. this study initially investigated the levels of cytokines in gingival tissue samples obtained from animals with established periodontitis. tnf- is well known for its involvement in inflammatory disorders, contributing to inflammation and pain, and plays a leading role in bone resorption in periodontitis.20 indeed, this inflammatory mediator is one of the detrimental factors for disease progression as it stimulates osteoclast activity by inducing rankl production, in addition to mmp production.17,21 simvastatin was previously suggested to inhibit tnf-.12 this study found that simvastatin does not affect tnf- production at 14 days after disease induction, as animals treated with this drug exhibited similar levels of this mediator in their gingival samples to those of the vehicle - treated group. however, fentoglu demonstrated that simvastatin inhibits tnf release in to the gum when administered for 3 months in vivo ; this suggests that in order to modulate this cytokine, simvastatin may have to be taken for longer periods than assessed in our study. also, we can not overrule the possible modulatory effects of this drug on the release of other proinflammatory cytokines in our model. indeed, recent studies showed that simvastatin anti - inflammatory effects are also related to its ability to reduce il-1 and il-6 levels and to increase the anti - inflammatory cytokine il-10.4 studies in il-10 knockout mice showed that these mice present with higher osteoclast activity and bone loss,23 and this has been linked to increased rankl expression.24 here, simvastatin increased the gingival release of il-10, reinforcing its protective effects in periodontitis. mmps, proteases that target the extracellular matrix, are released by infiltrating inflammatory cells such as macrophages and fibroblasts residing in the gum, and they are associated with remodeling of the soft and mineralized periodontal tissues.1 their expression is known to be regulated by cytokine production in periodontitis4,5 and other chronic diseases in which tissue destruction occurs.1 mmp gene polymorphisms have been recently linked to periodontitis susceptibility.25 our data show that simvastatin reduced gingival mmp-9 but did not affect mmp-2 levels in samples with established periodontitis. an inhibitory effect on mmp expression this drug was found to decrease mmp-9 mrna expression in gingival tissue samples from rats with periodontitis induced by aggregatibacter actinomycetemcomitans lps, and a similar effect was observed when simvastatin was incubated in vitro with human leukemia cells.26 also, patients treated with simvastatin present with lowered levels of circulating mpp-927 and reduced expression of this molecule in aortic wall samples.28 the effects of simvastatin on mmp-2 have been shown to be rather controversial, with some reports suggesting inhibition of this molecule by simvastatin29 and others suggesting a lack of effect for this drug on this pathway.26,30 it is possible though that mmp regulation by simvastatin differs according to the disease model investigated. in the case of periodontitis, mpp-2 has been linked with asymptomatic apical disease, while mmp-9 is associated with chronic gingival inflammation.31 overall, our data showed that simvastatin may be useful for treating gingival inflammatory conditions, and more specifically, periodontal disease, as repeated treatment reduces mmp-9 levels while increasing il-10 release into the inflamed gum. we suggest simvastatin be used as an aid therapy for chronic inflammatory diseases in which bone resorption occurs. | purposethe aim of this study was to evaluate the effect of simvastatin on the synthesis of cytokines tnf- and il-10 and metalloproteinase (mmps) 2 and 9 in a rat model of ligature - induced periodontitis.materials and methodstwenty wistar rats were used, and a cotton ligature was place in a subgingival position encircling the entire cervix of the first molar of the left (ipsilateral) side of the mandible. the right (contralateral) side of the mandible had no ligature placed and was used as control. after the ligature placement, animals were randomly assigned to two experimental groups (n=10) : 1) rats with ligature + vehicle (saline ; 10 ml / kg ; orally) and 2) rats with ligature + simvastatin (25 mg / kg ; orally). after 14 days of treatment, the animals were euthanized by anesthetic overdose and the gingival tissue was removed and homogenized in appropriate buffer. mmp-2 and -9 release as well as the il-10 and tnf- levels were detected by enzyme - linked immunosorbent assay. statistical comparison was performed by unpaired student s t - test, with p0.05). however, il-10 was upregulated in simvastatin - treated animals (1.8-fold increase) in comparison with the vehicle - treated group (p<0.05). simvastatin reduced the gingival levels of mmp-9 (64.3%) in comparison with vehicle - treated samples (p<0.05).conclusionoral treatment with simvastatin increased the release of il-10 and reduced the mmp-9 in ligature - induced periodontitis model in rats. |
as an important and rare complication of acute stsegment elevation myocardial infarction (stemi), free wall rupture is underrecognized 1. sometimes it can be subacute and may not be typical of an acute blowout rupture leading to death in few minutes 1, 2, 3, 4. the first two patients experienced a relatively long period from rupture to death, and had some similar and characteristic presentations : recurrent or persistent chest pain and stsegment elevation, and hypotension. all these characters can be explained by small rupture of free wall with slow bleeding, and have not been reported extensively. thus, we summarize it as subacute freewall rupture syndrome. fortunately, we applied the conception to the following third patient with similar clinical condition, and salvaged him successfully. a 40yearold man was in his normal condition until awakened by a sudden squeezing chest pain 5 h prior to admission to our hospital. electrocardiograph (ecg) at 5 h after the onset showed profound stsegment elevation in the leads i, avl, and v1v6 (fig. then, the patient was admitted to our hospital, and a loading dose of dual antiplatelet agents (aspirin 300 mg and clopidogrel 600 mg) were administered immediately. the vessel was reopened by ballooning, and a drugeluting stent (des) was implanted in the proximal lad, shown in figure 2. as a final result, intravenous tirofiban was given continuously at a speed of 0.1 g / kg / min after pci. however, 24 h after the procedure, his chest pain aggravated again with recurrent stsegment elevation in pericardial leads, shown as figure 1c. the pain was located in the same area, but sharper than before, and occasionally sharpened by deep inspiration with mild dyspnea, and his blood pressure (bp) ranged from 100 to 125/60 to 70 mmhg. then, subacute stent thrombosis was suspected, but he and his family refused further study by cag. then, he was treated with intensive antithrombotics, including aspirin, clopidogrel, and prolonged tirofiban. sixty hours after pci, his condition worsened with severe dyspnea, sweating, and hypotension. all the antithrombotics were discontinued except clopidogrel, considering the implanted stent at proximal lad. the drainage was 420 ml in the first day, total 580 ml in the following second day, but on the fourth day increased to 740 ml and hemoglobin dropped from the normal to 81 his bp dropped further, and then he was transferred to department of cardiac surgery, and exploratory pericardiotomy was performed. during the operation, no definite crevasse was found, but blood clot on the surface of his heart and a slow bleeding area on the lateral wall of lv, were repaired by patch suture technique. a series of ecgs of case 1, a 40yearold man on account of chest pain for 5 h. (a) ecg before primary pci showed stsegment elevation of 24 mm in pericardial leads. (b) ecg immediately after primary pci showed a partial resolution of stsegment elevation. (c) ecg 24h after primary pci showed a recurrent stsegment elevation in pericardial leads. (a) the arrow indicated the position of the total occlusion at proximal lad before pci on the spider view. (b) the occluded lad was reperfused after a drugeluting stent was implanted at the proximal of lad. the arrow indicated position of the stent. in this case, when recurrent chest pain with stsegment elevation reoccurred to him 24 h after primary pci, culprit artery reocclusion due to intrastent thrombosis was thought to be the unique cause. but there could be no culprit artery reocclusion suggested by no second peak of creatine kinase mb (ckmb) on continuous monitoring. till his bp dropped, acute cardiac tamponade caused by free wall rupture was taken into account, and confirmed by bedside ucg. a 69yearold man was admitted due to abrupt chest pain for 3 h. he had histories of hypertension, stroke, and digestive ulcer, but no diabetes. pe : clear mentality, supine, bp 75/45 mmhg, no rales in both lungs, hr 118 bpm, no edema. ekg showed sinus tachycardia, st segments of i, ii, avl, and v5v9 elevation of 13 mm, qs waves in leads of ii, iii, v6v9 (fig. lab testing : ddimer 1.2 mg / l, white blood cell 31.9 10/l, hemoglobin 150 g / l, cardiac troponin i (ctni) 7.5 ng / ml, ckmb 38.7 ng / ml. four hours after pci, the patient suddenly lost his consciousness with electromechanical dissociation, and the subsequent resuscitation was ineffective. ecg of case 2, a 69yearold man admitted on account of abrupt chest pain for 3 h. ecg showed that st segments of i, ii, iii, avf, avl, and v5v6 elevated 0.53 mm, and q waves formed in leads of ii, iii, avf, and v5v6. left coronary angiography of case 2 before and after primary pci. (a) the hollow arrow indicated the position of total occlusion at mid lcx before pci on the caudal 35. the four solid arrows show the fatpad sign. after review of the case in detail, several lines of clues to the cause of his death were found. firstly, the patient had 3 h chest pain, but the level of ctni (7.5 ng / ml) on admission was much higher than the level expected. secondly, he had persistent hypotension even treated by supplementation of normal saline, and successful reperfusion. finally, on review of the cag, differential density sign, a manifestation of pericardial effusion, was detected on the images on caudal 35 projection before pci (fig. a 78yearold man was admitted because of chest discomfort for 3 days, and sudden chest pain with dyspnea and dizziness for 1 h. he had a history of hypertension, no diabetes. pe : bp 80/40 mmhg, blurred mentality, no rales in lungs, hr 54 bpm, irregular, no edema, no pathological reflex. 5) showed sinus tachycardia with wenckebach atrioventricular block, stsegment elevation in leads ii, iii, avf, and v7v9 with inverted t waves. testing : ctni 9.94 ng / ml, ckmb 31.5 u / l, ddimer 0.1 mg / dl. ecg of case 3, a 78yearold man admitted because of chest discomfort for 3 days, and sudden chest pain with dyspnea and dizziness for 1 h. ecg on admission showed that sinus rhythm with wenckebach atrioventricular block, pathologic q waves, and st segments elevation in leads ii, iii, and avf with inverted t waves. note : left arm / right arm leads were reversal. according to his symptom, stemi was presumed to occur 1 h before admission, but pathological q wave with the inverted t waves suggested that the duration of the infarction might be longer than 1 h ; furthermore, his ctni was extremely high, therefore, 3 days duration of stemi was more likely. but why did he get worsened ? according to our previous experiences, he had chest pain mismatched with the elevation of ctni, persistent st elevation, and unexplainable hypotension. therefore, the diagnosis of free wall rupture was established, and cag was canceled. after 150 ml of blood effusion was drained out, his bp increased to the normal, his consciousness became clear. all antithrombolic agents were discontinued. within the first day, 320 ml blood effusion was drained from his pericardial cavity. in the following 3 days, the drainage was 80, 40, and 20 ml, respectively, and disappeared then. the draining tube was removed on the 11th hospital day, when ucg demonstrated no pericardial effusion. the patient accepted dual antiplatelet agents till 1 month later when absence of pericardial effusion was proven by another ucg. his right coronary artery (rca) was totally occluded, and reopened by ballooning. a 40yearold man was in his normal condition until awakened by a sudden squeezing chest pain 5 h prior to admission to our hospital. electrocardiograph (ecg) at 5 h after the onset showed profound stsegment elevation in the leads i, avl, and v1v6 (fig. then, the patient was admitted to our hospital, and a loading dose of dual antiplatelet agents (aspirin 300 mg and clopidogrel 600 mg) were administered immediately. the vessel was reopened by ballooning, and a drugeluting stent (des) was implanted in the proximal lad, shown in figure 2. as a final result, intravenous tirofiban was given continuously at a speed of 0.1 g / kg / min after pci. however, 24 h after the procedure, his chest pain aggravated again with recurrent stsegment elevation in pericardial leads, shown as figure 1c. the pain was located in the same area, but sharper than before, and occasionally sharpened by deep inspiration with mild dyspnea, and his blood pressure (bp) ranged from 100 to 125/60 to 70 mmhg. then, subacute stent thrombosis was suspected, but he and his family refused further study by cag. then, he was treated with intensive antithrombotics, including aspirin, clopidogrel, and prolonged tirofiban. sixty hours after pci, his condition worsened with severe dyspnea, sweating, and hypotension. all the antithrombotics were discontinued except clopidogrel, considering the implanted stent at proximal lad. the drainage was 420 ml in the first day, total 580 ml in the following second day, but on the fourth day increased to 740 ml and hemoglobin dropped from the normal to 81 his bp dropped further, and then he was transferred to department of cardiac surgery, and exploratory pericardiotomy was performed. during the operation, no definite crevasse was found, but blood clot on the surface of his heart and a slow bleeding area on the lateral wall of lv, were repaired by patch suture technique. a series of ecgs of case 1, a 40yearold man on account of chest pain for 5 h. (a) ecg before primary pci showed stsegment elevation of 24 mm in pericardial leads. (b) ecg immediately after primary pci showed a partial resolution of stsegment elevation. (c) ecg 24h after primary pci showed a recurrent stsegment elevation in pericardial leads. (a) the arrow indicated the position of the total occlusion at proximal lad before pci on the spider view. (b) the occluded lad was reperfused after a drugeluting stent was implanted at the proximal of lad. the arrow indicated position of the stent. in this case, when recurrent chest pain with stsegment elevation reoccurred to him 24 h after primary pci, culprit artery reocclusion due to intrastent thrombosis was thought to be the unique cause. but there could be no culprit artery reocclusion suggested by no second peak of creatine kinase mb (ckmb) on continuous monitoring. till his bp dropped, acute cardiac tamponade caused by free wall rupture was taken into account, and confirmed by bedside ucg. a 69yearold man was admitted due to abrupt chest pain for 3 h. he had histories of hypertension, stroke, and digestive ulcer, but no diabetes. pe : clear mentality, supine, bp 75/45 mmhg, no rales in both lungs, hr 118 bpm, no edema. ekg showed sinus tachycardia, st segments of i, ii, avl, and v5v9 elevation of 13 mm, qs waves in leads of ii, iii, v6v9 (fig. lab testing : ddimer 1.2 mg / l, white blood cell 31.9 10/l, hemoglobin 150 g / l, cardiac troponin i (ctni) 7.5 ng / ml, ckmb 38.7 ng / ml. four hours after pci, the patient suddenly lost his consciousness with electromechanical dissociation, and the subsequent resuscitation was ineffective. ecg of case 2, a 69yearold man admitted on account of abrupt chest pain for 3 h. ecg showed that st segments of i, ii, iii, avf, avl, and v5v6 elevated 0.53 mm, and q waves formed in leads of ii, iii, avf, and v5v6. left coronary angiography of case 2 before and after primary pci. (a) the hollow arrow indicated the position of total occlusion at mid lcx before pci on the caudal 35. the four solid arrows show the fatpad sign. after review of the case in detail, several lines of clues to the cause of his death were found. firstly, the patient had 3 h chest pain, but the level of ctni (7.5 ng / ml) on admission was much higher than the level expected. secondly, he had persistent hypotension even treated by supplementation of normal saline, and successful reperfusion. finally, on review of the cag, differential density sign, a manifestation of pericardial effusion, was detected on the images on caudal 35 projection before pci (fig. a 78yearold man was admitted because of chest discomfort for 3 days, and sudden chest pain with dyspnea and dizziness for 1 h. he had a history of hypertension, no diabetes. pe : bp 80/40 mmhg, blurred mentality, no rales in lungs, hr 54 bpm, irregular, no edema, no pathological reflex. 5) showed sinus tachycardia with wenckebach atrioventricular block, stsegment elevation in leads ii, iii, avf, and v7v9 with inverted t waves. testing : ctni 9.94 ng / ml, ckmb 31.5 u / l, ddimer 0.1 mg / dl. ecg of case 3, a 78yearold man admitted because of chest discomfort for 3 days, and sudden chest pain with dyspnea and dizziness for 1 h. ecg on admission showed that sinus rhythm with wenckebach atrioventricular block, pathologic q waves, and st segments elevation in leads ii, iii, and avf with inverted t waves. note : left arm / right arm leads were reversal. according to his symptom, stemi was presumed to occur 1 h before admission, but pathological q wave with the inverted t waves suggested that the duration of the infarction might be longer than 1 h ; furthermore, his ctni was extremely high, therefore, 3 days duration of stemi was more likely., he had chest pain mismatched with the elevation of ctni, persistent st elevation, and unexplainable hypotension. therefore, the diagnosis of free wall rupture was established, and cag was canceled. after 150 ml of blood effusion was drained out, his bp increased to the normal, his consciousness became clear., 320 ml blood effusion was drained from his pericardial cavity. in the following 3 days, the drainage was 80, 40, and 20 ml, respectively, and disappeared then. the draining tube was removed on the 11th hospital day, when ucg demonstrated no pericardial effusion. the patient accepted dual antiplatelet agents till 1 month later when absence of pericardial effusion was proven by another ucg. his right coronary artery (rca) was totally occluded, and reopened by ballooning. classified the anatomical characters of free wall rupture into four types according to 42case autopsy findings : type idirect rupture ; type iimulticanalicular rupture ; type iiirupture protected by an intraventricular thrombus or a pericardial symphysis ; type ivincomplete epicardial, endocardial, or intramyocardial rupture. 6 used the classification in their study on 51 cases of free wall rupture after stemi. the clinical forms of presentation of free wall rupture, reported by them, were the following : syncope followed by death (60%), shock (21%), transitory syncope (4%), and psychomotor troubling (4%) 6. it was shown that the clinical presentations are associated with the anatomical type of the rupture, sudden death tends to occur in cases with direct rupture without any protection, which is accepted as acute rupture. as for patients with free wall rupture resulting in slow bleeding, they could survive for a relatively long time, ranging from several hours to days. these three cases with subacute rupture of lv free wall after stemi shared common clinical features : (1) recurrent or persistent chest pain, typically pericarditic without a second peak of ckmb ; (2) recurrent or persistent elevation of st segments in leads corresponding to infarcted zone, or occasionally extending to adjacent zone ; (3) unexplainable hypotension. firstly, the chest pain in this syndrome is either recurrent or continuous from onset of stemi. 6 also noted that pain persistence or recurrence occurred in 63% of the patients with free wall rupture. if the chest pain is recurrent, it is prone to be confused with the ischemic chest pain caused by culprit artery reocclusion, because location of the pain tends to be as same as that at onset of stemi. however, the pain from free wall rupture is more sharper in quality, and more or less related to inspiration, which mimics the chest pain of pericarditis. inflammation on the visceral pericardium corresponding to infarcted zone, together with stimulation of the blood in pericardium might be the causes of the pain. but if the patient experiences a silent stemi, the pain caused by rupture is prone to be confused with new onset of stemi, like the patient of case 2. it is really a big pitfall for clinicians, because any antithrombotic, especially anticoagulants, can worsen the hemorrhage. if the pain is accompanied by hypotension and blurred mentality, it is impossible for the patient to describe the pain in detail. under this situation, however, the chest pain is often mismatched with ecg evolution, and levels of serum myocardial biomarkers. the q waves may emerge within the first hour of infarction, but most commonly develop 812 h into infarction. very early deep pathologic q waves, for example case 3, may signify a previous silent myocardial infarction (mi) with pericardial pain as its first manifestation 7. the values of troponins remain normal or mildly higher in most patients with acute cardiac events as long as 6 h after symptom onset 8, 9. the level of ckmb within 6 h after onset of pain was reported ranging from 1.1 to 6.7 ng / ml, whereas troponin i from 0.4 to 1.2 ng / ml 10. therefore, as case 2 and case 3, it would be mismatched that patient with chest pain of onset within 3 h had relatively high levels of ckmb and troponin. ecg is most important laboratory test for diagnosis of acute pericarditis, and is also important to diagnose cardiac rupture after stemi. characteristic ecg change in free wall rupture is persistent or recurrent stsegment elevation, mainly located in the infarcted zone, occasionally spreading to all leads, but avr as reported by raposo 4. 11 also reported some ecg features of heart rupture, such as persistent stsegment elevation with t waves failing to invert in the same leads. in some cases, secondary pericarditis surrounding the infarct zone and local ventricular wall expansion may be possible contributors to the st elevation. generally, persistent occlusion of infarctrelated artery, or no effective myocardial reperfusion is most likely cause of unresolved st elevation. additionally, there are still two other important complications of stemi manifested by persistent or recurrent st elevation, which need to be differentiated from free wall rupture. firstly, the majority of cases presenting with recurrent st elevation after stemi are caused by reocclusion of culprit artery 12. noninterventional measure to make sure the reocclusion is a second peak of ckmb detected by continuous monitoring of serum ckmb. persistent st elevation in infarcted zone was also observed in patients with left ventricular aneurysm 13. clinical presentations of lv aneurysm are often symptoms of heart failure with absence of chest pain and a second peak of ckmb. outward bulging of the ventricular wall during systole occasionally with mural thrombus confirmed by ucg is necessary to the diagnosis. if the amount of hemorrhage is not enough to cause hemodynamic compromise, bp may keep in the normal range. on the other hand, for patients with stemi, hypotension may also result from infarction of right ventricle, severe lv dysfunction, or even dehydration. if hypotension can not be explained by the known conditions, cardiac tamponade must be taken into account. reported that the incidence of persistent or recurrent chest pain, hypotension, ecg changes, was 63.5%, 94%, 40%, respectively. but when all the signs are integrated, the balance of sensitivity and false positive diagnosis can be improved. therefore, we strongly recommended that integrating all the signs into a syndrome to increase the clinician 's awareness to the fatal complication. bedside ucg is the most important measure to establish the diagnosis of free wall rupture. the pericardium does not allow sudden increases in the volume without a marked increase in the intrapericardial pressure, thus, a sudden increase in pericardial volume of 100200 ml, as in hemopericardium, may elevate pericardial pressure till 2030 mmhg with acute cardiac tamponade 14. in case 1 and case 3, the volume of first drainages was 170 and 150 ml, respectively. under this situation, cardiac silhouette under xray is an alternative way to detect pericardial effusion. fatpad sign is resulted from separation of retrosternal from epicardial fat line, with low sensitivity but high specificity to pericardial effusion 15., similar to fatpad sign, is increase in lucency at heart margin caused by slight difference in contrast between myocardium, epicardial fat, and pericardial fluid. additionally, diminished pulsation of the outer contour of pericardium with heart can also be observed under fluoroscopy. increasing cases were survived from the fatal complication by successful surgical or conservative therapy 2, 3, 4, 11, 16, 17. 16 concluded that surgery is superior to conservative management for patients presenting with free wall rupture. 11. believed that surgical intervention is mandatory, once hemorrhagic fluid is confirmed by pericardiocentesis. to our opinions, conservative management can be successfully carried out in elderly patients and patients at a high surgical risk, who have small infarction with slow bleeding and absence of other mechanical complications. additional to timely diagnosis, discontinuing any antithrombotics is an important measure to ensure the conservative management to succeed. now that antithrombotics are mandatory to patients with stemi, it is crucial to avoid further bleeding by discontinuing them. for patients with small infarction and stent in noncritical site otherwise, it will be a dilemma to determine whether antithrombotics should be discontinued or not like in case 1., terming subacute free wall rupture syndrome here is to help prompt detection and diagnosis of free wall rupture after stemi. as for its rarity video s1 chest xray testing taken from rao view showed a differential density sign. | key clinical messagecommon clinical features of subacute rupture left ventricular free wall after acute st segment elevation myocardial infarction are : (1) recurrent or persistent chest pain ; (2) recurrent or persistent st segment elevation ; (3) hypotension. integrating these signs into a syndrome can increase the clinician 's awareness to the fatal complication. |
using modern technologies is a common feature of today 's world. as one of the most widely used of these technologies in the modern world, the internet is playing an increasingly significant role in revolutionizing peoples ' lives. indeed, it is frequently used for on - line purchasing, data collection, chatting, communicating with others and so on. internet use has increased enormously in the last 50 years and now it seems that every aspect of people 's lives has been affected by the " global village ". although the internet offers many advantages in the era of global communication, its improper or excessive use can produce many negative consequences. excessive internet use, which is also called uncontrolled use of the internet, pathological internet use, net addiction or internet addiction, causes problems at work and in social life [3, 4 ]. internet addiction is generally defined as an uncontrollable desire to use the internet, the devaluation of time spent without connecting to the internet, intense nervousness and aggression in the event of deprivation, and progressive deterioration of social and family life. the growing number of studies conducted on internet addiction reveals that internet addiction disorder is a psychosocial disorder, the features of which include lack of patience, symptoms of isolation and emotional disorders and interruption of social relations. recent research has placed increasing emphasis on internet misuse and its consequences, both psychological and behavioral, among young people [7 - 10 ]. such consequences include the emergence of possible behavioral alterations, loss of control, school failure, social isolation and an increase in family conflict [6, 11 ]. several studies have reported correlations between internet addiction disorder (iad) and depression [12 - 17 ]. these studies have revealed that personality traits, self - esteem and psychiatric disorders are associated with internet addiction. young (1998) reported that the vast majority of internet addicts have a history of depression and anxiety. self - esteem has emerged as a factor associated with internet use and problematic internet use. in addition, research on self - esteem and use of the internet includes studies examining adolescents ' use of some social networking sites and its association with their self - esteem. these studies have shown that adolescents with low self - esteem tend to spend more time on social networking sites than those with higher selfesteem [20 - 22 ]. in the present study, our objective was to determine the prevalence of internet addiction and its relationship with depression and self - esteem among students. to this end, we investigated depression and self - esteem and their relationship with the internet addiction among students. the statistical population of the present study comprised all the male and female students studying at the islamic azad university of birjand. to select the statistical sample of the study, we applied a cluster sampling method, through which 408 students were chosen from among all the students at birjand azad university. the iat has 20 items associated with internet use, including psychological dependence, compulsive use, withdrawal symptoms and related problems of school, sleep, family and time management. for each item, a graded response can be selected (1 = " not at all " to 5 = " always "). the minimum score is 20, while the maximum is 100 ; the higher the score, the greater the level of internet addiction. as suggested by young, cut - off scores for the iat were used to classify internet users on the basis of the severity of their addictive behavior (young, 1998b). in the present study, the same cut - off scores were used : minimal users (scores 20 to 39) average online users who have complete control over their internet use, moderate users (scores 40 to 69) those experiencing occasional or frequent problems due to internet use, excessive users (scores 70 to 100) those who have significant problems caused by internet use. iat is the most famous measurement in the internet addiction field and has been used by many researchers [23- 25 ]. for example, in yang. 's study, the internal consistency (cronbach 's alpha) of iat was found to be 0.92, and its test - retest reliability proved satisfactory. moreover, widyanto and mcmurran (2004) reported that " the iat has high face validity ". the cooper smith self - esteem scale has 58 items, 8 of which numbers 6, 13, 20, 27, 34, 41, 48, 55 are lie detectors. the remaining 50 items are divided into four subscales : general self - esteem, social self - esteem (peers), family self - esteem (parents) and educational self - esteem (school). if the respondent scores more than 4 out of the 8 " lie detector " items, it means that the validity of the test is low, and that the subject has tried to portray himself to be better than he is. bahrampour ascertained that the reliability of this questionnaire was 0.90 and 0.92 for male and female students, respectively. each question contains four options, which are scored from 0 (none) to 3 (severe). the validity and reliability of the farsi version of bdi have been demonstrated in iran. the iat has 20 items associated with internet use, including psychological dependence, compulsive use, withdrawal symptoms and related problems of school, sleep, family and time management. for each item, a graded response can be selected (1 = " not at all " to 5 = " always "). the minimum score is 20, while the maximum is 100 ; the higher the score, the greater the level of internet addiction. as suggested by young, cut - off scores for the iat were used to classify internet users on the basis of the severity of their addictive behavior (young, 1998b). in the present study, the same cut - off scores were used : minimal users (scores 20 to 39) average online users who have complete control over their internet use, moderate users (scores 40 to 69) those experiencing occasional or frequent problems due to internet use, excessive users (scores 70 to 100) those who have significant problems caused by internet use. iat is the most famous measurement in the internet addiction field and has been used by many researchers [23- 25 ]. for example, in yang. 's study, the internal consistency (cronbach 's alpha) of iat was found to be 0.92, and its test - retest reliability proved satisfactory. moreover, widyanto and mcmurran (2004) reported that " the iat has high face validity ". the cooper smith self - esteem scale has 58 items, 8 of which numbers 6, 13, 20, 27, 34, 41, 48, 55 are lie detectors. the remaining 50 items are divided into four subscales : general self - esteem, social self - esteem (peers), family self - esteem (parents) and educational self - esteem (school). if the respondent scores more than 4 out of the 8 " lie detector " items, it means that the validity of the test is low, and that the subject has tried to portray himself to be better than he is. bahrampour ascertained that the reliability of this questionnaire was 0.90 and 0.92 for male and female students, respectively. each question contains four options, which are scored from 0 (none) to 3 (severe). the validity and reliability of the farsi version of bdi have been demonstrated in iran. six students (2.2%) had severe levels of internet addiction, 38.5% had medium internet addiction, and (59.3%) had no internet addiction. on comparing internet addiction between male (16.41 23.44) and female (18 29.76) students, the average difference was 3.54 and the mean internet addiction score was significantly lower in females than in males (p 0.01). multivariate regression analysis revealed that depression and selfesteem scores could significantly predict internet addiction scores (tabs. i, ii). regression model, variance analysis and regression statistical characteristics (depression). regression model, variance analysis and regression statistical characteristics (depression self - esteem) results of multiple regression analysis predicting internet addiction according to the above table, the correlation coefficient of internet addiction and depression score was 0.31, and the depression score was able to predict about 10% of the internet addiction score. in addition, the depression score could significantly predict the internet addiction score. on adding the self - esteem variable to the regression equation, this means that the self - esteem variable increases the predictive power of the internet addiction score by 1%, and the correlation of these variables is 0.33 with the internet addiction score. on the basis of the above table, it can be concluded that the ratio of regression variance to error variance is significant, which means that the self - esteem variable is significantly involved in the regression line. the table above shows that the calculated t is significant at a level of p < 0.001. thus, it can be concluded that the ratio of the slope, which is determined by the depression factor, to the standard error is significant. in addition, it can be concluded that the ratio of the slope, which is determined by the self - esteem factor, to the standard error is significant, while the internet addiction score has an inverse relationship with self - esteem. globally, an average ratio of 2 - 5 million internet addicts per 50 million regular users has been estimated. in other words, about 5 to 10 per cent of internet users have ia in a study on turkish college students, 9.7% of respondents were internet addicted. another study in iran also reported that 10.8% of medical students were internet addicted, 2.8% and 8% of whom had severe and moderate ia, respectively. the statistics yielded by different studies are very similar and slight differences may be attributed to differences in sample size and instruments. the present study showed that the rate of ia was significantly higher in male than in female students. although a few studies have reported higher rates in female students, the results of the present study are consistent with those of most of the previous studies, suggesting that male gender is a predictor of ia [33 - 35 ] ; indeed, in one study, the risk of ia was 3.5 times higher among male students than female students. in a review of ia, chou. concluded that male internet users were more at risk of ia owing to a stereotyped use of sexual contents ; however, female users may be asymptomatic or may present limited symptoms. it also seems that male students are more likely to become internet dependent because they are more experienced in using the internet, receive less parental supervision and use the internet for entertainment purposes more than females do the results of the present study revealed that self - esteem was significantly and negatively correlated with internet addiction among students. furthermore, self - esteem was found to be a significant predictor of internet addiction. in the literature, many studies have examined the association between self - esteem and pathological internet use [37 - 39 ]. based on the results of these studies, we can conclude that a negative relationship exists between these two variables. he states that the participants ' use of internet is highly associated with its perception as a coping style and a way of compensating some deficiencies, such as low self - esteem. according to him, it makes users feel better, as it allows them to assume a different personality and social identity. in other words, as can be seen, when individuals have low self - esteem, they may perceive the internet as a way of making up for these shortcomings ; increased internet use may, however, turn into a dependent relationship. as expected, depression positively predicted internet addiction. recent studies on internet addiction showed that internet addiction was related positively to a decrease in social interactions, depression, loneliness and lower self - esteem [40, 41 ]. thus, it can be said that this finding is consistent with those of other studies that have found a positive relationship between depression and internet addiction [41 - 44 ]. in addition, supportive data can be found in the studies of depressed individuals, who are more likely to engage in internet use. therefore, it appears that if individuals can reduce their internet addiction, they may reduce their depression level. first, because it was conducted at the university of birjand, its results can not be generalized to other regions and universities without further research. second, because some students manifested fatigue and impatience in answering the large number of questions in the questionnaire, their answers might have been false or distorted. the present study investigated the prevalence of internet addiction and its relationship with depression and self - esteem among students. these variables should be targeted for effective cognitive behavioral therapy in people with internet addiction. | summarybackground.the aim of the study was to investigate the relationship of self - esteem and depression with internet addiction in university students.methods.the present descriptive - analytic correlation study involved 408 students (150 female and 258 male) who had been selected by means of a cluster sampling method from among all the students studying in birjand islamic azad university. students were evaluated through the beck depression inventory (bdi), cooper smith self - esteem inventory (csei) and internet addiction test (iat).results.the results indicated that 40.7% of the students had internet addiction. a significant correlation emerged between depression, self - esteem and internet addiction. regression analysis indicated that depression and self - esteem were able to predict the variance of internet addiction to some extent.conclusions.it may be important to evaluate self - esteem and depression in people with internet addiction. these variables should be targeted for effective cognitive behavioral therapy in people with internet addiction. |
resolution of inflammation typically follows an ordered series of events orchestrated by different cell types. during the early stages of inflammation, leukocytes such as polymorphonuclear neutrophils (pmn) are the first immune cells to arrive at the site of inflammation. pmn are recruited by gradients of proinflammatory signals and usually reach peak numbers within 2448 hrs. pmn have short half - lives and are normally cleared from sites of inflammation by undergoing apoptosis. mobilized monocyte - derived macrophages extravasate to inflammatory tissue sites and clear apoptotic pmn in a nonphlogistic fashion by the process of efferocytosis. apoptotic pmn release find - me signals that are sensed by extravasated macrophages. following phagocytosis, apoptotic pmn provides resolution cues to macrophages by evoking distinct signaling events that block release of proinflammatory mediators thus allowing further engulfment of apoptotic cells. fleming and mosser note that mobilized macrophages are divided into three groups based upon their activation states [4, 5 ]. these include the m1, m2, and the recently described regulatory macrophages (mres). m1 macrophages, classically referred to as activated macrophages, secrete proinflammatory factors that mediate host defense against invading pathogens. m2 macrophages, termed alternatively activated macrophages, are considered to be anti - inflammatory [6, 7 ]. finally, mres macrophages secrete considerable amounts of anti - inflammatory cytokines that prevent inflammatory and autoimmune pathology [8, 9 ]. mres macrophages also secrete various lipid mediators that play critical roles in resolution of inflammation (see below). extensive new research has identified expression markers or phenotypic signatures for the various macrophage activation states in mice. they include gene expression changes in il-1, tnf, and inos for classical activation and arginase-1 (arg1), chitinase 3-like 3 (ym-1), and tgf for the alternatively activated macrophages. resolution of inflammation and restoration of normal tissue function prevent the development of complications of excessive inflammation, a process referred to as catabasis. catabasis is driven by synthesis and release of proresolution lipid mediators such as resolvins, protectins, and lipoxins. lipoxins and protectins are synthesized by lipoxygenase enzymes (such as 15-lipoxygenase (15-lox)). resolvins are derived from omega-3 polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid. russell and schwarze have reviewed the proresolution effects of proresolution mediators in a variety of inflammatory states. however their regulation by vitamin c (vitc, ascorbic acid, asca) has yet to be examined. vitc readily functions as one or two electron - reducing agents for many oxidants and serves as a primary chemical antioxidant in most cell types. it modulates complex biochemical pathways that form an essential part of normal metabolism of immune cells., vitc accumulates in millimolar quantities in immune cells such as pmn and macrophages in which intracellular vitc concentrations are typically 4060 fold higher than that present in circulation [17, 18 ]. in pmn, vissers and wilkie showed that intracellular vitc levels regulate neutrophil apoptosis. further, vitc contributes to the antioxidant defenses as well as normal pmn and macrophage function. oberritter and colleagues showed that intracellular concentrations of vitc in macrophages are in the low millimolar range in freshly prepared peritoneal macrophages and in vivo or in vitro activation of peritoneal macrophages results in a significant decline in their vitc content. moreover, mice deficient in vitc generate excessive proinflammatory responses upon infection with the virulent bacterium klebsiella pneumonia. in humans, vitc levels are significantly reduced in critically ill patients and specifically in patients with poorly resolving proinflammatory states (e.g., sepsis, systemic inflammatory response syndrome) [23, 24 ]. several studies performed in septic patients have found that plasma vitc levels correlate inversely with the incidence of organ failure and directly with survival [25, 26 ]. we recently showed that vitc attenuates inflammation and normalizes pmn function in septic mice [27, 28 ]. we further showed that parenteral vitc attenuates proinflammatory biomarkers and reduces mortality in human sepsis. however information is limited regarding the mechanism by which vitc regulates the progression and eventual resolution of inflammatory states. in the current study we examined the progression and resolution of inflammation using a murine thioglycollate (tg-)elicited peritonitis model in vitc sufficient and deficient mice. while humans lack l - gulono--lactone oxidase (gulo), the final enzyme in the biosynthesis pathway of vitc, mice express functional gulo, resulting in cells and tissues generally maintaining high levels of vitc thereby complicating the translatability of vitc studies in mice, to humans. in order to establish the studies more relevant to humans, our studies reveal that progression and resolution of tg - induced inflammation is significantly delayed in vitc deficient mice. in particular, the spatiotemporal profile of pro- and anti - inflammatory mediator production by tg - elicited macrophages was significantly different between the vitc sufficient and deficient mice. further, macrophage function and phenotype, as well as the antioxidant capacity of vitc deficient macrophages, was significantly impaired by the decline in intracellular vitc levels. infusion of parenteral vitc as ascorbic acid (asca) partly restored macrophage phenotype and function in vitc deficient mice. gulo mice were bred in - house from an established homozygous colony as previously described. in order to maintain their plasma vitc levels similar to that observed in humans, vitc sufficient mice were fed ad libitum with regular chow and water supplemented with vitamin c (0.33 g / l) for 1 week, followed by complete removal of dietary vitc for additional 2 weeks. we and others have shown that this reduced supplementation significantly decreases the concentration of vitc in immune cells, plasma, and organs [27, 31, 32 ]. injection of 1 ml aged, sterile 3% tg solution to 911-week old gulo mice. injection of vitc as asca (200 mg / kg in saline) for a further 3 or 5 days. mice were euthanized on day 3 or day 5, and the peritoneal cavity lavaged with 7 ml of hanks ' balanced salt solution (hbss) containing 1% bovine serum albumin (bsa). the lavage was centrifuged and the resulting leukocyte pellet was washed with hbss and resuspended in rpmi-1640 medium. cytochemical staining of peritoneal cells was performed using harleco hemacolor solution (emd millipore). pmns were then separated from macrophages by adherence to a plastic dish as described previously [28, 33 ]. peritoneal macrophages were plated at a density of 2 10 cells in 35 mm dishes in growth media (dmem, 10% fbs). human acute monocytic leukemia suspension cell line (thp-1) was obtained from atcc (manassas, va). thp-1 cells were maintained in rpmi-1640 medium containing 10% fbs according to the instructions supplied. for induction of macrophages, pma (100 nm) was added to the medium and cells were seeded at a density of 0.1 10 cells / cm into tissue culture dishes and maintained in a humidified atmosphere of 95% air and 5% co2. media containing pma were replaced every 2 days, and experiments started after 5 days in culture, when the cells were phenotypically macrophage. plasma and intracellular vitc levels of peritoneal macrophages seeded onto 35 mm dishes were measured using a fluorescence end - point assay adopted from vislisel.. briefly, 0.2 ml of cold 20% trichloroacetic acid (tca) and 0.2 ml of cold 0.2% dithiothreitol (dtt) were added to 0.1 ml of plasma, vortexed for 2 min, and centrifuged (10,000 g, 10 min, 4c). peritoneal macrophages were similarly extracted with tca and dtt and frozen at 70c for batch analysis. assay buffer containing 1 m sodium acetate, ph 5.5, and 1 mm tempol was added to each well and the plate was incubated for 10 minutes at room temperature. freshly prepared o - phenylenediamine (opda) solution (5.5 mm opda in acetate buffer of ph 5.5) was then added. after a further 30 min incubation in the dark, fluorescence was measured at an emission wavelength of 425 nm following excitation at 345 nm and values determined after comparison to a standard curve. intracellular asca levels were estimated spectrophotometrically from the standard curve and the intracellular concentrations derived from the measured amount of asca and the known macrophage cell volume. isolation of total rna and real - time qpcr analyses were performed as described previously. briefly total rna was extracted and purified using qiashredders and rneasy columns according to the manufacturer 's specifications (qiagen). total rna (1 g) was reverse - transcribed into cdna using the high capacity cdna reverse transcription kit. complimentary dna (cdna) was diluted (1 : 500) and real time qpcr performed using power sybr green qpcr master mix. primers were designed to anneal to sequences on separate exons or to span two exons. cycling parameters were 95c, 10 min, 40 cycles of 95c, 15 sec, and 60c, 1 min. all pcr assays were performed in triplicate. no template controls and no reverse transcriptase controls were included. the mrna expression in macrophages from a the mrna expression of all other samples was compared relative to this sample which was used as the baseline. 18s rrna was used as housekeeping gene against which all the samples were normalized for differences in the amount of total rna added to each cdna reaction and for variation in the reverse transcriptase efficiency among the different cdna reactions. automated gene expression analysis was performed using the comparative quantitation module of mxpro qpcr software (agilent). mouse macrophage and thp - i whole - cell extracts were isolated for western blot analysis as described previously. proteins were resolved by sds polyacrylamide gel electrophoresis (420%) and electrophoretically transferred to polyvinylidene fluoride membranes (0.2 m pore size). immunodetection was performed using chemiluminescent detection with the renaissance western blot chemiluminescence reagent plus (perkin elmer life sciences inc., blots were stripped using the restore western blot stripping buffer (pierce biotechnology inc., purified rabbit polyclonal antibodies to phospho - nfb p65 (ser276, cell signaling), nfb p65 (sc-109, santa cruz biotechnology), inos (sc-650, santa cruz biotechnology), and actin (sc-1616, santa cruz biotechnology) were used. optical densities of antibody - specific bands were determined using quantity one acquisition and analysis software (bio - rad, hercules, ca). mouse peritoneal lavage obtained on day 3 and day 5 from vitc sufficient or deficient mice following induction of tg - induced peritonitis was pelleted by centrifugation at 4c. cells were resuspended in facs buffer containing fc receptor block (cd16/cd32 ebioscience) for 10 min at 4c. aliquots of the suspension were incubated at 4c for 30 min (in the dark) with fluorescein isothiocyanate (fitc-)conjugated anti - mouse cd45 (ebioscience) and allophycocyanin (apc-)conjugated anti - mouse cd11b (ebioscience). all runs were performed on a bd accuri c6 flow cytometer (bd accuri cytometers, mi, usa) and analyzed using flowjo software (tree star, ashland, or). fluorescence microscopy for evaluation of mitochondrial reactive oxygen species (ros) in macrophages was performed using the cell - permeant probe mitotracker red cmxros as described by the manufacturer. briefly, macrophages from vitc sufficient or deficient mice were grown on ibidi 6-channel ibitreat -slide vi. following treatments (h2o2, 18 hours) culture media were aspirated and cells were fixed in 3.7% paraformaldehyde in pbs for 10 minutes at 4c. fluorescence imaging was performed using an olympus model ix70 inverted phase microscope (olympus america, melville, ny) outfitted with an ix - fla fluorescence observation system equipped with a tritc filter cube (530560 nm excitation and 590650 nm emission, chroma technology corp. fluorescence images were digitized and captured by a magnafire digital camera (optronics, goleta, ca). quantitative analysis of eicosanoids was performed as previously described by us with minor modifications [3843 ]. briefly, peritoneal lavage was clarified by centrifugation and 0.05% bht and 10 ng of each internal standard added. the internal standards used were (d4) 8-iso pgf2, (d11) 5-iso pgf2-vi, (d4) 6k pgf1, (d4) pgf2, (d4) pge2, (d4) pgd2, (d4) ltb4, (d5) lipoxin a4, (d5) resolvin d2, (d4) txb2, (d4) ltc4, (d5) ltd4, (d5) lte4, (d8) 5-hydroxyeicosatetranoic acid (5hete), (d8) 15-hydroxyeicosatetranoic acid (15hete), (d8) 14,15 epoxyeicosatrienoic acid, (d8) arachidonic acid, and (d5) eicosapentaenoic acid. the samples were mixed by vortexing and subjected to purification via solid phase extraction (spe) using a 24 port vacuum manifold (sigma - aldrich). strata - x spe columns (phenomenex) were washed with methanol (2 ml) and then dh2o (2 ml). the samples were applied to the column. thereafter the sample vials were rinsed with 5% meoh (2 ml), which was then used to wash the columns. the eluents were then dried under vacuum and reconstituted in lcms grade 50 : 50 etoh : dh2o (100 l) for eicosanoid quantitation via uplc esi - ms / ms analysis. a 14-minute reversed - phase lc method utilizing a kinetex c18 column (100 2.1 mm, 1.7 m) and a shimadzu uplc was used to separate the eicosanoids at a flow rate of 500 l / min at 50c. the column was first equilibrated with 100% solvent a (acetonitrile : water : formic acid (20 : 80 : 0.02, v / v / v)) for two minutes and then 10 l of sample was injected. solvent b (acetonitrile : isopropanol (20 : 80, v / v)) was increased in a linear gradient to 25% solvent b to 3 minutes, to 30% by 6 minutes, to 55% by 6.1 minutes, to 70% by 10 minutes, and to 100% by 10.1 minutes. 100% solvent b was held until 13 minutes and then was decreased to 0% by 13.1 minutes and held at 0% until 14 minutes. the eluting eicosanoids were analyzed using a hybrid triple quadrapole linear ion trap mass analyzer (absciex 6500 qtrap) via multiple - reaction monitoring in negative - ion mode. the mass spectrometer parameters used were curtain gas : 30 ; cad : high ; ion spray voltage : 3500 v ; temperature : 300c ; gas 1 : 40 ; and gas 2 : 60 ; declustering potential, collision energy, and cell exit potential were optimized per transition. statistical analysis was performed using sas 9.3 and graphpad prism 6.0 (graphpad software, san diego, ca, usa). in order to make the gulo mice vitc deficient, supplementation of water with asca was withdrawn as described in the methods section. within 3 weeks of removal of vitc supplementation, this decline was not associated with deleterious changes in weight or health status in the vitc deficient mice (data not shown). to determine whether vitc deficiency impacts the progression of peritoneal inflammation, vitc sufficient or deficient mice were injected with tg and the progression of inflammation was monitored on days 3 and 5 (as described in section 2). with asca (200 mg / kg) prior to harvest of peritoneal lavage (see section 2). administration of ascorbate for 3 days restored circulating plasma vitc concentrations in these mice to levels observed in the vitc sufficient mice (figure 1). in all 3 groups, the infiltration of inflammatory cells on day 1 was similar to that observed in wild type mice and was in agreement with our previous observations (table 2). as seen in table 2, there was also no difference in the total number of cells elicited from the peritoneal exudation of day 3 and day 5. however, significant differences in the cellular composition of the lavage were evident on day 3 and day 5 between the 3 groups. in the vitc sufficient mice group, mononuclear cells were the predominant cell type on days 3 and 5 (table 2). pmn numbers, which peaked on day 1, returned to baseline by days 3 and 5. in contrast, significantly elevated numbers of pmns persisted in the peritoneal exudates of vitc deficient mice on days 3 and 5 (table 2). infusion of asca reduced pmn numbers by day 3 with a significant decline in pmn numbers to baseline similar to the vitc sufficient mice by day 5 table 2. we previously observed that tg - elicited pmn from vitc deficient mice (on day 1) demonstrated increased expression of the proinflammatory genes tnf and il-1. here we examined the expression of multiple pro- and anti - inflammatory mediators originating from macrophages, the predominant cell type recruited to the inflamed peritoneum on days 3 and 5. as seen in figure 2, significant differences were evident in the magnitude of pro- and anti - inflammatory mediator expression on days 3 and 5. on day 3, increased expression of the proinflammatory mediators (il-1, tnf, and mcp-1) was observed in macrophages from vitc deficient mice when compared to macrophages from vitc sufficient mice (figure 2(a), (a), (c), and (e)). infusion of asca in the vitc deficient mice (figure 2(a), (a), (c), and (e)). in contrast, anti - inflammatory gene expression (ym1 and arg1, but not il-10) was elevated in macrophages from vitc sufficient mice (figure 2(a), (b), (d), and (f)). daily asca infusion induced ym1 expression in vitc deficient macrophages but failed to restore arg1 expression. il-10 expression on the other hand was significantly lowered by asca infusion on day 3 (figure 2(a), (b), (d), and (f)). on day 5 (figure 2(b)), proinflammatory gene expression remained persistently elevated in macrophages from vitc deficient mice (il-1 and mcp-1) but was attenuated by asca infusion. in contrast, anti - inflammatory gene expression in vitc deficient macrophages was significantly higher when compared to macrophages from vitc sufficient mice (arg1, il-10). asca infusion did not alter anti - inflammatory gene expression on day 5 although arg1 levels were now similar to that observed in vitc sufficient mice (figure 2(b), (b), (d), and (f)). canali. recently showed that in contrast with baseline physiological activation, exposure to a second hit such as an inflammatory stimulus results in a markedly different modulation of gene expression in human peripheral blood mononuclear cells in the presence or absence of vitc supplementation. to examine whether peritoneal macrophages would exhibit an altered modulation of gene expression, we exposed day 3 peritoneal macrophages from vitc sufficient and deficient mice to bacterial lipopolysaccharide (lps, 50 ng / ml). some macrophages were incubated with asca (3 mm, 16 hours) prior to lps exposure. as seen in figures 3(a) and 3(b), lps exposure resulted in a robust increase in expression of proinflammatory markers (il-1, tnf) in macrophages from vitc sufficient mice. proinflammatory gene expression was also induced in macrophages from vitc deficient mice, but the magnitude of induction was significantly greater than that observed in the vitc sufficient macrophages (figures 3(a) and 3(b)). importantly, exposure of vitc deficient macrophages to asca prior to lps significantly attenuated il-1 and tnf expression. increased nfb activation (figure 3(c)) and inos protein expression (figure 3(d)) was observed upon exposure of vitc deficient macrophages to lps (p < 0.05). macrophages undergo reprogramming to adopt a variety of functional phenotypes upon receiving differentiation cues from their surrounding environment. we examined whether macrophage vitc sufficiency or deficiency could influence macrophage function during resolution of acute inflammation. macrophages were isolated on day 3 following tg - mediated peritonitis from vitc sufficient or deficient mice and intracellular concentrations of vitc measured (as described in section 2). asca (200 mg / kg) prior to harvest of peritoneal lavage (see section 2). as seen in figure 4(a), in contrast, intracellular ascorbate levels were significantly depleted in macrophages from vitc deficient mice. administration of asca for 3 days also restored macrophage intracellular concentrations to levels observed in the vitc sufficient mice (figure 4(a)). their expression is associated with generation of proresolving lipid mediators and successful resolution of inflammation. therefore we examined gal-1 and 15-lox expression in day 3 and day 5 macrophages from vitc sufficient or deficient mice. as seen in figure 4(b), gal-1 and 15-lox expression was significantly induced in macrophages from vitc sufficient mice on day 3 when compared to macrophages from vitc deficient mice. asca infusion restored gal-1 expression in vitc deficient macrophages but did not affect 15-lox expression on day 3 (figure 4(b)). in contrast, gal-1 expression in vitc deficient macrophages was delayed and observed to be higher on day 5 following tg - induced peritonitis (figure 4(c)). 15-lox expression was induced by asca infusion on day 5 and was higher than that observed in macrophages from vitc sufficient or deficient mice. in agreement with the expression data seen above, resolvin (figure 4(d)) production was higher on day 5 in vitc deficient mice indicating delayed resolution of inflammation.. recently showed that gal-1 was selectively expressed in cd11b macrophages, and its expression declined significantly once these cells converted toward a cd11b phenotype. moreover, cd11b macrophages are the predominant subtype to depart the peritoneum. to determine whether vitc regulated reprogramming of peritoneal macrophages to proresolution cd11b phenotype we used flow cytometry to examine the distribution of cd11b and cd11b population on macrophages isolated on day 3 and day 5 following tg - induced peritonitis in vitc sufficient or deficient mice. as seen in figure 5, there was a significant transition from cd11b to a cd11b phenotype observed from day 3 to day 5 in the vitc sufficient macrophages. this was not evident in the macrophages from vitc deficient mice indicative of a delay in the resolution of tg - induced peritonitis in these mice. release of large amounts of ros during activation exposes macrophages themselves to oxidant stresses not encountered by most other cell types. to test whether vitc deficiency affected mitochondrial function in macrophages, we exposed peritoneal macrophages (day 3) from vitc sufficient or deficient mice to varying concentrations of h2o2 for 18 hours and stained the cells with mitotracker red cmxros as described in section 2. this probe is selectively retained by mitochondria, where it is oxidized to its fluorescent form. as seen in figures 6(a) and 6(e), control macrophages from vitc sufficient or deficient mice were stained brightly with the probe. oxidative stress from exposure to h2o2 decreased fluorescent staining in macrophages from both vitc sufficient and vitc deficient mice. however, the magnitude of decrease was significantly greater in macrophages from vitc deficient mice (figures 6(f)6(h)). this decrease was partially reversed by pretreatment of vitc deficient macrophages with asca (figures 6(j) and 6(k)). these studies indicate that vitc deficient macrophages sustain greater mitochondrial dysfunction when challenged with ros. to address whether the modulatory activities of vitc are effective in human monocyte / macrophages, we exposed thp-1 cells to bacterial lps and examined the mrna expression of the proinflammatory genes il-6, il-8, and tnf. since the culture medium in which thp-1 cells are grown contains no vitc, we increased intracellular concentrations of vitc by loading cells with asca prior exposure to lps. as seen in figure 7(a), exposure of thp-1 cells to lps resulted in a robust activation of mrna for il-6, il-8, and tnf. loading cells with asca did not affect baseline proinflammatory gene expression. however lps exposure of asca loaded cells resulted in significant attenuation of mrna expression of these proinflammatory genes. attenuation of mrna expression was likely achieved by reduction in activation of the transcription factor nfb following lps exposure (figure 7(b)). in this study, we examined the mechanism by which vitc regulates the resolution of sterile inflammation. using mice lacking the ability to synthesize vitc, we show that subnormal cellular vitc levels negatively impact the progression and resolution of sterile inflammation. in particular, our results demonstrate that low circulating vitc levels are associated with significant delays in the timing of resolution of inflammation. this apparent vitc - dependent process primarily occurs due to failure of macrophages to transition from a proinflammatory to a proresolving phenotype. the initial response to sterile inflammation was identical in vitc sufficientanddeficient mice. during the early proinflammatory phase no differences in the cell numbers or cell types however, by days 3 and 5, vitc deficient mice exhibited significant numbers of pmn in peritoneal exudates (table 2). spatiotemporal mrna profiling of macrophage - derived inflammatory mediators revealed dramatic differences in the magnitude of pro- and anti - inflammatory mediator gene expression (figure 2). macrophages from vitc sufficient mice displayed prominent anti - inflammatory phenotypes, while vitc deficient macrophages persistently expressed mrna for il-1, tnf, and mcp-1, findings characteristic of a proinflammatory phenotype. lps activation of day 3 macrophages from vitc deficient mice led to proinflammatory gene expression that was significantly greater in magnitude than that observed in vitc loaded macrophages (figure 3). lps stimulation was characterized by enhanced nfb activation and inos induction in vitc deficient macrophages (figure 3). importantly, on day 3, vitc sufficient macrophages demonstrated cues for reprogramming into resolution type macrophages, a vital step required for resolution of inflammation. in day 3 macrophages from vitc sufficient mice, expression of gal-1 and 15-lox mrna was robust (figure 4). in contradistinction, enhanced gal-1 and 15-lox mrna expression was delayed to day 5 in vitc deficient macrophages. the delays in resolution we observed in vitc deficient mice were confirmed by quantification of resolvins in peritoneal exudates ; increases of which were present only on day 5 (figure 4). further confirmation of altered spatiotemporal relationships was achieved by studying macrophage phenotypic changes by examining the distribution of cd11b on macrophages from vitc sufficient or deficient mice on day 3 and day 5 following tg - induced peritonitis (figure 5). phenotypic changes in macrophages were accompanied by alterations in macrophage function as demonstrated by the increased susceptibility of vitc deficient macrophages to mitochondrial dysfunction when exposed to reactive oxygen species (figure 6). in final studies, we employed the human monocyte / macrophage cell line thp-1, which lacks vitc in culture medium when cultured under standard conditions. we demonstrated increased proinflammatory gene expression in thp-1 when exposed to lps under vitc - deprived conditions. loading thp-1 cells with asca significantly attenuated mrna expression of proinflammatory genes via a mechanism likely involving reduced activation of the transcription factor nfb. vitc loading was effective both in vitro and in vivo since daily asca infusion following induction of peritonitis significantly restored macrophage phenotype and function in the vitc deficient mice. ganguly. initially reported that vitc deficiency affected migration of guinea pig macrophages under in vitro conditions. they later showed that ascorbate deficient peritoneal macrophages were more susceptible to h2o2-induced mitochondrial dysfunction and apoptosis. however no studies to date have examined macrophage function during resolution of inflammation in mice lacking the ability to synthesize their own vitc. our observation of persistence of pmn at the site of inflammation in vitc deficient mice is in agreement with our previous results and those of vissers and wilkie who used a similar tg model of peritonitis to show impairment in pmn apoptosis and clearance. it has been suggested that the engulfment of apoptotic cells is generally anti - inflammatory or immunologically silent due to the fact that it sequesters dying cells thus preventing release of potentially toxic cell contents into the local environment. based on the observations that pmn persists for up to 5 days in the peritoneum of vitc deficient mice (table 2), it is therefore possible that the apoptosis - resistant pmn can cause strong proinflammatory responses from the macrophages that extravasate to sites of inflammation. indeed strong and persistent proinflammatory responses were evident in vitc deficient macrophages elicited on day 3 and even day 5 (figure 2). efferocytosis, a process by which dead and/or dying cells are being engulfed and removed by other cells, has been reported to induce production of anti - inflammatory mediators from macrophages that suppress inflammation thereby silently clearing apoptotic cells and thus dampening proinflammatory responses. vitc sufficient mice exhibited anti - inflammatory mediator expression in macrophages early (day 3) in the post tg - induced inflammatory process, a phenomenon indicative of functional efferocytosis. in contrast, vitc deficient macrophages failed to upregulate anti - inflammatory mediator production until day 5 (figure 2). have shown that gal-1 promotes the generation of m2-like macrophages, which then favors tissue repair during early resolution of inflammation. ariel and timor demonstrated that gal-1 promotes generation of mres from m2 macrophages, which generates proresolving lipid mediators. this phenotype change promotes macrophage departure from peritoneal cavities with resolving inflammation, thus allowing return of tissue to homeostasis. moreover, gal-1 expression, which is enhanced in cd11b macrophages, declines sharply as cells revert to the cd11b phenotype. cd11b macrophage phenotypes, as noted previously, promote departure from peritoneal cavities with resolving inflammation. our findings (figures 4 and 5) which agree with the above studies implicate vitc as a critical regulator of macrophage transition during resolution of inflammation. expression and function of 12/15-lox produce key mediators (e.g., lipoxins, resolvins, protectins, and maresins) that promote resolution of proinflammatory pathologies. in particular, human and murine monocytes / macrophages expression of 15-lox is upregulated by efferocytosis with production of mediators such as rvd1, a mediator shown to promote the resolution of murine peritonitis [47, 57 ]. further, gal-1 directly promotes 15-lipoxygenase expression and activity in macrophages during the inflammatory and resolving phases of peritonitis. the earlier increases in gal-1 and 15-lox mrna expression in vitc sufficient macrophages (figures 4(b) and 4(c)) and the delayed resolvin production in the vitc deficient macrophages (figure 4(d)) indicate for the first time that vitc influences multiple processes leading to the resolution of inflammation. the findings in this mouse model have significant human relevance since vitc levels are subnormal in multiple human inflammatory disease states including sepsis, systemic inflammatory response syndrome (sirs), trauma, and cancer, among others. in a recently completed phase i trial (clinicaltrials.gov identifier nct01434121) of intravenous asca in critically ill patients with severe sepsis, we showed that septic patients exhibited abnormally low vitc plasma levels and that intravenous asca infusion could significantly increase circulating vitc levels. further, asca infusion significantly reduced the proinflammatory biomarkers c - reactive protein and procalcitonin as well as multiple organ dysfunction. our findings here add a previously unrecognized element to our understanding of the machinery that governs the resolution of inflammation. | introduction. macrophage reprogramming is vital for resolution of acute inflammation. parenteral vitamin c (vitc) attenuates proinflammatory states in murine and human sepsis. however information about the mechanism by which vitc regulates resolution of inflammation is limited. methods. to examine whether physiological levels of vitc modulate resolution of inflammation, we used transgenic mice lacking l - gulono--lactone oxidase. vitc sufficient / deficient mice were subjected to a thioglycollate - elicited peritonitis model of sterile inflammation. some vitc deficient mice received daily parenteral vitc (200 mg / kg) for 3 or 5 days following thioglycollate infusion. peritoneal macrophages harvested on day 3 or day 5 were examined for intracellular vitc levels, pro- and anti - inflammatory protein and lipid mediators, mitochondrial function, and response to lipopolysaccharide (lps). the thp-1 cell line was used to determine the modulatory activities of vitc in activated human macrophages. results. vitc deficiency significantly delayed resolution of inflammation and generated an exaggerated proinflammatory response to in vitro lps stimulation. vitc sufficiency and in vivo vitc supplementation restored macrophage phenotype and function in vitc deficient mice. vitc loading of thp-1 macrophages attenuated lps - induced proinflammatory responses. conclusion. vitc sufficiency favorably modulates macrophage function. in vivo or in vitro vitc supplementation restores macrophage phenotype and function leading to timely resolution of inflammation. |
details of phylogenetic and other evolutionary analyses18, vaccinia infection experiments24, genotypes of yeast strains and yeast growth assays15, 21 are presented in the full methods accompanying this paper. | distinguishing self from non - self is a fundamental biological challenge. many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis, and cytoskeletal dynamics1 - 5. other mimics interfere with immunity6, 7. poxviruses encode k3l, a mimic of eif2, which is the substrate of protein kinase r (pkr), an important component of innate immunity in vertebrates8, 9. the pkr - k3l interaction exemplifies the conundrum imposed by viral mimicry. to be effective, pkr must recognize a conserved substrate (eif2) while avoiding rapidly evolving substrate mimics like k3l. using the pkr - k3l system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. we find that pkr has evolved under dramatic episodes of positive selection in primates. the ability of pkr to evade viral mimics is partly due to positive selection at sites most intimately involved in eif2 recognition. we also find that adaptive changes on multiple surfaces of pkr produce combinations of substitutions that increase the odds of defeating mimicry. thus, while it can appear that pathogens gain insurmountable advantages by mimicking cellular components, host factors like pkr can compete in molecular arms races with mimics because of remarkable evolutionary flexibility at protein interaction interfaces challenged by mimicry. |
baboons and vervet monkeys live side by side with humans in game management areas in zambia, and this situation often leads to high levels of human baboon / monkey conflicts. the zambia wildlife authority is mandated by the zambian government to control the large numbers of these animals. we collected tissues and serum samples from baboons and vervet monkeys killed for pest management purposes with the permission of the zambia wildlife authority (certificate no. samples were obtained from 50 yellow baboons (papio cynocephalus) and 50 vervet monkeys (chlorocebus pygerythrus) in the mfuwe region (131630.2s, 314000.4e), eastern province, zambia, in 2009 and from 50 chacma baboons (p. ursinus) and 39 vervet monkeys (c. pygerythrus) in the livingstone region (17508.72s 254359.19e), southern province, zambia, in 2010 and 2011. the species were identified on the basis of morphologic characters and the mitochondrial cytochrome b (cytb) gene sequence. baboons with the primer set papio cytb1f (5-gatacgaaaaaccatcgctgt-3) and papio cytb2r (5-gctccatttctggtttacaag-3), as described (8), and from spleen dna of chlorocebus spp. monkeys with the primer set chlorocebus cytb1f (5-tgatatgaaaaaccaccgttgt-3) and chlorocebus cytb2r (5-gctttctttctgagttgtcctagg-3), designed in this study. total rna was extracted from 189 spleen tissue samples by using trizol reagent (life technologies, carlsbad, ca, usa) and screened for paramyxoviruses by seminested broad - spectrum rt - pcr of paramyxovirus polymerase (l) genes (9). amplification was carried out with the primers par - f1, par - f2, and par - r. the pcr product (584 bp) was subjected to direct sequence analysis, and the identified paramyxovirus was tentatively named zmls/2011. blast analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi) indicated that zmls/2011 shared 98% nt identity with the hpiv3 l gene. to increase the sensitivity of hpiv3 genome detection, we screened all 189 rna samples by rt - pcr, using the hpiv3 l gene specific primer sets hpiv3 l1f (5-atgggagaattcttcctcaagctc-3) and hpiv3 l2r (5-aatgcrgcaactgatggatcacc-3). an hpiv3 genome was detected in 3 (6%) of 50 chacma baboon samples and in 1 (2%) of 50 yellow baboon samples but not from any of the 89 vervet monkey samples. nucleotide sequences of all 4 amplicons (367 bp) were identical to zmls/2011. in an attempt to isolate virus from rt - pcr positive spleen, vero cells cultured in minimum essential medium supplemented with trypsin were injected with tissue homogenates ; however, after 3 passages, cytopathic effects were not observed. viral rna was also not detected in the culture supernatants from the cells. to confirm and classify zmls/2011 as a strain of hpiv3, we amplified and sequenced the genome of zmls/2011 using total rna sample positive for the rt - pcr screening. the obtained zmls/2011 sequence (15,298 bp) was deposited in the ddbj database (genbank / embl / ddbj entry ab736166). the hpiv3 genome encodes 6 structural proteins, n, p, m, f, hn, and l. all the corresponding open reading frames were found in the zmls/2011 genome. a phylogenetic analysis was performed by using mega5 and based on the deduced amino acid sequence of the full - length hn protein (10). the phylogenetic tree clearly established zmls/2011 within the lineage of hpiv3 and distinct from other known parainfluenza viruses related to it (figure 1). zmls/2011 is most closely related to hpiv3 strain riyadh 149/2009, isolated from a hospitalized child in saudi arabia in 2009 (11). these results indicated that zmls/2011 identified from the chacma baboon is a strain of hpiv3. phylogenetic analysis of the amino acid sequence of the hn protein of human parainfluenza virus type 3 (hpiv3). the phylogenetic tree was constructed on the basis of the deduced amino acid sequence of the full - length hn gene of zmls/2011 (gray shading) and known paramyxoviruses. recombinant n protein of zmls/2011 was expressed in escherichia coli and purified by histidine tag based affinity chromatography. the 189 serum specimens were screened by using the mini - protean ii multiscreen apparatus (bio - rad, hercules, ca, usa). mouse monoclonal hpiv antibody (mab819 ; millipore, billerica, ma, usa) served as the positive control. among the serum samples tested, 2 (4%) from 50 yellow baboons, 11 (22%) from 50 chacma baboons, and 6 (7%) from 89 vervet monkeys had hpiv3 antibodies (table ; figure 2). positive results were obtained from serum samples collected from animals in the mfuwe and livingstone regions. all 4 baboons positive for the hpiv3 genome were negative for hpiv3 antibodies (data not shown), suggesting that, at the time the samples were taken, these hpiv3 antibody negative baboons might have been in the acute stage of infection, before a detectable immune response had developed. rt - pcr, reverse transcription pcr. western blot analysis was performed by using serum specimens from vervet monkeys (lanes 14) and baboons (lanes 58) in the mfuwe (lanes 1, 2, 5, 6) and livingstone (lanes 3, 4, 7, 8) regions. results of representative antibody - negative (lanes 1, 3, 5, 7) and antibody - positive (lanes 2, 4, 6, 8) samples are shown. mock antibody (lane 9) and hpiv monoclonal antibody (lane 10) were used as negative and positive controls, respectively. the putative molecular mass of the recombinant n protein (white arrowhead) is 59 kda. these nonhuman primates are widely distributed in africa (8), and their habitats overlap those of humans, mainly in rural areas. a survey performed in kenya of humans with influenza - like illness and however, hpiv3 infection in humans in africa has been poorly studied, making zmls/2011 difficult to compare with an epidemic strain of hpiv3 among humans. in addition, many tourists visit the livingstone region and, in some instances, are harmed by the nonhuman primates attempting to grab food carried by humans. detection of hpiv3 in wild nonhuman primates might be related to these contacts. further epidemiologic studies of humans and wild nonhuman primates are needed to determine whether hpiv3 is transmitted between humans and wild nonhuman primates. serologic evidence of hpiv3 infection was obtained from baboons and vervet monkeys in 2 distinct geographic areas of zambia, but little is known about hpiv3 infection in wild nonhuman primates. in 1963, simian agent 10 (also known as simian virus 10) was isolated from the mouth of a samango monkey (cercopithecus mitis) in a laboratory in south africa (13). complete genome sequence analysis showed simian agent 10 as a strain of hpiv3 (14). experimental infections showed that many nonhuman primates including chimpanzees ; macaques ; and squirrel, owl, patas, and rhesus monkeys are sensitive to hpiv3 infection (1,15). these previous reports support our finding that wild nonhuman primates are susceptible to hpiv3 infection. | human parainfluenza virus type 3 (hpiv3) genome was detected in 4 baboons in zambia. antibody for hpiv3 was detected in 13 baboons and 6 vervet monkeys in 2 distinct areas in zambia. our findings suggest that wild nonhuman primates are susceptible to hpiv3 infection. |
study design and population - a prospective study was conducted from march - december 2012 at 14 health care institutions in cali, colombia. this city is one of the three largest in colombia with a total population of 2,294,653 inhabitants. dengue is considered hyperendemic due to the circulation of all four dengue serotypes. in 2010, one of the largest dengue epidemics hit the area with 11,047 cases, 5.7% severe dengue and 16 attributable deaths. the 14 health care institutions included in the study were selected as they represent different levels of care (primary, secondary and tertiary) and have permanent access to rapid dengue diagnostic tests. personnel at the participating institutions had been trained in dengue diagnosis and treatment in 2010 and 2011, therefore, the level of knowledge of dengue among physicians was considered high. exhaustive sampling in the participating institutions was performed by including all subjects (regardless of age, sex or signs and symptoms) attending any of the 14 health care centres who were seen by a physician and were clinically diagnosed with dengue or for whom a dengue test was ordered during the study period. the study was approved by the ethical review board of university of valle and family compensation fund of valle del cauca. rapid dengue diagnostic tests - rapid dengue diagnostics were routinely available to all 14 institutions at a central lab. in august 2012, the largest institution (named here as a) was moved to a new building with its own lab and could perform dengue tests locally. at all 14 institutions, dengue diagnostics could be ordered at the physicians discretion at any time and results were made available through the computerised central clinical record system. during the study, the rapid dengue diagnostic test for the simultaneous detection of dengue - specific igm and igg (standard diagnostics inc) was available at the laboratory facilities. the sd bioline dengue duo kits (standard diagnostics inc) that simultaneously detect igm, igg and ns1 were only occasionally available. samples were processed by experienced laboratory technicians following the manufacturer s instructions. the study personnel did not have any direct or indirect contact with physicians to avoid any potential bias. data collection and quality control - dengue cases were identified from clinical records with a diagnosis of dengue according to the codes of the international classification of diseases version 2010 (icd-10) and from the forms used to notify the national surveillance system, which is compulsory (who 2010). information on patient identification and demographics, health care institution, date and ward in which the subject was seen by the physician was provided by the centralised statistics department of the health care institutions. data on all subjects for whom a dengue diagnostic test was ordered and the results of those tests were provided by the computerised laboratory information system of the central laboratory. quality control of the data was performed by verifying respective patient identification numbers present in both databases. indirect quality control of the technician in charge of dengue at the central lab was performed at the beginning of the study with three blind samples prepared by the research team. the results of these three samples remained consistent and further training was not considered necessary. statistical analysis - clinical, laboratory and surveillance databases were merged and exported to stata 10 (stata corp lp, usa) for data analysis. a descriptive analysis was conducted to identify the relative frequencies of the following : severity of dengue, cases in which a diagnostic test was ordered and igm positivity index. the latter was defined as the percentage of igm positive results out of the total igm performed. dengue cases were considered to be clinically diagnosed if one of icd-10 codes for dengue, a90 or a91, were reported in the clinical record. the dates when subjects were clinically diagnosed and tested for dengue were compared to ensure the clinical diagnosis preceded the lab test. when these dates were the same, the first reported diagnosis on the date was considered. factors associated with ordering a dengue test in subjects with a clinical diagnosis of dengue were identified using contingency tables with the corresponding odds ratio (or) and 95% confidence interval (ci) and chi - squared test or the fisher exact test when necessary. for quantitative variables, multivariate logistic regression models were fitted to estimated adjusted or with their corresponding 95% cis.. repeated dengue tests and those performed in subjects without an icd-10 for dengue in their clinical records were analysed separately. it was assumed that the subjects clinical diagnosis was other than dengue when there were not matches between the lab database and both the dengue clinical and surveillance databases. for subjects for whom a dengue test was ordered, but whose clinical diagnosis and tests results were other than dengue, a descriptive analysis was performed. a total of 778 subjects had a clinical diagnosis of dengue, of whom 39 (5%) were classified as having severe dengue ; 465 (59.8%) were men and 275 (35.4%) were zero-14 years old. the majority of cases (51.9%) were reported by two (a and m) of the 14 institutions. institution a (26.6% of cases), a referral institution, was classified as offering a tertiary level of care, while the institution m (25.3% of cases) was classified as offering secondary level of care. subjects with severe cases were more frequently female, between 45 - 64.9 years old and seen at the emergency wards with the highest frequency in september. however, there were no statistically significant differences between severe and nonsevere cases regarding sex, age, institution, month or emergency consultation (table i). table icharacteristics of subjects with clinical diagnosis of denguecharacteristicdengue casesor (95% ci)p nonsevereseveretotal (n = 778)(n = 739 ; 95%)(n = 39 ; 5%) n (%) n (%) n (%) sex male465 (59.8)445 (60.2)20 (51.3)-0.2 female313 (40.2)294 (39.8)19 (48.7)1.4 (0.7 - 2.7) years of age median (range in years)20.8 (0.6 - 91.8)20.9 (0.6 - 91.8)19.4 (0.6 - 60.3)-0.9 0 - 4.972 (9.3)68 (9.2)4 (10.3)1 5 - 14.9203 (26.1)193 (26.1)10 (25.6)0.8 (0.2 - 3)0.8 15 - 44.9411 (52.8)392 (53.1)19 (48.7)0.8 (0.2 - 2.5)0.7 45 - 64.974 (9.5)68 (9.2)6 (15.4)1.5 (0.4 - 5.6)0.5 6518 (2.3)18 (2.4)0 (0)00.3institution a207 (26.6)199 (27)8 (20.5)10.6 b6 (0.7)6 (0.8)0 (0)0 c73 (9.4)70 (9.5)3 (7.7)1 (0.2 - 4.1) d40 (5.1)38 (5.1)2 (5.1)1.3 (0.2 - 6.4) e26 (3.3)25 (3.4)1 (2.5)1 (0.1 - 8.3) f23 (3)23 (3.1)0 (0)0 g81 (10.5)79 (10.7)2 (5.1)0.6 (0.1 - 3) h40 (5.1)35 (4.7)5 (12.8)3.5 (1 - 11.6) i23 (3)22 (39)1 (2.5)1.1 (0.1 - 9.5) j11 (1.4)10 (1.3)1 (2.5)2.4 (0.2 - 22) k36 (4.6)34 (4.6)2 (5.1)1.4 (0.3 - 7.2) l1 (0.1)1 (0.1)0 (0)0 m197 (25.3)183 (24.7)14 (36)2 (0.7 - 4.6) n14 (1.8)14 (1.9)0 (0)0ward outpatient408 (52.4)389 (52.6)19 (48.3)1 emergency370 (47.6)350 (47.4)20 (51.3)1.1 (0.6 - 2.2)0.6month march127 (16.3)120 (16.2)7 (18)10.1 april84 (10.8)79 (10.7)5 (12.8)1.1 (0.3 - 3.5) may57 (7.3)55 (7.4)2 (5.1)0.6 (0.1 - 3.1) june57 (7.3)55 (7.4)2 (5.1)0.6 (0.1 - 3.1) july56 (7.2)53 (7.1)3 (7.7)0.9 (0.2 - 4) august61 (7.8)61 (7.8)0 (0)0 september72 (9.2)72 (9.2)9 (23.1)2.4 (0.8 - 7) october80 (10.3)80 (10.3)5 (12.8)1.1 (0.3 - 3.7) november61 (7.8)61 (7.8)2 (5.1)0.6 (0.1 - 3) december123 (15.8)123 (15.8)4 (10.3)0.6 (0.1 - 2)total dengue tests 0393 (50.5)380 (51.4)13 (33.3)1 1320 (41.1)297 (40.2)23 (59)2.2 (1.1 -4.5)0.01 253 (6.8)50 (6.8)3 (7.7)1.7 (0.5 - 6.3)0.4 311 (1.4)11 (1.5)0 (0)00.5 51 (0.2)1 (0.1)0 (0)00.8ci : confidence interval ; or : odds ratio. at least one dengue diagnostic test was ordered in 386 (49.5%) subjects with slight monthly variations (figure). age and sex of subjects were not associated with the ordering of a dengue test, but with severity of disease, emergency consultation, institution and month. all of these factors remained independently associated in the multivariate model ; however, institution was withdrawn from the model as a correlation with emergency consultation existed (table ii). monthly trends in number of dengue rapid diagnostic tests requested and igm positive results. table iifactors associated with requesting a dengue rapid diagnostic test in subjects with clinical diagnosis of denguecharacteristicdengue test requestedor (95% ci)padjustedp yesno(n = 386)(n = 392)n (%) n (%) or (95% ci)sex male236 (61.1)229 (58.4)1 - female150 (38.9)163 (41.6)0.8 (0.7 - 1.2)0.4- years of age median (range in years)20.8 (0.6 - 79)20.9 (0.3 - 91.8)-0.3- 0 - 431 (8)41 (10.4)1 - 5 - 14104 (27)100 (25.3)1.4 (0.8 - 2.3)0.2- 15 - 44206 (53.4)205 (52.3)1.3 (0.8 - 2.2)0.2- 45 - 6439 (10.1)35 (9)1.5 (0.7 - 2.8)0.2- 656 (1.5)12 (3)0.6 (0.2 - 2)0.4- dengue classification nonsevere360 (93.2)379 (96.7)1 - severe26 (6.8)13 (3.3)2.1 (1 - 4.2)0.032.2 (1.1 - 4.5)0.02institution level of care primary147 (38.1)221 (56.4)1 secondary93 (24.1)104 (26.5)1.3 (0.9 - 1.9)0.09- tertiary146 (37.8)67 (17.1)3.3 (2.2 - 4.7) < 0.001- ward outpatient171 (44.3)237 (60.5)- - emergency215 (55.7)155 (39.5)2 (1.4 - 2.5) < 0.0011.9 (1.4 - 2.5) < 0.001month march46 (12)81 (20.7)1 1 april39 (10.1)45 (11.5)1.5 (0.8 - 2.6)0.11.4 (0.7 - 2.4)0.2 may19 (5)38 (9.7)0.8 (0.4 - 1.7)0.70.7 (0.4 - 1.5)0.5 june29 (7.5)28 (7.1)1.8 (0.9 - 3.4)0.061.7 (0.9 - 3.3)0.08 july29 (7.5)27 (6.9)1.9 (1 - 3.5)0.051.6 (0.8 - 3.1)0.1 august32 (8.3)29 (7.4)1.9 (1 - 3.6)0.031.9 (1 - 3.6)0.04 september35 (9)37 (9.4)1.6 (0.9 - 3)0.081.5 (0.8 - 2.7)0.2 october51 (13.2)29 (7.4)3 (1.7 - 5.5) < 0.0013.1 (1.7 - 5.5) < 0.001 november36 (9.3)25 (6.4)2.5 (1.3 - 4.7)0.0042.3 (1.2 - 4.4)0.008 december70 (18.1)53 (13.5)2.3 (1.4 - 3.8)0.0012 (1.2 - 3.4)0.006 a : withdrawn from the model because of colinearity with institution ; ci : confidence interval ; or : odds ratio. a : withdrawn from the model because of colinearity with institution ; ci : confidence interval ; or : odds ratio. the igm positivity index was 30% (114/386), but it varied monthly reaching up to 58% in november (figure). the igm positivity index was twice as high in severe dengue cases (46.1%-12/26) as in nonsevere dengue cases (28.3%-102/360) cases (p = 0.05). during the study period, 491 dengue tests were ordered for subjects who did not have an explicit clinical diagnosis of dengue in their clinical record, but another diagnosis (such as upper and lower respiratory tract infections, unspecified viral disease, meningitis, unspecified thrombocytopenia, human immunodeficiency virus, diarrhoea, malaria, leptospirosis, among others). in this group, more than one dengue test was ordered in 65 subjects : two tests in 53, three in 11 and five in one person. there was no difference in the frequency of repeated dengue tests between severe (3 - 7.7%) and nonsevere dengue cases (62 - 8.4%). of the 58 subjects with negative igm or igg results in the first test, igm seroconversion was observed in 15 subjects in the second test and three subjects in the third test. similar results were found with igg, with seroconversion observed in 15 subjects in the second test and three subjects in the third test. only two subjects were tested twice with ns1 and negative results occurred in both cases. changes from positive to negative igm or igg results were observed in two subjects. in the present study, we analyse the use of rapid dengue diagnostic tests under routine conditions in health care settings in an endemic area of colombia. an emerging pattern in this study was the use of rdts to either rule in or rule out dengue as a differential diagnosis. half of the subjects with a clinical diagnosis of dengue had a dengue test ordered (i.e., a test used to rule in dengue) but subjects with other clinical diagnosis were also tested for dengue (i.e., a test use to rule out dengue). the use of dengue tests to confirm dengue diagnosis is supported by the relatively high specificity of the currently available tests, which is required to rule in a diagnosis. the reported specificity of the two tests used in the study sites, namely sd bioline dengue igm / igg and sd bioline dengue duo ns1, igm / igg rdt, ranges from 86.8 - 92.3% and from 83.9 - 100%, respectively (who / tdr 2009, tricou. 2010, blacksell. however, to date, there is no evidence that the dengue test results influence the physicians behaviour or impact the prognosis of a subject for whom a clinical diagnosis of dengue was already made (andries. hence, it is necessary to further assess the impact and cost - effectiveness of implementing rapid dengue diagnostics tests in real - life settings, accounting for evidence - based decisions. rapid dengue diagnostic tests have shown to have a large variation in sensitivities ranging from 47 - 79.2% for igm / igg and from 78.4 - 93.9% for ns1/igm / igg (who / tdr 2009, osorio. 2010, gan. consequently, they are not suitable for ruling out dengue or for screening purposes. understanding why physicians decide to order dengue tests is therefore worth exploring, while using their input to improve current rapid diagnostic methods. the results show that severe dengue presentation, emergency consultation and month were all independently associated with the ordering of dengue diagnostic tests. first, due to the potential fatal consequences of misdiagnosis in a severe case, clinicians may consider confirmation of these cases to be more relevant than in the nonsevere cases or seek assurances for themselves and the patient / patient s family in the validity of a positive result when ordering a dengue diagnostic test. the need for assurance can be interpreted as defensive in case of possible litigation or ones own expectations of self - efficacy (i.e., performing as expected), while the need to reassure a worried patient / patient s family can be either real or perceived due to social pressure (van der weijden. secondly, rdts are expected to be used at bedside ; hence in the context of emergency care, rapid dengue diagnostic tests could be thought of as an accessible tool to speed up the differential diagnosis process and corresponding management (peeling. alternatively, this factor may be explained by the inclusion of emergency departments located in the secondary and tertiary levels of care where it is known that diagnostic tests are more commonly used to rule in a disease. in contrast, diagnostics are used more often to rule out a condition or to determine the need for referral in primary care centres (whiting. finally, the association between ordering rapid dengue diagnostic tests with the month of the year may suggest that clinicians use diagnostic tests to detect outbreaks of febrile diseases, which is relevant to countries such as colombia, where there is not a distinctive seasonality of dengue incidence. of the subjects meeting clinical criteria for dengue, the probability of in fact having dengue is lower in nonepidemic periods than during epidemics. hence, laboratory diagnosis may help physicians to adjust the predictive values of the clinical definition. this epidemiological reason for ordering dengue tests could be useful in routine care by adjusting of the predictive values of the clinical definitions of disease in subsequent patients or to increase awareness of diseases. hence, improved laboratory based surveillance that informs clinicians of dengue outbreak at the local level have been proposed to assist them (lorenzi. this epidemiological use of health technology is rarely recognised and could be added to the five categories of factors (diagnostic, therapeutic and prognostic, patient - related, doctor - related and policy and organisation - related) that influence ordering of diagnostic tests previously proposed (whiting. 2007). during field work, a dengue epidemic was not declared in the study site, but the igm positivity steadily increased reaching 58% in november. however, this increase in igm positivity preceded an epidemic that was declared early in 2013 in the study site. this finding further supports the monitoring of igm positivity as a potential tool for early outbreak detection (hati 2009). a cut - off point above which an igm positive index suggests a dengue epidemic will need to be validated. there were too few positive ns1 samples in the present study to assess this marker. performing more than one rdt in the same subject was useful to identify dengue cases through seroconversion of igm and igg specific antibodies, but further studies are required to assess the cost - effectiveness of this practice in the routine care. reasons for ordering diagnostic tests are multiple and complex. while our approach was eminently quantitative and allowed us to identify factors associated with the use of rapid dengue diagnostic tests, a qualitative approach would be complementary to identify other factors that are not measurable by quantitative methods. dengue diagnosis and classification was based on the icd-10 available in the computerised system, which does not include the most recent definitions proposed by world health organization (who / tdr 2012). hence, it was not possible to explore the patterns of use of rdts in cases classified as dengue with warning signs. time - series analysis was not performed because the number of monthly observations (n = 10) was considered insufficient to yield reliable results. finally, detailed information of signs, symptoms and onset of fever were not available and could not be explored for their association with ordering diagnostic tests. particularly, the latter is expected to be critical to the use of dengue rdt as the sensitivity of these tests varies with time. after data analysis, we were able to retrieve information on date of disease onset on 296 subjects from the dengue surveillance database at the local public health office. dengue rdts were performed in 109 of these subjects with a median of four (range 0 - 21) days of symptoms compared to five (range 0 - 180) days in subjects who were not tested. surveillance records also contain detailed information of signs and symptoms, but neither this nor dates of onset of disease were used in the analysis because this data was not validated and was unavailable for most study subjects. in conclusion, rapid dengue diagnostic tests are been used to both rule in and rule out disease. the latter highlights the need for improved sensitivity of currently available rapid dengue diagnostic tests. further studies, which consider the influence of nonevidence - based reasons for using health technology, such as reassurance for clinicians or patients / families as well as epidemiological reasons, are required to assess the cost - effectiveness of implementing rapid dengue diagnostic tests in routine care in endemic areas. | there is insufficient evidence of the usefulness of dengue diagnostic tests under routine conditions. we sought to analyse how physicians are using dengue diagnostics to inform research and development. subjects attending 14 health institutions in an endemic area of colombia with either a clinical diagnosis of dengue or for whom a dengue test was ordered were included in the study. patterns of test - use are described herein. factors associated with the ordering of dengue diagnostic tests were identified using contingency tables, nonparametric tests and logistic regression. a total of 778 subjects were diagnosed with dengue by the treating physician, of whom 386 (49.5%) were tested for dengue. another 491 dengue tests were ordered in subjects whose primary diagnosis was not dengue. severe dengue classification [odds ratio (or) 2.2 ; 95% confidence interval (ci) 1.1 - 4.5 ], emergency consultation (or 1.9 ; 95% ci 1.4 - 2.5) and month of the year (or 3.1 ; 95% ci 1.7 - 5.5) were independently associated with ordering of dengue tests. dengue tests were used both to rule in and rule out diagnosis. the latter use is not justified by the sensitivity of current rapid dengue diagnostic tests. ordering of dengue tests appear to depend on a combination of factors, including physician and institutional preferences, as well as other patient and epidemiological factors. |
angiotensin converting enzyme (ace) inhibitors are commonly used in children for treatment of hypertension and congestive cardiac failure due to their cardiac and renoprotective properties. captopril and enalapril are off - patent ace inhibitors and hence are considered more economical. side effects including hyperkalemia, cough, angioedema, and hypoglycemia have been reported with the use of several ace inhibitors, including enalapril. a 5-year - old boy presented to our institution with headache and vomiting since three days. there was no fever, visual complaints, drug intake, trauma, tuberculosis contact, oliguria, dysuria, or bowel complaints. it was a home delivery conducted at his native place by a trained dai. he was apparently well till present without any significant complaints. on admission, he was afebrile with a heart rate of 106/min, respiratory rate of 24/min, and blood pressure of 160/110 mmhg (> 95 percentile for age and sex). his height was 94 cm and weight was 13.4 kg (both below the fifth percentile for age). investigations revealed : hemoglobin 7.6 g / dl, total leucocyte count 7600/cumm, and platelet count 4.5 lac / cumm. blood urea nitrogen was 34 mg / dl, and serum creatinine was 1.4 mg / dl. arterial blood gas analysis revealed : ph 7.28, pco2 25 mmhg, and hco3 12.3 mmol / l. serum calcium was 7.2 mg / dl, alkaline phosphatase 872 iu / l, and phosphorous 5.1 mg / dl. / kg / day), nifedepine (0.5 mg / kg / dose), and enalapril 0.5 his blood pressure was well controlled with above medications. on day 4 of admission, he developed altered sensorium. his repeat serum sodium was 109 meq / l. as the patient was not on any diuretics, had no gastrointestinal losses and his hypertension was under control, a diagnosis of enalapril induced severe hyponatremia leading to altered sensorium was made. enalapril was omitted, and subsequently hydrallazine (2 mg / kg / day) was added for hypertension. intravenous hyponatremic correction was started and his serum sodium gradually became normal within 3 days. repeat investigations are shown in table 1. as per the world health organization collaborating centre for international drug monitoring and naranjo algorithm, the adverse event was probably / likely related to enalapril. dimercaptosuccinic acid (dmsa) scan, micturating cystourethrogram, and renal biopsy were planned and he was discharged after 10 days. enalapril is a derivative of proline but unlike captopril does not contain a sulfydryl group. as a prodrug, enalapril is metabolised to the active form enalaprilat by various esterases in the liver. it has a half - life of 35 h and is still detectable in the plasma after 96 h. the maximum inhibition of ace activity occurs with peak plasma concentrations of enalaprilat and is sustained for 10 h and reverses gradually. excretion is primarily by glomerular filtration, and hence the drug will accumulate in patients who have advanced renal failure. renin is the rate - limiting enzyme that cleaves four amino acids from the renin substrate, angiotensinogen, produced by the liver to form angiotensin i. angiotensin i is further cleaved of two amino acids by ace, which is present in plasma and in the walls of small blood vessels in the lungs, kidneys, and other organs, to form the octapeptide angiotensin ll. it is the primary effector molecule of the ras and acts through stimulation of specific cell - surface receptors (i.e., at1 and at2) in the arteries and various target tissues. it occurs by potentiation of plasma renin activity due to decrease in the level of angiotensin ii. the antidiuretic effects of vasopressin can play a key role in the development of hyponatremia. johnson. found that the systemic administration of angiotensin ii or its precursors directly stimulates the thirst center, with the resulting polydipsia having the potential of further lowering the serum sodium concentration. described a 60-year - old man with idiopathic dilated cardiomyopathy who developed hyponatremia on enalapril therapy. the authors concluded that severe symptomatic hyponatremia induced by the syndrome of inappropriate secretion of antidiuretic hormone should be considered a rare but possible complication associated with ace inhibitor therapy. we could not estimate serum renin and vasopressin in our patient due to financial constraints. it has also been found that administration of ace inhibitors is associated with decrease tubular reabsorption of sodium. furthermore, enalapril therapy results in the sustained increase in effective renal plasma flow due to a pronounced fall of vascular resistance. the combination of these factors could result in an increased natriuretic effect in patients receiving enalapril therapy. this case demonstrates the probable association between the development of severe hyponatremia and the administration of enalapril. | enalapril is an angiotensin converting enzyme inhibitor widely used in children for treatment of hypertension and congestive cardiac failure. we report a 5-year - old boy who developed severe hyponatremia and altered sensorium on enalapril therapy. the serum sodium gradually became normal within 3 days. the patient 's sensorium improved significantly on correction of hyponatremia. through this case, we highlight the importance of monitoring serum sodium in patients on enalapril therapy. |
endodontic treatment should be performed within the confines of the root canal, avoiding overextension of instruments and filling materials into the periradicular tissues. the most favorable healing is achieved when these principles are complied with, and extrusion of the filling material has been found to be associated with severe inflammatory reaction. apical constriction is considered to be the part of the root canal with the smallest diameter and is located 0.5 - 1.5 mm inside the apical foramen. it is generally accepted as the terminal point the endodontic treatment should extend to. the preservation of the apical constriction helps maintenance of the endodontic instruments, chemicals and filling materials within the root canal. however, this anatomical structure may not have formed in immature teeth or may be lost iatrogenically (e.g., instrumentation beyond the apical foramen), surgically (e.g., apical resection) or due to an inflammatory resorption. then, in such cases, a new apical stop (apical seat, apical matrix, apical ledge) should be created to confine the instruments and chemicals to the canal and to supply a barrier against which gutta - percha can be compacted. this apical stop is prepared through the use of successively larger files to the working length. while apical stop preparation can be done using conventional stainless - steel files, rotary niti files produced especially for the instrumentation of the apical part of the root canal can also be used for this purpose. besides proper instrumentation of the root canal, another goal of endodontic treatment is to hermetically obturate the root canal system. in this case, ingress of tissue fluids and microorganisms into the canal space and any possible microbial activity within the canal could be eliminated or at least reduced. it is possible that instrumentation - related factors such as the shape of the apical preparation achieved using different instrumentation systems or the debris remaining on the dentinal walls following instrumentation could affect the adaptation of the root canal filling material to canal walls and thus have an impact on microleakage. the ability of apical preparation instruments for preparing an apical stop at predetermined levels and the microleakage occurring following obturation of such prepared root canals has not been investigated so far. this study aimed to test the ability of two niti rotary apical preparation techniques used with an apex locator - integrated endodontic motor, and a stainless - steel manual technique to create a new apical stop at a predetermined level in teeth with disrupted apical constriction, and to assess microleakage following obturation. eighty - five extracted intact human mandibular permanent incisors were selected after examination with stereomicroscopy and digital radiography. all the teeth were free of cracks, had single root canals and mature apices. the root surfaces were scaled with a periodontal curette to remove the tissue debris and the teeth were stored in physiologic saline solution. the pulp was extirpated and a # 25 k - file was inserted into the root canal and extruded 1 mm past the apical foramen. the coronal two - thirds were enlarged using # 1 and # 2 gates - glidden burs. two rotary niti apical preparation techniques and a manual stainless - steel preparation technique were tested., san antonio, tx, usa) and s - apex (fkg dentaire, la chaux - de - fonds, switzerland). tri auto zx (j morita co., kyoto, japan), an electronic apex locator - integrated endodontic motor was used along with the niti techniques. the device was adjusted to the h (high torque) mode and set to 0.5 mm (distance short of the apical foramen) and used at auto apical reverse function. the manual instrumentation group employed stainless - steel k - files (dentsply / detrey, konstanz, germany) used with a standardized technique described previously. briefly, the file was used first with a quarter clockwise rotation followed by a pull - back motion and used repeatedly until it became loose at the working length. before instrumenting in the manual group, working lengths were determined using tri auto zx at the apex locator mode (emr mode) and the device set to 0.5 mm. the rubber stops of the hand - files were adjusted to the registered lengths. a # 25 k - file was used to maintain apical patency after each instrument. the root canals were irrigated with 10 ml of 5.25% naocl between each instrument change. final irrigation was with 10 ml of 17% edta for 60 sec, followed by 10 ml of naocl irrigation. thirty teeth instrumented as described(n=10 for each instrumentation technique) were allocated for testing the ability of each instrumentation technique to approximate to the predetermined level (0.5 mm short of the apical foramen). once the apical preparation was accomplished, the final instrument was reinserted into the root canal without excessive pressure and advanced to the level it reached freely, then fixed in place with a flowable composite resin. an ohmmeter (yfe, hsin - chu city, taiwan, r.o.c.) was used in order to measure the distance between the apical foramen and the level of the created apical stop. briefly, one pole of the ohmmeter was connected to the shank of the fixed instrument in the canal, and the other pole was connected to the shank of a # 15 finger spreader (figure 1). the spreader was inserted retrogradely from the apical foramen through the root canal and advanced until the display of the ohmmeter indicated a contact. the distance the spreader advanced until the contact was registered by marking on the spreader with an acetate marker. the distance between the tip of the spreader and the mark was measured under stereomicroscope using a digital caliper to an accuracy of 0.01 mm and this value was considered the distance between the apical foramen and the apical stop. no significant difference was found between the measurements of these investigators when paired t - test was performed (p=0.90). the average of the two measurements for each sample was calculated and the data were analyzed statistically using the anderson - darling normality test and the tamhane test at p=5% as the level of significance. the experimental setting showing the ohmmeter with the poles connected to the files forty - five teeth instrumented as described (n=15 for each instrumentation technique) were allocated for testing the microleakage after root - filling of the instrumented teeth. the root canals were dried using paper points and filled using gutta - percha and sealer (ah plus, dentsply / detrey, konstanz, germany) with the lateral condensation technique. the gutta - percha cones were coated with sealer before insertion into the canal. the teeth were checked radiographically (rvg, trophy radiologie, paris, france) from the labial and lateral aspects for the quality of the root filling (e.g. homogeneity of the filling or presence of a void). all root surfaces were covered with nail polish with care to avoid the apical foramen. the teeth were stored in physiological saline with 0.2% sodium azide solution until and between the leakage experiments. the root fillings were tested for leakage using a fluid filtration assembly described elsewhere. in this method, a pressure of 2 psi (0.136 atm) was applied with o2 to force water through voids within a filled canal. the fluid flow was quantified by observing the movement of an air bubble created within a 25 l micropipette. this micropipette located between the pressure source and the sample to be tested. measurements of the fluid movement were made at 2-min intervals for 8 min and averaged. the differences at each period between the groups were statistically evaluated using the tamhane test. the difference for each group between the 1-week and 3-month measurements was analyzed statistically using the paired samples t - test at p=5% as the level of significance. eighty - five extracted intact human mandibular permanent incisors were selected after examination with stereomicroscopy and digital radiography. all the teeth were free of cracks, had single root canals and mature apices. the root surfaces were scaled with a periodontal curette to remove the tissue debris and the teeth were stored in physiologic saline solution. the pulp was extirpated and a # 25 k - file was inserted into the root canal and extruded 1 mm past the apical foramen. the coronal two - thirds were enlarged using # 1 and # 2 gates - glidden burs. two rotary niti apical preparation techniques and a manual stainless - steel preparation technique were tested., san antonio, tx, usa) and s - apex (fkg dentaire, la chaux - de - fonds, switzerland). tri auto zx (j morita co., kyoto, japan), an electronic apex locator - integrated endodontic motor was used along with the niti techniques. the device was adjusted to the h (high torque) mode and set to 0.5 mm (distance short of the apical foramen) and used at auto apical reverse function. the manual instrumentation group employed stainless - steel k - files (dentsply / detrey, konstanz, germany) used with a standardized technique described previously. briefly, the file was used first with a quarter clockwise rotation followed by a pull - back motion and used repeatedly until it became loose at the working length. before instrumenting in the manual group, working lengths were determined using tri auto zx at the apex locator mode (emr mode) and the device set to 0.5 mm. the rubber stops of the hand - files were adjusted to the registered lengths. a # 25 k - file was used to maintain apical patency after each instrument. the root canals were irrigated with 10 ml of 5.25% naocl between each instrument change. final irrigation was with 10 ml of 17% edta for 60 sec, followed by 10 ml of naocl irrigation. thirty teeth instrumented as described(n=10 for each instrumentation technique) were allocated for testing the ability of each instrumentation technique to approximate to the predetermined level (0.5 mm short of the apical foramen). once the apical preparation was accomplished, the final instrument was reinserted into the root canal without excessive pressure and advanced to the level it reached freely, then fixed in place with a flowable composite resin. an ohmmeter (yfe, hsin - chu city, taiwan, r.o.c.) was used in order to measure the distance between the apical foramen and the level of the created apical stop. briefly, one pole of the ohmmeter was connected to the shank of the fixed instrument in the canal, and the other pole was connected to the shank of a # 15 finger spreader (figure 1). the spreader was inserted retrogradely from the apical foramen through the root canal and advanced until the display of the ohmmeter indicated a contact. the distance the spreader advanced until the contact was registered by marking on the spreader with an acetate marker. the distance between the tip of the spreader and the mark was measured under stereomicroscope using a digital caliper to an accuracy of 0.01 mm and this value was considered the distance between the apical foramen and the apical stop. no significant difference was found between the measurements of these investigators when paired t - test was performed (p=0.90). the average of the two measurements for each sample was calculated and the data were analyzed statistically using the anderson - darling normality test and the tamhane test at p=5% as the level of significance. the experimental setting showing the ohmmeter with the poles connected to the files forty - five teeth instrumented as described (n=15 for each instrumentation technique) were allocated for testing the microleakage after root - filling of the instrumented teeth. the root canals were dried using paper points and filled using gutta - percha and sealer (ah plus, dentsply / detrey, konstanz, germany) with the lateral condensation technique. the gutta - percha cones were coated with sealer before insertion into the canal. the teeth were checked radiographically (rvg, trophy radiologie, paris, france) from the labial and lateral aspects for the quality of the root filling (e.g. homogeneity of the filling or presence of a void). all root surfaces were covered with nail polish with care to avoid the apical foramen. the teeth were stored in physiological saline with 0.2% sodium azide solution until and between the leakage experiments. the root fillings were tested for leakage using a fluid filtration assembly described elsewhere. in this method, a pressure of 2 psi (0.136 atm) was applied with o2 to force water through voids within a filled canal. the fluid flow was quantified by observing the movement of an air bubble created within a 25 l micropipette. this micropipette located between the pressure source and the sample to be tested. measurements of the fluid movement were made at 2-min intervals for 8 min and averaged. the differences at each period between the groups were statistically evaluated using the tamhane test. the difference for each group between the 1-week and 3-month measurements was analyzed statistically using the paired samples t - test at p=5% as the level of significance. the distance from the apical foramen for each instrumentation technique was as follows [average distance mm(sd mm) ] : manual technique [0.59(0.11) ], lightspeed [0.91(0.45) ] and s - apex [1.18(0.29) ]. thus, average proximity to the predetermined level was : manual technique : 0.09 mm ; lightspeed : 0.41 mm and s - apex : 0.68 mm. there was a significant difference between the manual technique and s - apex (p0.05). however, the positive control group leaked significantly more than the other groups at either period (p0.05). sd = standard deviation the lower case letters (a, b) indicate statistical difference between the 1-week and 3-month measurements for each group. groups with the same lower case letter are not significantly different at the two measurement periods (p>0.05). leakage at 3 months was significantly greater for all techniques compared to 1-week (p0.05). the distance from the apical foramen for each instrumentation technique was as follows [average distance mm(sd mm) ] : manual technique [0.59(0.11) ], lightspeed [0.91(0.45) ] and s - apex [1.18(0.29) ]. thus, average proximity to the predetermined level was : manual technique : 0.09 mm ; lightspeed : 0.41 mm and s - apex : 0.68 mm. there was a significant difference between the manual technique and s - apex (p0.05). however, the positive control group leaked significantly more than the other groups at either period (p0.05). sd = standard deviation the lower case letters (a, b) indicate statistical difference between the 1-week and 3-month measurements for each group. groups with the same lower case letter are not significantly different at the two measurement periods (p>0.05). leakage at 3 months was significantly greater for all techniques compared to 1-week (p0.05). rotary niti instrumentation has gained popularity in endodontics in recent years. while preparation of the root canal in conventional technique is done using single type of instrument (e.g. successive size k - files), rotary niti instrumentation employs different and specific instruments for the preparation of the coronal, mid and apical thirds of the canal. lightspeed and s - apex are examples to the instruments designed for the apical third. this study tested how close these novel systems and a conventional manual technique would approximate to a predetermined level in teeth with disrupted apical constriction with a purpose to create a new apical stop. the major findings of this study were that all instrumentation techniques prepared slightly short of the predetermined level ; the manual technique prepared closest, and the s - apex technique prepared farthest to the predetermined level. however, in spite of these variations, leakage following obturation was comparable in all groups. the niti rotary instruments in this study were used along with tri auto zx, an electronic apex locator - integrated endodontic motor. previous studies employing tri auto zx at a setting of 0.5 found that measurements using hand - files or rotary niti files were slightly short of the predetermined level. the finding that approximation with s - apex to the predetermined level was less satisfactory than the other techniques can be explained by some factors. the most important factor is that the s - apex files were not used as per the manufacturer 's recommendations : while a rotational speed of 500 - 1000 rpm has been recommended by the manufacturer for the optimum operation of the s - apex files, the tri auto zx device provides a constant speed of 28050 rpm which can not be changed. rotation at slow speed may be the main reason of the inferior performance of the s - apex system. different from the others, s - apex has an inverted cone cutting portion. in the gradually narrowing canal of the mandibular incisor, this shape could have disabled the progress of the file to the predetermined level. one explanation for this may be that the manual control of a file was simpler than the control of a rotary file mounted on a handpiece. it was " controlled - preparation " to the initially established working length in the manual group. however, the rotary groups followed a more sophisticated technique : it was a dynamic process including the simultaneous measurement of the working length and rotation of the files. a recent study also reported inequivalent apical measurements obtained with motor - driven apex locators (using niti files) and their manual apex - locating modes (using stainless - steel files). however, we believe that the difference is not due to the alloys the files used in these techniques were made of. in two studies, for example, no significant difference was found between the electric length measurements achieved by the use of stainless - steel or niti rotary instruments in the same root canal. regarding these, the differences found in this study between the performances of the different instrumentation systems may be attributed to their designs, handling and to technical shortcomings. nevertheless, all the instrumentation techniques tested in this study, with small deviations from the predetermined level, are considered to be clinically acceptable. it has been suggested that best treatment results in cases with necrotic pulp were achieved when treatment procedures were terminated within 2 mm (0 to 2 mm) of the radiographic apex. the results of the study presented here indicated that the tested techniques could perform within this range. in spite of the varying deviations from the predetermined level, leakage was similar in all groups. this may be because there were small (in a millimeter scale only) differences between the groups regarding approximation to the predetermined level. another explanation is that the results of the fluid filtration experiment reflect the seal of the root canal filling as whole. even if increased leakage existed at the apical part, this could have been blockaded by the filling at the middle or coronal parts of the canal, and thus the fluid flow in the experimental assembly could have been impeded. leakage in all groups was significantly greater in the 3-month measurements than in the 1-week measurements. the finding that the leakage in obturated root canals increased with time is in agreement with previous studies where zinc oxide - eugenol - based and epoxy resin - based sealers were used. in conclusion, there were significant but minor differences (less than a millimeter) among the instrumentation techniques regarding approximation to 0.5 mm short of the apical foramen as the predetermined level. however, despite these differences, leakage was similar in all groups following obturation of the root canals. | objectivethe aim of this study was to investigate the ability of two niti rotary apical preparation techniques used with an electronic apex locator - integrated endodontic motor and a manual technique to create an apical stop at a predetermined level (0.5 mm short of the apical foramen) in teeth with disrupted apical constriction, and to evaluate microleakage following obturation in such prepared teeth. material and methods : 85 intact human mandibular permanent incisors with single root canal were accessed and the apical constriction was disrupted using a # 25 k - file. the teeth were embedded in alginate and instrumented to # 40 using rotary lightspeed or s - apex techniques or stainless - steel k - files. distance between the apical foramen and the created apical stop was measured to an accuracy of 0.01 mm. in another set of instrumented teeth, root canals were obturated using gutta - percha and sealer, and leakage was tested at 1 week and 3 months using a fluid filtration device. resultsall techniques performed slightly short of the predetermined level. closest preparation to the predetermined level was with the manual technique and the farthest was with s - apex. a significant difference was found between the performances of these two techniques (p<0.05). lightspeed ranked in between. leakage was similar for all techniques at either period. however, all groups leaked significantly more at 3 months compared to 1 week (p<0.05). conclusionsdespite statistically significant differences found among the techniques, deviations from the predetermined level were small and clinically acceptable for all techniques. leakage following obturation was comparable in all groups. |
the prevalence of allergic diseases has increased dramatically over the last few decades, with population prevalence rates reaching 30% in the industrialized nations that have led the epidemic. the defining feature of allergic disorders is their association with aberrant levels, and targets, of immunoglobulin e (ige) production. allergy is thought to result from maladaptive immune responses to ubiquitous, otherwise innocuous environmental proteins, referred to as allergens1. allergens, by definition, are proteins that have the ability to elicit powerful t helper lymphocyte type 2 (th2) responses, culminating in ige antibody production (atopy). while allergens represent a minute fraction of the protein universe that humans are routinely exposed to, allergenicity is a very public phenomenon, with the identical proteins behaving as allergens in different allergic patients. why specific proteins drive such aberrant t cell and b cell responses is a basic mechanistic question that has remained largely unanswered. allergens derive from a variety of environmental sources such as plants (trees, grasses), fungi (alternaria alternata), arthropods (mites, cockroaches), and other mammals (cats, dogs, cows). allergens constitute a diverse range of molecules, varying in size from small to large multi - domain proteins. as they are derived from complex living organisms they serve a broad range of functions in their respective hosts, from structural to enzymatic. for example, the common house dust mite allergens include several cysteine proteases (der p 1, der p 3), serine proteases (der p 3, der p 6, der p 9), chitinases (der p 15, der p 18), lipid - binding molecules (der p 2), and structural molecules such as tropomyosin (der p 10). some are species specific ; others are molecules with broad biochemical homology that are found in many species. much work has centered on the study of the allergen epitopes recognized by t and b cells. however, as there is no compelling evidence for common structural characteristics among the diverse t and b cell epitopes recognized in allergic responses 2,3,4, it appears doubtful that the presence of such b cell and t cell epitopes are sufficient to endow a protein with allergenic potential. other factors such as the size, glycosylation status, resistance to proteolysis, and enzymatic activity, have been suggested to play an important role in allergenicity. however for example, glycosylation appears not to be a common critical determinant of allergenicity as both glycosylated and non - glycosylated proteins act as food allergens. it may be that there are many structural paths to allergenicity, but the absence of any common structural motif or conformational sequence pattern leaves open the possibility that proteins with allergic potential exhibit a necessary commonality of biological function. indeed, it has recently been proposed that allergens are linked by their ability to activate the innate immune system of mucosal surfaces, triggering an initial influx of innate immune cells that subsequently drive th2-polarized adaptive immune responses. it should be noted that, reductive experimental systems aside, natural exposure is not to single, purified proteins, but to complex mixtures of molecules. it may well be that the innate immune - activating molecules are not identical to the proteins recognized by allergic responses, although this would still beg the question as to why those particular proteins are so recognized among the many present during exposure. in this review, we will address recent advances in our understanding of the diverse innate immune - activating properties of allergens that appear to endow them with a propensity for driving th2 immune responses. several allergens have cysteine or serine protease activity, including diverse allergens from arthopods [e.g., house dust mites 5 - 7, german cockroaches 8, fungi (alternaria alternata)9 and cladosporium herbarum 10, mammals (e.g., felis domesticus)11, plants (e.g. pollens from ragweed 12 ]. in addition, many forms of occupational allergy are associated with encounters with proteolytic enzymes such as those used in the manufacture of detergents (alkaline detergents)13, or in the food industry (papain)14. first, intrinsic protease activity appears to be linked with sensitization ability in several allergens. removal of proteases from a. fumigatus 15, german cockroach frass 16, american cockroach per a 10 antigen17, epi p1 antigen from the fungus epicoccum purpurascens 18 or cur 11 antigen from the mold curvularia iunata 19 was reported to decrease airway inflammation and airway hyperresponsiveness in mouse models of allergic asthma. secondly, direct exposure of mice to proteolytic enzymes such as papain can induce allergic sensitization 20. moreover, co - administration of active proteases from a. fumigatus with the tolerogenic antigen, ovalbumin (ova), resulted in allergic sensitization 15. as co - exposure to a tolergenic protein with a protease can induce allergic sensitization, proteases found in ambient air derived from bacterial and viral species may play accessory roles. lastly, subcutaneous injection of a serine protease inhibitor, nafamostat mesilate, during sensitization to house dust mite extracts blunted the development of allergic inflammation and airway hyperresponsiveness 21. firstly, protease activity may increase transepithelial access of allergens to critical cells of the innate immune response, such as dendritic cells (dcs). for example, the cysteine protease der p 1, can alter epithelial permeability through disruption of epithelial tight junctions and a reduction in zo-1 and occludin content 6. consistent with this, proteolytic enzymes from a number of tree and grass pollens have also been shown to degrade zo-1 and disrupt tight junctions 22. moreover, der p 1 has been shown to cleave -1-anti - trypsin, inhibiting its ability to protect the respiratory tract against serine proteases such as der p 3 and der p 9. this may disrupt the protease - anti - protease balance in mucosal tissues, enhancing the activity of both endogenous and exogenous proteases and leading to enhanced tissue damage and immune activation. secondly, the cysteine protease activity of several mite allergens (der p 1, der f 1) may directly impair innate defense mechanisms in the lung by degrading and inactivating lung surfactant proteins (sp) a and d 23. sp - a and ap - d are calcium - dependent carbohydrate - binding proteins with multiple innate immune functions, including bacterial agglutination and modulation of leukocyte functions. importantly, sp - d and sp - a have been shown to protect against aspergillus fumigatus - induced allergic inflammation in mice 24, 25, likely via binding to glucan moieties of inhaled allergens and facilitation of their clearance. proteases from mites, cockroaches, and fungi can increase the expression of cytokines, including interleukin (il)-6, il-8 and gm - csf 5, 6, 8, 9, which may lead to the recruitment, activation and/or enhanced survival of dcs at the mucosal surface. additionally, der p 1 has been shown to influence the expression of costimulatory molecules such as cd40 on dcs. der p 1 can cleave cd40 on human monocyte - derived dcs, resulting in inhibition of the production of the pivotal th1-differentiating cytokine, il-12 26. the suppression of cd40 signaling and il-12 production may induce a shift towards th2 responses. a similar effect has been observed with the mold aspergillus (asp). exposure of healthy human monocyte - derived dcs to asp induced their maturation and enhanced their ability to prime th2 immune responses in allogeneic nave t cells as compared with naive t cells primed with lps - activated dcs 27. when the proteolytic activity of asp was neutralized by chemical inactivation, asp failed to up - regulate costimulatory molecules on dcs, and these dcs did not prime a th2 response in naive t cells. the skewed th2 response was thought to occur as a result of suppressed il-12 production by asp - primed dcs. interestingly, although the exact mechanisms by which allergen - derived proteases influence the decision making capability of dcs are not well understood, recent studies suggest that protease containing allergens such as der p 1 can target two c - type lectins, dc - sign and dc - signr 28. loss of dc - sign expression following der p 1 treatment led to a reduction in its binding to its ligand, icam-3, on nave t cells, which is thought to be important in th1 signaling 28. thus the combined proteolytic activities of der p 1 on surface expression of molecules such as cd40, and dc - sign could have profound effects on the decision making capability of dcs, biasing adaptive immunes response towards a th2 pattern of response. recently, study of the occupational allergen, papain (commonly used in the food industry), has led to the novel hypothesis that proteases can directly prime th2 immune responses through actions on basophils 20. data suggest that papain can cleave a yet - to - be identified host sensor which, in turn, activates basophils to produce il-4, and thymic stromal lymphopoietin (tslp)driving th2 differentiation. this intriguing work supports the concept that host detection of protease activity associated with allergens may provide a unique pathway of innate immune activation. the generality of this pathway, as well as its molecular identification, remain to be defined. allergen - derived proteases can have direct effects on adaptive immune responses as well, through cleavage of molecules such as cd25, and cd23. specifically, der p 1 has been shown to be able to cleave the chain of the il-2 receptor (cd25) on human t cells 29 (figure 1). as a result, t cells exposed directly to der p 1 display markedly reduced th1 cytokine production and enhanced th2 cytokine production, something dependent on the protease activity or der p 1. cleavage of cd25 might also, of course, alter regulatory function, as il-2 stimulation is required for the maintenance of regulatory t cells in the periphery. the overall effect may be to shift the balance of immune responses from a tolerogenic response to one favoring a th2 pattern of response. allergenic proteases such as der p 1 have also been shown to be able to cleave the low affinity receptor for ige, cd23, from the surface of human b cells, releasing the soluble form of the receptor 30 (figure 1). as the membrane - bound form of the ige receptor is thought to act as a negative regulator of ige synthesis, der p 1 cleavage of cd23 could potentially disrupt the negative feedback signal and enhance ige synthesis, thereby amplifying the allergic response. anti - trypsin can inhibit this effect of der p 1 on cd23 cleavage, suggesting that disruption of the balance between proteases and protease inhibitors might play a role in allergic sensitization. it should be noted that whether intact proteases such as der p 1 actually gain functional access to lymphocytes in vivo remains an open question. the biological effects of some allergenic proteases may also be mediated through activation of the protease - activated receptor 2 (par2). pars (1,2,3,4) are a family of proteolytically activated g - protein coupled receptors. proteases cleave within the n - terminus of the receptors and expose a tethered ligand domain that binds and activates the cleaved receptor. several lines of evidence suggest that par2, in particular, may be important in allergic sensitization. it is expressed by many cells in the lung, including airway epithelial cells 31, fibroblasts 32, macrophages 33 and mast cells 34, and, importantly, patients with asthma have been shown to exhibit increased expression of par2 on respiratory epithelial cells 35. several house dust mite allergens (der p1, (30), der p 3, der p 9), along with german cockroach extract 36, have been shown to be able to cleave and activate par2 (figure 1). several reports have shown that activation of par2 by house dust mite extract 7, german cockroach 37, or the mold allergen pen c 13 38 leads to increased cytokine production by airway epithelia. specifically, airway epithelial cells were shown to increase the expression of tslp through the activation of par2 when treated with papain, trypsin or the fungus alterneria 9. as tslp drives dc polarization of nave t cells to a th2 phenotype, these results suggest that par2 activation may serve as a link between innate and adaptive immune responses. several mouse models of allergic inflammation have also underscored a potential role for par2 in allergic sensitization. for example, one recent study showed that tolerance to inhaled ova could be overcome by co - administration of par2-activating peptides to the airways, promoting allergic sensitization 39. other studies have shown that overexpression of par2 in mice renders them susceptible to allergic airway inflammation when sensitized and challenged locally with ova, as compared to wildtype controls 40. while these data strongly support the concept that protease activity may lead to airway allergic sensitization via par2 activation, there are a few reports suggesting that par2 activation may also reduce airway inflammation. for example, despite the fact that tlr4 (vide infra) and par2 signaling have been shown to exhibit cooperativity 41, par2-activating peptides have been reported to inhibit lipopolysaccharide(lps)-induced neutrophil influx into mouse airways 42. in a rabbit model of experimental asthma, sensitization to the pollen parietaria judaica, followed by allergen challenge in the presence or absence of a par2-activating peptide, led to par2-mediated attenuation of the development of airway hyperresponsiveness and airway eosinophilia 43. it is possible that the discrepancies in these results are due to the timing of par2 activation, with activation by exogenous proteases during generation of immune responses having different effects than that occurring in the midst of an ongoing inflammatory response. recent recognition of the critical roles played by innate pattern recognition receptors (prrs) such as toll - like receptors (tlrs), nod - like receptors (nlrs), rig - i - like receptors (rlrs) and c - type lectin receptors (clrs), in activation and instruction of antigen - presenting cells (apcs)44 has led to the exploration of the role of these pathways in allergic responses. it will be noted that, while tlr - driven activation of th1 responses by dcs is well - studied and -understood, the receptors and pathways driving th2 immune responses have been considerably less tractable to experimental investigation. epidemiological studies have reported an inverse correlation between high levels of bacterial products such as lps in the ambient environment during very early life and the subsequent development of atopy and allergic disease 45 - 47. it has been postulated, pace the hygiene hypothesis, that such exposures drive robust counter - regulatory tone in the developing immune system 48. lps exposure can also exacerbate established asthma, however, probably by direct stimulation of airway pro - inflammatory responses 49. experimental mouse models have provided mechanistic insight into the ability of lps exposure to regulate the development of allergic asthma. as predicted by the hygiene hypothesis, lps dose appears to be a critical variable. while airway sensitization with ova along with very low dose (3)-beta - d - glucan, and are commonly found in the cell walls of fungi, pollens, and certain bacteria. in plants, polymers of -glucans are thought to protect the developing pollen during meiosis, and are later destroyed by the enzyme (13)--d - glucanase to liberate the microspores. although -glucans are widely expressed, they are not found in mammalian cells. as such, they can act as pamps, triggering immune responses through activation of specific prrs. the immunostimulatory properties of -glucans have been recognized for decades, since their identification as the immunoactive component of mushrooms 63. for example, it has been reported that -glucan structures present in the peanut glycoallergen ara h 1 have th2 inducing characteristics 64 native, but not deglycosylated, ara h 1 was shown to activate human monocyte - derived dc and induce il-4 and il-13 secreting th2 cells. exposure to -glucans has also been shown to induce airway hyperresponsiveness in allergic humans 65. studies in guinea pigs have shown that direct delivery of (13)--d - glucan to the airways can induce the recruitment of lung eosinophils and lymphocytes 66. in mice, exposure to soluble -glucan isolated from candida albicans 67 markedly exacerbated ova - induced eosinophilic airway inflammation, concomitant with enhanced lung expression of th2 cytokines and il-17a. exposure to -glucans plus ova increased the number of cells bearing mhc class ii and the expression of apc - related molecules such as cd80, cd86, and dec205 on bone marrow derived dcs. in support of a role of -glucans in the recruitment and activation of dcs at mucosal surfaces, recent studies have shown that -glucans contained in house dust mite extracts and in moulds may initiate immune responses at the mucosal surface. house dust mite extract - mediated induction of the release of the chemokine, ccl20, which recruits immature dcs, by human airway epithelial cells in culture, was shown to occur through -glucan and syk - dependent signaling pathways 68 (figure 1). although the exact lectin receptor mediating these effects was not identified in these studies, the results suggested that -glucan moieties contained in house dust mite extracts might mediate early processes leading to immature dc recruitment to the airways. this concept is supported by another recent study that showed that dectin-2 receptor signaling pathways (dectin-2/fcrgamma / syk) mediated the production of cysteinyl leukotrienes in bone marrow - derived dcs following stimulation with house dust mite extracts or aspergillus 69 (figure 1). taken together, these findings identify the dectin-2/fcrgamma / syk axis as a novel receptor mediated pathway by which several potent allergens are recognized by innate immune cells at the airway surface, linking them with the development of th2-skewed adaptive immune responses. consistent with a role for lectins in driving th2 immune responses, blockade of the mannose receptor, an endocytic c - type lectin receptor, significantly reduced der p 1 uptake by dcs 70. these findings are consistent with previous findings suggesting that engagement of the mannose receptor by selected ligands on human dcs leads to the induction of a dc phenotype favoring th2 polarization 71. although the study of the role of glucans as th2-inducing pamps is only in its infancy, data to date suggest that carbohydrate moieties contained in common allergens act as strong th2 inducers via activation of variety of c - type lectin receptors on dcs. it has recently been shown that common allergenic pollen grains contain nicotinamide adenine dinucleotide phosphate (reduced) [nad(p)h ] oxidase activity as well as allergens 72. such pollen grains have been shown to significantly increase the levels of reactive oxygen species (ros) in cultured cells, and to be able induce allergic airway inflammation in experimental animals 73 (figure 1). pretreatment of these pollen grains with nad(p)h oxidase inhibitors attenuated their capacity to increase ros levels in airway epithelial cells and subsequent airway inflammation. similarly, pre - treatment of mice with antioxidants has been shown to prevent the development of pollen - driven asthma in mice. interestingly, delaying anti - oxidant treatment until after pollen challenge was ineffective, suggesting that the oxidase activity is of critical importance during the period of innate immune activation. although the mechanisms remain to be defined, it has been speculated that nadph oxidase activity initiates immune activation through its ability to recruit inflammatory cells, possibly through the induction of il-8 by p38 mapk 74. of interest, genetic polymorphisms in genes regulating oxidative stress several allergens have cysteine or serine protease activity, including diverse allergens from arthopods [e.g., house dust mites 5 - 7, german cockroaches 8, fungi (alternaria alternata)9 and cladosporium herbarum 10, mammals (e.g., felis domesticus)11, plants (e.g. pollens from ragweed 12 ]. in addition, many forms of occupational allergy are associated with encounters with proteolytic enzymes such as those used in the manufacture of detergents (alkaline detergents)13, or in the food industry (papain)14. first, intrinsic protease activity appears to be linked with sensitization ability in several allergens. removal of proteases from a. fumigatus 15, german cockroach frass 16, american cockroach per a 10 antigen17, epi p1 antigen from the fungus epicoccum purpurascens 18 or cur 11 antigen from the mold curvularia iunata 19 was reported to decrease airway inflammation and airway hyperresponsiveness in mouse models of allergic asthma. secondly, direct exposure of mice to proteolytic enzymes such as papain can induce allergic sensitization 20. moreover, co - administration of active proteases from a. fumigatus with the tolerogenic antigen, ovalbumin (ova), resulted in allergic sensitization 15. as co - exposure to a tolergenic protein with a protease can induce allergic sensitization, proteases found in ambient air derived from bacterial and viral species may play accessory roles. lastly, subcutaneous injection of a serine protease inhibitor, nafamostat mesilate, during sensitization to house dust mite extracts blunted the development of allergic inflammation and airway hyperresponsiveness 21. firstly, protease activity may increase transepithelial access of allergens to critical cells of the innate immune response, such as dendritic cells (dcs). for example, the cysteine protease der p 1, can alter epithelial permeability through disruption of epithelial tight junctions and a reduction in zo-1 and occludin content 6. consistent with this, proteolytic enzymes from a number of tree and grass pollens have also been shown to degrade zo-1 and disrupt tight junctions 22. moreover, der p 1 has been shown to cleave -1-anti - trypsin, inhibiting its ability to protect the respiratory tract against serine proteases such as der p 3 and der p 9. this may disrupt the protease - anti - protease balance in mucosal tissues, enhancing the activity of both endogenous and exogenous proteases and leading to enhanced tissue damage and immune activation. secondly, the cysteine protease activity of several mite allergens (der p 1, der f 1) may directly impair innate defense mechanisms in the lung by degrading and inactivating lung surfactant proteins (sp) a and d 23. sp - a and ap - d are calcium - dependent carbohydrate - binding proteins with multiple innate immune functions, including bacterial agglutination and modulation of leukocyte functions. importantly, sp - d and sp - a have been shown to protect against aspergillus fumigatus - induced allergic inflammation in mice 24, 25, likely via binding to glucan moieties of inhaled allergens and facilitation of their clearance. proteases from mites, cockroaches, and fungi can increase the expression of cytokines, including interleukin (il)-6, il-8 and gm - csf 5, 6, 8, 9, which may lead to the recruitment, activation and/or enhanced survival of dcs at the mucosal surface. additionally, der p 1 has been shown to influence the expression of costimulatory molecules such as cd40 on dcs. der p 1 can cleave cd40 on human monocyte - derived dcs, resulting in inhibition of the production of the pivotal th1-differentiating cytokine, il-12 26. the suppression of cd40 signaling and il-12 production may induce a shift towards th2 responses. exposure of healthy human monocyte - derived dcs to asp induced their maturation and enhanced their ability to prime th2 immune responses in allogeneic nave t cells as compared with naive t cells primed with lps - activated dcs 27. when the proteolytic activity of asp was neutralized by chemical inactivation, asp failed to up - regulate costimulatory molecules on dcs, and these dcs did not prime a th2 response in naive t cells. the skewed th2 response was thought to occur as a result of suppressed il-12 production by asp - primed dcs. interestingly, although the exact mechanisms by which allergen - derived proteases influence the decision making capability of dcs are not well understood, recent studies suggest that protease containing allergens such as der p 1 can target two c - type lectins, dc - sign and dc - signr 28. loss of dc - sign expression following der p 1 treatment led to a reduction in its binding to its ligand, icam-3, on nave t cells, which is thought to be important in th1 signaling 28. thus the combined proteolytic activities of der p 1 on surface expression of molecules such as cd40, and dc - sign could have profound effects on the decision making capability of dcs, biasing adaptive immunes response towards a th2 pattern of response. recently, study of the occupational allergen, papain (commonly used in the food industry), has led to the novel hypothesis that proteases can directly prime th2 immune responses through actions on basophils 20. data suggest that papain can cleave a yet - to - be identified host sensor which, in turn, activates basophils to produce il-4, and thymic stromal lymphopoietin (tslp)driving th2 differentiation. this intriguing work supports the concept that host detection of protease activity associated with allergens may provide a unique pathway of innate immune activation. the generality of this pathway, as well as its molecular identification, remain to be defined. allergen - derived proteases can have direct effects on adaptive immune responses as well, through cleavage of molecules such as cd25, and cd23. specifically, der p 1 has been shown to be able to cleave the chain of the il-2 receptor (cd25) on human t cells 29 (figure 1). as a result, t cells exposed directly to der p 1 display markedly reduced th1 cytokine production and enhanced th2 cytokine production, something dependent on the protease activity or der p 1. cleavage of cd25 might also, of course, alter regulatory function, as il-2 stimulation is required for the maintenance of regulatory t cells in the periphery. the overall effect may be to shift the balance of immune responses from a tolerogenic response to one favoring a th2 pattern of response. allergenic proteases such as der p 1 have also been shown to be able to cleave the low affinity receptor for ige, cd23, from the surface of human b cells, releasing the soluble form of the receptor 30 (figure 1). as the membrane - bound form of the ige receptor is thought to act as a negative regulator of ige synthesis, der p 1 cleavage of cd23 could potentially disrupt the negative feedback signal and enhance ige synthesis, thereby amplifying the allergic response. anti - trypsin can inhibit this effect of der p 1 on cd23 cleavage, suggesting that disruption of the balance between proteases and protease inhibitors might play a role in allergic sensitization. it should be noted that whether intact proteases such as der p 1 actually gain functional access to lymphocytes in vivo remains an open question. the biological effects of some allergenic proteases may also be mediated through activation of the protease - activated receptor 2 (par2). pars (1,2,3,4) are a family of proteolytically activated g - protein coupled receptors. proteases cleave within the n - terminus of the receptors and expose a tethered ligand domain that binds and activates the cleaved receptor. several lines of evidence suggest that par2, in particular, may be important in allergic sensitization. it is expressed by many cells in the lung, including airway epithelial cells 31, fibroblasts 32, macrophages 33 and mast cells 34, and, importantly, patients with asthma have been shown to exhibit increased expression of par2 on respiratory epithelial cells 35. several house dust mite allergens (der p1, (30), der p 3, der p 9), along with german cockroach extract 36, have been shown to be able to cleave and activate par2 (figure 1). several reports have shown that activation of par2 by house dust mite extract 7, german cockroach 37, or the mold allergen pen c 13 38 leads to increased cytokine production by airway epithelia. specifically, airway epithelial cells were shown to increase the expression of tslp through the activation of par2 when treated with papain, trypsin or the fungus alterneria 9. as tslp drives dc polarization of nave t cells to a th2 phenotype, these results suggest that par2 activation may serve as a link between innate and adaptive immune responses. several mouse models of allergic inflammation have also underscored a potential role for par2 in allergic sensitization. for example, one recent study showed that tolerance to inhaled ova could be overcome by co - administration of par2-activating peptides to the airways, promoting allergic sensitization 39. other studies have shown that overexpression of par2 in mice renders them susceptible to allergic airway inflammation when sensitized and challenged locally with ova, as compared to wildtype controls 40. while these data strongly support the concept that protease activity may lead to airway allergic sensitization via par2 activation, there are a few reports suggesting that par2 activation may also reduce airway inflammation. for example, despite the fact that tlr4 (vide infra) and par2 signaling have been shown to exhibit cooperativity 41, par2-activating peptides have been reported to inhibit lipopolysaccharide(lps)-induced neutrophil influx into mouse airways 42. in a rabbit model of experimental asthma, sensitization to the pollen parietaria judaica, followed by allergen challenge in the presence or absence of a par2-activating peptide, led to par2-mediated attenuation of the development of airway hyperresponsiveness and airway eosinophilia 43. it is possible that the discrepancies in these results are due to the timing of par2 activation, with activation by exogenous proteases during generation of immune responses having different effects than that occurring in the midst of an ongoing inflammatory response. recent recognition of the critical roles played by innate pattern recognition receptors (prrs) such as toll - like receptors (tlrs), nod - like receptors (nlrs), rig - i - like receptors (rlrs) and c - type lectin receptors (clrs), in activation and instruction of antigen - presenting cells (apcs)44 has led to the exploration of the role of these pathways in allergic responses. it will be noted that, while tlr - driven activation of th1 responses by dcs is well - studied and -understood, the receptors and pathways driving th2 immune responses have been considerably less tractable to experimental investigation. epidemiological studies have reported an inverse correlation between high levels of bacterial products such as lps in the ambient environment during very early life and the subsequent development of atopy and allergic disease 45 - 47. it has been postulated, pace the hygiene hypothesis, that such exposures drive robust counter - regulatory tone in the developing immune system 48. lps exposure can also exacerbate established asthma, however, probably by direct stimulation of airway pro - inflammatory responses 49. experimental mouse models have provided mechanistic insight into the ability of lps exposure to regulate the development of allergic asthma. as predicted by the hygiene hypothesis, lps dose appears to be a critical variable. while airway sensitization with ova along with very low dose (3)-beta - d - glucan, and are commonly found in the cell walls of fungi, pollens, and certain bacteria. in plants, polymers of -glucans are thought to protect the developing pollen during meiosis, and are later destroyed by the enzyme (13)--d - glucanase to liberate the microspores. although -glucans are widely expressed, they are not found in mammalian cells. as such, they can act as pamps, triggering immune responses through activation of specific prrs. the immunostimulatory properties of -glucans have been recognized for decades, since their identification as the immunoactive component of mushrooms 63. for example, it has been reported that -glucan structures present in the peanut glycoallergen ara h 1 have th2 inducing characteristics 64 native, but not deglycosylated, ara h 1 was shown to activate human monocyte - derived dc and induce il-4 and il-13 secreting th2 cells. exposure to -glucans has also been shown to induce airway hyperresponsiveness in allergic humans 65. studies in guinea pigs have shown that direct delivery of (13)--d - glucan to the airways can induce the recruitment of lung eosinophils and lymphocytes 66. in mice, exposure to soluble -glucan isolated from candida albicans 67 markedly exacerbated ova - induced eosinophilic airway inflammation, concomitant with enhanced lung expression of th2 cytokines and il-17a. exposure to -glucans plus ova increased the number of cells bearing mhc class ii and the expression of apc - related molecules such as cd80, cd86, and dec205 on bone marrow derived dcs. in support of a role of -glucans in the recruitment and activation of dcs at mucosal surfaces, recent studies have shown that -glucans contained in house dust mite extracts and in moulds may initiate immune responses at the mucosal surface. house dust mite extract - mediated induction of the release of the chemokine, ccl20, which recruits immature dcs, by human airway epithelial cells in culture, was shown to occur through -glucan and syk - dependent signaling pathways 68 (figure 1). although the exact lectin receptor mediating these effects was not identified in these studies, the results suggested that -glucan moieties contained in house dust mite extracts might mediate early processes leading to immature dc recruitment to the airways. this concept is supported by another recent study that showed that dectin-2 receptor signaling pathways (dectin-2/fcrgamma / syk) mediated the production of cysteinyl leukotrienes in bone marrow - derived dcs following stimulation with house dust mite extracts or aspergillus 69 (figure 1). taken together, these findings identify the dectin-2/fcrgamma / syk axis as a novel receptor mediated pathway by which several potent allergens are recognized by innate immune cells at the airway surface, linking them with the development of th2-skewed adaptive immune responses. consistent with a role for lectins in driving th2 immune responses, blockade of the mannose receptor, an endocytic c - type lectin receptor, significantly reduced der p 1 uptake by dcs 70. these findings are consistent with previous findings suggesting that engagement of the mannose receptor by selected ligands on human dcs leads to the induction of a dc phenotype favoring th2 polarization 71. although the study of the role of glucans as th2-inducing pamps is only in its infancy, data to date suggest that carbohydrate moieties contained in common allergens act as strong th2 inducers via activation of variety of c - type lectin receptors on dcs. it has recently been shown that common allergenic pollen grains contain nicotinamide adenine dinucleotide phosphate (reduced) [nad(p)h ] oxidase activity as well as allergens 72. such pollen grains have been shown to significantly increase the levels of reactive oxygen species (ros) in cultured cells, and to be able induce allergic airway inflammation in experimental animals 73 (figure 1). pretreatment of these pollen grains with nad(p)h oxidase inhibitors attenuated their capacity to increase ros levels in airway epithelial cells and subsequent airway inflammation. similarly, pre - treatment of mice with antioxidants has been shown to prevent the development of pollen - driven asthma in mice. interestingly, delaying anti - oxidant treatment until after pollen challenge was ineffective, suggesting that the oxidase activity is of critical importance during the period of innate immune activation. although the mechanisms remain to be defined, it has been speculated that nadph oxidase activity initiates immune activation through its ability to recruit inflammatory cells, possibly through the induction of il-8 by p38 mapk 74. of interest, genetic polymorphisms in genes regulating oxidative stress although allergens are a diverse group of molecules, it is becoming increasingly clear that their allergenicity likely resides in their ability to activate various innate immune pathways at mucosal surfaces, rather than in any structural similarities. complex allergens contain multiple innate immune activating components, which trigger the initial mucosal influx of innate immune cells that subsequently drive th2-polarized adaptive immune responses. although the study of innate activating properties of allergens is in its infancy, it is clear that a better molecular understanding of the fundamental origins of allergenicity may well lead to the development of new therapeutic strategies to effectively block allergen recognition and the ensuing inflammatory cascade. | allergic diseases, which have reached epidemic proportions, are driven by inappropriate immune responses to a relatively small number of environmental proteins. the molecular basis for the propensity of specific proteins to drive maladaptive, allergic responses has been difficult to define. recent data suggest that the ability of such proteins to drive allergic responses in susceptible hosts is a function of their ability to interact with diverse pathways of innate immune recognition and activation at mucosal surfaces. this review highlights recent insights into innate immune activation by allergens via proteolytic activity, engagement of pattern recognition receptors, molecular mimicry of tlr signaling complex molecules, lipid binding activity, and oxidant potential and the role of such activation in inducing allergic disease. a greater understanding of the fundamental origins of allergenicity should help define new preventive and therapeutic targets in allergic disease. |
indeed, osteoporosis is a disease of the skeletal system characterized by low bone mass and deterioration of bone tissue, which may lead to an increase risk of bone fractures, especially in the wrist, hip, and spine. in osteoporotic patients, bone mineral density (bmd) is 2.5 standard deviation below the average mineral density of young adults. the prevalence of osteoporosis increases with age. fragility fracture may occur in any part of the body particularly in hip, spine, and forearm ; the most dangerous of which is hip fracture. in 1990, in addition to fragility fracture, osteoporosis may increase the rate of hospitalization due to secondary complications. with increase life expectancy, osteoporosis is emerging as a serious health problem worldwide, especially in developing countries. several genetic and environmental factors may influence the development of osteoporosis, the most important of which are low physical activity, smoking, alcohol consumption, wasting, calcium malabsorption, vitamin d deficiency, previous bone fractures, using corticosteroids, hormonal agents, genetic factors, and female sex. like many developing countries, since osteoporosis increases with age, the disease may be considered as a health priority. several studies have investigated the prevalence of osteoporosis among general population in several parts of iran. however, the results have been inconsistent. this meta - analysis was conducted to estimate the overall prevalence of osteoporosis among iranian general population. dual - energy x - ray absorptiometry (dxa) is a means of measuring bmd. world health organization (who) has classified bmd based on t - score as follows : (a) normal : a value of bmd within 1 standard deviation of the young adult reference mean (t - score 1) ; (b) osteopenia : a value of bmd > 1 standard deviation below the young adult mean, but 1 standard deviation below the young adult mean, but 30 years had osteoporosis and 35% had osteopenia. furthermore, our findings indicated that prevalence of these diseases was increasing during the recent years. this means that the prevalence of osteoporosis and thus its associated complications is increasing with life expectancy and may become a critical public health problem in iran in the near future. the prevalence of osteoporosis and osteopenia was significantly higher during postmenopausal period than premenopausal period. this finding is mainly secondary to the nature of the disease and the fact that it mainly affects women after the menopause. furthermore, the overall prevalence of osteoporosis and osteopenia has been increasing in recent years. several evidences indicated the presence of a positive correlation between age and prevalence of osteoporosis. nonetheless, the prevalence of osteoporosis and osteopenia was lower in women during premenopausal period compared to men. this issue may result from random error due to limited number of studies (two studies) conducted in women during premenopausal period. another reason may be the mean difference of age between the two groups (45 years in premenopausal women versus 49 years in men). the prevalence of osteoporosis and osteopenia in the northern regions of iran was higher than in the southern regions. the northern parts of the country are mostly mountainous, while majority of the southern parts is covered by deserts. this issue may help the people who live in the southern parts of the country to receive more vitamin d than residences of the northern parts. furthermore, there are several other factors that can play a role in coetaneous vitamin d synthesis and explain the difference between the two regions. vitamin d3 is produced endogenously in the skin of humans when ultraviolet rays from sunlight strike the skin and trigger vitamin d synthesis. the extent of coetaneous vitamin d production is dependent on latitude, altitude, time, total ozone, clouds, aerosols, and surface reflectivity. for clear atmospheric conditions, no endogenous vitamin d production occurs at 51 latitude and higher during some periods of the year. at 70 clouds, aerosols, and thick ozone events reduce the duration of vitamin d synthesis considerably and can suppress vitamin d synthesis completely. thus, these factors may explain the difference between the prevalence of osteoporosis in the northern and southern parts of the country. there was evidence of heterogeneity (small p value of chi test and large i statistic) among the results of the included studies. the studies were conducted in different settings and hence different densitometry devices and the related measurement errors may be a major source for the prevalence variation. however, care must be taken in the interpretation of the statistical tests for heterogeneity. the chi test has low power when the sample size is small. on the other hand, the test has high power in detecting a small amount of heterogeneity that may be clinically unimportant when there are many studies in a meta - analysis. therefore, we can attribute part of the observed heterogeneity to the great number of studies (31 studies) included in the meta - analysis and the large sample size (34,814 participants). another reason that may explain the observed heterogeneity is the presence of inconsistency between the studies results. despite several studies that have been conducted recently to address the prevalence of osteoporosis in the general population, the results were however different even in age and sex subgroups. there were several limitations and potential biases in this meta - analysis as follows : first, only 31% of the included studies had high quality ; this issue may raise the possibility of the information bias. second, a considerable numbers of studies (34 studies) reported the prevalence of osteoporosis without specifying the anatomical region of the disease ; these studies were excluded from this meta - analysis as this issue might have introduced selection bias in our results. third, there were more women than men in the study (29,928 women versus 4886 men). since the prevalence of osteoporosis was higher in women than in men, this issue might have lead to overestimation of the prevalence of osteoporosis and osteopenia in the iranian general population. we retrieved 2492 studies up to april 2012, including 1732 references through searching international electronic databases, 654 references through searching national electronic databases, 99 references through checking reference lists, and seven references through personal contact with the study authors [table 1 and figure 1 ]. of 2492 retrieved references, 588 references were excluded because of duplication, 1766 references did not relate to the objective of this review, 102 references did not meet the eligibility criteria, and five references had small sample size. eventually, we included 31 studies in the meta - analysis that involved 34,814 participants ; 4886 men with mean age of 49.2 years ; and 29,928 women with a mean age of 52.5 years. characteristics of the included studies in meta - analysis ; repeated references show the prevalence of osteoporosis and osteopenia among different subgroups a flow diagram depicting the phases of retrieving articles, checking eligibility criteria, and including the articles into the meta - analysis we considered all studies addressed osteoporosis irrespective of the organ or body location, including spine, femur, hip, and arm. however, spine was the only organ of which osteoporosis was addressed by all studies. osteoporosis of hip and arm was addressed by only few studies. in this review, we have reported the osteoporosis of spine that represents all studies and that of femur, which was reported by most studies [table 1 ]. however, meta - analysis was performed based on osteoporosis of spine, which comprised all studies. the overall prevalence of osteoporosis in the lumbar spine, based on the random - effects model, was 0.17 (95% ci : 0.13, 0.20). furthermore, the prevalence of osteoporosis was 0.12 (95% ci : 0.08, 0.17) among men, 0.03 (95% ci : 0.01, 0.07) among premenopausal women, and 19% (95% ci : 14%, 24%) among postmenopausal women [figure 2 ]. the overall prevalence of osteopenia in the lumbar spine, based on random - effects model, was 0.35 (95% ci : 0.30, 0.39). in addition, the prevalence of osteopenia was 0.33 (95% ci : 0.25, 0.42) in men, 0.21 (95% ci : 0.05, 0.37) in premenopausal women, and 0.40 (95% ci : 0.31, 0.49) in postmenopausal women [figure 3 ]. a forest plot for the prevalence of osteoporosis in spine among iranian general population older than 30 years a forest plot for the prevalence of osteopenia in spine among iranian general population older than 30 years the studies were divided into three categories based on the quality of reporting using strobe checklist of items as follows : 10 studies (31.25%) had high quality ; 10 studies (31.25%) had intermediate quality, and 12 studies (37.50%) had low quality. the prevalence of osteoporosis and osteopenia was estimated based on the different qualities of the studies. according to these results, the prevalence of osteoporosis and osteopenia was overestimated by the studies with low quality [table 2 ]. subgroup analysis of prevalence of osteoporosis and osteopenia by anatomical site, quality of the included studies, year of studies conduction, geographical regions of the country and the mean age groups using chi test for heterogeneity the prevalence of osteoporosis and osteopenia was estimated based on the years of studies. according to the results, the overall prevalence of osteoporosis and osteopenia in the lumbar spine had increased significantly in recent years [table 2 ]. the prevalence of osteoporosis and osteopenia was estimated based on the geographical regions of the country and the mean age of the participants [table 2 ]. the prevalence of osteoporosis and osteopenia in the northern regions of the country was significantly higher than that in the southern regions. in addition, the prevalence of osteoporosis and osteopenia was much higher among people aged 50 - 69 years as compared to people aged 30 - 49 years. there was considerable heterogeneity among the included studies, so that the result of chi test for heterogeneity was highly significant (p 30 years had osteoporosis and 35% had osteopenia. furthermore, our findings indicated that prevalence of these diseases was increasing during the recent years. this means that the prevalence of osteoporosis and thus its associated complications is increasing with life expectancy and may become a critical public health problem in iran in the near future. the prevalence of osteoporosis and osteopenia was significantly higher during postmenopausal period than premenopausal period. this finding is mainly secondary to the nature of the disease and the fact that it mainly affects women after the menopause. furthermore, the overall prevalence of osteoporosis and osteopenia has been increasing in recent years. several evidences indicated the presence of a positive correlation between age and prevalence of osteoporosis. nonetheless, the prevalence of osteoporosis and osteopenia was lower in women during premenopausal period compared to men. this issue may result from random error due to limited number of studies (two studies) conducted in women during premenopausal period. another reason may be the mean difference of age between the two groups (45 years in premenopausal women versus 49 years in men). the prevalence of osteoporosis and osteopenia in the northern regions of iran was higher than in the southern regions. the northern parts of the country are mostly mountainous, while majority of the southern parts is covered by deserts. this issue may help the people who live in the southern parts of the country to receive more vitamin d than residences of the northern parts. furthermore, there are several other factors that can play a role in coetaneous vitamin d synthesis and explain the difference between the two regions. vitamin d3 is produced endogenously in the skin of humans when ultraviolet rays from sunlight strike the skin and trigger vitamin d synthesis. the extent of coetaneous vitamin d production is dependent on latitude, altitude, time, total ozone, clouds, aerosols, and surface reflectivity. for clear atmospheric conditions, no endogenous vitamin d production occurs at 51 latitude and higher during some periods of the year. at 70 clouds, aerosols, and thick ozone events reduce the duration of vitamin d synthesis considerably and can suppress vitamin d synthesis completely. thus, these factors may explain the difference between the prevalence of osteoporosis in the northern and southern parts of the country. there was evidence of heterogeneity (small p value of chi test and large i statistic) among the results of the included studies. the studies were conducted in different settings and hence different densitometry devices and the related measurement errors may be a major source for the prevalence variation. however, care must be taken in the interpretation of the statistical tests for heterogeneity. on the other hand, the test has high power in detecting a small amount of heterogeneity that may be clinically unimportant when there are many studies in a meta - analysis. therefore, we can attribute part of the observed heterogeneity to the great number of studies (31 studies) included in the meta - analysis and the large sample size (34,814 participants). another reason that may explain the observed heterogeneity is the presence of inconsistency between the studies results. despite several studies that have been conducted recently to address the prevalence of osteoporosis in the general population, the results were however different even in age and sex subgroups. there were several limitations and potential biases in this meta - analysis as follows : first, only 31% of the included studies had high quality ; this issue may raise the possibility of the information bias. second, a considerable numbers of studies (34 studies) reported the prevalence of osteoporosis without specifying the anatomical region of the disease ; these studies were excluded from this meta - analysis as this issue might have introduced selection bias in our results. third, there were more women than men in the study (29,928 women versus 4886 men). since the prevalence of osteoporosis was higher in women than in men, this issue might have lead to overestimation of the prevalence of osteoporosis and osteopenia in the iranian general population. the results of this meta - analysis indicated that osteoporosis and osteopenia are common problems among iranian general population older than 30 years, particularly among women living in the northern parts of the country. this evidence promises that osteoporosis and thus its associated complications will become a critical public health problem in iran in the near future. this issue should be the focus of special attention of policy maker who plan preventive and controlling programs. in addition, because of the considerable heterogeneity between the studies results, further evidence based on a national survey is needed to estimate the exact prevalence of osteoporosis and osteopenia the country. | background : several studies have investigated the prevalence of osteoporosis among general population in several parts of iran. however, the results have been inconsistent. this meta - analysis was conducted to estimate the overall prevalence of osteoporosis.materials and methods : international and national electronic databases were searched until april 2012, including web of knowledge, medline, scopus, ovid, sciencedirect, science information database, iranmedex, magiran, as well the relevant conference databases. the reference lists of included studies were screened as well. the cross - sectional studies addressing the prevalence of osteoporosis among iranian general population were retrieved irrespective of age and sex. bone mineral density (bmd) based on t - score was classified as follows : (a) normal (t - score 1) ; (b) osteopenia (2.5sd < t - score < 1sd) ; (c) osteoporosis (t - score 2.5). study quality was assessed using the recommended checklist of strobe.results:of 2598 retrieved studies, 31 studies comprising 34,814 people was used for meta - analysis. the overall prevalence of osteoporosis in lumbar spine was 0.17 (95% ci : 0.13, 0.20) and that of osteopenia was 0.35 (95% ci : 0.30, 0.39). the prevalence was higher in older age groups, in women, and in the northern regions of the country, with an increasing trend in recent years.conclusion:this meta - analysis indicated that osteoporosis and osteopenia are common problems among iranian population older than 30 years. furthermore, increasing trend of the diseases in recent years is promising a critical public health problem in iran in the near future. however, due to the heterogeneity between the studies results, further evidence based on a national survey is needed to estimate the exact prevalence of the diseases in the country. |
the singularity theorems [245, 139, 140, 141 ] state that einstein s equations will not evolve generic, regular initial data arbitrarily far into the future or the past. an obstruction such as infinite curvature or the termination of geodesics will always arise to stop the evolution somewhere. the simplest, physically relevant solutions representing for example a homogeneous, isotropic universe (friedmann - robertson - walker (frw)) or a spherically symmetric black hole (schwarzschild) contain space - like infinite curvature singularities. although, in principle, the presence of a singularity could lead to unpredictable measurements for a physically realistic observer, this does not happen for these two solutions. the surface of last scattering of the cosmic microwave background in the cosmological case and the event horizon in the black hole (bh) case effectively hide the singularity from present day, external observers. singularity is generic and the types of singularities that appear in generic spacetimes remain major open questions in general relativity. the questions arise quickly since other exact solutions to einstein s equations have singularities which are quite different from those described above. for example, the charged bh (reissner - nordstrm solution) has a time - like singularity. it also contains a cauchy horizon (ch) marking the boundary of predictability of space - like initial data supplied outside the bh. a test observer can pass through the ch to another region of the extended spacetime. the big bang in frw is classified as an asymptotically velocity term dominated (avtd) singularity [94, 165 ] since any spatial curvature term in the hamiltonian constraint becomes negligible compared to the square of the expansion rate as the singularity is approached. however, some anisotropic, homogeneous models exhibit mixmaster dynamics (md), 187 ] and are not avtd the influence of the spatial scalar curvature can never be neglected. for more rigorous discussions of the classification and properties of the types of singularities see [97, 240 ]. once the simplest. there has been significant analytic progress [244, 191, 219, 3 ]. however, until recently such methods have yielded either detailed knowledge of unrealistic, simplified (usually by symmetries) spacetimes or powerful, general results that do not contain details. to overcome these limitations, one might consider numerical methods to evolve realistic spacetimes to the point where the properties of the singularity may be identified. of course, most of the effort in numerical relativity applied to bh collisions has addressed the avoidance of singularities. numerical calculations, even more than analytic ones, require finite values for all quantities. ideally then, one must describe the singularity by the asymptotic non - singular approach to it. a numerical method which can follow the evolution into this asymptotic regime since the numerical study must begin with a particular set of initial data, the results can never have the force of mathematical proof. one may hope, however, that such studies will provide an understanding of the phenomenology of singularities that will eventually guide and motivate rigorous results. some examples of the interplay between analytic and numerical results and methods will be given here. in the following, we shall consider examples of numerical study of singularities both for asymptotically flat (af) spacetimes and for cosmological models. these examples have been chosen to illustrate primarily numerical studies whose focus is the nature of the singularity itself. in the af context, we shall consider two questions : the first is whether or not naked singularities exist for realistic matter sources. one approach has been to explore highly non - spherical collapse looking for spindle or pancake singularities. if the formation of an event horizon requires a limit on the aspect ratio of the matter such configurations may yield a naked singularity. analytic results suggest that one must go beyond the failure to observe an apparent horizon to conclude that a naked singularity has formed. another approach is to probe the limits between initial configurations which lead to black holes and those which yield no singularity at all (i.e. flat spacetime plus radiation) to explore the singularity as the bh mass goes to zero. this quest led naturally to the discovery of critical behavior in the collapse of a scalar field. in the initial study, the critical (choptuik) solution is a zero mass naked singularity (visible from null infinity). however, it is a non - generic one since fine - tuning of the initial data is required to produce this critical solution. in a possibly related study, christodoulou has shown that for the spherically symmetric einstein - scalar field equations, there always exists a perturbation that will convert a solution with a naked singularity (but of a different class from choptuik s) to one with a black hole. reviews of critical phenomena in gravitational collapse can be found in [46, 125, 130, 126 ]. the second question which is now beginning to yield to numerical attack involves the stability of the cauchy horizon in charged or rotating black holes. it has been conjectured [245, 73 ] that a real observer, as opposed to a test mass, can not pass through the ch since realistic perturbed spacetimes will convert the ch to a strong spacelike singularity. numerical studies [56, 92, 63 ] show that a weak, null singularity forms first as had been predicted [212, 202 ]. in cosmology, we shall consider both the behavior of the mixmaster model and the issue of whether or not its properties are applicable to generic cosmological singularities. although numerical evolution of the mixmaster equations has a long history, developments in the past decade were motivated by inconsistencies between the known sensitivity to initial conditions and standard measures of the chaos usually associated with such behavior [193, 223, 225, 28, 102, 62, 147, 216 ]. a coordinate invariant characterization of mixmaster chaos has been formulated which, while criticized in its details, has essentially resolved the question. in addition, a new extremely fast and accurate algorithm for mixmaster simulations has been developed. belinskii, khalatnikov, and lifshitz (bkl) long ago claimed [17, 18, 19, 22, 21 ] that it is possible to formulate the generic cosmological solution to einstein s equations near the singularity as a mixmaster universe at every spatial point. while others have questioned the validity of this claim, numerical evidence has been obtained for oscillatory behavior in the approach to the singularity of spatially inhomogeneous cosmologies [250, 43, 36, 41 ]. we shall discuss results from a numerical program to address this issue [42, 36, 32 ]. the key claim by bkl is that sufficiently close to the singularity, each spatial point evolves as a separate universe most generally of the mixmaster type. for this to be correct, the dynamical influence of spatial derivatives (embodying communication between spatial points) must be overwhelmed by the time dependence of the local dynamics. in the past few years, numerical simulations of collapsing, spatially inhomogeneous cosmological spacetimes have provided strong support for the bkl picture [42, 35, 44, 250, 43, 36, 41 ]. in addition, the method of consistent potentials (mcp) [123, 36 ] has been developed to explain how the bkl asymptotic state arises during collapse. new asymptotic methods have been used to prove that open sets exist with bkl s local behavior (although these are avtd rather than of the mixmaster type) [163, 173, 3 ]. recently, van elst, uggla, and wainwright developed a dynamical systems approach to g2 cosmologies (i.e. those with 2 spatial symmetries). the weak cosmic censorship conjecture requires a singularity formed from regular, asymptotically flat initial data to be hidden from an external observer by an event horizon. an excellent review of the meaning and status of weak cosmic censorship has been given by wald. counter examples have been known for a long time but tend to be dismissed as unrealistic in some way. the strong form of the cosmic censorship conjecture forbids timelike singularities, even within black holes. perhaps, the first numerical approach to study the cosmic censorship conjecture consisted of attempts to create naked singularities. many of these studies were motivated by thorne s hoop conjecture that collapse will yield a black hole only if a mass m is compressed to a region with circumference c 4m in all directions. (as is discussed by wald, one runs into difficulties in any attempt to formulate the conjecture precisely. for example, how does one define c and m, especially if the initial data are not at least axially symmetric ? if the hoop conjecture is true, naked singularities may form if collapse can yield c 4m in some direction. the existence of a naked singularity is inferred from the absence of an apparent horizon (ah) which can be identified locally. although a definitive identification of a naked singularity requires the event horizon (eh) to be proven to be absent, to identify an eh requires knowledge of the entire spacetime. while one finds an ah within an eh [166, 167 ], it is possible to construct a spacetime slicing which has no ah even though an eh is present. methods to find an eh in a numerically determined spacetime have only recently become available and have not been applied to this issue [179, 184 ]. a local prescription, applicable numerically, to identify an isolated horizon is under development by ashtekar. (see for example). in the best known attempt to produce naked singularities, shapiro and teukolsky (st) considered collapse of prolate spheroids of collisionless gas. (nakamura and sato had previously studied the collapse of non - rotating deformed stars with an initial large reduction of internal energy and apparently found spindle or pancake singularities in extreme cases.) st solved the general relativistic vlasov equation for the particles along with einstein s equations for the gravitational field. if no trapped surfaces were found, they concluded that there was no ah in that slice. they found that an ah (and presumably a bh) formed if c 4m > 1 everywhere, but no ah (and presumably a naked singularity) in the opposite case. in the latter case, the evolution (not surprisingly) could not proceed past the moment of formation of the singularity. in a subsequent study, st also showed that a small amount of rotation (counter rotating particles with no net angular momentum) does not prevent the formation of a naked spindle singularity. however, wald and iyer have shown that the schwarzschild solution has a time slicing whose evolution approaches arbitrarily close to the singularity with no ah in any slice (but, of course, with an eh in the spacetime). this may mean that there is a chance that the increasing prolateness found by st in effect changes the slicing to one with no apparent horizon just at the point required by the hoop conjecture. while, on the face of it, this seems unlikely, tod gives an example where a trapped surface does not form on a chosen constant time slice but rather different portions form at different times. he argues that a numerical simulation might be forced by the singularity to end before the formation of the trapped surface is complete. such a trapped surface would not be found by the simulations. in response to such a possibility, shapiro and teukolsky considered equilibrium sequences of prolate relativistic star clusters. the idea is to counter the possibility that an eh might form after the time when the simulation must stop. if an equilibrium configuration is non - singular, it can not contain an eh since singularity theorems say that an eh implies a singularity. however, a sequence of non - singular equilibria with rising i ever closer to the spindle singularity would lend support to the existence of a naked spindle singularity since one can approach the singular state without formation of an eh. they constructed this sequence and found that the singular end points were very similar to their dynamical spindle singularity. wald believes, however, that it is likely that the st slicing is such that their singularities are not naked a trapped surface is present but has not yet appeared in their time slices. another numerical study of the hoop conjecture was made by chiba.. rather than a dynamical collapse model, they searched for ah s in analytic initial data for discs, annuli, and rings. previous studies of this type were done by nakamura. with oblate and prolate spheroids and by wojtk1ewicz with axisymmetric singular lines and rings. the summary of their results is that an ah forms if c 4m 1.26. (analytic results due to barrabs. [10, 9 ] and tod give similar quantitative results with different initial data classes and (possibly) an alternative definition of c.) there is strong analytical evidence against the development of spindle singularities. it has been shown by chruciel and moncrief that strong cosmic censorship holds in af electrovac solutions which admit a g2 symmetric cauchy surface. garfinkle and duncan report preliminary results on the collapse of prolate configurations of brill waves. they use their axisymmetric code to explore the conjecture of abrahams that prolate configurations with no ah but large i in the initial slice will evolve to form naked singularities. garfinkle and duncan find that the configurations become less prolate as they evolve suggesting that black holes (rather than naked singularities) will form eventually from this type of initial data. set out to generalize previous results [246, 241 ] that formation of a singularity in a slice with no ah did not indicate the absence of an eh. they looked specifically at trapped surfaces in two models of collapsing null dust, including the model considered by barrabs.. they indeed find a natural spacetime slicing in which the singularity forms at the poles before the outermost marginally trapped surface (omts) (which defines the ah) forms at the equator. nonetheless, they also find that whether or not an omts forms in a slice closely (or at least more closely than one would expect if there were no relevance to the hoop conjecture) follows the predictions of the hoop conjecture. motivated by st s results, echeverria numerically studied the properties of the naked singularity that is known to form in the collapse of an infinite, cylindrical dust shell. while the asymptotic state can be found analytically, the approach to it must be followed numerically. the analytic asymptotic solution can be matched to the numerical one (which can not be followed all the way to the collapse) to show that the singularity is strong (an observer experiences infinite stretching parallel to the symmetry axis and squeezing perpendicular to the symmetry axis). a burst of gravitational radiation emitted just prior to the formation of the singularity stretches and squeezes in opposite directions to the singularity. one wonders then if dust collapse gives any information about singularities of the gravitational field. the blue curve shows the singularity and the red curve the outermost marginally trapped surface. note that the singularity forms at the poles (indicated by the blue arrow before the outermost marginally trapped surface forms at the equator (indicated by the red arrow). the blue curve shows the singularity and the red curve the outermost marginally trapped surface. note that the singularity forms at the poles (indicated by the blue arrow before the outermost marginally trapped surface forms at the equator (indicated by the red arrow). one useful result from dust collapse has been the study of gravitational waves which might be associated with the formation of a naked singularity. such a program has been carried out by harada, iguchi, and nakao [160, 161, 198, 137, 138, 159 ]. (nsn) [197 ] conjectured that even if naked spindle singularities could exist, they would either disappear or become black holes. this demise of the naked singularity would be caused by the back reaction of the gravitational waves emitted by it. while nsn proposed a numerical test of their conjecture, they believed it to be beyond the current generation of computer technology. chiba extended previous results to search for ah s in spacetimes without axisymmetry but with a discrete symmetry. the discrete symmetry is used to identify an analog of a symmetry axis to allow a prescription for an analog of the circumference. given this construction, it is possible to formulate the hoop conjecture in this case and to explore its validity in numerically constructed momentarily static spacetimes. it was found that, if the quantity c defined as the circumference is less than approximately 1.168, a common apparent horizon surrounds the multi - black - hole configuration. the results of all these searches for naked spindle singularities are controversial but could be resolved if the presence or absence of the eh could be determined. one could demonstrate numerically whether or not wald s view of st s results is correct by using existing eh finders [179, 184 ] in a relevant simulation. of course, this could only be effective if the simulation covered enough of the spacetime to include (part of) the eh. for a more detailed discussion of critical behavior see. since gundlach s living review article has appeared, the updates in this section will be restricted to results i find especially interesting. critical behavior was originally found by choptuik in a numerical study of the collapse of a spherically symmetric massless scalar field. for recent reviews we note that this is the first completely new phenomenon in general relativity to be discovered by numerical simulation. in collapse of a scalar field, essentially two things can happen : either a black hole (bh) forms or the scalar waves pass through each other and disperse. choptuik discovered that for any 1-parameter set of initial data labeled by p, there is a critical value p such that p > p yields a 1311. he found (1)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{bh } \approx c_f \left ({ p - p } \right)^\gamma, $ $ \end{document } where mbh is the mass of the eventual bh. the constant cf depends on the parameter of the initial data that is selected but 0.37 is the same for all choices. furthermore, in terms of logarithmic variables = ln r +, = ln(t0 - t0) + (t0 is the proper time of an observer at r = 0, where r is the radial coordinate, with t0 the finite proper time at which the critical evolution concludes, and is a constant which scales r), the waveform x repeats (echoes) at intervals of in if is resealed to -, i.e. x (-, -) x (,). the scaling behavior (1) demonstrates that the minimum bh mass (for bosons) is zero. the critical solution itself is a counter - example to cosmic censorship (since the formation of the zero mass bh causes high curvature regions to become visible at r =). the numerical demonstration of this feature of the critical solution was provided by hamad and stewart. this result caused hawking to pay off a bet to preskill and thorne [61, 170 ]. soon after this discovery, scaling and critical phenomena were found in a variety of contexts. abrahams and evans discovered the same phenomenon in axisymmetric gravitational wave collapse with a different value of and, to within numerical error, the same value of. (note that the resealing of r with e 30 required choptuik to use adaptive mesh refinement (amr) to distinguish subsequent echoes. abrahams and evans smaller (e 1.8) allowed them to see echoing with their 2 + 1 code without amr.) garfinkle confirmed choptuik s results with a completely different algorithm that does not require amr. he used goldwirth and piran s method of simulating christodoulou s formulation of the spherically symmetric scalar field in null coordinates. this formulation allowed the grid to be automatically resealed by choosing the edge of the grid to be the null ray that just hits the central observer at the end of the critical evolution. (missing points of null rays that cross the central observer s world line are replaced by interpolation between those that remain.) hamade and stewart have also repeated choptuik s calculation using null coordinates and amr. figure 3this figure is the final frame of an animation of type ii critical behavior in einstein - yang - mills collapse. this figure is the final frame of an animation of type ii critical behavior in einstein - yang - mills collapse. evans and coleman realized that self - similar rather than self - periodic collapse might be more tractable both numerically (since ode s rather than pde s are involved) and analytically. (note that self - similarity (homothetic motion) is incompatible with af [93, 108 ]. however, most of the action occurs in the center so that a match of the self - similar inner region to an outer af one should always be possible.) in a series of papers, hirschmann and eardley [144, 145 ] developed a (numerical) self - similar solution to the spherically symmetric complex scalar field equations. these are ode s with too many boundary conditions causing a solution to exist only for certain fixed values of. numerical solution of this eigenvalue problem allows very accurate determination of a. the self - similarity also allows accurate calculation of as follows : the critical p = p solution is unstable to a small change in p. at any time t (where t > 0 is increasing toward zero), the amplitude a of the perturbation exhibits power law growth : (2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a \propto \left ({ p - p } \right){\left ({ - t } \right)^ { - \kappa } }, $ $ \end{document } where > 0. at any fixed t, larger a implies larger mbh. equivalently, any fixed amplitude a = will be reached faster for larger eventual mbh. scaling arguments give the dependence of mbh on the time at which any fixed amplitude is reached : (3)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{bh } } \propto \left ({ - { t_1 } } \right),$$\end{document } where (4)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left ({ p - p } \right){\left ({ - { t_1 } } \right)^\kappa } \propto \delta.$$\end{document } thus (5)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{bh } } \propto { \left ({ p - p } \right)^{1/\kappa } }.$$\end{document } therefore, one need only identify the growth rate of the unstable mode to obtain an accurate value of = 1/. it is not necessary to undertake the entire dynamical evolution or probe the space of initial data. (while this procedure allowed hirschmann and eardley to obtain = 0.387106 for the complex scalar field solution, they later found that, in this regime, the complex scalar field has 3 unstable modes. this means [129, 126 ] that the eardley - hirschmann solution is not a critical solution. a perturbation analysis indicates that the critical solution for the complex scalar field is the discretely self - similar one found for the real scalar field. although the similarities among the critical exponents in the collapse computations suggested a universal value, maison used these same scaling - perturbation methods to show that depends on the equation of state p = k of the fluid in the evans - coleman solution. he reformulates the model as nonlinear hyperbolic boundary value problem with eigenvalue and finds = 3.4439. as with the self - similar solutions described above, the critical solution is found directly without the need to perform a dynamical evolution or explore the space of initial data. extended the renormalization group approach of and applied it to the continuously - self - similar case. gundlach completed his eigenvalue analysis of the choptuik solution to find the growth rate of the unstable mode to be = 0.374 0.001. self - similar critical behavior has been seen in string theory related axion - dilaton models [95, 134 ] and in the nonlinear -model. garfinkle and duncan have shown that subcritical collapse of a spherically symmetric scalar field yields a scaling relation for the maximum curvature observed by the central observer with critical parameters that would be expected on the basis of those found for supercritical collapse. have generalized the original einstein - scalar field calculations to the einstein - yang - mills (eym) (for su(2)) case. here something new was found. the critical solution is a regular, unstable solution to the eym equations found previously by bartnik and mckinnon. in type ii collapse, the minimum bh mass is zero with the critical solution similar to that of choptuik (with a different 0.20, 0.74). the type i behavior arises when the collapsing system has a metastable static solution in addition to the choptuik critical one. brady, chambers and goncalves [71, 52 ] conjectured that addition of a mass to the scalar field of the original model would break scale invariance and might yield a distinct critical behavior. phases seen in the eym case. the type ii solution can be understood as perturbations of choptuik s original model with a small scalar field mass. here small means that 1 where is the spatial extent of the original nonzero field region. (the scalar field is well within the compton wavelength corresponding to.) on the other hand, 1 yields type i behavior. the minimum mass critical solution is an unstable soliton of the type found by seidel and suen. the massive scalar field can be treated as collapsing dust to yield a criterion for bh formation. the choptuik solution has also been found to be the critical solution for charged scalar fields [132, 151 ]. as p p, q / m 0 for the black hole. numerical study of the critical collapse of collision - less matter (einstein - vlasov equations) has yielded a non - zero minimum bh mass [217, 201 ]. an astrophysical application of bh critical phenomena has been considered by niemeyer and jedamzik and yokoyama. they consider its implications for primordial bh formation and suggest that it could be important. the question is then why these critical phenomena should appear in so many collapsing gravitational systems. the discrete self - similarity of choptuik s solution may be understood as scaling of a limit cycle. (the self - similarity of other systems may be understood as scaling of a limit point.) garfinkle originally conjectured that the scale invariance of einstein s equations might provide an underlying explanation for the self - similarity and discrete - self - similarity found in collapse and proposed a spacetime slicing which might manifestly show this. in fact, he later showed (with meyer) that, while the originally proposed slicing failed, a foliation based on maximal slicing did make the scaling manifest. these ideas formed the basis of a much more ambitious program by garfinkle and gundlach to use underlying actual or approximate symmetries to construct coordinate systems for numerical simulations. an interesting toy model for general relativity in many contexts has been wave maps, also known as nonlinear models. evolved wave maps from the base space of 3 + 1 minkowski space to the target space s. they found critical behavior separating singular and nonsingular solutions. for some families of initial data, the critical solution is self - similar and is an intermediate attractor. for others, however, the static solution has several unstable modes and is therefore not a critical solution in the usual sense. bizo and tabor have studied yang mills fields in d + 1 dimensions and found that generic solutions with sufficiently large initial data blow up in a finite time and that the mechanism for blowup depends on d. husa. then considered the collapse of su(2) nonlinear sigma models coupled to gravity and found a discretely self - similar critical solution for sufficiently large dimensionless coupling constant. they also observe that for sufficiently small coupling constant values, there is a continuously self - similar solution. interestingly, there is an intermediate range of coupling constant where the discrete self - similarity is intermittent. until recently, he considered spherical and non - spherical perturbations of p = 1/3 perfect fluid collapse. only the original (spherical) growing mode survived. recently pretorius and choptuik numerically evolved circularly symmetric scalar field collapse in 2 + 1 anti - de sitter space. they found a continuously self - similar critical solution at the threshold for black hole formation. the bh s which form have btz exteriors with strong curvature, spacelike singularities in the interior. remarkably, garfinkle obtained an analytic critical solution by assuming continuous self - similarity which agrees quite well with the one obtained numerically. unlike the simple singularity structure of the schwarzschild solution, where the event horizon encloses a spacelike singularity at r = 0, charged and/or rotating bh s the extended spacetimes have an inner cauchy horizon (ch) which is the boundary of predictability. poisson and israel [212, 213 ] began an analytic study of the effect of perturbations on the ch. their goal was to check conjectures that the blue - shifted infalling radiation during collapse would convert the ch into a true singularity and thus prevent an observer s passage into the rest of the extended regions. by including both ingoing and back - scattered outgoing radiation, they find for the reissner - nordstrm (rn) solution that the mass function (qualitatively r however, ori showed both for rn and kerr [202, 203 ], that the metric perturbations are finite (even though r r diverges) so that an observer would not be destroyed by tidal forces (the tidal distortion would be finite) and could survive passage through the ch. a numerical solution of the einstein - maxwell - scalar field equations would test these perturbative results. for an excellent, brief review of the history of this topic see the introduction in. gnedin and gnedin have numerically evolved the spherically symmetric einstein - maxwell with massless scalar field equations in a 2 + 2 formulation. the initial conditions place a scalar field on part of the rn event horizon (with zero field on the rest). an asymptotically null or spacelike singularity whose shape depends on the strength of the initial perturbation replaces the ch. for a sufficiently strong perturbation, although they claim to have found that the ch evolved to become a spacelike singularity, the diagrams in their paper show at least part of the final singularity to be null or asymptotically null in most cases. brady and smith used the goldwirth - piran formulation to study the same problem. they follow the evolution of the ch into a null singularity, demonstrate mass inflation, and support (with observed exponential decay of the metric component g) the validity of previous analytic results [212, 213, 202, 203 ] including the weak nature of the singularity that forms. they find that the observer hits the null ch singularity before falling into the curvature singularity at r = 0. whether or not these results are in conflict with gnedin and gnedin is unclear. the collapse of a scalar field in a charged, spherically symmetric spacetime causes an initial rn ch to become a null singularity except at r = 0 where it is spacelike. the observer falling into the bh experiences (and potentially survives) the weak, null singularity [202, 203, 51 ] before the spacelike singularity forms. this has been confirmed by droz using a plane wave model of the interior and by burko using a collapsing scalar field. numerical studies of the interiors of non - abelian black holes have been carried out by breitenlohner. although there appear to be conflicts between the two groups, the differences can be attributed to misunderstandings of each other s notation. the current status of the topic of singularities within bh s includes an apparent conflict between the belief and numerical evidence that the generic singularity is strong, oscillatory, and spacelike, and analytic evidence that the singularity inside a generic (rotating) bh is weak, oscillatory (but in a different way), and null. figure 4figure 1 from is a schematic diagram of the singularity structure within a spherical charged black hole. figure 1 from is a schematic diagram of the singularity structure within a spherical charged black hole. (i.e. one which results from collapse) black hole is of the weak, null type described by ori [202, 203 ]. brady. performed an analysis in the spirit of belinskii. to argue that the singularity is of this type. they constructed an asymptotic expansion about the ch of a black hole formed in gravitational collapse without assuming any symmetry of the perturbed solution. to illustrate their techniques the best numerical evidence for the nature of the singularity in realistic collapse is hod and piran s simulation of the gravitational collapse of a spherically symmetric, charged scalar field [154, 153 ]. rather than start with (part of) a rn spacetime which already has a singularity (as in, e.g.,), they begin with a regular spacetime and follow its dynamical evolution. they observe mass inflation, the formation of a null singularity, and the eventual formation of a spacelike singularity. ori argues that the rotating black hole case is different and that the spacelike singularity will never form. some insight into the conflict between the cosmological results and those from bh interiors may be found by comparing the approach to the singularity in gowdy spatially inhomogeneous cosmologies (see section 3.4.2) with t and s s spatial topologies. early in the collapse, the boundary conditions associated with the s s topology influence the gravitational waveforms. eventually, however, the local behavior of the two spacetimes becomes qualitatively indistinguishable and characteristic of a (non - oscillatory in this case) spacelike singularity. this may be relevant because the bh environment imposes effective boundary conditions on the metric just as topology does. unfortunately, no systematic study of the relationship between the cosmological and bh interior results yet exists. replacing the af boundary conditions with schwarzchild - de sitter and rn - de sitter bh s (see [185, 186 ;, 211, 70 ] and references therein for background and extended discussions.) the stability of the ch is related to the decay tails of the radiating scalar field. numerical studies have determined these to be exponential [53, 70, 72 ] rather than power law as in af spacetimes. the decay tails of the radiation are necessary initial data for numerical study of ch stability and are crucial to the development of the null singularity. analytic studies had indicated that the ch is stable in rn - de sitter bh s for a restricted range of parameters near extremality. however, brady. have shown (using linear perturbation theory) that, if one includes the backscattering of outgoing modes which originate near the event horizon, the ch is always unstable for all ranges of parameters. numerical studies are needed to demonstrate the existence of a null singularity at the ch in nonlinear evolution. extension of these studies to af rotating bh s has yielded the surprising result that the tails are not necessarily power law and differ for different fields. frame dragging effects appear to be responsible. as a potentially useful approach to the numerical study of singularities, we consider hiibner s [156, 157, 155 ] numerical scheme to evolve on a conformal compactified grid using friedrich s formalism. it was also used to search for choptuik s scaling and failed to produce agreement with choptuik s results. this was probably due to limitations of the code rather than inherent problems with the conformal method. the first, called asymptotically velocity term dominated (avtd) [94, 165 ], refers to a cosmology that approaches the kasner (vacuum, bianchi i) solution as. (spatially homogeneous universes can be described as a sequence of homogeneous spaces labeled by. here we shall choose so that = coincides with the singularity.) an example of such a solution is the vacuum bianchi ii model which begins with a fixed set of kasner - like anisotropic expansion rates, and, possibly, makes one change of the rates in a prescribed way (mixmaster - like bounce) and then continues to = as a fixed kasner solution. in contrast are the homogeneous cosmologies which display mixmaster dynamics such as vacuum bianchi viii and ix [22, 187, 133 ] and bianchi vi0 and bianchi i with a magnetic field [178, 29, 177 ]. mixmaster dynamics describes an approach to the singularity which is a sequence of kasner epochs with a prescription, originally due to belinskii, khalatnikov, and lifshitz (bkl), for relating one kasner epoch to the next. some of the mixmaster bounces (era changes) display sensitivity to initial conditions one usually associates with chaos, and in fact mixmaster dynamics is chaotic [86, 194 ]. note that we consider an epoch to be a subunit of an era. in some of the literature, this usage is reversed. the vacuum bianchi i (kasner) solution is distinguished from the other bianchi types in that the spatial scalar curvature r, (proportional to) the minisuperspace (mss) potential [187, 227 ], vanishes identically. but r arises in other bianchi types due to spatial dependence of the metric in a coordinate basis. thus an avtd singularity is also characterized as a regime in which terms containing or arising from spatial derivatives no longer influence the dynamics. this means that the mixmaster models do not have an avtd singularity since the influence of the spatial derivatives (through the mss potential) never disappears there is no last bounce. symplectic numerical methods have proven useful in studies of the approach to the singularity in cosmological models. symplectic ode and pde [101, 189 ] methods comprise a type of operator splitting. details of the application to the gowdy model (pde s in one space and one time direction) are given elsewhere. for a field q(t) and its conjugate momentum p(t), the hamiltonian operator splits into kinetic and potential energy sub - hamiltonians. thus, for an arbitrary potential v (q), (6)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = \frac{1}{2}{p^2 } + v\left (q \right) = { h_1}\left (p \right) + { h_2}\left (q \right).$$\end{document } if the vector x = (p, q) defines the variables at time t, then the time evolution is given by (7)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\frac{{dx}}{{dt } } = { \left\ { { h, x } \right\}_{pb } } \equiv ax,$$\end{document } where { } pb is the poisson bracket. the usual exponentiation yields an evolution operator (8)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e^{a\delta t } = e^{a_1 \left ({ \delta t/2 } \right) } e^{a_2 \delta t } e^{a_1 \left ({ \delta t/2 } \right) } + o\left ({ \delta t^3 } \right)$$\end{document } for a = a1 + a2 being the generator of the time evolution. higher order accuracy may be obtained by a better approximation to the evolution operator [234, 235 ]. this method is useful when exact solutions for the sub - hamiltonians are known. for the given h, variation of h1 yields the solution (9)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q = q_0 + p_0 \delta t,\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;p = p_0, $ $ \end{document } while that of h2 yields (10)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q = q_0, \;\;\;\;\;\;\;\;\;\;\;\;\;\;p = p_0 - \frac{{dv } } { { dq}}|_{q_0 } \delta t.$$\end{document } note that h2 is exactly solvable for any potential v no matter how complicated although the required differenced form of the potential gradient may be non - trivial. one evolves from t to t + t using the exact solutions to the sub - hamiltonians according to the prescription given by the approximate evolution operator (8). extension to more degrees of freedom and to fields is straightforward [42, 30 ]. symplectic methods can achieve convergence far beyond that of their formal accuracy if the full solution is very close to the exact solution from one of the sub - hamiltonians. on the other hand, because symplectic algorithms are a type of operator splitting, suboperators can be subject to instabilities that are suppressed by the full operator. one currently popular method is iterative crank - nicholson (see which is, in effect, an implicit method without matrix inversion. it was first applied to numerical cosmology by garfinkle and was recently used in this context to evolve t symmetric cosmologies. as pointed out in [42, 35, 41, 43 ], spiky features in collapsing inhomogeneous cosmologies will cause any fixed spatial resolution to become inadequate. such evolutions are therefore candidates for adaptive mesh refinement such as that implemented by hern and stuart [143, 142 ]. belinskii, khalatnikov, and lifshitz (bkl) described the singularity approach of vacuum bianchi ix cosmologies as an infinite sequence of kasner epochs whose indices change when the scalar curvature terms in einstein s equations become important. they were able to describe the dynamics approximately by a map evolving a discrete set of parameters from one kasner epoch to the next [22, 74 ]. for example, the kasner indices for the power law dependence of the anisotropic scale factors can be parametrized by a single variable u 1. bkl determined that (11)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u_{n + 1 } = \left\ { { \begin{array}{{20}c } { u_n - 1,\;\;\ ; } \\ { \;\;\left ({ u_n - 1 } \right)^ { - 1 }, } \\ \end{array } } \right.\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\begin{array}{{20}c } { 2 \leqslant u_n, \;\;\;\ ; } \\ { 1 \leqslant u_n \leqslant 2. } \\ \end{array}$$\end{document } the subtraction in the denominator for 1 un 2 yields the sensitivity to initial conditions associated with mixmaster dynamics (md). misner described the same behavior in terms of the model s volume and anisotropic shears. a multiple of the scalar curvature acts as an outward moving potential in the anisotropy plane. kasner epochs become straight line trajectories moving outward along a potential corner while bouncing from one side to the other. a change of corner ends a bkl era when u (u - 1). numerical evolution of einstein s equations was used to explore the accuracy of the bkl map as a descriptor of the dynamics as well as the implications of the map [193, 223, 225, 28 ]. rendall has studied analytically the validity of the bkl map as an approximation to the true trajectories. later, the bkl sensitivity to initial conditions was discussed in the language of chaos [11, 172 ]. an extended application of bernoulli shifts and farey trees was given by rugh and repeated by cornish and levin. however, the chaotic nature of mixmaster dynamics was questioned when numerical evolution of the mixmaster equations yielded zero lyapunov exponents (le s) [102, 62, 147 ]. only an exponential divergence, characteristic of a chaotic system, will yield a positive exponent.) this issue was resolved when the le was shown numerically and analytically to depend on the choice of time variable [223, 27, 99 ]. although md itself is well - understood, its characterization as chaotic or not had been quite controversial. have shown (analytically and numerically) that md can arise in bianchi vi0 models with magnetic fields (see also). in essence, the magnetic field provides the wall needed to close the potential in a way that yields the bkl map for u. a similar study of magnetic bianchi i has been given by leblanc. cornish and levin (cl) identified a coordinate invariant way to characterize md. sensitivity to initial conditions can lead to qualitatively distinct outcomes from initially nearby trajectories. while the le measures the exponential divergence of the trajectories, one can also color code the regions of initial data space corresponding to particular outcomes. cl defined the following set of discrete outcomes : during a numerical evolution, the bkl parameter u is evaluated from the trajectories. the first time u > 7 appears in an approximately kasner epoch, the trajectory is examined to see which metric scale factor has the largest time derivative. however, one can easily invent prescriptions other than that given by cornish and levin which would yield discrete outcomes. it is not clear how the value of the fractal dimension as measured by cornish and levin would be affected. the cl prescription has been criticized because it requires only the early part of a trajectory for implementation. it replaces a single representative, infinitely long trajectory by (easier to compute arbitrarily many trajectory fragments. figure 5the algorithm of is used to generate a mixmaster trajectory with more than 250 bounces. the trajectory is shown in the rescaled anisotropy plane with axes /||. the rescaling fixes (asymptotically) the location of the bounces. the algorithm of is used to generate a mixmaster trajectory with more than 250 bounces. the trajectory is shown in the rescaled anisotropy plane with axes /||. the rescaling fixes (asymptotically) the location of the bounces. to study the cl fractal and ergodic properties of mixmaster evolution, one could take advantage of a new numerical algorithm due to berger, garfinkle, and strasser. symplectic methods are used to allow the known exact solution for a single mixmaster bounce to be used in the ode solver. standard ode solvers can take large time steps in the kasner segments but must slow down at the bounce tens of orders of magnitude improvement in speed are obtained while the accuracy of machine precision is maintained. in, the new algorithm was used to distinguish bianchi ix and magnetic bianchi vi0 bounces. this required an improvement of the bkl map (for parameters other than u) to take into account details of the exponential potential. so far, most recent effort in md has focused on diagonal (in the frame of the su(2) 1-forms) bianchi ix models. long ago, ryan showed that off - diagonal metric components can contribute additional mss potentials (e.g. a centrifugal wall). the most interesting recent developments in spatially homogeneous mixmaster models have been mathematical. despite the strong numerical evidence that bianchi ix, etc. models are well - approximated by the bkl map sufficiently close to the singularity (see, e.g.,), there was very little rigorous information on the nature of these solutions. recently, the existence of a strong singularity (curvature blowup) was proved for bianchi viii and ix collapse by ringstrm [221, 222 ] and for magnetic bianchi vi0 by weaver. a remaining open question is how closely an actual mixmaster evolution is approximated by a single bkl sequence [218, 222 ]. since the berger. algorithm achieves machine level accuracy, it can be used to collect numerical evidence on this topic. for example, it has been shown that a given mixmaster trajectory ceases to track the corresponding sequence of integers obtained from the bkl map (11) at the point where there have been enough era - ending (mixing) bounces to lose all the information encoded in finite precision initial data. find evidence of chaotic behavior in bianchi ix - brans - dicke solutions while cotsakis. have shown that bianchi ix models in 4th order gravity theories have stable non - chaotic solutions. barrow and levin find evidence of chaos in bianchi ix einstein - yang - mills (eym) cosmologies. bianchi i models with string - theory - inspired matter fields have been found by damour and henneau to have an oscillatory singularity. this is interesting because many other examples exist where matter fields and/or higher dimensions suppress such oscillations (see, e.g., recently, coley has considered bianchi ix brane - world models and found them not to be chaotic. finally, we remark on a successful application of numerical regge calculus in 3 + 1 dimensions. bkl have conjectured that one should expect a generic singularity in spatially inhomogeneous cosmologies to be locally of the mixmaster type (local mixmaster dynamics (lmd)) for a review of homogeneous cosmologies, inhomogeneous cosmologies, and the relation between them, see. the main difficulty with the acceptance of this conjecture has been the controversy over whether the required time slicing can be constructed globally [13, 122 ]. montani, belinskii, and kirillov and kochnev [175, 174 ] have pointed out that if the bkl conjecture is correct, the spatial structure of the singularity could become extremely complicated as bounces occur at different locations at different times. bkl seem to imply that lmd should only be expected to occur in completely general spacetimes with no spatial symmetries. however, lmd is actually possible in any spatially inhomogeneous cosmology with a local mss with a closed potential (in the sense of the standard triangular potentials of bianchi viii and ix). a class of cosmological models which appear to have local md are vacuum universes on t r with a u(1) symmetry. even simpler plane symmetric gowdy cosmologies [121, 26 ] have open local mss potentials. however, these models are interesting in their own right since they have been conjectured to possess an avtd singularity. one way to obtain these gowdy models is to allow spatial dependence in one direction in bianchi i homogeneous cosmologies. it is well - known that addition of matter terms to homogeneous bianchi i, bianchi vi0, and other avtd models can yield mixmaster behavior [168, 178, 177 ]. allowing spatial dependence in one direction in such models might then yield a spacetime with lmd. application of this procedure to magnetic bianchi vi0 models yields magnetic gowdy models [250, 247 ]. of course, gowdy cosmologies are not the most general t symmetric vacuum spacetimes [121, 82, 40 ]. restoring the twists introduces a centrifugal wall to close the mss. magnetic gowdy and general t symmetric models appear to admit lmd [247, 248, 41 ]. the past few years have seen the development of strong numerical evidence in support of the bkl claims. the method of consistent potentials (mcp) has been used to organize the data obtained in simulations of spatially inhomogeneous cosmologies [42, 35, 250, 43, 36, 33, 41 ]. the main idea is to obtain a kasner - like velocity term dominated (vtd) solution at every spatial point by solving einstein s equations truncated by removing all terms containing spatial derivatives. if the spacetime really is avtd, all the neglected terms will be subdominant (exponentially small in variables where the vtd solution is linear in the time) when the vtd solution is substituted back into them. for the mcp to successfully predict whether or not the spacetime is avtd, the dynamics of the full solution must be dominated at (almost) every spatial point by the vtd solution behavior. surprisingly, mcp predictions have proved valid in numerical simulations of cosmological spacetimes with one and two [42, 35, 41 ] spatial symmetries. in the case of u(1) symmetric models, a comparison between the observed behavior and that in a vacuum, diagonal blanch ! ix model written in terms of u(1) variables provides strong support for lmd since the phenomenology of the inhomogeneous cosmologies can be reproduced by this rewriting of the standard blanch ! ix md. polarized plane symmetric cosmologies have been evolved numerically using standard techniques by anninos, centrella, and matzner [5, 6 ]. the long - term project involving berger, garfinkle, and moncrief and their students to study the generic cosmological singularity numerically has been reviewed in detail elsewhere [37, 36, 32 ] but will be discussed briefly here. the gowdy model on t r is described by gravitational wave amplitudes p(,) and q(,) which propagate in a spatially inhomogeneous background universe described by (,). (we note that the physical behavior of a gowdy spacetime can be computed from the effect of the metric evolution on a test cylinder.) the time variable measures the area in the symmetry plane with = being a curvature singularity. the first is nonlinearly coupled wave equations for dynamical variables p and q (where, a = /a) obtained from the variation of (12)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c } { h = \frac{1 } { 2}\int\limits_0^{2\pi } { d\theta \left [{ \pi _ p^2 + e^ { - 2p } \pi _ q^2 } \right ] } \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\ ; } \\ { + \frac{1 } { 2}\int\limits_0^{2\pi } { d\theta \left [{ e^ { - 2\tau } \left ({ p,_\theta ^2 + e^{2p } q,_\theta ^2 } \right) } \right ] } = h_1 + h_2, } \\ \end{array } $ $ \end{document } where p and q are canonically conjugate to p and q respectively. if the model is, in fact, avtd, the approximation in the symplectic numerical scheme should become more accurate as the singularity is approached. these can be expressed as first order equations for a in terms of p and q. this break into dynamical and constraint equations removes two of the most problematical areas of numerical relativity from this model the initial value problem and numerical preservation of the constraints. for the special case of the polarized gowdy model (q = 0), p satisfies a linear wave equation whose exact solution is well - known. for this case avtd behavior is defined in as follows : solve the gowdy wave equations neglecting all terms containing spatial derivatives. if the approach to the singularity is avtd, the full solution comes arbitrarily close to a vtd solution at each spatial point as. as, the vtd solution becomes (13)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p\left ({ \theta, \tau } \right) \to \upsilon \left (\theta \right)\tau, \;\;\;\;\;\;\;\;\;\;\;\;q\left ({ \theta, \tau } \right) \to q_0 \left (\theta \right),$$\end{document } where v > 0. substitution in the wave equations shows that this behavior is consistent with asymptotic exponential decay of all terms containing spatial derivatives only if 0 v > 1 [we have shown that, except at isolated spatial points, the nonlinear terms in the wave equation for p drive v into this range [35, 37 ]. the exceptional points occur when coefficients of the nonlinear terms vanish and are responsible for the growth of spiky features seen in the wave forms [42, 35 ]. we conclude that generic gowdy cosmologies have an avtd singularity except at isolated spatial points [35, 37 ]. after the nature of the solutions became clear through numerical experiments, it became possible to use fuchsian asymptotic methods to prove that gowdy solutions with 0 > v > 1 and avtd behavior almost everywhere are generic. these methods have recently been applied to gowdy spacetimes with s s and s topologies with similar conclusions. one striking property of the gowdy models are the development of spiky features at isolated spatial points where the coefficient of a local potential term vanishes [42, 35 ]. recently, rendall and weaver have shown analytically how to generate such spikes from a gowdy solution without spikes. addition of a magnetic field to the vacuum gowdy models (plus a topology change) which yields the inhomogeneous generalization of magnetic bianchi vi0 models provides an additional potential which grows exponentially if 0 > v > 1. local mixmaster behavior has recently been observed in these magnetic gowdy models [250, 247 ]. garfinkle has used a vacuum gowdy model with s s spatial topology to test an algorithm for axis regularity. along the way, he has shown that these models are also avtd with behavior at generic spatial points that is eventually identical to that in the t case. comparison of the two models illustrates that topology or other global or boundary conditions are important early in the simulation but become irrelevant as the singularity is approached. gowdy spacetimes are not the most general t symmetric vacuum cosmologies. certain off - diagonal metric components (the twists which are g, g in the notation of (12)) have been set to zero. restoring these terms (see [83, 40 ]) yields spacetimes that are not avtd but rather appear to exhibit a novel type of lmd [41, 249 ]. the lmd in these models is an inhomogeneous generalization of non - diagonal bianchi models with mss potential walls [227, 169 ] in addition to the usual curvature walls. in, remarkable quantitative agreement is found between predictions of the mcp and numerical simulation of the full einstein equations. a version of the code with amr has been developed. (asymptotic methods have been used to prove that the polarized version of these spacetimes have avtd solutions.) moncrief has shown that cosmological models on t r with a spatial u(1) symmetry can be described by five degrees of freedom { x, z, a,,, } and their respective conjugate momenta { px, pz, p, p, r}. all variables are functions of spatial variables u, v and time. einstein s equations can be obtained by variation of (14)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c } { h = \oint { \oint { dudv\mathcal{h } } \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\ ; } } \\ { = \oint { \oint { dudv(\tfrac{1 } { 8}p_z^2 + \tfrac{1 } { 2}e^{4z } p_x^2 + \tfrac{1 } { 8}p^2 + \tfrac{1 } { 2}4^\phi r^2 - \tfrac{1 } { 2}p_\lambda ^2 + 2p_\lambda) } } } \\ { + e^ { - 2\tau } \oint { \oint { dudv\ { (e^\lambda e^{ab }), _ { ab } - (e^\lambda e^{ab }), _ a \lambda, _ b \;\;\;\;\;\;\;\;\;\;\;\;\ ; } } } \\ { \;\;\;\;\;\;\ ; + e^\lambda [(e^ { - 2z }), _ u x,_v - (e^ { - 2z }), _ v x,_u ] } \\ { \;\;\;\;\;\;\;\;\;\;\;\;\ ; + 2e^\lambda e^{ab } \phi, _ a \phi, _ b + \tfrac{1 } { 2}e^\lambda e^ { - 4\phi } e^{ab } \omega, _ a \omega, _ b \ } } \\ { = h_1 + h_2 \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\ ; } \\ \end{array } $ $ \end{document } here and are analogous to p and q while e is a conformal factor for the metric eab(x, z) in the u - v plane perpendicular to the symmetry direction. note particularly that h1 contains two copies of the gowdy h1 plus a free particle term and is thus exactly solvable. a spectral evaluation of derivatives which has been shown to work in gowdy simulations does not appear to be helpful in the u(1) case.) a particular solution to the initial value problem is used since the general solution is not available. current limitations of the u(1) code do not affect simulations for the polarized case since problematic spiky features do not develop. asymptotic methods have been used to prove that an open set of avtd solutions exist for this case. the mcp indicates that the term containing gradients of in (14) acts as a mixmaster - like potential to drive the system away from avtd behavior in generic u(1) models. numerical simulations provide support for this suggestion [37, 43 ]. whether this potential term grows or decays depends on a function of the field momenta. however, failure to enforce the constraints can cause an erroneous relationship among the momenta to yield qualitatively wrong behavior. there is numerical evidence that this error tends to suppress mixmaster - like behavior leading to apparent avtd behavior in extended spatial regions of u(1) symmetric cosmologies [30, 31 ]. in fact, it has been found that when the hamiltonian constraint is enforced at every time step, the predicted local oscillatory behavior of the approach to the singularity is observed. (note that in a numerical study of vacuum bianchi ix homogeneous cosmologies, zardecki obtained a spurious enhancement of mixmaster oscillations due to constraint violation [253, 147 ]. in this case figure 6behavior of the gravitational wave amplitude at a typical spatial point in a collapsing u(1) symmetric cosmology. behavior of the gravitational wave amplitude at a typical spatial point in a collapsing u(1) symmetric cosmology. mixmaster simulations with the new algorithm can easily evolve more than 250 bounces reaching ||, 10. the larger number of bounces quickly reveals that it is necessary to enforce the hamiltonian constraint. an explicitly constraint enforcing u(1) code it is well known that a scalar field can suppress mixmaster oscillations in homogeneous cosmologies. this was demonstrated numerically for magnetic gowdy and u(1) symmetric spacetimes andersson and rendall proved that completely general cosmological (spatially t) spacetimes (no symmetries) with sufficiently strong scalar fields have generic avtd solutions. garfinkle has constructed a 3d harmonic code which, so far, has found avtd solutions with a scalar field present. cosmological models inspired by string theory contain higher derivative curvature terms and exotic matter fields. damour and henneaux have applied the bkl approach to such models and conclude that their approach to the singularity exhibits lmd. finally, there has been a study of the relationship between the long wavelength approximation and the bkl analyses by deruelle and langlois. numerical investigation of singularities provides an arena for the close coupling of analytic and numerical techniques. the references provided here contain many examples of analytic results guided by numerical results and numerical studies to demonstrate whether or not analytic conjectures are valid. while the search for naked singularities in the collapse of highly prolate systems has yielded controversial results, a naked singularity was discovered in the collapse of spherically symmetric scalar fields. the numerical exploration of cosmological singularities has yielded strong evidence that the asymptotic behavior is local each spatial point evolves toward the singularity as a separate universe. this area of research then should begin to overlap with the studies of black hole interiors (see for example) | this living review updates a previous version [25 ] which is itself an update of a review article [31 ]. numerical exploration of the properties of singularities could, in principle, yield detailed understanding of their nature in physically realistic cases. examples of numerical investigations into the formation of naked singularities, critical behavior in collapse, passage through the cauchy horizon, chaos of the mixmaster singularity, and singularities in spatially inhomogeneous cosmologies are discussed. |
figure 2a shows the low - energy raman spectrum of the gnrs in figure 1. in the case of 4cnr, one broad peak appears at 230 cm (full width at half - maximum, fwhm 100 cm), which comprises at least three contributions. for 6cnr, 8cnr, and m - anr, the most intense peak is located at 130150 cm and its width is sharp for m - anr (30 cm), whereas it is larger for 8cnr and 6cnr (80100 cm). furthermore, depending on the exact gnr structure, the peak disappears at certain excitation energies. for instance, the peak is not seen at energies below 1.96 ev for 4cnr, while for 8cnr it is detected at 1.96 ev. other peaks with small intensity are also seen in the spectral range 400700 cm, as shown in figure 2a. this is different from the case of cnts and armchair gnrs grown on substrate, where the low energy region is dominated by a sharp breathing mode with frequency uniquely determined by the cnt / gnr lateral size and described by the zone - folding (zf) approximation. (a) acoustic and (b) optical region of the raman spectrum for the cove - shaped gnrs in figure 1. the 4cnr and 6cnr were excited at 2.4 ev, while 8cnr and m - anr were excited at 1.9 ev. (c) peak dispersion of 8cnr as a function of excitation energy for the g (top) and d peaks (middle), as well as for the rlbm (bottom). in order to gain further insights into the origin of the gnrs raman peaks at low energy, we have computed from first principles the raman spectra of several gnrs (see methods) by increasing the structure complexity in a stepwise manner. we first consider perfect zz - gnrs, corresponding to the cores of the cove - shaped ribbons studied here (indicated by the shaded areas in figure 1a c). finally, we compare the above systems with functionalized ones, as in the real samples, though simulated by using shorter alkyl chains (c4h9 instead of c12h25) to make the calculations more affordable. figure 3a shows the simulated raman spectrum of the 8cnr, while the spectra of the other gnrs are reported in supporting information. let us first focus on the h - passivated 8cnr, hereafter labeled 8cnr+h. the spectrum is characterized by a dominant peak at 183 cm, which falls in - between the rlbms of the zz - gnrs corresponding to the minimum and maximum width of this cove - shaped gnr, that is, 209 and 164 cm for the 8- and 10-zz gnrs, respectively. this behavior, common to all the gnrs studied here, can be understood in terms of an effective width model. indeed, these cove - shaped gnrs present a modulated structure with variable width (e.g., 4cnr core width ranges from 0.69 to 1.13 nm). however, we can define an effective width as the weighted average of the different gnr widths (see supporting information) and compare our first principles simulations with the results obtained by zf. figure 3b shows that the agreement is within less than 10 cm for gnrs wider than 15, as expected for this approximation. besides the rlbm, there are higher frequency modes (470680 cm), combining longitudinal and transverse components. these modes, not present in zz - gnrs, appear in cove - shaped gnrs in view of the different periodicity along the ribbon axis introduced by the additional benzo rings at the edge. (a) acoustic and (c) optical region of the raman spectrum of 8cnr, as resulting from ab initio dfpt simulations. the spectrum is shown for both hydrogen - terminated (+ h, green) and functionalized 8cnr (+ c4h9, dark green). the dashed lines indicate the position of the rlbm for 8- and 10-zgnrs (labeled 8-zz and 10-zz, respectively, light green) and the position of the g peak for 8-zgnr. (b) the frequency of the rlbm calculated from first principles for several h - passivated cove - shaped gnrs is compared to the result of the zf approximation (black curve) as a function of the gnr effective width. (d) the low - energy spectral region of the functionalized 8cnr is shown as a function of the chain length and compared with experimental data. the dashed line indicates the frequency of the rlbm for the h - terminated system. for convenience, we next functionalize the edges with short alkyl chains (8cnr + c4h9). we observe a further red - shift of the rlbm peak, which moves from 183 to 156 cm, close to that of the larger zz component of the 8cnr, that is, 10-zz gnr, even though the origin of the shift is rather different, as clarified by our analysis. the breathing does not involve the ribbon only but also part of the chain that moves in phase with the ribbon atoms. this causes a redshift as a result of an increase in the effective width. we further check the effect of the chains by varying the chain length, as shown in figure 3d. in addition to the effect explained above with a redshift that depends on the chain length, we find that, depending on both the chain length and the gnr width, the coupling with the chain modes can give rise to different subpeaks (see, e.g., the case of c8h17, figure 3d). the relaxation of the system symmetry allows for both a mixing of longitudinal (l), transverse (t), and normal (z) modes and the activation of modes otherwise forbidden. all of this can explain the broadening of the low - frequency peak observed experimentally, which is indeed different for gnrs of different width. figure 2b shows the first order raman spectrum of the ribbons measured at 2.41 ev with d and g peaks typical of c sp materials. however, the g peak, which corresponds to the high frequency e2 g phonon at, is up - shifted (1605 cm) and broader if compared to pristine graphene (fwhm 25 cm). similar results were observed in small graphite domains and pah due to the relaxation of the momentum conservation induced by finite size. moreover, we do not observe any splitting of the g peak, as usually found instead for cnts, where this mode, which is doubly degenerate in graphene, splits into a longitudinal - optical (lo) and a transverse - optical (to) component. our first principles simulations show that the g peak, located at 1618 cm for the 8cnr (figure 3c), is mainly due to the to mode with smaller contributions due to to overtones (within 10 cm) ; the lo mode is instead inactive in backscattering, as expected in purely zz - gnrs. the presence of the side chains does not alter significantly the nature and position of the main features in the high - energy region of the spectrum, mostly dictated by the edge morphology, as seen by comparing the curves in figure 3c. note however that a different position of the functional chain along the gnr edge, as in the case of 6cnr, can induce a distortion of the gnr backbone, thus influencing the peak position (see simulated spectra of 6cnr in supporting information). figure 2b also shows a prominent d peak that is characterized by a dominant component at about 13101330 cm with an intensity comparable to that of the g peak and by one or more shoulders at lower wavenumbers. the raman shifts of these features are in agreement with our first principles simulations, which show a structured peak in this region with two main components (at 1336 and 1357 cm for the 8cnr, figure 3c) corresponding to the breathing modes of six - atom rings. this behavior makes the spectrum resemble that of defective graphene. however, there are notable differences that allow us to clearly distinguish our gnrs from defective graphene. the most striking one is found by analyzing the energy dependence of the d peak. in graphene, the d peak, coming from to phonons around the brillouin zone (bz) edge k, is activated by an intervalley double resonance process in the presence of defects, and it is strongly dispersive with excitation energy due to a kohn anomaly at k. the typical d peak dispersion of graphene is 50 cm / ev. in the case of gnrs, we measure different d - peak dispersions for the different gnrs (see supporting information). in particular, due to its low band gap (1.2 ev) the 8cnr allows performing raman spectroscopy in a wide range of energies (from 1.3 to 2.54 ev) without any photoluminescence background hiding the raman peaks (figure 2c). if we fit the dispersion with two slopes, we get about 7 and 35 cm / ev for the low (1.8 ev) excitation energy region, respectively. a considerable dispersion is found also for the rlbm (figure 2c, bottom panel), while the g peak shows a very small dispersion at the limit of the spectrometer resolution (figure 2c, top panel). in addition to this, we observe the g+d combination mode and the 2 g mode, which are not observed in defective graphene. a systematic comparison between defective graphene and our gnrs is reported in the supporting information. to fully understand these experimental observations, one needs to consider that several factors come into play when we move from graphene to narrow gnrs. first, we have neither kohn anomalies (see calculated phonon dispersion in supporting information) nor linear electronic dispersion in such gnrs, which have semiconducting character, with an optical response dominated by excitonic effects, especially for excitation energies close to the optical gap. second, the k point folds onto in these cove - shaped gnrs ; as such, we do not need a double - resonance process to activate this mode but just a first - order process like in armchair gnrs, where the d peak is also observed. for the same reason, we are also able to observe the g+d mode. in addition, one would expect a nondispersive behavior for the d peak, similar to the g peak in graphene. the observed dispersion is thus related to some disorder - induced scattering, for example, due to edge functionalization, defects formed during the gnr production, or length distribution. in conclusion, we combined an experimental and theoretical analysis of the raman spectra of ultranarrow and structurally well - defined graphene nanoribbons with cove - type edges. the low - frequency spectral region contains the main fingerprints of these materials. by analyzing the differences with respect to other systems, such as an ideal zzgnr and the same gnr without lateral chains (h passivated), ab initio simulations show that the number of raman peaks and their position are crucially affected by edge modifications. the full description of cove - type edge and alkyl chains is fundamental to get an agreement with experiments because both contribute to the shift and splitting of the peaks as well as to a redistribution of the raman intensity. the rlbm is especially sensitive, not simply to the width, but also to the edge modulation and functionalization, making it very different from the ideal cases studied to date, where the rlbm does not show significant dependence on edge type. the high - energy spectral region appears similar to that of defective graphene with d and g peaks of comparable intensity. however, the presence of the d+g combination mode and the different d - peak dispersion allow us to clearly distinguish these gnrs from defected graphene and other graphitic materials. details on the preparation of 4cnr, 8cnr, and m - anr are described in refs (2830). raman measurements are performed with a combination of different spectrometers (witec confocal spectrometer, renishaw invia, and tk64000 by horiba). a 100 objective is used and the power on the sample is below 0.1 mw to avoid damage. the raman spectra have been measured with at least three accumulations and in at least three different points on the same sample. measurements were repeated with different spectrometers in different laboratories in different countries to avoid any experimental artifact. the samples are measured as powder in resonance condition [the optical gap is 1.9, 1.2, and 1.12 ev for 4-cnr, 8-cnr, and m - anr, respectively, whereas for 6cnr it is 1.8 ev ]. simulations are performed using a first principles plane - wave pseudopotential implementation of density functional theory and density functional perturbation theory (dfpt), as available in the quantum espresso package. raman intensities are calculated within the placzek approximation (non - resonant condition) by using the second - order response method in ref (50). this approach is known to give accurate raman shifts, whereas the relative intensity of the peaks can not be directly compared with resonance raman experiments. norm - conserving pseudopotentials are employed with a plane - wave cutoff energy of 70 ry. a vacuum region of 12 in the nonperiodic directions is introduced to prevent interaction between periodic images. the atomic positions are fully relaxed until forces are less than 5 10 au. phonon frequencies and raman tensor are calculated using a 16 1 1 k - point grid. details on the preparation of 4cnr, 8cnr, and m - anr are described in refs (2830). raman measurements are performed with a combination of different spectrometers (witec confocal spectrometer, renishaw invia, and tk64000 by horiba). a 100 objective is used and the power on the sample is below 0.1 mw to avoid damage. the raman spectra have been measured with at least three accumulations and in at least three different points on the same sample. measurements were repeated with different spectrometers in different laboratories in different countries to avoid any experimental artifact. the samples are measured as powder in resonance condition [the optical gap is 1.9, 1.2, and 1.12 ev for 4-cnr, 8-cnr, and m - anr, respectively, whereas for 6cnr it is 1.8 ev ]. simulations are performed using a first principles plane - wave pseudopotential implementation of density functional theory and density functional perturbation theory (dfpt), as available in the quantum espresso package. raman intensities are calculated within the placzek approximation (non - resonant condition) by using the second - order response method in ref (50). this approach is known to give accurate raman shifts, whereas the relative intensity of the peaks can not be directly compared with resonance raman experiments. norm - conserving pseudopotentials are employed with a plane - wave cutoff energy of 70 ry. a vacuum region of 12 in the nonperiodic directions is introduced to prevent interaction between periodic images. the atomic positions are fully relaxed until forces are less than 5 10 au. phonon frequencies and raman tensor are calculated using a 16 1 1 k - point grid. | bottom - up approaches allow the production of ultranarrow and atomically precise graphene nanoribbons (gnrs) with electronic and optical properties controlled by the specific atomic structure. combining raman spectroscopy and ab initio simulations, we show that gnr width, edge geometry, and functional groups all influence their raman spectra. the low - energy spectral region below 1000 cm1 is particularly sensitive to edge morphology and functionalization, while the d peak dispersion can be used to uniquely fingerprint the presence of gnrs and differentiates them from other sp2 carbon nanostructures. |
the world is facing an increasingly aging population which is presently placing heavy demand on health care services, and this continues into the future. there are growing expectations that e - health will be the solution for these demands. e - health refers to tools and services using information and communication technologies (icts) that can improve prevention, diagnosis, treatment, monitoring, and management and can benefit the entire community by improving access to care and quality of care and by making the health sector more efficient. the goal is to make e - health both more user - friendly and thus more widely accepted by involving patients in strategy, design, and implementation, as well as supporting the general increase in quality of life [2, 3 ]. european countries, such as norway, denmark, germany, greece, and portugal, show steady development in using the internet as a source for health information. in sweden, it is possible for citizens nationwide to use web - based e - health services offered by swedish public health care providers to receive general e - health information online, receive personalized web - based e - health services (e.g., online e - prescription renewal), ask their doctors questions online, obtain medical devices, and reschedule doctor appointments [6, 7 ]. the next generation of e - health systems is mobile health applications that are considered as the strongest contribution for the next generation e - health systems. these applications act closely with an individual and focus on serving the needs of the user by providing widespread access to relevant information and/or remote data capture, thus eliminating the need for the user to be physically linked to a network or restricted to a specific geographic location. e - health has been recommended for supporting the health conditions of older adults. for older adults, health is closely linked to aspects of quality of life, such as psychological well - being, independence, mobility, safety, and social involvement [1013 ]. in turn, these aspects are impacted by personal and environmental factors that are equated with psychosocial factors that are often challenged by health conditions which usually worsen with age. several self - reported evaluations of e - health services from the perspective of patients show a low effect on health related quality of life and psychological outcomes. clearly, investigating what impact e - health has on psychosocial issues among older adults matters to understanding how e - health can be supportive or counteractive to the health of older adults. in this sense, the full use and adoption of technology are related to its role, perceived usefulness, and meaning in an individualized context. benefits and lack of benefits affect the motivation for the introduction of new technology innovation among older adults. in a previous study, we found a low degree of e - health service use among older adults in sweden despite the extensive development of these services. the objective of this study was thus to investigate the anticipated psychosocial impact from present web - based - e - health services and future mobile health applications among older swedes. this study implemented a cross - sectional survey based on two scenarios, the psychosocial impact of assistive devices (piads) questionnaire and also background questions. the survey was distributed by post, and respondents answered the survey during telephone interviews or filled them in at home and returned by post. the study was approved by the regional ethical review board in ume, sweden [ref. a total of 650 individuals aged 55105 were randomly selected from the official identity and address registry for swedish residents. as shown in the flowchart of the inclusion process (figure 1), a total of 154 persons responded to the survey, 368 declined, and 128 could not be reached. the age range for those who participated in the study was 55 to 91 years. a sample failure analysis showed that respondents differed from nonrespondents regarding age (mean respondent age was 71.9, while mean nonrespondent age was 74.1) (p = 0.010) but not with regard to gender (p = 0.407) the two illustrated scenarios of this study 's survey were based on focus group discussions from the research project myhealth@age (20082010) [21, 22 ] developed with a researcher focusing on pervasive and mobile computing at the lule university of technology and ultimately, thereafter designed by a graphic designer. a scenario can be defined as a description of a possible set of events that might reasonably take place ; for our survey, scenarios focus on use, what people can do with a system, and the consequences for users. the present scenario illustrates existing web - based e - health services together with an explanatory text of how people are able to use web - based e - health services, if needed (see figure 2). the future scenario illustrates mobile health applications under development together with an explanatory text of how people in the future will be able to use mobile health applications, if needed (see figure 3). presently and within the next few years, you can / are able to, if necessary, renew your prescriptions and book appointments at health care centers on the internet, receive sms appointment reminders, receive advice from the online medical counseling service, contact health care staff by email, take your blood pressure, ecgs, and blood tests at home by yourself and send the results via the internet to health care centers, talk to health care staff about your test results using a web camera, receive advice on how you should exercise and what you should eat to maintain or attain good health. renew your prescriptions and book appointments at health care centers on the internet, receive sms appointment reminders, receive advice from the online medical counseling service, contact health care staff by email, take your blood pressure, ecgs, and blood tests at home by yourself and send the results via the internet to health care centers, talk to health care staff about your test results using a web camera, receive advice on how you should exercise and what you should eat to maintain or attain good health. in the future, you will be able to, if necessary, be in constant contact with a health professional via sensors that will alert your health care center if they detect any problems with a measured value, track your fitness improvement by measuring walking distance, pulse, and blood pressure automatically, recognize people and receive assistance in remembering their names with the help of special glasses, wear a personal safety alarm that can determine your exact position in the event of you fall outdoors and need assistance, use a walking stick that shows you the way, use sensors in your shoes to obtain better balance. be in constant contact with a health professional via sensors that will alert your health care center if they detect any problems with a measured value, track your fitness improvement by measuring walking distance, pulse, and blood pressure automatically, recognize people and receive assistance in remembering their names with the help of special glasses, wear a personal safety alarm that can determine your exact position in the event of you fall outdoors and need assistance, use a walking stick that shows you the way, use sensors in your shoes to obtain better balance. in responding to the illustrated scenarios (figures 2 and 3) the psychosocial impact of assistive devices scale (piads) questionnaire was used to measure the anticipated impact on psychosocial factors of present web - based e - health services and future mobile health applications. the piads measures aspects related to quality of life that refer both to the person and the environment. the scale is designed to assess the experienced or anticipated impact of assistive technological devices before using them, thus anticipating the successful use or rejection assistive technologies. piads has good internal consistency as well as strong construct and predictive validity [2527 ]. the questionnaire can be administered individually, in a group, or via a telephone interview. it consists of 26 items based on the user 's description of how devices impact quality of life. each item is rated on a scale ranging from 3 (i.e., maximum negative impact) to + 3 (i.e., maximum positive impact). ratings are presented as three separate subscores that describe user perceptions along three dimensions (i.e., competence, adaptability, and self - esteem), as well as a total score. the competence dimension is evaluated by questions concerning topics such as competence, productivity, usefulness, performance, and independence. the adaptability dimension is evaluated by questions concerning topics such as the ability to participate, willingness to make changes, eagerness to try new things, and ability to take advantage of opportunities. the self - esteem dimension is evaluated by questions concerning topics such as self - esteem, security, sense of power and control, and self - confidence. the survey 's background questions asked for respondents ' age, gender, marital or cohabitation status, education level, income, self - rated health status according to a visual analog scale ranging from 0 to 100 and derived from the eq-5d, and experience using ict applications during a one - year period. other questions inquired about use of the internet for obtaining information about health, physical activity, and diet, while other questions asked respondents to report their experience using ict for health care services (see table 1). a pretest, with three older adults who responded to the survey and provided information including an information letter, background questions, and scenarios with piads, was conducted. the pretest clarified the importance of focusing on anticipated events as opposed to experiences with web - based e - health services at the present and mobile health applications in the future when rating piads. a logbook was used by the telephone interviewers (i.e., the first author and an assistant) to collect spontaneous comments and feedback from respondents. the survey was distributed by post to 650 randomly selected individuals in batches of 100. this process was repeated until all 650 surveys were distributed. upon a recipient 's consent to participate, a telephone interview was immediately conducted. respondents were then asked to study the illustration of the web - based e - health services in the present scenario and read the written explanation in order to rate their anticipated psychosocial impact concerning the 26 items in the piads. the same procedure was repeated for the future scenario illustrating mobile health applications. during the telephone interviews, recipients who declined to participate were asked to provide data regarding their gender and age. recipients who could not be reached after five attempts by telephone were mailed a survey including a glossary to help them interpret the piads items (cf.) and asked to complete and return the survey to researchers using prepaid, preaddressed envelopes. parametric analyses were made for the variables age and self - rated health because they were ratio variables and normally distributed. all other variables were analyzed nonparametrically, as they were at either nominal or ordinal levels, or, as in the case of the piads scores, presented a skewed distribution. for analyses of the statistical inference of differences between two groups, a student t - test was used for the parametric dependent variables, a mann - whitney u - test for ordinal variables, and the -test for nominal variables (see the description of sampling failure analysis on page 5 and tables 3 and 4). when analyzing the differences in piads scores between the present and future scenarios (see table 2), the wilcoxon signed - rank test was used because of its dependent measurements. correlations between piads total scores and parametric and ordinal scale variables were analyzed by the nonparametric spearman 's rank correlation (see tables 3 and 4). all analyses followed standard procedures and considerations, and the significance level was set at 5%. as shown in table 2, data indicated that respondents anticipated positive psychosocial impacts for using web - based e - health services and mobile health applications regarding both present and future scenarios. only 19 respondents (12%) reported negative total piads scores for the present scenario and 14 (9%) for the future scenario (data not shown). more importantly, both total piads score and sub - scores were significantly higher for the future scenario than those for the present. among all scores, age was significantly related to the total piads scores regarding both web - based e - health service and mobile health application scenarios (see tables 3 and 4), while gender, marital or cohabitation status, income, and educational levels were not. general ict experience (mobile phone, sms, computer use, email, and internet use) was consistently and significantly associated with the psychosocial impact anticipated from the present scenario, but less consistently for the future scenario. at the same time, self - related health was associated with the anticipated psychosocial impact for the future scenario but not the present. experience with health related ict (sms, email, web, chat, blog, and audiovideo communication) was not shown to be associated with the anticipated psychosocial outcome of either scenario except regarding using the internet to retrieve health information in the present scenario. for both scenarios, a pattern of positive anticipation for the psychosocial impact of using web - based e - health services and mobile health applications is clear. mobile health applications resulted in a higher anticipated psychosocial impact when compared to web - based e - health services. contrary to our expectations, respondents did not rate more highly the anticipated psychosocial impact of the present scenario despite being familiar with its description of the services currently available in sweden [6, 30, 31 ]. on the whole, it seems that older adults may find meaning from the perspective of the usefulness of mobile health applications. the future scenario could be interpreted to suggest situations of independence and sociability for the mobile health application illustrate the transition of e - health services towards mobility and self - management. adaptability received the highest piads sub - score for both scenarios, particularly for mobile health applications in future scenario. such a result suggests that these tools are more related to the environment in the sense that mobile health applications can serve as adapters that liberate users and enable them to pursue activities in daily life. independence was found to be imperative among older adults in order to avoid social exclusion and is, in the sense of control and choice, of great importance when older adults use e - health services. in one study, older adults with functional limitations reported that they were afraid of losing control and that control was strongly connected to feelings of independence. from the logbook, we read the following comments about scenario representing mobile health applications. it would be fantastic if i could get that kind of help if i needed it ; do you really mean that this is being developed ?, and yes, it would be helpful. this scenario painted respondents a picture of the technology being adapted to users instead of users being adapted to the technology and was perceived to be well integrated and will allow users autonomy without feeling intruded upon (cf. these results could in turn reduce stigmatization (cf. [9, 38 ]). mobile applications thus seem to be an important service that promotes mobility, which in turn positively impacts the quality of life of older adults. the correlation between increased age and lower anticipated psychosocial impact may be expected, as previous research has shown a lower usage of ict and a decreased interest in ict communication with health care services at very old ages [31, 40 ]. the decreased psychosocial anticipations with increased age may be explained by the fact that the oldest adults perceive technical devices to be uninteresting because of low self - efficacy in relation to ict use. results also showed a connection between general ict use and the anticipation of web - based e - health services in the present scenario, as well as a connection between the use of sms messaging and the internet with the anticipation of mobile health applications in the future scenario. this result may confirm that web - based e - health services to some extent possess an image recognition factor similar to general usage. the mobile health applications in the future scenario, sms messaging, and the internet may be considered by the respondents to be more advanced, thus making the connection somewhat obvious. internal validity was strengthened by survey pretests, low internal data losses, a glossary explaining the piads items, and a standardized procedure during interviews. a few days after the first delivery of 100 surveys, all authors contacted five individuals by telephone for a total of 20 older adults in order to investigate the possibility that recipients needed clarification on the survey and interview procedures. in the area of e - health, there are, to our knowledge, no instruments for evaluating psychosocial outcomes. we had to search in comparable areas and for assistive technology, there are a plethora of scales for assessing the impact and outcomes of devices. piads was developed and used in the context of assistive technology as wheelchairs, hearing aids, and so forth [4244 ]. quebec user evaluation of satisfaction with assistive technology (quest 2.0) assesses users ' satisfaction of a device and assumes that an experience takes place with the device and that was not the case in our study. another one is matching person & technology (mpt), an instrument that helps professionals together with the consumer, based on the person 's goals, identify technologies which are desired and needed but not yet available. we chose to use piads as an instrument because the design of this instrument fits our purpose, to evaluate the anticipated psychosocial impact of the e - health scenarios described. e - health means tools and services using information and communication technologies to assist the user, with it being important to measure the anticipated impact that these tools and services will have on the lives of older users and their environment. this study marks the first time that this instrument has been used to investigate the anticipated psychosocial impact of e - health services. we consider piads to be a valid instrument for this purpose, as it has been used to evaluate other technologies that support personal health [4244 ]. we attempted to reduce the rate of nonresponses by making several telephone calls to recipients using a two - mode strategy (i.e., postal delivery and telephone interviews). doing so may have positively affected the response rates. at the same time, it is possible that the number of questions negatively affected response rates. other negative factors may include a lower interest level in the survey issues, health issues, and surveys in general. i am too old to be answering this ; i am not interested ; and i am too sick. other nonrespondents found the questions to be excessive in number or too difficult to answer. prestudy sample size calculations showed that in order to detect differences in piads scores between two unequally sized groups corresponding to at least moderate effect size, considering a significance level of 0.05 and a statistical power of 0.80, at least 319 respondents would be necessary. considering an expected nonresponse rate of 50%, we therefore asked 650 persons to participate. despite serious efforts to the contrary, the nonresponse rate was ultimately higher than expected (74%), which is an external validity threat for implying a potential nonresponse bias. this result also highlights possible type - ii errors regarding p values bordering on statistical significance (see, e.g., table 4). first, the sample was derived from random selections of the official swedish population registry. second, we have reason to believe that the nonresponse bias may not have been too critical. our data did not indicate large selection bias concerning gender and age, and the general ict experience in our sample did not differ substantially from previously published results. although high response rates are preferred to ensure sample - to - population representativeness, empirical data seem to show that low response rates are not necessarily connected to large bias. third, as the piads scores were consistently positive to a high degree, we could expect that a possible overestimation resulting from sampling bias is less likely to distort the overall picture. we found that the anticipated psychosocial impact was positive for web - based e - health applications in the present scenario and also for mobile health applications in the future scenario but was negatively correlated to an increase in age. such findings may be interpreted to be especially interesting and unique since they concern an entire population of older adults and are not limited to specific diagnostic groups or samples participating in ict trials. by contrast, our findings indicate that in a population of older people, e - health is likely to positively impact quality of life from a psychosocial perspective. considering these aspects can serve as an important contribution to facilitating technology diffusion in health care among older adults. in the future, we can expect a continued increase in general ict experiences among older adults, including the oldest, which will perhaps decrease the effects of advanced age on the anticipated impact. as long as differences persist, however, we must acknowledge the possible digital divide when implementing e - health. for the oldest members of the population, it is important to investigate their needs from a psychosocial perspective in order to tailor health care services that are meaningful in their particular context. | this study investigates the anticipated psychosocial impact of present web - based e - health services and future mobile health applications among older swedes. random sample 's of swedish citizens aged 55 years old and older were given a survey containing two different e - health scenarios which respondents rated according to their anticipated psychosocial impact by means of the piads instrument. results consistently demonstrated the positive anticipation of psychosocial impacts for both scenarios. the future mobile health applications scored more positively than the present web - based e - health services. an increase in age correlated positively to lower impact scores. these findings indicate that from a psychosocial perspective, web - based e - health services and mobile health applications are likely to positively impact quality of life. this knowledge can be helpful when tailoring and implementing e - health services that are directed to older people. |
neuro - ophthalmic emergencies such as giant cell arteritis, orbital apex syndrome (oas), cavernous sinus thrombosis, intracranial aneurysm and pituitary apoplexy require urgent recognition and management due to their vision - threatening and potentially life - threatening nature. patients suffering with oas have complete ophthalmoplegia, ptosis, decreased corneal sensation and visual loss (1). this serious ocular complication may be the result of paranasal sinus diseases and especially disease in the posterior ethmoid and sphenoid sinuses (2). early intervention and careful selection of the optimal therapeutic modalities must be considered according to the underlying causes and the extent of disease. we report here on a case of a diabetic patient who presented with acute visual loss and ophthalmoplegia that were complicated by sphenoid fungal balls. a 64-yr - old man presented to the emergency room with right visual loss after a 5-day history of rhinorrhea, diplopia and severe headache. the initial blood sugar was measured and it was too high to register by a blood glucose test strip and it was decreased to 485 mg / dl after an iv infusion of normal saline. his visual acuity in the right eye was counting fingers (50 cm) and it was 20/40 in the left eye. endoscopic examination detected edematous mucosa, brown - colored fungal balls and there was a mucopurulent discharge from the sphenoethmoidal recess. most of the orbital lesions were located at the retrobulbar area and the orbital apex, and inferior and medial rectus muscles were thickened. even though there was bony erosion of the posterior wall of the sphenoid sinus, there was no evidence of intracranial extension. increased signal intensities therefore, we immediately started systemic amphotericin b (50 mg / day for 21 days and 25 mg / day for 10 days) and insulin therapy to control his blood sugar. the patient was also started on intravenous ampicillin / sulbactam (6.0 g / day) and ceftriaxone sodium (2.0 g / day) to cover any possible bacterial infection. the patient elected to undergo endoscopic sinus surgery under local anesthesia on the 3rd hospital day. the sphenoid sinus was filled with thick mucus and brown - colored matted fungal balls. the mucosa around the ostium and the fungal materials were sent to the department of pathology for the determination of invasiveness. by the 4th postoperative day (pod), there was no improvement and the visual acuity decreased from finger count to light perception. the patient complained of severe headache, nausea and vomiting ; the follow - up computed tomography and cerebrospinal fluid (csf) study showed no evidence of meningitis or intracranial invasion. therefore, empirical corticosteroid therapy with prednisolone (pds, 55 mg for 7 days, 40 mg for 7 days, and 20 mg for 7 days) was started on the 9th pod. after the 3rd day of pds therapy, his headache and ophthalmic symptoms began to improve. with the exception of the ophthalmoplegia, the ptotic upper eyelid completely recovered and the visual acuity improved to 20/100 by the day of discharge (the 28th pod). the patient 's blood sugar was not well controlled with fluctuations being noted during the hospitalization. by 3 months, the right visual acuity improved to 20/63 and the extraocular muscle movement was completely normalized. a fungal ball is defined as the non - invasive accumulation of dense fungal hyphae in the sinus cavity (3). typically, this occurs in immunocompetent patients ; however, if immunodeficiency develops, then this fungal infection may become invasive (4). the treatment for invasive fungal sinusitis whether acute or chronic, is first reversing the source the patient 's immunocompromised status, if present, followed by systemic antifungal therapy and surgical debridement (3). because of the rare incidence of oas that 's caused by fungal infection, there has been no consensus on the proper therapeutic strategy when there has been no response to the aforementioned therapy. for this current case, the initial pathologic report was aspergillosis. with knowing that the fungus was non - invasiveness, an empirical trial of prednisolone (pds) therapy was started due to the aggravated symptoms and the lack of a response to both amphotericin b and surgical drainage. however, a detailed review of the slides that was done by a pathologist after patient 's discharge showed evidence of minimal tissue invasion (fig. 2), and we were quite surprised by this. therefore, we believed that the invasive fungi was eliminated by amphotericin b and surgery, so the pds therapy that was administered later did n't aggravate the fungal infection, but it had a critical role for controlling the orbital apex lesions, which may have been a secondary inflammatory reaction. accurate assessment of the orbital apex pathology is very important when choosing the therapeutic modalities, but this is sometimes very difficult in clinical practice. although we did not perform orbital apex biopsy, we considered the orbital apex lesion to be non - invasive disease for the following reasons : 1) the lack of response to amphotericin b and surgical drainage, and 2) the dramatic improvement after systemic corticosteroid treatment. we believe that previous antifungal and surgical treatments might reverse the becoming invasive to non - invasive form of fungal balls. the prognosis of oas secondary to fungal infection is poor, and neurological sequelae and fatal outcomes have been reported in the majority of the cases (3, 5 - 8). when reviewing the case reports, the possible causes of a poor prognosis include a delayed presentation, preexisting lateral sphenoid bony dehiscence, advanced tissue invasion, misdiagnosis and the inappropriate use of systemic corticosteroids. there was histopathological evidence of minimal tissue invasion in this case, and if treatment had been delayed, then fulminate invasion may have occurred. systemic corticosteroids at that stage are absolutely contraindicated. in conclusion, the changing characteristics and clinical behavior of fungal infection should be closely monitored to select the optimal therapeutic modalities. a future study on the proper clinical tools to identify a residual fungal infection after treatment is needed to help save the vision and often the life of patients who suffer with oas secondary to fungal infection. | orbital apex syndrome (oas) is a rare disease that presents with a complex of symptoms, including ophthalmoplegia, ptosis and visual loss. due to the poor prognosis, making a prompt diagnosis and administering the appropriate treatment must be initiated without delay if oas is suspected. we report here on a case of a patient with sphenoid fungal balls, and he presented with acute visual loss and ophthalmoplegia. |
chronic renal failure is a syndrome characterized by inability of the kidneys to perform adequately, owing to the progressive loss of function over a period of months to years. a variety of adverse influences (e.g., toxins, overdosed drugs, infectious agents, ischaemic insults, neoplasia) can damage the kidneys, either reversibly or irreversibly, to produce renal disease. however, in many cases, a specific underlying cause can not be identified. approximately, 75% or more diminution in functional renal mass results in compromised excretory function to the extent that a state of azotaemia develops [1, 2 ]. however, neither the magnitude of azotaemia alone can be used to determine whether the azotaemia is prerenal, renal, or postrenal nor it can be used to determine whether the disease process is acute or chronic, reversible or irreversible, and progressive or nonprogressive. the diagnostic approach in these renal affections is to localize the azotaemia. fortunately, prerenal azotaemia can be distinguished from acute intrinsic renal failure in many patients simply by providing generous vigorous efforts are required to identify the underlying cause of any active renal disease which may contribute towards progression to chronic renal failure and thus for therapeutics of treatable causes. a diagnosis of crf is usually based on a combination of compatible history, physical examination, laboratory data, and imaging studies, which play a crucial role in differentiating acute from chronic renal failure. the minimum database for renal affection includes bun, creatinine, urinalysis, hb, pcv, total protein, albumin, and electrolyte assay (especially potassium, sodium, total calcium, inorganic phosphate). contrast radiographic studies, although, help to identify or rule out potentially treatable causes of crf, but it is laborious and associated with the risk of radiographic contrast agent - induced nephropathy. ultrasonography, on the other hand is a noninvasive and quite useful for detection and characterization of renal parenchymal details. it is the technique of choice for reliable diagnosis of fluid - filled cystic lesions, renal mass lesions, hydronephrosis, and anatomic localization of uroliths (radiopaque and radiolucent). renal biopsy besides establishing definitive nephropathy offers an appropriate alternative to provide important diagnostic and prognostic information especially in cases when distinction between arf (reversible) and crf (irreversible) is not otherwise possible. once the crf is established, the goals of conservative medical management of crf are to control clinical signs of uraemia ; maintain adequate fluid, electrolyte, and acid / base balance ; provide adequate nutrition ; minimize progression of renal failure. however, because renal lesions that cause crf are irreversible and usually progressive, so no specific therapy is curative. 10 dogs (8 males, 2 female) aged 413 years, weighing between 10 kg and 26 kg, presented in teaching veterinary clinical complex, covas, palampur in the year 2007 - 2008 with primary indication of azotaemia were subjected to detailed clinical, haematobiochemical, urinalysis, and microbiological examination along with radiographical and ultrasonographical examination to narrow the differential diagnoses / establish the renal diagnosis. ultrasound - guided renal biopsy and histopathological examination for ascertaining definitive nephropathy although being recommended in many diseased dogs could not be undertaken because of poor owner compliance. the treatment regimen in progressive crf comprised of fluid therapy, wepox injection (human erythropoietin), anabolic steroid injection (nandrolone), whole blood transfusion, imferon injection (shreya), haemaccel injection (npil) and livadex injection (agrivet), ranitidine injection (zantec), antibacterials, syrup riconia (ranbaxy), gel mucaine (wyeth), and syrup geriforte (himalaya). haematological parameters included hb, pcv, tec, tlc, dlc, and biochemistry - comprised plasma alt, ast, bun, creatinine, total bilirubin, total protein, glucose, sodium, potassium, inorganic phosphate, and total calcium using standardized procedures. urine following transcutaneous cystocentesis was examined for presence of blood (rbc), protein, glucose, ketone bodies, ph, leucocytes (wbc), and specific gravity and colour by using uriscan 10 sgl strip in semiautomatic urine analyzer uroscan optima ii. besides, urine sediment microscopic examination was carried out for visualizing the presence of epithelial cells, crystals, rbcs, wbcs, casts, bacteria, and debris. radiographical examination was performed using 500 mas, 125 kvp x - ray machine whereas ultrasonography was carried out using a bpl ultrasound scanner (a gray scale, real time, b+m - mode scanner), with 3.5 mhz microconvex and 3.5 mhz curvilinear transducer. the selection of the transducer (scan head) was based on the transducer contact area and having the organ / region / lesion of interest within the focal zone of the transducer. based on the ultrasonographic structural characterization, the affections of kidney leading to chronic renal failure (crf) in majority of dogs were diagnosed in 10 clinical cases. end - stage kidneys (n = 4), hydronephrosis (n = 1), renomegaly (n = 1), nephritis (n = 1), nephrolithiasis (n = 1), nephrocalcinosis (n = 1), and renal cyst (n = 1). eight dogs so diagnosed with crf, however, died during the period of therapeutic stabilization that is, after 525 days of presentation. end - stage kidneys and associated crf were diagnosed in four dogs (3 males, 1 female) with mean age of 8.5 years (4.013.0 years) and average body weight of 14.25 kg (10.017.0 kg). the dogs were presented with history of inappetance / anorexia, vomition, progressive lethargy, and weight loss for last 3 days to 2 months. two dogs passed scanty and constipated faeces, whereas stool was loose in two other dogs. one dog which suffered with dental tarter and acute gingivitis had undergone ultrasonic dental scaling one - month before. physical examination revealed tucked - up abdomen in three dogs with markedly arched back in the female dog. conjunctival mucous membrane was icteric in two dogs, whereas it was pallor in other two dogs. glossitis with necrosis of distal portion of tongue and tongue protrusion was marked in one female dog (figure 1). neutrophilic leucocytosis was detected in one dog, whereas there was significant lymphocytosis with decreased total leucocyte count and total erythrocyte count in the female dog. biochemical profile (table 2) indicated severe azotemia in all the dogs, whereas hyperkalaemia and hyperphosphataemia were significant in three dogs. the dogs passed urine with 46 leucocytes and 24 rbcs / hpf, moderate crystalluria (oxalates and triple phosphate crystals) and with specific gravity in the range of 1.0071.012 (table 3). microbiological examination of the urine in one dog yielded streptococcus species with sensitivity to amikacin, gentamicin and nitrofurantoin. intravenous pyelogram was also obtained in one dog, but the nephrogram and pyelogram phase could not be visualized., the renal parenchyma could hardly be differentiated from the surrounding tissue (figures 2 and 3). all the four dogs were given treatment for progressive crf as mentioned but died after 1525 days of therapeutics. chronic renal failure secondary to hydronephrosis following urethrolithiasis was diagnosed in nine - year - old, intact male, gaddi cross dog, with body weight of 22 kg. the dog was presented with a three - day history of anorexia and inappetance and vomition for the last eleven days. the dog had urine retention with infrequent blood tinged urine dribbles for the last 4 days. he had acidotic odour from mouth with severe glossitis and necrosis of distal portions of tongue. on abdominal palpation, a large and firm bladder was noted. gentle aseptic urethral catheterization encountered resistance in pelvic urethra 2 cm (approximately) proximal to urethral opening. following survey radiograph, urethral obstruction was relieved by propulsion of the calculi into the urinary bladder by gently advancing urethral catheter. urinalysis revealed brownish red coloured urine with specific gravity of 1.030 (table 3). urine sediment examination revealed the microscopic field filled with rbcs, leukocytes, and epithelial cells with occasional epithelial casts and triple phosphates crystals (figure 4). on microbiological examination, no growth was observed. pneumocystogram (standing lateral), following urine removal, allowed visualization of two small radiopaque calculi that were pushed into the urinary bladder by the urethral catheter. sonograph demonstrated enlarged kidneys (left and right kidneys measuring 91 mm and 82 mm in length, resp.) with renal pelvic dilation, separation of the central renal sinus with anechoic space, and atrophy of renal medulla (figures 6 and 7). very classic bilateral hyperechoic medullary rim suggestive of nephropathy and moderately dilated ureters were also observed. imaging of the urinary bladder revealed hyperechoic and thickened ub wall with echogenic urine and blood clots. renomegaly with nephritis and associated crf were diagnosed in a two - year - old, intact male, german shepherd dog with body weight of 26 kg. physical examination revealed congested conjunctival mucous membrane and acidotic odour from mouth. on abdominal palpation, enlarged kidneys with mild lumbar pain were noted. the urine collected after transabdominal cystocentesis appeared grossly normal. the haematological profile (table 1) was unremarkable whereas plasma biochemistry (table 2) demonstrated severe azotaemia (bun = 183.6 mg / dl, creatinine = 17.5 mg / dl) and elevated total bilirubin. urinalysis revealed isosthenuria (urine - specific gravity : 1.012) with moderate glucosuria and proteinuria. abdominal lateral survey radiograph did not reveal any abnormality and kidneys could not be appreciated. sonograph demonstrated enlarged kidneys (left and right kidneys measuring 103 and 98 mm in length and 60 and 62 mm in width, resp.) with increased overall echogenicity and thickness of renal cortex. the medullary pyramids were also echogenic with poor corticomedullary definition (figures 8 and 9). the dog although was rendered treatment, it died after 16 days of presentation in the clinics. chronic nephritis and associated chronic renal failure (crf) was diagnosed in a 17-year - old, 6 kg body weight, intact female, pomeranian dog. the dog had history of vomition and inappetance for last three days with polydipsia and polyuria for last one week. mouth cavity examination revealed acidotic odour from mouth, dental tarter, and moderate gingivitis. urinalysis and urine sediment examination did not show any significant alteration except 2 - 3 wbcs and 24 rbcs / hpf with specific gravity of 1.020. ultrasonographic examination revealed bilateral markedly increased echogenicity of the renal cortex comparable with spleen (figures 10 and 11). crf following nephrolithiasis (bilateral) with associated nephritis and cystolithiasis was diagnosed in a 9-year - old, intact male, doberman pinscher dog with body weight of 18 kg. haemato - biochemical findings (tables 1 and 2) revealed mild neutrophilic leucocytosis and severe azotemia (bun-128 mg / dl, creatinine 5.4 mg / dl). increased alt and ast activity with hyperbilirubinemia was also noticed. urinalysis was unremarkable except 68 rbcs, and 46 wbcs / hpf, crystalluria and isosthenuria (1.013). abdominal lateral survey radiograph revealed two irregular radiopaque densities in the renal regions corresponding in shape with the renal pelvis (figure 12). ultrasonographic examination revealed bilateral large hyperechoic structures in the renal pelvis with intense distal acoustic shadowing and masking of the far calculi renal cortex (figure 13). besides, the hyperechoic medulla with loss of corticomedullary definition was also observed in both the kidneys. the dog, although on aggressive treatment, died after 7 days of presentation in the clinics. nephrocalcinosis was diagnosed in a six - year old, intact female, mixed breed dog with body weight of 16 kg. physical examination suggested pallor mucous membrane and deep septic wound on the lateral aspect of right forelimb. haemogram was unremarkable but the biochemical profile of the dog demonstrated mild azotemia and hypercalcaemia. ultrasonographic examination revealed diffuse, small, multiple hyperechoic structures in the renal parenchyma with distal acoustic shadowing (figure 13). the dog was rendered conservative medical treatment and recovered well after 7 days of treatment. two solitary cortical renal cysts were diagnosed in a 16-year - old (geriatric) male dog with body weight of 13 kg. the biochemical profile although in normal range had bun and creatinine values on higher side. ultrasonographic examination besides having hyperechoic medullary region revealed two small spherical structures in the right renal cortical region which contained anechoic fluid with distal acoustic enhancement (figure 14). the dog recovered well after conservative medical treatment, that is, fluid therapy, antibacterials, and inj. anorexia, progressive lethargy, and weight loss observed in dogs with end - stage kidneys may be attributed to accumulation of nitrogenous wastes, metabolic acidosis and changes in the gastrointestinal tract. vomition in such dogs results as the uraemic toxins act centrally to stimulate the chemoreceptor trigger zone, which, in turn, stimulate the vomiting center. polyuria and secondary polydipsia observed by the owners in their dogs occur when the renal disease has progressed severe enough to render 6675% of the nephrons nonfunctional thus compromising the urine concentrating ability. markedly arched back in the female dog and pain on abdominal palpation may be due to associated renal pathology. gingivitis, glossitis, and acidotic odour observed in all the dogs may result from alterations in oral flora caused by the local presence of uraemic toxins, which serve as substrate for ammoniagenesis in urease - producing bacteria. anaemia in all the dogs may be due to chronic renal disease which results from decreased production of erythropoietin and depressed erythrogenesis and/or shortened erythrocyte life span due to accumulation of uraemic toxins. renal failure results in the retention of uremic toxins and gastrin that damage the gastric mucosa and blood vessels of the gastric wall and result in increased acid production. anabolic steroids, nandrolone, have been administered to stimulate rbc production in anemic animals. decreased leukogram, characterized by lymphocytosis and nonregenerative anaemia in one dog, reflected the immunocompromised status of animal. severe azotemia observed in all the dogs is the result of compromised excretory function where more than 75% of the nephrons are rendered nonfunctional. hyperphosphatemia and hyperkalaemia observed in the present study in three dogs is the most consistent electrolyte disturbance in patients with renal failure. isosthenuria with significant azotaemia observed in the present study related with malfunctioning of more than 2/3rd of the nephrons [4, 5 ] and indicated intrinsic renal failure. survey radiographic visualization failure may be attributed to lack of retroperitoneal contrast due to emaciation as most of the animals were in poor body condition. the failure to visualize the nephrogram and pyelogram following intravenous pyelography in one dog showed the impaired renal excretory function. end - stage kidneys and associated chronic renal failure which is attributed to impaired glomerular filtration and tubular concentrating ability in chronic renal failure. contrast studies were avoided in other dogs because of poor body condition of the animals. the sonographic observations of decreased size of kidneys could be related to chronic renal failure, and the loss of architectural details has been a significant feature of renal diseases due to gradual loss of functional nephrons, over a few months to several years. complete loss of corticomedullary definition observed in the present study might be due to chronic interstitial nephritis which is a potential cause of end - stage kidneys with sonographic observations of small sized kidneys and loss of corticomedullary detail which makes their differentiation from surrounding tissue difficult. in the present study, clinicopathologic manifestation of renal disease and loss of corticomedullary definition on ultrasonographic examination diagnosed the end - stage kidneys, though the ultrasonographic features are not specific, and renal biopsy findings are confirmatory for the diagnosis of specific nephropathy. retrograde catheterization in this dog confirmed the urethral obstruction which subsequently led to the distention of the urinary bladder and hydronephrosis. the obvious uraemic signs particularly the acidotic odour, glossitis with necrosis of distal part of tongue, and severe azotaemia suggested the associated nephropathy. biochemical findings of marked azotaemia, hyperphosphatemia, and hyperkalaemia observed in the present study is a feature during oliguria and anuria of terminal crf. hypersthenuria with specific gravity of 1.030 in the present study might be a result of dehydration following uraemia. radiography provided information of enlarged kidneys and two small radiopaque calculi in pneumocystograph which otherwise of urethral origin were pushed into the urinary bladder. sonography demonstrated enlarged kidneys with renal pelvic dilation, separation of the central renal sinus with anechoic space, atrophy of renal medulla, and very classic bilateral hyperechoic medullary rim and moderately dilated ureters as the features of hydronephrosis. the sonographic features like hyperechoic medullary rim are suggestive of nephropathy and marked renal pelvic dilation with central anechoic space and atrophy of renal parenchyma following longstanding ureteral obstruction [10, 12 ] in hydronephrosis have been documented. hydronephrosis have also been sonographically categorized as narrowed renal parenchyma with its structures still discernible in mild hydronephrosis to more thin homogenous renal parenchyma in advanced hydronephrosis. according to this classification, ureteral obstruction due to calculi, ub tumour, ectopic ureter, and accidental ligation of ureter during ovariohysterectomy has commonly been documented as causes of hydronephrosis. however, in the present study, hydronephrosis was observed secondary to urethrolithiasis. ultrasonography in the present study proved handy in detecting the parenchymal alterations and avoided the use of more laborious, time consuming excretory urography in animal with poor body condition. renomegaly and associated nephritis leading to irreversible renal failure was diagnosed in a two - year - old, intact male, german shepherd dog. survey radiographic failure to visualize the kidneys may again be due to lack of retroperitoneal contrast due to emaciation [6, 14 ], as the dog had lost substantial body weight before presentation and had poor body status. sonograph demonstrated enlarged kidneys (left and right kidneys measuring 103 and 98 mm in length and 60 and 62 mm in width, resp.) with increased overall echogenicity and thickness of renal cortex. the maximum value of kidney length in dogs weighing between 25 and 29 kg has been reported to be 78 mm with average value of 69 mm. the observed renal length and width in the present study were substantially higher thus proving renomegaly with associated nephritis. the increased overall echogenicity particularly of renal medullary pyramids, thickness of renal cortex with poor corticomedullary definition as the sonographic features have also been reported in dogs affected with chronic nephritis. chronic nephritis and associated chronic renal failure (crf) were diagnosed in an intact female, pomerarian dog. ultrasonographic findings of bilateral markedly increased echogenicity of the renal cortex comparable with spleen and fairly defined corticomedullary junction observed in the present study have also been described in chronic nephritis as increased echogenicity of the renal cortex, rarely of the renal medulla ; with normal size in early stage to loss of corticomedullary definition with increased overall echogenicity and larger or smaller size in advanced stage. the distended gall bladder and hypoechoic hepatic parenchyma with occasional hyperechoic foci also suggested accompanied hepatitis in the affected dog. nephrolithiasis (bilateral) with associated nephritis and cystolithiasis was diagnosed in a 9-year - old, intact male, doberman pinscher dog. neutrophilic leucocytosis observed may be due the infectious nephritis as the nephroliths predisposes to bacterial infection by serving as a nidus or may be the result of inflammatory condition of liver as the liver panel was also altered. laboratory findings of severe azotaemia and isosthenuria (1.013) were in agreement with those reported by bush and may be attributed to nephrolith induced nephropathy. the irregular radiopaque densities in the renal regions corresponding to the shape of the renal pelvis observed in the present study have been referred as stag - horn calculi. ultrasonographic observations of highly reflective structures in the renal pelvis with intense distal acoustic shadowing and masking of the far calculi renal cortex with medullary compression have been well described [11, 17 ]. in the present study, however difficulty was encountered while imaging the highly reflective large calculi as it did not allow detecting the true echogenicity of the renal cortex and medulla, the extent of medullary compression and evaluation of most of the renal parenchyma. inappetance, vomition, anaemia, and mild azotemia observed in the present dog indicated although nonspecifically towards renal pathology. hypercalcaemia observed might be due merely to an increase in the amount of biologically inactive complexed calcium bound to phosphates, oxalates, and various other anions in their blood which are found in renal failure patients with hyperplastic parathyroid glands with altered set point for detection and response to blood ionized calcium. radiographic failure in visualization and ultrasonographic findings of diffuse, small, multiple hyperechoic structures in the renal parenchyma with distal acoustic shadowing in the present study have been well documented as focal or diffuse hyperechoic areas with or without distal acoustic shadowing which may be recognized ultrasonographically before being visualized radiographically. a fine hyperechoic medullary rim observed on the left kidney is a unique sonographic feature of nephropathy. the ultrasonographic characterization, strengthened by the haematobiochemical findings in the present study was suggestive of nephrocalcinosis. sonography in the present study diagnosed nephrocalcinosis in the early stage without specific laboratory indication and radiographic appreciation. two solitary cortical renal cysts were diagnosed in a 16-year - old (geriatric) male dog. ultrasonographic findings of two small spherical structures in the right renal cortical region which contained anechoic fluid with distal acoustic enhancement have been well described. the presence of solitary or multiple renal cysts without clinical symptoms are usually of congenital origin and in most cases are incidental findings while carrying out ultrasonographic examination. in conclusion, the affections of kidney, leading to chronic renal failure in majority of dogs were diagnosed in 10 clinical cases. end - stage kidneys (n = 4), hydronephrosis (n = 1), renomegaly (n = 1), nephritis (n = 1), nephrolithiasis (n = 1), nephrocalcinosis (n = 1), and renal cyst (n = 1). the significant ultrasonographic features included small kidneys with loss of corticomedullary demarcation making its differentiation from surrounding tissue difficult (end - stage kidneys) ; increased cortical echogenicity and/or increased overall renal echogenicity (nephritis) ; dilation of the renal pelvis, separation of the central renal sinus with anechoic space, atrophy of renal medulla, and very classic bilateral hyperechoic medullary rim (hydronephrosis) ; enlarged kidneys with increased overall echogenicity and thickness of renal cortex (renomegaly and associated nephritis) ; hyperechoic - mineralized structure with masking of far calculi renal cortex (nephrolithiasis) ; diffuse, small, multiple hyperechoic structures in the renal parenchyma with distal acoustic shadowing (nephrocalcinosis) and small spherical structures in the renal cortical region containing anechoic fluid with distal acoustic enhancement (renal cysts). in the present study, ultrasound proved to be a quick, convenient and sensitive modality in detecting alterations in renal size and parenchymal architecture. thus, it overcame many of the shortcomings of abdominal palpation and eliminated the need to perform the more laborious excretory urography and renal biopsy which are generally unnecessary in patients with well - defined historical, clinical, laboratory, and ultrasonographic evidence of renal failure. | the present study comprises of 10 dogs of either sex with primary indication of azotaemia. all the dogs were subjected to detailed clinical, haematobiochemical, urinalysis, and microbiological examination along with radiographical and ultrasonographical examination. based on the ultrasonographic structural abnormalities, the different renal affections associated with crf in majority of dogs were diagnosed. the different affections included end - stage kidneys (n = 4), hydronephrosis (n = 1), renomegaly (n = 1), nephritis (n = 1), nephrolithiasis (n = 1), nephrocalcinosis (n = 1), and renal cyst (n = 1). the significant ultrasonographic features in these affections included small kidneys with loss of corticomedullary demarcation (end - stage kidneys) ; increased cortical echogenicity (nephritis) ; dilation of the renal pelvis, separation of the central renal sinus with anechoic space, atrophy of renal medulla, (hydronephrosis) ; enlarged kidneys with increased overall echogenicity of renal cortex (renomegaly and associated nephritis) ; hyperechoic - mineralized structure with shadowing (nephrolithiasis) ; diffuse, small, multiple hyperechoic structures in the renal parenchyma with distal acoustic shadowing (nephrocalcinosis) ; small spherical intercortical anechoic structures fluid (renal cysts). in the present study, ultrasound proved to be a quick, convenient, and sensitive modality in detecting alterations in renal size and parenchymal architecture. all the dogs so diagnosed with crf were rendered conservative medical treatment to control clinical signs of uraemia ; maintain adequate fluid, electrolyte, and acid / base balance ; provide adequate nutrition ; minimize progression of renal failure. |
autism is a pervasive developmental disorder characterized by qualitative impairments in social skills, verbal and non verbal communication ; and restricted repetitive stereotyped behaviors. picture exchange communication system (pecs), a form of augmentative and alternative communication (aac), is a relatively newer intervention specially designed for children with autism based on the principles of applied behavior analysis (aba) and uses pictures instead of words to help children communicate. pecs leads to improvement in communication of children with autism who have difficulty in approaching another person and in pecs the child is made the incharge of the communication and since he is not expected to speak the initial approach becomes less intimidating. the operant mechanisms in these stereotypies are independent of the environment and therefore, these behaviors persist even in the absence of social consequences since the maintaining reinforcer reinforcer (self stimulatory sensory and perceptual consequences) is the direct result of the behavior. lagrow and repp classified the techniques of reducing stereotyped behaviors under four categories:(a) manipulation of setting conditions, drugs or antecedent events, (b) positive procedures ; (c) manipulation of sensory stimulation, and (d) aversive procedures. differential reinforcement of alternative behavior (dra) in which alternative adaptive responses are differentially reinforced in order to reduce maladaptive responding is the most widely used evidence - based behavioral technique for managing stereotypical behaviors of autism. however, all these techniques including dra and pecs have limited validity when used individually. the present case study provides insights into the process of combining pecs with these various traditional techniques toward effective management of communication and behavioral problems of autism. p, a seven - year - old male child, was brought by his parents with chief complaints of repetitive meaningless body movements, hand flapping, poor language / communication development, not managing his activities of daily living, not having peer group / family interactions. based on the history, clinical interview and assessments, he was diagnosed as having childhood autism. the baseline assessments were done using various rating scales including childhood autism rating scale (cars), vineland social maturity scale (vsms) and visual analogue scale (vas) as per clinician observation and parental reports. on vsms, he obtained a score of 59. on cars, it was revealed that he was partially dependent for all his basic activities of daily living, had marked language and communication deficits. the target behaviors identified during this stage included repeatedly moving his head, flapping his hands along with training in self help and communication skills. intervention process was carried out in thirty - two sessions over a period of three months using pecs along with traditional behavioral techniques such as dra, reprimand and task direction. the intervention program was so planned that it targeted the specific areas of improvement in his communication, self help skills and decrease behavioral problems. pecs was carried over the six phases : in phase 1 called physical exchange phase, the therapist worked as a communiation partner and based on reinforcer sampling, made a picture of the reinforcer which was placed under a clear container, so the child could see it, but not get it. when the child looked interested in the item, the therapist gave the child the picture card. then the child was prompted to hand the picture card back to the therapist who after receiving the card, verbalized the request aloud (" you want biscuit ! you can have it ! "). at this point, (expanding spontaniety phase), therapist moved slightly away from the child so that the child had to move toward the therapist to place the picture card in his hand. during phase 3 (discrimination training) the child was given more than one picture card and he had to choose a desired object, and then gave that card to the therapist. initially, he had a hard time distinguishing between the two pictures ; however, eventually he could do that without any difficulty. in the next phase of sentence structure, the child was given a card with the phrase this card now had to be used with the picture card showing what was desired. even though the child could not yet read, he gradually learnt to recognize the words as sight words on the cards. in phase 5 the child what do you want ? and the child had to hand a picture card to enable the child know how to communicate his desires. after the five phases, pecs was generalized to more than one therapist, and he was also taught how to communicate his experiences outside the therapy room. since on analysis it was found that most of his stereotyped behaviors were relatively independent of social consequences, techniques such as dra, reprimand and task direction were also used. using dra, he was reinforced contingent upon the performance of a desirable behavior mentioned in his activity schedule. when a pre set number of alternative behaviors were met, he was given an activity reward. in the initial phase, a fixed reinforcement schedule was followed which was later changed to variable interval schedule. the edible reinforcer was given intermittently, and replaced with verbal praise. throughout the sessions it involved calling his name and directing his attention back to the task at hand. since on analysis it was found that most of his stereotyped behaviors were relatively independent of social consequences, techniques such as dra, reprimand and task direction were also used. using dra, he was reinforced contingent upon the performance of a desirable behavior mentioned in his activity schedule. when a pre set number of alternative behaviors were met, he was given an activity reward. in the initial phase, a fixed reinforcement schedule was followed which was later changed to variable interval schedule. the edible reinforcer was given intermittently, and replaced with verbal praise. throughout the sessions it involved calling his name and directing his attention back to the task at hand. by the end of thirty - two therapy sessions, the child had shown approximately 60% improvement in the targeted behaviors as found on the vas (both parent and clinician rated). his behaviors of repeatedly moving his head, flapping his hands reduced to almost negligible along with an increase in his independence levels in carrying out self help activities and significant enhancement in his communication. this case study demonstrates the utility of pecs as an important adjunct to traditional behavioral techniques such as dra, reprimand and task direction in managing behavioral problems in a child with autism. these findings are consistent with the findings of previous studies whereby it has been reported that after training in pecs, problem behaviors often subside as the benefits of communication become more tangible. also, combination of these approaches further accelerated the management of behvioural problems along with an enhancement in communication and self help skills. the case study carries significant implications for non - pharmacological management of autism- instead of directly focusing on the stereotyped behavior, the need is to reinforce adaptive behavior by using behavioral techniques such as contingency management, differential reinforcement, activity scheduling, reprimand and task direction. since, the present work is based on single case study, there is a need to carry out research on a larger sample. further, a longer follow - up should be maintained to evaluate the efficacy of the treatment program. | communication skills deficits and stereotyped behaviors are frequently found among people with pervasive developmental disabilities like autism. these communication and behavioral oddities of autism are often considered to be difficult to treat and are challenging. picture exchange communication system (pecs) is a six - phase picture system based on applied behavior analysis and is specially designed to overcome these communication difficulties in children with autism by encouraging the child to be the communication initiator. the present paper throws light on the process of using pecs along with other traditional behavioral approaches in managing communication deficits and behavioral stereotypies in a seven - year - old male child diagnosed as having childhood autism. the identified target behaviors of repeated head turning, flapping his hands, poor communication skills were assessed using various rating scales including visual analogue scale as per clinician observation and parental reports and managed using pecs as an adjunct to traditional behavioral techniques of contingency management, differential reinforcement, task direction and reprimand. outcome was assessed using same tools after thirty - two sessions of interventions spread over three months. significant improvements of around 60% were observed in the target behaviors. |
in spite of the increased focus and interest generated in the area of controlled release and targeted drug delivery system in recent years, tablet dosage forms that are intended to be swallowed whole, disintegrate, and release their medicaments rapidly in the gastrointestinal tract still remain the formulation of choice from both a manufacturing as well as a patient acceptability point of view. thus, a drug given in the form of a tablet must undergo dissolution before being absorbed and eventually transported into systemic circulation. difficulties with and resistance to tablet - taking are most common in all patient groups and can exacerbate compliance problems and undermine treatment efficacy. physical problems with swallowing (dysphasia) can occur at any age but are particularly prevalent in geriatric, pediatric, and psychiatric patients. nonetheless, oral dosing remains the preferred mode of administration for many types of medication due to its simplicity, versatility, convenience, and patient acceptability. by considering the above points, patient convenience and compliance oriented research has resulted in bringing out safer and newer drug delivery systems ; one of such approaches is fast disintegrating drug delivery system. fast disintegrating drug delivery systems (fddds) are a new generation of formulations which combine the advantages of both liquid and conventional tablet formulations and, at the same time, they provide the convenience of a tablet formulation and also allow the ease of swallowing provided by a liquid formulation. fddds offer the luxury of much more accurate dosing than the primary alternative, oral liquids. recent advances in novel drug delivery systems (ndds) aim at enhancing the safety of a drug molecule while maintaining its therapeutic efficacy so as to achieve better patient compliance. us food and drug administration center for drug evaluation and research (cder) defines, in the orange book, a fdt as a solid dosage form containing medicinal substances, which disintegrates rapidly, usually within a matter of seconds, when placed upon the tongue. european pharmacopoeia described fdts as uncoated tablets intended to be placed in the mouth where they disperse rapidly before being swallowed and as tablets which should disintegrate within 3 minutes. fast disintegrating tablets (fdt) are also known as fast dissolving, mouth dissolving, rapid - dissolving, quick disintegrating, orally disintegrating, rapimelt, fast melt, orodispersible, melt - in - mouth, quick dissolving, porous tablets, and efvdas (effervescent drug absorption system). the bioavailability of drugs may be increased due to absorption of drug in oral cavity and also due to pregastric absorption of saliva containing dispersed drugs that pass down into the stomach. moreover, the amount of drug that is subjected to the first pass metabolism is reduced as compared to standard tablet. formulation of the drug chosen for the treatment of allergic cough and other respiratory disorders is available in market in conventional tablet and liquid dosage forms. tablets to be swallowed are resisted by pediatric patients and patient compliance is an issue with such dosage forms. hence they do not comply with the prescription, which results in high incidence of noncompliance and ineffective therapy. the benefits, in terms of patient compliance, rapid onset of action, increased bioavailability, and good stability make fast disintegrating tablets popular as a dosage form of choice in the current market. it is a nonsedative second generation antihistamine drug used in the treatment of seasonal allergic rhinitis, perennial allergic rhinitis, chronic urticaria, and atopic dermatitis and also used as adjuvant in seasonal asthma and allergic cough. cetirizine inhibits the release of histamine and of cytotoxic mediators from platelets, as well as eosinophil chemotaxis during the secondary phase of allergic response. due to sore throat conditions, the patient experiences difficulty in swallowing a tablet type of dosage form thus, fast disintegrating tablets would serve as an ideal dosage form pediatric patients who find it difficult to swallow the conventional tablets and capsules. hence an attempt was made for preparation of fast disintegrating tablet of cetirizine hydrochloride with an aim of improving / enhancing patient convenience and compliance, reducing the lag time and providing faster onset of action to relieve the allergic and respiratory disorders immediately. cetirizine hydrochloride was received as gift sample from trojan pharma, baddi, india. microcrystalline cellulose (avicel ph-102) sodium starch glycolate (primogel, explotab) and directly compressible mannitol (d - mannitol) were purchased from qualikems fine chem pvt. sodium saccharin was purchased from loba chemie, mumbai, and talc from nice chemicals private limited, hyderabad, india. all other chemicals and reagents which were of analytical grade were used. the most important parameter that needs to be optimized in the development of fast disintegrating tablets is the disintegration time. fast disintegrating tablets were prepared firstly using different excipients (binders and superdisintegrants) and then evaluated for various parameters like friability, hardness, and disintegration time to select the best combination for formulation of fast disintegrating tablets. the combination with the lowest disintegration time, optimum hardness, and friability was selected for further study. optimization of superdisintegrant sodium starch glycolate (primogel, explotab). for tablets and capsules which require rapid disintegration, the inclusion of the right superdisintegrant and in its optimum concentration is a prerequisite for optimal bioavailability. thus, the proper choice of superdisintegrant its consistency of performance are of critical importance to the formulation of rapidly disintegrating dosage forms. formulation f1f6 was prepared to study the effect of type and concentration of superdisintegrants in table 1. tablets were prepared by direct compression technique. weighed quantity of cetirizine hydrochloride with different concentration of superdisintegrant along with excipients was mixed in geometric progression in a dry and clean mortar. then the blend was passed through sieve number 60 for direct compression. the powder blend was then compressed into tablets using 8 mm punch in multi punch tablet compression machine (dhiman industries, india). optimization of polyvinylpyrrolidone (pvp k-30) or microcrystalline cellulose (avicel ph-102) as binder along with optimized concentration of superdisintegrant. the composition of fast disintegrating tablet is shown in table 2. weighed quantity of cetirizine hydrochloride with optimized concentration of sodium starch glycolate along with different concentration of binders (pvp k-30, mcc) along with excipients the powder blend was then compressed into tablets using 8 mm punch in multi punch tablet compression machine (dhiman industries, india). fast disintegrating tablets of cetirizine hydrochloride were prepared by direct compression method according to the formula given in table 3. weighed quantities of cetirizine hydrochloride along with optimized concentration of superdisintegrant and binder along with excipients were mixed in geometric progression in a dry and clean mortar. the powder blend was then compressed into tablets using 8 mm punch in multi punch tablet compression machine. twenty tablets were selected, weighed on digital weighting balance (ohaus, usa) and average weight was determined. then individual tablets were weighed and the individual weight was compared with an average weight as given in table 4. thickness of tablets was determined using vernier caliper (indian caliper industries, ambala, india). the crushing strength of the tablets was measured using a monsanto hardness tester (perfit). three tablets from each formulation batch were tested randomly and the average reading was noted. ten tablets were weighed and placed in a roche friabilator (veego, india) and the equipment was rotated at 25 rpm for 4 min. the percentage friability of the tablets was measured as per the following formula (1)percentage friability = initial weightfinal weightinitial weight100. the test was carried out on 6 tablets using digital tablet disintegration tester (veego, india). distilled water at 37c 2c was used as a disintegration media and the time in second taken for complete disintegration of the tablet with no palable massremaining in the apparatus was measured in seconds. time required for the upper surface of the tablet to become complete red was noted. ten tablets (200 mg) were powdered in mortar pestle and the blend equivalent to 5 mg of cetirizine hydrochloride was weighed and dissolved in 100 ml of 6.8 ph phosphate buffer solutions. the solution was sonicated, filtered through whatman filter paper, suitably diluted with 6.8 ph phosphate buffer and the drug content was analyzed by using double beam uv spectrophotometer (uv-1800 shimadzu) at 230 nm respectively. the release of from formulated fdts was determined using usp eight stage dissolution testing apparatus2 (paddle method) (lab, india). the dissolution test was performed using 500 ml of phosphate buffer solution, ph 6.8 at 37 0.5c and 50 rpm. a sample (5 ml) of the solution was withdrawn from the dissolution apparatus at specific time intervals and the samples were replaced with fresh dissolution medium. absorbance of these solutions was measured at 230 nm using a double beam uv spectrophotometer (uv-1800 shimadzu). cumulative percentage (%) of drug release was calculated using standard plot of cetirizine hydrochloride. ftir spectra of pure drugs and formulated fdt containing drug were recorded on ftir spectrophotometer (bruker, usa). the scanning range was from 4000 to 600 cm and the resolution was 1 cm. the scans were evaluated for presence of principal peaks of drug, shifting and masking of drug peaks, and appearance of new peaks due to excipient interaction. this spectral analysis was employed to check the compatibility of drugs with the excipients used. the selected formulations were closely packed in aluminum foils and then stored at 40 2c/75% rh 5% in stability chamber for 1 month and evaluated for their physical appearance, drug content, percent friability, and in vitro disintegration time at intervals of the 15th and 30th days. the present investigation was undertaken to formulate and evaluate fast disintegrating tablets of cetirizine hydrochloride by direct compression method using sodium starch glycolate as a superdisintegrant and mannitol as directly compressible diluent and sodium saccharin was used to enhance palatability. this grade of microcrystalline cellulose is granular in nature and thus displays excellent flow properties. to impart pleasant taste and improve mouth feel, sodium stearyl fumarate was employed as a lubricant instead of magnesium stearate not only because of the metallic taste of the latter, but also due to its water solubility and directly compressible features. superdisintegrants are generally used by formulation scientists for developing fdts or for improvement of solubility for drugs. the total 6 formulations (f1f6) were prepared using different concentration of sodium starch glycolate to study its effect on disintegration time. the results for optimization of superdisintegrant concentration in fdts by direct compression method are shown in table 5. from the evaluation parameters, it was observed that 4% sodium starch glycolate was the optimum concentration for rapid tablet disintegration on the basis of the least disintegration time observed with f3 formulation. the superdisintegrant action of sodium starch glycolate resulted in hydrophilicity and swelling which in turn causes rapid disintegration. it absorbs water rapidly and swells in water to the extent of 200300% and disintegrates rapidly. sodium starch glycolate is used as superdisintegrant in tablet formulation at a concentration of 46%. above 8% disintegration times the binders such as polyvinylpyrrolidone (pvp k-30) or microcrystalline cellulose were optimized with superdisintegrant concentration to further study the effect of binders on the disintegration time as well as on hardness and friability of tablets of the formulation. total 14 formulations (f1f14) were prepared using different concentration of polyvinylpyrrolidone (pvp k-30) or microcrystalline cellulose to study its effect on disintegration time of formulations. the results for optimization of different binder in fdts by direct compression method are shown in table 6. from the evaluation parameters, it was observed that disintegration time of the formulation was further decreased and tablet hardness, friability with in ip limits. the least disintegration time was observed in f8 formulation, that is, 1% mcc, as compared to f2 formulation, that is, 2% pvp k-30. water soluble materials such as pvp k-30 tend to dissolve rather than disintegrate, while insoluble materials like mcc generally produce rapidly disintegrating tablets. due to the presence of porous morphology wicked into these pathways through capillary action and ruptures the interparticulate bonds causing the tablet to break apart. therefore 1% microcrystalline cellulose was selected as optimum binder concentration selected for final formulation of cetirizine hydrochloride fdt. final formulation of cetirizine hydrochloride fdt was tested for all the official tests of tablet and was found to be within limits as shown in table 7. percent weight variation was well within the acceptable limit for uncoated tablets as per indian pharmacopoeia. it is well known to formulation scientists that the tablets with more hardness show longer disintegration time. since mechanical integrity is of paramount importance in successful formulation of fdts, hence the hardness of tablets the friability of cetirizine hydrochloride fdt was less than 1% which is acceptable according ip criteria. no tablet from ten tablets lies out of the range of 85115% of the label claim. these results indicated that the dosage form had uniform distribution and proper dose of the active ingredient. the wetting time and disintegration time were practically good for formulation. according to ip, the dispersible tablet must disintegrate within 3 minutes, but the formulated fdts showed low dt indicating suitability of formulation for mouth dissolving tablet. in vitro dissolution studies showed that more than 50% of the drug was released from the formulation within 5 minutes. the rapid drug dissolution might be due to easy breakdown of particle by superdisintegrant action. from in vitro dissolution data, it was observed that 94.74 2.48% of cetirizine hydrochloride released in 16 minutes as shown in figure 1 indicates that the tablet complies as per ip specifications, that is, 85%110%. the results obtained with ir studies showed that there was no interaction between the drug and other excipients used in the formulation. the ftir of cetirizine hydrochloride had shown intense band at 757.13 cm, 1317.62 cm, 1055.66 cm and 1184.57 cm corresponding to the presence of functional groups such as aliphatic chlorocompound, carboxylic acid, alkyl substituted ether and tertiary amine. the ftir of cetirizine hydrochloride fdt formulation shown intense bands at 758.41 cm, 1312.37 cm, 1078.32 cm and 1181.48 cm indicates no change in the functional groups such as aliphatic chlorocompound, carboxylic acid, alkyl substituted ether, and tertiary amine confirming undisturbed structure of cetirizine hydrochloride, which indicates no drug - excipient interaction as shown in figure 2. in the present study, stability studies were carried out on formulated fdts (formulated in three primary batches), wrapped in aluminium foil to prevent the formulation from exposure to light to simulate the aluminum packaging, that is, alu alu packing, of drug products, and stored in air - tight containers which is impermeable to solid, liquid, and gases, under the following condition for onemonth period as prescribed by ich guidelines for accelerated stability study. during the stability studies, the product is exposed to normal condition of temperature and humidity. however the studies will take a longer time and hence it would be convenient to carry out accelerated stability studies, where the product is stored under extreme condition of temperature and humidity. result is obtained after 1 month of stability studies at room temperature and at ambient humidity. the result of the stability study indicated that there were not many differences observed in hardness, disintegration time, drug content uniformity, and friability before and after the storage period at room temperature and at ambient humidity, but, at temperature of 40 2c/75% rh 5% relative humidity, hardness was increased with time, prolonging the dt of the tablet ; the probable reason was loss of moisture from tablets, but, in all cases, dt is within the specified ip limit (within 3 min). fast disintegrating tablet is a promising approach with a view of obtaining faster action of the drug and would be advantageous in comparison to currently available conventional dosage forms. the prime objective of the study was to develop cetirizine hydrochloride fast disintegrating tablet by using commonly available excipients and conventional technology. from the above study, it was concluded that, by employing commonly available pharmaceutical excipients, such as superdisintegrants, hydrophilic and swellable excipients and proper filler, a fast disintegrating tablet of cetirizine hydrochloride can be developed which can be commercialized. | recent developments in fast disintegrating tablets have brought convenience in dosing to pediatric and elderly patients who have trouble in swallowing tablets. the objective of the present study was to prepare the fast disintegrating tablet of cetirizine hydrochloride for allergic and respiratory disorders. as precision of dosing and patient 's compliance become important prerequisite for a long - term treatment, there is a need to develop a formulation for this drug which overcomes problems such as difficulty in swallowing, inconvenience in administration while travelling, and patient 's acceptability. hence, the present investigation was undertaken with a view to develop a fast disintegrating tablet of cetirizine hydrochloride which offers a new range of products having desired characteristics and intended benefits. superdisintegrants such as sodium starch glycolate were optimized. different binders were optimized along with optimized superdisintegrant concentration. the tablets were prepared by direct compression technique. the tablets were evaluated for hardness, friability, weight variation, wetting time, disintegration time and uniformity of content. optimized formulation was evaluated by in vitro dissolution test, drug excipient compatibility and accelerated stability study. it was concluded that fast disintegrating tablets of cetirizine hydrochloride were formulated successfully with desired characteristics which disintegrated rapidly, provide rapid onset of action, and enhance the patient convenience and compliance. |
biological therapies can no longer be disregarded in the management of inflammatory bowel disease (ibd) and have become - together with the 5-aminosalicylates (5-asas), methotrexate, and purine analogues (azathioprine and 6-mercaptopurine) - the cornerstone of ibd treatment. corticosteroids, though abandoned more and more, may still be used as efficacious induction agents or as a bridge to the action of immunomodulators (or as both). given that the mentioned drugs, with the exception of 5-asas, affect the immunity of the patient and that there is increasing use of these drugs in ibd, safety concerns include the occurrence of (opportunistic) infections, and physicians should take active measures to prevent or treat these infections. moreover, as many patients now enjoy a very good quality of life as a consequence of the therapeutic advances made, travel and vaccination advice become part of the follow - up of patients with ibd. prevention of opportunistic infection includes recognizing risk factors for infection, closely monitoring patients during therapy (clinical and lab), and vaccinating where possible. risk factors for opportunistic infections include combined immunomodulator use, including corticosteroids, older age, malnutrition, and comorbidities. the best - documented infectious complication of anti - tumor necrosis factor (anti - tnf) agents (infliximab, adalimumab, and certolizumab pegol) is the reactivation of latent tuberculosis (tb). by february 2003, active tb was reported in 350 of more than 400,000 patients treated with infliximab, a cumulative incidence of 0.46 per 1000 patient - years. most cases will occur within the first 2 months after initiation of therapy and are associated with a substantial rate of mortality. therefore, all patients who will undergo treatment with an anti - tnf agent should be questioned and evaluated for latent tb with a tuberculin skin test or interferon - gamma release assay and chest x - ray. recommendations for screening tb and treatment are proposed by national scientific organizations and authorities, and their national guidelines should be consulted by physicians. the largest long - term study on safety of anti - tnf from clinical practice was done in 743 infliximab - treated ibd patients and an equally sized control group of non - anti - tnf - treated ibd patients. with a follow - up of as long as 14 years, concomitant steroid therapy was the only independent risk factor for infections in the infliximab group, with a 2.69-fold (95% confidence interval [ci ] 1.18 - 6.12) increased risk in the case of co - administration of corticosteroids together with infliximab (p = 0.018). likewise, a recent cohort study from british columbia, canada, showed that corticosteroids tripled the risk of clostridium difficile infections (relative risk 3.4, 95% ci 1.9 - 6.1) compared with other immunosuppressant agents. the european crohn s and colitis organisation (ecco) consensus on opportunistic infections in ibd brought 30 ibd and infectious disease specialists together. their guidelines recommend serology testing for specific viruses and administration of a number of vaccinations shortly following the diagnosis of ibd. the reason for doing this at an early stage is that at least half of the patients will require immunomodulators or anti - tnf (or both) during their further disease course and live - attenuated vaccines (yellow fever and varicella being the two most important ones) can not be administered during this treatment. second, immunomodulators or biological agents (or both) may attenuate the response to vaccination. figure 1 summarizes which serologies should be checked and which vaccines are recommended. besides the serologies that should be checked (figure 1), a baseline laboratory exam, including neutrophil, eosinophil, and lymphocyte blood count and c - reactive protein and hiv serology, nobody questions vaccination for influenza and pneumococcus pneumoniae, however, vaccinating for hepatitis b virus (hbv) and hepatitis c virus (hcv) has generated debate. more recently, a nationwide spanish study of more than 2000 consecutively recruited patients with ibd showed, in contrast to previous reports, that the prevalence rates of hbv and hcv infection were similar to those of the general population. however, the frequency and severity of liver dysfunction appear to be significantly higher in hbv - infected patients than in hcv - infected patients. in the study from the spanish geteccu (grupo espaol de enfermedades de crohn y colitis ulcerosa), hbv infection in patients with ibd resulted in liver dysfunction in more than one - third (36%) of patients, including six cases of hepatic failure. liver dysfunction in hcv was less frequent (15.7%), and only one patient developed hepatic failure. the treatment with two or more immunosuppressants was an independent predictor of hbv reactivation (odds ratio 8.75, 95% ci 1.16 - 65.66). interestingly, the studies that have reported on the degree of effective vaccination for hbv show that only a minority of the patients with ibd (range 12 - 48%) are effectively vaccinated. we recommend that hbv and hcv serology be checked and that patients negative for hbv receive vaccination. in hcv - positive patients under long - term treatment with immunomodulators or biologicals (or both), aminotransferases and hcv viral load should be followed up. the figure depicts the serology and vaccination to be performed in inflammatory bowel disease patients before immunomodulators or biological therapy (or both) are started, as recommended by the consensus guidelines of the european crohn s and colitis organisation. anti - tnf, anti - tumor necrosis factor ; hbsag+, positive for the surface antigen of hbv ; hbv, hepatitis b virus ; vzv, varicella zoster virus. in comparison with the more severe (opportunistic) infections, benign infections such as the common cold, bronchitis, warts, and herpes are found more frequently. in a prospective cohort study with a follow - up of 207 patient - years, no difference in upper respiratory tract infections was found between azathioprine - taking patients and those not on purine analogues. in contrast, the rates of incidence of herpes flares and warts were significantly increased in ibd patients receiving azathioprine. where possible, stopping therapy may be recommended if flares are too frequent or severe. if therapy needs to be continued to maintain disease remission, daily oral acyclovir (400 mg twice a day) is an option to prevent frequent recurrences of herpes simplex disease. ibd patients who need immunomodulators or biological therapy should receive appropriate counseling regarding the risk of infections and should receive a number of vaccinations : first, all patients starting on an anti - tnf agent should undergo tuberculin skin testing or an interferon - gamma release assay as well as a chest x - ray to rule out latent tb ; if the assay is positive, anti - tb treatment should be initiated at least 2 weeks prior to therapy. the ecco consensus guidelines on opportunistic infections furthermore recommend serology testing for varicella zoster virus (vzv) and hbv and vaccination for varicella (in case of negative serology for vzv and no history of chickenpox), hbv, influenza (yearly), and pneumococcal polysaccharide (every 3 - 5 years). a baseline laboratory exam, including neutrophil, eosinophil, and lymphocyte blood count, c - reactive protein, and hiv serology, should be performed at the time of diagnosis, and routine blood monitoring every 2 - 4 months should be continued while the patient is on immunosuppressive agents. mild viral infections like warts and herpes are more frequent under immunomodulators or anti - tnf but, in general, are no reason to stop therapy. if such an infection is too invalidating for the patient, daily oral acyclovir (400 mg twice a day) is an option in case of herpes simplex disease. | the current medical therapy used in crohn s disease and ulcerative colitis comprises drugs that interfere with immune response and therefore caution is needed for infectious side effects, and where possible, strategies to prevent their occurrence should be undertaken. last year, international consensus guidelines on this topic were published by the european crohn s and colitis organisation. |
the most commonly affected site is the lung, with a reported incidence ranging from 7% to 28% (1). unusual presentations included bilateral spontaneous pneumothoraces with predominantly cystic lesions (2 - 4). metastatic uterine ess presenting as a solitary lung nodule is also unusual and only a few cases were documented histologically (1, 5). a case of bilateral reticulonodular infiltrates corresponding to a lymphangitic growth pattern was documented (1). we experienced a case of pulmonary metastases of uterine ess that presented intraalveolar micronodules, which is a rare pattern of metastasis not described in english literature. a 37-yr - old woman with increased extent of small random micronodules and ground glass opacities (ggo) in both lungs with dyspnea visited the hospital. she had been diagnosed with ess in the uterus which had been removed due to postpartum bleeding 4 yr ago. one year later, both of her ovaries and vaginal stump were removed due to the recurred uterine ess. at that time, chest computed tomography (ct) showed miliary pattern of micronodules in both lung fields with symptom of dyspnea american thoracic society (ats) grade i. chemotherapy with southwest oncology group (swog) protocol and ifosphamide were performed. during the 3 yr follow - up period, no significant change was found in pulmonary micronodular densities, but occasionally, ggo was found on chest ct with symptoms of dyspnea ats grade ii and fever (fig. these ggo and her symptoms improved after corticosteroid therapy and the nature of ggo was clinically regarded as alveolar hemorrhage or intraalveolar fibroblastic polyps. video assisted thoracotomy biopsy was performed to evaluate the pulmonary micronodules and ggo. under gross examination, 2). these nodules consisted of short spindle cells arranged in ill - defined whorls and the neoplastic cells had bland nuclear features and eosinophilic cytoplasm (fig. the histologic features of the lung nodules were identical to that of low grade uterine ess obtained from hysterectomy specimen. immunohistochemically, these neoplastic cells showed strong nuclear staining for estrogen receptor (novocastra, newcastle, u.k., 1:100 dilution) (fig. 3, inset) and progesterone receptor (novocastra, 1:40 dilution), and diffuse cytoplasmic staining for cd10 (novocastra, 1:40 dilution). the patient was well and asymptomatic when last seen, 8 months post thoracotomy, and the ggo was improved with repeated tamoxifen and ifosphamide therapies. ess represents approximately 15 - 25% of all uterine sarcomas and are separated into high and low grades, depending primarily on the mitotic index (more or less than 10 mitoses per 10 high power fields, respectively). distant metastases may develop after long tumor - free intervals and the lung is the most commonly affected site. the most common growth pattern of pulmonary metastatic ess, previously described in the literatures, is well circumscribed nodules with entrapped air spaces lined by non - neoplastic respiratory epithelium (1). the unusual growth patterns are solitary nodule, satellite with infiltrative margins, lymphangitic pattern and bilateral spontaneous pneumothoraces associated with predominantly cystic lesions mimicking lymphangioleiomyomatosis, which can contribute to the diagnostic dilemma (1 - 5). this is especially true if clinicians and/or pathologists are unaware of a prior diagnosis of uterine ess. prior misdiagnosis of a uterine tumor can also be misleading, emphasizing the need to review previous surgical specimens, particularly gynecologic specimen in female patients with unusual mesenchymal lung neoplasms (6, 7). the micronodules of the present case did not show entrapped respiratory epithelium and were mainly located in the intraalveolar spaces growing out from alveolar walls suggesting hematogeneous spread. this pattern of metastases of uterine ess is interesting and has been not reported in english literatures. we assumed that the lesions were caused by lodging of microtumor emboli through the pulmonary artery. the metastatic ess demonstrated the same range of histologic features seen in primary uterine ess. the differential diagnosis includes benign metastasizing leiomyoma, sclerosing hemangioma, solitary fibrous tumor, hemangiopericytoma, and lymphangioleiomyomatosis in small biopsies. immunostaining for estrogen receptor and progesterone receptor are positive in nearly all cases of uterine ess. diffuse immunoreactivity for cd10 in uterine ess can also be helpful in separating them from uterine smooth muscle neoplasms, which are usually negative. here, we report a case of unusual pulmonary metastatic uterine ess in a 37-yr - old female with a diffuse micronodule and ggo on chest ct. pulmonary metastases of low grade ess may have an excellent prognosis and little effect on survival. | pulmonary metastases of uterine endometrial stromal sarcoma (ess) are uncommon. the patterns of uterine ess metastasis to the lung are multiple pulmonary nodules, single nodule, or cystic lesions. pulmonary intraalveolar micronodular metastases of uterine ess are unusual and have not been reported. we experienced a case of metastatic uterine ess presenting as pulmonary diffuse micronodules with ground glass opacities on chest computed tomography of a 37-yr - old woman who previously underwent hysterectomy due to low grade ess of the uterus four years ago. the histologic findings of video assisted thoracotomy biopsy showed numerous intraalveolar polypoid micronodules protruding from the alveolar septums. all tumor nodules were composed of short spindle cells arranged in ill - defined whorls, and nuclear feature and sparse cytoplasm were seen in uterine ess. immunohistochemically, these cells showed strong nuclear staining for estrogen receptor and progesterone receptor, and diffuse cytoplasmic staining for cd10. |
the early 1990 s marked the discovery and publication of numerous accounts concerning a host of protein products of genes in the 3540 kda range consisting of ctgf / ccn-2 (connective tissue growth factor), nov, wisp-2 and wisp-3, cyr-6. these and now some others have now been classified as the ccn family of genes after the early suggestion that members of this family of genes acted as growth regulators (peer 1993 ; moussad and brigstock 2000 ; brigstock 2003). ctgf / ccn-2 in particular has in the past been referred to as a growth factor as its earlier name suggests, however the collective work of a number of laboratories and in particular the development of knockout mice has confirmed that ctgf / ccn-2 for example more accurately serves as a modifier of the signals transmitted by other molecules - such as tgf- (moussad and brigstock 2000 ; brigstock 2003 ; leask and abraham 2006 ; ivkovic. it has also emerged and is fairly broadly accepted within the ccn community that the best designation for this molecule is it is now widely accepted that ccn-2 exerts important homeostatic control of connective tissues by virtue of its ability to mediate extracellular matrix processes. ccn-2 proteins modulate mitosis, cellular migration, binding of other growth factors, wound healing and have varying influence concerning either pro- or anti - apoptotic signaling depending upon the cells and tissues involved (leask and abraham 2006 ; gygi. 2003 ; croci. 2004 ; hishikawa. 2000). over - expression of ccn-2 leads to significant morbidity such as is the case with fibrotic diseases such as may be the case with scleroderma and fibrosis affecting other organ systems such as the liver, kidney, lung, pancreas and others (moussad and brigstock 2000 ; ivkovic. 2003 ; perbal 2004 ; ming - jyun. furthermore, elevated ccn2 expression has been reported to play a crucial role in migratory / invasive processes in breast cancer (pai - sheng. on the other hand, human lung adenocarcinoma cells stably transfected with vectors containing ccn2 and injected into nude mice were found to resist metastatic activity. in this study it was theorized that ccn-2 induced inhibition of metastasis may have been the result of inhibiting vegf - a dependent angiogenesis - perhaps as a consequence of increasing the degradation of hif-1 (cheng - chi. therefore it has emerged that ccn-2 has widely diverse functions and is associated with a broad range of physiological activities. one area that has received far less attention has been the dynamic role of ccn-2 in development, particularly with respect to the varying influence of this molecule from early embryogenesis through to the mature organism in various tissue compartments and its contribution to health and disease as a function of its changing expression patterns. the notochord is common to all higher vertebrates as a developmentally vital structure. during development stiffness to the developing embryo - as well as to function as a source of differentiation signals to local undifferentiated mesenchymal tissues (ming - jyun. 2006 ; smits and lefebvre 2003 ; marcelle. 1999 ; barrionuevo. 2006 ; hirano and najita 1995). the notochord is of mesodermal origin and forms during the gastrulation phase of the developing embryo where the early notochord begins as at the primitive pit and ultimately becomes a rod - like structure that is enveloped within a sheath composed of predominantly extracellular matrix, collagens and proteoglycans, all of which are similar to the composition of cartilage and mesenchymal / fibrous tissues (smits and lefebvre 2003 ; barrionuevo. the notochord most closely resembles cartilage in terms of its tissue type and as such the stiffness it provides during development provides for an axial skeleton - like structure until somitogenesis provides for the development of the axial skeleton (stemple 2005). signals secreted by the notochord are required in order for sclerotomal cells to migrate, condense and differentiate - all of which are central to the formation of the vertebral column and the developing spinal cord (hirano and najita 1995). the spinal cord is formed from condensation of neural crest cells that migrate and lie dorsal to the notochord, forming the neural tube and ultimately the spinal cord (echelard. the terminal stage of notochord development in higher animals is marked by the notochord segmenting during vertebrogenesis and in the vertebral regions it is largely replaced by bone and in the intervertebral areas it forms the centre of the intervertebral disc - the nucleus pulposus (stemple 2004, 2005). a number of factors are involved in the complex process of notochordal evolution specifically including the expression of the brachury gene t that codes for the t - box transcription factor - commonly referred to as t, as well as sonic hedgehog (shh), noggin and pax1 all of which play essential roles that induce differentiation of the sclerotome (barrionuevo 2006 ; ghosh 1988a). the t gene is expressed in all mesodermal cells and is expressed within the notochord until formation of the developing embryonic body is completed (herrmann and kispert 1994). t however is expressed only transiently by other mesodermal cells demonstrating the directed activity of this gene in notochordal development (herrmann and kispert 1994). in mutant mouse studies it has been found that t is necessary for the maintenance of the notochord and that t seems to be a controlling factor in cell motility and/or cell adhesion (herrmann and kispert 1994). interestingly t protein is able to switch the fate of cells from an ectodermal to a ventral mesodermal destination allowing ventral mesodermal cells to respond to diffusible dorsal signal factors. as the following sections detail, there is an early role for ccn-2 in development and in the formation of the notochord in particular. it is therefore an attractive notion that ccn-2 may well interact with such important differentiation and signaling pathways such as those involving t protein as well as other critical transcription factors such as those encoded by the sox genes. for example, it has been shown that ccn-2 interacts with wnt signaling in xenopus development and that it is able to inhibit canonical signaling involving -catenin as well as non - canonical signaling probably via interaction with the wnt receptor complex (mercurio. further evidence of the complex interaction of ccn-2 with development is that overexpression of ccn-2 in xenopus results in inhibition of neural crest migration - perhaps by virtue of wnt pathway inhibition (mercurio. the reader is referred to the detailed report by mercurio for further details concerning ccn2 activity and wnt signaling (mercurio. once the sclerotome is formed, in addition to t, the notochord is known to be heavily influenced by several key transcription factors encoded by sox5, sox6 and sox9 genes (smits and lefebvre 2003 ; barrionuevo. the sox expression pattern found in the notochord has similarities to that observed in cartilage where sox 5, 6 and 9 are coordinated and linked (lefebvre. 2001). the sox genes encode a number of dna - binding proteins that are considered to be important relatively late in development as opposed to the earlier stages where they are considered to have some redundancy (smits and lefebvre 2003). in general, the sox dna binding proteins are thought to perform their various functions in a sterically - defined manner whereby they act as architectural proteins and organize chromatin structure and assemble other dna - bound transcription factors into larger more complex multi - protein units (wegner 1999). it is likely that sox proteins link proteins to signal transduction pathways (such as these multi - protein units) which by themselves may not be capable of inducing signal transduction. sox5 and 6 gene transcripts are known to be expressed in cartilage but no other tissues in the developing mouse and have some kind of co - operative function with sox9-considered to be the master chondrogenic factor (smits and lefebvre 2003 ; lefebvre. sox5 and 6 are considered to be crucial to the survival of notochordal cells within the disc nucleus pulposus, a potentially vital pathway inherent within the cells of animals that retain their notochordal cells into adulthood (moussad and brigstock 2000 ; smits and lefebvre 2003). indeed sox5 and 6 mutant mice demonstrate aberrant removal of notochordal cells from the intervertebral areas and do so later than wild type embryos in mice - the theory being that this delay may be associated with a delay in cartilage development and/or apoptosis of notochordal cells (smits and lefebvre 2003). these sox5 and 6 mutant mice display a failure to form a nucleus pulposus, along with severely distorted vertebral column development confirming the pivotal role that these genes play in directing the development, maturation and survival of the disc nucleus pulposus (smits and lefebvre 2003). in what is likely a more upstream effect, mutant mice lacking sox9 expression fail to develop a normal notochord however the survival of notochordal tissue in the presence of normal sox9 signaling poses a strong argument for the importance of this transcription factor in notochordal cell maintenance in addition to its known role in chondrogenesis and likely the cooperative interaction of sox 5, 6, and 9 (moussad and brigstock 2000). in addition, jun (a major transcription factor essential for embryonic development) elicits anti - apoptotic signaling critical to the survival of notochord cells (croci. 2004 ; marcelle. 1999). however, the precise mechanisms at play in the pivotal morphological evolution of notochordal cells whereby they become located within the nucleus pulposus of the intervertebral disc (ivd) are largely unknown - as is the role played by these cells within the disc (moussad and brigstock 2000 ; pai - sheng (2007). clearly there is a delicate and precise ballet involved with the development of this anatomically central structure whereby aberrant expression of any of these elements create a highly disturbed phenotype. with respect to the role played by ccn-2 during development, a recent study introduced anti - sense oligonucleotides into zebrafish embryos to in order to specifically knockdown ccn-2 expression (ming - jyun 2006). further these investigators also injected a series of ccn-2 promoter - driven green fluorescent protein (gfp) plasmids into one - cell - stage zebrafish embryos in order monitor the expression of ccn-2. the targeted knockdown of ccn-2 led to a malformed and distorted notochord in the developing embryo and many of the embryos died in early developmental time periods (ming - jyun 2006). the promoter - driven gfp signal was detected in the midline, floor plate and head section of the notochord of the developing embryos confirming the expression of ccn-2 during embryological development in general and the notochord specifically. the detection of ccn2 linked gfp within the midline, floor plate and head section of these developing embryos is all the more intriguing when taken in context with the findings that t is considered to be involved with cellular functions concerning cell adhesion (herrmann and kispert 1994). ccn-2 is well known to be highly involved in cell growth, migration and adhesion and although there are no direct references in this regard, the detection of gfp - linkedccn-2 in the developing notochord provides for the notion that an axis involving ccn-2 and t in notochord development and maintenance may be more complex than is currently understood. these findings are of considerable relevance in the context of the lethal phenotype generated by the deletion of ccn-2 in mice that results in severe skeletal dimorphisms that is also inconsistent with life shortly after birth (ivkovic. these ccn-2 deletion mutants are born with severe skeletal abnormalities, malformed rib cages and die due to poor extracellular matrix synthesis, defective cartilage and associated respiratory failure (ivkovic. however, there is as of yet no detailed description of the fate of the intervertebral disc nucleus pulposus - in particular what transpires in terms of notochordal cell development apart from impaired skeletogenesis. with respect to initial notochordal signaling, it is widely recognized in studies involving zebrafish, drosophila and chicks that shh is a key inductive signal for ventral patterning of the cns and that a gradient of signaling seems to exist between dorsal ectodermal and ventral mesodermal signaling in cns development (horne 2004). the floor plate releases shh which results in neuronal patterning that are directly related to the gradient of shh signals and their interaction with undifferentiated mesenchymal cells. it was as a consequence of these important experiments that shh became known as a classic morphogen. it has been reported in other studies that the ccn-2 regulatory region has a shared homology within the zebra fish shh gene ; further lending support to the essential involvement of ccn2 in key developmental processes involving embryological developmental patterning (ming - jyun. it has been reported that coincident with the reduction in the number of notochord cells, the nucleus pulposus is seen to undergo typical internal disorganization accompanied by degenerative changes (ghosh 1988b ; thompson. however, animals that retain notochordal cells within their ivd np do not develop degenerative disc disease nearly as early or as severely as those animals that do not retain these cells. there are natural occurring sub - species of canine that are unique in that one, the non - chondrodystrophic (ncd) canine is protected from developing degenerative disc disease (ddd), whereas the chondrodystrophic (cd) canine (beagles, dachshunds, poodles) suffer significant and early ddd (braund 1975). the single most critical difference between these two sub - species is that the non - chondrodystrophic canine maintains its population of notochordal cells within the nucleus pulposus (np) of the ivd for most of the life of the animal whereas the cd canine contains sparse notochordal cells by even 1 year of age. during the course of harvesting notochordal cells from canine sources the experience of this writer is such that 30 lumbar ivd np s harvested from five (5) 812 month old ncd canines (6 lumbar discs each) will typically yield 404510 notochordal cells. eight (8) cd canines on the other hand using 6 lumbar ivd nps obtained from each will typically yield only 3410 notochordal cells. the disorganization and degenerative changes witnessed in notochordal cell - poor animals strongly suggests that notochord cells when present, act as matrix guardians that continue to secrete growth and other factors therefore modulating a homeostatic protective action on the disc matrix (aguiar. aguiar, johnston and oegema were the first amongst a very few to suggest that notochordal cells were able to biochemically contribute to ivd homeostasis by developing a crude conditioned medium and evaluating proteoglycan production in bovine disc - derived np cells (aguiar. this first study by this group was followed by our own studies of this relationship. in one study we reported that totally serum - free notochordal cell conditioned medium (nccm) obtained from ncd notochordal cells could in a dose - dependent fashion, upregulate proteoglycan production and in a non - dose - dependent fashion increase cell proliferation in bovine caudal disc np cells (erwin and inman 2006). in a second study, also using bovine caudal disc np as the target cells, we demonstrated upregulated gene expression for the essential extracellular matrix proteoglycans aggrecan and versican after 24-hour culture with totally serum - free notochordal cell conditioned medium (erwin. in addition, gene expression for hyaluronic acid synthase-2, the enzyme essential for hyaluronic acid synthesis production was also upregulated in bovine disc np cells. using lc - ms / ms mass spectroscopy we identified the presence of ccn-2 within the conditioned medium (erwin. after we detected ccn-2 within the notochordal cell conditioned medium, we cultured bovine np cells with serum - free notochordal cell conditioned medium (nccm) and measured doses of recombinant human ccn2 (rccn2) (a kind gift from dr. david brigstock, ohio state university) and compared aggrecan gene expression by the bovine np cells. we found that 100200 ng / ml of rccn2 induced upregulation of aggrecan gene expression to exactly the same level of expression as our serum free nccm derived from ncd canine - source notochordal cells (fig. we have recently followed - up our mass spectroscopy findings and have verified the presence of ccn2 in ncd notochordal cell lysates using western blotting techniques (antibody a kind gift from dr. it appears from our initial studies that notochordal cells secrete the pro - form of ccn2 that is activated likely within the cytoplasm to in turn act in a paracrine fashion upon nucleus pulposus cells. the question remains concerning the possible autocrine activity of ccn2 since it is known to fulfill both functions - and may well do so within the notochordal cell - rich milieu of the ncd canine nucleus pulposus (fig. chondrocytes were cultured for 24 h with either, dmem, 50 ng / ml, 100 ng / ml, 200 ng / ml rctgf and nccm. amplicons were generated from the reverse transcription and subsequent pcr using aggrecan specific primers of 1 g total rna harvested from treated chondrocytes (trizol). the above results reflect mean gene expression ratios of between 5 and 9 separate experiments all from at least three separate sources of chondrocytes and notochord cells. dmem and ctgf 50 were repeated three times, ctgf 100 and 200 were repeated eight times, and nccm was repeated nine times. 210 g notochordal cell lysates run in parallel using rabbit anti - ctgf western blot (black arrowhead) lanes 12. the notochordal cell secretes a pro - form of ccn2 that is likely activated within the cytoplasm to the active form. the possible role of tgf- within this notochordal cell / nucleus pulposus cell milieu is a question at the present. ccn2 is known to be a downstream effector molecule of tgb- signaling - however the specific role of tgf- within this pathway has not yet been tested. notochordal cells secrete a unique form of aggrecan themselves (depicted as notochordal cell secreted increased proteoglycan synthesis). also, soluble factors secreted by notochordal cells activate nucleus pulposus cells to increase their proteoglycan production (indicated as increased proteoglycan synthesis by nucleus pulposus cells in red) aggrecan gene expression (normalized to hprt) for disc - derived chondrocytes. chondrocytes were cultured for 24 h with either, dmem, 50 ng / ml, 100 ng / ml, 200 ng / ml rctgf and nccm. amplicons were generated from the reverse transcription and subsequent pcr using aggrecan specific primers of 1 g total rna harvested from treated chondrocytes (trizol). the above results reflect mean gene expression ratios of between 5 and 9 separate experiments all from at least three separate sources of chondrocytes and notochord cells. dmem and ctgf 50 were repeated three times, ctgf 100 and 200 were repeated eight times, and nccm was repeated nine times. 2006) 10 g notochordal cell lysates run in parallel using rabbit anti - ctgf western blot (black arrowhead) lanes 12. control lanes were murine -cells, lanes 34 schematic depicting notochordal cell interaction with nucleus pulposus cells. the notochordal cell secretes a pro - form of ccn2 that is likely activated within the cytoplasm to the active form. the possible role of tgf- within this notochordal cell / nucleus pulposus cell milieu is a question at the present. is known to be a downstream effector molecule of tgb- signaling - however the specific role of tgf- within this pathway has not yet been tested. notochordal cells secrete a unique form of aggrecan themselves (depicted as notochordal cell secreted increased proteoglycan synthesis). also, soluble factors secreted by notochordal cells activate nucleus pulposus cells to increase their proteoglycan production (indicated as increased proteoglycan synthesis by nucleus pulposus cells in red) it has been reported that the np cells of non - chondrodystrophic canines (notochordal cell - rich) produce proteoglycans that migrate considerably further from the cell prior to becoming immobilized within the extracellular matrix than do proteoglycans produced from notochordal cells of chondrodystrophic dogs (12). in the case of the chondrodystrophic canine, this process may be impeded and therefore hinder the production of a protective peri - cellular matrix and lead to a loss of these otherwise anabolically important cells and increase the rate of degenerative change within the matrix. these differences in proteoglycan biology may explain both structurally and functionally that the notochordal cell - rich disc may afford superior characteristics than the notochordal cell - poor chondrodystrophic disc (stemple 2005). apoptosis or programmed cell death is essential to tissue re - modeling and the normal metabolic process of growth, development and tissue homeostasis. ccn2has been described as having both pro - apoptotic and anti - apoptotic properties, depending upon the type of cells and culture environment studied (lefebvre. it has been reported that inhibiting ccn2 production induces apoptosis of rhabdomyosarcoma cells by removing autocrine ccn2 signaling whereas the addition of ctgf significantly increased cell survival (wegner 1999). on the other hand, it has been reported that ccn2 induces apoptosis via the type i caspase-3 pathway in human aortic smooth muscle cells (ghosh 1988b). the notochordal cell - rich ivd np resists degenerative change whereas notochordal cell - poor discs do not. perhaps part of the resistance to degenerative change on the part of notochordal cell - rich discs is afforded by pro - survival signals secreted by these cells. since ctgf is known to promote ecm synthesis, cell proliferation, proteoglycan and collagen production, our discovery that notochordal cells secrete ccn2 may figure prominently in the resistance of certain species to the development of ddd as well as possibly fulfilling an anti - apoptotic function. the ongoing secretion of ccn-2 on the part of notochordal cells found within the mature mammalian intervertebral disc such as the ncd canine is a fascinating phenomenon. however, unlike fibrotic disease whereby upregulated ccn-2 signaling induces pathological consequences it may be that within the ivd nucleus pulposus ccn-2 signaling is a highly adaptive response that provides a favorable environment for long - term homeostasis. the likely genetic mechanisms responsible for the ongoing survival of notochordal cells within the ncd canine disc as opposed to the cd canine disc are not known. further, the heterogeneity of nucleus pulposus and notochordal cells continue to poorly characterized - there is no definitive marker for notochordal cells. the role played by ccn-2 in the development of the notochord has been partially established in mice and lower animals such as zebrafish. this tends to occur at times when the developing embryo is in a state of flux, such as when it lacks a mature circulatory system. however little is known concerning the biology of notochordal cells that naturally persist within the avascular ivd nucleus pulposus within the mature mammal such as the is the case of the ncd and cd canines. it is likely that the multifunctional role played by ccn-2 exerts other effects upon the extracellular matrix milieu and may be a key player in the maintenance of a healthy compartmentalized tissue such as the ivd nucleus. the ivd is an immune privileged, avascular tissue compartment which is a very different environment than is encountered in most other parts of the body. however there continues to be no explanation for the persistence of notochordal cells within this compartment by some animals and not others. yet it may be that certain essential molecular and signaling switches are activated in the development and maturation of the notochord in some animals such as the ncd canine thus intimately involving ccn-2. such molecular switching in turn may allow these cells to survive through maturity and in so doing, preserve matrix integrity within the fully differentiated tissue compartment such as the ivd nucleus pulposus. to date however this question remains a perplexing and fascinating area yet to be explored. the notochord is common to all higher vertebrates as a developmentally vital structure. during development stiffness to the developing embryo - as well as to function as a source of differentiation signals to local undifferentiated mesenchymal tissues (ming - jyun. 2006 ; smits and lefebvre 2003 ; marcelle. 1999 ; barrionuevo. 2006 ; hirano and najita 1995). the notochord is of mesodermal origin and forms during the gastrulation phase of the developing embryo where the early notochord begins as at the primitive pit and ultimately becomes a rod - like structure that is enveloped within a sheath composed of predominantly extracellular matrix, collagens and proteoglycans, all of which are similar to the composition of cartilage and mesenchymal / fibrous tissues (smits and lefebvre 2003 ; barrionuevo. the notochord most closely resembles cartilage in terms of its tissue type and as such the stiffness it provides during development provides for an axial skeleton - like structure until somitogenesis provides for the development of the axial skeleton (stemple 2005). signals secreted by the notochord are required in order for sclerotomal cells to migrate, condense and differentiate - all of which are central to the formation of the vertebral column and the developing spinal cord (hirano and najita 1995). the spinal cord is formed from condensation of neural crest cells that migrate and lie dorsal to the notochord, forming the neural tube and ultimately the spinal cord (echelard. the terminal stage of notochord development in higher animals is marked by the notochord segmenting during vertebrogenesis and in the vertebral regions it is largely replaced by bone and in the intervertebral areas it forms the centre of the intervertebral disc - the nucleus pulposus (stemple 2004, 2005). a number of factors are involved in the complex process of notochordal evolution specifically including the expression of the brachury gene t that codes for the t - box transcription factor - commonly referred to as t, as well as sonic hedgehog (shh), noggin and pax1 all of which play essential roles that induce differentiation of the sclerotome (barrionuevo 2006 ; ghosh 1988a). the t gene is expressed in all mesodermal cells and is expressed within the notochord until formation of the developing embryonic body is completed (herrmann and kispert 1994). t however is expressed only transiently by other mesodermal cells demonstrating the directed activity of this gene in notochordal development (herrmann and kispert 1994). in mutant mouse studies it has been found that t is necessary for the maintenance of the notochord and that t seems to be a controlling factor in cell motility and/or cell adhesion (herrmann and kispert 1994). interestingly t protein is able to switch the fate of cells from an ectodermal to a ventral mesodermal destination allowing ventral mesodermal cells to respond to diffusible dorsal signal factors. as the following sections detail, there is an early role for ccn-2 in development and in the formation of the notochord in particular. it is therefore an attractive notion that ccn-2 may well interact with such important differentiation and signaling pathways such as those involving t protein as well as other critical transcription factors such as those encoded by the sox genes. for example, it has been shown that ccn-2 interacts with wnt signaling in xenopus development and that it is able to inhibit canonical signaling involving -catenin as well as non - canonical signaling probably via interaction with the wnt receptor complex (mercurio. 2004). further evidence of the complex interaction of ccn-2 with development is that overexpression of ccn-2 in xenopus results in inhibition of neural crest migration - perhaps by virtue of wnt pathway inhibition (mercurio. the reader is referred to the detailed report by mercurio for further details concerning ccn2 activity and wnt signaling (mercurio. once the sclerotome is formed, in addition to t, the notochord is known to be heavily influenced by several key transcription factors encoded by sox5, sox6 and sox9 genes (smits and lefebvre 2003 ; barrionuevo. the sox expression pattern found in the notochord has similarities to that observed in cartilage where sox 5, 6 and 9 are coordinated and linked (lefebvre. the sox genes encode a number of dna - binding proteins that are considered to be important relatively late in development as opposed to the earlier stages where they are considered to have some redundancy (smits and lefebvre 2003). in general, the sox dna binding proteins are thought to perform their various functions in a sterically - defined manner whereby they act as architectural proteins and organize chromatin structure and assemble other dna - bound transcription factors into larger more complex multi - protein units (wegner 1999). it is likely that sox proteins link proteins to signal transduction pathways (such as these multi - protein units) which by themselves may not be capable of inducing signal transduction. sox5 and 6 gene transcripts are known to be expressed in cartilage but no other tissues in the developing mouse and have some kind of co - operative function with sox9-considered to be the master chondrogenic factor (smits and lefebvre 2003 ; lefebvre. sox5 and 6 are considered to be crucial to the survival of notochordal cells within the disc nucleus pulposus, a potentially vital pathway inherent within the cells of animals that retain their notochordal cells into adulthood (moussad and brigstock 2000 ; smits and lefebvre 2003). indeed sox5 and 6 mutant mice demonstrate aberrant removal of notochordal cells from the intervertebral areas and do so later than wild type embryos in mice - the theory being that this delay may be associated with a delay in cartilage development and/or apoptosis of notochordal cells (smits and lefebvre 2003). these sox5 and 6 mutant mice display a failure to form a nucleus pulposus, along with severely distorted vertebral column development confirming the pivotal role that these genes play in directing the development, maturation and survival of the disc nucleus pulposus (smits and lefebvre 2003). in what is likely a more upstream effect, mutant mice lacking sox9 expression fail to develop a normal notochord however the survival of notochordal tissue in the presence of normal sox9 signaling poses a strong argument for the importance of this transcription factor in notochordal cell maintenance in addition to its known role in chondrogenesis and likely the cooperative interaction of sox 5, 6, and 9 (moussad and brigstock 2000). in addition, jun (a major transcription factor essential for embryonic development) elicits anti - apoptotic signaling critical to the survival of notochord cells (croci. 2004 ; marcelle. 1999). however, the precise mechanisms at play in the pivotal morphological evolution of notochordal cells whereby they become located within the nucleus pulposus of the intervertebral disc (ivd) are largely unknown - as is the role played by these cells within the disc (moussad and brigstock 2000 ; pai - sheng (2007). clearly there is a delicate and precise ballet involved with the development of this anatomically central structure whereby aberrant expression of any of these elements create a highly disturbed phenotype. with respect to the role played by ccn-2 during development, a recent study introduced anti - sense oligonucleotides into zebrafish embryos to in order to specifically knockdown ccn-2 expression (ming - jyun 2006). further these investigators also injected a series of ccn-2 promoter - driven green fluorescent protein (gfp) plasmids into one - cell - stage zebrafish embryos in order monitor the expression of ccn-2. the targeted knockdown of ccn-2 led to a malformed and distorted notochord in the developing embryo and many of the embryos died in early developmental time periods (ming - jyun 2006). the promoter - driven gfp signal was detected in the midline, floor plate and head section of the notochord of the developing embryos confirming the expression of ccn-2 during embryological development in general and the notochord specifically. the detection of ccn2 linked gfp within the midline, floor plate and head section of these developing embryos is all the more intriguing when taken in context with the findings that t is considered to be involved with cellular functions concerning cell adhesion (herrmann and kispert 1994). ccn-2 is well known to be highly involved in cell growth, migration and adhesion and although there are no direct references in this regard, the detection of gfp - linkedccn-2 in the developing notochord provides for the notion that an axis involving ccn-2 and t in notochord development and maintenance may be more complex than is currently understood. these findings are of considerable relevance in the context of the lethal phenotype generated by the deletion of ccn-2 in mice that results in severe skeletal dimorphisms that is also inconsistent with life shortly after birth (ivkovic. these ccn-2 deletion mutants are born with severe skeletal abnormalities, malformed rib cages and die due to poor extracellular matrix synthesis, defective cartilage and associated respiratory failure (ivkovic. however, there is as of yet no detailed description of the fate of the intervertebral disc nucleus pulposus - in particular what transpires in terms of notochordal cell development apart from impaired skeletogenesis. with respect to initial notochordal signaling, it is widely recognized in studies involving zebrafish, drosophila and chicks that shh is a key inductive signal for ventral patterning of the cns and that a gradient of signaling seems to exist between dorsal ectodermal and ventral mesodermal signaling in cns development (horne 2004). the floor plate releases shh which results in neuronal patterning that are directly related to the gradient of shh signals and their interaction with undifferentiated mesenchymal cells. it was as a consequence of these important experiments that shh became known as a classic morphogen. it has been reported in other studies that the ccn-2 regulatory region has a shared homology within the zebra fish shh gene ; further lending support to the essential involvement of ccn2 in key developmental processes involving embryological developmental patterning (ming - jyun. it has been reported that coincident with the reduction in the number of notochord cells, the nucleus pulposus is seen to undergo typical internal disorganization accompanied by degenerative changes (ghosh 1988b ; thompson. however, animals that retain notochordal cells within their ivd np do not develop degenerative disc disease nearly as early or as severely as those animals that do not retain these cells. there are natural occurring sub - species of canine that are unique in that one, the non - chondrodystrophic (ncd) canine is protected from developing degenerative disc disease (ddd), whereas the chondrodystrophic (cd) canine (beagles, dachshunds, poodles) suffer significant and early ddd (braund 1975). the single most critical difference between these two sub - species is that the non - chondrodystrophic canine maintains its population of notochordal cells within the nucleus pulposus (np) of the ivd for most of the life of the animal whereas the cd canine contains sparse notochordal cells by even 1 year of age. during the course of harvesting notochordal cells from canine sources the experience of this writer is such that 30 lumbar ivd np s harvested from five (5) 812 month old ncd canines (6 lumbar discs each) will typically yield 404510 notochordal cells. eight (8) cd canines on the other hand using 6 lumbar ivd nps obtained from each will typically yield only 3410 notochordal cells. the disorganization and degenerative changes witnessed in notochordal cell - poor animals strongly suggests that notochord cells when present, act as matrix guardians that continue to secrete growth and other factors therefore modulating a homeostatic protective action on the disc matrix (aguiar. aguiar, johnston and oegema were the first amongst a very few to suggest that notochordal cells were able to biochemically contribute to ivd homeostasis by developing a crude conditioned medium and evaluating proteoglycan production in bovine disc - derived np cells (aguiar. this first study by this group was followed by our own studies of this relationship. in one study we reported that totally serum - free notochordal cell conditioned medium (nccm) obtained from ncd notochordal cells could in a dose - dependent fashion, upregulate proteoglycan production and in a non - dose - dependent fashion increase cell proliferation in bovine caudal disc np cells (erwin and inman 2006). in a second study, also using bovine caudal disc np as the target cells, we demonstrated upregulated gene expression for the essential extracellular matrix proteoglycans aggrecan and versican after 24-hour culture with totally serum - free notochordal cell conditioned medium (erwin. in addition, gene expression for hyaluronic acid synthase-2, the enzyme essential for hyaluronic acid synthesis production was also upregulated in bovine disc np cells. using lc - ms / ms mass spectroscopy we identified the presence of ccn-2 within the conditioned medium (erwin. after we detected ccn-2 within the notochordal cell conditioned medium, we cultured bovine np cells with serum - free notochordal cell conditioned medium (nccm) and measured doses of recombinant human ccn2 (rccn2) (a kind gift from dr. david brigstock, ohio state university) and compared aggrecan gene expression by the bovine np cells. we found that 100200 ng / ml of rccn2 induced upregulation of aggrecan gene expression to exactly the same level of expression as our serum free nccm derived from ncd canine - source notochordal cells (fig. we have recently followed - up our mass spectroscopy findings and have verified the presence of ccn2 in ncd notochordal cell lysates using western blotting techniques (antibody a kind gift from dr. it appears from our initial studies that notochordal cells secrete the pro - form of ccn2 that is activated likely within the cytoplasm to in turn act in a paracrine fashion upon nucleus pulposus cells. the question remains concerning the possible autocrine activity of ccn2 since it is known to fulfill both functions - and may well do so within the notochordal cell - rich milieu of the ncd canine nucleus pulposus (fig. chondrocytes were cultured for 24 h with either, dmem, 50 ng / ml, 100 ng / ml, 200 ng / ml rctgf and nccm. amplicons were generated from the reverse transcription and subsequent pcr using aggrecan specific primers of 1 g total rna harvested from treated chondrocytes (trizol). the above results reflect mean gene expression ratios of between 5 and 9 separate experiments all from at least three separate sources of chondrocytes and notochord cells. dmem and ctgf 50 were repeated three times, ctgf 100 and 200 were repeated eight times, and nccm was repeated nine times. 210 g notochordal cell lysates run in parallel using rabbit anti - ctgf western blot (black arrowhead) lanes 12. the notochordal cell secretes a pro - form of ccn2 that is likely activated within the cytoplasm to the active form. the possible role of tgf- within this notochordal cell / nucleus pulposus cell milieu is a question at the present. ccn2 is known to be a downstream effector molecule of tgb- signaling - however the specific role of tgf- within this pathway has not yet been tested. notochordal cells secrete a unique form of aggrecan themselves (depicted as notochordal cell secreted increased proteoglycan synthesis). also, soluble factors secreted by notochordal cells activate nucleus pulposus cells to increase their proteoglycan production (indicated as increased proteoglycan synthesis by nucleus pulposus cells in red) aggrecan gene expression (normalized to hprt) for disc - derived chondrocytes. chondrocytes were cultured for 24 h with either, dmem, 50 ng / ml, 100 ng / ml, 200 ng / ml rctgf and nccm. amplicons were generated from the reverse transcription and subsequent pcr using aggrecan specific primers of 1 g total rna harvested from treated chondrocytes (trizol). the above results reflect mean gene expression ratios of between 5 and 9 separate experiments all from at least three separate sources of chondrocytes and notochord cells. dmem and ctgf 50 were repeated three times, ctgf 100 and 200 were repeated eight times, and nccm was repeated nine times. 2006) 10 g notochordal cell lysates run in parallel using rabbit anti - ctgf western blot (black arrowhead) lanes 12. control lanes were murine -cells, lanes 34 schematic depicting notochordal cell interaction with nucleus pulposus cells. the notochordal cell secretes a pro - form of ccn2 that is likely activated within the cytoplasm to the active form. the possible role of tgf- within this notochordal cell / nucleus pulposus cell milieu is a question at the present. ccn2 is known to be a downstream effector molecule of tgb- signaling - however the specific role of tgf- within this pathway has not yet been tested. notochordal cells secrete a unique form of aggrecan themselves (depicted as notochordal cell secreted increased proteoglycan synthesis). also, soluble factors secreted by notochordal cells activate nucleus pulposus cells to increase their proteoglycan production (indicated as increased proteoglycan synthesis by nucleus pulposus cells in red) it has been reported that the np cells of non - chondrodystrophic canines (notochordal cell - rich) produce proteoglycans that migrate considerably further from the cell prior to becoming immobilized within the extracellular matrix than do proteoglycans produced from notochordal cells of chondrodystrophic dogs (12). in the case of the chondrodystrophic canine, this process may be impeded and therefore hinder the production of a protective peri - cellular matrix and lead to a loss of these otherwise anabolically important cells and increase the rate of degenerative change within the matrix. these differences in proteoglycan biology may explain both structurally and functionally that the notochordal cell - rich disc may afford superior characteristics than the notochordal cell - poor chondrodystrophic disc (stemple 2005). apoptosis or programmed cell death is essential to tissue re - modeling and the normal metabolic process of growth, development and tissue homeostasis. ccn2has been described as having both pro - apoptotic and anti - apoptotic properties, depending upon the type of cells and culture environment studied (lefebvre. it has been reported that inhibiting ccn2 production induces apoptosis of rhabdomyosarcoma cells by removing autocrine ccn2 signaling whereas the addition of ctgf significantly increased cell survival (wegner 1999). on the other hand, it has been reported that ccn2 induces apoptosis via the type i caspase-3 pathway in human aortic smooth muscle cells (ghosh 1988b). the notochordal cell - rich ivd np resists degenerative change whereas notochordal cell - poor discs do not. perhaps part of the resistance to degenerative change on the part of notochordal cell - rich discs is afforded by pro - survival signals secreted by these cells. since ctgf is known to promote ecm synthesis, cell proliferation, proteoglycan and collagen production, our discovery that notochordal cells secrete ccn2 may figure prominently in the resistance of certain species to the development of ddd as well as possibly fulfilling an anti - apoptotic function. the ongoing secretion of ccn-2 on the part of notochordal cells found within the mature mammalian intervertebral disc such as the ncd canine is a fascinating phenomenon. however, unlike fibrotic disease whereby upregulated ccn-2 signaling induces pathological consequences it may be that within the ivd nucleus pulposus ccn-2 signaling is a highly adaptive response that provides a favorable environment for long - term homeostasis. the likely genetic mechanisms responsible for the ongoing survival of notochordal cells within the ncd canine disc as opposed to the cd canine disc are not known. further, the heterogeneity of nucleus pulposus and notochordal cells continue to poorly characterized - there is no definitive marker for notochordal cells. the role played by ccn-2 in the development of the notochord has been partially established in mice and lower animals such as zebrafish. this tends to occur at times when the developing embryo is in a state of flux, such as when it lacks a mature circulatory system. however little is known concerning the biology of notochordal cells that naturally persist within the avascular ivd nucleus pulposus within the mature mammal such as the is the case of the ncd and cd canines. it is likely that the multifunctional role played by ccn-2 exerts other effects upon the extracellular matrix milieu and may be a key player in the maintenance of a healthy compartmentalized tissue such as the ivd nucleus. the ivd is an immune privileged, avascular tissue compartment which is a very different environment than is encountered in most other parts of the body. however there continues to be no explanation for the persistence of notochordal cells within this compartment by some animals and not others. yet it may be that certain essential molecular and signaling switches are activated in the development and maturation of the notochord in some animals such as the ncd canine thus intimately involving ccn-2. such molecular switching in turn may allow these cells to survive through maturity and in so doing, preserve matrix integrity within the fully differentiated tissue compartment such as the ivd nucleus pulposus. to date however this question remains a perplexing and fascinating area yet to be explored. there are many open questions concerning the role played by ccn-2 both in development and in maturity in a host of physiological processes. taken together with a number of other reports ctgf / ccn-2 appears to play a pivotal role within numerous signaling pathways that are important in development, cellular differentiation, cell migration, proliferation, cell adhesion, and a host of other crucial cellular regulatory functions. as highlighted earlier in this paper gfp - conjugated ccn2 expression has been detected very early in development in the zebrafish notochord. the detection of ccn2 activity at the critical time when the notochord is undergoing its earliest organization and differentiation when taken in context with the capacity for ccn2 to suppress wnt signaling suggests the tantalizing notion that ccn2 could occupy an unknown yet pivotal role much earlier in development than current studies have shown. the situation involving ccn2 within the canine adult intervertebral disc nucleus pulposus represents something of an enigma ; perhaps in this case mature tissue within an isolated and avascular tissue compartment provides a unique physiological niche whereby under certain circumstances, sustained ongoing secretion of ccn-2 is more friend than foe. | the growth regulating factor ctgf / ccn-2 is an integral factor in growth and development, connective tissue maintenance, wound repair and cell cycle regulation. it has recently been reported that ctgf / ccn-2 is involved in very early development having been detected in early notochord formation in zebrafish using ctgf / ccn-2 promoter - driven green fluorescent protein (gfp) plasmids. in these studies fluorescence was detected early in the developing embryos, a finding of considerable significance in that ctgf / ccn-2 deficient mutant mice die early after birth due to severe cartilage and skeletal dysplasia and respiratory failure. such findings confirm the importance of ctgf / ccn-2 in development and of the necessary and sufficient role of this molecule in formation of the skeleton, extracellular matrix and chondrogenesis. of particular relevance to the relationship between the notochordal cell and ctgf / ccn-2 there is a remarkable sub - species of canine, the non - chondrodystrophic canine that is protected from developing degenerative disc disease (ddd). these animals are unique in that they preserve the population of notochordal cells within their disc nucleus (np) and these cells secrete ctgf / ccn-2. we have detected ctgf / ccn-2 within conditioned medium developed from the notochordal cells of these animals (nccm) and used this conditioned medium to demonstrate robustly increased proteoglycan production. the addition of recombinant human ctgf / ccn-2 to totally serum - free media containing cultures of bovine np cells replicated the robustly increased aggrecan gene expression found with nccm alone strongly suggesting the importance of the effect of ctgf / ccn-2 in notochordal cell biology within the disc nucleus of non - chondrodystrophic canines. the chondrodystrophic canine, another sub - species on the other hand are almost totally devoid of notochordal cells and they develop ddd profoundly and early. these two sub - species of canine reflect a naturally occurring animal model that is an excellent example of differential notochordal cell survival and possible associated developmental differences in extracellular maintenance. |
in recent years the prevalence of gastrointestinal conditions, such as gastro - oesophageal reflux disease, has increased (1, 2). these changes in prevalence have been attributed to changes in lifestyle and an increase in the prevalence of smoking, hypertension, obesity and psychological stresses in the general population (3). this increase in prevalence of gastrointestinal conditions has led to an increase in the demand for endoscopic procedures (2). endoscopic procedures are considered safe but they are not without risk of serious complications such as perforation of a viscous. studies from the united states suggested that more than 98% of colonoscopies were performed with sedation (6, 7). by contrast, endoscopy is commonly performed without sedation or anesthesia in many european countries (2). the level of patient satisfaction with gi endoscopic procedure is an important criterion to indicate the level of expertise in endoscopy (8). like other aspects of medicine, there have been several studies to investigate the level of satisfaction in patient underwent upper endoscopy and colonoscopy. these studies have been designed to identify opportunities to improve the quality of elective procedures (1, 811). (12) and others (7, 13) introduced a high - quality sedation with newer treatments such as propofol alone and in combination with older therapies. nowadays, the level of satisfaction with anesthesia and sedation during gi endoscopy is a matter of concern and is an important factor in decision making about this procedure (10). in iran, use of sedation and anesthesia in endoscopic procedures is not common and the level of patient 's satisfaction and knowledge of physician has not been investigated. the aim of this study was to assess satisfaction level and its related factors among patients who had an anesthesia during upper endoscopy or colonoscopy. this prospective cross - sectional study was conducted on gi endoscopic patients in resalat hospital in tehran, iran. all patients were at least 18 years old and had been referred for elective outpatient upper endoscopy or colonoscopy in the endoscopy department of the hospital between june, 2010, and october, 201. the study was approved by the ethics committee of the army university of medical sciences. the patients were assured that their private information would be kept confidential and a written informed consent was obtained from them. the initial intravenous dose of propofol was 1.01.4mg / kg, followed by additional bolus doses if necessary. thereafter, it was administered intravenously at 1.02.0mg / kg / hour continuously during the procedure and additional doses (0.40.6mg / kg) were administered if the patient began to move. at the beginning of the procedure, supplemental oxygen (2 l / minute via nasal cannula) and 15 mg of intravenous pentazocine were administered. to evaluate patient 's satisfaction with anesthesia, we used a translation from the patient satisfaction with sedation instrument (pssi) (10). response options were presented on a 7-point likert scale (range, 7 [very satisfied ] to 1 [very dissatisfied ]). questions were categorized into 4 sub scales : anesthesia delivery (2 items), procedural recall (4 items), side effects (10 items) and global satisfaction (4 items). statistical analyses were performed using spss v.16 (spss, chicago, illinois, usa) software. descriptive statistics was included frequency and percentage for categorical variables, and mean [sd ] for continuous variables. a total of 379 patients (159 men and 220 women) were included the study. 81% of patients had at least a high school diploma or higher levels of education. characteristics of patient population (n=379) upper gi endoscopy was the major procedure : 337 patients (92.3%) underwent upper endoscopy and 19 patients (5.2%) had a colonoscopy procedure. both procedures were performed in 9 patients (2.4%). (table 2) summary of post procedure questionnaire results the pssi subscale scores and total satisfaction (question 1 - 16) score showed good internal consistency reliability. the reliabilities for the subscale scores were 0.936 for procedural recall, 0.818 for anesthesia delivery, and 0.983 for side effects ; for the total satisfaction (question 1 - 16) score, reliability was 0.92. the mean score of satisfaction with anesthesia delivery, procedural recall, side effects, global satisfaction, and total satisfaction (question 1 - 16) were 6.15 1.23, 5.65 1.48, 5.24 1.16, 5.01 1.29, and 5.46 1.14, respectively (table 3). anesthesia satisfaction in detail there was a significant difference in patients satisfaction level when results were compared according to employment (p=0.02) and education (p=0.01). when considering results by employment status ; mean satisfaction levels were (from the highest to the lowest respectively) : retired (5.90.8), student (5.60.9), employee (5.51.2), private business (5.41.0), unemployed (5.40.9), and housekeeper (5.11.1). the level of satisfaction was significantly associated with the level of education, so that patients with graduate level of education were more satisfied than other categories of education, especially illiterate patients. but there were no significant difference between patient 's satisfaction and their gender, age, residence (urban or rural), prior experience of anesthesia, and the procedure which they underwent. in recent years, satisfaction of patients with endoscopic procedures has became important in health care systems and is considered as a critical criteria to evaluate a physician 's ability in performing of this mild invasive procedure (14). as endoscopic procedures are used for both screening and therapeutic purposes, the quality of the procedure, including patient satisfaction, is important. there are a few studies to evaluate the level of patients satisfaction and its related factors (15, 16). in our country, experience with anesthesia for gi endoscopy is developing and we are unaware of any standardized documentation to assess a patients satisfaction with the anesthesia. in view of this, we applied a previously validated questionnaire to assess patient satisfaction with anesthesia for endoscopy (10). cronbach 's alpha for this translated version was 0.92 which indicated it was highly valid for measuring the satisfaction in our patients. our results showed that patients satisfaction was significantly associated with the educational achievement and differed significantly according to employment. the level of satisfaction was positively correlated with the level of education so that highest satisfaction level was obtained in graduated patients. factors that may explain increased satisfaction among the more educated patients undergoing procedures may include an increased understanding of the procedure, leading to reduced anxiety, compared to those with a lower level of education. the highest satisfaction was found among retired patients and the lowest score was seen in housekeepers. one may assume that retired patients may have had a previous high level of educational attainment, or lower expectations of health care provision. surprisingly, there was no correlation between patients satisfaction and their gender, age, place of living and even prior experience of anesthesia. this is contrary to previous studies that found younger age as well as female gender was associated with patient dissatisfaction (17, 18). it seems that the satisfaction level of patients is more related to their knowledge than their physical and environmental situation. it was performed in a single iranian health care center and confounding factors such as the quality of bowel preparation and duration of endoscopic procedure were not assessed. in conclusion, our results identified two factors relevant to the satisfaction with endoscopic procedures : level of education and employment status. it is recommended to perform studies in this field to determine and evaluate other factors that may have influence on patients satisfaction with anesthesia in endoscopy. | aimthe purpose of this study was to assess satisfaction level and related factors among patients who had an anesthesia during endoscopic procedures ; and also validate a questionnaire for evaluating satisfaction with anesthesia.backgroundthe level of patient satisfaction with gi endoscopic procedure is an important criterion to indicate the level of expertise in endoscopy.patients and methodswe performed a prospective descriptive study at resalat hospital, tehran, iran. three hundred seventy nine elective patients undergoing anesthesia for gi endoscopy procedure in 2010 were recruited. a 20-item questionnaire was used to evaluate the satisfaction with the anesthesia. the questionnaire was answered within 72 hours after the procedure. the satisfaction was graded into four major groups : anesthesia delivery, procedural recall, side effects and global satisfaction.resultsthe level of satisfaction with anesthesia and its related factors were determined. the mean score of satisfaction with anesthesia delivery, procedural recall, side effects, global satisfaction, and total satisfaction (question 1 - 16) were 6.15 1.23, 5.65 1.48, 5.24 1.16, 5.01 1.29, and 5.46 1.14, respectively. there was a significant difference in patients satisfaction level between different jobs (p=0.02) as well as different levels of education (p=0.01).conclusionhigher educational level was accompanied with greater satisfaction. the highest satisfaction score was seen among retired patients and the lowest level was found in housekeepers. |
the study comprised 2 cohorts of patients : an imaging cohort, consisting of subjects with stable angina and mi, and an outcome cohort, comprising patients enrolled in the global registry of acute coronary events (grace).18 consecutive patients with acute st - segment elevation mi (stemi), non - stemi,19 and stable angina pectoris scheduled for invasive coronary angiography were recruited from the royal infirmary of edinburgh, as described previously.20 patients with mi fulfilled the criteria for type 1 mi according to the universal definition of mi.19 stemi was defined as new st - segment elevation at the j point in 2 contiguous leads with the following cut points : 0.1 mv in all leads except v2 to v3, for which the thresholds were 0.2 mv for men and 0.25 mv for women. consecutive patients with stable angina pectoris were recruited if they had typical symptoms of exertional anginal chest pain, had previously documented coronary artery disease (> 70% stenosis of at least 1 major epicardial coronary artery), and were scheduled for invasive coronary angiography. patients were excluded if they had suffered an acute coronary syndrome within the previous 3 months. other exclusion criteria were age 250 mol / l), known contrast allergy, and inability to provide informed consent. studies were performed with the approval of the local research ethics committee, in accordance with the declaration of helsinki, and with the written informed consent of each participant. all patients underwent a comprehensive baseline clinical assessment including evaluation of their cardiovascular risk factor profile. blood was drawn from all participants for evaluation of plasma c - reactive protein (crp) concentrations, which were measured using the multigent crp vario assay (archi - tech csystems assay ; abbott laboratories). all patients underwent pet - ct imaging of the thorax with a hybrid scanner (biograph mct ; siemens medical systems) using 18f - fdg as well as coronary calcium scoring and ct angiography of the aorta and coronary arteries.2022 subjects were administered a target dose of 200 mbq 18f - fdg intravenously and subsequently rested in a quiet environment for 90 minutes. a low - dose attenuation - correction ct scan (50 mas, 120 kev with care dose 4d) was then performed, followed by pet imaging of the thorax, covering 1 pet bed for 20 minutes. patients were asked to observe a low - carbohydrate, high - protein, and high - fat diet and to refrain from alcohol intake for at least 24 hours prior to the 18f - fdg scan to minimize cardiac uptake. following acquisition of the pet data, an electrocardiogram - gated breath - hold ct scan (non contrast enhanced, 40 mas / rotation, 120 kv ; siemens medical systems) was performed for calcium scoring. anonymized pet - ct data sets were presented in a random order on an osirix workstation (64 bit ; version 5.5.1 ; osirix imaging software) to trained observers (n.v.j., a.s.) who were blinded to the patients clinical status.18 to aid image analysis, pet images were fused with the ct angiograms, and regions of interest were drawn around the thoracic aorta on serial 3-mm axial slices. within these regions, mean and maximum tracer activities were measured using standard uptake values and corrected for blood - pool activity in the superior vena cava to provide tissue - to - background ratios (tbrs).2024 the ascending aorta was defined as the segment of the aorta from the lower level of the right pulmonary artery up to the last slice at which the aorta maintained its circular cross - sectional appearance. the descending aorta was defined similarly as the region extending up from the tip of the diaphragm to the last circular slice. the aortic arch was defined as the region of aorta connecting the ascending and descending aortas (figure1). aortic radiotracer uptake was quantified using the method of fayad.25 in brief, the following measures of uptake were measured on axial slices across the aorta as a whole and within each region : tbrmax, the average of the maximum tbr values measured on each axial slice ; tbrmean, the average of the mean tbr values measured on each slice ; max tbr, the maximum uptake tbr value in any axial section ; and tbrmds, the most diseased segment, defined as the highest maximal tbr value averaged over 3 consecutive slices.25 thirty patients were selected randomly to test the repeatability of 18f - fdg measurements in the aorta. fifteen patients from each cohort were selected, and 2 trained readers (n.v.j., s.p.l.) quantified aortic activity independently. activity within nonvascular tissue was assessed using oval - shaped regions of interest drawn within paraspinal muscle (area 7 cm) on 5 consecutive axial slices. the aorta was segmented into ascending aorta (a), arch of aorta (b), and descending aorta (c), as shown. regions of interest were drawn around the aorta on axial slices to provide measures of radiotracer uptake. ct analysis was performed on a dedicated cardiovascular workstation (vitrea ; vital images). vessel - specific and total agatston calcium scores were calculated, as described previously,21 for the coronary arteries, the aorta, and its different regions using a threshold of 130 hounsfield units.26 full details of the grace methods have been published previously.27,28 the edinburgh cohort of the prospectively maintained grace database was used to identify 1003 patients admitted with an acute coronary syndrome between january 20, 2003, and june 9, 2009.29 to be eligible, patients (aged > 18 years) had to be admitted with an acute coronary syndrome as a presumptive diagnosis and had to have at least 1 of the following conditions : electrocardiographic changes consistent with acute coronary syndrome, serial increases in biomarkers of cardiac necrosis, or documented coronary artery disease. for consistency, only patients who had plasma troponin i concentrations quantified (abbott laboratories) in a standardized accredited laboratory were included. exclusion criteria were secondary myocardial injury precipitated or accompanied by a significant comorbidity, trauma, or surgery. information regarding patient demographic characteristics, medical history, timing and occurrence of acute coronary symptoms, clinical characteristics, electrocardiographic findings, treatment approaches, and in - hospital outcomes was collected through completion of a standardized proforma.27,28 the baseline and peak troponin i concentrations during admission were recorded, and patients were placed in tertiles based on their peak troponin i to reflect the degree of myocardial injury and the size of their infarct.30 the primary end point of the analysis was early recurrent mi following the index admission, defined as recurrent type 1 mi within 30 days of index admission.18 to avoid confounding with the index presentation, only those patients having recurrent mi beyond the first 24 hours after presentation were analyzed, as described previously.18 we also examined the factors associated with late recurrent mi as an exploratory end point, defined as a recurrent type 1 mi > 30 days following index admission. as a prespecified end point of our previously reported trial (clinicaltrials.gov identifier nct01749254),20 we explored 18f - fdg uptake in remote aortic atheroma of patients with recent mi or stable coronary heart disease. nonparametric data were presented as median (interquartile range [iqr ]) and compared using the mann altman method and intraclass correlation coefficients with 95% cis. the fisher exact test or the chi - square test was used for analysis of categorical variables. correlation of parametric data was assessed using the pearson correlation coefficient, and the spearman rank correlation was used for nonparametric data. patients in the registry cohort were placed in tertiles according to their peak troponin concentration. meier curves were used to estimate the distribution of early recurrent mi across the tertiles. univariate analysis was undertaken to identify associations with early (30 days) and late (> 30 days) recurrent mi that were then entered into the multivariate logistic regression model based on a univariate association of p 70% stenosis of at least 1 major epicardial coronary artery), and were scheduled for invasive coronary angiography. patients were excluded if they had suffered an acute coronary syndrome within the previous 3 months. other exclusion criteria were age 250 mol / l), known contrast allergy, and inability to provide informed consent. studies were performed with the approval of the local research ethics committee, in accordance with the declaration of helsinki, and with the written informed consent of each participant. all patients underwent a comprehensive baseline clinical assessment including evaluation of their cardiovascular risk factor profile. blood was drawn from all participants for evaluation of plasma c - reactive protein (crp) concentrations, which were measured using the multigent crp vario assay (archi - tech csystems assay ; abbott laboratories). all patients underwent pet - ct imaging of the thorax with a hybrid scanner (biograph mct ; siemens medical systems) using 18f - fdg as well as coronary calcium scoring and ct angiography of the aorta and coronary arteries.2022 subjects were administered a target dose of 200 mbq 18f - fdg intravenously and subsequently rested in a quiet environment for 90 minutes. a low - dose attenuation - correction ct scan (50 mas, 120 kev with care dose 4d) was then performed, followed by pet imaging of the thorax, covering 1 pet bed for 20 minutes. patients were asked to observe a low - carbohydrate, high - protein, and high - fat diet and to refrain from alcohol intake for at least 24 hours prior to the 18f - fdg scan to minimize cardiac uptake. following acquisition of the pet data, an electrocardiogram - gated breath - hold ct scan (non contrast enhanced, 40 mas / rotation, 120 kv ; siemens medical systems) was performed for calcium scoring. anonymized pet - ct data sets were presented in a random order on an osirix workstation (64 bit ; version 5.5.1 ; osirix imaging software) to trained observers (n.v.j., a.s.) who were blinded to the patients clinical status.18 to aid image analysis, pet images were fused with the ct angiograms, and regions of interest were drawn around the thoracic aorta on serial 3-mm axial slices. within these regions, mean and maximum tracer activities were measured using standard uptake values and corrected for blood - pool activity in the superior vena cava to provide tissue - to - background ratios (tbrs).2024 the ascending aorta was defined as the segment of the aorta from the lower level of the right pulmonary artery up to the last slice at which the aorta maintained its circular cross - sectional appearance. the descending aorta was defined similarly as the region extending up from the tip of the diaphragm to the last circular slice. the aortic arch was defined as the region of aorta connecting the ascending and descending aortas (figure1). aortic radiotracer uptake was quantified using the method of fayad.25 in brief, the following measures of uptake were measured on axial slices across the aorta as a whole and within each region : tbrmax, the average of the maximum tbr values measured on each axial slice ; tbrmean, the average of the mean tbr values measured on each slice ; max tbr, the maximum uptake tbr value in any axial section ; and tbrmds, the most diseased segment, defined as the highest maximal tbr value averaged over 3 consecutive slices.25 thirty patients were selected randomly to test the repeatability of 18f - fdg measurements in the aorta. activity within nonvascular tissue was assessed using oval - shaped regions of interest drawn within paraspinal muscle (area 7 cm) on 5 consecutive axial slices. the aorta was segmented into ascending aorta (a), arch of aorta (b), and descending aorta (c), as shown. regions of interest were drawn around the aorta on axial slices to provide measures of radiotracer uptake. ct analysis was performed on a dedicated cardiovascular workstation (vitrea ; vital images). vessel - specific and total agatston calcium scores were calculated, as described previously,21 for the coronary arteries, the aorta, and its different regions using a threshold of 130 hounsfield units.26 consecutive patients with acute st - segment elevation mi (stemi), non - stemi,19 and stable angina pectoris scheduled for invasive coronary angiography were recruited from the royal infirmary of edinburgh, as described previously.20 patients with mi fulfilled the criteria for type 1 mi according to the universal definition of mi.19 stemi was defined as new st - segment elevation at the j point in 2 contiguous leads with the following cut points : 0.1 mv in all leads except v2 to v3, for which the thresholds were 0.2 mv for men and 0.25 mv for women. consecutive patients with stable angina pectoris were recruited if they had typical symptoms of exertional anginal chest pain, had previously documented coronary artery disease (> 70% stenosis of at least 1 major epicardial coronary artery), and were scheduled for invasive coronary angiography. patients were excluded if they had suffered an acute coronary syndrome within the previous 3 months. other exclusion criteria were age 250 mol / l), known contrast allergy, and inability to provide informed consent. studies were performed with the approval of the local research ethics committee, in accordance with the declaration of helsinki, and with the written informed consent of each participant. all patients underwent a comprehensive baseline clinical assessment including evaluation of their cardiovascular risk factor profile. blood was drawn from all participants for evaluation of plasma c - reactive protein (crp) concentrations, which were measured using the multigent crp vario assay (archi - tech csystems assay ; abbott laboratories). all patients underwent pet - ct imaging of the thorax with a hybrid scanner (biograph mct ; siemens medical systems) using 18f - fdg as well as coronary calcium scoring and ct angiography of the aorta and coronary arteries.2022 subjects were administered a target dose of 200 mbq 18f - fdg intravenously and subsequently rested in a quiet environment for 90 minutes. a low - dose attenuation - correction ct scan (50 mas, 120 kev with care dose 4d) was then performed, followed by pet imaging of the thorax, covering 1 pet bed for 20 minutes. patients were asked to observe a low - carbohydrate, high - protein, and high - fat diet and to refrain from alcohol intake for at least 24 hours prior to the 18f - fdg scan to minimize cardiac uptake. following acquisition of the pet data, an electrocardiogram - gated breath - hold ct scan (non contrast enhanced, 40 mas / rotation, 120 kv ; siemens medical systems) was performed for calcium scoring. anonymized pet - ct data sets were presented in a random order on an osirix workstation (64 bit ; version 5.5.1 ; osirix imaging software) to trained observers (n.v.j., a.s.) who were blinded to the patients clinical status.18 to aid image analysis, pet images were fused with the ct angiograms, and regions of interest were drawn around the thoracic aorta on serial 3-mm axial slices. within these regions, mean and maximum tracer activities were measured using standard uptake values and corrected for blood - pool activity in the superior vena cava to provide tissue - to - background ratios (tbrs).2024 the ascending aorta was defined as the segment of the aorta from the lower level of the right pulmonary artery up to the last slice at which the aorta maintained its circular cross - sectional appearance. the descending aorta was defined similarly as the region extending up from the tip of the diaphragm to the last circular slice. the aortic arch was defined as the region of aorta connecting the ascending and descending aortas (figure1). aortic radiotracer uptake was quantified using the method of fayad.25 in brief, the following measures of uptake were measured on axial slices across the aorta as a whole and within each region : tbrmax, the average of the maximum tbr values measured on each axial slice ; tbrmean, the average of the mean tbr values measured on each slice ; max tbr, the maximum uptake tbr value in any axial section ; and tbrmds, the most diseased segment, defined as the highest maximal tbr value averaged over 3 consecutive slices.25 thirty patients were selected randomly to test the repeatability of 18f - fdg measurements in the aorta. fifteen patients from each cohort were selected, and 2 trained readers (n.v.j., s.p.l.) quantified aortic activity independently. activity within nonvascular tissue was assessed using oval - shaped regions of interest drawn within paraspinal muscle (area 7 cm) on 5 consecutive axial slices. the aorta was segmented into ascending aorta (a), arch of aorta (b), and descending aorta (c), as shown. regions of interest were drawn around the aorta on axial slices to provide measures of radiotracer uptake. ct analysis was performed on a dedicated cardiovascular workstation (vitrea ; vital images). vessel - specific and total agatston calcium scores were calculated, as described previously,21 for the coronary arteries, the aorta, and its different regions using a threshold of 130 hounsfield units.26 full details of the grace methods have been published previously.27,28 the edinburgh cohort of the prospectively maintained grace database was used to identify 1003 patients admitted with an acute coronary syndrome between january 20, 2003, and june 9, 2009.29 to be eligible, patients (aged > 18 years) had to be admitted with an acute coronary syndrome as a presumptive diagnosis and had to have at least 1 of the following conditions : electrocardiographic changes consistent with acute coronary syndrome, serial increases in biomarkers of cardiac necrosis, or documented coronary artery disease. for consistency, only patients who had plasma troponin i concentrations quantified (abbott laboratories) in a standardized accredited laboratory were included. exclusion criteria were secondary myocardial injury precipitated or accompanied by a significant comorbidity, trauma, or surgery. information regarding patient demographic characteristics, medical history, timing and occurrence of acute coronary symptoms, clinical characteristics, electrocardiographic findings, treatment approaches, and in - hospital outcomes was collected through completion of a standardized proforma.27,28 the baseline and peak troponin i concentrations during admission were recorded, and patients were placed in tertiles based on their peak troponin i to reflect the degree of myocardial injury and the size of their infarct.30 the primary end point of the analysis was early recurrent mi following the index admission, defined as recurrent type 1 mi within 30 days of index admission.18 to avoid confounding with the index presentation, only those patients having recurrent mi beyond the first 24 hours after presentation were analyzed, as described previously.18 we also examined the factors associated with late recurrent mi as an exploratory end point, defined as a recurrent type 1 mi > 30 days following index admission. as a prespecified end point of our previously reported trial (clinicaltrials.gov identifier nct01749254),20 we explored 18f - fdg uptake in remote aortic atheroma of patients with recent mi or stable coronary heart disease. nonparametric data were presented as median (interquartile range [iqr ]) and compared using the mann altman method and intraclass correlation coefficients with 95% cis. the fisher exact test or the chi - square test was used for analysis of categorical variables. correlation of parametric data was assessed using the pearson correlation coefficient, and the spearman rank correlation was used for nonparametric data. patients in the registry cohort were placed in tertiles according to their peak troponin concentration. meier curves were used to estimate the distribution of early recurrent mi across the tertiles. univariate analysis was undertaken to identify associations with early (30 days) and late (> 30 days) recurrent mi that were then entered into the multivariate logistic regression model based on a univariate association of p 50 000 ng / l ] ; p 4-fold higher among patients in the highest troponin tertile compared with the lowest, whereas risk was doubled in the middle tertile compared with the lowest (table4 and figure3). the variables with univariate association of p 30 day) recurrent mi data are shown as relative risk (95% ci). patients were placed in tertiles according to their peak plasma troponin i concentrations (tertile 1, 220 ng / l ; tertile 2, 230 to 6130 ng / l ; tertile 3, 6140 ng in contrast, recurrent late mi was not associated with troponin tertiles on univariate analysis (tertile 2 versus 1 : relative risk 1.07 [95% ci 0.64 to 1.79 ], p=0.79 ; tertile 3 versus 1 : relative risk 0.80 [95% ci 0.46 to 1.39 ], p=0.43) but rather was more closely related to traditional predictors of mi (table4). overall, 40 patients with stable angina and 40 with mi underwent 18f - fdg pet imaging. the median time between hospitalization and 18f - fdg pet imaging was 11 days (iqr 8 to 17 days) in patients with mi. compared with patients with stable angina, patients with mi were younger, had less extensive coronary artery disease (table1), and had lower coronary artery calcium scores (coronary artery calcium score : stable angina 599 agatston units [au ; iqr 60 to 1302 au ] ; mi 159 au [iqr 42 to 456 au ] ; p=0.006) (tables1 and 2). although apparently higher aortic calcium scores were noted in patients with stable angina, this difference did not reach statistical significance (aortic calcium scores ; stable angina 538 au [iqr 4 to 1870 au ] ; mi 135 au [iqr 0 to 805 au ] ; p=0.12) (table2). baseline characteristics of patients with coronary artery disease data are shown as meansd or number (percentage) except as noted. acei indicates angiotensin converting enzyme inhibitor ; arb, angiotensin receptor blocker ; bmi, body mass index ; cabg, coronary artery bypass grafting ; ctca, computed tomography coronary angiography ; cva, cerebrovascular accident ; hdl, high - density lipoprotein ; iqr, interquartile range ; ldl, low - density lipoprotein ; mi, myocardial infarction ; nstemi, non st - segment elevation myocardial infarction ; pci, percutaneous coronary intervention ; stemi, st - segment elevation myocardial infarction ; tia, transient ischemic attack. aortic calcium scores and 18f - fdg tissue - to - background ratios in patients with stable angina and myocardial infarction 18f - fdg, 18f - fluorodeoxyglucose ; ct, computed tomography ; iqr, interquartile range ; max tbr, highest value tissue - to - background ratios in any axial slice ; mean tbrmax, average of the maximum tissue - to - background ratios across all slices in the segment ; mean tbrmean, average of the mean tissue - to - background ratios for all slices in the segment ; nstemi, non st - segment elevation myocardial infarction ; stemi, st - segment elevation myocardial infarction ; tbrmds, tissue - to - background ratio in the most diseased segment. the reproducibility of tbr measurements in the aorta for tracer activity was excellent, with no fixed or proportional biases and with narrow limits of agreement (table s1). in contrast to the calcium scores, 18f - fdg uptake was 20% higher in the aortas of patients with recent mi than those with stable coronary artery disease (table2 and figure2). this finding was consistent in all regions of the aorta assessed (entire thoracic aorta, ascending aorta, aortic arch, descending aorta ; all p 50 000 ng / l ] ; p 50 000 ng / l ] versus 3800 ng / l ] ; p 4-fold higher among patients in the highest troponin tertile compared with the lowest, whereas risk was doubled in the middle tertile compared with the lowest (table4 and figure3). the variables with univariate association of p 30 day) recurrent mi data are shown as relative risk (95% ci). patients were placed in tertiles according to their peak plasma troponin i concentrations (tertile 1, 220 ng / l ; tertile 2, 230 to 6130 ng / l ; tertile 3, 6140 ng / l). in contrast, recurrent late mi was not associated with troponin tertiles on univariate analysis (tertile 2 versus 1 : relative risk 1.07 [95% ci 0.64 to 1.79 ], p=0.79 ; tertile 3 versus 1 : relative risk 0.80 [95% ci 0.46 to 1.39 ], p=0.43) but rather was more closely related to traditional predictors of mi (table4). using 18f - fdg pet imaging, in patients with recent mi, we demonstrated increased metabolic activity in remote aortic atherosclerotic plaques that correlated with the degree of myocardial necrosis and exceeded that observed in patients with stable coronary disease who had a greater atherosclerotic burden. using the grace registry, we explored the clinical relevance of these findings to assess whether infarct size and the associated increase in atherosclerotic inflammation could predict recurrent coronary atherothombotic events in everyday clinical practice. intriguingly, patients with the largest infarcts had a > 4-fold increase in the risk of early recurrent mi, with baseline tertiles of plasma troponin concentration emerging as an independent predictor of these events. consequently, we provided clinical data to support the hypothesis that mi exacerbates systemic atherosclerotic inflammation, destabilizes remote atheromatous plaque, and causes an increase in early recurrent atherothrombotic events. preclinical data have indicated that mi induces a macrophage - driven proinflammatory state31 that directly increases inflammation in remote atheroma and induces further atherosclerosis.12,32,33 in a mouse model, mi induced by coronary artery ligation increased splenic monocyte motility,12 with these cells exiting the spleen en masse and migrating both to the injured myocardium and, crucially, to remote atherosclerotic plaque.34 in addition, sympathetic stimulation following infarction increased the production and liberation of hematopoietic stem and progenitor cells from the bone marrow, resulting in further increases in circulating monocytes and the accumulation of macrophages within regions of remote atheroma.12 the net result was a marked accumulation of macrophages in remote atherosclerotic plaque following mi that resulted in acceleration of the disease process in these areas. the first aim of the current study was to test whether this proinflammatory phenomenon also occurred in humans, using 18f - fdg as a marker of vascular macrophage inflammatory activity. because we could not measure 18f - fdg uptake before and after mi, we prospectively compared uptake between patients with stable coronary heart disease and those with recent mi.18 despite a comparable or lower overall aortic and coronary plaque burden, aortic 18f - fdg uptake was 20% higher in patients who had sustained a recent mi. indeed, the aortic 18f - fdg uptake was consistently increased across all regions of the thoracic aorta. in contrast, paraspinal muscle uptake was similar between the cohorts, indicating a specific vascular and atherosclerotic response rather than generalized nonspecific inflammation across all tissues. based on clinical factors and peak plasma troponin concentration, patients with stemi had larger infarcts and demonstrated greater increases in aortic 18f - fdg uptake than those with non - stemi. we observed good correlation between peak plasma troponin i concentrations and aortic 18f - fdg activity, suggesting that the association may be causal. next we investigated whether the observed increases in vascular inflammatory activity were of clinical importance in actual practice. our imaging cohort was not large enough to address this question, so we turned to data from the well - established grace registry. in > 1000 patients, we demonstrated that the size of the initial mi emerged as an independent predictor of early recurrent mi over and above traditional risk factors, with a 4-fold increase in these events among those with the biggest infarcts. this finding indicates that the increased vascular inflammation associated with mi translates into adverse clinical events, perhaps due to plaque destabilization and the associated increased risk of rupture. interestingly, our data also suggest that the clinical effects of this systemic inflammatory response are only transitory, with no association between infarct size and later recurrent events after 30 days, by which time the inflammation will have subsided and plaques begun to stabilize once more. our data are also consistent with those of the smaller (n=378) evaluation of mcc-135 for left ventricular salvage in acute myocardial infarction (evolve) randomized controlled trial,35 which also observed an increased early event rate in patients in the highest tertile of troponin concentration, with a trend for an increase in recurrent mi. our findings are supported by recently reported studies.3639 patients with acute coronary syndromes have increased plaque vulnerability in nonculprit lesions, with increased incidence of thin - capped fibroatheromas and adherent thrombus.39 furthermore, patients with stemi have accelerated plaque progression in nonculprit lesions on follow - up angiography,37 whereas stenting of such lesions at the same time as culprit lesions reduces adverse cardiovascular events.38 moreover, patients with unstable coronary disease have higher metabolic carotid plaque activity compared with patients with stable disease, suggesting a panvascular inflammatory process, as indicated by our study.36 taken together, our data offer clear support for the hypothesis that mi begets early recurrent mi due to upregulated macrophage - mediated inflammation in remote atherosclerotic plaque. it would also offer an explanation for the increased incidence of ischemic stroke following mi and would explain why immediate reperfusion for stemi and the resultant limitation in myocardial injury confer greater reduction in the risk of recurrent events than revascularization after infarct completion.10,40 although our data support the hypothesis that mi results in remote arterial inflammation, it could also be argued that more widespread and intense atherosclerotic inflammation occurs prior to the precipitation of mi and that this is the explanation for the association. the correlation between infarct size and the inflammatory signal would support the former explanation ; however, to resolve this issue, 18f - fdg imaging would be required both before and after mi, which, given the unpredictable nature of these events, is extremely challenging in the clinical context. we also acknowledge that we did not directly measure mi size and used peak plasma troponin concentration as a surrogate measure ; however, peak plasma troponin concentration has a strong correlation with infarct size (r=0.740, p<0.001)30,35 and is a valid surrogate marker, especially when applied to large data sets such as the registry cohort used in this study. finally, we were unable to measure systemic markers of inflammation such as crp in our outcome cohort ; however, crp is well known to correlate with troponin release after mi,4143 and indeed this was observed in our imaging cohort. in summary, we demonstrated that the presence and extent of mi are associated with increased aortic atherosclerotic inflammation and early recurrent mi, supporting the hypothesis that mi begets mi in humans. the study was funded by the chief scientist office, scotland (etm/160) and the british heart foundation (pg/12/8). dweck, mills and newby are supported by the british heart foundation (ch/09/002, fs/10/024, fs/10/026). the wellcome trust clinical research facility and the clinical research imaging centre are supported by nhs research scotland (nrs) through nhs lothian. | backgroundpreclinical data suggest that an acute inflammatory response following myocardial infarction (mi) accelerates systemic atherosclerosis. using combined positron emission and computed tomography, we investigated whether this phenomenon occurs in humans.methods and resultsoverall, 40 patients with mi and 40 with stable angina underwent thoracic 18f - fluorodeoxyglucose combined positron emission and computed tomography scan. radiotracer uptake was measured in aortic atheroma and nonvascular tissue (paraspinal muscle). in 1003 patients enrolled in the global registry of acute coronary events, we assessed whether infarct size predicted early (30 days) and late (> 30 days) recurrent coronary events. compared with patients with stable angina, patients with mi had higher aortic 18f - fluorodeoxyglucose uptake (tissue - to - background ratio 2.150.30 versus 1.840.18, p 50 000 ] versus 3800 [1000 to 9200 ] ng / l, p<0.0001) and greater aortic 18f - fluorodeoxyglucose uptake (2.240.32 versus 2.020.21, p=0.03) than those with non st - segment elevation mi. peak plasma troponin concentrations correlated with aortic 18f - fluorodeoxyglucose uptake (r=0.43, p=0.01) and, on multivariate analysis, independently predicted early (tertile 3 versus tertile 1 : relative risk 4.40 [95% ci 1.90 to 10.19 ], p=0.001), but not late, recurrent mi.conclusionsthe presence and extent of mi is associated with increased aortic atherosclerotic inflammation and early recurrent mi. this finding supports the hypothesis that acute mi exacerbates systemic atherosclerotic inflammation and remote plaque destabilization : mi begets mi.clinical trial registrationurl : https://www.clinicaltrials.gov. unique identifier : nct01749254. |
compound distal tibia fractures have high incidence of nonunions and have varied presentation as far as status of fibula is concerned. if fibula is sufficiently healthy we can use it for bridging the nonunion of tibia. we present a case of 20 year old female with compound and segmental tibia fracture. primary stablisation by external fixation and later cast brace application achieved union at the proximal end of the segmental fragment with non union at the distal end. this was then treated with bridge grafting of fibula and screw fixation of fibula to the tibia. its infrequent to find fibula spanning across the tibia nonunion, however when available this can be used as bridge graft to promote healing. distal tibia is common sites of aseptic non unions and delayed unions, especially in compound wounds with extensive soft tissue injury. two factors dictate the further management in aseptic cases, mobility at fracture site and healing response from the bone. in cases with fibrous union or a stiff non union only bone grafting with immobilization may be adequate to achieve union.3 in cases with abnormal mobility an internal fixation is required. compression plates can be used for angulated distal fractures ; and ring fixators are preferred in cases with bone loss. in certain cases, specially with poor local skin condition, distal fibula can be used to bridge the nonunion tibia site and provide advantage of both bone grafting and internal fixation. such use was first described in 2005 by james and santrock in an abstract presented at foot and ankle surgery course 2008. we describe successful use of similar technique in non infected tibia nonunion in 20 year old male. her left leg was crushed under a truck tyre and she presented with a grade iiib compound fracture tibia with a 20 cm by 8 cms wound over the anteromedial side of lower third of tibia, extending over to the medial side of ankle joint and foot. radiographs of the leg showed a distal third tibia fracture with a segmental piece of tibia which was further comminuted. the fibula fracture was at the same level as tibia and was short oblique fracture. patient presented immediately after the injury and debridement was done within 2 hours of injury. two further debridements were needed and at 4 weeks the wound was healthy enough to perform a cross leg flap. at 3 weeks post flap surgery, the external fixator was removed due to multiple pin track infections and the limb was immobilized in a long leg cast. at 3 and half months radiographs showed that the segmental tibia fragment was showing good union response at the proximal end ; however the distal end appeared to be rounded with attempt to callus formation which is non bridging (fig. abnormal mobility at the fracture site indicated a need to stabilize the fracture, however soft tissue condition precluded any form of internal fixation. at that time the fibula fracture was completely united. a decision to do a posterior bone grafting along with bridge fibula grafting lateral approach was taken and cancellous graft taken from the same side (right) iliac crest was filled in the interosseous membrane between tibia and fibula by at the site of the fracture, just proximal and just distal to it. two 3.5 mm cortical screws were passed from the fibula to into the tibia at the site where the graft ends proximally and distally. this was done to make a rigid strut with cancellous graft in the middle (fig. 3). patient was put in a long knee brace and advised non - weight bearing for a period of 8 weeks. at two months after bridge grafting good callus was seen bridging the fracture and the patient was given a patella tendon bearing cast and was started on partial weight bearing. the ankle movement was 10 degree dorsiflexion from neutral and 20 degree plantar flexion from neutral position at that time. patient resumed her activities at 8 months post trauma while screw removal was done at 18 months post injury. she was walking full weight bearing and was able to carry out all her activities. ankle dorsiflexion was restricted to 10 but the plantar flexion was around 50 (fig. 4). there was no history of pain or discharge from the injury site. the radiograph showed the fracture outline to have a silhouetted appearance with good union on posterior and medial side with good consolidation of the entire area of the bridge graft between tibia and fibula (fig. 5). 20 year old female presenting with compound fracture of tibia treated with debridement and external fixator. three and half months post injury radiographs showing segmental tibia fragment with good union response at the proximal end ; however the distal end appeared to be rounded with non bridging callus. radiograph showing bridge fibula graft done with stabilization using two screws and iliac crest cancellous bone graft between the tibia and fibula. intact fibula can be appreciated to add a strut support to the tibia clinical photographs five years post bridge grafting showing good ankle range. bridge grafting of fibula has limited indications but can provide initial stability and achieve good cross union between the bones. only one such case is described previously and present case demonstrates the usefulness of bridge grafting which should be kept in mind in such cases with complicated bony and soft tissue injuries. he performed posterolateral cancellous bone grafting between tibia and fibula creating a cross union between the bones. after him many have reported the success of this method ; however the indication is limited to fibrous nonunions with limited mobility and no requirement of additional stability. in cases where additional stability was required transfer of ipsilateral fibula by osteotomy at both ends and fixation of the fibular strut graft across the non union this is similar to huntington procedure which was done in two steps [9 - 11 ]. in our case this could have been achieved by an intramedullary nail, however an additional open procedure for bone grafting and bone graft harvesting would have been needed. another option was internal fixation using a compression plate, however the soft tissue envelope on the medial side was very thin to allow any surgery on the medial side. a ring fixator could have helped the patient with compression at the fracture site and bone grafting, however issues with pin tracts for which the external fixator was removed would have again raised problems. with these options and problems with them, we tried the concept of bridge grafting as described by james and shr. a similar concept was utilized by weinberg to fill up defect in war injuries. the fibula was united in slight angulation towards the tibia thus we could utilized the fibula as an internal fixation device without osteotomy. we fixed the fibula at ends across the non union site, which acted as the working length and stabilizing the non union site. additional bone graft in the interosseous space consolidated over a period of time to form a stable tibia fibular synostosis. reckling and waters commented that formation of this synostosis facilitates tibia union and also does not affect the knee and ankle joint in long term. at 5 years. the limitation of dorsiflexion may be because of loss of interosseous space and also the slight procurvatum at the tibia union site. thus in conclusion in non union tibia, use of fibula bridge graft with interosseous cancellous graft may be an option in select cases like one described here. careful selection of patient should be considered while making a clinical decision and in select cases good results can be expected within limits of the local biological potentional distal fibular nonunions are commonly seen however it is infrequent to find fibula spanning across the tibia nonunion. when such scenario presents the described technique can be used as bridge graft to promote healing. | introduction : compound distal tibia fractures have high incidence of nonunions and have varied presentation as far as status of fibula is concerned. if fibula is sufficiently healthy we can use it for bridging the nonunion of tibia.case report : we present a case of 20 year old female with compound and segmental tibia fracture. primary stablisation by external fixation and later cast brace application achieved union at the proximal end of the segmental fragment with non union at the distal end. this was then treated with bridge grafting of fibula and screw fixation of fibula to the tibia. five years follow shows good clinical and functional outcome without any complications.conclusion:its infrequent to find fibula spanning across the tibia nonunion, however when available this can be used as bridge graft to promote healing. |
aminophosphines of the type pr2nhr containing a direct polar p(iii)n bonds have received considerable attention in recent years as versatile ligands for transition metals. they are accessible in large quantities through the use of relatively simple condensation processes from inexpensive starting materials, i.e., primary amines and pr2cl compounds which contain dialkyl or diaryl substituents as well as achiral and chiral p o and p thus, variations of electronic, steric, and stereochemical parameters may be achieved in a very facile fashion. due to their soft / hard donor atoms as well their acidic nh hydrogen, these polyfunctional ligands exhibit numerous coordination modes as illustrated in scheme 1. as middle and late transition metals m are concerned pr2nhr ligands typically coordinate in (p)-fashion i, while (n)-coordination is unknown. upon deprotonation of the pr2nhr ligands, anionic amidophosphines [pr2nr ] are readily obtained which exhibit a higher affinity toward electropositive metals due to their increased nucleophilicity at the n - site. thus, in conjunction with early transition metals m, amidophosphine ligands were shown to display (p, n) coordination ii, and, albeit less common, also (n)-coordination iii, while in the presence of both early and middle / late transition metals, amidophosphine ligands were shown to act as bridging ligand thereby forming heterobimetallic complexes of the type iv. besides interesting structural features, for instance, we have recently shown that in vinylidene and allenylidene complexes [rucp(pph2nhr)2(c(c)n = chr) ] and [rutp(pph2nhr)2(c(c)n = chr) ] (n = 0, 1 ; r = ph, n - pr ; r = alkyl, aryl ; tp = trispyrazoloylborate) an intramolecular addition of the nhr moiety to the -carbon of the cumulene moiety takes place (scheme 2) resulting in the formation of novel four - membered aza - phospha - carbenes (scheme 3, a). complexes of the types [rucp(pph2nhph)(ch3cn)2 ] and [rucp(pr2nhr)(ch3cn)2 ] (r = ph, i - pr, r = ph, c6f5) have been found to react with terminal alkynes and diynes to give amido butadiene complexes involving pr2nhr ligand migration (scheme 3, b). reported the synthesis of the dinuclear ruthenium complex [ru2(co)3(-pph2)(-ph2pnmepph2)((c, p)c(o)nmepph2) ] containing a carboxamido - phospha - ruthen - acyclic moiety (scheme 3, c). reported on the preparation of half sandwich complexes of the type [mcp(co)2(c(o)n(s - nhptbu2)ptbu2) ] (m = cr, mo, w) by reacting [mcp(co)3h ] with ptbu2pn = s = nptbu2 (scheme 3, d). as iron complexes are concerned, [fecp(co)2(pph2nhnme2 ] bearing a hydrazinophosphine ligand, which is closely related to pr2nhr ligands, was shown to react with nbuli to give the carboxamido - phospha - ferracycle [fecp(co)((c, p)-(co)-nnme2-pph2) ] (scheme 3, e). in the present paper we report on the synthesis of iron(ii) complexes containing aminophosphine ligands of the type pr2nhr with r = ph, ipr, r = ipr, tbu, cy. we describe the reactivity of these complexes yielding, upon treatment with strong bases, novel cyclic four - membered carboxamido - phospha - ferracycle formed via intramolecular addition of the amine moiety of the bifunctional aminophosphine ligand according to scheme 4. treatment of [fecp(co)2cl ] with 1 equiv of the amidophosphine ligands [r2pnr ] (r = ph, ipr, r = ipr, tbu, cy), prepared in situ by the reaction of r2pnhr with nbuli in thf at 20 c, afforded complexes of the type [fecp(co)((c, p)-(co)nipr - pph2) ] (1a), [fecp(co)((c, p)-(co)-ntbu - pph2) ] (1b), and [fecp(co)((c, p)-(co)-ncy - pipr2) ] (1c) in reasonable isolated yields (4050%) (scheme 5). although no intermediate could be detected spectroscopically, it is most likely that the deprotonated r2pnhr ligand first forms a complex with a (p)-bound rather than a (n)-bound amidophosphine. it has to be noted that (n) coordinated [pr2nr ] ligands to late transition metals are, according to our knowledge, unknown. subsequently, intramolecular, chelate assisted, nucleophilic attack of the amido moiety of the pr2nr ligand at one of the two co ligands took place resulting in the formation of novel four - membered carboxamido - phospha - ferracycle. this is also supported by the fact that 1a is readily formed if the cationic dicarbonyl complex [fecp(co)2(pph2nhipr) ] with either br (2) or bf4 (2) as counterions was treated with kotbu as base according to scheme 6. in the absence of base, even at elevated temperatures, no reaction took place. complexes 2 and 2 were readily obtained by the treatment of [fecp(co)2br ] with 1 equiv of the pph2nhipr ligand in the absence or presence of nabf4, respectively, in thf at room temperature (scheme 6). all complexes are thermally robust orange solids that are air stable both in the solid state and in solution for several days. their identity was unequivocally established by h, c{h } and p{h } nmr, ir spectroscopy, and elemental analysis. in addition, the molecular structures of complexes 1a and 2 were determined by x - ray crystallography. 1 and 2 with selected bond distances and angles reported in the captions. complexes 1a c display a single resonance in the p{h } nmr at 111.6, 113.0, and 140.0 ppm, respectively. in the c{h } nmr spectra, the cp ring gives rise to a singlet in the range of about 8082 ppm. the co ligands exhibit a low - field doublet at 220.9, 221.0, and 221.0 ppm with coupling constants jpc of 24.426.4 hz, while the resonance of the carboxamido carbon atoms give rise to a doublet centered at 200.4 (jpc = 39.0 hz), 201.1 (jpc = 45.8 hz), and 199.8 ppm (jpc = 37.8 hz), respectively. in the ir spectrum, the characteristic co stretching frequency of the co ligand and the carboxamido unit was observed at 1937, 1919, and 1914 cm and 1617, 1605, and 1618 cm, respectively. for comparison, the ir stretching frequency of the carboxamido unit in [fecp(co)((c, p)-(co)-nnme2-pph2) ] was found at 1642 cm. complex 1a adopts a typical three legged piano stool conformation with the c and p atoms of the c(o)nipr - pph2 moiety and the c atom of the co ligand as the legs (fig. 1). the iron carbon bond distances fe1c6 and fe1c7 are 1.739(1) and 1.981(1), respectively, the latter being typical for an iron carbon single bond. thus, also in agreement with the nmr and ir spectroscopic data, 1a is best described as a carboxamido complex (a) rather than an aminocarbene complex (b) (scheme 7). the fe1-p1 bond distance of 2.1773(3) is typical for iron phosphine complexes but slightly shorter than fe1p1 = 2.2223(4) in the parent complex 2 (fig the c7fe1p1 bite angle of the chelating c(o)nipr - pph2 ligand is 69.43(4). for comparison, in the related complex [fecp(co)((c, p)-(co)-nnme2-pph2) ] since acyl and carbamoyl ligands are typically nucleophilic at the carbonyl oxygen, i.e., resonance structure b may play a role, the reaction with carbon - based electrophiles may lead to aminocarbene complexes. it has to be kept in mind however that an electrophilic attack may occur also at the n atom of the carboxamido moiety resulting in p treatment of 1a with [me3o]bf4 (1 equiv) at room temperature for 4 h in ch2cl2 as the solvent results in the formation of the aza - phospha - carbene complex [fecp(co)((c, p) = c(ome)-nipr - pph2) ] (3) in 58% isolated yield (scheme 8). this class of complexes belongs to a rare series of transition metal complexes in which the carbene moiety is part of a four - membered chelate ligand coordinated in a (c, p) mode. complex 3 was characterized by elemental analysis and by h, c{h }, and p{h } nmr spectroscopy. characteristic features comprise, in the c{h } nmr spectrum, a marked low - field doublet resonance at 238.0 ppm (d, jcp = 34.8 hz) assignable to the carbene carbon atom of the four - membered aza - phospha - carbene moiety. the carbonyl resonance gives rise to a doublet centered at 216.3 ppm (d, jcp = 22.2 hz). the p{h } nmr spectrum of 3 reveals a singlet at 115.0 ppm (cf. 111.6 ppm in 1a). on the other hand, protonation of 1a with the brnsted acid [hnet3 ] led to clean formation of complex 2a in 95% isolated yield (scheme 8). n bond cleavage and reformation of the co and ph2pnhipr ligands. in search of related iron systems where carboxamido - phospha - cycles may also be formed via nucleophilic attack of a (p)-coordinated pr2nhr ligand, we prepared complexes of the type cis, trans, cis-[fe(co)2(ph2pnhipr)2(br)2 ] (4a) and cis, trans, cis-[fe(co)2(ph2pnhtbu)2(br)2 ] (4b). these compounds were readily obtained as red, air stable solids by reacting cis-[fe(co)4(br)2 ] with 2 equivs of the respective pr2nhr ligands in 85 and 88% yield (scheme 9) in ch2cl2 at room temperature. in these complexes the co and bromide ligands are in a mutual cis position, whereas the phosphine ligands are trans to one another. treatment of 4a with 2 equivs of kotbu in the presence of co in thf at room temperature afforded the isomeric complexes trans-[fe(co)2((c, p)-(co)nipr - pph2)(ph2pnhipr)br ] (5a) and cis-[fe-(co)2((c, p)-(co)nipr - pph2)(ph2pnhipr)br ] (5a) in a roughly 5:1 ratio in 50% overall yield. in agreement with experimental data dft / b3lyp calculations confirm that the isomer with a trans co arrangement is slightly more stable by 2.5 kcal / mol (free energy) than the corresponding cis isomer. in the case of 4b only one isomer, viz trans-[fe(co)2((c, p)-(co)-ntbu - pph2)(ph2pnhtbu)br ] (5b), was formed in 40% yield (scheme 9). these reactions again involve attack of the amido moiety of a deprotonated pr2nhr ligand on coordinated co. it has to be mentioned that the yields of 5a and 5b were independent of whether 1 or 2 equivs of kotbu were used, i.e., complexes such as [fe(co)2((c, p)-(co)nipr - pph2)2 ], were not observed. complexes 5a and 5b are thermally robust red solids that are air stable in the solid state but slowly decompose in solution. their identity was unequivocally established by h, c{h } and p{h } nmr, ir spectroscopy, and elemental analysis. in the c{h } nmr spectrum of the major isomer 5a the most noticeable resonances are a low - field signal of the two trans co ligands observed as triplet centered at 212.4 ppm with a p c coupling constants of 22.7 hz. the resonance of the carboxamido moiety give rise to a doublet of doublets centered at 206.0 ppm with p c couplings constant of 9.6 and 13.4 hz. the p{h } nmr spectrum of 5a reveals two doublets centered at 95.6 and 85.8 ppm with a large coupling constant of 84.7 hz which is indicative for the phosphorus atoms being in a mutual trans position. most h and c{h } nmr of resonances of the minor isomer 5a ' could not be reliably assigned since they were superimposed by the signals of the major isomer 5a. in the p{h } nmr spectrum, however, 5a also exhibits two doublets centered at 94.8 (d, jpp = 115.9 hz) and 90.0 ppm (d, jpp = 115.9 hz). the large coupling constant again is consistent with a trans - p, p configuration. similar nmr spectra were observed for 5b, and are thus not discussed here. in the ir spectrum of 5a and 5b the two co ligands give rise to two bands at 1966 and 1960 cm (5a) and 1955 and 1950 cm (5b) which can be assigned to the asymmetric co stretching frequency (cf 2143 cm in free co). as expected the symmetric co stretching frequency is ir inactive which is also confirmed by dft / b3lyp calculations. the appearance of two the resonances, which are only 56 cm apart, may be due to intermolecular interactions, e.g. cohn bonds, in the solid state. the co vibration of the carboxamido phospha cycle is shifted to lower wavenumbers observed at 1619 and 1616 and cm, respectively. the scaled calculated frequencies co together with the experimentally observed values are given in table 1 and show a reasonably good agreement. the molecular structure of complex 5a was determined by x - ray crystallography. a structural view is depicted in fig. 3 with selected bond distances and angles reported in the caption. the geometry about the metal center is distorted octahedral with the two phosphorus and carbon atoms in trans position and the bromide and carbon atom of carboxamido moiety atoms in cis position (fig. the chelate system fe1p1n1(c31o1)fe1 resembles closely in bond lengths and bond angles the corresponding system in complex 1a, except for the bond fe1-p1, which is 2.1773(3) in 1a while it is 2.2480(9) in 5a due to the trans - influence of phosphorus p2 (fe1p2 = 2.2651(9)). the short intramolecular hydrogen bond n2h2no1 in 5a (n2o1 = 2.839(4)) has no effect on the bond angle fe1-c31o1 = 134.2(2) as evident from the corresponding angle 133.8(1) in complex 1a which lacks this interaction. in the present study iron(ii) complexes featuring one or two aminophosphine ligands of the type pr2nhr with r = ph, ipr and r = ipr, tbu, cy were synthesized. we demonstrated that upon treatment of [fecp(co)2x ] (x = cl, br) with the anionic amidophosphine ligands [r2pnr], or upon deprotonation of the pr2nhr ligand in complexes [fecp(co)2(pr2nhr2) ] and cis, trans, cis-[fe(co)2(ph2pnhr)2(br)2 ] complexes featuring four - membered carboxamido - phospha - ferracycles were obtained. in the course of these reactions the highly nucleophilic amido nitrogen atom reacted readily in an intramolecular fashion with the electrophilic carbon atom of a co ligand. we have further demonstrated that the carboxamido - phospha - ferracycles react with the carbon - based electrophile [me3o ] to afford an aza - phospha - carbene. this is a relatively rare type of transition metal complexes in which the carbene moiety is part of a four - membered chelate ligand coordinated in a (c, p) mode. in the presence of protons, the ligands pph2nhipr, pph2nhtbu, and pph2nhcy and the complexes [fecp(co)2cl ], of [fecp(co)2br ], and cis-[fe(co)4br2 ] were prepared according to the literature. the deuterated solvents were purchased from aldrich and dried over 4 molecular sieves. h, c{h }, and p{h } nmr spectra were recorded on bruker avance-250 and avance-300 dpx spectrometers and were referenced to sime4 and h3po4 (85%), respectively. a solution of pph2nhipr (300 mg, 1.23 mmol) in thf (20 ml) was cooled to 20 c and nbuli (500 l, 1.23 mmol, 2.5 m solution in n - hexane) was slowly added. the solution was stirred for 2 h at 20 c and an additional hour at room temperature. after that, [fecp(co)2cl ] (262 mg, 1.23 mmol) was added and the mixture was stirred for 8 h at room temperature. the residue was redissolved in toluene (10 ml) and the solution was filtered through celite. after removal of the solvent under reduced pressure, an orange solid was obtained which was washed with n - pentane (10 ml) and dried under vacuum. : c, 63.03 ; h, 5.29 ; n, 3.34. found : c, 63.09 ; h, 5.12 ; n, 3.28. h nmr (, cdcl3, 20 c) : 7.87 (m, 4h, ph), 7.51 (m, 6h, ph), 4.38 (s, 5h, cp), 3.49 (m, 1h, ch(ch3)2), 1.29 (d, j = 5.8 hz, 3h, ch(ch3)2), 0.99 (d, j = 6.7 hz, 3h, ch(ch3)2). c{h } nmr (, cdcl3, 20 c) : 220.9 (d, jpc = 26.0 hz, co), 200.4 (d, jpc = 39.0 hz, nco), 137.2 (d, jpc = 40.6 hz, ph), 133.8 (d, jpc = 13.5 hz, ph), 131.8 (d, jpc = 2.7 hz, ph), 130.7 (d, jpc = 13.5 hz, ph), 130.4 (d, jpc = 2.7 hz, ph), 128.6 (d, jpc = 9.5 hz, ph), 128.4 (d, jpc = 7.5 hz, ph), 82.1 (s, cp), 50.7 (d, jpc = 8.1 hz, ch(ch3)2), 23.0 (s, ch(ch3)2), 21.4 (s, ch(ch3)2). p{h } nmr (, cdcl3, 20 c) : 111.6. ir (atr, 25 c) : 1937 (c = o), 1617 (co). this complex was prepared analogously to 1a with [fecp(co)2cl ] (400 mg, 1.55 mmol) and ph2pnhtbu (400 mg, 1.55 mmol), and nbuli (630 l, 1.48 mmol, 2.5 m solution in n - hexane) as starting materials. calcd for c23h24feno2p : c, 63.76 ; h, 5.58 ; n, 3.23. h nmr (, cdcl3, 20 c) : 7.90 (m, 4h, ph), 7.48 (m, 6h, ph), 4.39 (s, 5h, cp), 1.20 (s, 9h, c(ch3)3). c{h } nmr (, cdcl3, 20 c) : 221.0 (d, jpc = 24.4 hz, co), 201.1 (d, jpc = 45.8 hz, nco), 136.4 (d, jpc = 39.3 hz, ph), 133.8 (d, jpc = 41.3 hz, ph), 132.7 (d, jpc = 12.5 hz, ph), 131.2 (d, jpc = 3.2 hz, ph), 130.7 (d, jpc = 11.7 hz, ph), 130.3 (d, jpc = 3.2 hz, ph), 128.6 (d, jpc = 7.4 hz, ph), 128.4 (d, jpc = 5.8 hz, ph), 82.8 (s, cp), 65.8 (s, ch(ch3)2), 29.5 (s, ch(ch3)2). ir (atr, 25 c) : 1919 (co), 1605 (co). this complex was prepared analogously to 1a with [fecp(co)2cl ] (370 mg, 1.75 mmol), ph2pnhcy (376 mg, 1.75 mmol), and nbuli (700 l, 1.75 mmol, 2.5 m solution in n - hexane) as starting materials. calcd for c19h30feno2p : c, 58.48 ; h, 7.49 ; n, 3.59. found : c, 58.53 ; h, 7.40 ; n, 3.62. h nmr (, cdcl3, 20 c) : 4.61 (s, 5h, cp), 2,93 (m, 1h, nch), 2.54 (m, 1h, ch(ch3)2), 2.18 (m, 1h, ch(ch3)2), 1,69 (bs, 4h, cy), 1,53 (bs, 6h, cy), 1.32 (d, j = 11.0 hz, 3h, ch(ch3)2), 1.30 (s, 1h, cy), 1.25 (d, j = 7.3 hz, 3h, ch(ch3)2). c{h } nmr (, cdcl3, 20 c) : 221.0 (d, jpc = 26.4 hz, co), 199.8 (d, jpc = 37.8 hz, nco), 80.8 (s, cp), 58.5 (d, jpc = 8.1 hz, nc), 32.7 (d, jpc = 71.9 hz, ch(ch3)2), 30.6 (d, jpc = 19.3 hz, cy), 27.9 (d, jpc = 15.9 hz, cy), 26.8 (s, cy), 25.3 (s, cy), 20.4 (d, jpc = 5.7 hz, ch(ch3)2), 18.7 (s, cy), 18.0 (d, jpc = 5.74 hz, ch(ch3)2). ir (atr, 25 c) : 1914 (co), 1618 (co). to a solution of [fecp(co)2br ] (1.00 g, 3.89 mmol) in thf (10 ml) ph2pnhipr (995 mg, 4.01 mmol) was added and the reaction mixture was stirred overnight at room temperature. calcd for c22h23brfeno2p : c, 52.94 ; h, 4.44 ; n, 2.81. found : c, 53.04 ; h, 4.39 ; n, 2.85. h nmr (, cdcl3, 20 c) : 7.61 - 7.53 (m, 10h, ph), 5.96 (d, j = 10.2 hz, 1h, nh), 5.27 (s, 5h, cp), 2.99 (bs, 1h, ch(ch3)2), 1.16 (d, j = 5.4 hz, 6h, ch(ch3)2).). c{h } nmr (, cdcl3, 20 c) : 210.3 (d, jpc = 27.1 hz, co), 135.0 (d, jpc = 59.1 hz, ph), 131.7 (d, jpc = 10.4 hz, ph), 131.3 (d, jpc = 10.7 hz, ph), 129.0 (d, jpc = 10. 9 hz, ph), 88.7 (s, cp), 48.8 (d, jpc = 9.6 hz, ch(ch3)2), 24.8 (d, jpc = 3.52 hz, ch(ch3)2). ir (atr, 25 c) : 2040 (co), 1993 (co). this complex was prepared analogously to 2 with [fecp(co)2br ] (350 mg, 1.65 mmol) and ph2pnhipr (400 mg, 1.65 mmol) as starting materials but in the presence of nabf4 (182 mg, 1.65 mmol). n, 2.76. found : c, 52.14 ; h, 4.08 ; n, 2.80. a solution of [fecp(co)2(ph2pnhipr)]bf4 (2a) (220 mg, 0.44 mmol) in thf (10 ml) was treated with kotbu (55 mg, 0.48 mmol) and was stirred for 8 h. the solvent was removed under vacuum and the crude product was redissolved in toluene and filtered through celite. after removal of the solvent under reduced pressure, 1a was obtained which was washed with n - pentane (10 ml) and dried under vacuum. (500 mg, 1.19 mmol) in ch2cl2 (10 ml) was treated with [me3o]bf4 (177 mg, 1.19 mmol). after stirring for 4 h on removal of the solvent, 5 was obtained as an orange solid which was washed with n - hexane, and dried under vacuum. calcd for c35h39bbrf4fen2o3p : c, 53.27 ; h, 4.98 ; n, 3.55. h nmr (, cdcl3, 20 c) : 7.58 (m, 10h, ph), 4.80 (s, 5h, cp), 4.33 (s, 3h, och3), 3.93 (m, 1h, ch(ch3)2), 1.40 (d, j = 6.4 hz, 3h, ch(ch3)2), 0.92 (d, j = 6.4 hz, 3h, ch(ch3)2). c{h } nmr (, cdcl3, 20 c) : 238.0 (d, jpc = 34.8 hz, fec), 216.3 (d, jpc = 22.2 hz, co), 134.7 (d, jpc = 22.3 hz, ph), 134.4 (d, jpc = 14.2 hz, ph), 134.2 (d, jpc = 22.2 hz, ph), 132.8 (d, jpc = 2.59 hz, ph), 131.9 (d, jpc = 2.5 hz, ph), 131.3 (d, jpc = 11.1 hz, ph), 129.9 (d, jpc = 11.3 hz, ph), 83.1 (s, cp), 64.9 (s, och3), 55.8 (d, jpc = 5.5 hz, ch(ch3)2), 21.8 (s, ch(ch3)2), 20.8 (s, ch(ch3)2). ir (atr, 25 c) : 2004 (co). a solution of 1a (500 mg, 1.19 mmol) in ch2cl2 (10 ml) was treated with [hnet3]cl (165 mg, 1.19 mmol). after stirring for 4h, insoluble materials were removed by filtration through celite. on removal of the solvent, 2 was obtained as a yellow solid which was collected on a glass frit, washed with n - hexane, and dried under vacuum. yield : 514 mg (95%). to a solution of cis-[fe(co)4br2 ] (1.00 g, 3.05 mmol) in ch2cl2 (10 ml) ph2pnhipr (1.52 g, 6.25 mmol) was added at 0 c and the mixture was stirred overnight at room temperature. after removal of the solvent under reduced pressure, an orange solid was obtained which was washed with diethyl ether (10 ml) and dried under vacuum. calcd for c32h36br2fen2o2p2 : c, 50.69 ; h, 4.79 ; n, 3.69. h nmr (, cdcl3, 20 c) : 7.99 (bs, 8h, ph), 7.48 (bs, 12h, ph), 3.42 (bs, 2h, nh), 2.29 (m, 2h, ch(ch3)2), 0.91 (d, jpc = 6.3 hz, 12h, ch(ch3)2). c{h } nmr (, cdcl3, 20 c) : 212.7 (t, jpc = 22.7 hz, co), 133.6 (d, jpc = 25. hz, ph), 133.0 (dd, jpc = 5.1 hz, ph), 131.7 (dd, jpc = 5.4 hz, ph), 130.8 (s, ph, 127.9 (dd, jpc = 4.6 hz, ph), 46.4 (dd, jpc = 4.9 hz, ch(ch3)2), 24.7 (s, ch(ch3)2). ir (atr, 25 c) : 2041 (co), 1986 (co). this complex was prepared analogously to 4a with cis-[fe(co)4br2 ] (845 mg, 2.58 mmol) and ph2pnhtbu (1.33 g, 5.16 mmol) as starting materials. calcd for c34h40br2fen2o2p2 : c, 51.94 ; h, 5.13 ; n, 3.56. found : c, 52.04 ; h, 5.20 ; n, 3.46. h nmr (, acetone - d6, 20 c) : 8.36 (bs, 8h, ph), 7.48 (bs, 12h, ph), 3.7 (bs, 2h, nh), 0.87 (s, 18h, c(ch3)3). c{h } nmr (, cdcl3, 20 c) : 213.3 (t, jpc = 24.0 hz, co), 134.2 (dd, jpc = 25.7 hz, ph), 133.5 (dd, jpc = 5.3 hz, ph), 130.7 (s, ph), 127.7 (dd, jpc = 4.7 hz, ph), 55.5 (s, c(ch3)3), 31.7 (s, c(ch3)3). p{h } nmr (, acetone - d6, 20 c) : 72.2. ir (atr, 25 c) : 2035 (co), 1980 (co). a schlenk tube was charged with 4a (400 mg, 0.53 mmol) and kotbu (122 mg, 1.06 mmol). under a co atmosphere thf (10 ml) the red solution was then filtered through celite and the solvent was removed under reduced pressure. after evaporation of the solvent in vacuo, a red solid was obtained which was washed with n - pentane (10 ml) and dried under vacuum. calcd for c33h35brfen2o3p2 : c, 56.19 ; h, 5.00 ; n, 3.97. found : c, 56.00 ; h, 5.12, n, 4.01. nmr (, cdcl3, 20 c) : 7.85 (d, j = 6.79 hz, 8h, ph), 7.50 (d, j = 8.38 hz, 8h, ph), 7.43 (s, 4h, ph), 4.70 (bs, 1h, nh), 3.46 (m, 1h, ch(ch3)2), 3.12 (m, 1h, ch(ch3)2), 1.31 (d, j = 6.4 hz, 6h, ch(ch3)2), 0.94 (d, j = 6.4 hz, 6h, ch(ch3)2). c{h } nmr (, cdcl3, 20 c) : 212.4 (t, jpc = 22.7 hz, co), 206.0 (dd, jpc = 9.6 hz, jpc = 13.4 hz, nco), 135.1 (d, jpc = 37.5 hz, ph), 133.3 (d, jpc = 12.1 hz, ph), 133.1 (d, jpc = 13.5 hz, ph), 131.8 (s, ph), 130.2 (s, ph), 128.7 (d, jpc = 11.1 hz, ph), 128.0 (d, jpc = 11.1 hz, ph), 45.7 (t, jpc = 12.7 hz, ph), 25.5 (s, ch(ch3)2), 22.0 (s, ch(ch3)2). p{h } nmr (, cdcl3, 20 c) : 95.6 (d, jpp = 84.7 hz), 85.8 (d, jpp = 84.7 hz). ir (atr, 25 c) : 1966 (co), 1960 (co), 1616 (co). minor isomer cis-[fe(co)2((c, p)-(co)nipr - pph2)(ph2pnhipr)br ] (5a) : most h and c nmr resonances are superimposed by the signals of the major isomer and could not reliably assigned. p{h } nmr (, cdcl3, 20 c) : 94.8 (d, jpp = 115.9 hz), 90.0 (d, jpp = 115.9 hz). this complex was prepared analogously to 5a with 4b (0.70 g, 0.89 mmol) as starting material. calcd for c35h39brfen2o3p2 : c, 57.32 ; h, 5.36 ; n, 3.82. found : c, 57.40 ; h, 5.29 ; n, 3.79. h nmr (, cdcl3, 20 c) : 7.98 (bs, 8h, ph), 7.42 (bs, 12h, ph), 4.84 (bs, 1h, nh), 3.46 (m, 1h, ch(ch3)2), 1.28 (s, 9h, c(ch3)3), 1.05 (s, 9h, c(ch3)3). c{h } nmr (, cdcl3, 20 c) : 213.3 (dd, jpc = 23.4 hz, jpc = 22.9 hz co), 206.0 (dd, jpc = 9.6 hz, jpc = 13.4 hz, nco), 136.2 (d, jpc = 47.7 hz, ph), 136.1 (d, jpc = 47.7 hz, ph), 134.5 (d, jpc = 10.8 hz, ph), 132.8 (d, jpc = 10.8 hz, ph), 132.6 (d, jpc = 11.6 hz, ph), 131.4 (d, jpc = 2.3 hz, ph), 129.9 (d, jpc = 2.3 hz, ph), 128.5 (d, jpc = 10.6 hz, ph), 127.6 (d, jpc = 10.0 hz, ph), 62.3 (d, jpc = 7.6 hz, c(ch3)3), 55.9 (d, jpc = 13.9 hz, c(ch3)3), 32.1 (d, jpc = 3.1 hz, c(ch3)3), 29.5 (d, jpc = 1.5 hz, c(ch3)3). p{h } nmr (, cdcl3, 20 c) : 90.4 (d, jpp = 79.3 hz), 87.3 (d, jpp = 79.3 hz. ir (atr, 25 c) : 1955 (c = o), 1950 (co),1619 (co). single crystals for x - ray diffraction were obtained as follows : [fecp(co)((c, p)-(co)nipr - pph2) ] (1a), by vapor diffusion of n - pentane into a thf solution, [fecp(co)2(ph2pnhipr)]br (2) by vapor diffusion of et2o into a ch2cl2 solution ; trans / cis-[fe(co)2((c, p)-(co)nipr - pph2)(ph2pnhipr)br ] (5a/5a ') by vapor diffusion of et2o into a ch2cl2 solution. the color of the crystals varied from orange to red brown. x - ray diffraction data were collected at t = 100 k on a bruker kappa apex-2 ccd diffractometer with an oxford cryosystems cooler using graphite - monochromatised mo - k radiation (= 0.71073) and fine sliced - and -scans covering complete spheres of the reciprocal space. after data integration with program saint corrections for absorption and detector effects were applied with the program sadabs the structures were solved by direct methods (shelxs97) and refined on f with the program shelxl97 non - hydrogen atoms were refined anisotropically. most h atoms were placed in calculated positions and thereafter refined as riding. in (5a) br was partly substituted by co and vice versa (84% trans- and 16% in cis - dicarbonyl configuration), and the subordinately occupied sites were refined with distance and displacement parameter restraints. all crystal structures were checked with the program platon molecular graphics was generated with program mercury crystal data and experimental details are given in table 2. calculations were performed using the gaussian 09 software package, and the b3lyp functional without symmetry constraints. the optimized geometries were obtained with the stuttgart / dresden ecp (sdd) basis set to describe the electrons of the iron atom. for all other atoms | treatment of [fecp(co)2cl ] with 1 equiv of the amidophosphine ligands li[r2pnr ] (r = ph, ipr, r = ipr, tbu, cy) afforded complexes of the type [fecp(co)(2(c, p)-(c 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 o)nipr - pph2) ] (1a), [fecp(co)(2(c, p)-(co)-ntbu - pph2) ] (1b), and [fecp(co)(2(c, p)-(co)-ncy - pipr2) ] (1c) in 4050% yields. complex 1a was also formed when [fecp(co)2(pph2nhipr)]+ (2) was reacted with 1 equiv of kotbu. these complexes feature a four - membered carboxamido - phospha - ferracycle as a result of an intramolecular nucleophilic attack of the amidophosphine ligand on coordinated co. upon treatment of 1a with the electrophile [me3o]bf4 the aminocarbene complex [fecp(co)(2(c, p)c(ome)-nipr - pph2)]+ (3) was obtained bearing an aza - phospha - carbene moiety. upon treatment of cis, trans, cis-[fe(co)2(ph2pnhipr)2(br)2 ] (4a) and cis, trans, cis-[fe(co)2(ph2pnhtbu)2(br)2 ] (4b) with kotbu the carboxamido - phospha - ferracycles trans-[fe(co)2(2(c, p)-(co)-nipr - pph2)(ph2pnhipr)br ] (5a) and trans-[fe(co)2(2(c, p)-(co)-ntbu - pph2)(ph2pnhtbu)br ] (5b) were formed in moderate yield. finally, representative structures were determined by x - ray crystallography. |
we used data collected in the national health interview survey (nhis) from 1986 to 1996 and linked to the national death index (ndi) for mortality through 31 december 2002. the nhis is a continuous, annual, household survey conducted by the national center for health statistics. the survey uses a stratified cluster probability sampling design to collect information from a representative sample of the civilian, noninstitutionalized u.s. population. each subsample is administered one of six checklists of chronic conditions and respondents are asked to indicate the presence or absence of each condition specified on the particular list assigned to them. the nhis and ndi are linked using a probabilistic matching algorithm to determine the vital status of all nhis participants aged 18 years. it is estimated that the matching methods correctly identify > 9% of all living nhis respondents and 96% of those who died, with no substantial difference according to age, sex, race / ethnicity, or socioeconomic status (16). for each participant who died by 3 december 2002, available data included information on the quarter and year of death and on the underlying causes of death classified according to the icd-10. educational attainment was used as the main indicator of sep because unlike income and occupation, education is unlikely to be affected by poor health in adulthood. detailed information on the highest level of school completed was collected and the variable was categorized as (high school not completed), high school degree (high school diploma or general equivalency diploma), and more than high school degree (some college, vocational, or technical school ; associate 's degree ; bachelor 's, master 's, or professional degree). race / ethnicity was self - reported. participants with diabetes were those who reported themselves or whose proxy reported that they had diabetes in the past 12 months. participants were considered at risk for death during the period between the time of nhis interview and either the quarter of their death, the quarter of their 85th birthday, or the fourth quarter of 2002, whichever occurred first. direct standardization was used to estimate age- and sex - standardized mortality rates overall and according to educational level among subjects with and without diabetes, using the whole population (regardless of diabetes) as the standard. educational disparities in mortality were measured using multivariate cox regression models controlling for time - updated age, sex, race / ethnicity, and survey year. terms of interaction between education and diabetes status were included in the models to measure differences in the magnitude of educational disparities between subjects with and without diabetes. first, hazard ratios (hrs) associated with educational level were computed, using the highest level of education as reference. whereas hrs are easy to interpret, comparisons of hrs across various groups of the population are complicated by different distributions of educational level across these subgroups. indeed, the advantages conferred by, e.g., holding a high school degree probably differ across age, sex, or race / ethnicity strata. the use of the relative index of inequality (rii) as a measure of educational inequalities overcomes this problem by providing a continuous measure of inequalities that accounts simultaneously for the size and relative position of educational groups (13). it does so by using a specific measure of individuals ' relative educational position (i.e., the mean proportion of the overall population that has an educational level higher than his / her own). for example, each individual in the lowest educational group is assigned a value corresponding to the proportion of the population with middle or high education, plus half of the proportion of the population with low education. this is therefore a continuous measure, taking the value 0 for someone at the top of the educational scale and 1 for someone at the bottom. the rii, obtained by regressing mortality on this new indicator, is the predicted ratio of mortality rates at the two extremes of the educational scale. we calculated the rii overall (using individuals ' educational position relative to the whole population as indicator of education) and separately across age, sex, and race / ethnicity strata (using individuals ' educational position relative to the population within their strata as indicator of education). absolute educational disparities in mortality were estimated by the slope index of inequality (sii), corresponding to the slope coefficient obtained by regressing mortality on the indicator of relative educational position defined above. the sii is the predicted difference in mortality rates between the two extremes of the educational scale. we accounted for the complex sampling design and data weighting of nhis in estimating standardized mortality rates but not in estimating associations between education and mortality. all statistical analyses were performed using stata / se 10.0 (stata, college station, tx). for each participant who died by 3 december 2002, available data included information on the quarter and year of death and on the underlying causes of death classified according to the icd-10. educational attainment was used as the main indicator of sep because unlike income and occupation, education is unlikely to be affected by poor health in adulthood. detailed information on the highest level of school completed was collected and the variable was categorized as less than high school degree (high school not completed), high school degree (high school diploma or general equivalency diploma), and more than high school degree (some college, vocational, or technical school ; associate 's degree ; bachelor 's, master 's, or professional degree). race / ethnicity was self - reported. participants with diabetes were those who reported themselves or whose proxy reported that they had diabetes in the past 12 months. participants were considered at risk for death during the period between the time of nhis interview and either the quarter of their death, the quarter of their 85th birthday, or the fourth quarter of 2002, whichever occurred first. direct standardization was used to estimate age- and sex - standardized mortality rates overall and according to educational level among subjects with and without diabetes, using the whole population (regardless of diabetes) as the standard. educational disparities in mortality were measured using multivariate cox regression models controlling for time - updated age, sex, race / ethnicity, and survey year. terms of interaction between education and diabetes status were included in the models to measure differences in the magnitude of educational disparities between subjects with and without diabetes. first, hazard ratios (hrs) associated with educational level were computed, using the highest level of education as reference. whereas hrs are easy to interpret, comparisons of hrs across various groups of the population are complicated by different distributions of educational level across these subgroups. indeed, the advantages conferred by, e.g., holding a high school degree probably differ across age, sex, or race / ethnicity strata. the use of the relative index of inequality (rii) as a measure of educational inequalities overcomes this problem by providing a continuous measure of inequalities that accounts simultaneously for the size and relative position of educational groups (13). it does so by using a specific measure of individuals ' relative educational position (i.e., the mean proportion of the overall population that has an educational level higher than his / her own). for example, each individual in the lowest educational group is assigned a value corresponding to the proportion of the population with middle or high education, plus half of the proportion of the population with low education. this is therefore a continuous measure, taking the value 0 for someone at the top of the educational scale and 1 for someone at the bottom. the rii, obtained by regressing mortality on this new indicator, is the predicted ratio of mortality rates at the two extremes of the educational scale. we calculated the rii overall (using individuals ' educational position relative to the whole population as indicator of education) and separately across age, sex, and race / ethnicity strata (using individuals ' educational position relative to the population within their strata as indicator of education). absolute educational disparities in mortality were estimated by the slope index of inequality (sii), corresponding to the slope coefficient obtained by regressing mortality on the indicator of relative educational position defined above. the sii is the predicted difference in mortality rates between the two extremes of the educational scale. we accounted for the complex sampling design and data weighting of nhis in estimating standardized mortality rates but not in estimating associations between education and mortality. all statistical analyses were performed using stata / se 10.0 (stata, college station, tx). we identified 86,817 adults aged 3584 years at the time of the nhis interview, who had been asked about the presence of diabetes and for whom ndi - linked data were available. of these, we excluded 863 with missing data on educational attainment and 87 who died within the quarter following interview, yielding a final sample of 85,867 individuals. the median follow - up time was 10.5 years (range one - quarter to 16.8 years). at baseline, 5,007 (5.6%) participants reported having diabetes ; they accounted for 43,295 person - years of follow - up. regardless of diabetes status, participants who did not complete high school and high school graduates were older and more likely to be women than those with more than a high school degree. participants with less than a high school degree were also more likely to be non - hispanic blacks or hispanics (table 1). characteristics of 85,867 participants with and without diabetes, according to educational level data are %, unless otherwise indicated. of 15,351 participants who died, 2,188 (14.0%) had diabetes at baseline. cvd accounted for 46.6% of the causes of death among participants with diabetes versus 40.2% among those without. major non - cvd causes of death were cancers (17.6% of deaths), diabetes (14.7%), and respiratory conditions (5.9%) among participants with diabetes and cancers (29.7%) and respiratory conditions (10.1%) among those without. all - cause, cvd, and non - cvd mortality rates were 340.0, 150.7, and 189.3 per 10,000 person - years, respectively, in adults with diabetes versus 136.9, 52.1, and 84.8 per 10,000 person - years, respectively, in those without. 1, all - cause, cvd, and non - cvd mortality rates were inversely associated with educational level in both adults with and without diabetes. age- and sex - standardized all - cause, cvd, and non - cvd mortality rates (95% cis) according to educational level among adults with and without diabetes. as shown in table 2, the inverse relationship between education and mortality risk was statistically significant among adults with diabetes even after accounting for age, sex, race / ethnicity, and survey year. overall, the risk of all - cause mortality was 28% higher in diabetic adults with the lowest versus the highest position on the educational scale, as measured by the rii. this inverse relationship between education and mortality risk in adults with diabetes reflected marked educational differences in the risk of cvd mortality. conversely, the risk of non - cvd mortality did not differ significantly across education strata in adults with diabetes. relative and absolute educational disparities in all - cause, cvd, and non - cvd mortality among adults with and without diabetes data are hr / rii / sii (95% ci). hr : hazard ratio of death (reference category : individuals with more than a high school degree). rii : ratio of mortality rates of individuals with the highest and lowest educational level in the population. sii : difference between mortality rates of individuals with the highest and lowest educational level in the population. all measures are adjusted for age, sex, race / ethnicity, and survey year ; p < 0.05 for interaction between educational level and diabetes status. evidence for the existence of an inverse educational gradient in all - cause and cvd mortality risk was found in both diabetic adults aged 3564 years and in their older counterparts, in diabetic men and women, and in white and black diabetic adults (fig. the magnitude of educational disparities in all - cause, cvd, and non - cvd mortality was significantly lower in adults with diabetes compared with their nondiabetic counterparts (table 2). relative index of inequality (adjusted for survey year and, if appropriate, for age, sex, and race / ethnicity) (95% cis) in all - cause, cvd, and non - cvd mortality among adults with diabetes by age (a), sex (b), and race / ethnicity (c). as shown in table 2, the difference in the estimated risk of all - cause mortality between diabetic adults with the lowest versus the highest position on the educational scale, as measured by the sii, was 503.0 deaths per 10,000 person - years. this difference was largely driven by educational disparities in cvd mortality, accounting for 401 excess deaths per 10,000 person - years of follow - up. these absolute educational disparities in all - cause and cvd mortality were greater in adults with diabetes than in their nondiabetic counterparts. in contrast, absolute educational disparities in non - cvd mortality did not differ in magnitude according to diabetes status. we identified 86,817 adults aged 3584 years at the time of the nhis interview, who had been asked about the presence of diabetes and for whom ndi - linked data were available. of these, we excluded 863 with missing data on educational attainment and 87 who died within the quarter following interview, yielding a final sample of 85,867 individuals. the median follow - up time was 10.5 years (range one - quarter to 16.8 years). at baseline, 5,007 (5.6%) participants reported having diabetes ; they accounted for 43,295 person - years of follow - up. regardless of diabetes status, participants who did not complete high school and high school graduates were older and more likely to be women than those with more than a high school degree. participants with less than a high school degree were also more likely to be non - hispanic blacks or hispanics (table 1). characteristics of 85,867 participants with and without diabetes, according to educational level data are %, unless otherwise indicated. cvd accounted for 46.6% of the causes of death among participants with diabetes versus 40.2% among those without. major non - cvd causes of death were cancers (17.6% of deaths), diabetes (14.7%), and respiratory conditions (5.9%) among participants with diabetes and cancers (29.7%) and respiratory conditions (10.1%) among those without. all - cause, cvd, and non - cvd mortality rates were 340.0, 150.7, and 189.3 per 10,000 person - years, respectively, in adults with diabetes versus 136.9, 52.1, and 84.8 per 10,000 person - years, respectively, in those without. 1, all - cause, cvd, and non - cvd mortality rates were inversely associated with educational level in both adults with and without diabetes. age- and sex - standardized all - cause, cvd, and non - cvd mortality rates (95% cis) according to educational level among adults with and without diabetes. as shown in table 2, the inverse relationship between education and mortality risk was statistically significant among adults with diabetes even after accounting for age, sex, race / ethnicity, and survey year. overall, the risk of all - cause mortality was 28% higher in diabetic adults with the lowest versus the highest position on the educational scale, as measured by the rii. this inverse relationship between education and mortality risk in adults with diabetes reflected marked educational differences in the risk of cvd mortality. conversely, the risk of non - cvd mortality did not differ significantly across education strata in adults with diabetes. relative and absolute educational disparities in all - cause, cvd, and non - cvd mortality among adults with and without diabetes data are hr / rii / sii (95% ci). hr : hazard ratio of death (reference category : individuals with more than a high school degree). rii : ratio of mortality rates of individuals with the highest and lowest educational level in the population. sii : difference between mortality rates of individuals with the highest and lowest educational level in the population. all measures are adjusted for age, sex, race / ethnicity, and survey year ; p < 0.05 for interaction between educational level and diabetes status. evidence for the existence of an inverse educational gradient in all - cause and cvd mortality risk was found in both diabetic adults aged 3564 years and in their older counterparts, in diabetic men and women, and in white and black diabetic adults (fig. 2). however, such a gradient was not found among hispanic adults with diabetes. the magnitude of educational disparities in all - cause, cvd, and non - cvd mortality was significantly lower in adults with diabetes compared with their nondiabetic counterparts (table 2). relative index of inequality (adjusted for survey year and, if appropriate, for age, sex, and race / ethnicity) (95% cis) in all - cause, cvd, and non - cvd mortality among adults with diabetes by age (a), sex (b), and race / ethnicity (c). as shown in table 2, the difference in the estimated risk of all - cause mortality between diabetic adults with the lowest versus the highest position on the educational scale, as measured by the sii, was 503.0 deaths per 10,000 person - years. this difference was largely driven by educational disparities in cvd mortality, accounting for 401 excess deaths per 10,000 person - years of follow - up. these absolute educational disparities in all - cause and cvd mortality were greater in adults with diabetes than in their nondiabetic counterparts. in contrast, absolute educational disparities in non - cvd mortality did not differ in magnitude according to diabetes status. our results suggest that differences in educational position produce substantial disparities in mortality risk in u.s. adults with diagnosed diabetes regardless of age, sex, and race / ethnicity. in relative terms, these disparities are weaker than in nondiabetic adults. however, in absolute terms, adults with diabetes suffer the greatest mortality burden from low educational position, with a difference of over 500 deaths per 10,000 person - years of follow - up between the two extremes of the educational scale. these disparities are mainly driven by cvd mortality, a cause of death for which many effective preventive measures are available. strengths of this study, which lend weight to these conclusions, include a nationally representative cohort large enough to afford multiple stratified multivariate analyses and long - term follow - up that is nearly 100% complete. the accuracy of diabetes self - report has been reported to be high overall and improves with educational level (17). moreover, approximately one - third of u.s. adults with diabetes are estimated to be undiagnosed (18), a rate possibly higher among people with low education (19). this suggests that self - reported cases of diabetes may underrepresent the milder cases (i.e., those either undiagnosed or diagnosed but underreported), especially among people with a low education. consequently, educational health disparities measured within adults with diagnosed diabetes may be more marked than those occurring in the whole population of people with diabetes. additionally, although educational health disparities may differ according to diabetes type (7), nhis does not attempt to distinguish between type 1 and type 2 diabetes. however, since type 2 diabetes accounts for the large majority (9095%) of cases in the u.s. mortality rates provided in the study were estimated accounting for sampling weights, thus they are representative of the u.s. population. however, we could not account for data weighting in estimating associations between education and mortality because we were unable to calculate correct sampling weight for bootstrap analyses and thus unable to provide an accurate estimate of rii and sii variances. complementary analyses show that regardless of diabetes status, point estimates of hrs and rii do no substantially differ whether calculations are based on weighted or unweighted data. though, sii estimates in adults with diabetes appear greater using weighted rather than unweighted data (600.3 vs. 503.0 deaths per 10,000 person - years for all - cause mortality), suggesting that absolute educational disparities in mortality among u.s. adults with diabetes may be underestimated in our study. by showing that the risk of mortality differs according to educational attainment, both in relative and in absolute terms, our results provide strong evidence for the existence of educational disparities in mortality in u.s. thereby, the present study suggests that socioeconomic disparities in health previously reported among people with diabetes in europe (5,6) occur in the u.s. underlying pathways may involve a large range of factors, including patient factors (e.g., health behaviors, material conditions, or psychosocial factors) as well as characteristics of the providers, the community, and the health care system (6). given the major burden of diabetes in the u.s. across the various socioeconomic strata of the population adults with diabetes are mainly driven by differences in the risk of death from cvd causes. including deaths with diabetes as the underlying cause in the definition of cvd deaths did not change this finding (data available on request). in addition, such disparities among adults with diabetes are substantial in all age, sex, and race / ethnicity strata except hispanic adults. the absence of educational health disparities among hispanics has been reported among the general population as well (20), suggesting that its underlying mechanisms are likely to be independent of diabetes status. although they are substantial, educational disparities in mortality in adults with diabetes appear to be smaller than disparities in nondiabetic adults. such difference has also been reported in italy (7) and in finland (8), two countries with equitable access to health services. one possible explanation is that diabetes management levels off disparities in health care and health behaviors across the various educational groups. our findings suggest that such a salutary role of diabetes management may occur as well in the context of the u.s. health care system, a hypothesis supported by a recent study (21) showing that concurrently with major improvement in diabetes management over the past decade, there has been limited widening of educational health disparities in the u.s. diabetic population. whether this arises from the specificities of diabetes management itself or from its beneficial consequences in terms of enhancing health care access and use deserves further studies. we found that in diabetic adults absolute disparities in mortality are strong, specifically from cvd - related causes, and, in contrast to relative disparities, greater than in the nondiabetic population. indeed, we found that diabetic adults who hold the lowest position on the educational scale suffer 503 excess deaths per 10,000 person - years of follow - up than those with the highest position, a gap 73% higher than in the nondiabetic population. the results were even more striking for cvd mortality, with a gap 319% higher in the diabetic versus the nondiabetic population. the contrasted results we obtained using either relative or absolute measures of disparities stem from the fact that the burden of cvd mortality is dramatically higher in adults with versus without diabetes. this finding highlights the relevance of using both relative and absolute measures of inequalities to adequately assess health disparities and suggests that educational health disparities among adults with diabetes have a major public health impact. in summary, we have shown that the risk of mortality differs substantially according to educational level among people with diabetes in the u.s. although relative educational disparities in mortality are less marked in adults with diabetes than in those without, their absolute impact is greater and translates into a major mortality burden., especially among the most deprived categories of the population, this suggests that reducing social health inequalities among people with diabetes is likely to have a major public health impact. future research should determine pathways underlying these educational disparities with an eye toward developing strategies to eliminate them. | objectiveto measure relative and absolute educational disparities in mortality among u.s. adults with diabetes and to compare their magnitude with disparities observed within the nondiabetic population.research design and methodsa total of 85,867 individuals (5,007 with diabetes), aged 3584 years, who participated in the national health interview survey from 1986 to 1996 were followed for mortality through 31 december 2002. relative and absolute educational disparities in all - cause, cardiovascular disease (cvd), and non - cvd mortality were measured.resultsin relative terms, the risk of all - cause mortality was 28% higher in diabetic adults with the lowest versus the highest position on the educational scale (relative index of inequality 1.28 [95% ci 1.081.53 ]). this inverse relationship reflected marked disparities in cvd mortality and was found in all age, sex, and race / ethnicity groups except hispanics. although substantial, this relative educational gradient in mortality among adults with diabetes was smaller than in the nondiabetic population. in absolute terms, diabetic adults with the lowest position on the educational scale suffered 503 excess deaths per 10,000 person - years of follow - up compared with those with the highest position. these absolute disparities were stronger than in the nondiabetic population. the results were even more striking for cvd mortality.conclusionsthe risk of mortality differs substantially according to educational level among individuals with diabetes in the u.s. although relative educational disparities in mortality are weaker in adults with versus without diabetes, their absolute impact is greater and translates into a major mortality burden. |
cephalometry has been widely used for the diagnosis, planning, and evaluation of craniofacial growth and development, and the follow up of longitudinal studies for different orthodontic therapies. conventional cephalometric records, as part of the orthodontic documentation, include lateral cephalograms. with the introduction of new technologies, such as digital radiographies, and cone beam computed tomography (cbct) scans, it has become necessary to validate the images generated from these exams to afford comparisons. because these imaging methods conventional and digital cephalograms, and images similar to cephalograms obtained from cbct scans have not been compared, new images may not be used to evaluate the growth and longitudinal results of orthodontic therapies in relation to conventional cephalograms. traditional imaging methods have been questioned due to a higher probability of errors while identifying landmarks, or making hand - traced measurements, and for the large amount of time consumed for the evaluations. moreover a number of studies have compared the efficiency of programs that carried out evaluations of digitized cephalograms with those of manual tracing methods, and asserted that the digital method can make linear and angular measurements in an efficient manner. such results, however, are not unanimous in the literature. in addition, since cephalometric analyses are subject to human judgment, and because of errors of different magnitudes such as landmark identification, measurement methods, and quality of radiographic exams, methods are sought that will minimize such errors. new technologies are emerging, aiming at improving the quality of such evaluations. despite the countless advantages of the current programs, no consensus exists regarding the best way to accomplish the migration from manual tracing to digital tracing. since the change to digital methods is eminent, professionals must be prepared, so that the transition is accomplished in the safest way possible. before adopting new methods in scientific research, the comparison of traditional exams with those using digital images, and with those obtained from cbct scans is fundamental, with a view to making this transition from bidimensional to tridimensional methods. thus, the aim of this study was to evaluate whether a difference exists between cephalometric measurements based on manual tracings (mt), digitized lateral cephalograms (dlc), and in lateral cephalograms obtained from cbct scans (lc - cbct). the protocol of this study was approved by the ethics in research of the university north of paran. the sample size for each group was calculated based on an alpha significance level of 0.05 and a beta of 0.2 to achieve 80% of power. fifty patient exams [conventional lateral cephalograms and cone beam computer tomography (cbct) scans ] from a radiological clinic were selected. for the manual tracings, the cephalograms were traced manually, and evaluated according to the conventional method, for the digitized lateral cephalograms, the cephalograms were digitized and measured by using the dolphin imaging 11 program (dolphin imaging, chatsworth, ca, usa), and for the lateral cephalograms from the cbct, the cephalometric measures were made on images similar to lateral cephalograms obtained from the cbct scans by using the same program as that used with the dlc. the lateral cephalograms were obtained from the same orthopantomograph op 100 (instrumentarium corp tusula, finland) machine (17.6 s., 77 kvp, and 12 to 14 ma), with a 10% rate of magnification and with patients placed at 1.52 m away from the cephalostat. all measurements were made by the same examiner (g.o). for the mt, the measurements were made in a darkened room, using ultraphan paper (size 8"x10 ", thickness of 0.03, gac, a negatoscope and a 0.5 pentel mechanical pencil (figure 3a). definitions of abbreviations of the less usual cephalometric variables used cephalometric variables : 1, 1.na ; 2, 1-na ; 3, impa ; 4, a - nperp ; 5, pog - nperp ; 6, 1.nb ; 7, 1-nb ; 8, ul - e ; 9, ll - e ; 10, stc ; 11, ban / ptgn ; 12, sn / occlusal plane a) manual tracing ; b) digitized lateral cephalograms ; c) lateral cephalograms from cone - beam computed tomography (cbct) for the dlc, the same 50 cephalograms that were used for the mt were digitized on a scanner which is proper for radiographs (hp 4500, 600 dpi), and using the ruler for a 100 mm calibration as recommended by the manufacturer of the dolphin program. prior to the measurement of the cephalometric magnitudes, the examiner was allowed to treat the images, so as to improve the brightness and contrast, in order to better identify the structures. once the treatment of the images was completed, the measurements were made (figure 3b). cbct scans were performed using an i - cat tomography (imaging sciences, hatfield, pa, usa), with an fov of 22x16 cm, 40 s, 0.4 voxel, 120 kvp, and 36 mas. for the lc - cbct, prior to making the measurements, a standardization for each image was made, with a conventional lateral skull, and a view of the right side of the patients. these reconstructed images were lined up, with the orbits parallel to the horizontal plane, and with a corrected head 's rotation. after the alignment of the skull, an image similar to the right lateral cephalograms was generated, and stored in a jpeg format (1360x2045 - 8 bits). the landmarks were established and the measurements generated automatically by the software (figure 3c). with the cbct scans, in addition to the resources used previously for the dlc (variation of brightness and contrast), the dolphin program afforded image filters as additional tools, which were used, to make it easier to see the anatomic repairs (figure 4). filters of the software dolphin imaging thirty days after the first evaluation (t1), all the exams were retraced by the same examiner (t2). the error of the method was carried out for each variable individually, by using the paired t test and the dahlberg formula. all the cephalometric variables were normally distributed. for comparison between the methods, analysis of variance (anova) all the tests were made using the statistical for windows v.5.1 (statsoft inc., tulsa, ok, usa) program, with a 5% level of significance (p<0.05). thirty days after the first evaluation (t1), all the exams were retraced by the same examiner (t2). the error of the method was carried out for each variable individually, by using the paired t test and the dahlberg formula. all the cephalometric variables were normally distributed. for comparison between the methods, analysis of variance (anova) was used, followed by the tukey test and pearson 's correlation coefficient. all the tests were made using the statistical for windows v.5.1 (statsoft inc., tulsa, ok, usa) program, with a 5% level of significance (p<0.05). the results obtained showed a systematic error for 7 of the variables in the mt (sna ; pog - nb ; ans - me ; 1-na, ul - e ; ll - e, and gl'-sls - pog ') and 6 variables in the dlc (co - a ; ans - me ; impa ; 1-na ; 1-nb, and gl'-sls - pog '). the range of casual errors for the mt varied from 0.63 to 2.38, and 0.52 to 3.00 for the dlc (tables 1 and 2), with most of the variables below 2 or 2 mm. no systematic errors were detected for the lc - cbct, and the range of casual errors varied from 0.27 to 0.91 (table 2). results of systematic and casual errors investigation for manual tracings sd = standard deviation statistically significant at p<0.05 results of systematic and casual errors investigation for digitized lateral cephalograms sd = standard deviation statistically significant at p<0.05 pearson 's correlation analysis proved significant among the three methods studied (table 3). six variables showed a statistically significant difference (sna ; co - a ; anb ; ul - e ; is - na ; and gl'-sls - pog ') among the methods (table 4). measurements made in the dlc had a statistically greater sna than those made in the mt and lc - cbct. regarding maxillary length (co - a), the measurements performed using the mt and dlc were statistically greater than the lc - cbct. with regards to the evaluation of the maxillomandibular relationship, the dlc was greater than that evaluated on the mt. the protrusion of the upper incisor was lower in the dlc than the measurements made on the mt and lc - cbct. regarding the evaluation of the soft tissue, the dlc showed statistically lower values than the mt and lc - cbct for variable gl'-sls - pog ', and for variable ul - e, the dlc had lower values compared to the lc - cbct. results of systematic and casual errors investigation for lateral cephalograms from cone - beam computed tomography (cbct) sd = standard deviation statistically significant at p<0.05 correlations between methods (pearson correlations) statistically significant at p<0.05 variations between the cephalometric and cbct methods reflect the technical differences inherent to each system. in a cephalostat, the distance between the midsagittal plane of the head and the radiation source is fixed, as is the distance from the midsagittal plane to the film. in cbct devices, these differences may lead to variations in magnifications and distortion. for angular measurements, however, absolute distances between landmarks can show differences between methods, especially if they are located in different tomographic planes, as previously reported. one problem in this study was that none of the 3 imaging methods could be taken as the gold standard. for this reason, our focus was on the reproducibility of the parameters obtained on the 3 types of cephalograms. the results of the analyses showed that the measurements performed were independent of the type of image used (conventional, scanned or cbct). the cephalometric analyses performed on cbct cephalograms were more reproducible than the measurements made on both the conventional and the digital cephalograms (tables 1, 2 and 3). the first reason might be because errors of projection present in the conventional cephalograms, and, therefore, the identification of landmarks of bilateral structures offer some inaccuracy. to convert a conventional cephalometric film into a digital format by scanning can result in image distortion. this is the reason why the cephalogram data sets must be prepared so that the structures may be clearly seen. in the mt, the measurements that showed a statistically significant difference were the sna(1.82), pog - nb (1.45 mm), ans - me (0.94 mm), 1-na (0.86 mm), ul - e (0.94 mm), ll - e (0.84 mm), and gl'-sls - pog ' (1.65 mm) (table 1). these alterations may be justified by the difficulty of locating the a point, the anterior nasal spine, and the perfect identification of the tip of the nose in all evaluated cephalograms, since these landmarks are of subjective location and low radiopacity. furthermore, the mechanical errors introduced by drawing lines between landmarks manually and by measuring with a ruler and protractor were common in conventional cephalometric analyses. although a radiographic film is quite stable and can retain information for many years, due to its physical nature, it is not always a dependable means of filing. film deterioration has been a major source of information loss in craniofacial biology ; therefore, digital archiving of lateral cephalograms is a valuable method for orthodontic clinics. today, due to technological advancements and the need for data mobility the dlc demonstrated statistically significant differences in 6 cephalometric measurements ; co - a (1.96 mm) ; ans - me (1.12 mm) ; impa (2.02) ; 1-na (1.90 mm) ; 1.nb (2.49), and gl'-sls - pog ' (1.19) (table 2). previous studies have already reported errors between manual and digitized tracings similar to those found in this study, but concluded that although these values are statistically significant, they do not present clinical relevance (2 degrees or 2 mm). the digitalization process of scanners changes the nature of the image from an analog form to a digital form. however, the use of computers for cephalometric analyses does not increase the measurement error when compared with hand tracing. most studies evaluating the accuracy of on - screen computer tracing software have transferred conventional cephalometric films to a digital format by scanning, a procedure that may result in image distortion. (2001) indicate that 75 dpi is enough for scanning lateral cephalograms. during landmark digitalization, magnification was often used to identify certain structures more accurately. in several instances, the magnification caused significant pixelation and blurriness of the image, increasing the difficulty of accurate identification. a higher scanning dpi was suggested to assist in circumventing this problem, and for this reason all radiographs were scanned at 600 dpi in this study. if the film is scanned and transferred to digital format, such as in the present study, the quality of the original film is one of the most important criteria in validating the results, and all radiographs offered excellent quality. the measurements of the lc - cbct did not show significant differences between t1 and t2, thus affording more reliable tracings (table 3). this greater reproducibility may be justified by the possibility of using image filters tools that proved to be essential for the correct identification of anatomic landmarks. previous studies have stated that the degree of error is related to the identification of landmarks, which depends especially on the level of experience of the examiner, on the definition of the landmark itself, and on the density and clearness of the images. nevertheless, there are other ways of reducing errors, such as, care when carrying out exams, standardization of the analysis of cephamometric measurements, and, more recently, the possibility of using specific software for analysis and planning in orthodontics. the dolphin software affords the enhancement of the cephalograms, which is advantageous especially while precisely marking soft tissue landmarks. although a significant difference was found for some of the variables in the mt and dlc methods, the pearson 's correlation analysis proved significant among the three methods studied, for all magnitudes evaluated. (2010), wherein cephalometric measurements obtained from digitized lateral cephalograms were compared to those obtained from cbct, using the same program as that used in this study. thus, although individually, the first two methods showed significant differences for some of the variables, identifying cephalometric landmarks seems to get increasingly easier by using tools that make the contrast between anatomic structures clearer. therefore, the three methods may be used safely, in that the precision of the lc - cbct must be emphasized. the direct landmark identification method with monitor - displayed images has the following advantages : excellent repeatability and reproducibility, time saving because of no tracing, and efficiency because of no accessory equipment and supplies to print out hard copies of the digital images. although errors in identification of the 3d craniofacial structures with a 2d approach have been addressed, cephalometry has been and still is a valuable method to diagnose and evaluate the treatment outcomes of orthodontic patients. the importance of testing different available methodologies to make cephalometric analyses must be pointed out. these studies afford excellent results during the inevitable transition from analog to digital records. in the current study, only six of the 20 cephalometric variables (sna ; co - a ; anb ; 1-na ; ul - e, and stc) offered statistically significant differences between methods (table 5). but for soft tissue variables, all the measurements that showed statistically significant differences were related to the a point. this point lies at the edge of the skeletal structures and most of the time is the least reliable. although the human eye can determine the edge of a skeletal structure with precision, it is not always easy to trace the intended target with sufficient accuracy. the idea behind the software feature investigated in this study was that a cursor and the software tools could facilitate this coordination during landmark digitization and facilitate the measurements. furthermore, differences between soft tissue measurements were found, because some information can be obtained by enhancing the contrast or creating a mask that can highlight the soft tissue profile. intergroup comparisons of the cephalometric variables among the 3 methods evaluated (anova and tukey tests) different letters indicate statistically significant differences statistically significant at p<0.05 in contrast to conventional cephalograms, errors due to malposition of the patient during image acquisition could be corrected in cbct data sets by interactive adjustment. the innate 3d characteristics of the cbct data sets allow the generation of virtually countless reformatted images, and orthogonal cephalograms (parallel x - rays). furthermore, it is possible to represent the right and left parts of skulls separately, avoiding superimposition of the bilateral structures. however, the cbct scans are valued when 3d morphology is necessary, and should be used only when the inherent 3d information could improve the diagnosis and treatment plan. cbct demands a higher radiation dose than traditional cephalometric images. for these reasons, its use should be limited to specific indications, such as patients with impacted teeth, or those with facial asymmetries or craniofacial anomalies, in which cbct is better able to quantify the differences between the right and the left side of craniofacial structures. cbct data sets can provide undistorted 3d morphology, making it possible to identify craniofacial structures more naturally. however, landmark identification in 3d is not simple. to obtain a high level of precision is very important, especially when new tools are used in scientific research, since image visualization errors would result in altered diagnoses, and, thus, in erroneous plans of treatment., there should be concerns that persons who are inappropriately trained to read images, regardless of the method used, will misinterpret the data with consequent misdiagnoses and inappropriate patient treatment. 1- all the methods tested proved to be reliable and practical for scientific research, with clinically acceptable differences between the manually and digitally traced radiographs. | objective : the aim of this study was to compare the reliability of three different methods of cephalometric analysis.material and methods : conventional pretreatment lateral cephalograms and cone beam computed tomography (cbct) scans from 50 subjects from a radiological clinic were selected in order to test the three methods : manual tracings (mt), digitized lateral cephalograms (dlc), and lateral cephalograms from cbct (lc - cbct). the lateral cephalograms were manually analyzed through the dolphin imaging 11.0(tm) software. twenty measurements were performed under the same conditions, and retraced after a 30-day period. paired t tests and the dahlberg formula were used to evaluate the intra - examiner errors. the pearson 's correlation coefficient and one - way analysis of variance (anova) tests were used to compare the differences between the methods. results : intra - examiner reliability occurred for all methods for most of the measurements. only six measurements were different between the methods and an agreement was observed in the analyses among the 3 methods. conclusions : the results demonstrated that all evaluated methodologies are reliable and valid for scientific research, however, the method used in the lateral cephalograms from the cbct proved the most reliable. |
now that the human genome and the hapmap projects have been completed, the international scientific community is turning its attention to the 1000 genomes project, an international collaboration between china, germany, the uk, and the usa. the initial goal was to discover most of the genetic variation that occurs at a population frequency greater than 1% by deep sequencing at least 1,000 individuals from different worldwide populations using next - generation platforms and technologies. the genomes of approximately 2,000 individuals, from at least 20 different populations representing africa, europe, east asia, and the americas, are being collected and sequenced. for some populations, trios (both biological parents and an adult child) have been collected. many of the samples, including some from the children, are going to be densely genotyped using genome - wide arrays. the goal of this study design is to reconstruct the parental chromosomal phase using the information provided by the child. promising results have already arisen from the pilot 1 analysis, which has been referred to as the ' 1000 genomes low coverage pilot '. this analysis consists of 180 individuals from the four original hapmap populations sequenced at 2 to 4 coverage, meaning that an average number of two to four sequences are generated for every genomic position. in this initial phase, more than 9 million new single - nucleotide poly mor phisms (snps), many novel indels (insertions / deletions), and some large structural variants have been identified. the results that arise from the completion of this project will lead to a great leap in our knowledge of human genetic variation. at first, the results will allow scientists to identify population - specific genetic variation at an unprecedented degree of resolution. of the 9 million novel snps identified so far from pilot 1 analysis alone, approximately 8 million are seen in only one hapmap population. most common variants have already been identified ; the novel variants are disproportionately rare and thus more likely to be observed in only one of the studied populations. the identification of these variants will help the development of new population - specific genotyping arrays. this will maximize genome coverage while minimizing the ascertainment bias that affects currently available arrays, especially for non - european populations. this unbiased survey of polymorphism in diverse groups will also help improve the imputation methods for genetic variants that are not represented on current arrays. in addition to improving the resolution of population genetics, the clinical implications are endless. for example, genome - wide association studies (gwass) are now routinely used to identify genomic regions associated with common human diseases. however the reason for this is that the human genome contains regions of strong linkage disequilibrium, and a disease - associated locus can encompass several genes and multiple tightly associated polymorphisms. in addition, current arrays emphasize so - called ' tag snps ', or snps that are highly correlated with their local linkage disequilibrium structure to provide more even coverage across the genome. these snps can usually be found in introns or intergenic regions, not coding regions or known regulatory elements that are likely to be functional. this makes it difficult to pinpoint causal variants by association mapping using genotyping arrays alone. deep sequencing studies will identify novel or rare functional variants, thereby allowing scientists to find all potential disease - causing variants and genes. however, the strong associations among genetic variants in a given genomic region will require experimental studies to be performed to determine which of the associated genetic variants are actually functionally causal. recent work has shown on a small level that resequencing of candidate regions from gwass can often uncover rarer variants with higher effects and more direct functional consequences in common diseases. the unprecedented level of sequence information that will arise from the 1000 genomes project will also improve our knowledge of genomic configurations that were shaped by evolutionary processes. for example, analyses of the distribution of snp density along chromosomes will inform us about chromosomal regions that are more susceptible to selective pressures or differential patterns as a result of the expansion of humans throughout different continents. recent findings from exonic resequencing have shown that patterns of population - specific rare, deleterious mutations (such as those that cause mendelian recessive diseases) in coding regions can be largely explained by historical processes affecting specific populations. however, understanding how these processes occur, which genes are affected, and which other populations in the future could have a higher prevalence of diseases caused by rare variants requires a large - scale resource for validation of population genetics methods, a resource such as the 1000 genomes project. the samples that are included in the 1000 genomes project do not have identifying information, phenotypes, or clinical data available. the project is providing a resource about human genetic variation that will be used in many studies of particular phenotypes, such as complex diseases or drug response. the availability of full genome sequences from worldwide samples will directly improve the accuracy of direct - to - consumer genetic ancestry tests. medical applications, such as determining drug efficacy and/or toxicity and prediction of disease and prognosis, will need further deep sequencing in clinical projects before public benefits from these new technologies are developed. the attention from the media will probably increase the public 's expectations of the 1000 genomes project and its potential applications. however, scientists should take advantage of the increased public awareness to highlight the importance of genetic research and to encourage the participation of all communities in future research. larger follow - up studies will be needed to achieve the required statistical power to establish conclusions in datasets that will include billions of variables integrating genomic data with future transcriptomic, proteomic, and epigenetic information. the recent improvements in sequencing technology, which allow the sequencing of samples for the 1000 genomes project, presage the forthcoming availability of whole genome sequences at affordable prices. the sequencing of individual genomes raises concerns about potential threats to privacy and other ethical issues. although the usa recently passed the genetic infor mation nondiscrimination act (gina), prohibiting genetic discrimination in employment and health insurance, the protection of individual rights varies from country to country. for example, the european commission strongly recommends the prevention of discrimination as a result of genetic testing in insurance and employment. however, the eu does not have uniform legislation regarding whether it is legal for insurance companies or employers to access genetic testing results or other medical records. rather, the decision on whether to legislate the use of genetic test results has been left to the discretion of each individual country. the information that the 1000 genomes project and the next generation of deep sequencing platforms will provide is unprecedented and will have important implications for genetics and healthas we move closer and closer to the era of the ubiquitous personal genome as part of our medical record. despite our best intentions, history has demonstrated that it will be very difficult to get the genie back into the bottle once it is opened. although there are potential social costs associated with linking informative genetic data to individuals or populations, we believe that these potential costs are outweighed by the benefits in terms of diagnosis and research. scientists should continue to use the genetic diversity that exists within humans as starting points for further research. rather, we should keep rigorous safeguards in place to ensure that scientific advancements proceed and serve to benefit all humanity. finally, it is important to establish a fluent communication with the general public and engage participation in genetic research. gina : genetic information nondiscrimination act ; gwas : genome - wide association study ; snp : single - nucleotide polymorphism. we acknowledge the support of national institutes of health (hl078885, hl088133, u19 ai077439, and es015794), flight attendant medical research institute (famri), and the rwj amos medical faculty development award to egb, beatriu de pinos postdoctoral grant (2006 bp - a 10144) to mv, the ucsf chancellor 's fellowship to cg, the sandler center for basic research in asthma and the sandler family supporting foundation. we also thank lisa brooks and jean mcewen (nih / nhgri and the 1000 genomes project) for their usef | the 1000 genomes project, an international collaboration, is sequencing the whole genome of approximately 2,000 individuals from different worldwide populations. the central goal of this project is to describe most of the genetic variation that occurs at a population frequency greater than 1%. the results of this project will allow scientists to identify genetic variation at an unprecedented degree of resolution and will also help improve the imputation methods for determining unobserved genetic variants that are not represented on current genotyping arrays. by identifying novel or rare functional genetic variants, researchers will be able to pinpoint disease - causing genes in genomic regions initially identified by association studies. this level of detailed sequence information will also improve our knowledge of the evolutionary processes and the genomic patterns that have shaped the human species as we know it today. the new data will also lay the foundation for future clinical applications, such as prediction of disease susceptibility and drug response. however, the forthcoming availability of whole genome sequences at affordable prices will raise ethical concerns and pose potential threats to individual privacy. nevertheless, we believe that these potential risks are outweighed by the benefits in terms of diagnosis and research, so long as rigorous safeguards are kept in place through legislation that prevents discrimination on the basis of the results of genetic testing. |
parkinson 's disease (pd) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in the subsequent loss of function of the basal ganglia circuit. the molecular pathogenesis of pd is believed to be associated with mitochondrial dysfunction, oxidative stress, and activation of the apoptotic cascade. the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp) induces permanent parkinsonism in humans via its metabolite mpp (1-methyl-4-phenylpyridinium) [2, 3 ]. mpp has been shown to induce a pd - like pathology in animals and cellular models by selective and potent inhibiting of complex 1 of the mitochondrial electron transport chain [4, 5 ]. mpp - induced neuronal death is mediated by impairment of the mitochondrial membrane potential and opening of the mitochondrial permeability transition pore [6, 7 ]. elevation in the reactive oxygen species (ros) level has also been involved in mpp - induced cytotoxicity [810 ]. activation of the apoptotic cascade may play a role in mpp - induced cell death by altering mitochondrial membrane permeability and controlling the release of cytochrome c from mitochondria [11, 12 ]. caspase-3 activation by released cytochrome c has been shown to involve mpp - induced apoptosis [9, 13, 14 ]. once activated, caspase-3 will induce nuclear dna condensation and fragmentation and, ultimately, apoptosis. a number of antioxidants, such as xanthones, have been demonstrated to have a protective effect on vulnerable neurons under oxidative stress conditions [1618 ]. the fruit hull of mangosteen (garcinia mangostana linn.), a tropical fruit, has been demonstrated to exert an antioxidative effect. alpha - mangostin was shown to induce a protective effect in cardiac reperfusion damage by attenuation of oxidative stress. neuroprotective activities of alpha - mangostin against h2o2-induced oxidative stress have been demonstrated in ng108 - 15 neuroblastoma cells. this xanthone ameliorated iodoacetate - induced cell death in primary cultures of cerebellar granule neurons by reducing ros formation. alpha - mangostin was also shown to attenuate the neurotoxicity induced by beta - amyloid oligomers in sk - n - sh neuroblastoma cells and primary rat cerebral cortical neurons [22, 23 ]. the antioxidative property of alpha - mangostin is probably mediated by its modulatory effect on the activity of glutathione peroxidase. although alpha - mangostin has been reported to possess potential neuroprotective properties, there is insufficient information on its protective effects in a pd cellular model. this study aims to investigate whether alpha - mangostin could protect sh - sy5y neuroblastoma cells from mpp - induced apoptosis and the possible underlying mechanisms. the sh - sy5y human neuroblastoma cells were cultured in a 1 : 1 mixture of dulbecco 's modified eagle medium (dmem) and nutrient mixture ham 's f12 medium and supplemented with 10% heat - inactivated fetal bovine serum (fbs), 1 mm sodium pyruvate, 0.1 mm nonessential amino acid, 1.5 g / l sodium bicarbonate, 100 units / ml penicillin, and 100 g / ml streptomycin. all media and supplements were purchased from gibco (gaithersburg, md, usa). cells were maintained at 37c in a humidified atmosphere of 5% co2. in the experiment the number of cells to be subcultured was assessed under a phase - contrast microscope based on the exclusion of trypan blue dye. the cultured cells were maintained for 2 days to allow for adhering on the plates. louis, mo, usa), or a combination of -mangostin and mpp according to the experiment design. sh - sy5y cells were seeded onto a 96-well plate at a density of 8 10 cells / well in 200 l of medium and incubated at 37c under 5% co2 in a humidified incubator for 2 days. after exposure to mpp (1000 m), -mangostin (10 m), or a combination of both for 24 hours, cell viability was measured by mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay (sigma - aldrich, st. this method was based on the reduction of tetra ring of mtt by mitochondrial dehydrogenases with nadh in the active mitochondria, yielding a blue formazan product, which can be measured spectrophotometrically. after incubation, 20 l of mtt (5 mg / ml) was added to each well and the cells were cultured for another 4 hours, then medium was removed, and 100 l of dmso (sigma - aldrich, st. louis, mo, usa) was added to each well to dissolve the formazan. the color reaction was measured at wavelength 570 nm with a reference at 690 nm using the versamax tunable microplate reader with softmax pro software (molecular devices, sunnyvale, ca, usa). sh - sy5y cells were seeded on coverslips and fixed with 4% paraformaldehyde in 0.01 m pbs (ph 7.4) for 20 minutes at room temperature and then rinsed three times with pbs. after being washed with pbs, the cells were stained with 1 mg / ml hoechst 33258 (sigma - aldrich, st. louis, mo, usa) in pbs for 20 minutes at room temperature and then washed again. hoechst 33258 (bisbenzimide) preferentially binds to triplet adenine and thymine base pairs in the minor groove outside the double helix, which allows one to observe the morphological change in the nuclei of apoptotic cells. nuclear morphology was examined under a laser scanning confocal microscopy (olympus fv1000, olympus, tokyo, japan) with excitation wavelength 556 nm and emission wavelength 573 nm. sh - sy5y cells were seeded onto 6-well plates at a density of 12 10 cells / well in 2 ml of medium and then incubated at 37c under 5% co2 in a humidified incubator for 24 hours. after exposure to mpp (1000 m), -mangostin (10 m), or the combination of mpp (1000 m) and -mangostin (10 m) for 24 hours, cells were trypsinized and centrifuged at 2,500 rpm, 4c for 5 minutes. the pellet was washed twice with cold pbs and resuspended at a concentration 1 10 cells / ml. apoptotic cells were detected using an fitc annexin - v / dead cell apoptosis kit (molecular probes, eugene, or, usa). cells were incubated with 5 l of fitc annexin - v and 1 l of the 100 g / ml propidium iodide (pi) at room temperature for 15 minutes. the apoptotic cells were analyzed with a facscalibur flow cytometer (bd biosciences, san jose, ca, usa), measuring the fluorescence emission at 530 nm (fl1) and > 575 nm (fl3). both early apoptotic (annexin - v - positive and pi - negative) and late apoptotic (annexin - v - positive and pi - positive) cells were included in cell death determinations. the production of ros was determined by measuring the intensity of fluorescence emitted by cell - permeable fluorescent dyes, dihydrorhodamine 123 (dhr123) and dihydroethidium (dhe), purchased from invitrogen (eugene, or, usa). dhr123 is oxidized to a fluorescent rhodamine-123 by intracellular hydrogen peroxide and peroxynitrite, while dhe is oxidized by superoxide anion to ethidium. sh - sy5y cells were seeded on 6-well plates at a density of 12 10 cells / well and incubated at 37c under 5% co2 in a humidified incubator for 48 hours. after exposure to mpp (1000 m), -mangostin (10 m) or the combination of mpp (1000 m) and -mangostin (10 m) for 48 hours, cells were washed twice with hanks buffer salt solution (hbss) and trypsinized by 0.25% trypsin / edta and centrifuged at 2,000 rpm for 7 minutes. the pellets were incubated with 15 m dhr123 and 10 m dhe for 20 minutes at 37c in the dark. thereafter, cells were washed with hbss before cell fixation in 1% paraformaldehyde and the mean fluorescence intensity (mfi) of green (fl1, dhr) or red (fl2, dhe) fluorescence was determined using a facscalibur flow cytometer. sh - sy5y cells were seeded onto 6-well plates at a density of 12 10 cells / well and incubated at 37c under 5% co2 in a humidified incubator for 24 h. after exposure to mpp (1000 m), -mangostin (10 m) or the combination of mpp (1000 m) and -mangostin (10 m) for 24 hours, cells were trypsinized and centrifuged at 2,500 rpm, 4c for 5 minutes. total mrna was extracted from the pellet using paris kit according to the supplier 's instructions. the quantity and purity of rna were determined by optical density measurements at od a260/a280 ratio with 1.8 or above using nanodrop 2000 spectrophotometer (thermo fisher scientific inc., wilmington, de, usa). the integrity of rna was confirmed by running 0.5 g of rna samples on 1% agarose gel. later, the cdna was synthesized from 1 g of rna using masterscript rt - pcr system (5 prime, gaithersburg, md, usa), according to the manufacturer 's instruction and stored at 20c until assay. kapa sybr fast qpcr kit (kapa biosystems, woburn, ma, usa) was used for real - time pcr quantification. the 20 l real - time pcr reaction mixture contained 20 ng cdna template, 10 l of 1x kapa sybr fast qpcr master mix, 200 nm of forward and reverse primers, and pcr - grade water. the sequences of the primers for the qrt - pcr are as follows : p53 : sense, 5-ggaggttgtgaggcgctgg-3 ; antisense, 5-cacgcacctcaaagctgttc-3 ; bax : sense, 5-cccgagaggtctttttccgag-3 ; antisense, 5-ccagcccatgatggttctgat-3 ; bcl-2 : sense, 5-catgtgtgtggagagcgtcaa-3 ; antisense, 5-gccggttcaggtactcagtca-3 ; -actin : sense, 5-tgcagaggatgattgctgac-3 ; antisense, 5-gaggactccagccacaaaga-3. the reaction was performed in performed in the applied biosystems 7500 real - time pcr system (applied biosystems, foster city, ca, usa) with the pcr cycling conditions as follows : 3 minutes enzyme activation at 95c, 40 cycles of 3 seconds initial denaturation at 95c and annealing / extension at 58c for 32 sec. melting curve analysis was performed for verify specificity of each primer after pcr to ensure amplification specificity. the threshold cycle (ct) number was determined and used in the comparative ct method. sh - sy5y cells were plated into 6-well plates at a density of 12 10 cells / well and incubated overnight. after exposure to mpp (1000 m), -mangostin (10 m) or the combination of mpp (1000 m) and -mangostin (10 m) for 24 hours under 5% co2 in a humidified incubator at 37c, cells were trypsinized by 0.25% trypsin / edta and centrifuged at 4,000 rpm, 4c for 5 minutes. the pellets were washed with cold pbs, resuspended and incubated on ice for 30 minutes. then cells were vortexed and centrifuged at 15,000 rpm for 15 minutes at 4c. the supernatant was collected and total protein concentration was determined using a bca protein assay. an equal amount of 30 g proteins from each experiment group were separated by 15% sds - polyacrylamide gel electrophoresis. the membrane was blocked with a 5% skim milk for 2 hours, washed with tbst buffer, and incubated with 1 : 1000 dilution of cleaved caspase-3 primary antibodies (cell signaling technology, denvers, ma, usa) or 1 : 5000 dilution of monoclonal -actin primary antibodies (sigma - aldrich, st. after washing, the membrane was incubated with 1 : 10000 dilution of hrp - conjugated goat polyclonal anti - rabbit igg (abcam, cambridge, uk) as the secondary antibody for cleaved caspase-3 and 1 : 5000 hrp - conjugated goat anti - mouse igg (invitrogen, eugene, or, usa) as the secondary antibody for -actin for 2 hours at room temperature before being developed using ecl prime western blotting detection (ge healthcare, buckinghamshire, uk). the membrane was reproved with anti -actin antibody to control for equal loading of protein. the signal intensities were determined by densitometry using image - j software (national institutes of health, bethesda, maryland, usa). statistical analyses were performed with one - way anova test followed by a post hoc analysis (tukey 's multiple comparison test) using graphpad prism 5 software for windows (graphpad software, inc., san diego, ca, usa). all values were presented as mean standard error of the mean (mean sem) for each group. to investigate the influence of alpha - mangostin on neuronal cell viability, we treated sh - sy5y cells with various concentrations of alpha - mangostin for 24 hours and examined cell viability with the mtt assay. exposure of sh - sy5y cells to alpha - mangostin induced a reduction in cell viability in a concentration - dependent manner. cell viability significantly decreased when cells were treated with 10 m alpha - mangostin for 24 hours, compared with that of control (p < 0.01 ; figures 1 and 2). treatment of 20 and 40 m alpha - mangostin led to loss of cell viability by more than 50% (p < 0.001 ; figure 1). when sh - sy5y cells were exposed to 2.510 m alpha - mangostin in the presence of 1000 m mpp for 48 hours, the co - treated cells showed a significant increase in cell viability in a concentration - dependent manner, compared to those treated with 1000 m mpp alone (figure 3). based on these results, 10 m alpha - mangostin was utilized in later experiments. apoptotic cells were quantified with pi and annexin - v dual staining using flow cytometry. the annexin - v / pi population consisted primarily of normal healthy cells, while annexin - v / pi cells were considered to be in the early stage of apoptosis, and annexin - v / pi cells were those considered to be in the necrosis / late apoptosis stage (figure 4(a)). after exposure to 1000 m mpp for 24 hours, the percentage of apoptotic cells increased (p < 0.001 ; figure 4(b)). cotreatment with 10 m alpha - mangostin in the presence of 1,000 m mpp for 24 hours significantly alleviated the apoptosis when compared to those treated with mpp only (p < 0.001). treatment with 10 m alpha - mangostin decreased the number of apoptotic nuclei (figure 5). alpha - mangostin significantly decreased the percentage of apoptotic nuclei when compared to mpp treatment alone (p < 0.001 ; figure 5(e)). to evaluate whether ros play an important role in the attenuation effect of alpha - mangostin on mpp - induced apoptosis in sh - sy5y cells, cells were exposed to 1000 m mpp and 10 m alpha - mangostin plus 1000 um mpp for 6 hours, and intracellular ros production was assessed as dhe and dhr123 fluorescence using flow cytometry. the result shows that cotreatment with 10 m alpha - mangostin in the presence of 1000 m mpp significantly decreased intracellular ros levels as measured by the mean fluorescence intensity (mfi) of dhe (p < 0.001 ; figure 6(a)) and dhr123 (p < 0.001 ; figure 6(b)), compared to mpp treatment alone. expression of mrna of bax and bcl-2 was investigated using real - time quantitative rt - pcr analysis. bax expression significantly increased in 1000 m mpp - treated cells, compared to that of control cells (figure 7(a)). cotreatment of 10 m alpha - mangostin and 1000 m mpp for 24 hours significantly decreased the bax expression, compared to mpp treatment alone (p < 0.01). treatment with 1000 m mpp significantly decreased bcl-2 expression, while cotreatment with 10 m alpha - mangostin significantly increased bcl-2 expression (p < 0.01 ; figure 7(b)). as a result, bax / bcl-2 expression was significantly increased in sh - sy5y cells treated with 1000 m mpp for 24 hours (p < 0.001 ; figure 7(c)), whereas bax / bcl-2 expression was significantly lowered in cells cocultured in 10 m alpha - mangostin and 1000 m mpp (p < 0.001). the expression of p53 mrna was significantly increased in 1000 m mpp - treated sh - sy5y cells after 24-hour exposure when compared to unexposed cells (p < 0.001 ; figure 7(d)). cotreatment of 10 m alpha - mangostin and 1000 m mpp significantly reduced p53 expression when compared to cells cultured in mpp alone (p < 0.01). to investigate the effect of alpha - mangostin on the caspase-3 protein, a critical executioner of apoptosis activation of caspase-3 requires cleavage of the protein at asp175 into the activated p17 and p19 fragments. after a 24-hour treatment of sh - sy5y cells with 1000 m mpp, a significant increase in activated caspase-3 protein when sh - sy5y cells were exposed to 10 m alpha - mangostin in the presence of 1000 m mpp, the cotreated cells showed a significant decrease in activated caspase-3 compared to the mpp treatment alone (p < 0.001). the present study has, for the first time, demonstrated that alpha - mangostin rescues apoptosis in dopaminergic sh - sy5y cells treated with mpp, a cellular model of parkinson 's disease. the results suggest that cytoprotection of alpha - mangostin against mpp - induced apoptosis may be associated with the reduction of ros production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. mpp is a neurotoxin used to generate animal and cellular models of parkinson 's disease. mpp has been shown to increase ros in neuroblastoma cells [9, 25, 26 ] and induce superoxide anion (o2) production via inhibition of mitochondrial complex i [10, 27 ]. the presence of antioxidant enzymes such as superoxide dismutase protects against mpp toxicity in neuronal cell lines and dopaminergic neurons in primary culture. previous studies showed that alpha - mangostin reduced ros formation [21, 24, 30 ]. in the present study, dhe and dhr123 dhe is used extensively to monitor superoxide production, perhaps the most specific dye that is retained well by cells [31, 32 ]. our results suggest that alpha - mangostin attenuated the oxidative effect of mpp through the reduction of superoxide production. dhr123 is used for the detection of peroxide and peroxynitrite (onoo). in vitro, our dhr123 oxidation assay suggests that treatment with alpha - mangostin also decreased the production of h2o2 and onoo induced by mpp. ros are elevated in cells undergoing apoptosis and antioxidants have been shown to protect neuronal cells against apoptosis induced by a variety of apoptotic agents. ros activate caspase-3 and caspase-3-like proteases in various cell types including neuronal cells, leading to nuclear condensation and dna fragmentation [36, 37 ]. ros also induce apoptosis, probably by decreasing expression of bcl-2, an antiapoptotic molecule [9, 38 ]. bcl-2 and bax may control the mitochondrial permeability transition pore, which can influence the passage of cytochrome c and other apoptosis - inducing factors that trigger the activation of caspase cascade and result in apoptosis. mangosteen extract has been shown to protect sk - n - sh neuroblastoma cells against an a-induced increase in caspase-3 activity. here, we showed that alpha - mangostin decreased caspase-3 activation, decreased bax mrna expression, and increased bcl-2 mrna expression induced by mpp in dopaminergic sh - sy5y neuroblastoma cells. a previous study on cisplatin - induced apoptotic death showed that alpha - mangostin attenuates the increase in p53 expression induced by cisplatin. proapoptotic bax, directly induced by p53, can overcome the antiapoptotic effect of bcl-2, whereas p53 can directly inhibit bcl-2. in the present study, treatment with 1000 m mpp significantly increased the expression of p53. as a result, increased p53 induced bax expression and inhibited bcl-2 expression, leading to the apoptosis of sh - sy5y cells. our results suggest that alpha - mangostin might reduce the effect of mpp on bax and bcl-2 expression by attenuating p53 expression. caspase-3 has been shown to be involved in the apoptotic events occurring in the mitochondrial - dependent pathway, which are associated with nuclear condensation and dna cleavage. caspase-3 also induces phosphatidylserine externalization from the internal to external leaflets of the plasma membrane. phosphatidylserine exposure on the external leaflet of the plasma membrane can be bound with annexin - v and is widely observed during apoptosis. our dna staining and pi / annexin - v dual staining supported the role of alpha - mangostin in protection against nuclear changes and phosphatidylserine externalization induced by mpp treatment in sh - sy5y neuroblastoma cells. alpha - mangostin is an antioxidant among the most abundant bioactive xanthones found in the mangosteen pericarp, which has long been used in traditional medicine to treat diarrhea, dysentery, infected wounds, and chronic ulcers. it has a broad range of bioactivities, such as antioxidant, anti - inflammation, anticancerogenic, and antihistaminergic effects [39, 4447 ]. alpha - mangostin has a cardioprotective effect against myocardial infarct and reperfusion injury in rats ; such an effect was related to low levels of lipid peroxidation, a decrease in protein carbonylation and the preservation of high glutathione content [19, 48 ]. recently, alpha - mangostin has been shown to inhibit and dissociate the beta - amyloid aggregation, which could contribute to its effect of attenuating beta - amyloid oligomers - induced neurotoxicity in a cellular model of alzheimer 's disease. this study indicates that alpha - mangostin protects sh - sy5y cells against mpp - induced apoptosis. the underlying mechanism could be attributed to its antioxidative properties and thus modulating the apoptotic process. the results showed that alpha - mangostin potentially possesses neuroprotective effects in a cellular model of pd. alpha - mangostin is a small lipid - soluble molecule with the potential to pass the blood - brain barrier. | in vitro studies have shown that extracts from mangosteen (garcinia mangostana linn.) act as antioxidants and cytoprotective agents against oxidative damage. the protective effect of alpha - mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of parkinson 's disease (pd), has not been investigated. this study aims to investigate whether alpha - mangostin could protect sh - sy5y neuroblastoma cells from mpp+-induced apoptosis. the effects of alpha - mangostin on mpp+-induced cell death were evaluated with a cell viability assay, staining for nuclear dna morphology, flow cytometry for apoptotic cells and reactive oxygen species (ros) production, quantitative real - time pcr for the expression of p53, bax, and bcl-2, and western blot analysis for cleaved caspase-3. concomitant treatment with alpha - mangostin attenuated the effect of mpp+ on cell viability and apoptotic cell death. alpha - mangostin reduced ros formation induced by mpp+. bax / bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha - mangostin and mpp+. the cotreated cells showed a significant decrease in activated caspase-3 compared with mpp+ treatment alone. our data suggest that cytoprotection of alpha - mangostin against mpp+-induced apoptosis may be associated with the reduction of ros production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. |
in vitro transcription reactions, particularly those mediated by t7 rna polymerase, have become a cornerstone of modern rna biochemistry and biophysics. these cell - free transformations facilitate the preparation of short as well as long native rna transcripts using synthetic and plasmid - derived dna templates. while of rather general utility, t7 rna polymerase requires a specific promoter for optimal transcription and tends to also be rather sensitive to sequence composition, particularly next to its consensus promoter. numerous studies have analyzed the initiation and elongation stages in these processes and, practically, identified optimal sequences at the transcript s guanosine residues are frequently found at positions + 1 and + 2, where altered sequences typically suffer from significantly diminished transcription efficiency. in vitro prepared transcripts therefore almost exclusively possess a gtp at their 5 end, with 15 out of 17 reported promoters initiating transcription with pppg and 13 with pppgpg. a formidable contemporary challenge, which is compounded by the constraints outlined above, is the need to modify rna transcripts, typically with fluorescent probes, for diverse biochemical and biophysical applications. this problem has been tackled in several ways, including the development of orthogonal base pairing systems, which require, however, the synthesis of modified dna templates in addition to the necessary complementary modified triphosphates. the fastidious behavior of t7 rna polymerase has also limited the modification position, with most appearing remote to the promoter, where the enzymatic process is believed to be beyond its vulnerable initiation phase. replacing the initiating gtp at position + 1 with alternatives such as gmp and guanosine, some with modifications on the monophosphate or ribose, has been explored with different levels of success. transcription initiation with gtp analogs, where the heterocyclic nucleus has been altered has not, to our knowledge, been explored. this has motivated the study reported here, where newly synthesized gtp, a highly isomorphic and emissive analog, has been investigated as a gtp surrogate in t7 rna transcription reactions. the recent completion of an emissive rna alphabet, a fluorescent ribonucleoside set comprised of highly emissive purine and pyrimidine analogs, all derived from thieno[3,4-d]pyrimidine, presents unique opportunities for the generation of modified rna constructs. despite the high structural resemblance to their native counterparts, it was unclear at the onset of this project whether or not polymerases could accommodate and effectively incorporate these modified nucleosides into oligonucleotides. as discussed above, particularly challenging is the initiation of in vitro transcription reactions with gtp analogs, which is inevitable in this context. in this contribution we critically assess the ability of t7 rna polymerase to initiate rna transcription and elongate the nascent modified transcript using gtp (2), a highly emissive and isomorphic gtp analog, and compare the results to its performance with the native triphosphate. we demonstrate that the modified nucleotide is capable of initiating and maintaining transcription reactions, leading to the formation of fully modified and highly emissive transcripts. importantly, to assess proper folding and function of rna transcripts where all g residues have been replaced with a synthetic analog, we explore modified hammerhead ribozymes, catalytic rna assemblies, which are likely to be exceedingly sensitive to such alterations. the fully modified enzyme and substrate of the hammerhead ribozyme are efficiently transcribed, and the impact of replacing all g residues with g is evaluated. we further demonstrate that the emissive transcripts can be used to monitor the ribozyme - mediated cleavage reaction in real time. the 5-triphosphate of g was synthesized from the parent nucleoside using freshly distilled pocl3 and tributylammonium pyrophosphate (scheme 1). the triphosphate was purified by ion - exchange chromatography and reverse - phase hplc (see experimental section). final treatment with chelex 100 afforded the analytically pure nucleotide [p nmr 9.98 (d, j = 20.1 hz, p), 10.64 (d, j = 18.6 hz, p), 22.64 (t, j = 18.6 hz, p) ]. reagents and conditions : (i) pocl3, (meo)3po, 0 c ; (ii) tributylammonium hydrogen pyrophosphate in dmf, bu3n, 0 c. transcription reactions with the analytically pure gtp (2) and t7 rna polymerase were performed to first analyze its enzymatic incorporation into short rna oligonucleotides. a short dna promoter template duplex was used to discern the ability of gtp to initiate transcription and be incorporated during the elongation phase (figure 1). the dna template 3 is terminated with a single t at the 5 end so a lone a is directed to the 3 end of the transcript. when trace amounts of -p atp are used, only successfully transcribed full length labeled rna products would be visible, whereas short failed transcripts would be undetected after gel electrophoresis (figure 1). t7 promoter and template 3 depicting the enzymatic incorporation reaction using natural ntps and gtp or gtp resulting in transcripts 4 or 5. the t7 promoter and template 3 were annealed, and transcribed in the presence of natural ntps or with gtp replacing gtp and a trace of -p atp. a phosphorimage revealed a full length 10-mer product (transcript 5) using gtp that corresponded to the natural triphosphates transcript 4 (figure 2a). the overall yield of transcript 5, containing four g residues, compared to the natural unmodified transcript 4 was 70 3%, indicating that the average individual incorporation was 91 1%. next, a large - scale transcription reaction was run and uv shadowing was used to visualize all products. comparing the transcription reactions with gtp to reactions with gtp illustrates that the desired product and abortive transcripts appear almost identical (compare lanes 1 and 2, respectively, in figure 2b). importantly, when visualizing the gel under uv illumination (302 nm), the product and initiation phase truncated transcripts are highly fluorescent (right figure 2b). following extraction, the isolated yield of the g modified 10-mer (transcript 5) was 78 12% compared to the native 10-mer (transcript 4), averaging 94 2% per g incorporation. the transcripts were characterized by esi (figure s1a) and digested using s1 nuclease and dephosphorylated. the nucleoside mixtures were subjected to hplc - ms analysis. comparing the chromatogram obtained for the native 10-mer (4, figure s3b) to that of the modified one (5, figure 3) (a) small scale transcription using trace -p atp. lane 1 : control transcription reaction in the absence of gtp or gtp. lane 2 : control reaction in the presence of all natural ntps. lane 3 : reaction in the presence of equimolar concentration of gtp and gtp. incorporation efficiencies of gtp transcripts are reported with respect to transcription in the presence of gtp. all reactions were performed in triplicate, and the errors reported are the standard deviations. (b) large scale transcription reaction using template 3 with all natural ntps (lane 1 and 1) or atp, utp, ctp, and gtp (lanes 2 and 2, 78 12% isolated yield) with uv light at 254 nm (on tlc plate) and 302 nm (pl). hplc - ms traces of the (a) mixture of nucleosides used as a standard and (b) digestion results of transcript 5. digestion of 12 nmol of transcript was carried out using s1 nuclease for 2 h at 37 c and followed by dephosphorylation with alkaline phosphatase for 2 h at 37 c. the ribonucleoside mixture obtained was analyzed by reverse - phase analytical hplc, using a mobile phase of 06% acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 12 min ; flow rate 1 ml / min. to test the runoff transcription of longer constructs and assess the function of the resulting transcripts, longer dna templates (6 and 7) were used to generate hammerhead ribozymes : the natural all native substrate (s), the modified substrate (g - s), the natural enzyme (e), and the modified enzyme (g - e) (figure 4). these transcription reactions were executed only on a large scale, and the rna transcripts (s, g - s, e, and g - e) were isolated after polyacrylamide gel electrophoresis (figure s4), characterized by esi (figure s1b and s1c), and then digested (figure s3c and s3d). as before, the full length products and all short failed transcripts in transcription reactions using gtp are highly emissive (figure s4). t7 promoter and templates 6 and 7 depicting the enzymatic incorporation reaction using natural ntps and gtp or gtp resulting in transcripts s, thg - s, e, or thg - e. g is underlined and bolded blue in the transcripts g - s and g - e. following purification, the natural substrate (s) and g - substrate (g - s) were dephosphorylated with alkaline phosphatase and 5 labeled with t4 polynucleotide kinase according to standard protocols. the assembled ribozymes were then tested for the anticipated strand cleavage in all different combinations (figure 5), using conditions similar to those previously published, where the ribozyme cleavage reaction was initiated by mixing equal volumes of buffered solutions containing mgcl2 of substrate with enzyme. the reactions contained excess enzyme to obtain pseudo first - order kinetic rate constants (k2). single turnover reactions at 31 c contained 0.3 m substrate (including a trace of 5-p labeled material) and 3 m enzyme, in 50 mm tris - hcl ph 7.0, 200 mm nacl, and 10 mm mgcl2. the rate constants obtained for the native hh ribozyme s and e (figure 5a) and the substrate - modified one g - s and e (figure 5b) were 0.15 0.1 min and 0.12 0.1 min, respectively (figure 6b). the unmodified hh enzyme e cleaved 87% and 86% of the substrates s and g - s, respectively, at 20 min. the fully modified g - containing enzyme (g - e) showed no cleavage of s or g - s at 31 c (figures 5c, 5d, and 6a). reactions of g - e with the natural and modified substrates at slightly elevated temperatures (37 c) did show a small amount of sequence specific cleavage, estimated at about 2% after 40 min (figure s5). hammerhead ribozymes and cleavage reactions of (a) natural substrate and enzyme (s and e), (b) modified substrate and natural enzyme (g - s and e), (c) natural substrate and modified enzyme (s and g - e), and (d) modified substrate and modified enzyme (g - s and g - e). (a) hh ribozyme cleavage reaction results were followed by p radioactive labeling of substrate strands s and g - s. s and p1, and g - s and g - p1 indicate substrate and product strands (figure 5). all reactions were conducted at 31 c and contained 0.3 m substrate (including a trace of 5-p labeled material), 3 m enzyme, 50 mm tris ph 7.0, 200 mm nacl, and 10 mm mgcl2. the reactions were quenched at the given times (t in min) and resolved by gel electrophoresis on a denaturing 20% polyacrylamide gel with 7 m urea. (b) initial kinetics of s and e and g - s and e. the pseudo - first - order rate constants (k2) of the cleavage reactions are determined as the slope of ln(fraction cleaved) versus time. (c) ribozyme - mediated cleavage curves as determined by p data for s and e and g - s and e. fraction cleaved (s / s0) was determined by dividing the amount of cleaved substrate by the sum of the full length and cleaved substrate. the cleavage of g - s by the native enzyme e was monitored with nonradiolabeled material using steady - state fluorescence spectroscopy under the same conditions as the experiments performed with the radiolabeled constructs but in a slightly larger volume (see figure s6 for absorption and emission spectra of g - s). mixing the substrate with the enzyme in a fluorescence cuvette at 31 c gave final concentrations of 0.3 m substrate and 3 m enzyme in 50 mm tris - hcl ph 7.0, 200 mm nacl, and 10 mm mgcl2. the fluorescence intensity, monitored at 450 nm, increased during the reaction (figure 7a and 7c). alternatively, the fully modified enzyme g - e mixed with the native substrate s, which showed no measurable strand cleavage at 31 c when monitored using the p labeled substrate, displayed minimal fluorescence intensity changes (figure 7b and 7d). the fraction cleaved of g - s from the radioactive and fluorescence experiments was normalized and showed a similar trend (figure s7). importantly, page analysis confirms the presence of two fluorescent products g - p1 and g - p2 (see figure s8). fluorescence spectra of (a) g - s and e (blue) (excitation 470 nm, emission 425485 nm, slit widths 8 nm) and (b) s and g - e (black) (excitation 470 nm, emission 425485 nm, slit widths 4 nm), where t = 0 min and t = 20 min spectra have thicker lines. all reactions were conducted at 31 c and contained 0.3 m substrate, 3 m enzyme, 50 mm tris ph 7.0, 200 mm nacl, and 10 mm mgcl2. the fluorescence intensity shown at 450 nm over 20 min of the (c) cleavage of g - s by e (blue) and (d) mixing of s and g - e (black). as has been previously demonstrated, fluorescent nucleoside analogs can tremendously facilitate the study of nucleic acid folding, recognition, and catalysis, including the enablement of real time assays. of particular significance is the modification of oligonucleotides with isomorphic nucleoside analogs, as their high similarity to their canonical counterparts and nonperturbing nature frequently results in faithful folding and function. a case in point is our recent study of emissive mrnas, where specific g residues have been surgically replaced by g using solid - phase synthesis. these modified rna constructs were recognized by the ribosome and capable of fluorescently reporting discrete steps in translation. here we set to explore the ability of gtp to support the initiation step of in vitro transcription reactions and then elongate the nascent rna transcripts to yield full length products in which all g residues are replaced by g. we then evaluate the impact of this rather dramatic modification on the function of the hh ribozyme as a prototypical functional rna system. the commonly used t7 promoters end with c, so one could not test a single g or g incorporation in g - containing transcripts where a g residue is remote to the promoter. we therefore initially used a short oligonucleotide 3 that has been previously used by our lab to evaluate t7 rna polymerase s ability to produce short modified transcripts (figure 1). transcription reactions using template 3 in the presence of gtp, ctp, atp, and utp but no gtp (figure 2a, lane 3) yielded full length products, which suggested that gtp could indeed initiate such transcription reactions. the high incorporation efficiency of the g modification at an average yield of 91 1% per incorporation demonstrated its structural and functional similarity to g. it is likely, however, that the incorporation yield at positions + 1 and + 3 is lower than those at positions + 6 and + 8, due to their proximity to the promoter. capitalizing on these positive results, the transcription reactions were scaled up to provide large quantities of full length products for analytical characterization. indeed, ms analysis and enzymatic digestion reactions followed by hplc - ms analysis further confirmed that gtp was indeed recognized by t7 rna polymerase, and efficiently utilized during the initiation and elongation phases (figure 3). the intense fluorescence of the abortive transcripts provided another positive indication that gtp was indeed the agent responsible for initiating transcription (figure 2b). dna templates (e.g., 6 and 7), yielding medium rna transcripts, also demonstrated that gtp is efficiently incorporated during the initiation and elongation phases. much longer transcripts, which include multiple g residues at the 5-end, as well as several consecutive g residues, have also been transcribed in high yields, with an average incorporation yield of 95% per gtp (see figure s9). we submit that these observations validate g as a true isomorphic nucleoside analog of g, which is faithfully recognized by the polymerase, while retaining high selectivity for wc pairing. although, in principle, multiple alterations within an rna transcript might be functionally detrimental, the intense emission of these per - modified transcripts suggests potential utility (see below). additionally, established protocols in rna biochemistry allow ligations of rna fragments into larger constructs, which suggest that short g - containing rnas can be ligated to longer native ones for certain applications. in addition to gtp being a substrate for t7 rna polymerase, we tested the fully modified oligonucleotides, specifically g - s and g - e, for their ability to function as components in a hh ribozyme, as a prototypical catalytically active rna. the substrates (s and g - s) were first radioactively labeled to visualize their ribozyme - mediated cleavage. incidentally, this successful labeling demonstrates that the 5-end of the transcript g - s does not hamper alkaline phosphatase - mediated dephosphorylation and t4 polynucleotide kinase - mediated phosphorylation. these observations are of significance, as they suggest that modifying rna transcript with g does not hinder enzymatic transformations by commonly used molecular biological agents. the unmodified hh enzyme e cleaves its native substrate s and the fully modified one g - s with similar rates, indicating that replacing all g residues with g in the hh substrate does not substantially interfere with ribozyme catalysis. in contrast, the fully modified hh enzyme g - e showed very little cleavage ability of either the native substrate s or the corresponding modified g - s. this suggests that the substitution of g for g interferes with either the folding or catalysis of the hh enzyme. as the modified substrate is almost fully duplexed and effectively cleaved by the native enzyme, attention is then focused on the relevant residues in the enzyme that are proposed to be involved in catalysis : g8, g10.1, and g12 (figure 5c). in the proposed ribozyme cleavage mechanism, the n7 of g10.1 coordinates a divalent metal ion, which appears to be involved in the phosphodiester cleavage reaction. the lack of the imidazole ring and hence of n7 in g may therefore explain the severely attenuated catalytic activity of the fully modified enzyme g - e. additionally, the nucleobase of g12 is involved in the cleavage reaction where the putatively deprotonated n1 position acts as a base to abstract the proton on the o2 of c17, which then nucleophilically attacks the adjacent 3 phosphate, eventually leading to strand cleavage. since the pka of the n1 position of g within the folded ribozyme likely differs from that of g the sugar and not the nucleobase is proposed to be involved in catalysis, making it less likely that modification at this position is directly hindering cleavage. importantly, the cleavage of the modified hh substrate g - s by the native enzyme e can be observed using steady - state fluorescence spectroscopy, demonstrating the utility of such emissive transcripts for monitoring rna related processes in real time (figure 7a and 7c). a significant fraction of the large increase seen in fluorescence intensity likely originates from the cleavage of g - s by e because inactive combinations (such as that of s and g - e) exhibit much smaller fluorescence intensity changes (figure 7b and 7d). we note, however, that changes in emission intensity observed when mixing such fluorescent rna strands represent multiple events, including the annealing, folding, and mg coordination, as well as strand cleavage and dissociation. this is supported by the small fluorescence increase seen when the s and g - e are mixed to generate an inactive hh ribozyme, which likely reflect only conformational changes and metal coordination (figure 7b and 7d). notably, however, when the normalized data generated by p labeling is compared to the fluorescence - generated rates for g - s and e, similar overall trends and rates are seen (figure s6). while these deficiencies can likely be circumvented by monitoring the process using a stopped - flow kinetic apparatus (thus separating the fast events from the relatively slow cleavage reaction), we emphasize that monitoring catalytic rnas using simple benchtop steady - state fluorescence spectrometers can be an effective way to probe substrate cleavage and inhibition. it greatly reduces the overall experimental time, and unlike p - monitored reactions, which are not monitored in real - time, can provide additional insight into conformational changes, hybridization, and magnesium binding events, among others. gtp is found to be accepted by t7 polymerase as a faithful gtp surrogate. this highly isomorphic nucleoside triphosphate can initiate transcription, as well as be incorporated during the elongation phase in short and longer oligonucleotides. importantly, such modified transcripts, which contain a pppg at their 5-end, are also successfully dephosphorylated with alkaline phosphatase and then phosphorylated with t4 polynucleotide kinase, illustrating that other terminus - modifying enzymes tolerate this modification as well. to investigate the impact of g substitution on rna function, we tested hh ribozyme combinations where the substrate and enzyme were per - modified with g. a hh ribozyme containing a fully modified substrate g - s hybridized to a native enzyme e undergoes efficient phosphodiester bond cleavage that can be monitored either with radioactively labeled substrate followed by page or, in real time, using gs fluorescence. in contrast, the fully modified hh enzyme g - e displayed very little cleavage ability with either s or g - s. for example, the inactivity of the fully g modified hh enzyme suggests utility of this nucleoside in probing specific mechanistic questions in rna catalysis. moreover, due to their reliable hybridization and wc pairing, highly isomorphic but potentially inactive hh constructs could provide useful tools for structural analysis. as such, the observation that gtp is accepted by t7 polymerase as a faithful gtp surrogate (in both its initiation and elongation phases) and that other enzymes, commonly used in molecular biology, accept such per - modified strands as viable substrates opens up numerous creative opportunities to utilize such modified nucleosides and oligonucleotides, both as structural as well as fluorescent tools. even 2-aminopurine, an extensively employed emissive a isoster, fails to perform in certain cases. we feel, however, that g can be an extremely useful probe due to its unique structural features and favorable photophysical characteristics. oligonucleotides were purified by polyacrylamide gel electrophoresis and desalted on sep - pak (waters corporation). ntps and the ribonuclease inhibitor (ribolock) were obtained from fermentas life science. radiolabeled -p atp (10mci / ml, 3000 ci / mmol) and -p atp (10mci / ml, 6000 ci / mmol) were obtained from perkinelmer. chemicals for preparing buffer solutions were purchased from fisher biotech (enzyme grade). autoclaved 0.1% depc treated water was used in all biochemical reactions and fluorescence titrations. small molecule mass spectra (ms) were recorded at the university of california, san diego, chemistry and biochemistry mass spectrometry facility, utilizing an agilent 6230 hr - esi - tof mass spectrometer. reverse - phase hplc (vydac c18 column) purification and analysis were carried out using an agilent 1200 series instrument. steady - state fluorescence experiments were carried out in a microfluorescence cell (125 l) with a path length of 1.0 cm (hellma gmbh & co. kg, mllheim, germany) on a horiba jobin yvon (fluoromax-3) spectrometer. mass spectra for oligonuceotides were obtained on a thermofinnigan lcq deca xp at trilink biotechnologies, inc. tris(tetrabutylammonium) hydrogen pyrophosphate (0.99 g, 1.1 mmol) in a 10 ml round - bottom flask, and g (60 mg, 0.20 mmol) in a 25 ml round - bottom flask, were separately coevaporated with anhydrous pyridine and dried. trimethyl phosphate (2 ml) was added to g and cooled in an ice bath to 0 c. phosphoryl chloride (46 l, 0.5 mmol) was added slowly, and the reaction was stirred for 2 h at 0 c, resulting in a pinkish brown solution. the coevaporated tris(tetrabutylammonium) hydrogen pyrophosphate was dissolved in 2 ml of anhydrous dmf and added to the g reaction mixture. then tributyl amine (0.26 mmol, 1.1 mmol) was added and the reaction was kept stirring at 0 c for 40 min. to the reaction mixture was added 6 ml of 1 m triethylammonium bicarbonate buffer (teab), and the mixture was stirred briefly. the mixture was then transferred to a separatory funnel and washed with 10 ml of etoac. the organic layer was then back - extracted with 5 ml of 1 m teab. the aqueous layers were combined and concentrated under reduced pressure at room temperature to afford an oily yellow residue. the residue was dissolved in 10 ml of 0.05 m ammonium bicarbonate buffer and loaded onto a deae sephadex a25 anion - exchange column kept in a cold room at 4 c. the column was eluted using a gradient mixer with 0.050.5 m of ammonium bicarbonate buffer. a fraction collector was used to collect 260 fractions that were about 8 ml (220 drops). the fractions containing the triphosphate were evaporated under reduced pressure at 10 c, and then the residue was lyophilized. to purify the triphosphate further, the residue was run on another deae sephadex a25 anion - exchange column and eluted with 0.060.6 m ammonium bicarbonate buffer. after lyophilization, the triphosphate was treated with 25 mg of chelex 100 for 15 min with occasional shaking, and then filtered. the triphosphate was further purified by hplc (phenomenex synergi fusion - rp 80a c18 column, 4 m, 250 10 nm, 04% acetonitrile in 50 mm teab buffer, ph 6.0, 30 min). h nmr (500 mhz, d2o) 8.10 (d, j = 4.7 hz, 1h), 5.164.97 (m, 1h), 4.34 (s, 1h), 4.21 (dd, j = 11.5, 9.5 hz, 4h) ; p nmr (202 mhz, d2o) 9.98 (d, j = 20.1 hz, p), 10.64 (d, j = 18.6 hz, p), 22.64 (t, j = 18.6 hz, p) ; hr esi - ms (negative ion mode) [c11h15n3o14p3s ] calculated 537.9493, found 537.9500 ; esi - ms (negative ion mode) [c11h15n3o14p3s ] calculated 538.24, found 537.98 and 559.96 as [m 2h + na ]. single strand dna templates were annealed to an 18-mer t7 rna polymerase consensus promoter sequence in te buffer (10 mm tris - hcl, 1 mm edta, 100 mm nacl, ph 7.8) by heating a 1:1 mixture (10 m) at 90 c for 3 min and cooling the solution slowly to room temperature. transcription reactions were performed in 40 mm tris - hcl buffer (ph 7.9) containing 500 nm annealed templates, 10 mm mgcl2, 10 mm dithiothreitol (dtt), 10 mm nacl, 2 mm spermidine, 1 u/l rnase inhibitor (ribolock), 1 mm gtp or 1 mm gtp, 1 mm ctp, 1 mm utp, 20 m atp, 2 ci -p atp (800 ci / mmol stock), and 2.5 u/l t7 rna polymerase (fermentas) in a total volume of 20 l. after 3 h at 37 c, reactions were quenched by adding 10 l of loading buffer (7 m urea in 1 tbe with 0.05% bromophenol blue and 0.05% xylene cyanol), heated to 75 c for 3 min, and 10 l was loaded onto an analytical 20% denaturing polyacrylamide gel. transcription efficiencies are reported with respect to transcription in the presence of natural nucleotides. transcription efficiencies were determined from three independent reactions, and the errors reported represent standard deviations. to preparatively isolate rna and for enzymatic digestions large scale transcription reactions using template 3 were performed in a 250 l reaction volume under similar conditions, with the following changes. the reaction contained 1 mm atp, ctp, and utp, 1 mm gtp or gtp, 15 mm mgcl2, 500 nm template, 1500 units t7 rna polymerase, and 250 units of ribolock. after incubation for 5 or 6 h at 37 c, the precipitated magnesium pyrophosphate was removed by centrifugation. the mixture was heated at 75 c for 3 min and loaded onto a preparative 20% denaturing polyacrylamide gel. the gel was uv shadowed ; appropriate bands were excised, extracted with 0.5 m ammonium acetate, and desalted on a sep - pak column. concentrations of the rna transcript were determined using absorption spectroscopy at 260 nm using the following extension coefficients : c, 7200 ; u, 9900 ; g ; 11500 ; a, 15400 ; and g, 5517 lmol cm. to preparatively isolate rna and for enzymatic digestions large scale transcription reactions using template 6 and 7 were performed in a 250 l reaction volume under similar conditions, with the following changes. the reaction contained 2 mm ctp, utp, and atp, 2 mm gtp or gtp, 20 mm mgcl2, 500 nm template, 6 u/l (1500 u) t7 rna polymerase, and 1 u/l (250 u) of ribolock. after incubation for 5 or 6 h at 37 c, the precipitated magnesium pyrophosphate was removed by centrifugation. the mixture was heated at 75 c for 3 min, and loaded onto a preparative 20% or 15% denaturing polyacrylamide gel. the gel was uv shadowed ; appropriate bands were excised, extracted with 0.5 m ammonium acetate, and desalted on a sep - pak column. concentrations of the rna transcript were determined using absorption spectroscopy at 260 nm as described above. all transcripts (12 nmol of 4, 5, s, g - s, e, g - e) were incubated with s1 nuclease in reaction buffer (promega) for 2 h at 37 c. the reaction was further treated with alkaline phosphatase and dephosphorylation buffer (promega) for 2 h at 37 c. the ribonucleoside mixture obtained was analyzed by reverse - phase analytical hplc with an agilent column eclipse xdb - c18 (5 m, 4.6 150 mm). mobile phase : 06% acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 12 min ; flow rate 1 ml / min. mass spectra as raw data were taken by esi mass spectrometer in negative ion mode with xcalibur software version 1.3, and the raw esi - ms m / z data were deconvoluted by promass for xcalibur version 2.5 sr-1. the running buffer was 10 mm tert - butylamine in 70% acetonitrile in water. all deconvoluted mass spectra are in figure s1 and an example of raw m / z data and deconvoluted spectra are shown in figure s2. the rna transcripts s and g - s (12.9 pmol and 12.3 pmol, respectively) in 3 l of 10 dephosphorylation buffer and 1 l of calf intestinal alkaline phosphatase in a total volume of 30 l were incubated at 37 c for 2 h. water (70 l) was added and the reaction mixture extracted with 100 l of phenol : chloroform (chcl3):isoamyl alcohol (iaa) = 25:24:1. the water layer was extracted with chloroform (100 l). the rna in the aqueous layer was precipitated with 6 l of glycoblue, 20 l of 10 m nh4oac, and 400 l of etoh and put in dry ice bath for 1 h, followed by centrifugation at 14,000 rpm for 20 min and removal of the supernatant. the pellet was washed 4 with 50 l of cold 70% etoh. the pellet was air - dried for 30 min and then dissolved in 38 l of water. five l of 10 kinase buffer, 1 l of dithiothreitrol, 5 l of -p atp, and 1 l of t4 polynucleotide kinase, were added and the reaction was heated to 37 c for 2 h. the rna was then precipitated (2 l of glycoblue, 10 l of 10 m nh4oac, and 200 l of etoh) and washed (1 with 25 l of cold 70% etoh), similar to the above procedure. the pellet was dissolved on 1 tbe 7 m urea loading buffer, and then the rna was resolved by gel electrophoresis on a denaturing 20% polyacrylamide gel. the rna was cut out and extracted with water overnight, filtered, and then concentrated using a speed vac. cleavage reactions were conducted in a total reaction volume of 34 l for the natural enzyme (e) and 22 l for the g - enzyme (g - e) with the substrate (s) or g - substrate (g - s) for radiography. for the fluorescence - based experiments, the reactions were carried out at 31 c in a buffer containing 50 mm tris - hcl (ph 7.0) and nacl (200 mm). buffered solutions of the substrate (0.6 m with traces of 5-p labeled substrate) and enzyme (6 m) were denatured separately by heating to 90 c for 90 s and cooled to room temperature over 10 min to allow for refolding. mgcl2 (to make a final concentration of 10 mm) was added to both the enzyme and substrate, and both were equilibrated at 31 c for 10 min. the cleavage reaction was then initiated by manually mixing equal volumes of the modified or natural substrate (0.6 m) with the enzyme or modified enzyme (6 m) in a heat block at 31 c, to give final concentrations of 0.3 m of the substrate and 3 m of the enzyme and 10 mm mgcl2. for initial data points (time = 0), following initiation of the reaction, 4 l aliquots were removed at designated time periods and quenched with 12 l of urea containing loading buffer (7 m urea, 1 tbe, and 0.05% bromophenol blue, and xylene cyanol ff). the tubes were heated to 90 c for 90 s and loaded on a 20% polyacrylamide with 7 m urea gel. corresponding bands were quantified on a personal molecular imager and analyzed with quantity one software (biorad). rate constants (k2) were calculated as the slope of ln(1 s / s0) versus time, where s / s0 is the fraction of cleaved substrate. for experiments utilizing a radioactively labeled substrate, s / s0 was determined by dividing the amount of cleaved substrate by the sum of the full length and cleaved substrates. | the fastidious behavior of t7 rna polymerase limits the incorporation of synthetic nucleosides into rna transcripts, particularly at or near the promoter. the practically exclusive use of gtp for transcription initiation further compounds this challenge, and reactions with gtp analogs, where the heterocyclic nucleus has been altered, have not, to our knowledge, been demonstrated. the enzymatic incorporation of thgtp, a newly synthesized isomorphic fluorescent nucleotide with a thieno[3,4-d]pyrimidine core, is explored. the modified nucleotide can initiate and maintain transcription reactions, leading to the formation of fully modified and highly emissive rna transcripts with thg replacing all guanosine residues. short and long modified transcripts are synthesized in comparable yields to their natural counterparts. to assess proper folding and function, transcripts were used to assemble a hammerhead ribozyme with all permutations of natural and modified enzyme and substrate strands. the thg modified substrate was effectively cleaved by the natural rna enzyme, demonstrating the isomorphic features of the nucleoside and its ability to replace g residues while retaining proper folding. in contrast, the thg modified enzyme showed little cleavage ability, suggesting the modifications likely disrupted the catalytic center, illustrating the significance of the hoogsteen face in mediating appropriate contacts. importantly, the ribozyme cleavage reaction of the emissive fluorescent transcripts could be followed in real time by fluorescence spectroscopy. beyond their utility as fluorescent probes in biophysical and discovery assays, the results reported point to the potential utility of such isomorphic nucleosides in probing specific mechanistic questions in rna catalysis and rna structural analysis. |
a 44-year - old female presented with excessive involuntary eye blinking with a 5-month history. she had a history of febrile convulsion with a frequency of 5 or 6 times per year during infancy. when she was 7 years old, she was diagnosed with epilepsy and started medication with aeds. semiology revealed complex partial seizures composed of vacant staring and automatism with occasionally secondary generalized tonicclonic seizures. she was recently treated with phenobarbital (maintenance dose of 60 mg / day) in combination with primidone (250 mg / day) at a local clinic, which decreased her seizure frequency to twice yearly. five months prior to visiting our clinic, she was treated with ltg (lamictal at 25 mg / day) following discontinuation of previous aeds at another hospital. the dosage of ltg was increased slowly to 200 mg / day, and her seizures were well controlled. however, 5 months after ltg treatment, an involuntary eye blinking developed. no other medication (including digestives) was prescribed and no procedure was performed when this symptom developed. she visited our hospital because of persistent excessive involuntary eye blinking for the previous 5 months. the initial neurological examination revealed excessive eye blinking with a frequency of about twice per second that was not accompanied with blepharospasm. she could not voluntarily control her eye blinking, and the frequency of eye blinking did not change during extraocular movement at any direction or during conversation. cranial nerve function including pupil light reflex and extraocular movement, facial motor and sensory functions, corneal reflex, and glabella reflex were all normal. video monitoring combined with electroencephalography (eeg) with extra electrodes 1 cm lateral to both orbits showed normal findings without epileptiform discharges. eye blinking on electromyography (emg) continued at a frequency of 1.7 times / second during eeg monitoring (fig. we changed ltg to topiramate (topamax at 100 mg / day), with the dosage subsequently being increased to 200 mg / day. we changed topiramate to valproic acid (depakote er at 500 mg / day) due to intolerable side effects of a poor appetite and tingling sensation. after treatment of valproic acid and cessation of ltg, the episodes of involuntary eye blinking resolved completely without an seizures. repeated eeg monitoring revealed that the frequency of eye blinking had decreased (to once every 2 seconds) (fig. 1-b), and excessive eye blinking was absent at a 7-month follow - up. eye blinking is fundamental to corneal wetting and eye protection, and consists of two physiologic components : (1) inhibition of sustained activity of the levator palpebrae superioris muscles and (2) concurrent activation of the orbicularis oculi muscles.4 the eye - blinking signal is generated in the reticular formation of the rostral pons and regulated by the substantia nigra, superior colliculus, occipital cortex via the lateral geniculate body, and optic radiation. the thalamus and cerebellum are also involved in the production of eye blinking.5 an abnormal eye blinking rate is observed in many diseases involving the central nervous system. the rate of eye blinking is usually decreased in parkinson 's disease, but increased in schizophrenia, huntington 's disease, and blepharospasm.5 in depression, eye blinking can be either increased or decreased.5 our patient did not show any cognitive or motor deficits except for the excessive involuntary eye blinking, and this was not induced by seizure based on the eeg findings being normal during excessive eye blinking. excessive involuntary eye blinking began in our patient after 5 months of administration with ltg and disappeared completely after the cessation of ltg, which indicates a close association between the condition and ltg. tremor (postural or kinetic) is one of common side effects of aeds such as valproic acid and phenytoin.6,7 chorea was reported in several patients receiving phenytoin or valproic acid.8,9 ltg - induced eyelid movement disorder is extremely rare. verma.10 reported a patient who developed blepharospasm 4 months after beginning the administration of ltg, with it disappearing 1 month after the cessation of ltg. however, to our knowledge, involuntary eye blinking associated with ltg has not been reported previously. neuroleptics are representative of the drugs that induce abnormal movement, acting on dopamine receptors directly and leading to postsynaptic dopamine supersensitivity and a postsynaptic increase in the number of dopamine receptors.5 the mechanism of ltg - induced involuntary movement has been unclear. ltg does not directly affect striatal dopamine receptors, and hence the pathophysiological mechanism of ltg - induced involuntary movement differs from that induced by neuroleptics. one possible explanation is that ltg inhibits presynaptic excitatory neurotransmitters, which induce the striatal dopaminergic system secondarily. for example, amantadine, a subtype n - methyl - d - aspartate receptor (nmda) blocker, exerts antiparkinsonian effects via inhibition of nmda receptors, which secondarily facilitates the presynaptic release of striatal dopamine. although ltg has no effect of nmda receptor blockade, it may indirectly influence the dopaminergic system by inhibiting some types of excitatory neurotransmitters. zipp.11 reported five patients with parkinson 's disease who were successfully treated with ltg, which suggests that ltg can influence the dopaminergic system indirectly via an unknown mechanism, even though it is not associated with the direct inhibition of dopamine or glutamate receptors. further investigations are needed to clarify the exact pathophysiological mechanism of ltg - induced involuntary movement. in conclusion the results presented here indicate that unrepresentative side effects such as involuntary eye blinking should be considered when ltg is administered. | a wide variety of movement disorders can be induced by the administration of antiepileptic drugs. a 44-year - old female was admitted with involuntary excessive eye blinking that manifested 5 months after beginning the administration of lamotrigine for control of complex partial and secondarily generalized seizures. the involuntary eye blinking persisted while taking lamotrigine, and disappeared 1 month after the cessation of lamotrigine. the development of atypical involuntary eye blinking in this case may have resulted from the inhibition of excitatory neurotransmitters by lamotrigine, which led to secondary dysfunction of the dopaminergic system. |
paragangliomas are rare neural crest tumours of sympathetic (generally catecholamine producing) or parasympathetic (rarely catecholamine producing) origin. emergency surgeries have been cancelled on table in patients demonstrating unstable haemodynamics during induction, and subsequent investigations confirmed the presence of a pre - operatively unsuspected pheochromocytoma. in a post - mortem series, 27% of the patients with an undiagnosed pheochromocytoma died during or shortly after unrelated surgery. life - threatening events may follow resection in an unsuspected and, hence, unprepared patient with a retroperitoneal catecholamine - secreting tumour. paraganglioma in a haemodynamically asymptomatic patient, detected intraoperatively due to haemodynamic volatility, is a rare presentation, as in our case. a 59-year - old female, 45 kg, with pain abdomen for 1 month and altered bowel habits (flatulence, bloating sensations) for 1 week, was diagnosed to have a retroperitoneal duodenal duplication cyst on computerised tomography (ct) [figure 1 ] and posted for laparotomy and resection. routine blood and urine investigations, liver and thyroid function tests, electrocardiogram (ecg) and echocardiogram were normal. high - resolution computed tomography scan showing tumour (0.625 mm retro reconstruction images were studied for diagnosis) on the pre - operative night, tab. an 18 g thoracic epidural catheter was placed at the level of the t9 - 10 space with a 3 cm segment within the epidural space. epidural was activated with total bolus dose of 14 ml of bupivacaine 0.125% with fentany l.2 g / ml. after 15 min, ga was induced with inj. thiopentone 200 mg, vecuronium 6 mg and fentanyl 100 g and maintained with oxygen, n2o (nitrous oxide) and sevoflurane with intermittent positive - pressure ventilation (ippv). the retroperitoneal mass was globular, cystic, 10 cm8.5 cm and situated between the aorta and the inferior vena cava. while dissecting the cyst, there appeared sudden tachycardia, 125/ min and hypertension, 235/134 mmhg. this was followed by ventricular bigeminy initially and, later, idioventricular rhythms with varying, altered pulse beats. lignocaine 80 mg iv was injected, the plane of anaesthesia deepened with increasing concentrations of sevoflurane and additional doses of propofol 50 mg and fentanyl 75 g were administered. as hypertension persisted, nitroglycerine infusion, 2 g / kg / min, was started after an initial bolus of 50 g. the patient was stabilised at a pulse rate of 54/ min and bp of 161/82 mmhg, 12 min following the crisis, but a grossly elevated st of 2.5 mm persisted [table 1 ]. nitroglycerine infusion was titrated, the requirement remaining around 26 g / kg / min for the first half an hour of the crisis, and tapered off subsequently. epidural infusion with bupivacaine 0.125% was continued till the first episode of hypotension (80/56 mmhg), which appeared at the 75 min of crisis. epidural opioid (tramadol 50 mg in 12 ml of saline, 0.9%) in 8-hourly bolus doses was continued for post - operative analgesia along with inj. diclofenac 75 mg, i m, twice daily. recorded vital readings, with st changes in the immediate post - operative period, serum electrolytes showed potassium 3.9 meq / l, sodium 134 meq / l and blood sugar 139 mg / dl. ecg showed no st elevation, but t inversions in the lateral leads were noted. repeat ecg and ckmb were normal at the 10 h, with normalised t waves. a second episode of hypotension (80/60 mmhg) was observed at the 17 h, and managed with fluids (hetastarch 6% and saline 0.9%). the 24-h urinary vanillyl mandelic acid (vma) study done on urine collected on day 1 of surgery showed an elevated value (17 mg / d). probable diagnosis of a catecholamine - secreting tumour was made only after observation of intraoperative haemodynamic events. in michelle and colleagues study of 143 patients, where the perianaesthetic risks and outcomes of pheochromocytoma and paraganglioma resection were assessed, less than 2% of the patients were intraoperatively diagnosed as in our case. intraoperative episodes of hypertension with other cardiovascular events like tachycardia, sweating, pulmonary oedema and acidosis in the absence of an obvious cause should alert one to the possibility of a missed neural crest tumour. occurrence of arrhythmias with non - q antero - inferior wall infarction in a hypertensive patient during resection of a pre - operatively undiagnosed abdominal paraganglioma under halothane anaesthesia has been reported. notwithstanding the possible correlation between the use of halothane and the arrhythmias in the above - reported case, paraganglioma or pheochromocytoma should be considered in any patient with hypertension and ischaemic heart disease undergoing resection of an uncharacterised retroperitoneal tumour. previous studies suggest that intraoperative dysrrhythmias occur less often with pre - operative beta - blocked patients, although sustained hypertension can still occur. patients may not enter the phase of crisis that was observed in our case. our patient did not have past history of hypertension, flushing, headache or any other related symptoms suggestive of a catecholamine - secreting neoplasm. our patient had a lower thoracic epidural catheter in situ, which was activated 30 min prior to the incision, and this probably suppressed the incision - related hypertensive response. successful use of combined regional and ga is rarely reported in the literature. by blocking parietal pain signals, the regional technique would be expected to partially mute the catecholamine response, although catecholamines could yet act at the vascular level and overcome the blockade. a combined epidural with ga may be a better choice for a retroperitoneal tumour resection than ga alone. a theoretical disadvantage of the combined approach however may be the blunted induction and incision response itself that may not give the surgical team an opportunity to abort the procedure, pending further evaluation. control of hypertensive episodes can be successfully achieved with the use of sodium nitroprusside or nitroglycerine infusions, and their dosage can also be titrated. st elevations following arrhythmias with normal myocardial enzymes may be indicative of acute coronary spasms in response to circulating vasoactive agents. hypotension in our case did not immediately follow the ligation of the tumour vessels, in contrast to others reports, and neither did she require vasopressors post - operatively. management of circulating blood volume in these patients is essential pre - operatively ; however, an increased circulating blood volume may not prevent hypotension. two factors might have influenced the hypotension episode ; first being myocardial depression following arrhythmias ; second, the use of metaprolol. use of -blockers alone is not recommended in an unprepared patient, although not all agree with the same. no specific tumour markers were studied pre - operatively as the working diagnosis was a retroperitoneal cyst. a 123i - labeled metaiodobenzylguanidine scan for diagnosis of neuroendocrine tumours however, the intraoperative diagnosis was confirmed by histopathological report of paraganglioma and high urinary vma within the first day following surgery. a possibility of men type iia too should be suspected, which our patient did not suffer from. our case report highlights the importance of suspecting and appropriately investigating for a paraganglioma or pheochromocytoma in every case of retroperitoneal tumour, even in a haemodynamically asymptomatic patient. it behoves the anaesthesiologist to be well prepared in such cases for management of acute life - threatening perioperative events. malignant hyperthermia, thyrotoxic crisis and central anticholinergic syndrome may be some of the differential diagnoses one can consider for volatile intraoperative haemodynamic events. | excision of a suspected retroperitoneal, duodenal duplication cyst was performed in a pre - operatively normotensive patient under combined epidural and general anaesthesia. intraoperatively, the cystic tumour was discovered to be a retroperitoneal mass, free from duodenal or adrenal origin. development of severe arrhythmias, st segment changes and hypertensive spikes during cyst handling and dissection suggested the possibility of a catecholamine - secreting tumour. these were managed effectively with pharmacological agents. subsequently, histopathology of the specimen revealed a paraganglioma. vasoactive tumour has to be suspected in every patient undergoing anaesthesia for retroperitoneal cystic lesion. |
mycobacterium (m.) paratuberculosis is the etiologic agent of chronic enteritis of ruminants, known as paratuberculosis or johne 's disease. m. paratuberculosis is a gram - positive, acid - fast bacillus that belongs to the m. avium complex. it grows very slowly and requires mycobactin j, an iron - chelating cell wall component produced by most other mycobacteria, for growth in vitro. as a result identification of m. paratuberculosis depends on mycobactin - dependent growth and detection of the species - specific is 900 insertion sequence by polymerase chain reaction (pcr). like other mycobacteria, the cell wall of m. paratuberculosis is lipid - rich and consists of several layers. the main components of the cell wall are lipoarabinomannan (lam) and arabinomannan (am). it has been reported that lam is highly immunogenic and reacts with sera from infected cattle. several proteins that induce a humoral immune response in infected cows are produced by m. paratuberculosis. these include 17 kda, 34 kda, and 400 kda proteins, and the antigen 85 complex, which consists of four proteins. these protein antigens have been investigated in an effort to find antigens that react specifically with sera from animals infected with m. paratuberculosis, and not those infected with other mycobacterial species. however, these antigens share epitopes with m. avium. it is believed that young calves are infected by m. paratuberculosis via the oral route, through contaminated feces, colostrum, or milk. only 10 - 15% of infected cows develop clinical disease, usually after two or more years of infection. however, subclinically infected animals may shed bacilli intermittently in their feces, spreading infection in the herd. cattle with clinical johne 's disease exhibit decreased production, diarrhea, and weight loss. these animals usually shed bacilli in their feces and have detectable antibodies in their serum. the host response to m. paratuberculosis infection results in granulomatous lesions in the small intestine. the intestinal wall subsequently undergoes progressive thickening, which is caused by hypoproteinemia and edema due to decreased intravascular osmotic pressure. it is not clear which bacterial factors trigger multiplication of m. paratuberculosis, or which host responses control m. paratuberculosis infection. after ingestion by young calves, it is thought that the bacilli enter intestinal tissue through m cells in the peyer 's patches of the small intestine. fibronectin bound to these receptors can, in turn, bind to integrins on m cells and mediate the uptake of m. paratuberculosis. after crossing the intestinal epithelial layer, macrophages are known to have several receptors that are involved in the uptake of mycobacteria [7,73,75,85 - 87 ]. these receptors include complement receptors (cr1, cr3, and cr4), immunoglobulin receptors (fcr), the mannose receptor, and scavenger receptors. human macrophages exhibit decreased uptake of m. avium following the addition of anti - cr3 antibodies. serum opsonization of m. tuberculosis increases the uptake of bacilli by human monocytes, while blocking cr3 with specific antibody decreased the uptake of bacilli by about 87%. likewise, the uptake of m. paratuberculosis by murine macrophages was inhibited by preincubation with anti - cr3 monoclonal antibody. similarly, opsonization of m. paratuberculosis with serum from normal adult cows or from cows with clinical paratuberculosis enhanced the uptake of bacilli by bovine mononuclear phagocytes. these observations suggest that complement opsonization is important to the uptake of m. paratuberculosis by bovine mononuclear phagocytes. it has also been reported that mononuclear phagocytes can synthesize and secrete complement proteins that opsonize particles for phagocytosis. it is very important to understand the survival mechanisms of m. paratuberculosis in bovine mononuclear phagocytes if we are to develop more effective ways to control johne 's disease. because infected animals develop clinical symptoms of johne 's disease relatively slowly, infected cows presumably can suppress multiplication of m. paratuberculosis and delay or prevent the development of johne 's disease. for example, complement receptor cr1-mediated uptake of particles does not stimulate the production of superoxide anion. mannose receptor - mediated uptake of pathogenic or nonpathogenic mycobacteria does not activate nadph oxidase in human macrophages, and selective receptor blockade did not alter the intracellular survival of m. tuberculosis in human macrophages. cr3-mediated binding and uptake of m. tuberculosis by macrophages does not seem to affect the intracellular fate of bacilli. there was no difference in bacterial burden or granulomatous response between wild - type and complement component c3-deficient mice following m. avium infection. opsonization of m. paratuberculosis with normal serum from adult cows, or serum containing specific antibodies against m. paratuberculosis, increased the uptake of bacilli by bovine mononuclear phagocytes. however, intracellular survival of m. paratuberculosis in the bovine mononuclear phagocytes was not affected. toll - like receptors (tlr) are pattern - recognition receptors that detect microbes or microbial components and initiate inflammatory responses. antigen - presenting cells (apcs), which include dendritic cells and macrophages, express tlr receptors and initiate an immune response, and then bind to pathogen - associated molecular patterns (pamps) of microbes. the 19 kda lipoprotein of m. tuberculosis activates murine and human macrophages through tlr2, and this, in turn, activates a signaling pathway that kills intracellular bacilli. this tlr2-mediated mycobactericidal effect is dependent on the production of nitric oxide in murine macrophages and enhanced expression of vitamin d receptor in human macrophages. tlr ligands such as lipopolysaccharide (lps), salmonella dublin, and listeria monocytogenes activate bovine mononuclear phagocytes and induce the production of reactive oxygen intermediates (rois). although there have been no reports of how tlr activation alters the intracellular survival of m. paratuberculosis, pretreatment of j774 cells and bovine monocytes with lps and ifn- slightly decreased the number of viable intracellular m. paratuberculosis bacteria. during the process of phagocytosis by macrophages, the nadph oxidase complex on the plasma membrane produces a series of rois, including superoxide anion, hydrogen peroxide, and hydroxyl radical. however, there have been confilicting reports on the mycobactericidal effect of rois against m. tuberculosis. bovine macrophages produce superoxide anion following stimulation with phorbol 12-myristate 13-acetate (pma), zymosan, or lps. however, bovine monocytes and macrophages do not stimulate much roi production after m. paratuberculosis infection, and ifn- activation of bovine monocytes and macrophages did little to increase the release of rois. m. paratuberculosis secretes superoxide dismutase, which is a possible protective mechanism for intracellular bacilli. however, we need more evidence to clarify the role of rois on the intracellular fate of m. paratuberculosis in bovine mononuclear phagocytes. nitric oxide and other reactive nitrogen intermediates (rnis) are known to be major mycobactericidal molecules, especially in mice. after activation with ifn- and tnf-, murine macrophages produce significantly increased amounts of rni. mycobacteria have the ability to inhibit recruitment of nitric oxide synthase to mycobacteria - containing phagosomes. ifn- activation of bovine macrophages did not result in increased nitric oxide production, whereas ifn--activated murine macrophages produced significant amounts of nitric oxide (45 - 83 m) at 72 and 96 h after treatment. lps, listeria monocytogenes, and salmonella dublin all enhanced the production of nitric oxide (20 to 70 m) by bovine macrophages. in contrast, m. paratuberculosis - infected bovine macrophages and monocytes produced small amounts of nitric oxide (2 - 3 m), and ifn- treatment increased nitric oxide production only to 6 - 8 m. stimulation of monocytes with ifn- or ifn- and lps increased the production of nitric oxide, but the amount produced was inadequate to kill intracellular m. paratuberculosis. in the granulomatous lesions of bovine johne 's disease, the immunoreactivity of inducible nitric oxide synthase (inos) is weak and localized at or near the intestinal crypts. chemically - generated nitric oxide kills extracellular m. paratuberculosis, but bovine mononuclear phagocytes do not produce enough nitric oxide to kill intracellular m. paratuberculosis. although the inhibition of nitric oxide production resulting from the addition of n - monomethyl - l - arginine (nmma) increases the intracellular survival of m. tuberculosis in ifn--pretreated murine macrophages, nmma treatment does not promote intracellular survival of m. paratuberculosis in bovine monocytes. these data suggest that nitric oxide might not be a major mycobactericidal mechanism with which to control m. paratuberculosis in infected cattle. this acidic condition is maintained by a membrane atp - dependent proton pump, the vacuolar h+-atpase. phagosomal maturation is also inhibited by retention of the small gtp - binding protein, rab5, and by reduced recruitment of early endosomal autoantigen 1 (eea1) to mycobacterial phagosomes. live m. paratuberculosis cells inhibit phagosome - lysosome fusion and phagosomal acidification in the j774 murine macrophage cell line, but killed m. paratuberculosis cells do not have this effect. similar results were seen in bovine mononuclear phagocytes, in which killed m. paratuberculosis cells were associated with greater phagosome - lysosome fusion than live m. paratuberculosis cells. pretreatment with ifn- and lps enhanced phagosome - lysosome fusion in murine macrophages infected with m. avium or m. bovis bcg. treatment of m. paratuberculosis - infected j774 cells with ifn- and lps also enhanced phagosome - lysosome fusion and the killing of intracellular bacilli. however, the effect of ifn- and lps treatment on the maturation of phagosomes containing m. paratuberculosis in bovine mononuclear phagocytes is unknown. there have been few investigations of the molecular mechanisms of phagosomal maturation and of the mycobacterial molecules that inhibit phagosome maturation in bovine mononuclear phagocytes. after infection with m. paratuberculosis, although gene expression of tnf- was identified in ileal tissues of cattle infected with m. paratuberculosis, no difference was seen between uninfected and infected cattle. tnf- treatment of murine macrophages infected with m. paratuberculosis resulted in either enhanced or decreased viability of intracellular bacilli, depending on the tnf- concentrations and the lengths of incubation. no report has yet been published on the effect of tnf- on intracellular survival of m. paratuberculosis in bovine mononuclear phagocytes. although m. avium is antigenically and genetically very similar to m. paratuberculosis, it is generally considered to be relatively nonpathogenic in cattle. bovine macrophages expressed greater amounts of il-10 mrna following infection with m. paratuberculosis than with m. avium. the il-10 gene is expressed to a greater extent in intestinal tissues and lymph nodes from cows clinically infected with m. paratuberculosis than in subclinically infected or healthy cows. bovine macrophages infected with m. paratuberculosis produce il-10, and neutralization of il-10 enhances the killing of intracellular bacilli. another report showed greater production of il-10 from peripheral blood mononuclear cells (pbmc) isolated from cows with clinical johne 's disease than from healthy cows. addition of il-10 increased intracellular survival of m. paratuberculosis in pbmcs isolated from healthy cows. neutralization of il-10 also increased the production of ifn- by bovine peripheral blood mononuclear cells after infection with m. paratuberculosis, and enhanced phagosome acidification and apoptosis of bovine macrophages. however, the general role of il-10 in resistance to mycobacterial infection is not clear. il-10/mice did not show greater resistance to acute m. tuberculosis infection than did wild - type mice. apoptosis is a process of programmed cell death that is characterized by dna fragmentation, nuclear chromatin condensation, compacting of cellular organelles, and membrane blebbing. it has been suggested that apoptosis of mycobacteria - infected macrophages induces the intracellular killing of bacilli, but that necrotic macrophage death does not induce the killing of mycobacteria. there have been reports that apoptotic stimuli like fas ligand, tnf-, picolinic acid, atp, and the mycobacterial 19 kda lipoprotein can kill intracellular mycobacteria in mononuclear phagocytes. however, it is not clear whether apoptosis of infected macrophages is required for intracellular killing of mycobacteria, nor is it clear whether stimuli that induce intracellular killing of bacilli are distinct from those that trigger apoptosis. m. tuberculosis induced 30 - 50% apoptosis of infected human alveolar macrophages at 2 and 4 days after infection, and this occurred via a tnf--dependent mechanism. interestingly, pathogenic m. tuberculosis evades apoptosis of macrophages by inducing the secretion of tnf - r2 from infected macrophages that, in turn, is dependent on the production of il-10. m. paratuberculosis induced 18 to 27% apoptosis of infected bovine monocytes at 6 and 48 h after infection, with live bacilli causing greater apoptosis than heat - killed m. paratuberculosis. in that paper, however, the authors did not quantify changes in the intracellular survival of m. paratuberculosis. more recently, evidence has indicated that the longer survival of m. paratuberculosis within bovine monocytes in vitro (4 to 8 days) results in morphological changes that may reflect reduced differentiation or survival of the infected monocytes. extracellular atp can kill intracellular mycobacteria, presumably through the activation of purinergic receptors on the surface of infected mononuclear phagocytes. atp is released by nonlytic and lytic mechanisms from both resting cells, as well as by cells undergoing apoptotic or necrotic cell death. atp released from lymphocytes or mononuclear phagocytes in foci of mycobacterial infection might activate purinergic receptors on infected mononuclear phagocytes. extracellular atp is known to be cytotoxic to macrophages, and triggers the killing of mycobacteria in murine and human macrophages [26,31,52 - 54,68 ]. this response reflects increased intracellular calcium and phospholipase d (pld) activity following atp activation of p2x7 receptors. it is not clear whether macrophage cytotoxicity is required for the mycobactericidal effect of atp to occur. bovine macrophages express mrna for the p2x7 receptor, and the addition of atp to bovine macrophages infected with m. bovis bcg induced the killing of intracellular bacilli. elimination of extracellular atp by the addition of apyrase increased the survival of m. paratuberculosis - infected bovine monocytes, but unexpectedly reduced the survival of bacilli. similarly, the addition of atp or benzyl - atp reduced the survival of m. paratuberculosis - infected monocytes, but not the survival of the bacilli themselves. thus, m. paratuberculosis may differ from other mycobacterial species in terms of how the presence of atp affects the intracellular survival of bacilli within bovine mononuclear phagocytes. in this review, we have described possible intracellular survival mechanisms of m. paratuberculosis in infected macrophages, mainly in comparison to m. tuberculosis and m. avium. m. paratuberculosis seemed to have shared strategies with other closely - related mycobacteria for intracellular survival, but it also has unique mechanisms. it is important to clarify the intracellular survival tactics of m. paratuberculosis in order to understand the pathogenesis of johne 's disease and develop means by which to prevent this disease. however, it is difficult to investigate the survival mechanisms of m. paratuberculosis in infected macrophages because of the long incubation period required to count visible colonies in media and the limited availability of bovine reagents to use for this purpose. with the development of new technologies and vigorous efforts, these survival mechanisms will be better elucidated in the future, and will allow us to understand the pathogenesis of this, one of the most challenging bacteria to study. | johne 's disease is a condition that refers to chronic granulomatous enteritis in ruminants. it is believed that survival and replication of mycobacterium (m.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of johne 's disease. however, it is not clear how m. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of johne 's disease. investigating the intracellular fate of m. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). existing animal models also have limitations. despite those obstacles, there has been progress in understanding the intracellular survival tactics of m. paratuberculosis and the host response against them. in this review, we compare known aspects of the intracellular survival tactics of m. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes. |
during ontogeny, hematopoietic cells home to various organs and settle within defined microenvironments containing favorable factors. these cells remain until the local conditions are modified and then gradually disappear, moving to other subsequent permissive niches that exhibit specific receptors and extracellular matrix (ecm) proteins (dzierzak 1999 ; cumano and godin 2001). first, transient hematopoietic cells are generated in the vitelline islands in the yolk sac at 7 days post - coitum (dpc). at 8 dpc, the intra - embryonary development of blood cells begins in the aorta - gonads - mesonephros region (agm), and then these hematopoietic stem cells spread to the circulation and are attracted by fetal liver endothelium (dzierzak and medvinsky 1995 ; dzierzak 1999). at 11 dpc, in the fetal liver, the hematopoietic cells proliferate at an exponential rate and differentiate properly under the influence of the favorable environment created by the hepatic cells (morrison. these cells secrete extracellular molecules and also express surface molecules that induce the differentiation process in blood cells, thereby creating various proliferation niches. the hepatocytes participate in the cell fate determination of hematopoietic lineages acting as a stroma for blood cells (arias and stewart 2002). at this time, hepatic cells express large amounts of -fetoprotein (afp) and low amounts of albumin (houssaint 1980 ; gualdi. 1996 ; zaret 2002). on the other hand, hematopoietic cells expand greatly and also secrete large amounts of oncostatin m (osm) into the extracellular space, inducing the maturation of the hepatocytes. at the same time, afp expression decreases, whereas albumin expression increases in the hepatocytes, denoting the maturation process (kinoshita and miyajima 2002). hepatocytes express various molecules on their surface and secrete other proteins into the ecm ; these substances are favorable to the local arrival of other hematopoietic cell lineages, promoting the progression of the differentiation process (kinoshita. as soon as the liver becomes more mature, it becomes an unfavorable environment for hematopoiesis, and, consequently, the blood cells depart from the hepatic sinusoids and move away to the circulation once again. at this time, the bone marrow expresses a stromal - derived factor gradient (sdf-1, cxcl-12), converting the bone marrow into a definitive organ for hematopoiesis in the adult. the constitution of environments for hematopoiesis in the various organs depends mainly on the local expression of ecm proteins, which define distinct niches favorable to the different blood lineages. although, some niches have previously been characterized in adult mammal bone marrow, only a small amount of fragmented information exists regarding the hematopoietic environment in the fetal liver. for example, fibronectin has been described as being important for sinusoid development and hepatocyte differentiation in the fetal liver (snchez. matrix metalloproteinases (mmps) are known to be responsible for the degradation and remodeling of the ecm and are also crucial, during ontogeny, in many processes such as cellular growth, morphogenesis, angiogenesis, and wound repair (vu and werb 2000 ; pardo and selman 2005). the presence of mmp1, mmp2, mmp3, mmp7, mmp9, and mmp13 acting as a negative feedback of i, iii, and iv collagen synthesis has been detected during fetal liver development (quondamatteo. this work has aimed to describe morphologically the sequential characteristics of murine fetal liver niches, with an attempt at discriminating favorable and unfavorable environments for the settlement and migration of hematopoietic cells from 12 dpc to 0 day post - partum. all procedures with mice were performed according to the ethical recommendation of the ethics commitee of the fundao oswaldo cruz (oswaldo cruz foundation). eight female and eight male swiss webster mice from the animal laboratory center of the fundao oswaldo cruz (cecal) were matched, each couple being housed in individual cages and receiving food and water ad libitum. on the day after breeding, females with a positive vaginal plug defining 0 dpc were individually isolated. the gestational age was determined from the time of the appearance of the vaginal plug, together with histological parameters as defined in the mouse development atlas of kaufman (1992). the histological analysis was carried out by comparing several morphological aspects of each animal with the characteristics described in the above - mentioned atlas, because fetal development from the same mother presents a range of developmental timing according to kaufman (1992) and in our experience. newborn mice had their gestational age determined after birth, which was established as 0 day post - partum (0 dpp). eight pregnant female swiss webster mice were killed by 5% co2 in a closed atmosphere on the following gestational ages : 12, 13, 14, 15, 16, 17, 18 dpc, and 0 dpp (one pregnant female / gestational age). all the fetuses were decapitated, and in those older than 14 dpc, the livers were isolated. the material was fixed in carson s modified millonig s phosphate - buffered formalin, ph 7.4 (carson. 1973). the specimens were further histologically processed through increasing ethanol concentrations (70%, 95%, and 100%, for 1 h each), cleared in xylene (2 hours), and paraffin embedded. sections of 7 m in thickness were dewaxed with xylene (three times), hydrated with ethanol (three times each in 100%, 95%, 70%, and 50%) and stained with hematoxylin and eosin, lennert s giemsa (lennert 1978), masson s trichrome, sirius red at ph 10.2 (eosinophils ; bogomoletz 1980 ; luque and montes 1989), gomori s reticulin (reticulin fibers), and periodic acid schiff (pas)/alcian blue (ab) at ph 1.0 and 2.5 (acidic and sulfated glycosaminoglycans). other slides were stained with naphthol as - d chloroacetate esterase (ncae ; sigma - aldrich ; 91-c kit) and labeled with fast red for the visualization of granulocytic cells. all slides were analyzed by bright - field microscopy (zeiss axiovert 200 m) with a digital camera (zeiss axiocam hrc) or with confocal laser microscopy (lsm 510-meta). sections were dewaxed with xylene (three times) and hydrated in decreasing ethanol concentrations (three times each in 100%, 95%, and 70%) and distilled water. antigenic retrieval was carried out in 0.01 m citrate buffer ph 6.0 in a microwave oven for 10 min. subsequently, fetus sections were incubated overnight at 4c with antibodies against fibronectin (ab-10, rb-077-r7, labvision), mmp-1 (rb-9225-r7, labvision), mmp-9 (rb-9234-r7, labvision), or afp (sc 8108, santa cruz). on the following day, sections were washed in phosphate - buffered saline (pbs) at ph 7.4 and incubated at 37c for 1 h with secondary antibody (alexafluor488, invitrogen). nuclei were then labeled with dapi (4,6-diamidino-2-phenylindole ; invitrogen ; 5 mg / ml) for 30 min, and sections were counterstained with evans blue (1:10,000) for 1 min, differentiated in pbs, and mounted with glycerol - paraphenylenediamine (sigma - aldrich). the slides were analyzed by confocal laser microscopy (lsm 510 meta, zeiss) ; set - ups including an ar488 laser and bp505 - 550 filter (alexafluor488), hene543 laser and lp560 filter (evans blue), and 405 laser and lp420 filter (dapi) were used. all procedures with mice were performed according to the ethical recommendation of the ethics commitee of the fundao oswaldo cruz (oswaldo cruz foundation). eight female and eight male swiss webster mice from the animal laboratory center of the fundao oswaldo cruz (cecal) were matched, each couple being housed in individual cages and receiving food and water ad libitum. on the day after breeding, females with a positive vaginal plug defining 0 dpc were individually isolated. the gestational age was determined from the time of the appearance of the vaginal plug, together with histological parameters as defined in the mouse development atlas of kaufman (1992). the histological analysis was carried out by comparing several morphological aspects of each animal with the characteristics described in the above - mentioned atlas, because fetal development from the same mother presents a range of developmental timing according to kaufman (1992) and in our experience. newborn mice had their gestational age determined after birth, which was established as 0 day post - partum (0 dpp). eight pregnant female swiss webster mice were killed by 5% co2 in a closed atmosphere on the following gestational ages : 12, 13, 14, 15, 16, 17, 18 dpc, and 0 dpp (one pregnant female / gestational age). all the fetuses were decapitated, and in those older than 14 dpc, the livers were isolated. the material was fixed in carson s modified millonig s phosphate - buffered formalin, ph 7.4 (carson. 1973). the specimens were further histologically processed through increasing ethanol concentrations (70%, 95%, and 100%, for 1 h each), cleared in xylene (2 hours), and paraffin embedded. sections of 7 m in thickness were dewaxed with xylene (three times), hydrated with ethanol (three times each in 100%, 95%, 70%, and 50%) and stained with hematoxylin and eosin, lennert s giemsa (lennert 1978), masson s trichrome, sirius red at ph 10.2 (eosinophils ; bogomoletz 1980 ; luque and montes 1989), gomori s reticulin (reticulin fibers), and periodic acid schiff (pas)/alcian blue (ab) at ph 1.0 and 2.5 (acidic and sulfated glycosaminoglycans). other slides were stained with naphthol as - d chloroacetate esterase (ncae ; sigma - aldrich ; 91-c kit) and labeled with fast red for the visualization of granulocytic cells. all slides were analyzed by bright - field microscopy (zeiss axiovert 200 m) with a digital camera (zeiss axiocam hrc) or with confocal laser microscopy (lsm 510-meta). sections were dewaxed with xylene (three times) and hydrated in decreasing ethanol concentrations (three times each in 100%, 95%, and 70%) and distilled water. antigenic retrieval was carried out in 0.01 m citrate buffer ph 6.0 in a microwave oven for 10 min. subsequently, fetus sections were incubated overnight at 4c with antibodies against fibronectin (ab-10, rb-077-r7, labvision), mmp-1 (rb-9225-r7, labvision), mmp-9 (rb-9234-r7, labvision), or afp (sc 8108, santa cruz). on the following day, sections were washed in phosphate - buffered saline (pbs) at ph 7.4 and incubated at 37c for 1 h with secondary antibody (alexafluor488, invitrogen). nuclei were then labeled with dapi (4,6-diamidino-2-phenylindole ; invitrogen ; 5 mg / ml) for 30 min, and sections were counterstained with evans blue (1:10,000) for 1 min, differentiated in pbs, and mounted with glycerol - paraphenylenediamine (sigma - aldrich). the slides were analyzed by confocal laser microscopy (lsm 510 meta, zeiss) ; set - ups including an ar488 laser and bp505 - 550 filter (alexafluor488), hene543 laser and lp560 filter (evans blue), and 405 laser and lp420 filter (dapi) were used. at 12 dpc, fetal liver showed hematopoietic cells with many megakaryoblasts and a large amount of red blood cells at various stages of maturation uniformly distributed among the hepatocytes (fig. pas - ab ph 1.0 stain was seen as a delicate mesh around primitive larger liver portal vessels (fig. furthermore, many mitotic cells in various stages were observed, in contrast to the few apoptotic cells (fig. reticular fibers were noticed in the submesothelium layer of the capsule and inside the liver in which thin fibers were found around vessels (fig. 1mouse fetal liver at 12 days post - coitum (dpc). a erythroid lineage cells (thin white arrows) : some are still nucleated inside blood vessels. note the megakaryocytes (black arrows) and some mitotic cells (white arrowheads). bar 30 m. b erythropoietic foci (black chevrons) together with some immature myeloid cells : promyelocyte (black arrowhead) and myelocytes (black arrows). bar 10 m. c reticular fibers (black arrows) in the submesothelium layer from the capsule and inside the liver bar 5.5 m mouse fetal liver at 12 days post - coitum (dpc). a erythroid lineage cells (thin white arrows) : some are still nucleated inside blood vessels. note the megakaryocytes (black arrows) and some mitotic cells (white arrowheads). bar 30 m. b erythropoietic foci (black chevrons) together with some immature myeloid cells : promyelocyte (black arrowhead) and myelocytes (black arrows) c reticular fibers (black arrows) in the submesothelium layer from the capsule and inside the liver. bar 5.5 m fibronectin expression was observed in the sinusoid and venula walls and around the parenchymal and/or hematopoietic cells, displaying a punctiform and discontinuous pattern ; the capsule was weakly positive (fig. 2mouse fetal liver at 12 dpc (green alexafluor488, blue evans blue, red dapi [4,6-diamidino-2-phenylindole ]). b matrix metalloproteinase-1 (mmp-1) expression in endothelial cells of a venule (small fat arrow), in the periphery of some immature and intravascular cells (arrowhead), and in erythroid cells in the parenchyma (arrows). c mmp-9 expression in parenchymal erythroid aggregates (arrows) and in the mesothelial layer (arrowhead). bars 40 m mouse fetal liver at 12 dpc (green alexafluor488, blue evans blue, red dapi [4,6-diamidino-2-phenylindole ]). b matrix metalloproteinase-1 (mmp-1) expression in endothelial cells of a venule (small fat arrow), in the periphery of some immature and intravascular cells (arrowhead), and in erythroid cells in the parenchyma (arrows). c mmp-9 expression in parenchymal erythroid aggregates (arrows) and in the mesothelial layer (arrowhead). bars 40 m mmp-1 expression occurred in the periphery of cells, probably from the erythroid lineage, and appeared a little more intensely in venulae and in some immature and intravascular cells (fig. mmp-9 was more evident in vessel walls and surrounding some groups of small cells, probably of erythroid lineage, whereas its expression in the mesothelial layer was weaker (fig. immature erythroid cells were predominant, with megakaryoblasts, megakaryocytes, and many cells in mitosis (fig. vascular and trabecular vessels were better defined, and more hematopoietic cells were present inside the liver (fig. pas staining continued to be expressed in the conjunctive tissue, around vessel walls, and in the capsule (fig. proteoglycans positive for pas - ab ph 1.0 were detected in the capsular mesothelium and in the venular endothelium (fig. a erythroblasts, normoblasts, myeloblasts, and metamyelocytes (black arrows), megakaryoblasts, megakaryocytes, and many cells in mitosis (black arrowheads). b high - power magnification showing megakaryocytes (stars), erythroid cells (fat arrow), monocyte (thin arrow), and mitotic figures (arrowheads). insert in d delicate layer of highly sulfated proteoglycans around cells (small arrow) and endothelium. a erythroblasts, normoblasts, myeloblasts, and metamyelocytes (black arrows), megakaryoblasts, megakaryocytes, and many cells in mitosis (black arrowheads). b high - power magnification showing megakaryocytes (stars), erythroid cells (fat arrow), monocyte (thin arrow), and mitotic figures (arrowheads). sirius red ph 10.2 stain. insert in d delicate layer of highly sulfated proteoglycans around cells (small arrow) and endothelium. bar 7.5 m immunostaining for fibronectin was seen in endothelial and subendothelial cells, presenting a more exacerbated punctiform pattern than that at 12 dpc. it was also detected with higher intensity in some small cellular groups (seemingly erythroid) and with weak linear expression in the subcapsular region (fig. 4mouse fetal liver at 13 dpc (green alexafluor488, blue evans blue, red dapi). a fibronectin in erythroid cells (arrowhead), in endothelial (fat arrow) and subendothelial cells, and in the subcapsular region (chevron). b mmp-1 in the cytoplasm of erythroid cells in the parenchyma and in blood vessels. c mmp-9 dispersed through the parenchyma and presented in venulae walls with a granular pattern. bars 40 m mouse fetal liver at 13 dpc (green alexafluor488, blue evans blue, red dapi). a fibronectin in erythroid cells (arrowhead), in endothelial (fat arrow) and subendothelial cells, and in the subcapsular region (chevron). b mmp-1 in the cytoplasm of erythroid cells in the parenchyma and in blood vessels. c mmp-9 dispersed through the parenchyma and presented in venulae walls with a granular pattern. bars 40 m expression of mmp-1 was seen in the cytoplasm of erythroid cells but not in the capsule (fig. mmp-9 was more disperse than at the previous time point, appearing throughout the parenchyma and occurring in the venula walls with a granular pattern (fig. the fetal liver was still profusely occupied by erythroid cells, but they were more mature, and inside the vessels, red blood cells without a nucleus predominated (fig. megakaryocytes were also more differentiated and presented a weak reaction to pas - ab ph 2.5 or 1.0 (fig. the surface of the venular endothelium and mesothelium exhibited a linear pattern of pas - ab ph 1.0 (fig. the mesothelium, under a superficial layer of proteoglycan, presented a light pas reaction. a intermediary normoblasts, erythroid cells (some with a nucleus), megakaryoblasts (arrows) and monocytes (arrowhead). b pas - positive reaction in hepatocytes around intrahepatic venulae (arrow) and discontinuous subendothelial cells. note the mesothelium with a light pas reaction, and the linear pattern of ab ph 1.0 proteoglycans in the surface of the venular endothelium and around cells. c note some reticular fibers inside the liver (arrows), but the capsular region is without reticular fibers. d fibronectin in sinusoids, in portal veins, and in the subcapsular region (green alexafluor488, red evans blue, blue dapi). e myeloid cells expressing naphthol as - d chloroacetate esterase (ncae) in the parenchyma (green gill s 3 hematoxylin, red fast red). insert in e myeloid cells expressing ncae in the subcapsular region (green gill s 3 hematoxylin, red fast red). a intermediary normoblasts, erythroid cells (some with a nucleus), megakaryoblasts (arrows) and monocytes (arrowhead). b pas - positive reaction in hepatocytes around intrahepatic venulae (arrow) and discontinuous subendothelial cells. note the mesothelium with a light pas reaction, and the linear pattern of ab ph 1.0 proteoglycans in the surface of the venular endothelium and around cells. c note some reticular fibers inside the liver (arrows), but the capsular region is without reticular fibers. d fibronectin in sinusoids, in portal veins, and in the subcapsular region (green alexafluor488, red evans blue, blue dapi). e myeloid cells expressing naphthol as - d chloroacetate esterase (ncae) in the parenchyma (green gill s 3 hematoxylin, red fast red). insert in e myeloid cells expressing ncae in the subcapsular region (green gill s 3 hematoxylin, red fast red). bar 20 m fibronectin was only seen in vessel walls including the sinusoids, being stronger in portal veins in which it was concentrically arranged (fig. granulocytes were histochemically demonstrated with ncae close to the capsule and dispersed in the parenchyma (fig. the liver was still highly settled by erythroid cells and also showed an increase in megakaryocytes (fig. they were located distant from each other or were beginning to form trabeculae (fig. d fibronectin in the capsular submesothelial layer, sinusoids, and venula wall (green alexafluor488, blue evans blue, red dapi). bars 30 m mouse fetal liver at 15 dpc. a intense erythroid and megakaryocytic proliferation d fibronectin in the capsular submesothelial layer, sinusoids, and venula wall (green alexafluor488, blue evans blue, red dapi). bars 30 m fibronectin expression was intense with a granular pattern around venulae, forming a concentric arrangement. it was also positive in the conjunctive of the submesothelial capsule, in the sinusoid walls, delimiting hepatic trabeculae with many erythroid cells (fig. both mmp-1 and mmp-9 were negative. at 16 dpc, the presence of megakaryocytes and erythroid cells was still intense, but the erythroid lineage cells were more mature with many normoblasts. the granulocyte populations, predominantly neutrophils with few eosinophils, were seen in all extension of the subcapsular region and in the stroma around large portal veins (fig. hepatocytes were more mature with a marked amount of pas - positive granules (glycogen ; fig. 8d). some isolated spherical cells with basophilic cytoplasm and metachromasy following giemsa staining indicated the possible presence of mast cell precursors. eosinophils were sparsely seen inside the parenchyma and were always mature, without eosinopoietic foci (fig. 7a). some of the large cells with a doughnut - shaped nucleus seemed to be granulocyte - macrophage cells. portal veins were stained only with masson s trichrome and did not express reticular fibers as shown by gomori s reticulin (fig. mesothelial cells were positive for ab ph 1.0, and the pas reaction was more reduced in the venula walls (fig. mature neutrophilic and eosinophilic cells (star) in the conjunctive tissue around the large portal veins. 8mouse fetal liver at 16 dpc. a ncae in neutrophil foci in the subcapsular region (green gill s 3 hematoxylin, red fast red). b mmp-1 expression in the cytoplasm of neutrophils (green alexafluor488, blue evans blue, red dapi). c granular fibronectin expression in the venulae and parenchyma (green alexafluor488, blue evans blue, red dapi). bars 30 m mouse fetal liver at 16 dpc. a erythroid lineage cells with many normoblats. mature neutrophilic and eosinophilic cells (star) in the conjunctive tissue around the large portal veins. a ncae in neutrophil foci in the subcapsular region (green gill s 3 hematoxylin, red fast red). b mmp-1 expression in the cytoplasm of neutrophils (green alexafluor488, blue evans blue, red dapi). c granular fibronectin expression in the venulae and parenchyma (green alexafluor488, blue evans blue, red dapi). bars 30 m fibronectin expression at 16 dpc was lower than that at 15 dpc in the wall of venulae, in the submesothelial conjunctive tissue of the capsule, and within the whole parenchyma, showing a dotted pattern in sinusoid walls (fig. the expression of ncae easily allowed the visualization of the preferential location of neutrophil foci in the subcapsular region (fig. 8a), and mmp-1 expression was intense in the cytoplasm of neutrophils (fig. the erythroid lineage was significantly more highly differentiated, showing many polychromatic and orthochromatic normoblasts that were evenly distributed throughout the liver, sometimes forming small aggregates around portal veins. the relative density of the erythroid cells when compared with hepatocytes was lower than that observed in livers from previous stages. an expressive reduction was also noted in the number of megakaryocytic cells, and the red blood cells inside the circulation were enucleated. we also noticed monocyte clusters with undifferentiated cells presenting an immunoblastic - like aspect around the large portal veins (fig. hepatocytes were more mature, with many vacuolizations and pas - positive granules inside them, forming distinct trabeculae.. a lower relative density of erythroid cells compared with previous stages, showing predominance of normoblasts. c fibronectin expression in the venulae walls, in the capsular submesothelial layer, and in perivascular small erythroid cells (green alexafluor488, blue evans blue, red dapi). bar 30 m mouse fetal liver at 17 dpc. a lower relative density of erythroid cells compared with previous stages, showing predominance of normoblasts. c fibronectin expression in the venulae walls, in the capsular submesothelial layer, and in perivascular small erythroid cells (green alexafluor488, blue evans blue, red dapi). bar 30 m fibronectin expression had a granular pattern in the venular and sinusoidal walls and in the submesothelial conjunctive tissue of the capsule (fig. neutrophils were clearly discernable because of their high expression of ncae, and immunostaining for mmp-1 was intense in neutrophils and was occasionally also located around vessels. at 18 dpc, erythroid and megakaryocytic lineages showed a similar pattern to that at 17 dpc, but with greater rarefaction of the hemopoietic component (fig. neutrophil and eosinophil lineages were more frequent, mainly in the large portal spaces and in the subcapsular region (fig. these cells were also seen in small portal spaces in which collagen began to be deposited. some neutrophils and eosinophils reticular fibers were noted in the capsule, in large portal spaces, and occasionally in the parenchyma (fig. d fibronectin expression in sinusoids and in the connective submesothelial tissue (green alexafluor488, blue evans blue, red dapi). e mmp-1 in the cytoplasm of neutrophils in the large portal spaces (green alexafluor488, blue evans blue, red dapi). eosinophilic lineages associated with late erythroblasts in large portal spaces and in the subcapsular region. d fibronectin expression in sinusoids and in the connective submesothelial tissue (green alexafluor488, blue evans blue, red dapi). e mmp-1 in the cytoplasm of neutrophils in the large portal spaces (green alexafluor488, blue evans blue, red dapi). bar 30 m mmp-1 was intense in the cytoplasm of neutrophils (fig. fibronectin was less intense, with a granular pattern in the venular and sinusoidal walls and in the submesothelial connective tissue of the capsule (fig. erythroid cells formed small isolated foci throughout the liver ; these foci consisted of basophilic, polychromatic, and mainly orthochromatic normoblasts, and apoptotic cells were sometimes seen (fig. the number of megakaryocytic cells was higher than at 18 dpc, with many megakaryoblasts. myeloid cells, especially neutrophils, were more numerous, displaying several foci mainly in the large portal spaces and in the subcapsular region (fig. eosinophils were less frequent than at 18 dpc, and immunoblast - like cells (uncharacterized immature cells) were sparse throughout the parenchyma. mast cells were rare and scattered in the parenchyma or located in the portal spaces. an impressive increase occurred in the number of reticular fibers, making a three - dimensional network in the parenchyma and exhibiting higher density in the portal and capsular regions (fig. hepatocytes showed a diffuse microgoticular steatosis with pas - positive neutral glycoprotein surrounding fat vacuoles (fig. no collagen increase was observed in the connective tissue of the intrahepatic venulae and portal spaces (fig. a marked decrease in the erythroid lineage (white arrow), with a few limited proliferative foci. insert in a presence of myeloid cells in the subcapsular region and of vacuolated hepatocytes. b myeloid proliferative focus (white star) in the connective tissue of the portal space. c reticular fibers forming a three - dimensional network in the parenchyma and capsular regions (arrows). e fibronectin showing a granular pattern in the venula wall and sinusoids (green alexafluor488, blue evans blue, red dapi). f mmp-1 expression in the cytoplasm of neutrophils (green alexafluor488, blue evans blue, red dapi). bar 30 m mouse liver at 0 dpp. a marked decrease in the erythroid lineage (white arrow), with a few limited proliferative foci. insert in a presence of myeloid cells in the subcapsular region and of vacuolated hepatocytes. bar 20 m. b myeloid proliferative focus (white star) in the connective tissue of the portal space. c reticular fibers forming a three - dimensional network in the parenchyma and capsular regions (arrows). e fibronectin showing a granular pattern in the venula wall and sinusoids (green alexafluor488, blue evans blue, red dapi). f mmp-1 expression in the cytoplasm of neutrophils (green alexafluor488, blue evans blue, red dapi). bar 30 m fibronectin expression was similar to that observed in other developmental stages, showing a granular pattern in the venular and sinusoidal walls and in the submesothelial connective tissue of the capsule (fig. bars 30 m a negative control with anti - rabbit alexafluor488 (cartilage). bars 30 m table 1 summarizes the main molecules analyzed in this work, and table 2 presents a semiquantified analysis of myeloid, megakaryocytic, and erythroid lineages during murine fetal liver development. table 1summary of extracellular matrix components in the murine fetal liver from 12 days post - coitum (dpc) to 0 day post - partum (dpp), showing some markers of liver maturity (pas periodic acid schiff staining, mmp matrix metalloproteinase). the intensity of each element was scored from + to + + + + or as negativedevelopment agefibronectinreticulin fiberspas (hepatocytes)mmp-1mmp-912 dpc+ (granular)+++ (erythroid)+ (erythroid)+ (endothelium)+++ (endothelium)13 dpc+/++ (linear)++++++ (erythroid)+++ (erythroid)- (endothelium)+ (endothelium)14 dpc+/++ (trabecular)negative++negativenegative15 dpc++ (trabecular)negative+++negativenegative16 dpc++ (trabecular)negative++++neutrophilsnegative17 dpc+++ (trabecular/ vascular)negative++++neutrophilsnegative18 dpc+++ (trabecular/ vascular)+++++neutrophilsnegative0 dpp+++ (trabecular/ vascular)++++++neutrophilsnegativetable 2semiquantified analysis of three hematopoietic lineages present in the murine fetal liver during embryogenesis. the presence of each lineage was scored from + to + + + + development ageerythroidmegakaryocyticmyeloid12 dpc+++++++++13 dpc+++++++++14 dpc++++++++++15 dpc+++++++++(with eosinophils)16 dpc++++++++++17 dpc+++++++++18 dpc++++++0 dpp+++ summary of extracellular matrix components in the murine fetal liver from 12 days post - coitum (dpc) to 0 day post - partum (dpp), showing some markers of liver maturity (pas periodic acid schiff staining, mmp matrix metalloproteinase). the intensity of each element was scored from + to + + + + or as negative semiquantified analysis of three hematopoietic lineages present in the murine fetal liver during embryogenesis. here, we describe the development of fetal liver as a hematopoietic organ from 12 dpc, when the primordium begins, to the first day of birth (0 dpp). the results show that : (1) fetal liver, during ontogeny, presents two basic phases, the one being an immature phase, which is predominantly endodermic (before 14 dpc), and the other being a more mature phase (endodermic - mesenchymal) during which hepatocyte maturation, a better definition of trabecular and vascular patterns, and an increase in the mesenchymal connective tissue in the capsule, in the venular walls, and in the parenchyma interstitium occur (after 15 dpc) ; (2) the hepatocytes act as the fundamental stroma for the erythroid lineage (trabecular hematopoiesis) in the immature fetal liver (endodermic stromal support) ; (3) the extracellular molecule fibronectin appears to be more closely related to hepatocyte maturation and the definition of the trabecular pattern of the fetal liver ; (4) reticulin fibers, which biochemically correspond to collagen type iii or fibronectin or to undefined non - collagenic glycoproteins (unsworth. 1982), are not relevant to fetal hematopoiesis and arise only in the late period of the more mature phase of the fetal liver ; (5) the appearance of myeloid cells in the fetal liver is related to an increase of the mesenchymal stroma, especially around the thick branches from the portal vein ; (6) ncae is the best histochemical marker for the more immature neutrophils foci, whereas the mature neutrophils are positive for mmp-1 ; (7) the decay of hepatic hematopoiesis (uninhabitable phase) is coincident with hepatic maturity. in the literature, only a few studies can be found that concern the interaction between hematopoietic cells with hepatic endothelial and stromal cells, and that stress the importance of this kind of study to a better understanding of hematopoietic embryology. this study has therefore dealt with the sequential appearence of hematopoietic cells, hepatocyte maturation, metalloproteinase expression by hematopoietic cells, and ecm composition during liver development. at 12 dpc, many progenitor cells begin to home toward the fetal liver in which they then locally differentiate into erythroid and megakaryocytic cells. at this time, hepatocytes are still immature and act as stromal cells for the hematopoietic lineage until 18 dpc. the fetal liver follows the four main morphological steps of other lymphoid and lymphohematopoietic organs : (1) the mesenchymal - endodermic pre - hematopoietic phase ; (2) hematopoiesis establishment ; (3) differentiation and specialization of the organ ; (4) expansion or the terminal phase (lenzi and lenzi 1986). here, we have sequentially described some of these development steps in vivo, integrating the hematopoietic process with the components and arrangement of ecm. the expression of collagens and proteoglycans is low but constant from 12 to 18 dpc. fibronectin expression is higher and more constantly expressed, especially around vessels, in the wall of veins and sinusoids, being a marker of blood vessels and hepatocyte trabeculae maturity. (2000) have shown that only fibronectin facilitates the formation of elongated cord - like structures, reminiscent of liver plate organization, modulating fetal hepatocyte morphology, growth, and differentiation. our data do not indicate that the output of hematopoietic mature cells from the liver is related to tissue fibronectin levels, in view of its constant expression during the various periods of liver development. in vitro, the integrin vla4 (very late antigen 4) mediates the adhesion of hemopoietic progenitors to bone marrow stroma through an interaction with its ligands vcam-1 (vascular cell adhesion molecule 1) and the cs1 moiety of fibronectin, as observed by craddock. (1997b) ; however, these authors have observed that, in contrast to vcam-1, the cs1 moiety of fibronectin is not a significant ligand in vla4-mediated progenitor trafficking in vivo. hence, its major role seems to be devoted to hepatocyte maturation, and not to hematopoietic cell adherence in the fetal liver. the appearance of reticulin fibers in the fetal liver, after a transitory phase, is belated, occurring after 17.5 dpc, as has also been seen by amenta and harrison (1997). the scarcity of fibers in the early periods of liver development is probably advantageous for hematopoiesis, favoring intrahepatic cell mobility and attachment to hepatocytes, which seem to act as the main stromal cells to hematopoietic cells. the late appearance of the collagen network appears to be involved with the requirement for a physical scaffold to support the gradual and vast hepatic enlargement and the maturation of hepatocytes. brill. (2002) have observed that adult hepatocyte ecm better supports the expression of adult genes, whereas fetal hepatocyte ecm induces the expression of fetal genes. considering the scarcity of ecm in the liver, we suggest that fetal hematopoiesis is essentially controlled by paracrine cell - cell contact, rather than being under the influence of the ecm. indeed, hematopoietic environments usually have a tiny amount of fibrillar ecm and are more dependent on proteoglycan components. (2002) have provided evidence that heparin sulfate is the active element in adult ecm, and chondroitin sulfate in fetal ecm. we have demonstrated that erythroid cells appear in the fetal liver at 12 dpc and proliferate in clusters among hepatocytes (trabecular erythopoiesis), being sometimes difficult to discriminate in the liver parenchyma from the hematopoietic cells. the erythroid lineage evolves gradually from nucleated to anucleated cells only after 14 dpc. in humans, megakaryocytic cells are seen near erythroid cells in the sinusoids and hepatic hilum from 6th and 7th weeks, respectively (h.l. zamboni (1965a, 1965b) has observed megakaryocytes after 12 weeks, when hepatic hematopoiesis is effective with all lineages. in mouse, we have detected these cells near hepatic sinusoids from 12 dpc. at 15 dpc, the number of megakaryocytic cells decreases, and these cells persist at low levels until 18 dpc. (2000) have shown in vitro the effect of sdf-1 on megakaryocyte migration binding to cxcr4. this mechanism might contribute to the homing process during ontogeny, when these cells migrate many times, from the vitelline yolk to the fetal bone marrow. neutrophils and eosinophils have been detected in the fetal liver from 12 dpc. at 1517 dpc, they are seen around and near large sinusoids. from 1618 dpc this late detection of myeloid cells in the fetal liver can be explained by the following mechanisms : (1) the stem cells committed to both cellular lineages might quiescently persist in the fetal liver until a favorable microenvironment arises in the subcapsular region and around portal vein walls, which express ecm - mesenchyma dependence ; or (2) this type of stem cell arrives late in the fetal liver, coming from an undefined organ. this second hypothesis is unlikely, because hematopoiesis stops at 11.5 dpc in the yolk sac and placenta and at 12 dpc in the agm. thomas and yoffey (1964), enzan. (1978), emura. (1983), and sohn. ((1978) have reported granulocytic cells such as neutrophils and eosinophils after the cell culture of human fetal liver from 10 to 13 weeks of gestation and have drawn attention to a topographic association between the various stages of these cells and the hepatic mesenchyma. here, we have described the presence of neutrophilic granulocytes in the connective stroma (collagenic and reticular fibers) in the capsule and in the subcapsular region of fetal mouse liver from 14 to 17 dpc, as shown by ncae. after 15 dpc, an increase of neutrophilic granulocytes and the appearance of eosinophils have been observed in connective tissue around the wall of the large vessels of the portal system. for instance, some wound healing caused by esophageal varices in patients infected with schistosoma mansoni exhibit a regenerative process similar to post - necrotic cirrhosis only in the liver periphery (andrade 1967). (2003) have described an association between hematopoietic and mesenchymal cells in the liver, but they have not analyzed the ecm of the liver ; this aspect deserves further investigation in order to improve our understanding of its contribution to the proliferation and differentiation of several hematopoietic lineages in vivo and the formation of a special environment for hematopoiesis. mature or immature neutrophils depart from the liver when the environment is no longer favorable to them, migrating to other suitable environments through the circulation (intravasation and extravasation). papayannopoulou and craddock (1997) have shown that hematopoietic cells injected into mice are not specifically taken up by bone marrow but occur in the circulation and are widely spread in many organs such as the liver, lungs, kidneys, spleen, and bone marrow. subsequently, blood cells are seen in the spleen and bone marrow, indicating a retention and proliferation of these cells in such niches. hematopoietic stem cells in mouse fetal liver double in number daily from 12.5 to 14.5 dpc but then decrease in number at 15.5 (ikuta and weissman 1992 ; morrison. dpc, the fetal liver (+ /+ or + /sl mouse) contains 1,200 and 2,300 thy-1 lin sca-1 cells, respectively, at a relatively constant percentage of 0.03% (ikuta and weissman 1992). at 15 dpc, however, rather then doubling to 10,200 by 16 dpc, only 4,350 fetal liver hematopoietic stem cells have been found. the rest of the expected cells occur in the circulation for progressive settling in the spleen and bone marrow (christensen. hence, the stem cell decrease in the fetal liver at 15.5 dpc seems to be a consequence of hepatocyte differentiation (kinoshita. the expression of mmp-1 is weak in the fetal liver endothelium and erythroid lineage at 12 dpc, becoming higher in erythroid cells at 13 dpc. mature neutrophils from 16 dpc to 0 dpp always express mmp-1 with a high intensity. (2005) have verified that human neutrophil collagenase is one of the proteins in the secondary granules of late granulocytic cells, whereas mmp-9 has been detected only in the liver endothelium and erythroid cells in the immature liver (12 and 13 dpc), being completely negative after 15 dpc. these results reveal that both mmp-1 and mmp-9 contribute to the beginning of erythropoiesis, but the mechanisms involved are still unknown. (2005) have shown that mmp-9 is not required for erythropoietin and granulocyte colony - stimulating factor (g - csf) mobilization. 2002) have confirmed that mmp-9 promotes the release of the cell - soluble kit ligand (skitl) by bone marrow, a ligand that favors the homing of endothelial and hematopoietic cells from quiescent to proliferative niches. mmps not only induce the break - up of ecm molecules, but also allow the release of cytokines (vu and werb 2000), such as the vascular endothelial growth factor, which might play a role in angiogenesis (bergers. in contrast, mmp and timp (tissue inhibitor of mmp) play other roles, acting as growth factors and removing cellular receptors (guedez. many other investigations have demonstrated that timp-1 and -2 have many functions such as anti - protease activity and as growth factors, acting as enhancers of the erythroid lineage (docherty. 1985 ; hayakawa. 1990 ; stetler - stevenson. 1992 ; chesler. krane (1995) have described a physiological function of mmp-1 in bone remodeling. in addiction, neutrophils are reported to express mmp-1 in chronic obstructive lung disease (cawston. 2001) and in inflammatory reactions in dentary pulp lesions (shin. 2002). mmp-1 might also play a role in destroying or disturbing the basal membrane of the vessels during cell diapedesis, especially in the migration from fetal liver to bone marrow during ontogeny, with a similar mechanism of metastatic homing (brinckerhoff. 2000 ; yana and seiki 2002 ; seiki 2003 ; seiki and yana 2003 ; pardo and selman 2005). the diffusion of hematopoietic cells during ontogenetic development makes use of similar processes as inflammatory, tumor, and fungal cell spreading, all of which present the following : (1) a primary source (stem cell, primary tumor or focus), (2) local proliferation, (3) detachment from the primary source, (4) intravasation, (5) intravascular motility, (6) extravasation, (7) entrance to the organ parenchyma, (8) metastatic or metastatic - like proliferation influenced by paracrine growth factors, (8) repetition of the cycle from step 3 (de vita. 2004 ; mendes - giannini. 2000). in conclusion, the intensity and location of hematopoietic lineages change during the process of fetal liver maturation. we have been able to correlate the myeloid lineage with the mesenchymal stroma (around portal vessel walls and subcapsules) and erythroid development with the endodermal stroma (hepatocytes). | embryonic hematopoiesis occurs via dynamic development with cells migrating into various organs. fetal liver is the main hematopoietic organ responsible for hematopoietic cell expansion during embryologic development. we describe the morphological sequential characteristics of murine fetal liver niches that favor the settlement and migration of hematopoietic cells from 12 days post - coitum (dpc) to 0 day post - partum. liver sections were stained with hematoxylin and eosin, lennert s giemsa, sirius red ph 10.2, gomori s reticulin, and periodic acid schiff / alcian blue ph 1.0 and ph 2.5 and were analyzed by bright - field microscopy. indirect imunohistochemistry for fibronectin, matrix metalloproteinase-1 (mmp-1), and mmp-9 and histochemistry for naphthol as - d chloroacetate esterase (ncae) were analyzed by confocal microscopy. the results showed that fibronectin was related to the promotion of hepatocyte and trabecular differentiation ; reticular fibers did not appear to participate in fetal hematopoiesis but contributed to the physical support of the liver after 18 dpc. during the immature phase, hepatocytes acted as the fundamental stroma for the erythroid lineage. the appearance of myeloid cells in the liver was related to perivascular and subcapsular collagen, and ncae preceded mmp-1 expression in neutrophils, an occurrence that appeared to contribute to their liver evasion. thus, the murine fetal liver during ontogenesis shows two different phases : one immature and mainly endodermic (15 dpc) with the maturation of hepatocytes, a better definition of trabecular pattern, and an increase in the connective tissue in the capsule, portal spaces, and liver parenchyma. the decrease of hepatic hematopoiesis (migration) coincides with hepatic maturation. |
although necrotizing fasciitis is rare, 0.4 cases every 0.1 million adults, it is a serious life - threatening infectious disease which causes severe destructive necrosis in soft tissues and skin tissues. in addition, as the mortality rate is very high even when the necrotic tissues are extensively resected, early diagnosis and intensive surgical treatment play important roles in reducing the mortality rate. however, early diagnosis is difficult because lesions are initiated in deep subcutaneous tissues and spread via the fascia without external clinical symptoms. this mainly takes place after the skin damage in the pelvic limb has occurred and sometimes occurs due to hematogenous spread as the result of secondary bacteremia. necrotizing fasciitis can occur anywhere in the body ; it commonly takes place in the limbs, but can also occur in abdominal walls, perianal regions, inguinal regions, and wounds caused by surgeries. only a couple of cases in which necrotizing fasciitis has occurred in femoral regions primarily without skin damage, including cases associated with direct perforation caused by colorectal cancers, have been reported [3 - 6 ]. therefore, the authors, based on their experience, report the case of a patient with necrotizing fasciitis in femoral regions due to rectal perforation caused by rectal - cancer surgery and by complications of radiation therapy. a 66-year - old male patient visited orthopedics due to pains in the right femoral regions and pelvis that had started a month earlier. a week before, the patient had visited the emergency room as he was experiencing more severe pains in the right femoral regions and pelvis, as well as a fever that had started a day before. four years earlier, the patient had received a lower anterior resection and a resection of the right lobe because of colorectal cancer and liver metastasis (american joint committee on cancer stage t4n2m1, stage iv). after the surgery, adjuvant chemotherapy was performed eight times with capecitabine and radiation therapy at 5,580 cgy ; 31 fractions were carried out in the pelvis. moreover, no unusual findings were observed in the family history, and there was no history of drinking or smoking history. vital signs at the time of his visit indicated 107/77 mmhg, 90/min, 38.1, and 18/min for blood pressure, pulse rate, body temperature, and respiration rate, respectively. no unusual findings were found on the chest auscultation, and the abdomen was soft without oppressive and rebound pains. severe pains in the right femoral regions and ion the side of the abdomen, as well as an erythematous rash, oppressive pains, and crepitus in the right femoral regions, were palpated in the patient. general blood tests showed leukocytes of 10,440/mm, hemoglobin at 12.6 g / dl, a platelet count of 149,000/mm, and an erythrocyte sedimentation rate of 50 mm / hr ; c - reactive protein was elevated (191 mg / dl). general chemical tests resulted in a total protein content of 5.5 g / dl, albumin at 3.1 g / dl, total bilirubin at 1.0 mg / dl, an aspartate aminotransferase / alanine aminotransferase ratio of 55/191 iu / l, alkaline phosphatase at 58 iu / l, r - glutamyl transpeptidase at 14 iu / l, lactate dehydrogenase at 762 u / ml, and a blood urea nitrogen / creatinine ratio of 16.1/0.6 mg / dl ; also, increases in creatine kinase (ck, 8,424 iu / l), ck - mb (20.14 ng / ml), and myoglobin (1,325 ng / ml) were noted. the patient showed unique necrotizing fasciitis in the magnetic resonance imaging (mri) ; a gluteus maximus muscle, a thick fascia in the exterior section of the right femoral regions, an edema in surrounding soft tissues, and fascial gas were observed (fig. 1). resection of extensive necrotic tissues and dissection of epidermis and subcutaneous tissues were immediately carried out, and the abscesses were discharged by inserting drainage tubes. ceftriaxone, clindamycin, and amikacin were administered as initial antibiotics against all gram - positive, gram - negative, and anaerobic bacteria. enterococcus senstive to bacteroides thetaiotaomicron and vancomycin were identified in the blood and necrotic lesions culture tests, respectively. thus, the antibiotics were replaced with meropenem and vancomycin. at the same time, electrolytes were corrected, and fluids were replenished. five days after the fascial resection, excrement was detected in the lesions of the right femoral regions (fig. 2), and mri was carried out in the rectal area because the presence of a rectal fistula was suspected. pus of 8 cm in diameter had formed in the presacral space, and it was connected to the right gluteus maximus muscle (fig. we were able to observe that the pus was discharging from a gap between the fascia resected region and the rectum (fig. performed two months earlier, a severe ulcer with edema and rubefaction had been observed in the connected region between the rectum and the sigmoid colon (fig. since radiation colitis and rectal perforation were considered at that time, we suggested that the patient undergo an ileostomy, but he refused. the rectal perforation was thought to have been caused by a conventional ulcer in the anastomosis region. in order to treat the necrotizing fasciitis radically, we recommended one more time that the patient undergo an ileostomy, but he refused again. in the bacteria cultures performed on the wound, as klebsiella pneumonia resistant to carbapenem and enterococcus faecium resistant to multiresistant acinetobacter baumannii and vancomycin were identified, the antibiotics were replaced with tigecycline. although tigecycline was administered for 16 days, 30 days after the fascial resection, the patient died due to septic shock. necrotizing fasciitis is a serious infection in which bacteria mainly invade subcutaneous soft tissues and rapidly spread out with deep fascia. however, as oppressive pain, rubefaction, and edema can also occur without necrosis of skin tissues in the early stage, differentiating between cellulitis, abscesses, etc. necrotizing fasciitis can occur anywhere in the body, and it commonly takes place in the limbs, with the pelvic limb being the most common place for necrotizing fasciitis to occur. it also takes place in abdominal walls, perianal regions, inguinal regions, and surgical wounds. rarely is a case of gas gangrene caused by basic enteropathy reported, and those that are mostly originate from the large intestine. the causes of death in necrotizing fasciitis are septicemia, abnormalities of blood clotting, acute renal failure, acute lung failure, multiple organ dysfunction, etc.. necrotizing fasciitis is classified into two types : type 1 includes streptococcus and mainly occurs in the abdominal and the inguinal regions due to a mixed infection of gram - negative rods, anaerobic bacteria, etc. type 2 is a monomicrobial infection in which serotype a streptococcus is the main bacteria ; it is unique because fasciitis is observed in the limbs. the present case was considered to be combined necrotizing fasciitis : type 1 microbiologically because the bacteria identified in the present case were anaerobic bacteria originating from the intestine and enteric bacteria and type 2 because it was found in the femoral regions. so far, very limited studies have investigated necrotizing fasciitis occurring in the femoral regions and associated with colorectal cancers. reported a case of necrotizing fasciitis in the femoral regions, complicated by an exterior pelvic abscess, in a patient with sigmoid colon cancer. in addition, highton. investigated a case of necrotizing fasciitis in the femoral regions that had been complications due to a piriformis muscle cause by a rectal - cancer perforation, and liu. reported a case of necrotizing fasciitis, caused by a rectal - cancer perforation, on both sides of the pelvic limb. although not in the femoral regions, boo. investigated a case, similar to the present case, of necrotizing fasciitis in the perineal region that had occurred after surgery and radiation therapy in a patient with colorectal cancer. if necrotizing fasciitis is to be treated, necrotic tissues should be resected extensively as early as possible and reobservation of the lesions and detachment of the necrotic tissues should be carried out repeatedly. a wide range of antibiotics against all gram - positive, gram - negative and anaerobic bacteria should be administered at the time of diagnosis. then, the antibiotics should be replaced with others that are more sensitive based upon the results of repeated bacteriologic examination. also, electrolyte imbalance should be corrected, and sufficient fluid therapy should be carried out. antimicrobial monotherapy does not lead to good results in necrotizing fasciitis because antibiotics are inhibited from penetrating into tissues due to the formation of microcirculation in the blood clots around lesions ; in this case, the necrotic tissues had to be resected completely. most cases of acute radiation colitis in the early stages are temporary and can be easily treated whereas chronic damage in the late stage occurs in 6 months, generally, several years, and even 30 years after the radiation therapy. the symptoms start with bloody diarrhea, abdominal pain, and tenesmus, and more serious problems, including ulcers, bleeding, stenosis, perforation etc. a radiation dose of more than 5,000 cgy is generally known to cause the development of digestive tract complications. besides the radiation dose, systemic diseases, including diabetes, hypertension, and cardiovascular diseases that can cause vascular lesions, may also be causes of digestive tract damage. furthermore, chemotherapy is a factor that aggravates digestive tract complications, and many anticancer drugs are known to increase the effects of radiation. in the present case, the patient, who had diabetes and rectal cancer, had damage in the digestive tract due to causes such as rectal cancer surgery, chemotherapy, and radiation therapy at a high dose (more than 5,000 cgy). also, chronic radiation proctitis had occurred in the surgical anastomotic region, and a rectal fistula had formed due to ulcer perforation, indicating that necrotizing fasciitis might have taken place. abdominal infection spreads to the femoral regions via various pathways : anterior or medial femoral infections via the psoas major muscle, obturator foramen, and femoral sheath, and exterior femoral infections via the sacrosciatic notch. in the present case, colonic contents were considered to have reached the femoral regions directly through the fistula after the rectal perforation, thereby causing necrotizing fasciitis. additionally, resistance against the antibiotics developed very quickly ; initially, enterococcus sensitive to b. thetaiotaomicron and vancomycin was identified, so the patient was treated with meropenem and vancomycin. after 2 weeks, k. pneumonia resistant to carbapenem and e. faecium resistant to multi - resistant a. baumannii and vancomycin were identified. in conclusion, the diagnosis of necrotizing fasciitis in the patient with radiation colitis was delayed in the present case because no symptoms, such as fever or rubefactions, that would have indicated the infection, existed, even though the patient had complained of pains in the femoral regions and pelvis a month earlier. therefore, if patients who receive rectal cancer surgeries and have radiation colitis claim to be experiencing pains in the pelvis or femoral regions, that the possibility of secondary complications due to rectal perforation inducing necrotizing fasciitis in the femoral regions should be considered. furthermore, if necrotizing fasciitis primarily takes place in the femoral regions without skin damage, or if intestinal microflora, enterococci, etc. are detected in the wound culturing, examinations such as abdominal computed tomography should be carried out because those organisms might possibly have originated from the intraperitoneal cavity or the intestine. | necrotizing fasciitis usually occurs after dermal injury or through hematogenous spread. to date, few cases have been reported as necrotizing fasciitis of the thigh secondary to rectal perforation in rectal cancer patients. a 66-year - old male complained of pelvic and thigh pain and subsequently developed necrotizing fasciitis in his right thigh. four years earlier, he had undergone a low anterior resection and radiotherapy due to of rectal cancer. an ulcerative lesion had been observed around the anastomosis site during the colonoscopy that had been performed two months earlier. pelvic computed tomography and sigmoidoscopy showed rectal perforation and presacral abscess extending to buttock and the right posterior thigh fascia. thus, the necrotizing fasciitis was believed to have occurred because of ulcer perforation, one of the complications of chronic radiation colitis, at the anastomosis site. when a rectal - cancer patient complains of pelvic and thigh pain, the possibility of a rectal perforation should be considered. |
study participants were members of the women s genome health study (wghs), a prospective genetic evaluation study in initially healthy u.s. study participants were health professionals who were age 45 years and older and free of major chronic disease including cancer and cardiovascular disease at study entry (19921995). information on baseline variables including medical history and dietary and lifestyle factors was self - reported on questionnaires. participants provided a baseline blood sample and consented to ongoing analyses linking blood - derived observations with risk factor profiles and disease. the study was approved by the institutional review board of brigham and women s hospital (boston, ma). genotype data were available for 22,054 women.we excluded women with missing rs8050136 genotype (n = 3) or bmi (n = 376), resulting in 21,675 women of european ancestry for this analysis. caloric information was missing for 555 women, and physical activity information was missing for one woman ; these women were included in other analyses. bmi was calculated as weight divided by the square of the height (kg / m) and used to define categories of bmi (normal 2550th, > 5075th, > 7590th, and > 90th percentiles) to evaluate the effect of a range of values of physical activity and caloric intake. statistical tests for interaction were obtained using likelihood - ratio tests that compared models with and without the interaction term. associations and interactions were also similarly examined in four combinations of physical activity and caloric intake levels in order to examine the joint effects of physical activity and caloric intake on the fto gene related risk. study participants were members of the women s genome health study (wghs), a prospective genetic evaluation study in initially healthy u.s. study participants were health professionals who were age 45 years and older and free of major chronic disease including cancer and cardiovascular disease at study entry (19921995). information on baseline variables including medical history and dietary and lifestyle factors was self - reported on questionnaires. participants provided a baseline blood sample and consented to ongoing analyses linking blood - derived observations with risk factor profiles and disease. the study was approved by the institutional review board of brigham and women s hospital (boston, ma). genotype data were available for 22,054 women.we excluded women with missing rs8050136 genotype (n = 3) or bmi (n = 376), resulting in 21,675 women of european ancestry for this analysis. caloric information was missing for 555 women, and physical activity information was missing for one woman ; these women were included in other analyses. bmi was calculated as weight divided by the square of the height (kg / m) and used to define categories of bmi (normal 2550th, > 5075th, > 7590th, and > 90th percentiles) to evaluate the effect of a range of values of physical activity and caloric intake. statistical tests for interaction were obtained using likelihood - ratio tests that compared models with and without the interaction term. associations and interactions were also similarly examined in four combinations of physical activity and caloric intake levels in order to examine the joint effects of physical activity and caloric intake on the fto gene related risk. the frequency of the fto risk - allele (a) was 0.40, and hardy - weinberg equilibrium was met (= 0.02, d.f. = 1, p = 0.88). the mean age of participants was 54.2 years, with mean bmi of 25.9 (sd 5.0) kg / m. each fto a allele was significantly associated with a mean bmi difference of + 0.52 kg / m (se 0.05 kg / m, p 0.001). there were statistically significant genotype associations with all the reported obesity phenotypes ; a / a homozygotes had higher values for all obesity phenotypes compared with heterozygotes, consistent with an additive mode of inheritance. there were statistically significant associations for fto with obesity - related traits such as hypertension and type 2 diabetes (p 0.001). there were no genotype differences in the total met - hours / week or number of hours spent walking. there were no associations with total caloric intake or intake from specific nutritional components of food (fats, carbohydrates, legumes, or fruits). associations were found for higher intake of two nutritional components (protein and cereal) with the a allele. characteristics of the study population by fto genotype values shown for variables are means (sd) or median (interquartile range : 25th to 75th percentile) and percentage. kg / m), overweight (bmi 2529.9 kg / m), and obese (bmi > 30 a / a, homozygous carriers of the a allele ; a / c, heterozygous carriers ; c / c, noncarriers. there were statistically significant effects of the fto a allele within all prespecified percentile categories of physical activity and caloric intake (fig. although there was no direct relationship between genotype and either physical activity or caloric intake (as shown in table 1), both activity and intake modified the association of genotype with obesity (significant interaction terms). specifically, larger effects of the a allele on bmi were noted in women who were less active or had higher intake (fig. 1). furthermore, the effects of activity and intake on modifying the association of fto with bmi were independent of each other, since the associations remained essentially unchanged after mutual adjustment. the statistical tests for these interactions were significant before and after adjusting for the other lifestyle factor (p before adjustment : 2550%, > 5075%, the p values within each percentile category for the association of the a allele with mean bmi (starting with 90%) were 1,679 kcal / day ; low intake, 1,679 kcal / day ; inactive, 8.8 met - hours / week ; active, > 8.8 met - hours / week. p indicates the significance of the interaction across the four categories defined by caloric intake and physical activity. p value indicates the significance of the association for having one copy of the fto a allele with bmi (continuous variable, kg / m) within each category of caloric intake and physical activity. odds ratio (or), 95% ci, and associated p value for likelihood of obesity (categorical variable, bmi 30 kg / m) per a allele within each category of caloric intake and physical activity. when bmi was categorized according to obesity status (bmi 30 kg / m), the a allele was associated with greater odds of obesity in all categories except the active / low intake participants, which was borderline significant (p = 0.07). when compared with noncarriers of the a allele, inactive / high intake women had 39% greater likelihood of obesity associated with each copy of the a allele, whereas active / low intake carriers had only 13% greater likelihood (p = 0.003). the prevalence of diabetes was low in this study (2.6%), and the overall results were very similar to the results among the subgroup of women without diabetes. among women with diabetes, significant association for fto with bmi and obesity were only seen among inactive / high intake women, with each a allele associated with a mean bmi difference of + 1.67 kg / m, p = 0.04, and an increased risk of obesity (odds ratio 1.61, 95% ci 0.962.69 ; p = 0.07). statistically significant interactions were noted for physical activity and caloric intake in relation to bmi and obesity among the subgroups of women with and without diabetes. generally similar results were obtained when physical activity and caloric intake were categorized according to u.s. federal guideline - based recommended levels of physical activity and caloric intake (9,10) instead of medians. table 3 shows odds ratios per a allele of type 2 diabetes among women stratified by combinations of activity and intake. among inactive / high intake women, each copy of the a allele was associated with significantly greater likelihood of type 2 diabetes (odds ratio 1.36, 95% ci 1.071.73) compared with noncarriers. to test whether the association of fto with diabetes may be mediated through its effect on bmi, we further adjusted the analyses in table 3 for bmi and found that the associations with diabetes became attenuated and nonsignificant. difference in risk of type 2 diabetes per fto a allele according to combinations of physical activity and caloric intake high intake, > 1,679 kcal / day ; low intake, 1,679 kcal / day ; inactive, 8.8 met - hours / week ; active, > 8.8 met - hours / week. p indicates the significance of the interaction across the four categories defined by caloric intake and physical activity. odds ratio (or), 95% ci, and associated p value for likelihood of type 2 diabetes per a allele within each category of caloric intake and physical activity. caucasian middle - aged women, variation in the rs8050136 fto genotype was associated with higher bmi, obesity phenotypes, and obesity - related conditions including hypertension and type 2 diabetes. there was statistically significant modification of this risk by lifestyle, with the largest magnitude of effect noted in women with both low levels of physical activity and high caloric intake. healthier lifestyle blunted but did not completely eliminate this associated fto - related genetic risk. the allele frequency and the modest strength of association with obesity in this study was similar to previously reported studies in populations of european descent (4,18). each a allele in this study was associated with an increase in mean bmi of + 0.52 kg / m, consistent with previously reported increases in the range of 0.40 to 0.66 kg / m (2). importantly, we found that the effect of the risk alleles ranged from as much as 1.18 kg / m per allele in inactive / high intake women to as little as 0.24 kg / m per allele in active / low intake women. the main finding of this study is that modifiable behaviors that define healthy or unhealthy lifestyles may amplify or blunt the genetic risk of increased weight associated with fto gentoype. we found interactions between fto, physical activity, and caloric intake, and combinations of these lifestyle choices. prior studies have been inconsistent in reporting associations or interactions between physical activity, caloric intake, and fto risk alleles and have not examined the combined effects of activity and intake on fto - related risk. several studies have found that the effects of fto risk alleles are accentuated by lower levels of physical activity (5,8) and blunted by physical activity (6). however, one recent study in a large cohort showed no interaction between fto risk alleles and physical activity (7). studies have been unclear as to whether increased caloric intake may be a mechanism by which fto predisposes to obesity (1921), and a recent study reported that dietary fat and carbohydrate intake modified the obesogenic effect of fto (8). the current study extends our knowledge of this topic by reporting an interaction across a combination of lifestyle choices (activity and intake) and fto genotype. the rs8050136 polymorphism is likely not causal, since other polymorphisms in the first intron of the fto gene that are in linkage disequilibrium with rs8050136 have been found to be associated with obesity phenotypes (3). it is unclear whether these changes influence fto expression or splicing, or tag another genetically mechanistic region. it should be noted that the effect of fto on bmi and type 2 diabetes is modest, and the clinical applications, if any, of genetic testing remain to be determined. at this time, lifestyle interventions that emphasize both increased physical activity and dietary interventions that help individuals obtain and maintain an ideal body weight should be recommended to all individuals (9,10,22). physical activity, caloric intake, and obesity variables were assessed by self report. however, the effect of the fto a allele on bmi in this study was similar to studies with more objective measurements of adiposity. under - reporting of energy intake and overestimation of physical activity (23,24) is a documented phenomenon among obese individuals, and misclassification of this nature would be expected to bias our results toward the null. data on energy intake were obtained from food frequency questionnaires, which may underestimate absolute energy intake. by contrast, the relative ordering of subjects (ranked by medians or percentile cut - points) has good reproducibility, as assessed by correlation coefficients of 0.6 or greater (16). we only had data on caucasian women, which may limit the generalizability of these findings. although our study is large and had detailed phenotyping and genotyping, it is cross - sectional in design. finally, the bmi variance explained by the fto allele is modest and the missing heritability is possibly explained by other common polymorphisms and rare variants. strengths of this study include the combined information on activity, dietary intake, and other risk factors in a large cohort of women that allowed for a comprehensive look at the lifestyle by gene interactions at fto with obesity. given the large number of participants in our study, we were able to examine the effect of fto across a wide range of values of activity, intake, and obesity phenotypes. in this study, we found that carriers of the rs8050136 a allele in the fto gene have a greater risk of both obesity and its related conditions including type 2 diabetes. physical activity and caloric intake had separate and additive effects on the fto - associated genetic risk. finally, this study shows that healthier lifestyle behaviors relating to caloric intake and physical activity may blunt the deleterious effects of this common genetic variant. | objectivevariation in the fat mass and obesity - associated (fto) gene is associated with obesity. the extent to which separate and combined effects of physical activity and caloric intake modify this association remains unclear.research design and methodsfto polymorphism rs8050136 was measured, and physical activity, caloric intake, and anthropometrics were self - reported in 21,675 apparently healthy caucasian women.resultsthe effect of the risk allele (a) on bmi was larger among inactive or higher intake women, with additive effects of inactivity and high intake on the associated genetic risk. specifically, each a allele was associated with mean bmi difference of + 0.73 (se 0.08) kg / m2 among inactive women (median, 8.8 met - hours / week), compared with + 0.31 (0.06) kg / m2, p 8.8 met - hours / week). similarly, each a allele was associated with mean bmi difference of + 0.65 (0.07) among high intake women (> median, 1,679 kcals / day), compared with + 0.38 (0.07) kg / m2, p = 0.005, among low intake women (1,679 kcals / day). among inactive / high intake women, each a allele was associated with mean bmi difference of + 0.97 (0.11) kg / m2 vs. + 0.22 (0.08) kg / m2 among inactive / low intake women, p < 0.0001. among inactive / high intake women, each a allele carried increased risk of obesity (odds ratio 1.39, 95% ci 1.271.52) and diabetes (odds ratio 1.36, 95% ci 1.071.73).conclusionsin this study, lifestyle factors modified the genetic risk of fto on obesity phenotypes, particularly among women who were both inactive and had high intake. healthier lifestyle patterns blunted but did not completely eliminate the associated genetic risk. |
leukodystrophies are congenital demyelinating disorders causing damage to the myelin sheath, an insulating membrane that surrounds nerve cells in the brain. there are forty different types and adrenoleukodystrophy (ald) is an x - linked inherited disorder with a prevalence of 1 in 20,000 - 50,000 individuals worldwide. people with x - linked adrenoleukodystrophy (x - ald) accumulate high levels of saturated, very long chain fatty acids (vlcfa) in the brain and adrenal cortex. the loss of myelin and the progressive dysfunction of the adrenal gland are the primary characteristics of x - ald. while nearly all patients with x - ald suffer from adrenal insufficiency, also known as addison 's disease, the neurological symptoms can begin either in childhood or in adulthood. the childhood cerebral form is the most severe, with onset between ages 4 and 10. the most common symptoms are usually behavioral changes such as abnormal withdrawal or aggression, poor memory, and poor school performance. other symptoms include visual loss, learning disabilities, seizures, poorly articulated speech, difficulty swallowing, deafness, disturbances of gait and coordination, fatigue, intermittent vomiting, increased skin pigmentation and progressive dementia. there are a couple of case reports in india on this disorder, but none relating the symptoms to attention deficit hyperactivity disorder (adhd). we are presenting this interesting case of x - ald, childhood onset, initially presenting to psychiatric outpatients department with features of adhd. a 7-year - old boy was brought to the psychiatry outpatient department by his mother. no concerns noted in the school or at home until 5 years of age. when he progressed to year one in school, problems started. teachers complained that he had been inattentive, does nt sit at one place and disruptive in the class. his scholastic performance declined, and mother started having difficult time at home as well. he showed no interest in homework, could nt remember what has been taught, more importantly started to forget what he has learnt in the past 2 years. the teachers have advised consulting a psychiatrist and that he may need a special school for further education as his behavior was unmanageable. she however tried changing the schools twice before bringing him for consultation, but his behavior problems got worse. during the assessment the boy was very impatient, constantly trying to leave the interview room, running up and down the stairs, distractible, and uncooperative. the assessment was done on two occasions, and neuropsychologist was involved who administered rating scales to screen and assess the severity of behavioral problems. the boy had hyper - pigmented lesions in his face, but it was overlooked at that point. he was started on methylphenidate 5 mg initially and titrated to 10 mg. as his behavior was very disruptive, putting himself at risk of accidental harm, he was started on risperidone 0.5 mg. two weeks later he presented with fever and an episode of generalized tonic clonic seizures. he presented again the following week with another episode of generalized tonic - clonic seizures with fever and altered consciousness. brain was done which revealed symmetrical t2-hyperintense white matter changes mainly in the posterior periventricular area. pediatric neurologist and endocrinologist were involved, plasma assay of vlcfa done and a diagnosis of ald was made. probing family history revealed similar problems in his cousin who died at the age of ten. literature search has shown a couple of case reports from united states where ald was misdiagnosed as adhd. this is the first time we hear about this rare neurological condition presenting as adhd in india. it never struck in our mind that symptoms of adhd could be secondary to another neurological disorder in this boy given the typical nature and onset of the presentation. furthermore, we overlooked the hyperpigmentation in the face initially until he was seen by endocrinologist after the mri helped with the diagnosis. the presence of facial hyperpigmentation and occurrence of tonic clonic seizures during the course of the illness should alert the clinicians look for these rare but important genetic cause. we explained the course and prognosis of the condition to the parents and given appropriate psychological support. the boy was also referred to special education and occupational therapy as part of the treatment plan. | x - linked adrenoleukodystrophy (x - ald) is one the leukodystrophies causing a progressive decline in neurological function mainly affecting the children. the most common symptoms are changes in behavior, including social withdrawal or aggression, poor memory or poor scholastic performance. here, we present a 7-year - old boy who presented with symptoms of inattention and hyperactivity and later turned out to be a case of x - ald. |
the prevalence of excessive weight and obesity (ewo) has increased worldwide, and the problem includes mexico.1)2) ewo are known independent factors in the development of heart failure3)4)5) since they lead to a cardiomyopathy state through inflammatory and hormonal mechanisms as a result of cardiac steatosis ; this refers to the pathological accumulation of triglycerides in myocytes which in turn, leads to reactive oxygen species release that foster fibrosis formation, hypertrophy and myocardial apoptosis.6) moreover, excessive accumulation of epicardial fat fosters a pro - inflammatory state.7) also, individuals with ewo develop a hyperdynamic state that in the long - run, leads to ventricular remodeling and dysfunction.8) ventricular remodeling and dilation may condition mechanical asynchrony, a marker reflecting heart failure progression in several populations.9) previous studies have documented the presence of interventricular asynchrony in female obese, diabetic patients.10) schuster.11) demonstrated the existence of intraventricular asynchrony in 10 morbidly obese individuals but that reverted after 8 weeks of physical training. echocardiography is useful in the evaluation of synchrony, both by tissue analysis (tissue and speckle tracking) as well as in its tridimensional modality. it is also a validated method in the evaluation of epicardial fat.12)13) the aim of this study was to evaluate mechanical synchrony in subjects with ewo compared with eutrophic individuals, as well as to analyze the relation between the linear quantity of epicardial fat - determined by echocardiography - and ventricular mechanical synchrony. participants with a normal physical examination, electrocardiogram and baseline echocardiogram were included. those with a history of heart disease or any other relevant pathology, receiving any type of drug or harboring an infection, were excluded ; subjects with a sub - optimal acoustic window or that refused to participate in accordance with the informed consent were also excluded. all individuals were asked to fast for 8 hours before the procedures, and this study was approved by our institutional ethics committee and all the participants gave informed consent. participants were divided into two groups according to their body mass index (bmi) : subjects with a bmi > 25 kg / m were cases and formed the ewo group while subjects with a bmi 25 excess weight was defined as a bmi > 25 but 15 mm). its thickness was measured perpendicularly on the free wall of the right ventricle at end - systole in 3 cardiac cycles at the point on the free wall of the right ventricle at which the ultrasound beam is oriented in a perpendicular manner, using the aortic annulus as an anatomic landmark. for midventricular parasternal short - axis assessment, maximum epicardial fat thickness was measured from 2d images on the right ventricular free wall along the midline of the ultrasound beam perpendicular to the interventricular septum at mid chordal and tip of the papillary muscle level, as anatomic landmarks.13) according to their distribution, continuous variables were expressed as means and standard deviations or medians and minimum - maximum intervals ; comparisons were obtained with student 's t test or mann - whitney 's u test, respectively. the relation between variables was analyzed with pearson 's coefficient and potential confusers, by multiple lineal regression. weight (w) : obtained in a single well - calibrated scale while only wearing a hospital gown. height (h) : measured with the patient barefoot, with his back to the stadiometer. the abdominal perimeter was measured at the mid - point between the inferior costal margin and the iliac crest. hip circumference was measured in three points : posterior (maximum gluteal extension), anterior (at the pubic level), and lateral (at the level of the femoral greater trochanters). an ie33 echocardiography machine (philips, andover, ma, usa), was used, with a 5x matrix transducer and q - lab version 9.0. based on the guidelines,14) interventricular (vv) synchrony evaluation : it was studied with pulsed doppler of the outflow tracts ; it is defined as the time difference in onset of flow between both ventricles and is determined as the time period from the initiation of the qrs complex to the initiation of left ventricular flow onset vs. that of the right ventricle. atrio - ventricular (av) synchrony, the percentage of time occupied by ventricular filling in the cardiac cycle, was also evaluated with pulsed doppler of the mitral valve. intraventricular synchrony, the analysis of the regional time periods of ventricular mechanical activation, was measured by color - coded tissue characterization, following the recommendations of a consensus of experts.15) peak systolic velocity, time between the electrocardiographic q wave and the systolic peak (q - s time), and the time between the electrocardiographic q wave and the beginning of the e ' wave, were measured in the basal and mid - segments of 4, 3, and 2 chambers (total of 12 segments). the standard deviation of the q - s times in the 12 segments was obtained in order to calculate the yu index.16) longitudinal strain was obtained in bidimensional images of the same projections. the systolic synchrony index (ssi) was calculated on the basis of the threedimensional (3d) complete volume acquisition that is, the analysis of time dispersion vis visa minimal regional volume of 16 segments. standard parasternal long - axis and short - axis views from two - dimensional (2d) images were obtained. epicardial fat was identified as the relatively echo - free space between the outer wall of the myocardium and the visceral layer of pericardium ; epicardial fat thickness can be also appear as hyperechoic space, if in large amount (> 15 mm). its thickness was measured perpendicularly on the free wall of the right ventricle at end - systole in 3 cardiac cycles at the point on the free wall of the right ventricle at which the ultrasound beam is oriented in a perpendicular manner, using the aortic annulus as an anatomic landmark. for midventricular parasternal short - axis assessment, maximum epicardial fat thickness was measured from 2d images on the right ventricular free wall along the midline of the ultrasound beam perpendicular to the interventricular septum at mid chordal and tip of the papillary muscle level, as anatomic landmarks.13) according to their distribution, continuous variables were expressed as means and standard deviations or medians and minimum - maximum intervals ; comparisons were obtained with student 's t test or mann - whitney 's u test, respectively. the relation between variables was analyzed with pearson 's coefficient and potential confusers, by multiple lineal regression. a total of 55 subjects were included in the study ; 17 (30.9%) were controls (bmi < 25 kg / m) and 38 (69.09%) were cases (bmi 25 kg / m). the average age of the case group was 30 and that of controls, 26 (p = 0.017). table 1 summarizes anthropometric measurements, past history and the main findings in the baseline electrocardiogram and echocardiogram. epicardial fat was greater in the ewo group, p = 0.001. left atrial volume was different in both groups, also being greater in the ewo group, p = 0.001. furthermore, the systolic and diastolic ventricular volumes as well as the baseline right ventricular diameter were also greater in the ewo group. using conventional criteria in the evaluation of synchrony (responders criteria), we found neither intraventricular asynchrony according to yu 's index, no ssi, and no av nor vv asynchrony in either group (table 1). there was a correlation between the amount of epicardial fat and the bmi, r = 0.78, p < 0.001. the relationship between epicardial fat and yu 's index was r = 0.53, p < 0.001 (fig. 1). finally, we established that the greater the bmi, the greater the ssi, r = 0.48, p < 0.001 (fig. inter - observer reproducibility was evaluated between the two echocardiographists participating in the study : there was good concordance, with an intra - class correlation coefficient of 0.75, in the determination of peak systolic velocity, 0.86 in the q - s time estimates, 0.97 in the ssi and 0.88 in the 3d left ventricular ejection fraction. bland - altman analysis of yu 's index proved that measurements by both echocardiographists were similar (difference of -1.81 ms, p = 0.289). this study, for the first time, demonstrates that there is an association between the bmi, the amount of epicardial fat and intraventricular mechanical asynchrony. since both epicardial fat and bmi related to the time dispersion of the ventricular contraction as measured with yu 's index, this suggests that the greater the amount of epicardial fat and bmi, the greater the ventricular asynchrony. this supports the theory that ventricular asynchrony is another pathophysiological mechanism that may contribute to the ventricular dysfunction of obesity cardiomyopathy. other authors have reported that vv asynchrony correlates with the amount of body fat.17) intraventricular asynchrony was described in a study previous to ours, in 2 patients with morbid obesity and it reverted after weight loss.18) in kramer 's study in mice, obesity was shown to cause intraventricular asynchrony and decrease strain and torsion.19) the heart 's electrical activity is a quick process that occurs in the first 40 milliseconds and is followed by the synchronic mechanical activation of all myocardial segments. pathological entities inducing abnormalities in its structure and function may lead to dispersion in the timing of ventricular contraction reflecting the fact that some ventricular regions contract earlier while others do so later within the same cardiac cycle.20) in ewo, there are structural myocardial changes resulting from the excessive intracellular accumulation of free and intermediate fatty acids such as ceramides, both toxic and contributors to cardiomyocyte death through oxidative and non - oxidative pathways ; they also generate fibrosis.7)21)22) therefore, the amount of intra - myocardial lipids leading to cardiac steatosis contributes to cardiac dysfunction.23)24)25) obesity also affects the hemodynamic behavior of the heart8) by increasing the total circulating volume ; obese individuals thus have an increased cardiac output.8)26) this study found that patients with ewo have significantly larger intra - cavitary volumes when compared with controls. the amount of adipocytes within myofibrils is associated with a greater ventricular mass23)27) but our study did not reveal greater myocardial mass ; this may result from the fact that most of our participants only had excess weight while in other studies, obese individuals were predominant. this suggests an evolutionary sequence of events in obese cardiomyopathy in which the first step is an increase in cavity volumes followed by an increase in ventricular mass ; this second step could well be an advanced marker of pathological ventricular remodeling. krishnan.28) analyzed this same concept and concluded that cavity dilation in conjunction with an increased ventricular mass are part of a sequence of events that culminate in ventricular dysfunction. an early sign of this ventricular dysfunction is the presence of abnormalities in the deformation and the longitudinal strain rate;29)30) our findings are in agreement since we detected a decrease in the myocardial longitudinal strain properties in our ewo group. the mechanisms by which obesity leads to heart failure are multiple, ranging from hemodynamic, metabolic and inflammatory abnormalities to the direct toxic effect of adipocytes on the myocardium.7)31)32) we can now further suggest that intraventricular asynchrony is another implicated mechanism. finally, we must emphasize that this study did not include patients with associated confusers such as hypertension or diabetes, among others, so the observed differences are explained by ewo, and underscore the impact of these diseases on the myocardium. the study was primarily conducted in subjects with excess weight and there are few obese individuals ; this results from the difficulty in recruiting obese subjects with no associated comorbidities. the cohort number needs to be increased to confirm these observations and understand the long - term impact of our results. intraventricular mechanical asynchrony is associated to both ewo and the lineal amount of epicardial fat as measured by echocardiography ; hence, intraventricular asynchrony may be one more pathophysiological component implicated in the evolution to heart failure in the ewo population. the study was primarily conducted in subjects with excess weight and there are few obese individuals ; this results from the difficulty in recruiting obese subjects with no associated comorbidities. the cohort number needs to be increased to confirm these observations and understand the long - term impact of our results. intraventricular mechanical asynchrony is associated to both ewo and the lineal amount of epicardial fat as measured by echocardiography ; hence, intraventricular asynchrony may be one more pathophysiological component implicated in the evolution to heart failure in the ewo population. | backgroundexcessive weight and obesity (ewo) are independent factors in the development of heart failure ; they lead to a state of myocardiopathy via inflammatory and hormonal mechanisms. if excessively accumulated, epicardial fat favors a proinflammatory state. ventricular asynchrony is a marker of heart failure progression and has been poorly studied in ewo. the objective was evaluate the relation between epicardial fat, body mass index (bmi) and mechanical synchrony measured by echocardiography, in healthy individuals with ewo.methodswe included 55 healthy individuals between the ages of 18 and 35, 17 had a bmi 25 kg / m2 (ewo group) (69.09%), anthropometric measurements, transthoracic echocardiogram and synchrony evaluation were obtained.resultsleft atrial volume, telediastolic and telesystolic left ventricular volumes and the baseline volume of the right ventricle were greater in the ewo group (20 ml / m2 vs. 15 ml / m2, p = 0.001 ; 106 ml vs. 82 ml, p = 0.0149 vs. 32 ml, p = 0.001 and 34 mm vs. 31 mm, p = 0.02, respectively). the yu index also correlated with epicardial fat, r = 0.53, p < 0.01, whereby the greater the amount of epicardial fat, the greater the dispersion timing of ventricular activation. the systolic synchrony index also correlated with the bmi, p = 0.01.conclusionmechanical intraventricular asynchrony is associated to ewo and the amount of epicardial fat ; hence, asynchrony may be one more factor leading to heart failure in ewo individuals. |
about 70% of stroke patients experience altered arm function, and about 40% are left with a nonfunctional arm.1 arm weakness limits the individual s activities of daily living and impacts negatively on quality of life.2 the greatest recovery in upper limb function is achieved in the 6 months following a stroke, and this period offers the best opportunity for rehabilitation interventions. the main predictor of success in physical therapy is the amount of therapy undertaken, and guidelines for patients recommend high intensity repetitive motions.3 rehabilitative therapy has focused on practicing normal movements to encourage return of relevant function and inhibit compensatory habits that may prevent further recovery. more recently, there has been an emphasis on strengthening, practice of functional tasks, and forcing use of the weak limb to complete everyday activities using constraint - induced movement therapy.46 the national stroke guidelines endorse 45 minutes of daily physiotherapy in the rehabilitation phase of stroke recovery.1 physiotherapy, however, is both labor - intensive and expensive, and service provision varies widely. at present, only 50% of people with stroke receive the rehabilitation to meet their needs during the crucial first 6 months.1 furthermore, although therapists prescribe a home exercise regimen, only about 31% of patients complete the recommended exercise.7 rehabilitation after stroke using virtual reality therapy is of increasing interest.811 a cochrane review on the effect of virtual reality on arm function found limited evidence that it may be beneficial in improving arm function and activities of daily living when compared with the same dose of conventional therapy. the authors conclusion was that present studies were too small and too few to draw conclusions and more randomized controlled trials (rcts) were needed.12 nintendo wii sports (wii) is commercially available and offers task specific training that is repetitive and motivating13,14 and may be a useful adjunct to rehabilitation.1517 wii games include tennis, golf, boxing, baseball, and bowling activities, which are played using a remote control held in the same way that real world objects such as the handle of a racket are held and manipulated. the research into its efficacy is limited, although it is being used by stroke therapists.11 pilot studies have suggested faster task performance following use of wii, with no wii-related side effects or injuries.16,17 the trial of wii in stroke (twist) is a multicenter, pragmatic, parallel group, rct with blinded outcome assessment, incorporating a qualitative study and cost - effectiveness analysis (table 1). the primary outcome in the rct is arm function as measured by the action research arm test (arat) at 6 weeks.18 the arat assesses ability to handle objects and tests grasp, grip, pinch, and gross movement of elbow and shoulder of the affected arm.19,20 secondary outcome measures include occupational performance, stroke impact, and severity. the canadian occupational performance measure (copm) is used to detect change in self - perception of occupational performance over time.21 the stroke impact scale (sis) assesses change in impairments, disabilities, and handicaps following a stroke.22 the modified rankin scale (mrankin) measures disability following stroke.23 these outcome measures will be applied at baseline, 6 weeks, and 6 months. arm function will be assessed at 6 months using the arat and motor activity log-14 (mal14). the mal14 ascertains how well the participant has performed day to day tasks using their affected arm.24,25 participants will be asked to complete a diary to capture a daily record of duration of arm exercises and adverse events. the diary is brief and enables the participant or carer to record daily exercise time, adverse events, and visits from the national health service (nhs), private carers, and social services staff. baseline data collection will include demographic data, type of stroke using the oxfordshire community stroke project classification,26 affected arm strength using the medical research council scale of muscle strength,27 and cognitive function using the montreal cognitive assessment.28 outcome measures will be purchased (arat, copm) or permission to use in study will be obtained from authors (mal14, eq-5d 3l, mrankin, and sis). the qualitative study will explore participants and carers experiences of using the wii and barriers to its implementation. the health economic analysis will estimate the incremental cost per quality - adjusted life year (qaly) gained from the cost perspective of health care and personal social services. a total of 240 participants will be recruited to the rct across ten sites in the uk by the local research practitioners. once participants have given written informed consent, outcome measures will be assessed at baseline, 6 weeks, and 6 months. a research therapist will either install the wii in the participant s home and instruct them on the use of the wii, or will advise the participant on tailored arm exercises to be performed during the 6-week intervention phase. all participants will be instructed to perform warm - up exercises prior to tailored arm exercises or wii exercises to minimize the risk of injury. the research therapist will visit the patient prior to the 6-week assessment to collect participant s diary and wii if applicable. the research therapist will remind participants not to disclose their treatment allocation during the subsequent assessment visits by the research practitioner. participants will be telephoned by a non - blinded member of the local research team on a weekly basis to encourage them to complete their diary and enable them to express any concerns they may have had about the study. all research staff will be trained at site initiation in all outcome measures use of the trial website, case report form (crf) completion and safety considerations. eligible participants with arm weakness following stroke will be randomized to one of two groups. the intervention group will be instructed to undertake up to 45 minutes of exercise, in a seated position, using the wii game daily for 6 weeks at home. the wii console and game will be provided on loan to trial participants as part of the study for the duration of the intervention period. the control group will be instructed to undertake up to 45 minutes of tailored arm exercises, in a seated position, daily for 6 weeks at home. both groups will continue to receive usual care and rehabilitation as provided by their services in the area. allocation to intervention will be determined by use of a web - based randomization service, accessed by the research therapist prior to the setup visit. allocation will be stratified by center, baseline arat score (scores 049 and 5057), and previous use of a wii. the research team will visually inspect the wii and clean with antiseptic wipes prior to allocation to the next participant in the intervention group. serious adverse events will be reported by the site s principal investigator (pi) to the clinical trials unit (ctu) and reported on a monthly basis to the trial steering committee (tsc), chief investigator (ci), and sponsor. minor adverse events thought not to be related to the trial will not be reported. for the primary outcome of arat score at 6 weeks, the literature suggests a standard deviation of about 15 and that a clinically important effect is six points.29 for a two - tailed t - test at the 5% level and with 80% power, completed data are required on 99 patients per group. to allow for attrition a statistical analysis plan will be prepared by the study statistician, in advance of any statistical analysis of the study data. participants choosing to withdraw from either intervention arm will be followed up where possible, and reasons for withdrawal, if elicited, will be reported according to the consort (consolidated standards of reporting trials) guidelines.30 baseline characteristics will be summarized using suitable descriptive statistics. the principal analysis will be analysis of covariance with terms for treatment group and stratification variables. a further analysis will replace baseline arat score group (as stratified) by the actual arat scores in order to explore the robustness of conclusions. in each case, least - squares mean treatment differences and their 95% confidence intervals will be reported. both analyses will be repeated for the secondary outcome measure of arat at 6 months, and other continuous outcome measures. the interactions between treatment group and the stratification variables will be examined and allowed for in the analyses as appropriate. all audio recordings will be transcribed verbatim, and the transcribed interviews will be analyzed using thematic analysis.31 the field notes and observation data obtained during the interviews will be included in the analysis process. themes will be drawn from the data using constant comparison methods ; participants accounts will be compared and contrasted to enhance interpretation. if data are unclear, findings will be presented to participants to allow further comment in order to aid interpretation. the qualitative researcher will use a computer - aided program to manage the data. for trustworthiness, the lead qualitative researcher will analyze a subset of five transcripts. any unresolved disagreements will be resolved by discussion with the ci, ensuring that data will be presented in such a way as to maintain blinding of treatment group. the economic evaluation will estimate the incremental cost per qaly gained from the perspective of health and social care utilization. the costs of the wii equipment will be the price paid annuitized over a useful life of 3 years assuming the equipment could be used by eight clients per year. the main source of information on resource use will be the participant diaries which will include prompts derived from the service receipt inventory (sri).32 the sri will also be used retrospectively at 6 months. when an assessment of need is completed during the trial, details of agreed care package will be recorded. unit costs of health and social care services will be taken from standard published sources.33,34 health - related quality of life will be assessed by eq-5d 3l35 and valued using the york tariff.36 the economic evaluation will provide within - trial estimates of the incremental cost - effectiveness ratio. sampling variation will be analyzed using probabilistic sensitivity analysis, reported as a scatterplot on the cost - effectiveness plane. sensitivity analysis to estimate the effects of parameter uncertainty will include an exploration of the sensitivity of results to differences in the costs and effects of different provisions of rehabilitation services. data will be recorded on study - specific data - collection forms (crfs). persons authorized to collect and record trial data at each site will be listed on the study site delegation log and authorized by the pi. completed crfs will be checked and signed at the research site by the research practitioner or research therapist (as applicable) before being sent to the ctu for double - data entry onto a password - protected database accessed via the internet. the ci will be responsible for the overall running of the study and will be in regular contact with the principle investigators at the other trial centers. each trial center will have a pi, a research administrator, a research practitioner, and a research therapist to conduct this study. the ctu will coordinate trial activities and assist with overall trial management and monitoring. a data - monitoring committee will not be convened due to the nature of the intervention and short duration of the trial. the trial - management group (tmg) will include the ci, ctu trial coordinator, trial statistician, and other members of the team and will meet regularly to monitor progress. the tsc will include a representative from the funder and a lay person. early on in the recruitment phase, these were 1) that a proportion of patients were assessed as having the highest possible arat score at baseline and 2) the slow recruitment rate. further to this, the research therapists asked that patients with severe shoulder subluxation be excluded, as pain precluded participants from playing the wii or doing arm exercises. in response to these concerns, the tsc recommended baseline arat stratification for randomization was changed from 30 to 50 points and the mal14 be added as a secondary outcome measure at 6 months follow - up as a second outcome measure for arm function. the tsc also recommended some changes to the eligibility criteria which were implemented by the tmg : to exclude patients with symptomatic shoulder subluxation, to broaden the inclusion criteria to include patients with non - dominant weakness, and to remove the requirement for participants to have supervision during their exercises. finally, the number of participating centers was increased from seven to ten, and the tmg applied to the funder for an extension to the recruitment period in order to maximize the chances that the trial could recruit the full sample size. all changes to the protocol were approved by the sponsor, the research ethics committee, and the funder. the primary outcome in the rct is arm function as measured by the action research arm test (arat) at 6 weeks.18 the arat assesses ability to handle objects and tests grasp, grip, pinch, and gross movement of elbow and shoulder of the affected arm.19,20 secondary outcome measures include occupational performance, stroke impact, and severity. the canadian occupational performance measure (copm) is used to detect change in self - perception of occupational performance over time.21 the stroke impact scale (sis) assesses change in impairments, disabilities, and handicaps following a stroke.22 the modified rankin scale (mrankin) measures disability following stroke.23 these outcome measures will be applied at baseline, 6 weeks, and 6 months. arm function will be assessed at 6 months using the arat and motor activity log-14 (mal14). the mal14 ascertains how well the participant has performed day to day tasks using their affected arm.24,25 participants will be asked to complete a diary to capture a daily record of duration of arm exercises and adverse events. the diary is brief and enables the participant or carer to record daily exercise time, adverse events, and visits from the national health service (nhs), private carers, and social services staff. baseline data collection will include demographic data, type of stroke using the oxfordshire community stroke project classification,26 affected arm strength using the medical research council scale of muscle strength,27 and cognitive function using the montreal cognitive assessment.28 outcome measures will be purchased (arat, copm) or permission to use in study will be obtained from authors (mal14, eq-5d 3l, mrankin, and sis). the qualitative study will explore participants and carers experiences of using the wii and barriers to its implementation. the health economic analysis will estimate the incremental cost per quality - adjusted life year (qaly) gained from the cost perspective of health care and personal social services. a total of 240 participants will be recruited to the rct across ten sites in the uk by the local research practitioners. once participants have given written informed consent, outcome measures will be assessed at baseline, 6 weeks, and 6 months. a research therapist will either install the wii in the participant s home and instruct them on the use of the wii, or will advise the participant on tailored arm exercises to be performed during the 6-week intervention phase. all participants will be instructed to perform warm - up exercises prior to tailored arm exercises or wii exercises to minimize the risk of injury. written instructions for both groups the research therapist will visit the patient prior to the 6-week assessment to collect participant s diary and wii if applicable. the research therapist will remind participants not to disclose their treatment allocation during the subsequent assessment visits by the research practitioner. participants will be telephoned by a non - blinded member of the local research team on a weekly basis to encourage them to complete their diary and enable them to express any concerns they may have had about the study. all research staff will be trained at site initiation in all outcome measures use of the trial website, case report form (crf) completion and safety considerations. eligible participants with arm weakness following stroke will be randomized to one of two groups. the intervention group will be instructed to undertake up to 45 minutes of exercise, in a seated position, using the wii game daily for 6 weeks at home. the wii console and game will be provided on loan to trial participants as part of the study for the duration of the intervention period. the control group will be instructed to undertake up to 45 minutes of tailored arm exercises, in a seated position, daily for 6 weeks at home. both groups will continue to receive usual care and rehabilitation as provided by their services in the area. allocation to intervention will be determined by use of a web - based randomization service, accessed by the research therapist prior to the setup visit. allocation will be stratified by center, baseline arat score (scores 049 and 5057), and previous use of a wii. the research team will visually inspect the wii and clean with antiseptic wipes prior to allocation to the next participant in the intervention group. serious adverse events will be reported by the site s principal investigator (pi) to the clinical trials unit (ctu) and reported on a monthly basis to the trial steering committee (tsc), chief investigator (ci), and sponsor. minor adverse events thought not to be related to the trial will not be reported. for the primary outcome of arat score at 6 weeks, the literature suggests a standard deviation of about 15 and that a clinically important effect is six points.29 for a two - tailed t - test at the 5% level and with 80% power, completed data are required on 99 patients per group. to allow for attrition a statistical analysis plan will be prepared by the study statistician, in advance of any statistical analysis of the study data. participants choosing to withdraw from either intervention arm will be followed up where possible, and reasons for withdrawal, if elicited, will be reported according to the consort (consolidated standards of reporting trials) guidelines.30 baseline characteristics will be summarized using suitable descriptive statistics the principal analysis will be analysis of covariance with terms for treatment group and stratification variables. a further analysis will replace baseline arat score group (as stratified) by the actual arat scores in order to explore the robustness of conclusions. in each case, least - squares mean treatment differences and their 95% confidence intervals will be reported. both analyses will be repeated for the secondary outcome measure of arat at 6 months, and other continuous outcome measures. the interactions between treatment group and the stratification variables will be examined and allowed for in the analyses as appropriate. all audio recordings will be transcribed verbatim, and the transcribed interviews will be analyzed using thematic analysis.31 the field notes and observation data obtained during the interviews will be included in the analysis process. themes will be drawn from the data using constant comparison methods ; participants accounts will be compared and contrasted to enhance interpretation. if data are unclear, findings will be presented to participants to allow further comment in order to aid interpretation. the qualitative researcher will use a computer - aided program to manage the data. for trustworthiness, the lead qualitative researcher will analyze a subset of five transcripts. any unresolved disagreements will be resolved by discussion with the ci, ensuring that data will be presented in such a way as to maintain blinding of treatment group. the economic evaluation will estimate the incremental cost per qaly gained from the perspective of health and social care utilization. the costs of the wii equipment will be the price paid annuitized over a useful life of 3 years assuming the equipment could be used by eight clients per year. the main source of information on resource use will be the participant diaries which will include prompts derived from the service receipt inventory (sri).32 the sri will also be used retrospectively at 6 months. when an assessment of need is completed during the trial, details of agreed care package will be recorded. unit costs of health and social care services will be taken from standard published sources.33,34 health - related quality of life will be assessed by eq-5d 3l35 and valued using the york tariff.36 the economic evaluation will provide within - trial estimates of the incremental cost - effectiveness ratio. if appropriate, modelling beyond the term of the trial will be undertaken. sampling variation will be analyzed using probabilistic sensitivity analysis, reported as a scatterplot on the cost - effectiveness plane. sensitivity analysis to estimate the effects of parameter uncertainty will include an exploration of the sensitivity of results to differences in the costs and effects of different provisions of rehabilitation services. data will be recorded on study - specific data - collection forms (crfs). persons authorized to collect and record trial data at each site will be listed on the study site delegation log and authorized by the pi. completed crfs will be checked and signed at the research site by the research practitioner or research therapist (as applicable) before being sent to the ctu for double - data entry onto a password - protected database accessed via the internet. the ci will be responsible for the overall running of the study and will be in regular contact with the principle investigators at the other trial centers. each trial center will have a pi, a research administrator, a research practitioner, and a research therapist to conduct this study. the ctu will coordinate trial activities and assist with overall trial management and monitoring. a data - monitoring committee will not be convened due to the nature of the intervention and short duration of the trial. the trial - management group (tmg) will include the ci, ctu trial coordinator, trial statistician, and other members of the team and will meet regularly to monitor progress. early on in the recruitment phase, the tmg reported two emerging issues of concern to the tsc. these were 1) that a proportion of patients were assessed as having the highest possible arat score at baseline and 2) the slow recruitment rate. further to this, the research therapists asked that patients with severe shoulder subluxation be excluded, as pain precluded participants from playing the wii or doing arm exercises. in response to these concerns, the tsc recommended baseline arat stratification for randomization was changed from 30 to 50 points and the mal14 be added as a secondary outcome measure at 6 months follow - up as a second outcome measure for arm function. the tsc also recommended some changes to the eligibility criteria which were implemented by the tmg : to exclude patients with symptomatic shoulder subluxation, to broaden the inclusion criteria to include patients with non - dominant weakness, and to remove the requirement for participants to have supervision during their exercises. finally, the number of participating centers was increased from seven to ten, and the tmg applied to the funder for an extension to the recruitment period in order to maximize the chances that the trial could recruit the full sample size. all changes to the protocol were approved by the sponsor, the research ethics committee, and the funder. twist is the first uk rct assessing the feasibility, cost effectiveness, and acceptability of wii in stroke rehabilitation. | introductionmany stroke patients experience loss of arm function requiring rehabilitation, which is expensive, repetitive, and does not always translate into real life. nintendo wii sports (wii) may offer task - specific training that is repetitive and motivating. the trial of wii in stroke (twist) is designed to investigate feasibility, efficacy, and acceptability using wii to improve affected arm function for patients after stroke.methodthis is a randomized controlled trial (rct), incorporating a qualitative study and health economics analysis that compares playing wii versus arm exercises in patients receiving standard rehabilitation in a home setting within 6 months of stroke with a motor deficit of less than 5 on the mrc (medical research council) scale (arm). in this study, we expect to randomize 240 participants.outcome measuresprimary outcome is change in affected arm function at 6 weeks follow - up in intervention and control group using the action research arm test. secondary outcomes include occupational performance using the canadian occupational performance measure, quality of life using the stroke impact scale, cost effectiveness analysis, and a qualitative study investigating factors that influence use of wii for patients and carers.conclusiontwist is the first uk rct assessing the feasibility, cost effectiveness, and acceptability of wii in stroke rehabilitation. the trial has been registered with isrctn 06807619 and uk crn 11030. results of the study will be published after completion of study in august 2014. |
posterior reversible encephalopathy syndrome (pres) has been well reported over the years since it was first described by hinchey in 1996.1 the typical clinical signs consist of various associations of seizure activity, consciousness impairment, headaches, visual abnormalities, and focal neurological signs, as well as radiologic findings of focal vasogenic edema involving both the grey and white matter. however, the syndrome is most commonly reported in association with acute hypertension, pre - eclampsia, eclampsia, renal disease, sepsis, and exposure to immunosuppressants.25 here, we report a case of pres in a 62-year - old well controlled hypertensive lady who suffered an inadvertent dural puncture while undergoing an emergency laparotomy for ischemic colitis. to the best of our knowledge, this is the first case study of pres in a surgical patient with an accidental dural puncture. a 62-year - old woman presented acutely with severe sudden onset left - sided abdominal pain and several episodes of diarrhea with blood mixed in with stool. her past medical history included coeliac disease and hypertension that was well controlled with ramipril 10 mg daily. on admission, observations were : temperature 36.4c, blood pressure 115/50 mmhg, heart rate 75 bpm, respiratory rate 16, and oxygen saturations of 99% breathing on air. examination revealed localized peritonitis of the left abdomen, and rectal examination showed altered blood with no stool. a computed tomography (ct) scan of her abdomen and pelvis showed an appearance consistent with colitis involving the transverse and sigmoid colon, the distribution suggesting ischemic colitis. flexible sigmoidoscopy the following day showed an ischemic splenic flexure, and the decision was made for the patient to undergo a laparotomy. a low thoracic epidural was attempted for postoperative pain relief ; however, the procedure was abandoned after one attempt due to a dural tap. consequently, the patient had general anesthesia with systemic opioid analgesia and a rectus sheath block. intraoperative findings revealed an ischemic left colon and proximal sigmoid colon ; therefore, she underwent a hartmann s procedure. intraoperatively, the patient remained hemodynamically stable, with an average blood pressure of 120/60 mmhg. postoperatively, the patient was commenced on a morphine patient - controlled analgesia for pain relief and was anti - coagulated for the ischemic colitis. investigations towards underlying conditions likely to have caused the ischemic colitis were also carried out. screening for presence of the anti - phospholipid antibody, jak2 mutation, and thrombophilia proved negative. her blood pressure rose from an average of 150/80 mmhg on postoperative day 1 to 190/80 mmhg on postoperative day 3, despite remaining on her usual dose of ramipril. on postoperative day 3, the patient complained of a frontal headache and cloudy vision. the headache and rising blood pressure was initially thought to be due to postoperative pain and dural tap ; on postoperative day 3, she was commenced on a second antihypertensive, amlodipine. on postoperative day 4, headache, severe hypertension, and worsening vision continued. neurological examination revealed no focal abnormality ; however, fundoscopy showed blurred discs but no retinal hemorrhages or exudates. the patient was commenced on bisoprolol and underwent an urgent ct head scan (figure 1). the ct head scan revealed bilateral areas of low attenuation in the left occipital lobe and to a lesser extent in the right occipital lobe. there were also some patchy areas of low attenuation in the right and left cerebellar hemispheres. a subsequent magnetic resonance imaging scan of the head and magnetic resonance venography (figure 2) showed patchy white matter edema most markedly in the occipital lobes but extending anteriorly to reach the frontal lobes, consistent with pres. despite these findings the patient was loaded with intravenous phenytoin, intubated, and blood pressure was tightly controlled with a mean arterial pressure of 85 mmhg. blood pressure continued to be tightly controlled with regular antihypertensives, and she remained on phenytoin to prevent further seizures. the patient continued to improve and was discharged 21 days postoperatively with blood pressure controlled (systolic readings of 120130 mmhg) on her usual dose of ramipril plus 10 mg amlodipine daily. the only residual neurological defects that she experienced on discharge were minor visual disturbances and memory problems. pres is a clinico - neuroradiological entity initially described by hinchey in 1996.1 the typical clinical signs consist of headache, visual disturbances, seizures, and altered mental state, which are associated with distinctive neuroimaging findings of posterior cerebral white matter edema. most reported cases have been recognized to be in association with acute hypertension, immunosuppressive therapies, sepsis, renal failure, pregnancy, and systemic lupus erythematous.25 the pathophysiology of pres continues to be debated ; however, regardless of the mechanism of initiation, it involves the development of edema in affected areas of the brain.6 two contradicting main theories, resulting in vasogenic cerebral edema as a result of disruption to the blood brain barrier from cerebral blood perfusion disturbances, have been proposed. one involves impaired cerebral autoregulation leading to an increase in cerebral blood flow, and the other involves endothelial dysfunction with cerebral hypoperfusion from vasoconstriction.7 pres is usually reversible ; however, it can lead to irreversible neurological damage or even death as a result of progressive cerebral edema, or intracerebral hemorrhage.6,8 treatment for pres by symptom control and resolution of the underlying implicating factor is critical as soon as the condition is recognized, to avoid the risk of permanent neurological insult.9 our patient had onset of a frontal headache without postural features 3 days postoperatively following an emergency laparotomy with an inadvertent dural tap during anesthesia and a postoperative ileus. she was noted to be hypertensive, with a mean arterial pressure of > 120 mmhg at the same time. these symptoms were thought to be due to a post - dural tap headache, malabsorption of antihypertensive medication due to a postoperative ileus and uncontrolled pain. alfery stated that the diagnosis of a post - dural headache should not be formed unless a headache with postural features is present. in this scenario, a diagnosis of pres was not suspected and was only established in this case following neuroimaging. we acknowledge that there is a degree of overlap between the diagnosis of pres and hypertensive encephalopathy, and perhaps blood pressure should have been controlled more aggressively with intravenous blood pressure agents from the start due to malabsorption of oral medications due to a postoperative ileus. however, did uncontrolled hypertension lead to pres ? or, did pres lead to hypertension ? our patient started to complain of headaches prior to changes in her blood pressure, and fundus examination did not reveal retinal hemorrhages and exudates, which are characteristic of hypertensive encephalopathy,11 therefore swaying to a diagnosis of pres leading to uncontrolled hypertension. the occurrence of pres following epidural or spinal anesthetic has been described before in patients with uncontrolled preoperative hypertension, parturient patients, or those who have received chemotherapy.1216 could pres have been attributed to the dural puncture, and should it be considered as a differential diagnosis of post - dural puncture headache ? in 1956, prior to when pres was first described, vandam and dripps17 reported symptoms of visual disturbances such as diplopia or cortical blindness after dural puncture. a possible theory leading to the development of pres following a post - dural puncture headache is through the monro - kellie hypothesis, leading to cerebral hyperperfusion and disruption of the autoregulatory effect of the blood brain barrier, culminating in cerebral vasogenic edema.18 following a dural puncture, the rate of cerebrospinal fluid (csf) loss is greater than that of csf production. the loss of csf results in intracranial hypotension leading to vasodilation of cerebral blood vessels as a compensatory mechanism (monro - kellie hypothesis) to increase cerebral blood volume. magnetic resonance imaging scans performed in the presence of a post - dural puncture headache frequently reveal sagging of the intracranial structures and may demonstrate meningeal enhancement.19 the meningeal enhancement is thought to be characteristic of vasodilation of thin - walled vessels in response to the intracranial hypotension.19 the white matter edema in patients with pres is usually localized in posterior areas. this might be explained by heterogeneity of the autonomic innervation of the intracranial arterioles, with less nerve representation around the vessels in the posterior cerebral circulation, which are therefore more vulnerable to the effects of hyperperfusion.20 here we describe the management of a well - controlled hypertensive patient who developed pres following an inadvertent dural puncture during an emergency laparotomy for ischemic colitis. we are unable to give the exact pathogenesis of pres in this case, but feel that preexisting hypertension and an inadvertent dural tap are the main contributing factors resulting in cerebral vasogenic edema. we believe that the awareness of pres is essential to all specialties, especially anesthesiology, surgery, and obstetrics, due the nonspecific clinical scenario developed in patients with pres. it is important to be familiar with this syndrome as well as the possible causative factors, including dural taps, in order to establish early diagnosis and treatment of this condition and reduce adverse outcomes. | posterior reversible encephalopathy syndrome (pres) is a clinico - neuroradiological syndrome characterized by various symptoms of neurological disease. it has commonly been reported in association with acute hypertension, pre - eclampsia, eclampsia, sepsis, and exposure to immunosuppressants. here, we report on a normotensive woman who developed a severe frontal headache, visual disturbances, and hypertension 3 days after undergoing an emergency laparotomy for ischemic colitis during which she suffered an inadvertent dural puncture. neuro - imaging revealed features consistent with pres. the patient went on to make a good recovery, being discharged 21 days postoperatively, with only minor visual disturbances and memory problems. this case highlights the importance of awareness of pres to all specialties. on reviewing the literature, we feel that pres may be a potential differential diagnosis to post - procedural neurological symptoms in those patients undergoing routine procedures such as spinal anesthetics or lumbar punctures. |
a common and serious complication of type 1 diabetes mellitus (t1 dm) is diabetic autonomic neuropathy [1, 2 ]. cardiovascular autonomic neuropathy (can) is a subset of diabetic autonomic neuropathy characterized by impaired autonomic control of the cardiovascular (cv) system. for instance, can has been reported to increase the mortality of diabetic patients by a factor of 3.45. clinically, the most common methods for assessing can are heart rate variability (hrv) analysis and baroreflex sensitivity (brs) [3, 5, 6 ]. in t1 dm, aspects of the baroreflex arc can be impaired, such that both baroreceptor activity and excitability are blunted [8, 9 ] and the aortic depressor nerves undergo axonal atrophy. as well, autonomic efferents, primarily of the parasympathetic nervous system (psns), have decreased activity, reduced responsiveness, and decreased neurochemical activity in the heart [10, 11 ]. impairment of central nervous system regions has also been reported as the limiting factor of brs [12, 13 ]. reduced heart rate variability (hrv) is often the earliest symptom of can. whether measured by time domain analysis or by frequency domain analysis and whether in clinical or experimental t1 dm, hrv is consistently reported to be reduced in t1 dm [5, 1417 ]. exercise has been demonstrated to be an effective means of improving deficits in hrv and brs in both clinical and experimental t1 dm [1821 ]. such improvements have been attributed to improved insulin sensitivity, increased endogenous antioxidant and anti - inflammatory mediators, and improved autonomic control of the cv system [2224 ]. despite similar reductions in hrv and brs, there are marked differences in early - stage changes to other cv parameters between clinical and experimental t1 dm. specifically, in clinical t1 dm, increases in heart rate (hr) and blood pressure (bp) are commonly reported in early autonomic neuropathy [1, 3, 14, 24, 2628 ]. in contrast, experimental stz - induced t1 dm is regularly associated with decreased bp and hr, beginning shortly after diabetes induction [15, 16, 2931 ]. due to these opposing initial changes in bp and hr, exercise training is often observed to produce contrasting outcomes on cv parameters in experimental and clinical t1 dm, namely, increased bp and hr in experimental t1 dm and decreased bp and hr in clinical t1 dm [19, 25, 3234 ]. as a result, both the increase and decrease of these cv factors are concurrently cited as exercise - mediated improvements to can with little consideration of the fact that the changes are opposed between these two contexts of t1 dm. this is important because if animal models do not accurately reproduce t1 dm pathology, then the outcomes of experimental studies may not translate to the treatment of human can, as the mechanisms underlying the pathology and exercise modifications may differ. another important difference between experimental and clinical t1 dm is the common omission of insulin treatment in experimental diabetes leading to severe hyperglycemia ranging from roughly 17 to 25 mm blood glucose concentrations ([bg ]) [15, 16, 29, 30 ]. as the severity and duration of hyperglycemia have been shown to influence the degree of diabetic neuropathy, acute and steep elevations of [bg ] in stz - induced t1 dm may not only cause early onset neuropathy to the psns but also cause acute neuropathy of the sympathetic nervous system (sns) and directly affect the sinoatrial (sa) node. these changes may mediate the observed reduction in bp and hr that arise acutely in experimental t1 dm and in late - stage clinical t1 dm [2, 3538 ]. indeed, intensive insulin therapy has been shown to restore bp and hr to non - t1 dm levels in stz - induced t1 dm rats [25, 39 ]. yet, despite the use of insulin therapy in clinical t1 dm, it is often the case that chronic, moderate hyperglycemia is maintained as a result of difficulties in regulating [bg ] in response to dynamic influences on glycemic control, such as food intake and exercise [40, 41 ]. this is often resultant of a tendency to err on the side of moderate hyperglycemia in order to circumvent the acute discomfort and danger associated with hypoglycemic episodes, which occur more frequently with diabetic neuropathy due to the impairment of the glucagon response [40, 42, 43 ]. to address this, our laboratory established a model of t1 dm using a multiple low - dose stz - treatment and insulin therapy to replicate the moderate hyperglycemia observed in clinical t1 dm. in our previous studies that employed this model, we observed impairments in glucose tolerance, vascular responsiveness, cardiac function, and bone health, which were improved with high intensity aerobic exercise training [4447 ]. the purpose of the current study was to investigate whether our model of multiple low - dose stz - induced t1 dm with insulin therapy would induce deficits in cardiovascular autonomic function more representative of clinical t1 dm, and if high intensity aerobic training could prevent those deficits. we hypothesised that (1) our model of stz - induced t1 dm would elicit indices of can, including a blunted brs (bradycardia and tachycardia response), lowered hrv and intrinsic heart rate, increased vascular sympathetic tone, and increased mean arterial pressure, and (2) high intensity aerobic exercise training would prevent or ameliorate the indications of can. the protocols used in this investigation were approved by the university of western ontario council on animal care and conformed to the guidelines of the canadian council on animal care. eight - week - old male sprague - dawley rats were obtained from charles river laboratories canada (saint - constant, quebec). the rats were housed in pairs and maintained on a 12-hour dark / light cycle at a constant temperature (20 1c) and relative humidity (50%). sixty - four rats were randomly assigned to one of four groups as follows : (1) sedentary control (c, n = 16) ; (2) exercised control (cx, n = 16) ; (3) sedentary t1 dm (d, n = 16) ; (4) exercised t1 dm (dx, n = 16). all functional and blood endpoint measures were acquired 24 hours after the final exercise bout. upon arrival rats subsequently, t1 dm was induced over five consecutive days by multiple intraperitoneal (ip) injections of 20 mg / kg streptozotocin (stz, sigma - aldrich) dissolved in a citrate buffer (0.1 m, ph 4.5). diabetes was confirmed by blood glucose measurements greater than or equal to 18 mm on two consecutive days. if necessary, subsequent 20 mg / kg stz injections were administered until diabetes was confirmed. following the confirmation of diabetes, insulin pellets (1 pellet ; 2 u insulin / day ; linplant, linshin canada, inc., insulin pellet doses were then monitored for 1 week and adjusted (0.5 pellets) in order to obtain daily nonfasting blood glucose concentrations in the moderate hyperglycemic range of 917 mm. insulin dose was determined by multiplying the total quantity of pellet implanted (0.5 pellet increments) by the amount of insulin released per pellet (2 units of insulin / day / pellet) divided by the body weight (kg) of the rat. blood was obtained from the saphenous vein by venous puncture with a 30-gauge needle and measured via freestyle lite blood glucose monitoring system (abbott diabetes care inc., intravenous glucose tolerance tests (ivgtt) were performed on all animals prior to t1 dm induction (pre - t1 dm) and at the end of week 10 of the exercise training period. rats were fasted for approximately 8 to 12 hours prior to the assay and did not perform exercise on the day of their ivgtt. a sterile - filtered dextrose solution (50% dextrose, 50% ddh2o) was injected (1 g / kg) into the lateral tail vein of the conscious rat. following dextrose infusion, blood glucose was measured at 5 minutes, at 10 minutes, and then at 10-minute intervals thereafter until blood glucose levels plateaued. prior to the initiation of the exercise training program, rats were familiarized with the exercise equipment on two consecutive days. the familiarization consisted of two 15-minute sessions of running at progressive treadmill speeds up to 30 meters per minute (m / min). the treadmill was a custom - built apparatus fabricated by the physical plant at university of western ontario and has been used in many previous studies [4447 ]. the exercise training program consisted of 1 hour of motor - driven treadmill running per day at 27 m / min with a 6-degree incline, 5 days per week, for 10 weeks. the exercise intensity was determined based on earlier research that investigated oxygen uptake in rats at various treadmill speeds. the chosen intensity was found to represent approximately 7585% vo2max [48, 49 ]. to achieve a surgical plane of anesthesia, rats were placed in an induction chamber circulating 4% isoflurane (96% o2). once motor reflexes were undetectable, rats were transferred to a nosecone delivering 3% isoflurane (97% o2) and placed on a hot water pad (37c). rats were cannulated with saline - infused polyethylene (pe90) catheters in the right jugular vein and carotid artery and each catheter was attached to a three - way stopcock. the jugular vein catheter was used for drug infusions and the carotid artery catheter was connected in series with a pressure transducer (px272, edwards life sciences, irvine, california, usa) for arterial blood pressure measurements. at the end of the preparative surgery, rats were injected ip with a 25 mg / kg cocktail of urethane (16 mg / ml) and -chloralose (100 mg / ml), an anesthetic cocktail that has been shown to have the least inhibition of baseline cv control and autonomic function. a total of 10 ml of urethane/-chloralose was made, 5 ml of which was diluted to 50% with ddh2o and was used as needed to maintain anesthesia throughout data collection. isoflurane anesthesia was gradually removed, whereby urethane/-chloralose was the primary anesthesia used during data collection. heart rate (hr), systolic blood pressure (sbp), and mean arterial pressure (map) were determined from the blood pressure pulse waveform and were collected while the rats were under urethane/-chloralose anesthesia in the supine position. data were obtained using a powerlab data acquisition system, digitized, and recorded at 1000 hz using the bundled labchart 7 pro software (adinstruments, colorado springs, co, usa). prior to drug infusions, 5 minutes of spontaneous electrocardiogram data was sampled at 1000 hz and analyzed with labchart hrv analysis software (adinstruments). time domain analysis of the standard deviation between normal peak pulses of the pressure pulse waveform (sdnn) was quantified as a measure of the total variability of the hr. frequency domain analysis of the high frequency (hf) band of the fast fourier transform (fft) of the data was assessed as an index of parasympathetically mediated hrv. baroreflex sensitivity (brs) was assessed using the modified oxford technique [51, 52 ]. the brs was quantified using the slope of the linear regression line representing the linear portion of the sigmoidal heart rate - systolic blood pressure relationship (hr { bpm}/sbp { mmhg }) after rapid bolus injections (~5 s) of phenylephrine (pe, 12 g / kg, 10 g / ml) and sodium nitroprusside (snp, 60 g / kg, 110 g / ml) dissolved in ddh2o. (2007). for each drug, the catheter was first filled with a 0.2 ml volume to ensure accuracy of the drug dose. after a stable baseline was obtained, a bolus injection of snp was rapidly infused and the reflex sns mediated tachycardia response was measured. the analysis began at the onset of sbp decrease after snp infusion and ended when sbp reached its nadir. this was followed by a saline flush to washout any remaining snp in the catheter. after a stable baseline was reestablished, this same procedure was then followed using pe to measure psns mediated reflex bradycardia, except that analysis began at the onset of sbp increase and ended when sbp reached its zenith. responses to pe and snp were plotted separately and only regression lines (slopes) with correlation coefficients (r) 0.70 and p 0.05). the glucose clearance rate (kg) of the diabetic groups (d and dx) decreased from pre - t1 dm to week 10 of training (p < 0.05), whereas kg of the cx group increased (p < 0.05 ; figure 2(a)). both diabetic groups had significantly lower kg values than both the control groups (c and cx) at week 10 (p < 0.05 ; figure 2(a)). the amount of insulin supplementation that the dx group received was significantly less than the amount the d group received at week 10 (p < 0.05 ; figure 2(b)). for resting hr and map, there was not a significant difference between groups at week 10 (figures 3(a) and 3(b), resp.). however, for the intrinsic heart rate (ihr), there was main effect of both exercise and t1 dm, where t1 dm decreased the ihr, while exercise increased ihr (p < 0.05, figure 3(c)). further, within the t1 dm groups (d and dx), exercise increased ihr, while within the nonexercised groups (c and d) t1 dm decreased ihr (p < 0.05). total hrv at week 10, as measured by the standard deviation of the normal pulse wave peaks (sdnn), was not significantly different between groups (figure 4(a)). however, there was a main effect of exercise on the hf contribution to hrv, where exercise increased hf hrv (p < 0.05, figure 4(b)). particularly, within the t1 dm groups (d and dx), exercise increased hf hrv (p < 0.05). in response to snp infusion, there was not a significant difference between groups in the tachycardia brs response (figure 5(a)). however, a significant interaction between t1 dm and exercise was observed for brs during the bradycardia response to phenylephrine (p < 0.05, figure 5(b)). more specifically, within the t1 dm groups (d and dx) exercise prevented the reduction in brs that was observed in the d group (p < 0.05). an interaction between t1 dm and exercise was observed for the prazosin - induced change in map (p < 0.05, figure 6(a)). within the nonexercised groups (c and d), t1 dm resulted in an increased change in map (p < 0.05). within the t1 dm groups (d and dx) exercise prevented the increased change in map observed in the d group (p < 0.05). there was also a main effect of t1 dm on [npy ] (p < 0.05, figure 6(b)). within the nonexercised groups (c and d) and exercised groups (cx and dx), serum [npy ] this study demonstrated that a multiple low - dose stz model with moderate hyperglycemia, maintained using insulin therapy, produced deficits in cardiovascular autonomic function without inducing the resting bradycardia or hypotension typical of other stz models. this study also showed that high intensity aerobic exercise training can prevent deficits of cardiovascular autonomic function caused by t1 dm. furthermore, because [bg ] was held within a moderate hyperglycemic range, the observed exercise - mediated improvements to indications of can were independent of changes in [bg ] and, instead, may primarily have been the result of improvements to other aspects of glucoregulation and/or the preservation of autonomic nervous system function. although we found time domain analysis of total hrv, as measured by the sdnn, did not demonstrate differences between groups, frequency domain analysis exposed a reduction in the hf power in the d group compared with the dx group. since the hf power corresponds to the level of vagally mediated parasympathetic hrv, these results demonstrate not only the detrimental effects of t1 dm on autonomic cardiac control but also the benefits of exercise training toward ameliorating those effects. these findings are similar to those of other experiments of both experimental [16, 21 ] and clinical diabetes [18, 56, 57 ]. (2009) reported that stz - induced t1 dm reduced the hf component of hrv, which was improved by exercise. also, they found that the vagal tonus of the control exercised rats did not differ from sedentary controls. likewise, chen. (2008) reported that children with t1 dm who performed a high level of physical activity did not differ from controls in hrv ; however, children with t1 dm who had low level of physical activity had significantly reduced hrv compared to both active children with t1 dm and non - t1 dm children. thus, the current study provides support that exercise can be an effective means to improve hrv in t1 dm. both tachycardia and bradycardia responses were studied in the context of brs analysis in order to explore the control features related to unloading or loading of the baroreceptors, respectively. some discrepancy exists between different experimental models of t1 dm and their impact on brs measures. investigations using the hyperglycemic non - obese diabetic (nod) t1 dm mouse model have shown elevations in brs measures rather than attenuated responses. in contrast, tachycardic - snp and bradycardic - pe responses have been shown to be lower in stz - induced t1 dm hyperglycemic rats in comparison to non - t1 dm controls but were improved with exercise training. in the current study, the slope of the hypotensive tachycardia response was not significantly different between any of the groups suggesting that responses to baroreceptor unloading are not affected by t1 dm or exercise. however, t1 dm reduced the bradycardia response to baroreceptor loading, which was nullified by concurrent exercise training. these findings are in line with previous reports demonstrating a bradycardia change in pe - brs without an accompanying change in snp - brs, which was improved following aerobic exercise. discrepancies in brs responses in t1 dm models seem to be closely associated with both the duration and the severity of diabetes. a recent study examining the time - course of brs changes in response to stz - induced hyperglycemia reported that alteration of the snp - brs was not evident until 12 weeks of diabetes, while a change in pe - brs was evident as early as 4 weeks after induction. interestingly, the animals in the aforementioned study were moderately hyperglycemic (1618 mm), suggesting that the severity of the hyperglycemia may play a role in the progression of this neuropathy. vinik and ziegler (2007) reported that poor glycemic control and duration of diabetes play a central role in progression of cardiovascular autonomic neuropathy. yet, it is not clear what role insulin therapy may play in the neuropathy. insulin supplementation to stz - induced t1 dm rats can modify the changes in brs sensitivity evident at 48 weeks of t1 dm. indeed, in clinical t1 dm patients, intensive therapy is well documented to slow the progression and delay the appearance of abnormal autonomic function. however, the current study provides evidence that the ability of exercise to ameliorate cardiovascular autonomic dysfunction may be independent of its ability to reduce [bg ], which challenges the direct relationship between [bg ] and can suggested by previous studies [6769 ]. the ivgtt performed at the conclusion of the 10-week exercise period demonstrated an increased glucose clearance rate (kg) and therefore glucose tolerance, in the cx group compared to the preexercise training period. however, in both the sedentary and exercise diabetic groups there was an equal decline in kg to nearly the same rate. this decrease was significantly different from pre - t1 dm values and the week 10 values of the c and cx groups. while this would normally indicate that both of the diabetic groups developed equally impaired glucose tolerance, it was also the case that the dx group required approximately half of the dosage of exogenous insulin compared to the d group to maintain their [bg ] in the 917 mm range. with double the insulin dose, it is likely that the total serum insulin over a given time during ivgtt would have been greater in the d than dx group, and with their kg being equal, that would indicate that there was a greater insulin sensitivity in the diabetic exercise group [70, 71 ]. together, these ivgtt results demonstrate that exercise training improved glucose tolerance and insulin sensitivity. furthermore, since the [bg ] of the diabetic groups in this study was held in a constant range, any abovementioned exercise - induced improvements to cv autonomic function would not have been mediated through a reduction in systemic [bg ] but may have been the result of improvements in insulin sensitivity and glucose utilization [72, 73 ]. this should be borne in mind when considering the effects of diabetes and exercise on indices of cv autonomic function, such as hrv and brs. an alternative mechanism by which exercise can influence brs was reported by bernardi. they demonstrated that a reduced parasympathetic brs in patients with t1 dm was improved by both oxygen supplementation and deep breathing to the same degree, which indicated the increased respiration and oxygen delivery resultant of exercise could have been mediating increases in brs. this led the authors to suggest that hypoxia in t1 dm functionally restrains parasympathetic activity. however, reduced brs could also be attributed to defects in the baroreceptors, baroreceptor afferent nerves, cns structures, or efferent fibres of the baroreflex circuit [7, 8, 61 ]. in the present study, the finding that the tachycardia response of the baroreflex was unimpaired by t1 dm, while the bradycardia response was, suggests that the afferent arm and central regulators of the baroreflex were not dysfunctional and that the observed decrement of baroreflex bradycardia may have been caused partly by alterations in efferent parasympathetic outflow [8, 29 ]. another interesting outcome of the current study was the alteration of sympathetic vasomotor control in the d group, which was also modified by concurrent exercise training. in this study, prazosin treatment resulted in a drop in map that was approximately twofold greater in the d group compared to the c and dx groups, which is indicative of a much greater sympathetic contribution to the maintenance of baseline vascular resistance [75, 76 ]. similarly, martinez - nieves and dunbar (1999) reported that male t1 dm rats had a greater decrease in map after a bolus injection of prazosin compared to their control cohorts. however, they postulated that an elevated prazosin response could be the result of increased 1-adrenergic receptor sensitivity. yet, in this study, the finding that treatment with pe, an 1-adrenergic receptor agonist, did not result in a greater peak sbp, nor a greater percent increase in sbp from baseline in the t1 dm group (data not shown), argues against a receptor - based sensitivity mechanism and, rather, suggests that efferent sympathetic outflow may have been elevated in the d group. however, we can not determine the mechanism that resulted in prazosin showing a preferential decrease in map in the d group versus dx or c based on the data in this study. yet, in line with the current results, such elevations in resting sympathetic activity would make activation of the brs response to snp - induced hypotension more difficult. the conclusion above regarding sympathetic hyperactivity in the d group is supported by measurements of neuropeptide y [npy ] obtained in this study. [npy ] is coreleased with norepinephrine from perivascular and cardiac sympathetic nerve terminals during sympathetic activation [78, 79 ]. in clinical t1 dm, a diabetes - related decrease in [npy ] is attributed to impaired sympathetic function, whereas increased [npy ] is attributed to sympathetic overactivity [7981 ]. in the current study, serum [npy ] was greater in both of the t1 dm groups in comparison to their control groups. thus, despite the ability of exercise to preserve reflex cardiac function in t1 dm, hyperglycemia itself appears to have impacted basal vascular adrenergic activity in both t1 dm groups. as both t1 dm groups were maintained at equally elevated [bg ], there may have been a correspondingly similar stimulation of peripheral sympathetic activation and npy release [79, 82, 83 ]. despite improvements by exercise training to deficits of cardiovascular autonomic function, no observable statistical differences in either map or hr were evident between any of the groups. indeed, it has been shown that alterations in autonomic function occur before or without alterations in map and hr and are uncorrelated to changes in sympathetic tone. the observed changes in basal sympathetic activity may assist in the maintenance of blood pressure, ventricular function, and cardiac output during the early stage of diabetes, which is supported by our findings that inhibition of sympathetic activity results in a greater decrease in map in diabetic rats than normal rats. in that respect, we previously reported that although t1 dm animals demonstrated significant alterations in myocardial dimensions and structure, measurements of cardiac performance (ejection fraction, fractional shortening, and cardiac output measurements) were unchanged. to evaluate the heart rate of these animals without neural influence, we measured the beat rate of denervated hearts using the isolated langendorff technique. we found that the ihr of the d group was lower than both c and dx groups, which would support the notion that decreased ihr masked the effects of sympathetic overactivity in the current study. further, it supports evidence that stz - induced diabetes may have a direct effect on heart rate by modifying the heart itself [86, 87 ]. interestingly, in some studies, insulin therapy was only able to partially reverse bradycardia and it was shown that stz - treatment itself could lengthen the action potential duration in the sa node, slowing the hr. however, if hyperglycemia or stz directly affected cardiac muscle or the sa node and caused a decreased ihr in the d group, it is also the case that exercise training rescued or prevented the deficit, as the ihr of the dx group was not different from the cx group. thus, previous experimental t1 dm studies that reported that stz - induced bradycardia and hypotension were caused by can, and that exercise - induced normalization of hr and bp was evidence of improvements in autonomic function, may really have been observing changes in intrinsic cardiac function which were independent of autonomic control. such changes could instead have been due to depressed sarcoplasmic reticulum function or impaired calcium handling [87, 89, 90 ]. therefore, the direct effects of stz on the heart and ihr require further examination and should be taken into consideration in future studies that investigate the autonomic regulation of cv function in stz - induced t1 dm models. an important consideration regarding the design of the current study was the use of anesthetized rats. in order to accurately reflect cardiovascular parameters in such a state, we selected an anaesthetic regime that provides the lowest level of influence on baseline and reflexive cv control attainable in rodent models. a light plane anesthesia 0.51.2 g / kg has been shown to maintain the integrity of the cardiovascular system, where higher doses of urethane (above 1.5 g / kg) can produce hypotension and bradycardia, as well as high rates of mortality [91, 92 ]. in the current study we used a minimal dose of 25 mg / kg, which was reported in previous studies by our laboratory to have little influence on neurovascular blood flow measures [9395 ]. that being said, it can not be determined to what extent, if at all, the autonomic nervous system was augmented by the urethane - chloralose treatment in comparison to conscious animals. further work examining a comparison of our anesthesia regime with freely moving conscious animals (using telemetry devices) will better address this matter. in this study, t1 dm induced indications of parasympathetic withdrawal, sympathetic overactivity, and, despite a decreased ihr, no change in resting map or hr. however, concurrent exercise training with t1 dm maintained the sensitivity of the parasympathetically mediated baroreflex bradycardia, prevented an increase in vascular sympathetic tone, maintained a higher bodyweight, and prevented a decrease in ihr. the ability of exercise training to preserve parasympathetic function in this model of t1 dm indicates that the exercise - mediated improvements to parasympathetic function are independent of alterations in [bg ]. however, the finding that [npy ] remained elevated suggests that hyperglycemia has a direct impact on adrenergic activity. taken together, our t1 dm model of progressive stz induction and insulin treatment induced autonomic impairments similar to those observed in clinical t1 dm and demonstrates the novelty of this model for investigating the effectiveness of high intensity aerobic exercise training as a means to prevent the progression of can in t1 dm. thus, although not examined in this study, the mechanisms that underlie the physiological changes caused by t1 dm and exercise can be the focus of future investigations using this model. | indices of cardiovascular autonomic neuropathy (can) in experimental models of type 1 diabetes mellitus (t1 dm) are often contrary to clinical data. here, we investigated whether a relatable insulin - treated model of t1 dm would induce deficits in cardiovascular (cv) autonomic function more reflective of clinical results and if exercise training could prevent those deficits. sixty - four rats were divided into four groups : sedentary control (c), sedentary t1 dm (d), control exercise (cx), or t1 dm exercise (dx). diabetes was induced via multiple low - dose injections of streptozotocin and blood glucose was maintained at moderate hyperglycemia (917 mm) through insulin supplementation. exercise training consisted of daily treadmill running for 10 weeks. compared to c, d had blunted baroreflex sensitivity, increased vascular sympathetic tone, increased serum neuropeptide y (npy), and decreased intrinsic heart rate. in contrast, dx differed from d in all measures of can (except npy), including heart rate variability. these findings demonstrate that this t1 dm model elicits deficits and exercise - mediated improvements to cv autonomic function which are reflective of clinical t1 dm. |
continuous increase in population and industries has led to elevated releases of toxic substances into the environment. these substances seriously influence the metabolism of living organisms that can cause permanent if not lethal damage, subsequently, placing a potential hazard to the ecosystem. global environmental organizations, such as, the environmental protection agency (epa), have made the removal of heavy metal contaminates including chromium and other toxic substances from aquatic systems an important issue for environmental engineering and other technical areas. moreover, there is a need for the development of efficient and cost effective processes for the treatment of discharge streams and recovery of valuable components [1, 2 ]. as an inorganic contaminant, the transitional metal, chromium, exists in the oxidation states cr(iii) and cr(vi). it is most often produced by industrial processes such as electroplating, stainless steel, protective coatings on metal, magnetic tapes, and pigments for paints, cement, paper, and rubber, as a composition of floor covering and other materials [48 ]. as a result of industrial and waste disposal locations, chromium has been released to the environment via leakage and poor storage during manufacturing or improper disposal practices [9, 10 ]. due to the elemental nature of chromium and its solubility and mobility characteristics in the environment, the environmental protection agency (epa) has set an enforceable standard level called a maximum contaminant level (mcl) for chromium, which is designated as 0.1 mg l. hexavalent chromium is a toxic element having severe outcome on public health which is considered to be a mutagen, teratogen, and carcinogen and whose exposure, ingestion, or inhalation may also cause respiratory failure and ulcerations of the body [1214 ]. drinking water that contains more than 0.05 mg l chromium is considered to be very toxic for living beings. hence, the effective removal or reduction of cr(vi) to cr(iii) from contaminated water and wastewater is very important. metal oxides such as zno are essential for the development of efficient and smart materials. zno nanoparticles can be found in vast applications including pharmaceutical and cosmetic, rubber manufacture, and electronic materials. other conventional and biomass derived adsorbents that have been used in the removal of heavy metals are activated carbon, wheat bran, activated neem leaf powder, mussel shell and walnut shell powder, and peanut shells. depending upon their particle size and concentration, there are some advantages for using the aforementioned nonconventional adsorbents ; they can remove up to 90% of the adsorbent. the adsorbents are also readily available and require less maintenance and supervision. on the other hand, they do require further treatment before they can be used to extract the heavy metal of choice. the advantages and interesting properties of zno such as low cost and uv - blocking render them being a very good candidate for the adsorption studies. since the past decade, there has been an increased amount of activity regarding the toxicity of metal oxide nanoparticles and other bulk materials as to how they interact with both living and nonliving organisms. illustrated using commercially available nonwoven polyethylene microfibers as a substrate material where zno nanorods were grown by a method called chemical bath deposition. fabricated antibacterial poly(d, l - lactide) nanofibers impregnated with zinc oxide nanoparticles to investigate the morphological, mechanical, and antibacterial properties. capitalizing upon this idea of fibrous substrates, from research, it was found that nanofibers are an exciting new class of material with diameters ranging from a few nanometers up to micrometers [5, 30 ]. also, there are other characteristics as high surface area to volume ratio, tensile strength, and flexibility that make these fibers an excellent candidate for applications that encompasses surface reactions to take place such as separation / filtration membranes and surface - supported chemical reactions [31, 32 ]. these fibers are typically on an order of magnitude of two or three times smaller than fibers conventionally produced. the electrospinning technique was employed in the preparation and formation of the polymer nanofibers in the form of a nonwoven mat. this technique is a simple and versatile method of producing a fairly large - scale amount of fibers from a variety of materials. these fibers, which are nonmechanical and electrostatic, involve the utilization of a rather high electrostatic field to charge polymer solution 's surface droplet, thus inducing the ejection of a liquid jet to be deposited onto a collector 's plate. in order to utilize these materials, we report the fabrication of highly flexible nanofibers of poly - l - lactide (plla) by electrospinning, which was used as a substrate for the growth of radially oriented zno nanowires by the chemical bath deposition method. assembled nanostructured polymeric nanofibrous mat retains the flexibility and high surface area ; it has the additional functionality of zno nanorods. as a direct result of this assembled nanostructure, we devised a simple setup for adsorption and desorption of cr(vi) ions from the single metal aqueous solution in a continuous flow mode. poly - l - lactide (mw = 100000), chloroform, sodium hydroxide, and hydrochloric acid were purchased from sigma - aldrich. all chemicals were analytical grade reagents and used as received without further purification. high purity water with a resistivity of 18 m cm was used throughout all the experiments. chloroform was used to dissolve the polymer (7 wt%) while stirring for 24 h. the sample was loaded into a syringe composed of a stainless steel needle (0.8 mm in diameter). the collector 's plate was covered with aluminum foil and was connected with the cathode of a voltage supply. a voltage of 9 - 10 kv was applied between the needle and the collector. the needle containing the polymer solution was mounted to a syringe pump (cole - parmer) in a horizontal direction. the distance from the tip of the needle to the collector is 10 cm with a flow rate of 1.0 ml h. bestowing high voltage power supply, the potential was applied to the hypodermic needle (needle nokor admix) on the end of the syringe. for a total time of 30 min, the plla nanofibers were immersed into colloidal zno suspension prepared by modifying a method described by bahnemann. nanofibers were immersed into a mixed aqueous solution of 20 mm each of zn(no3)2 and hexamine and heated to 75c for 6 h. the prepared plla - zno assembled nanostructured material was washed followed by drying in a vacuum oven for 1 h. the plla - zno assembled nanostructured material so prepared was inserted into a glass tube. this column was connected to a 100 ml glass bottle (reservoir) via a peristaltic pump. the adsorption experiments were performed by the mixing of k2cr2o7 in aqueous solution with 0.01 m nano3 and pumped at a flow rate of 2.0 ml min and total time of 1500 minutes. after each designated time, a sample was taken from the reservoir and prepared for analysis. the amount of cr(vi) ions in the supernatant was determined with the inductively coupled plasma (icp - oes icap 6000 series thermo scientific). in the aqueous phase, the amount of cr(vi) adsorbed per unit mass of adsorbent qe (mg g) was calculated using(1)qe = c0cevm, where c0 is the initial chromium concentration (mg l) and ce is the chromium concentration in the aqueous phase at equilibrium (mg l). v is the total aqueous volume (l) and m is the mass of the zno - plla nanofibers (g). for the desorption experiment, the cr(vi) solution the amount of cr(vi) released from the zno nanorod - plla nanofiber into the supernatant was determined. the recovery efficiency (r) of cr(vi) is calculated as(2)r%=cdescads100,where cdes is the amount of cr(vi) released into the solution and cads is the amount of cr(vi) adsorbed onto the zno - plla nanocomposite. sugunan. reported that the growth of zno nanorods is dependent upon a set of parameters and growth conditions such as concentration and temperature, which directly affect the diameter and length of the nanofibers. morphology and microstructure of the nanocomposite have been investigated by scanning electron microscopy (sem). figure 1(a) presents a micrograph of electrospun plla nanofibers and figure 1(b) shows these nanofibers with assembled zno. it can be observed in figure 1 that the growth of zno nanorods and fabrication of plla nanofibers are intact and uniform without degradation. the cross section of plla nanofibers ranged from few tens of nanometers to 4 m. moreover, the cross section of the nanocomposite has increased up to 12 m, which is dependent on the parameters such as growth conditions and physiochemical properties (temperature and concentration) of zno nanorods. one parameter that can have a consequential impact on the adsorption of cr(vi) is ph. the uptake of cr(vi) by this highly flexible nanofiber nanocomposite from solutions was studied as a function of contact time, ph, and cr(vi) concentration. the oxidation states of chromium range from 2 to + 6 ; in nature, it is mainly found in two oxidation states cr(iii) and cr(vi). cr(iii) and cr(vi) differ in many of their properties such as ph, charge, physiochemical characteristics, mobility, and toxicity. cr(vi) speciation exists in solution as chromate (cro4), bichromate (hcro4), and dichromate (cr2o7) [3, 4 ]. in this study, cr(vi) was the only species in the solution considered as our starting material which was k2cr2o7. for each sample, 2 - 3 wavelengths were selected to ensure sensitivity, improved precision, and detection limits while maintaining minimal interference from other elements is not accounted. the axial plasma orientation was used for the analysis of cr(vi) ions in solutions. the amount of cr(vi) adsorbed on the flexible nanofiber nanocomposite platform as a function of ph in the range of 2 to 9 was experimentally determined and the data are shown in figure 2. the optimum ph for the adsorption of cr(vi) species by the flexible zno - plla nanofiber nanocomposite is 3.5. at low ph, better adsorption of cr(vi) was achieved which may be attributed to the presence of large amount of hydrogen ions (h), as also reported in the literature [5, 35 ]. the adsorption of cr(vi) in the acidic ph range is similar to the work of avila. who investigated the adsorption of cr(vi) using surface functional polyacrylonitrile (pan) nanofibers at ph = 2. the ph level can neutralize the negatively charged zno adsorbent surface, subsequently reducing the hindrance to the diffusion of cr(vi) anions. conversely, at increased ph values, the abundance of hydroxide ions (oh) increases hindrance to diffusion of cr(vi) anions, thereby reducing the removal efficiency. in alkaline solution, the surface charge of zno is negative. with such chromium species as neutral h2cro4 and the main goal of performing zeta potential is to gain understanding on the dispersion behavior of the material. as illustrated in figure 3, there is a change in zeta potential as the zno nanoparticles are titrated against naoh as the point of zero charge (pzc) for zno is being determined. it can be observed that as the zno reaches the pzc, the surface charge of the oxide becomes neutral. subsequently, very little to no adsorption of the cr(vi) was obtained. as the ph of the metal oxide approaches the acidic regime, the predominant form of cr(vi), as an ion, is hcro4 ; therefore, the surface becomes more positively charged, where the maximum adsorption of cr(vi) in solution can occur. other studies have reported that the adsorption of the zinc species affects the sign and the magnitude of the surface charge of the zinc oxide nanoparticles [38, 39 ]. at higher positive pzc, the dominant species were reported to be zn. in order to assess the optimal contact time for the adsorption of cr(vi) species, the experiments were performed at ph = 3.5, which was found to be the ultimate ph for this study as shown in figure 2. reported that the rapid adsorption of metals on nanoparticle surface was due to the external surface adsorption. several batch experiments were performed in order to determine the time required to reach equilibrium. after a certain period, the adsorption curve reached a plateau region where no more cr(vi) species were adsorbed. based on this observation, the optimum contact time for adsorption of cr(vi) species at this ph value was determined to be 1300 minutes. figure 4 illustrates the effect on the amount of cr(vi) adsorbed as a function of time. the equilibrium can be obtained when the maximum interaction of the zno - plla nanofiber nanocomposite occurred at a time of 1300 minutes and initial ph of 3.5 resulting in maximum adsorption of 3.5 mg g of cr(vi) adsorbed. for the cr(vi) species, at acidic ph the (hcro4) species has been known to be dominant. it is important to note that the guideline value, derived from tolerable daily intake, daily consumption of water, and body weight, is 0.05 mg thus, methods for drinking water management by means of adsorption zno - plla nanofibers in the small concentration ranges have appeared to be effective. as shown in figure 2, the pzc for zno nanostructures was found to be 10. the zeta potential was highly influenced by ph. the surface charge on zno was increased positively as the ph became more acidic. when zno is introduced into solutions composed of negative species, adsorption can readily occur. as the zeta potential curve moves toward higher ph, the charge density changes such that the electrostatic interaction is no longer applicable ; instead the system within solution shows more of a repulsive scenario. the effect of repulsive interaction can be most beneficial in systems, where the interest is regeneration of the nanomaterial to be reused for several cycles of adsorption and desorption operations. as the zno is attached to the plla nanofiber, the amount of zn species dissolved in the solution was found to be less than 0.25 mg l, showing a stable performance for adsorption. to regenerate the adsorbent nanocomposite nanofiber platform, it is essential that all adsorbed cr(vi) species are desorbed so that the platform can be reused as this step is crucial for recovery of the species for recycling in other processes. it also can give rise to information regarding whether cr(vi) is reversibly or irreversibly adsorbed onto the zno - plla nanocomposite. the recovery of cr(vi) species from zno - plla nanofiber nanocomposite was tested at ph = 12. in the alkaline regime, the oh ions interact with the cr(vi) ions attached to the platform thereby releasing the cr(vi) into solution, thereby indicating that the desorption process was reversible. figure 5 shows that the maximum recovery of cr(vi) from the zno - plla nanofiber nanocomposite into solution was 60%. this could be attributed to the fact that the sites on the material are initially occupied with cr(vi) and when introduced to ph of 12 the cr(vi) ions leave the sites and are released back into solution. metal recovery and reuse of the adsorbent can become economically viable in the overall process. in this work, plla and zno - plla nanofiber nanocomposite were successfully fabricated and have been shown to be an effective material in the removal of highly toxic cr(vi) species. given the optimum adsorption conditions, the adsorption equilibrium can be reached after several hours. the removal of cr(vi) using the nanocomposite was highly dependent on ph. at ph electrostatic and surface attractions between zno and cr(vi) species control the interactions between the flexible nanocomposite platform and the cr(vi) species. in the basic ph range solution, cr(vi) can be regenerated and the zno - plla nanofiber nanocomposite can be reused. a 60% desorption efficiency of cr(vi) the zno - plla nanofiber nanocomposite has shown great potential in applications such as water and wastewater treatment for the removal of heavy metal ions of cr(vi) species. | nanomaterials of zno - plla nanofibers have been used for the adsorption of cr(vi) as a prime step for the purification of water. the fabrication and application of the flexible zno - plla nanofiber nanocomposite as functional materials in this well - developed architecture have been achieved by growing zno nanorod arrays by chemical bath deposition on synthesized electrospun poly - l - lactide nanofibers. the nanocomposite material has been tested for the removal and regeneration of cr(iv) in aqueous solution under a continuous flow mode by studying the effects of ph, contact time, and desorption steps. the adsorption of cr(vi) species in solution was greatly dependent upon ph. sem micrographs confirmed the successful fabrication of the zno - plla nanofiber nanocomposite. the adsorption and desorption of cr(vi) species were more likely due to the electrostatic interaction between zno and cr(vi) ions as a function of ph. the adsorption and desorption experiments utilizing the zno - plla nanofiber nanocomposite have appeared to be an effective nanocomposite in the removal and regeneration of cr(vi) species. |
the development of anemia during critical illness is hematologically similar to the anemia of chronic disease / inflammation (acd), except that the onset is generally acute. blunting of endogenous erythropoietin production, mediated through the action of inflammatory cytokines, is thought to be the most important cause of this common syndrome. because low serum iron and elevated serum ferritin levels are also clinical features of acd, investigators once believed that altered iron metabolism was responsible for this condition. this early hypothesis has merit because iron utilization plays a key role in anemia and can affect the response to endogenous or exogenously administered erythropoietin. nutritional deficiencies (folic acid, vitamin b12, and iron) can possibly contribute to the etiology of early anemia in the intensive care unit (icu). in a study conducted by rodriguez and colleagues, 13% of critically ill patients were identified as having potentially correctable nutritional abnormalities, with 9% having laboratory values consistent with iron deficiency (i.e. iron / total iron binding capacity < 15%, with a ferritin level < 100 ng / ml). the relevant iron parameters that characterize anemia in chronic disease or critical illness, as compared with those in iron deficiency anemia and normal individuals, are summarized in table 1. in acd - type disorders, absorption of iron is actually stimulated and there are adequate stores of iron in the bone marrow. release of iron from macrophages in the reticulo - endothelial system is defective, ferritin concentrations are increased, and serum transferrin levels are normal, rather than elevated. this ' functional iron deficiency ', when diagnosed by cytometry, is present in 35% of patients on admission to the icu. disturbed iron metabolism from enhanced immune activation has also been documented in surgical icu patients and in patients with multiple organ dysfunction syndrome. iron parameters characteristic of anemia in chronic disease or critical illness acd, anemia of chronic disease / inflammation ; % saturation = (iron / total iron binding capacity) 100 ; stfr, serum transferrin receptor ; tfr - f index, stfr / ferritin. values derived from rodriguez and coworkers, hudson and comstock, miller and coworkers, and punnonen and coworkers. regardless of whether iron supply is limited as a result of nutritional or functional factors, when the iron available to developing erythroblasts is insufficient to meet the demands of heme synthesis, maturing red cells may contain suboptimal hemoglobin. to prevent the synthesis of hypochromic red cells, administration of iron in conjunction with erythropoietin therapy should be considered in the critically ill patient, but when, how much, and by what route should iron be given ? in a recent review of best practice and research in clinical hematology, it was noted that iron uptake into developing erythroblasts begins at an early stage and is completed before late erythroblastosis. precisely when to administer iron with erythropoietin therapy has not been systematically investigated. in a study examining health - related quality of life in rheumatoid arthritis patients receiving treatment with erythropoietin, all 28 patients who completed the study responded to treatment with erythropoietin. however, 82% developed functional iron deficiency and were well supported with a mean absolute dose of 710 560 mg intravenous iron sucrose. in that 12-week study, iron support was not initiated until the deficiency was noted (after a mean of 4.5 weeks of treatment). in other small studies conducted in elderly or critically ill patients, iron support was initiated concurrently with erythropoietin therapy. of note, daily intravenous iron therapy alone or in combination with folic acid did not result in a significant increase in reticulocyte count among critically ill patients. administration of iron in critically ill patients is regarded by some as undesirable because free iron may promote bacterial growth and have detrimental effects in patients who are immunosuppressed or susceptible to infection and sepsis. iron overload is most commonly associated with chronic transfusions in patients with refractory anemia because there is no physiological mechanism to excrete iron ; chelation therapy is generally required to remove it. this constitutes an important reason for careful consideration of iron dosing and route of administration in the critically ill patient. when given by the parenteral route, iron has been implicated in increased susceptibility to bacterial infection [11 - 14 ]. this effect is believed to be related to the influence of iron on the immune system. the cytokine - mediated defect in iron release from macrophages in humans is said to have evolved as a primitive mechanism of defense against microbial pathogens to limit their access to iron. studies on the effects of iron on host defense suggest that increased iron availability, seen with increased transferrin saturation, leads to less efficient bacterial killing of various pathogens by plasma. this finding was corroborated by hoen and colleagues at serum ferritin levels greater than 500 ng / ml, but this was later refuted when they found no association between serum ferritin and infection in a large prospective trial. in these studies it is not clear whether high ferritin levels are associated with iron overload or an acute phase reaction, or both. in light of this rather limited evidence, some advocate that these observations should not limit iron utilization in patients with chronic kidney disease (ckd). additional studies in critically ill patients are needed to determine whether there is a clinically relevant link between infection, septicemia, and iron availability. studies of iron supplementation are plentiful in the ckd literature, in pregnancy, and in the pediatric setting. however, even in the well studied ckd population, the dosing, route, and frequency of iron administration are controversial [21 - 23 ]. concomitant use of intravenous iron was deemed superior to oral iron in patients with chronic renal failure treated with erythropoietin. total dose infusion therapy versus low dose intravenous iron replacement were equally effective in hemodialysis patients receiving erythropoietin therapy. in pregnancy, the intravenous route of iron administration was similar to the oral route in terms of measured hemoglobin increases and toxicity, but intravenous iron increased ferritin concentrations significantly more than did oral iron. studies in premature infants have attempted to address the dosage and timing of iron supplementation. in neonates with anemia of prematurity, high dose iron (16 mg / kg per day) was no more effective than low dose (8 mg / kg per day) during erythropoietin therapy. early iron supplementation in infants was safe and prevented red blood cell transfusions as compared with late iron supplementation ; no erythropoietin was given in that study. because there is substantial need for iron in erythropoietin - stimulated erythroid progenitors, tarng and colleagues recommended maintenance of serum ferritin and transferrin saturation levels over 300 ng / ml and 30%, respectively. they also asserted that, in uremic patients, oral iron is unlikely to keep pace with iron demand, rendering the response to erythropoietin suboptimal. thus, intravenous iron therapy is believed to be the most appropriate choice, leading to reductions in erythropoietin dosage and costs. although oral supplements are usually the first line of treatment because of cost and convenience, oral iron may be poorly absorbed, associated with increased gastrointestinal distress, and may not maintain iron stores in critically ill patients receiving erythropoietic therapy. studies designed to examine the optimal dose and route of iron supplementation in critically ill patients are lacking. in otherwise healthy adults treated with erythropoietin before elective surgery, intravenous iron (200 mg iron sucrose twice weekly) was superior to daily oral iron (160 mg iron sulfate). pain and discoloration at the injection site, as well as reports of sarcoma at the site of injection of iron dextran, have discouraged administration of iron by the intramuscular route. iron dextran has also been associated with important incidents of anaphylactic reaction ; however, the newest formulations are considered safer. according to zager and colleagues, parenteral iron formulations are highly potent pro - oxidants that can induce tubular and endothelial cell death. in in vitro animal studies, each formulation demonstrated a markedly different toxicity profile, with iron sucrose being the most toxic, followed by iron gluconate, then iron dextran. correct timing of infusion and dose are important to avoiding oversaturation of physiologic transport mechanisms. iron administered via total parenteral nutrition (tpn) solutions has been advocated in some publications. however, product literature states that iron formulations should not be mixed with other medications or in tpn. the administration of iron through tpn should not be regarded as a first - line option in critically ill patients until safety, physical compatibility, and efficacy data become available in the literature. parenteral iron formulations how effective is iron alone in the treatment of anemia or acd type disorders ? with regard to reducing the need for transfusions, iron has not proven very effective in improving the capacity for autologous blood donation or in reducing transfusion requirements. iron was helpful in reducing erythropoietin requirements for the maintenance of hemoglobin in end - stage renal disease patients on hemodialysis, but a similar study has not been done in icu patients. after 1 month of treatment, intravenous iron alone was sufficient to increase hematocrit in patients with ckd, but the response was less than that noted in the group administered erythropoietin plus iron. in orthopedic surgery patients, those treated with iron alone did not experience a significant increase in erythropoietic variables over baseline values, and the effects of erythropoietin were seen irrespective of the route (oral or intravenous) of iron administration. two studies of erythropoietin in the critically ill were conducted in which patients were treated with iron therapy, but neither of these two studies addressed the preferred route of iron administration. oral iron (at least 150 mg / day elemental iron) or parenteral iron (if unable to take oral formulation or inadequate response to iron therapy defined by transferrin saturation < 20% and serum ferritin < 100 ng / ml) were administered to all patients starting on study day 1 for 24 weeks. the question has been raised as to whether iron therapy is really needed as an adjuvant to erythropoietin therapy. iron supplementation was found unnecessary in one small, limited observational study of critically ill surgical patients undergoing erythropoietin treatment, because iron store deficiency was not noted during the course of their icu stay. however, the majority of clinical evidence shows that, if properly balanced, iron therapy can help to optimize erythropoietin treatment. furthermore, iron deficiency is also the most common cause of resistance to erythropoietin treatment in ckd. finally, adjuvant therapy, such as ascorbic acid, to increase oral iron absorption and physiologic utilization has been used in many clinical ckd settings and must be tested in critically ill patients. another clinical concern related to iron supplementation in the icu is its risk to affect the physiological redox potential. iron is a mineral that is essential to cellular homeostasis, but an excess of iron can affect physiology and lead to cell injury. during biologic stress, free radicals are formed. these radicals can have detrimental effects at different cellular levels (such as nucleic acid modification) and are involved in many biologic processes that can damage lipid and protein membranes. the role of oxidative stress secondary to iron - derived free radicals and depressed antioxidant reserves has also been associated with pathologic damage in humans. free hydroxyl radical formation is catalyzed by iron via the fenton reaction, which involves ferrous iron and peroxide. although the clinical significance of this biologic reaction and the formation of reactive oxygen species is still debated, stress oxidation is certainly a viable reason to avoid iron overload and to monitor iron parameters during iron supplementation and critical illness. biologic tissues exposed to higher concentrations of free iron may be prone to oxidative damage. in summary, clinicians must first understand that the majority of the clinical data available are derived from the ckd population. the final decision to administer supplemental iron will depend ultimately on the etiology of anemia in the critically ill patient. because iron deficiency is noted in a small subset of critically ill patients, iron supplementation following identification of such patients is appropriate. if anemia is multifactorial, as is usually the case in a critically ill patient, then iron alone may not be sufficient to stimulate erythropoiesis but may be useful as an adjunct with erythropoietin. although not specifically evaluated in the critically ill, studies in pregnant females, neonates, and patients with renal disease suggest that higher iron doses are not more effective than lower ones. therefore, until proven otherwise, clinicians should probably monitor iron parameters on a regular basis if they elect to administer iron and erythropoietin therapies concomitantly. based on the ckd population, these parameters may help in optimizing therapy while preventing iron overload. specific populations of icu patients (i.e. those with severe infection or sepsis) may not benefit from iron therapy because of possibly increased risk for iron - mediated infection, although this association is still very controversial and is not based on robust clinical evidence. the optimal dose, route, and timing of iron administration in critically ill patients, especially when given concurrently with erythropoietin therapy, remains an open issue that requires further study. the author is a member of both the speakers bureau and advisory board for ortho biotech products, l.p. acd = anemia of chronic disease / inflammation ; ckd = chronic kidney disease ; icu = intensive care unit ; tpn = total parenteral nutrition. | derangements of iron metabolism may be present in critically ill patients who develop anemia during a stay in the intensive care unit. iron supplementation may be appropriate, especially if an underlying nutritional disorder is present. it may be even more critical to replace iron when erythropoietin therapy is used because of the consumption of iron stores that occurs during heme synthesis. iron therapy is not without risks, and controversy persists regarding the potential for iron overload and infections. clinical trials that define the optimal dose, route, and timing of iron administration in critically ill patients are lacking. however, studies of iron supplementation in chronic kidney disease, pregnancy, and anemia of prematurity may provide some guidance about approaches to treatment. clinical evidence and limitations that can assist clinicians in managing iron therapy in the intensive care unit are presented. |
in recent years, breast augmentation with non - invasive approaches such as autologous fat grafting become more popular. several synthetic materials have been used for breast augmentation, either resorbable, like hyaluronic acid, or non - resorbable, like polyacrylammide hydrogel (paag). however, biomaterial injections for large volume breast augmentation are limited as they presents several limitations involving patient safety and high costs. currently, many efforts have been performed in the field of tissue engineering and regenerative medicine for the realization of an ideal biomaterial to be used as scaffold in combination with stem cells for breast augmentation. nevertheless, to date no synthetic scaffold has been approved to be combined with stem cells for breast augmentation in humans. current techniques for breast augmentation and reconstruction include implant positioning, autologous tissue transfer and fat grafting. however, several synthetic substances are available on the market, and not all of them are approved for medical use. the aim of this study was to share with other plastic surgeons the case of a patient presenting complications after breast augmentation with an unknown synthetic substance containing methacrylate. a 33 years old chinese patient came to our institution presenting breast deformities, lumps, and chest pain (fig. the initial manifestation was pain, swelling and right breast leakage of a yellow material. a thorough examination of the patient s history revealed previous breast augmentation performed in china six years before through bilateral breast injections with an unknown substance. she referred several times to the procedure as lipofilling with hospital fat but when asked, she affirmed to have received neither liposuction nor fat excision because she were too slim. the patient was not aware about the details of the procedure, and language barriers further limited the communication. on examination, breasts resulted asymmetric with irregular contours and visible lumps. the right breast appeared deflated and smaller then the left one, and presented an hyperchromic scar above the infra - mammary fold indicated by patient as the site of previous breast discharge. the left breast was larger then the right one, presenting edema, firmness and visible lumps. the most noticeable was located medially, showing gel migration toward sternal region (fig. palpation of the right breast did not detect any particular sign, while in the left breasts it produced intense pain. under pressure palpation lumps could be displaced producing a sense of fluctuation, and the indentation persists after releasing the pressure. before operative procedure, the implanted material was localized with ultrasounds. the patient was informed that complete removal of the substance would likely be not possible in one session, therefore she was encouraged to come back if symptoms would persist or represent. an open procedure under general anesthesia was undertaken, and surgical drainage was performed through hemi - periareolar incisions. in the right breast 2) while the rest of the material was drained and squeezed out (fig. the implant pocket was realized in the no gel plane, and implants were placed in the submuscular plane. after surgery, the patient was invited to multiple follow - up controls including clinical examinations and breast ecography. patient s recovery was uneventful, with no recurrence during 30 months of follow - up (fig. the dispersion was centrifuged and the precipitate was subjected to solubility test in organic solvents, re - suspended in acetone and n - hexane, boiled in sodium dodecyl sulfate (sds) in the presence of -mercaptoethanol, and, after centrifugation, the precipitate was treated with sulfuric acid. chemical analysis revealed that no protein band and peptide were detectable and that the material had an organic nature not constituted by the silicone polymer. over the past decades, plastic surgeons have searched for an ideal biological or synthetic substance for breast augmentation. several non - absorbable materials are available on the market and few of those contain acrylates, a family of polymers made from acrylate monomers, including acrylammide (in the form of polyacrylammide hydrogel paag) or methacrylate (in the form of polymethyl - methacrylate pmma, ethyl - methacrylate ema or hydroxyethyl - methacrylathe hema). products containing methacrylate are commercialized as artefill (suneva medical, san diego, usa), dermalive (dermatech, france), metacrill (private lab, rio de janeiro, brazil), and rhegecoll (dermabiol institute of kuhra vital gmbh, lucerne, switzerland) ; among those only artefill is approved by fda for nasolabial folds. nevertheless, several unapproved manufactured materials can be available for injections in many regions like china, latin america or eastern europe, and often their components remain unknown. to the best of our knowledge, this is the first case reporting the use of methacrylate for large volume injections to the breast : in the medical literature several studies describing breast injection with permanent fillers are available, including paag, dimethylpolisiloxane (liquid silicon) and paraffin, but no data on the use of methacrylate for breast augmentation have been previously published. the only reported breast application of methacrylate consisted on small volume injections to the nipple in order to improve its projection after breast reconstruction. one of the most challenging aspects for a surgeon facing this cohort of patients is the inability to completely remove the injected material. some authors argue that permanent fillers in the breast, like paag, can be easily removed by aspiration with a suction cannula. nevertheless, we considered blunt aspiration extremely difficult because of the semisolid consistence of methacrylate and also unpredictable as it might leaves residual material in the breast tissues, therefore an open procedure with surgical debridement is recommended. the inflammation of mammary ducts during lactation in patients with permanent fillers has been previously reported after paag injection, and it has been postulated that the blocked lactiferous ducts may lead to inflammation and galactocele formation. furthermore, we hypothesize that the mechanical suction performed by the child during lactation can cause material displacement in the breast tissue and rupture of the capsule that surrounds it, causing material leakage and inflammation. therefore, asymptomatic patients who underwent breast injection with permanent biomaterials should avoid breastfeeding. injection of synthetic non resorbable biomaterials to the breast can causes irreversible damages, necessitating debridement procedures and breast reconstruction. with a growing demand for non - invasive cosmetic surgery, a growing population of untrained and unlicensed personnel performing cosmetic surgery has been reported in many countries where there are no laws that restrict the use of cosmetic procedures to physicians with appropriate training and with approved materials. in north america and western europe experience with non - resorbable filler for breast augmentation however, migratory streams might expose surgeons without any experience with these injections to manage patients with the related complications. written informed consent was obtained from the patient for publication of this case report and accompanying images. | highlightsdifferent biomaterials have been used for breast augmentation, including paraffin, dimethilpolysiloxane (liquid silicon), polyacrylammyde hydrogel and hyaluronic acid.in many countries, unregulated manufactured materials are available for biomedical use.permanent filler to the breast can lead to severe complications.complete removal of permanent fillers is challenging and require open surgical procedure.female patients who underwent permanent injection to the breast should avoid breast feeding. |
over the past 10 years, small handheld personal digital assistants (pdas) were replaced by the popular smartphone but now by devices with a much larger landscape tablet computers, such as the apple ipad and samsung galaxy tablet. mobile technology has rapidly become the norm in the preclinical years of medical education because it affords flexible, wireless, and mobile access to endless amounts of medical content and knowledge (5). since 2009, a growing number of medical schools have implemented mobile device medical curriculums (6). many of these medical schools purchase devices for students and pre - load them with electronic textbooks and the medical school curriculum. other medical schools simply require or recommend mobile devices to students for purchase (7). the ways in which mobile technologies are being used in clinical practice is also expanding. at least 75% of us physicians are currently using ios mobile devices and technology (8). healthcare organizations are now integrating their electronic health records (ehrs) with mobile devices, such as the ipad. the use of ipads has been embraced by chief medical information officers (cmio) in many hospitals across the country and has created a more efficient workflow for practitioners (9). this expansion of access has afforded ready availability of the ehr at the bedside for patient education, electronic prescription writing, laboratory, and x - ray order as well as access to evidence - based practice tools for patient care (10). with thousands of medical apps available at the tap of a finger on the screen, the ipad has enabled even more opportunities for evidence - based decision support in real - time at the point of care. research related to the use of mobile technology for patient care is not new (11). while initial reports of ipad use by medical professionals are promising (12), there are no in - depth research studies (i.e., beyond questionnaires) that explore the use of the ipad in clerkships by third - year medical students. the purpose of this year - long mixed methods study was to further understand medical students use of the ipad during their internal medicine clerkship. specifically, we sought to answer the following questions : in what ways did the students use mobile technology for learning and clinical decision support?what apps did the students use in the care of patients?did the amount of time spent and the students expertise in using mobile technology grow overtime ? in what ways did the students use mobile technology for learning and clinical decision support ? what apps did the students use in the care of patients ? did the amount of time spent and the students expertise in using mobile technology grow overtime ? following approval by the university of georgia institutional review board, we invited 37 third - year medical students from the partnership medical school campus (gru / uga medical partnership) who rotated to a local community hospital (st. mary 's hospital) to participate in the study. all students were issued a wi - fi only, third - generation ipad with 64 gb of storage. management of the ipads was enabled by a mobile device management (mdm) program that required passcodes, provided lost device tracking and remote device erasure capabilities. all students were required to sign an ipad agreement attesting to the terms and conditions when using the ipad to be hipaa compliant. students were free to install additional applications on their ipads purchased with their own funds. each device was pre - loaded with a variety of applications (e.g., pdf expert, visualdx, penultimate), as well as bookmark links to pubmed, the medical school library and medlineplus. training by members of the research team involved extensive review of the technology as well as case - based scenarios to practice use of the apps. broad topics covered during the training included : 1) basic use of the ipad, 2) utilization of productivity apps, and 3) how to access appropriate medical knowledge resources and apps for clinical decision support. this year - long mixed methods study involved quantitative and qualitative data collection (13) from 37 third - year medical students. four data collection instruments were used : 1) beginning and end - of - year questionnaires, 2) ipad usage logs, 3) weekly rounding observations, and 4) weekly semi - structured medical student interviews. the team of five researchers collected data over a 12-month period (july 2012june 2013). we administered a baseline questionnaire prior to beginning the internal medicine clerkship which included demographic questions and eight additional questions assessing the students past and present use of mobile technology and apple computers. a second questionnaire was administered at the completion of the 48 weeks of clerkships and included demographics questions and additional questions that related to their experience with the ipad over the past year. students logged weekly the types and amount of time used on medical resources and apps on the ipad in the care of patients. the time used for the apps ranged from 1 min to a few hours depending on the application. we conducted weekly, 1-hour observations at the hospital as students rounded with their preceptors. researchers were able to observe directly how the ipad was being utilized in real - time and recorded notes on a structured observation log. weekly semi - structured, one - on - one interviews (face - to - face, phone or facetime) were conducted with each medical student during the internal medicine clerkship. the end - of - the week interviews with students ranged from 10 to 30 min. descriptive statistics were generated for the questionnaires as well as for the apps and resources reported in the ipad usage and observation logs. inductive analysis (14) was used by a member of the research team to code the open - ended responses on the ipad usage and observation logs, as well as the weekly medical student interviews. following a robust data analysis protocol using microsoft word (15) by a member of the research team, following approval by the university of georgia institutional review board, we invited 37 third - year medical students from the partnership medical school campus (gru / uga medical partnership) who rotated to a local community hospital (st. mary 's hospital) to participate in the study. all students were issued a wi - fi only, third - generation ipad with 64 gb of storage. management of the ipads was enabled by a mobile device management (mdm) program that required passcodes, provided lost device tracking and remote device erasure capabilities. all students were required to sign an ipad agreement attesting to the terms and conditions when using the ipad to be hipaa compliant. students were free to install additional applications on their ipads purchased with their own funds. each device was pre - loaded with a variety of applications (e.g., pdf expert, visualdx, penultimate), as well as bookmark links to pubmed, the medical school library and medlineplus. training by members of the research team involved extensive review of the technology as well as case - based scenarios to practice use of the apps. broad topics covered during the training included : 1) basic use of the ipad, 2) utilization of productivity apps, and 3) how to access appropriate medical knowledge resources and apps for clinical decision support. this year - long mixed methods study involved quantitative and qualitative data collection (13) from 37 third - year medical students. four data collection instruments were used : 1) beginning and end - of - year questionnaires, 2) ipad usage logs, 3) weekly rounding observations, and 4) weekly semi - structured medical student interviews. the team of five researchers collected data over a 12-month period (july 2012june 2013). we administered a baseline questionnaire prior to beginning the internal medicine clerkship which included demographic questions and eight additional questions assessing the students past and present use of mobile technology and apple computers. a second questionnaire was administered at the completion of the 48 weeks of clerkships and included demographics questions and additional questions that related to their experience with the ipad over the past year. students logged weekly the types and amount of time used on medical resources and apps on the ipad in the care of patients. the time used for the apps ranged from 1 min to a few hours depending on the application. we conducted weekly, 1-hour observations at the hospital as students rounded with their preceptors. researchers were able to observe directly how the ipad was being utilized in real - time and recorded notes on a structured observation log. weekly semi - structured, one - on - one interviews (face - to - face, phone or facetime) were conducted with each medical student during the internal medicine clerkship. the end - of - the week interviews with students ranged from 10 to 30 min. descriptive statistics were generated for the questionnaires as well as for the apps and resources reported in the ipad usage and observation logs. inductive analysis (14) was used by a member of the research team to code the open - ended responses on the ipad usage and observation logs, as well as the weekly medical student interviews. following a robust data analysis protocol using microsoft word (15) by a member of the research team, all of the students indicated they used the ipad in a variety of ways to assist in the care of patients. these activities are intimately linked ; therefore, the results are reported together in two major areas : 1) clinical decision support and 2) student learning and productivity. the students reported using the ipad at all stages of patient care : before, during, and after patient encounters. two primary uses were indicated in the data : obtaining real - time patient data via the ehr and finding additional information for clinical decision support. most students, as reported in all four sources of data, indicated daily use of the ehr to obtain real - time patient data. this use is well summarized in these quotes from weekly interviews when talking about the use of the ipad on rounds : i feel like when we 're doing rounds, to have access to the medical record if there 's a lab value or something that i forgot to check or needed to pull up, i can use the ipad, what 's useful for that is i can use it to look up stuff which is really helpful on rounds when i have to go present my patients i 've used it a lot for accessing the medical records. i feel like when we 're doing rounds, to have access to the medical record if there 's a lab value or something that i forgot to check or needed to pull up, i can use the ipad, what 's useful for that is i can use it to look up stuff which is really helpful on rounds when i have to go present my patients i 've used it a lot for accessing the medical records. these quotes as well as the other data indicate the value of the ipad for enabling real - time access to the ehr to assist with clinical decision support. identifying medical knowledge resources for clinical decision support various medical knowledge resources were used including library resources and a multitude of ipad apps. library resources were the fifth highest used resource as reported by 65% of the students on the end - of - year questionnaire. as a student detailed in an interview : i mainly used it to get access to the journals and i did n't need to get it through the school [physically ]. and so what was very helpful from the library was that by logging on, i could get the pdf [of the article ]. i mainly used it to get access to the journals and i did n't need to get it through the school [physically ]. and so what was very helpful from the library was that by logging on, i could get the pdf [of the article ]. more details about use of apps on the ipad are reported later in the article ; the data from the interviews indicated that easy and ready access to information was an important use of the ipad by the students in their internal medicine clerkship. while patient care accounted for the vast majority of the use of the ipad, students also indicated using the ipad for personal learning and productivity throughout the day. the ipad usage and observation logs, and end - of - year questionnaire data indicated that the highest productivity use of the ipad were email, note taking and word processing (e.g., quickoffice, notepad). the high value of easy access to productivity tools was also explained during weekly interviews, for example : i had been using it primarily as a study tool with my pdfs on it i was still doing research through first consult, dynamed and epocrates but now i 'm taking patient histories on my wireless keyboard on the ipad and i 'm presenting the patient histories off of the ipad to the preceptor, as well as just like doing research on the go rather than sitting at home using it. i had been using it primarily as a study tool with my pdfs on it i was still doing research through first consult, dynamed and epocrates but now i 'm taking patient histories on my wireless keyboard on the ipad and i 'm presenting the patient histories off of the ipad to the preceptor, as well as just like doing research on the go rather than sitting at home using it. the most commonly reported resources included question banks (e.g., usmle, kaplan), medical knowledge resources, and documents related to the medical school curriculum. as students explained during weekly interviews and in the end - of - year questionnaires : i use my ipad as an ebook reader for my textbooks.the ipad was very useful for doing practice questions and reading while studying.i use it for studying, i read my textbooks on there and i do review questions, because i can carry it around and have an infinite number of review questions, and my textbooks, anywhere in the hospital. i use it for studying, i read my textbooks on there and i do review questions, because i can carry it around and have an infinite number of review questions, and my textbooks, anywhere in the hospital. as the data indicated, the students reported multiple uses of their ipads for personal learning and productivity. the third - year medical students used a multitude of apps for clinical decision support in the care of patients. for example, the top apps reported in the end - of - year questionnaires are summarized in fig. the top three apps reported as widely used by the students on the end - of - year questionnaire included epocrates, pdfexpert, and visualdx. top apps recorded in the ipad usage logs included micromedex, dynamed, and epocrates. twenty - three of the 37 students also reported loading additional apps, with the top three being : 1) first consult, 2) drawmd, and 3) usmle world q bank. student - loaded apps reported by more than one student are listed in table 1. additional apps loaded by more than one medical student onto the ipad the overall usefulness of the apps and ipad for clinical decision support is best conveyed in open - ended responses to the end - of - year questionnaire as well as in the weekly interviews. as described by these quotes from students : initially i was using micromedex a lot to review mechanisms or look up side effects of drugs i used visualdx a lot when looking at rashes ; i used it a couple of times with patients to see if the images i had looked similar to what their rash initially looked like. as time went by i learned that epocrates was a great tool for looking up recommended treatments and quick facts about diseases ; even helped my preceptors look up recommended dose of medications or alternative treatments for certain situations.the ipad made it easy to look up a patient 's or physician 's question without leaving the room, or within minutes of leaving. the accessibility of information meant we were all more informed more quickly, and that our knowledge of labs, cultures, and studies could always be up to date at the time of rounding initially i was using micromedex a lot to review mechanisms or look up side effects of drugs i used visualdx a lot when looking at rashes ; i used it a couple of times with patients to see if the images i had looked similar to what their rash initially looked like. as time went by i learned that epocrates was a great tool for looking up recommended treatments and quick facts about diseases ; even helped my preceptors look up recommended dose of medications or alternative treatments for certain situations. the ipad made it easy to look up a patient 's or physician 's question without leaving the room, or within minutes of leaving. the accessibility of information meant we were all more informed more quickly, and that our knowledge of labs, cultures, and studies could always be up to date at the time of rounding as illustrated in these quotes, not only were students using the ipad to assist their own learning, they used them to assist with patient education and to provide real - time access to information for their preceptors. all of the students indicated they used the ipad in a variety of ways to assist in the care of patients. these activities are intimately linked ; therefore, the results are reported together in two major areas : 1) clinical decision support and 2) student learning and productivity. the students reported using the ipad at all stages of patient care : before, during, and after patient encounters. two primary uses were indicated in the data : obtaining real - time patient data via the ehr and finding additional information for clinical decision support. most students, as reported in all four sources of data, indicated daily use of the ehr to obtain real - time patient data. this use is well summarized in these quotes from weekly interviews when talking about the use of the ipad on rounds : i feel like when we 're doing rounds, to have access to the medical record if there 's a lab value or something that i forgot to check or needed to pull up, i can use the ipad, what 's useful for that is i can use it to look up stuff which is really helpful on rounds when i have to go present my patients i 've used it a lot for accessing the medical records. i feel like when we 're doing rounds, to have access to the medical record if there 's a lab value or something that i forgot to check or needed to pull up, i can use the ipad, what 's useful for that is i can use it to look up stuff which is really helpful on rounds when i have to go present my patients i 've used it a lot for accessing the medical records. these quotes as well as the other data indicate the value of the ipad for enabling real - time access to the ehr to assist with clinical decision support. identifying medical knowledge resources for clinical decision support various medical knowledge resources were used including library resources and a multitude of ipad apps. library resources were the fifth highest used resource as reported by 65% of the students on the end - of - year questionnaire. as a student detailed in an interview : i mainly used it to get access to the journals and i did n't need to get it through the school [physically ]. and so what was very helpful from the library was that by logging on, i could get the pdf [of the article ]. i mainly used it to get access to the journals and i did n't need to get it through the school [physically ]. and so what was very helpful from the library was that by logging on, i could get the pdf [of the article ]. more details about use of apps on the ipad are reported later in the article ; the data from the interviews indicated that easy and ready access to information was an important use of the ipad by the students in their internal medicine clerkship. while patient care accounted for the vast majority of the use of the ipad, students also indicated using the ipad for personal learning and productivity throughout the day. the ipad usage and observation logs, and end - of - year questionnaire data indicated that the highest productivity use of the ipad were email, note taking and word processing (e.g., quickoffice, notepad). the high value of easy access to productivity tools was also explained during weekly interviews, for example : i had been using it primarily as a study tool with my pdfs on it i was still doing research through first consult, dynamed and epocrates but now i 'm taking patient histories on my wireless keyboard on the ipad and i 'm presenting the patient histories off of the ipad to the preceptor, as well as just like doing research on the go rather than sitting at home using it. i had been using it primarily as a study tool with my pdfs on it i was still doing research through first consult, dynamed and epocrates but now i 'm taking patient histories on my wireless keyboard on the ipad and i 'm presenting the patient histories off of the ipad to the preceptor, as well as just like doing research on the go rather than sitting at home using it. the most commonly reported resources included question banks (e.g., usmle, kaplan), medical knowledge resources, and documents related to the medical school curriculum. as students explained during weekly interviews and in the end - of - year questionnaires : i use my ipad as an ebook reader for my textbooks.the ipad was very useful for doing practice questions and reading while studying.i use it for studying, i read my textbooks on there and i do review questions, because i can carry it around and have an infinite number of review questions, and my textbooks, anywhere in the hospital. i use it for studying, i read my textbooks on there and i do review questions, because i can carry it around and have an infinite number of review questions, and my textbooks, anywhere in the hospital. as the data indicated, the students reported multiple uses of their ipads for personal learning and productivity. the students reported using the ipad at all stages of patient care : before, during, and after patient encounters. two primary uses were indicated in the data : obtaining real - time patient data via the ehr and finding additional information for clinical decision support. most students, as reported in all four sources of data, indicated daily use of the ehr to obtain real - time patient data. this use is well summarized in these quotes from weekly interviews when talking about the use of the ipad on rounds : i feel like when we 're doing rounds, to have access to the medical record if there 's a lab value or something that i forgot to check or needed to pull up, i can use the ipad, what 's useful for that is i can use it to look up stuff which is really helpful on rounds when i have to go present my patients i 've used it a lot for accessing the medical records. i feel like when we 're doing rounds, to have access to the medical record if there 's a lab value or something that i forgot to check or needed to pull up, i can use the ipad, what 's useful for that is i can use it to look up stuff which is really helpful on rounds when i have to go present my patients i 've used it a lot for accessing the medical records. these quotes as well as the other data indicate the value of the ipad for enabling real - time access to the ehr to assist with clinical decision support. identifying medical knowledge resources for clinical decision support various medical knowledge resources were used including library resources and a multitude of ipad apps. library resources were the fifth highest used resource as reported by 65% of the students on the end - of - year questionnaire. as a student detailed in an interview : i mainly used it to get access to the journals and i did n't need to get it through the school [physically ]. and so what was very helpful from the library was that by logging on, i could get the pdf [of the article ]. i mainly used it to get access to the journals and i did n't need to get it through the school [physically ]. and so what was very helpful from the library was that by logging on, i could get the pdf [of the article ]. more details about use of apps on the ipad are reported later in the article ; the data from the interviews indicated that easy and ready access to information was an important use of the ipad by the students in their internal medicine clerkship. while patient care accounted for the vast majority of the use of the ipad, students also indicated using the ipad for personal learning and productivity throughout the day. the ipad usage and observation logs, and end - of - year questionnaire data indicated that the highest productivity use of the ipad were email, note taking and word processing (e.g., quickoffice, notepad). the high value of easy access to productivity tools was also explained during weekly interviews, for example : i had been using it primarily as a study tool with my pdfs on it i was still doing research through first consult, dynamed and epocrates but now i 'm taking patient histories on my wireless keyboard on the ipad and i 'm presenting the patient histories off of the ipad to the preceptor, as well as just like doing research on the go rather than sitting at home using it. i had been using it primarily as a study tool with my pdfs on it i was still doing research through first consult, dynamed and epocrates but now i 'm taking patient histories on my wireless keyboard on the ipad and i 'm presenting the patient histories off of the ipad to the preceptor, as well as just like doing research on the go rather than sitting at home using it. the most commonly reported resources included question banks (e.g., usmle, kaplan), medical knowledge resources, and documents related to the medical school curriculum. as students explained during weekly interviews and in the end - of - year questionnaires : i use my ipad as an ebook reader for my textbooks.the ipad was very useful for doing practice questions and reading while studying.i use it for studying, i read my textbooks on there and i do review questions, because i can carry it around and have an infinite number of review questions, and my textbooks, anywhere in the hospital. i use it for studying, i read my textbooks on there and i do review questions, because i can carry it around and have an infinite number of review questions, and my textbooks, anywhere in the hospital. as the data indicated, the students reported multiple uses of their ipads for personal learning and productivity. the third - year medical students used a multitude of apps for clinical decision support in the care of patients. for example, the top apps reported in the end - of - year questionnaires are summarized in fig. the top three apps reported as widely used by the students on the end - of - year questionnaire included epocrates, pdfexpert, and visualdx. top apps recorded in the ipad usage logs included micromedex, dynamed, and epocrates. twenty - three of the 37 students also reported loading additional apps, with the top three being : 1) first consult, 2) drawmd, and 3) usmle world q bank. student - loaded apps reported by more than one student are listed in table 1. additional apps loaded by more than one medical student onto the ipad the overall usefulness of the apps and ipad for clinical decision support is best conveyed in open - ended responses to the end - of - year questionnaire as well as in the weekly interviews. as described by these quotes from students : initially i was using micromedex a lot to review mechanisms or look up side effects of drugs i used visualdx a lot when looking at rashes ; i used it a couple of times with patients to see if the images i had looked similar to what their rash initially looked like. as time went by i learned that epocrates was a great tool for looking up recommended treatments and quick facts about diseases ; even helped my preceptors look up recommended dose of medications or alternative treatments for certain situations.the ipad made it easy to look up a patient 's or physician 's question without leaving the room, or within minutes of leaving. the accessibility of information meant we were all more informed more quickly, and that our knowledge of labs, cultures, and studies could always be up to date at the time of rounding initially i was using micromedex a lot to review mechanisms or look up side effects of drugs i used visualdx a lot when looking at rashes ; i used it a couple of times with patients to see if the images i had looked similar to what their rash initially looked like. as time went by i learned that epocrates was a great tool for looking up recommended treatments and quick facts about diseases ; even helped my preceptors look up recommended dose of medications or alternative treatments for certain situations. the ipad made it easy to look up a patient 's or physician 's question without leaving the room, or within minutes of leaving. the accessibility of information meant we were all more informed more quickly, and that our knowledge of labs, cultures, and studies could always be up to date at the time of rounding as illustrated in these quotes, not only were students using the ipad to assist their own learning, they used them to assist with patient education and to provide real - time access to information for their preceptors. in the end - of - year questionnaire, the majority of the third - year medical students (71% ; n=20) reported that the amount of time they spent using the ipad for clinical decision support grew overtime (see fig. strongly agree or agree, the majority (58% ; n=11) reported using their ipad many times a day. the 18% of students who disagreed provided insights into why their use did not grow, ranging from the size and weight of the ipad to the use of other electronic devices (e.g., desktop computers, iphone). students also reported that the fast pace of rounds or lack of access to a stable and consistent wireless network impacted their use. amount of time spent and perceived expertise using the ipad the majority of the third - year medical students (75% ; n=21) also reported that their expertise in using the ipad for clinical decision support grew overtime (see fig. all students (n=28) indicated that their level of expertise in using the ipad at the end - of - year was expert or intermediary. those who indicated strongly agree or agree provided insights as to why their expertise grew over time. one major theme was that the students comfort level with the use and navigation increased over time, contributing to the growth in expertise using the ipad. i did become much more comfortable using a number of medical apps ; i also felt more comfortable using my ipad with my preceptor around. secondly, students indicated that they learned how to use specific apps to meet specific needs. as described by a student : i learned to use the apps that were appropriate for each case more effectively. as time went by i knew which apps would have the information that i was looking for. as time went by i knew which apps would have the information that i was looking for. expertise in using the ipad allowed students to use the ipad in various clinical settings beyond internal medicine. as one student reported : by the end of the year i was able to use the ipad in any clinical setting. initially started as a personal reference tool, then a presentation tool with peers and preceptors, then a patient education tool. the students not only became more comfortable with use, some also started thinking about the use of the ipad as just what you do : everyday use of the ipad is a habit now, and i do not have to think about where to find information and how to use it. the 11% (n=3) who disagreed with their expertise growing over time stated they did not choose to use the ipad (e.g., worried about losing it, like paper and books, do n't have time to learn how to use it). while it is important to recognize that not all students grew in use and/or expertise with the ipad, it is noteworthy that the majority of the students did grow in both areas. this is promising for continued use of mobile technologies for real - time clinical decision support. this year - long, mixed methods study provides substantial evidence that medical students used mobile technology to support their clinical decision support and learning in the care of patients. although numerous studies have been conducted using surveys as their primary methodology, this study significantly extends the data collected by studying the use of the ipad using weekly usage and observation logs, interviews and questionnaires. this study supports the consistent conclusions of multiple studies 14, (16), that tablet computers are being used to enhance patient care and learning in clinical contexts for students and residents. this study finds that students primary uses were to obtain real - time patient data via the ehr during rounds and accessing other medical knowledge resources (e.g., apps, library databases, e - textbooks) to support clinical decisions and personal learning. in addition, students used mobile technology to access productivity apps for note taking during and after rounds, e - mail, and studying. these uses were generally consistent with the findings of a national survey (16) of third - year us medical students that found the highest uses for medical reference apps (47%), usmle preparation (49%), and e - books (43%), but relatively lower use for ehr access (23%). in contrast yet consistent with ellaway 's (17) findings that student choices are shaped by the learning context, evidence from this study indicates that the ehr use was much higher because it was conducted in a hospital setting and preceptors also were given ipads. one supposition is that the tablet computers ability to display large, high - quality images enables a clearer reading of the ehr, radiographic images, as well as articles and e - textbooks than do other mobile devices (2, 18) providing the necessary affordances to use mobile technology for real - time clinical decision support. this study found that the primary applications that students used for clinical decision support were epocrates, pdf expert, micromedex, dynamed, and visualdx in addition to more traditional library resources. the students indicated that the use of the apps enabled easy access to information for clinical decision support and their learning by connecting knowledge to real - time needs. as found in the boruff and storie study (2), epocrates was a favorite resource because it provided quick facts on diseases, drugs and treatments. integrating mobile technology into the medical student 's daily workflow was essential for learning and clinical decision support. consistent with boruff and storie 's (2) finding that 70% of third- and fourth - year medical students used mobile devices at least once a day, the majority of the students in this study reported using their ipad many times a day as it became a seamless part of their work. the primary reasons indicated for the students increased usage were that practice using the ipad shortened access time, increased their comfort level using it in patient care, and improved their knowledge of useful resources and apps. the importance of practice using the ipad is supported by lombardo and honisett 's (19) results from a pediatric clerkship that found 70% of students agreed that the ipad was useful in achieving the learning objectives of the clerkship and 84% agreed the technology skills acquired by using the ipad would be useful in future medical careers. in our study, the students who did not report an increase in use and expertise indicated alternative ways to access information. this study has several limitations : 1) the 37 students were the first class to rotate in one community hospital during a third - year internal medicine clerkship, 2) the wireless network in the community hospital that served all users (including patients and visitors) was unstable and lacked the robustness to support consistent mobile technology use, and 3) the ipad usage logs were self - report and could have been more accurate with automatic tracking of use. the current study indicates that developing expertise in using mobile technology and various apps was critical for effective and efficient support of real - time clinical decisions. although this study used ios devices, certainly other mobile technology devices (e.g., windows, android) may have produced similar results. however, due to the limited medical - related apps on other platforms, healthcare providers are choosing ios devices (8). several recommendations result from the study and include : 1) encouraging medical students to use mobile technology to access medical knowledge resources including the ehr ; 2) providing data service capability and mid - level storage capacity on each device ; 3) integrating quarterly app training to increase effectiveness in clinical decision support ; and 4) providing tablets that fit into a white coat pocket. future research could include the impact of mobile technology on 1) patient outcomes and length of stay, 2) establishing patient / doctor rapport, 3) student performance on shelf tests and usmles, and 4) the preceptors experience with students use of mobile technology. ethical approval was granted by the institutional review board at the university of georgia, april 2012, 2012 - 108951. initial results of this study were presented at the following events : elearn, october 2012, montreal, quebec, canadainstitute for evidence - based health professions, january 2013, athens gacollege of education, uga faculty research conference, february 2013, athens, gast mary 's continuing medical education, march 2013, athens, gaamerican educational research association, april 2014, philadelphia, pa elearn, october 2012, montreal, quebec, canada institute for evidence - based health professions, january 2013, athens ga college of education, uga faculty research conference, february 2013, athens, ga st mary 's continuing medical education, march 2013, athens, ga american educational research association, april 2014, philadelphia, pa the authors have not received any funding or benefits from industry or elsewhere to conduct this study. | purposedespite widespread use of mobile technology in medical education, medical students use of mobile technology for clinical decision support and learning is not well understood. three key questions were explored in this extensive mixed methods study : 1) how medical students used mobile technology in the care of patients, 2) the mobile applications (apps) used and 3) how expertise and time spent changed overtime.methodsthis year - long (july 2012june 2013) mixed methods study explored the use of the ipad, using four data collection instruments : 1) beginning and end - of - year questionnaires, 2) ipad usage logs, 3) weekly rounding observations, and 4) weekly medical student interviews. descriptive statistics were generated for the questionnaires and apps reported in the usage logs. the ipad usage logs, observation logs, and weekly interviews were analyzed via inductive thematic analysis.resultsstudents predominantly used mobile technology to obtain real - time patient data via the electronic health record (ehr), to access medical knowledge resources for learning, and to inform patient care. the top four apps used were epocrates, pdf expert, visualdx, and micromedex. the majority of students indicated that their use (71%) and expertise (75%) using mobile technology grew overtime.conclusionsthis mixed methods study provides substantial evidence that medical students used mobile technology for clinical decision support and learning. integrating its use into the medical student 's daily workflow was essential for achieving these outcomes. developing expertise in using mobile technology and various apps was critical for effective and efficient support of real - time clinical decisions. |
interaction - dependent pcr (idpcr) is a solution - phase method to identify binding partners from combined libraries of small - molecule ligands and targets in a single experiment. binding between dna - linked targets and dna - linked ligands induces formation of an extendable duplex. extension links codes that identify the ligand and target into one selectively amplifiable dna molecule. in a model selection, idpcr resulted in the enrichment of dna encoding all five known proteinligand pairs out of 67 599 possible sequences. |
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patients with cervical spinal cord injury or stroke often have impaired proprioception of upper limbs and reduced muscle strength and range of movement (rom). proprioception training, muscle strength training (mst), and rom training are usually implemented in physical therapy programs for patients with cervical spinal cord injury and stroke1. a number of research studies have focused on increasing muscle strength and/or rom of upper limbs. after stroke, the function of the hemiplegia side is always greatly affected and part or all of the upper limb s motor function disappears. therefore, the handedness exchange is very important for patients with hemiplegia to improve activities of daily living. patients subdominant hands proprioception training has been used in clinics for handedness exchange. and after cervical spinal cord injury, patients upper limb proprioception is evaluated to know the extent of the disability and its effects on activities of daily living. besides, it has also been used as a part of treatment for cervical spinal cord injury as well as practical functional treatment in occupational therapy rehabilitation programs. along with muscle strength, proprioception sense is also decreased in patients with cervical spinal cord injury or stroke. the joint position error (jpe) test is considered the primary measure of upper limb proprioception and has been widely used as an outcome indicator for patients with cervical spinal cord injury and hemiplegia2.. in clinical treatments and researches, resistance to wrist joint flexion is used to enhance the strength of the radialis and ulnar extensor muscle of the wrist, whereas in clinical treatments, isotonic contraction is used. neuromuscular joint facilitation (njf) is a new therapeutic exercise based on kinesiology that integrates the facilitation element of proprioceptive neuromuscular facilitation (pnf) and joint composition movements, aiming to improve movements of the joint through passive, active, and resistance exercises4. njf is used to increase strength, flexibility and rom5, and improve wrist joint functions. njf uses the same motion pattern as pnf, but the location of resistance of njf is different. the proximal resistance is applied to the scaphoid or triangular bone in wrist patterns. the purpose of this study was to examine immediate effects of strength training and njf distal resistance training in wrist joints by using writing time and evaluation of proprioception using the jpe test. the subjects characteristics are detailed in table 1table 1.subject characteristicsm sd n=24age (yrs)24.2 3.1height (cm)169.7 6.5weight (kg)65.3 12.6. all of the subjects were right - handed. the purpose and contents of this research were explained to the subjects, and they gave their informed consent to participate in the study. the study was approved by the research ethics committee of china rehabilitation research center (irb no. the subjects sat on chairs and placed their left upper limbs on tables in front. the subjects shoulder joints were flexed at 45 and their elbow joints were flexed at 45. two isotonic contractions were performed on wrist joints extension : the wrist joint extension muscle strength training (mst) and the wrist joint extension pattern of njf. resistance was applied to the highest level possible that allowed subjects to complete the isotonic exercise. 1. the other hand of the examiner was placed on the distal forearm to fixate the wrist joint. 2. njf group : the wrist extension - radial drift (erd) pattern and wrist extension - ulnar drift (eud) pattern of njf were performed. in the erd pattern, one hand of the examiner was placed against the distal second dorsal metacarpal and traction and resistance were increased. the examiner s other hand was on the triangular bone, which was moving distally when wrist joints extended. in the eud pattern, one hand of the examiner was placed against the distal fifth dorsal metacarpal and traction and resistance were increased. the examiner s other hand was on the scaphoid, which was moving distally when wrist joints extended. when the subjects performed the wrist joint extension pattern, traction and resistance were applied throughout the process by two hands. in both mst and njf groups, the interventions were carried out ten times. in the njf, traction and resistance were applied five times on the scaphoid and five times on the triangular bone6. there was a 1-hour rest period between interventions in the mst and njf groups, and two interventions and tests were performed within 1 day. all trials were performed at random. before and after one intervention of mst and njf, jpe and the writing time were measured. in the jpe test, the rom measurement equipment (bioval 4.51, sycomore 8.51, rm ingenierie, france) was used. the fixed and mobile arms were the vertical axis of the radius and fifth metacarpal ; the extension angles of wrist joints were recorded by the computer. the subjects were asked to close their eyes, and the examiner extended the subject s wrist joints at random angles. the subjects wrist joints were put back to tables, and they were asked to extend their wrists at approximately the same angles as in the initial extension. each measurement was carried out five times, and the average value was used for analysis. in the writing time evaluation, the subjects were asked to write the uppercase english alphabet by left hand on a4 paper. there were writing grids of which each area was 1 cm on a4 paper. two - way repeated - measures analysis of variance (anova) was used to test for statistically significant differences, and the factors were intervention and group. if any significant interaction was found, the paired t - test was performed to compare the outcome indicators before and after the intervention. two - way anova revealed significant interactions among the jpes and the writing time of the two groups, indicating that the changes between the groups were significantly different (table 2table 2.intervention effects of the jpe and writing time of different treatments for the wrist jointthe error of wrist flexion angle ()the writing time (s)a. befor of mst group5.8 d41.3 9.1c > dd. after of njf group1.4 0.533.4 2.3p<0.05 ; p<0.01). the errors in wrist extension angle repetition were reduced, and the writing time was increased by njf intervention. compared with that of the mst group, the njf group s errors in wrist extension angle repetition were reduced, and the writing time was decreased significantly. these results can be attributed to the improvement in functions of periarticular muscles of subdominant hands wrist joints due to the application of the proximal resistance. the alignment of the wrist joint capsule, the functions of periarticular muscles of wrist joints, and the wrist position sense were improved ; therefore, the jpe and the writing time were decreased in the njf group. in the njf resistance exercise, the midcarpal joints were hustled using the proximal resistance on the scaphoid or triangular bone7. these results suggest that the wrist joint proprioception and functions can be improved by njf together with proximal resistance training, which can be used as a new form of exercise for improving functions of subdominant hands wrist joints. | [purpose ] the purpose of this study was to examine immediate effects of strength training and njf distal resistance training in wrist joints by using writing time and evaluation of proprioception using the jpe test. [subjects and methods ] the subjects were 12 young healthy people (24.2 3.1 y, 169.7 6.5 cm, 65.3 12.6 kg). two isotonic contraction techniques were applied on the wrist joint : wrist joint extension muscle strength training (mst) and the wrist joint extension pattern of njf. the uppercase english alphabet writing time and joint position errors of the left upper limb were measured before and after one intervention session of mst and njf. [results ] the decrease in errors in wrist extension angle repetition and the writing time represented the improvement resulting from njf. [conclusion ] this result suggests that the subdominant hands wrist joint proprioception and writing function can be improved by njf together with proximal resistance training. |
the present analysis is based on data from the dortmund nutritional and anthropometric longitudinally designed study (donald), an ongoing open cohort study conducted at the research institute of child nutrition in dortmund, germany (12). briefly, since 1985, detailed data on diet, growth, development, and metabolism have been collected from > 1,300 healthy children. participants are recruited in the city of dortmund and surrounding communities via personal contacts, maternity wards, or pediatric practices. on average, 40 infants are newly recruited every year and first examined at the age of 3 months. each child returns for three more visits during the first year, two in the second, and then annually until adulthood. since 2005, participants over the age of 18 years are invited for subsequent examinations with fasting blood withdrawal. the study was approved by the ethics committee of the university of bonn, and all examinations are performed with written parental and adult participants consent (12). because of the open cohort design, many children had not yet reached younger adulthood, and among those who did age varied from 18 to 36 years. at the time of this analysis, one measurement of insulin and glucose was available for 319 participants (mean age 22.7 years), who were term (3643 weeks gestation) singletons with a birth weight 2,500 g. alt and ggt values were available for 309 participants. of these, 229 participants (for homa analysis) and 221 (for alt and ggt analysis), respectively, had provided at least two plausible 3-day weighed dietary records during the adolescent baseline period (chronological age : girls 914 years, boys 1015 years), allowing the estimation of habitual dietary intake. participants who consistently underreported their energy intake (i.e., they had provided more implausible than plausible food records) were excluded from the study (n = 20) (13). a 3-day weighed dietary record was considered plausible when the total recorded energy intake was adequate in relation to the basal metabolic rate (13). for inclusion in the study sample, participants also had to have anthropometric measures taken in adolescence and adulthood as well as information on relevant covariates. this resulted in a final sample of 226 participants for analysis of insulin or related outcomes and of 214 for the liver enzymes. venous blood samples were drawn after an overnight fast, centrifuged within 15 min, and frozen at 80c in the research institute. for the present analysis, blood samples were transported to the technical laboratory of the german diabetes center to determine serum activities of alt and ggt using the cobas c311 analyzer (roche, mannheim, germany). serum insulin concentrations were measured with an immunoradiometric assay in the laboratory for translational hormone analytics in pediatric endocrinology at the university of giessen. based on these values, homa - ir and secretion (homa of -cell function [homa- ]) were calculated (14). from the age of 2 years onward, standing height is measured to the nearest 0.1 cm using a digital stadiometer (harpenden, crymych, u.k.). body weight is measured to the nearest 100 g with an electronic scale (seca 753e ; seca weighing and measuring systems, hamburg, germany). skinfold thicknesses are measured from the age of 6 months onward at four different sites (suprailiacal, subscapular, biceps, and triceps) on the right side of the body to the nearest 0.1 mm using a holtain caliper (holtain, crosswell, u.k.). waist circumference in younger adulthood was measured at the midpoint between the lower rip and the iliac crest to the nearest 0.1 cm. sex- and age - specific sd scores (sds) were calculated for the adolescent bmi values using the german bmi standards (15). for definition of overweight during puberty, percentage body fat (% bf) for pubescent children was derived using the equations of slaughter. (17), and excess body fatness was defined according to the % bf standard (18). for estimation of % bf in adulthood, equations of durnin and womersley were used (19). during 3 days, the participants or their parents weighed and recorded all foods and beverages consumed as well as leftovers to the nearest 1 g using electronic food scales (initially, soehnle digita 8000 ; leifheit, nassau, germany ; now, wedo digi 2000 ; werner dorsch, mnster / dieburg, germany). for this analysis, dietary variables were calculated as individual means of the 3-day weighed dietary records using lebtab (20), the in - house database. as we aimed to describe the habitual dietary intake, an individual average intake during puberty was calculated from at least two records (average of 5 records per participant). each carbohydrate - containing food recorded in the dietary records was assigned a published gi value (21) (based on glucose as a reference food) according to a standardized procedure (22). the carbohydrate content (in grams) of each consumed food was then multiplied by the food s gi to obtain the respective gl. the overall dietary gi is obtained by dividing total daily gl by total daily carbohydrate intake. the following foods were defined as added sugars : white sugar, brown sugar, raw sugar, corn syrup, corn syrup solids, high - fructose corn syrup, malt syrup, maple syrup, pancake syrup, fructose sweetener, liquid fructose, honey, molasses, anhydrous dextrose, and crystal dextrose (23). whole - grain intake was estimated by assigning whole - grain content in grams to each carbohydrate - containing food using the respective recipe and ingredient information available at the time of recording. the definition of whole grain followed the whole - grain label statements of the u.s. food and drug administration (24). baseline characteristics of the study population are presented by sex - specific tertiles of dietary gi. tests for differences between these tertiles were performed using anova for normally distributed continuous variables, kruskal - wallis test for non normally distributed continuous variables, and test for categorical variables. for analysis of the prospective association between carbohydrate nutrition during puberty and risk markers for type 2 diabetes in younger adulthood, as the outcome variables were not normally distributed, homa - ir was log transformed prior to analysis, and liver enzymes alt and ggt were log transformed twice to obtain normal distribution. all dietary variables except dietary gi were energy adjusted using the residual method. to account for age - dependent nutritional differences, we standardized all variables by age - group and sex (mean sd 0 1). covariates considered as potentially affecting the association between carbohydrate nutrition and risk markers of type 2 diabetes were birth weight, gestational age, breast - feeding for > 2 weeks, firstborn child (yes / no), bmi sds or % bf at baseline, maternal overweight (bmi 25 kg / m), high maternal educational status (12 years of schooling), maternal occupation (yes / no), smoking in the household, parental history of diabetes (yes / no [questionnaire based ]), physical activity level (light, moderate, or high [questionnaire based ]), and intakes of protein (total, animal, or vegetable) and fat (total and saturated fat). vice versa adjustment for added sugar, fiber, and gi was also considered. each potential confounder was initially examined separately and included only if it 1) substantially altered the association of the principal dietary variables with the outcome in the unadjusted models (> 10%), 2) significantly predicted the outcome, or 3) improved the coefficient of determination (> 5%). in the basic model (model a), sex and age were included, since age at blood withdrawal in younger adulthood varied considerably (1835 years). in a second model (model b), we further adjusted for early life and socioeconomic as well as other nutritional factors. finally, we ran a conditional model (additionally including waist circumference in younger adulthood) to assess whether the observed associations are partly attributable to effects of carbohydrate nutrition on body composition. verification of the linear regression modeling assumptions showed that these were appropriate for the analyzed longitudinal data. as associations between carbohydrate nutrition and risk markers of type 2 diabetes did not differ by sex (p for interaction > 0.2), data were pooled for analysis. all statistical analyses were carried out using sas procedures (version 9.1.3 ; sas institute, cary, nc). venous blood samples were drawn after an overnight fast, centrifuged within 15 min, and frozen at 80c in the research institute. for the present analysis, blood samples were transported to the technical laboratory of the german diabetes center to determine serum activities of alt and ggt using the cobas c311 analyzer (roche, mannheim, germany). serum insulin concentrations were measured with an immunoradiometric assay in the laboratory for translational hormone analytics in pediatric endocrinology at the university of giessen. based on these values, homa - ir and secretion (homa of -cell function [homa- ]) from the age of 2 years onward, standing height is measured to the nearest 0.1 cm using a digital stadiometer (harpenden, crymych, u.k.). body weight is measured to the nearest 100 g with an electronic scale (seca 753e ; seca weighing and measuring systems, hamburg, germany). skinfold thicknesses are measured from the age of 6 months onward at four different sites (suprailiacal, subscapular, biceps, and triceps) on the right side of the body to the nearest 0.1 mm using a holtain caliper (holtain, crosswell, u.k.). waist circumference in younger adulthood was measured at the midpoint between the lower rip and the iliac crest to the nearest 0.1 cm. sex- and age - specific sd scores (sds) were calculated for the adolescent bmi values using the german bmi standards (15). for definition of overweight during puberty, percentage body fat (% bf) for pubescent children was derived using the equations of slaughter. (17), and excess body fatness was defined according to the % bf standard (18). for estimation of % bf in adulthood, equations of durnin and womersley were used (19). during 3 days, the participants or their parents weighed and recorded all foods and beverages consumed as well as leftovers to the nearest 1 g using electronic food scales (initially, soehnle digita 8000 ; leifheit, nassau, germany ; now, wedo digi 2000 ; werner dorsch, mnster / dieburg, germany). for this analysis, dietary variables were calculated as individual means of the 3-day weighed dietary records using lebtab (20), the in - house database. as we aimed to describe the habitual dietary intake, an individual average intake during puberty was calculated from at least two records (average of 5 records per participant). each carbohydrate - containing food recorded in the dietary records was assigned a published gi value (21) (based on glucose as a reference food) according to a standardized procedure (22). the carbohydrate content (in grams) of each consumed food was then multiplied by the food s gi to obtain the respective gl. the overall dietary gi is obtained by dividing total daily gl by total daily carbohydrate intake. the following foods were defined as added sugars : white sugar, brown sugar, raw sugar, corn syrup, corn syrup solids, high - fructose corn syrup, malt syrup, maple syrup, pancake syrup, fructose sweetener, liquid fructose, honey, molasses, anhydrous dextrose, and crystal dextrose (23). whole - grain intake was estimated by assigning whole - grain content in grams to each carbohydrate - containing food using the respective recipe and ingredient information available at the time of recording. the definition of whole grain followed the whole - grain label statements of the u.s. baseline characteristics of the study population are presented by sex - specific tertiles of dietary gi. tests for differences between these tertiles were performed using anova for normally distributed continuous variables, kruskal - wallis test for non normally distributed continuous variables, and test for categorical variables. for analysis of the prospective association between carbohydrate nutrition during puberty and risk markers for type 2 diabetes in younger adulthood, multivariable linear regression models were used. as the outcome variables were not normally distributed, homa - ir was log transformed prior to analysis, and liver enzymes alt and ggt were log transformed twice to obtain normal distribution. all dietary variables except dietary gi were energy adjusted using the residual method. to account for age - dependent nutritional differences, we standardized all variables by age - group and sex (mean sd 0 1). covariates considered as potentially affecting the association between carbohydrate nutrition and risk markers of type 2 diabetes were birth weight, gestational age, breast - feeding for > 2 weeks, firstborn child (yes / no), bmi sds or % bf at baseline, maternal overweight (bmi 25 kg / m), high maternal educational status (12 years of schooling), maternal occupation (yes / no), smoking in the household, parental history of diabetes (yes / no [questionnaire based ]), physical activity level (light, moderate, or high [questionnaire based ]), and intakes of protein (total, animal, or vegetable) and fat (total and saturated fat). vice versa adjustment for added sugar, fiber, and gi was also considered. each potential confounder was initially examined separately and included only if it 1) substantially altered the association of the principal dietary variables with the outcome in the unadjusted models (> 10%), 2) significantly predicted the outcome, or 3) improved the coefficient of determination (> 5%). in the basic model (model a), sex and age were included, since age at blood withdrawal in younger adulthood varied considerably (1835 years). in a second model (model b), we further adjusted for early life and socioeconomic as well as other nutritional factors. finally, we ran a conditional model (additionally including waist circumference in younger adulthood) to assess whether the observed associations are partly attributable to effects of carbohydrate nutrition on body composition. verification of the linear regression modeling assumptions showed that these were appropriate for the analyzed longitudinal data. as associations between carbohydrate nutrition and risk markers of type 2 diabetes did not differ by sex (p for interaction > 0.2), data were pooled for analysis. all statistical analyses were carried out using sas procedures (version 9.1.3 ; sas institute, cary, nc). subjects who were excluded from the study sample because of missing information (dietary intake data or covariates) (n = 93) did not differ from those included (n = 226) with respect to early life factors or anthropometric or metabolic characteristics in younger adulthood (data not shown). participants with a higher dietary gi during adolescence were more likely to be exposed to smoking in the household (table 1). there were no other differences in anthropometric, early life, or socioeconomic factors during puberty between the dietary gi tertiles. regarding data from younger adulthood, participants with a higher dietary gi during puberty had higher alt and ggt values (table 1). in terms of nutritional intake data during puberty, those in the higher dietary gi tertiles consumed less (animal) protein, (fruit) fiber, and whole grain, as well as more added sugar, especially from drinks (table 2). demographic, anthropometric, birth, and socioeconomic characteristics by sex - specific tertiles of dietary glycemic index : donald, germany baseline nutritional data by sex - specific tertiles of dietary glycemic index : donald, germany the amount of carbohydrates, dietary gl, added sugar, fiber, and whole - grain intake during puberty was not associated with homa - ir in younger adulthood (table 3). a higher dietary gi during puberty was prospectively related to higher values of homa - ir in multivariable analysis (p for trend = 0.03 [model a ]). this association was not explained by baseline bmi, early life or socioeconomic factors, or protein or fiber intake (p for trend = 0.03 [model b ]). no prospective associations were observed between carbohydrate nutrition and homa- (p for trend 0.2) (data not shown). homa - ir in younger adulthood by tertiles of carbohydrate nutrition parameters during puberty a higher dietary gi was also independently associated (adjustment for baseline bmi and socioeconomic and nutritional factors) with higher values of both alt (p for trend = 0.02 [model b ]) and ggt (p for trend = 0.04 [model b ]) (fig. 1). amount of carbohydrates, dietary gl, total added sugar, dietary fiber, and whole - grain intake were not related to liver enzymes. higher intakes of added sugar from drinks during puberty were independently related to higher levels of ggt in adulthood (p for trend = 0.04 [model b ]) (data not shown). (units / l) (b) levels in younger adulthood by energy - adjusted tertiles of dietary glycemic (gi) (mean dietary gi across tertiles [t ] : tertile 1, 53.5 ; 2, 56.2 ; and 3, 58.5) during puberty (baseline) for 214 subjects. data are geometric means (95% ci) adjusted for sex, age (categorical 19, > 19, 25, and > 25 years), bmi sds at baseline, socioeconomic factors (maternal overweight), energy (residuals), and protein and fiber intake. see the text for results from the conditional model additionally considering waist circumference in younger adulthood. note that the slight u - shape in a results from illustration of least square means by gi tertiles, the association is linear, and all assumptions of linear regression modeling are met. we also examined the association between carbohydrate nutrition and fasting insulin levels ; similarly, this analysis revealed a prospective positive relation for dietary gi only (p for trend = 0.045). further adjustment for breast - feeding status, birth weight, physical activity level, or parental history of type 2 the additional inclusion of waist circumference in adulthood attenuated the associations between dietary gi and risk markers of type 2 diabetes toward a trend (conditional model [table 3 ]). the corresponding mean predicted alt and ggt values in sex - specific tertiles of gi were 16.7 units / l (95% ci 15.318.4), 16.3 units / l (15.017.8), and 18.0 units / l (16.419.9) (p for trend = 0.07) and 14.1 units / l (12.715.7), 14.0 units / l (12.615.5), and 16.6 units / l (14.818.7) (p for trend = 0.09), respectively. this study provides new epidemiological evidence of a detrimental role of postprandial glycemic excursions during puberty for risk markers of type 2 diabetes in younger adulthood. dietary gi was the only feature of carbohydrate nutrition that was consistently related to different diabetes risk markers. as a low - gi diet is characterized by an average of 45 (25), the dietary gi in the present sample (56.0 2.4) can be considered moderate. the association between dietary gi and diabetes risk seen in our study is in accordance with observational evidence in adulthood linking dietary gi to risk of developing type 2 diabetes (3,4). our study is, however, the first to suggest that this association emerges already during puberty. in view of the relatively large 95% cis a 5-unit increase of dietary gi was accompanied by a 9% increase in homa - ir and an 11% increase in alt values. this is in line with evidence from large observational studies, where moderate gi differences between extreme quantiles were also associated with relatively large differences in type 2 diabetes risk (3). importantly, there was no strong correlation between homa - ir, alt, and ggt in our study (r < 0.4), which argues against the possibility of chance findings. of note, the relation between dietary gi and diabetes risk markers appeared to be partly attributable to body composition, since associations were attenuated toward a trend in the conditional model. nonetheless, a trend was maintained, suggesting an additional mechanism independent of body composition. in fact, a previous analysis of ours did not reveal an independent association between gi during puberty and body composition in younger adulthood (26). another mechanism by which dietary gi may affect diabetes risk independently of body composition is oxidative stress : increased postprandial glycemia can exert prooxidative and proinflammatory effects (27). in turn, impaired mitochondrial function may cause both hepatocyte injury and subsequently increased release of alt and ggt (28) and contribute to insulin resistance independently of hepatic lipid content (29). moreover, excessive postprandial glycemia increases the strain on -cell mass, which can be particularly detrimental in a phase of decreased insulin sensitivity such as puberty (30). our data indicate a long - term relevance of dietary gi for both systemic and hepatic insulin resistance, as reflected by associations with homa - ir and insulin as well as ggt and alt. moreover, in our healthy sample, habitual dietary gi seems to be of long - term relevance for insulin sensitivity only, since gi was not prospectively related to -cell function (e.g., homa-). the results of our study dismiss the relevance of total carbohydrate intake for later insulin sensitivity and corroborate the rising awareness that carbohydrate quality is more important for risk of type 2 diabetes than carbohydrate quantity at least for healthy persons. we can not, however, exclude the possibility that lower carbohydrate intake may offer some benefits for obese adolescents, since they can not adapt appropriately to high - carbohydrate diets by increasing their insulin sensitivity and may, hence, need to increase insulin secretion further (31). we observed no prospective association between consumption of added sugar from drinks or fiber intake and adult type 2 diabetes risk markers except for an association between added sugar from drinks and ggt. observational studies in adults support a relation of both consumption of sugar - sweetened beverages (32) and cereal fiber (33) to type 2 diabetes risk, while mechanistic studies point to specific benefits of viscous fiber on insulin sensitivity (34)., confounding is less likely because the donald population is comparably homogeneous with a higher socioeconomic status. in addition, benefits of higher fiber intakes are partly attributed to lower postprandial glycemia. this response is, however, better described by dietary gi : in a recent study using 121 foods and 13 meals, postprandial glycemia was related to gi and gl but not fiber content (35). it is therefore possible that exposure to postprandial glycemia during puberty (as estimated by dietary gi) is of particular relevance for diabetes risk in younger adulthood, whereas other mechanisms linking fiber intake to diabetes risk become more important in later adulthood. the main strengths of our study are its prospective design and the detailed repeated measurements of dietary intake during puberty. assessment of dietary intake during puberty is notoriously difficult, but the present analysis was based on an average of five dietary records during puberty (range 26 per participant), which allowed estimation of habitual dietary intake. comparisons of our carbohydrate - intake data with other studies in adolescents showed similar intake levels with respect to total carbohydrate, added sugar (36,37), and dietary gi (38). the availability of data on several potential confounders, such as parental characteristics, including self - reported parental history of type 2 diabetes, further strengthens our analysis. however, we can not preclude residual confounding, resulting from imprecisely measured or unmeasured confounding factors. first, risk markers of type 2 diabetes were only measured once in younger adulthood. second, the relatively elaborate donald study design results in a socioeconomic status above average, and extremes of diet or behavior might not be represented, which is likely to introduce selection bias. thirdly, estimation of the dietary gi from the gi values of individual foods is discussed controversially (10,39). however, in contrast to most epidemiological studies using food - frequency questionnaires, the gi estimates in this study stem from direct assignment of gi values to all carbohydrate - containing foods recorded during 3 days (22). relating our results to those from other studies, the lack of data on the longer - term influence of adolescent nutrition on later health becomes very evident. our study provides new evidence for a long - term impact of postprandial glycemic excursions during puberty on later diabetes risk. the absence of such associations for other measures of carbohydrate quality suggests that advice focusing solely on dietary fiber and added sugar intake is insufficient. further large - scale studies, preferably in at - risk populations (e.g., overweight or insulin - resistant adolescents) are needed to support the present findings and confirm their public health relevance. in conclusion, our data indicate that a habitually higher dietary gi during puberty may adversely affect risk markers of type 2 diabetes in younger adulthood. advice for preferred selection of low - gi carbohydrates during puberty may need to be incorporated into preventive dietary recommendations given to adolescents. | objectivecarbohydrate nutrition during periods of physiological insulin resistance such as puberty may affect future risk of type 2 diabetes. this study examined whether the amount or the quality (dietary glycemic index [gi ], glycemic load [gl ], and added sugar, fiber, and whole - grain intake) of carbohydrates during puberty is associated with risk markers of type 2 diabetes in younger adulthood.research design and methodsthe analysis was based on 226 participants (121 girls and 105 boys) from the dortmund nutritional and anthropometric longitudinally designed study (donald) with an average of five 3-day weighed dietary records (range 26) during puberty (girls, age 914 years ; boys, age 1015 years) and fasting blood samples in younger adulthood (age 1836 years) (average duration of follow - up 12.6 years). multivariable linear regression was used to analyze the associations between carbohydrate nutrition and homeostasis model assessment insulin resistance (homa - ir) as well as the liver enzymes alanine aminotransferase (alt) and -glutamyltransferase (ggt) (n = 214).resultsa higher dietary gi was prospectively related to greater values of homa - ir (ptrend = 0.03), alt (ptrend = 0.02), and ggt (ptrend = 0.04). after adjustment for sex, adult age, baseline bmi, and early life and socioeconomic factors as well as protein and fiber intake, predicted mean homa - ir values in energy - adjusted tertiles of gi were 2.37 (95% ci 2.162.60), 2.47 (2.262.71), and 2.59 (2.352.85). the amount of carbohydrates, gl, and added sugar, fiber, and whole - grain intake were not related to the analyzed markers.conclusionsour data indicate that a habitually higher dietary gi during puberty may adversely affect risk markers of type 2 diabetes in younger adulthood. |
obesity has been labelled as global epidemics by who and reached to alarming situation in india as about 5% of country 's population is affected by morbid obesity. long - term weight loss with caloric restriction alone or coupled with exercise have not promising results. bariatric surgery has evolved as a boon for morbidly obese patients who have failed every attempt to achieve their target weight loss simply by diet and exercise. one of the most commonly performed bariatric procedures is roux - en - y gastric bypass (rygb), which is now considered as gold standard for weight loss. recent development in the field of bariatric surgery is the introduction of da vinci robotic surgical system. a procedure like gastric bypass or a mini gastric bypass has a gastric component and an intestinal component. in robotic surgeries working in multiple quadrants will involve complicated port positions with change in position of the patient multiple times, with multiple docking of the robotic arms thereby wasting a lot of precious time under anaesthesia. most of the gastrointestinal (gi) surgeries are performed robotic - assisted through multiple quadrant approach. it amounts to a waste of time and effort on the part of the surgeon and the team. anaesthesia time in bariatric surgery is very critical in morbidly obese patients, as they have increased risk of developing pulmonary complications postoperatively. robotic rygb in a single quadrant by single docking without putting additional ports reduces the surgical time as it abates the need to change the position of the patient multiple times during the procedure. in this study, we describe and elucidate short - term results of robotic rygb using single docking - single quadrant technique. single docking - single quadrant technique was used for rygb surgery. written informed consent was taken from each patient. another 12 mm port was placed at right mid clavicle line and parallel to optical trocar. this non - docking port was used for assistance and staplers were fired across from this port. furthermore, if any special instrument is to be introduced which is non - robotic then, it is done by this port. further, two 8 mm robotic ports were placed one on the left side in mid axillary line and the other on the right side in mid clavicular line below rib margins for docking of 2 (right side) and 3 (left side) robotic arm. another 8 mm trocar was placed in left mid - clavicular line exactly parallel to optical trocar. finally, nathanson liver retractor (cook medical, usa) was placed just below the xiphisternum before docking. the minimum distance in between the trocars should be 8 cm [figure 1 ]. the complete robotic technique is divided into two parts : (a) laparoscopic part and (b) robotic part. the first step in a robotic rygb is the division of the greater omentum [figure 2 ]. the patient was kept in supine position. after a diagnostic laparoscopy to access the bowel and hiatus, the omentum was divided starting from the base of the transverse colon till the left subtotal angle. the ligament of treitz was identified when the transverse colon is raised [figure 3 ]. marking of the biliopancreatic and alimentary limb of the bowel by a black silk thread marking stitch was done. the biliopancreatic limb was marked at 80 cm, and alimentary limb was marked at 120 cm. division of greater omentum identification of duodenojejunal junction the biliopancreatic limb was clipped with the thread to the left subtotal area so that it does not fall down after giving a steep head up position before docking. the position of the patient was maintained supine at this stage [figure 4 ]. marking of biliopancreatic and alimentary limb the robotic part requires re - positioning of the patient. this position was remains constant till the end of the procedure [figure 5 ]. steep head up position in robotic part the robotic docking process was streamlined with the help of the assistant team, which need not to be scrubbed for the procedure [figure 6 ]. the first step in robotic the pouch was made by dividing the gastric omentum just below the left gastric pedicle [figure 7 ]. the pars flaccida approach is most commonly done to make a gastric pouch at our centre. after entering into the lesser sac, we use a blue load of 6 cm to make a horizontal fire [figure 8 ]. the 36 fr bougie was used to calibrate the pouch, and two vertical firings were made with blue reloads of 60 mm and gastric pouch of size approximately 30 ml was made [figure 9 ]. once the pouch was made, the loop of bowel was clipped at the subcostal area at 80 cm of its length. this was done with a fourth layer taking antimesenteric part of the bowel with posterior wall of stomach pouch [figure 10 ]. gastrostomy was made with a hook to create a defect of size 2.5 cm and an enterotomy of size 2.5 cm [figure 11 ]. the second and a final layer were taken to completely close the gastrostomy enterotomy defect. pars flaccida approach for gastric pouch making of gastric pouch gastric pouch of size 30 ml anastomosis between the loop and the gastric pouch a 3.0-barbed suture (quill, angiobiotech, usa) for closure of gastrostomy enterotomy defects was used [figure 12 ]. after the loop pouch jejunal anastomosis ; the loop was divided close to the anastomosis avoiding a candy cane [figure 13 ]. an enterotomy was made at the alimentary limb of size 2 cm and also at the biliopancreatic limb. a linear cutter stapler was used for making an anastomosis of size 6 cm and enterotomy defects were closed [figure 14 ]. the internal hernia defect was then closed with a non - absorbable silk 2.0 (ethicon biosurgery, jhonson and jhonson, india) and finally the petersons space was closed with the same suture. closure of gastrostomy enterotomy defect loop was divided close to anastomosis stapler is used for making an anastomosis and the enterotomy defects were closed closure of internal hernia and petersons space the laparoscopy was done in the initial part of the procedure to perform basic steps such as division of omentum, performing a diagnostic laparoscopy, arranging the bowel and marking the bowel. these steps can also be performed using the robot but to simplify the procedure and to avoid the waste of time, these steps were done laparoscopically. the amount of blood loss, duration of surgery and other parameters were documented intraoperatively. patients were followed up and documentation of weight loss, body mass index (bmi) change at 6 months and 12 months postoperatively was done. in which the time <3 months after surgery is used to define as early complications and complications later than 3 months are documented as late. another 12 mm port was placed at right mid clavicle line and parallel to optical trocar. this non - docking port was used for assistance and staplers were fired across from this port. furthermore, if any special instrument is to be introduced which is non - robotic then, it is done by this port. further, two 8 mm robotic ports were placed one on the left side in mid axillary line and the other on the right side in mid clavicular line below rib margins for docking of 2 (right side) and 3 (left side) robotic arm. another 8 mm trocar was placed in left mid - clavicular line exactly parallel to optical trocar. finally, nathanson liver retractor (cook medical, usa) was placed just below the xiphisternum before docking. the minimum distance in between the trocars should be 8 cm [figure 1 ]. the complete robotic technique is divided into two parts : (a) laparoscopic part and (b) robotic part. the first step in a robotic rygb is the division of the greater omentum [figure 2 ]. the patient was kept in supine position. after a diagnostic laparoscopy to access the bowel and hiatus, the omentum was divided starting from the base of the transverse colon till the left subtotal angle. the ligament of treitz was identified when the transverse colon is raised [figure 3 ]. marking of the biliopancreatic and alimentary limb of the bowel by a black silk thread marking stitch was done. the biliopancreatic limb was marked at 80 cm, and alimentary limb was marked at 120 cm. division of greater omentum identification of duodenojejunal junction the biliopancreatic limb was clipped with the thread to the left subtotal area so that it does not fall down after giving a steep head up position before docking. the position of the patient was maintained supine at this stage [figure 4 ]. marking of biliopancreatic and alimentary limb the robotic part requires re - positioning of the patient. this position was remains constant till the end of the procedure [figure 5 ]. steep head up position in robotic part the robotic docking process was streamlined with the help of the assistant team, which need not to be scrubbed for the procedure [figure 6 ]. the pouch was made by dividing the gastric omentum just below the left gastric pedicle [figure 7 ]. the pars flaccida approach is most commonly done to make a gastric pouch at our centre. after entering into the lesser sac, we use a blue load of 6 cm to make a horizontal fire [figure 8 ]. the 36 fr bougie was used to calibrate the pouch, and two vertical firings were made with blue reloads of 60 mm and gastric pouch of size approximately 30 ml was made [figure 9 ]. once the pouch was made, the loop of bowel was clipped at the subcostal area at 80 cm of its length. this was done with a fourth layer taking antimesenteric part of the bowel with posterior wall of stomach pouch [figure 10 ]. gastrostomy was made with a hook to create a defect of size 2.5 cm and an enterotomy of size 2.5 cm [figure 11 ]. the second and a final layer were taken to completely close the gastrostomy enterotomy defect. pars flaccida approach for gastric pouch making of gastric pouch gastric pouch of size 30 ml anastomosis between the loop and the gastric pouch a 3.0-barbed suture (quill, angiobiotech, usa) for closure of gastrostomy enterotomy defects was used [figure 12 ]. after the loop pouch jejunal anastomosis ; the loop was divided close to the anastomosis avoiding a candy cane [figure 13 ]. an enterotomy was made at the alimentary limb of size 2 cm and also at the biliopancreatic limb. a linear cutter stapler was used for making an anastomosis of size 6 cm and enterotomy defects were closed [figure 14 ]. the internal hernia defect was then closed with a non - absorbable silk 2.0 (ethicon biosurgery, jhonson and jhonson, india) and finally the petersons space was closed with the same suture. closure of gastrostomy enterotomy defect loop was divided close to anastomosis stapler is used for making an anastomosis and the enterotomy defects were closed closure of internal hernia and petersons space another 12 mm port was placed at right mid clavicle line and parallel to optical trocar. this non - docking port was used for assistance and staplers were fired across from this port. furthermore, if any special instrument is to be introduced which is non - robotic then, it is done by this port. further, two 8 mm robotic ports were placed one on the left side in mid axillary line and the other on the right side in mid clavicular line below rib margins for docking of 2 (right side) and 3 (left side) robotic arm. another 8 mm trocar was placed in left mid - clavicular line exactly parallel to optical trocar. finally, nathanson liver retractor (cook medical, usa) was placed just below the xiphisternum before docking. the minimum distance in between the trocars should be 8 cm [figure 1 ]. the complete robotic technique is divided into two parts : (a) laparoscopic part and (b) robotic part. the first step in a robotic rygb is the division of the greater omentum [figure 2 ]. the patient was kept in supine position. after a diagnostic laparoscopy to access the bowel and hiatus, the omentum was divided starting from the base of the transverse colon till the left subtotal angle. the ligament of treitz was identified when the transverse colon is raised [figure 3 ]. marking of the biliopancreatic and alimentary limb of the bowel by a black silk thread marking stitch was done. the biliopancreatic limb was marked at 80 cm, and alimentary limb was marked at 120 cm. division of greater omentum identification of duodenojejunal junction the biliopancreatic limb was clipped with the thread to the left subtotal area so that it does not fall down after giving a steep head up position before docking. the position of the patient was maintained supine at this stage [figure 4 ]. marking of biliopancreatic and alimentary limb the robotic part requires re - positioning of the patient. this position was remains constant till the end of the procedure [figure 5 ]. the robotic docking process was streamlined with the help of the assistant team, which need not to be scrubbed for the procedure [figure 6 ]. the pouch was made by dividing the gastric omentum just below the left gastric pedicle [figure 7 ]. the pars flaccida approach is most commonly done to make a gastric pouch at our centre. after entering into the lesser sac, we use a blue load of 6 cm to make a horizontal fire [figure 8 ]. the 36 fr bougie was used to calibrate the pouch, and two vertical firings were made with blue reloads of 60 mm and gastric pouch of size approximately 30 ml was made [figure 9 ]. once the pouch was made, the loop of bowel was clipped at the subcostal area at 80 cm of its length. this was done with a fourth layer taking antimesenteric part of the bowel with posterior wall of stomach pouch [figure 10 ]. gastrostomy was made with a hook to create a defect of size 2.5 cm and an enterotomy of size 2.5 cm [figure 11 ]. the second and a final layer were taken to completely close the gastrostomy enterotomy defect. pars flaccida approach for gastric pouch making of gastric pouch gastric pouch of size 30 ml anastomosis between the loop and the gastric pouch a 3.0-barbed suture (quill, angiobiotech, usa) for closure of gastrostomy enterotomy defects was used [figure 12 ]. after the loop pouch jejunal anastomosis ; the loop was divided close to the anastomosis avoiding a candy cane [figure 13 ]. an enterotomy was made at the alimentary limb of size 2 cm and also at the biliopancreatic limb. a linear cutter stapler was used for making an anastomosis of size 6 cm and enterotomy defects were closed [figure 14 ]. the internal hernia defect was then closed with a non - absorbable silk 2.0 (ethicon biosurgery, jhonson and jhonson, india) and finally the petersons space was closed with the same suture. closure of gastrostomy enterotomy defect loop was divided close to anastomosis stapler is used for making an anastomosis and the enterotomy defects were closed closure of internal hernia and petersons space the laparoscopy was done in the initial part of the procedure to perform basic steps such as division of omentum, performing a diagnostic laparoscopy, arranging the bowel and marking the bowel. these steps can also be performed using the robot but to simplify the procedure and to avoid the waste of time, these steps were done laparoscopically. the amount of blood loss, duration of surgery and other parameters were documented intraoperatively. patients were followed up and documentation of weight loss, body mass index (bmi) change at 6 months and 12 months postoperatively was done. the early and late complications were defined by standardised classification by clavien. in which the time <3 months after surgery is used to define as early complications and complications later than 3 months are documented as late. any complication requiring surgical intervention out of 140 patients who underwent robotic rygb using single docking - single quadrant technique, follow - up till 1 year could be made in 120 patients whereas 20 patients were lost to follow - up. out of 120 patients, 49.16% (59) of patients were male and 50.83% (61) were females. the mean pre - operative weight of 117.41 27.14 kg and the mean bmi was 43.75 8.01 [table 1 ]. the total mean operative time, including the laparoscopic, robotic and console time for the complete procedure, was 97.48 23.79 min. as the complete procedure consists of laparoscopic and robotic part ; the mean laparoscopic time was 19.61 5.50 min, docking time of 7.53 5.40 min and mean console time of 70.37 14.76 min. demographic and clinical characteristics of patients in our first seven cases, it was documented that the docking time was higher which ranged from 20 to 30 min (mean 24.28 min) of docking but there was gradual decreasing trend in docking time and finally reduced to just 47 min [figure 16 ]. the docking was done with four robotic arms one of which was used for the optical port. docking and console time it was found that there was only one early minor complication of minor wound infection with no early major complication. one case of mortality was documented 7 days post - surgery with a suspected cause of pulmonary embolism. two cases of late complication of stoma stenosis at 9 months post - operative in one case and 11 months post - operative in other case were documented on upper gi endoscopy. complication and operative details weight loss patterns of all the patients were observed at 6 months and 1-year post - surgery. the mean weight at 6 months was 87.83 14.32 kg, with a mean bmi of 32.75 3.19 kg / m and at 1 year mean weight was 83.86 143. 40 kg with mean bmi of 31.04 3.91 kg / m [table 3 ]. comparative analysis of body weight and body mass index mean excess body weight loss at 6 and 12 months of follow - up was 58.1 4.5 and 66.8 5.1 kg. the robotic surgery has evolved as the newer paradigm in minimal access surgery and it envisages the advantage of having a three - dimensional vision with articulating instruments. the surgeon sits on the console in the most comfortable way without stress on his shoulders and torque on arms even on most super obese patients. usually, in laparoscopy there is huge torque on hands of surgeon that makes suturing difficult and inaccurate. the major reason why this has happened is the accuracy with minimal damage to surrounding tissue specifically the prostatic nerve plexus. this helps in avoiding many complications such as erectile dysfunction, incontinence and premature ejaculation. robotics in bariatric surgery has still not come into the mainstream, and the major reason for that is the cost of equipment and cumbersome procedure involving multiple dockings and complicated port position. most of the studies published comparing the cost between a robotic bariatric procedure to a laparoscopic procedure reveal that the cost involved in the robotic bariatric procedure is more although the return to activity is faster. as oppose to the robotic surgery cost as per them is 5427 usd and that of laparoscopic is 5494 usd. this is possible according to them because of the lesser use of costly staplers while doing a robotic gastric bypass where suturing can abate the use of staplers in anastomoses. cirocchi. done a meta - analysis and included 22 studies in the quantitative analysis of robotic bariatric surgery. this study includes one randomised control trial, 9 clinical control trial and 12 case series. fourman and saber have described in their meta - analysis that in a total of 6 studies, 684 patients with a mean pre - operative bmi of 47.8 kg / m underwent robotic rygb. the mean reported follow - up time in 4 of 6 studies was 10.5 months. the major complications that they documented were gastrojejunal strictures, marginal ulcers, anatomic leak, bleeding, myocardial infarction, clostridium difficile infection, bowel perforations and internal hernia. they have concluded that out of a total of 3337 patients in 9 studies, 1382 were kept in the robotic arm and 1956 in laparoscopic arm. the mean operative time in robotic arm was 211.9 min and in laparoscopic arm was 185.1 min. however, in their analysis, they found out three studies with a significantly lower operating time for robotic gastric bypass. the length of stay was 5 days for robotic surgery and 7.1 days for laparoscopic surgery. if we compare our results with these previous reports, we have a significantly lower rate of complications with just one early minor wound infection and two gastro jejunostomy stricture which did not require any surgical treatment but were managed by endoscopic dilatation. lower mean operative time was achieved in our series of patients as we do not change the position of the patient and ports after docking is done. as compared previous reports complication rates were very low in present series. the length of stay in their case series ranged from a minimum of 2 days to 4 days that is comparable with our experience. the present study has focused more on technique and the simplification rather than comparison of robotic gastric bypass with our conventional bypass. they have essentially used 6 ports and similar port position but the technique required a median operating time of 169 min. they have compared this time with their conventional gastric bypass timing and it has turned out to be significantly lesser in robotic. they have attributed this to effective suturing and anastomosis in robotic surgery. the standard 5-port technique utilised by us for robotic gastric bypass the lesser operative time, and lower complications rate in our series is attributed to our simplified technique. as mentioned in other studies, the average docking time and console time has shown a decreasing trend with passage of time. the shortcoming of our study is that we did not compare our clinical outcomes of robotic gastric bypass with our laparoscopic gastric bypass performed during the same time. robotic rygb is a safe and feasible technique. with the single quadrant, single docking approach as performed at our centre, we have eliminated wastage of time and decreased over all operative time and time under anaesthesia. rygb when done with da vinci robotic surgical system has proven to be safe and effective procedure. single docking - single quadrant technique shows a lesser mean operative time with minimal complication rate. robotic bariatric surgery is still not for the prime time but with several innovations and rapid developments to follow it might get more popular and cost - effective. | background : roux - en - y gastric bypass (rygb) is one of the most widely performed bariatric surgeries in the world. performing an rygb by a da vinci surgical system is a new advancement. the aim of this study is to describe single docking - single quadrant technique and its short - term results.materials and methods : between january 2013 and december 2013, 140 robotic rygb were performed. the rygb was performed through single docking, single quadrant approach. the data were analysed retrospectively. intra- and post - operative details of every patient were documented. follow - up was done as per protocol at 6 months ; 1 and 2 years. in total, 120 patients completed the follow - up protocol as per our database.results:mean age of the patients was 42.7 12.11 years. ratio of males : females were equal. the mean operative time was 97.48 23.79 min. early mortality was seen 7 days post - surgery. two late complications were documented with no late mortality. the average length of stay was 2.89 1.06 days. average blood loss was 55.79 11.91 ml. there was no hospital re - admission after the surgery.conclusion:single docking - single quadrant technique is simple, effective and time saving without having complicated port position, multiple docking with minimal complications. |
dendritic spines are specializations of glutamatergic synapses. they have been the object of theoretical and experimental studies for more than a century. first identified by ramn y cajal, their role in synaptic transmission is still under study. morphologically, spines are clearly identified as tiny protrusions, about one micron long, with a mushroom - like shape, although this static description does not reflect their great variability in size and shape. spines are dynamic structures that undergo morphological changes in a developmental and activity - dependent manner [2, 3 ]. in this sense, neurons are able to control synaptic efficiency by adjusting the size and density of spines, which have been accepted, in turn, as important regulators of synaptic plasticity, learning, and memory formation [57 ]. in addition, abnormal spine shape and density have been associated with different pathologies, such as alzheimer 's disease, epilepsy, down 's syndrome, and fragile x syndrome [8, 9 ]. a motile actin cytoskeleton provides the required molecular substrate for the dynamic nature of spines. at the ultrastructural level, the actin state depends on the coordinated action of several actin binding proteins [8, 14 ]. said equilibrium, that is, the proportion between filamentous and monomeric actin, can be quantified employing frap (fluorescence recovery after photobleaching), fret (fluorescence resonance energy transfer), or photoactivated actin. ongoing actin polymerization exerts a direct control over membrane receptor composition and the stability of the postsynaptic density, and it has been suggested that actin might serve as an anchor place in the synapse [18, 19 ]. supporting this notion, spines contain discrete locus of polymerization often associated with postsynaptic density and receptor trafficking. besides neurons, glial cells also play an important role in synapse physiology and development. during synaptogenesis, glial cells release cholesterol and thrombospondins to increase synapse number and functionality. astrocytes not only regulate the synaptic microenvironment by removing or releasing neurotransmitters into the extracellular space ; they can also directly modulate synaptic transmission, synaptic plasticity [22, 23 ], and neurodegeneration. astrocyte interaction with synaptic spines requires physical contact, as demonstrated in electron microscopy reconstructions where 57% of the spines in a mature hippocampus are associated with astrocytes. in organotypic hippocampal cultures, astrocytes rapidly extend and retract fine processes that associate and release from dendritic spines. furthermore, astrocyte protrusions are essential in the maturation and stabilization of newly forming spines, and thus astrocyte contact enhances both lifetime and morphological maturation of spines. here, we employed frap techniques to study actin spine dynamics in dissociated cultures of rat hippocampal neurons and organotypic slices. our results reveal an unexpectedly high degree of variability regarding actin dynamics in individual spines. moreover, the spine population was segregated into two groups, according to their recovery velocity rate. additionally, we show that the presence of astrocytes in the culture can regulate actin cytoskeleton dynamics, in that, spines growing in the presence of astrocytes present a higher actin dynamics than those growing in the absence of astrocytes. finally, we described a simple protocol to demonstrate the presence of polymerization hot spots within the spine structure. primary cultures of hippocampal neurons were dissociated from postnatal (p0-p1) rat pups as previously described. in brief, hippocampal neurons were grown in culture media consisting of neurobasal medium supplemented with 0.5 mm glutamine, 50 mg / ml penicillin, 50 units / ml streptomycin, 4% fbs, and 4% b27 supplement (all from invitrogen). cells were plated in mattek chambers with a 12 mm glass coverslip center (mattek, usa), previously coated with poly - d - lysine (50 g / ml) and laminin (4 g / ml). on days 4, 7, 14, and 21 in vitro (div), 500 l (from a total of 2000 l) of the culture medium was replaced with 520 l of new, fresh medium. two types of cultures were used, depending on the density of astrocytes. under regular conditions, after the astrocytes grew to form a monolayer (usually after four days to a week in culture), a concentration of 4 m of cytosine - d - arabinofuranoside (sigma) was added to prevent glial cell overgrowth (this condition was referred to as ast high). in the second type of cultures, the inhibitor was added after 2 days in vitro, to obtain cultures growing in the near - absence of glial cells (we referred to this type of cultures as all neurons studied grew in ast high conditions, unless otherwise indicated. prior to plating, neurons were transfected with a vector plasmid encoding for the yfp / gfp fused to the n - terminus of chicken -actin gene, under the control of the platelet - derived growth factor enhancer / promoter region (pdgf ; vector kindly provided by drs. transfection was performed by neuronal electroporation, using the electroporation rat hippocampal neuron kit from amaxa according to the manufacturer 's instructions or with a biorad cell electroporator system (exponential discharge protocol with the following parameters : 220 v and 950 mf and resistance fixed to infinitum ; cells and plasmids were mixed in biorad electroporation buffer). in both protocols, 10 g of plasmidic dna hippocampal slices were prepared from young rats of both sexes (postnatal days 6 - 7) as previously described. briefly, after dissection of the hippocampi in ice - cold gassed (5% co2/95% o2) dissection solution (in mm : 10 glucose, 4 kcl, 24 nahco3, 234 sucrose, 0.5 mgcl26h2o, 0.7 cacl22h2o, and 0.03 phenol red at ph 7.4), 400 m transverse slices were prepared using a tissue slicer. slices were transferred to slice culture inserts (millipore) and cultured in culture medium (minimum essential media (mem) supplemented with 20% (v / v) horse serum, 1 mm glutamine, 1 mm cacl2, 2 mm mgso4, 1 mg / l insulin, 0.0012% (w / v) ascorbic acid, 30 mm hepes, 13 mm glucose and 5.2 mm nahco3 at ph 7.25, and a final osmolarity of 320 the recombinant egfp - actin was delivered into slices using the sindbis virus, as previously described. recombinant protein expression was typically 1224 h. images were taken either with a tcs - sp5 or a tcs - sl laser - scanning confocal spectral microscopes (both from leica microsystems heidelberg, gmbh). live images were acquired using a 63x oil immersion objective lens (na 1.32), with a pixel size of 58 nm 58 nm. the confocal pinhole was set at 4.94 airy units to minimize changes in fluorescence due to gfp / yfp - actin moving away from the focus plane. frap experiments were performed using the following protocol : 10 single prebleach scans were acquired at 225300 ms intervals, followed by 10 bleach scans at full laser power, over a circular area of 2 m in diameter. during the postbleach period, 250 scans were acquired at 225300 ms intervals, followed by 10 images acquired at 1 s time intervals. in order to resolve the initial fast recovery, some experiments were performed using the leica fly mode acquisition ; bleaching was performed during the x fly forward scan at 100% laser power. during the backward scan, fluorescence was read out with laser intensity set to imaging values (185 ms interval). postbleach images (3060) were acquired at the same time interval. to avoid significant photobleaching, the excitation intensity was attenuated to ~5 to 8% of the laser power during image acquisition. background fluorescence was measured in a random field outside of the dendrite and subtracted from all the measurements. dendrite fluorescence was determined for each image and compared with the initial dendrite fluorescence to determine the spontaneous signal lost during imaging. the fluorescence signal measured in the region of interest (roi) and normalized to the change in dendrite fluorescence was determined to be irel = it / i0t0/tt, where it is the average intensity in the region of interest at time t ; i0 is the average intensity in the region of interest during prebleach, t0 is the dendrite intensity during prebleach, and tt is the dendrite intensity at time t. the introduction of the correction factor (t0/tt) accounts for possible small fluctuations in total fluorescence intensity caused by the bleach itself and yields a more accurate estimate of the fluorescence measured in the roi. the net fluorescence recovery (mobile fraction, mf) measured in the region of interest was determined as mf = (fend fpost)/(fpre fpost), where fend is the roi mean intensity at the steady - state, fpost represents roi intensity after photobleaching, and fpre is the mean roi intensity prebleach. each individual spine recovery curve was fitted by a two - component exponential equation, although the initial fast component, driven by diffusion, was negligible in most of the recordings. therefore, the recovery time constant (tau,) was calculated from the fitting to a monoexponential curve. this protocol permits linear scans of 200300 nm width at 1-ms intervals. for these experiments, three consecutive scans or jobs were acquired (each consisted of 2000 lines 512 pixels in width) : an initial prebleach job (2000 lines), a bleach protocol (2000 lines at maximal laser power), and a final 6 x jobs (2000 lines each), to account for a recovery time of 12 seconds. to avoid significant photobleaching, excitation intensity was attenuated to ~5 to 8% of the laser power during image acquisition. organotypic hippocampal slices (37 div) expressing egfp - actin were perfused with acsf at 30c. two - photon fluorescence images were obtained with a zeiss lsm510 laser - scanning microscope using a 63x water immersion objective and a mai tai deepsee (spectra physics) 910 nm laser as light source for excitation. for frap experiments, images were acquired every 200 ms for 2.7 min (810 images). after 3 images, the egfp - actin signal from dendritic spines was photobleached with one iteration of high laser intensity. fluorescence values at the spine were normalized to those of the adjacent dendrite to compensate for ongoing bleaching during imaging. fluorescence values and the spine area were analyzed using image j. immunocytochemical analysis was performed as follows : cultures were rinsed in phosphate buffer saline (pbs) and fixed for 15 min in 4% paraformaldehyde in pbs. coverslips were then washed three times in pbs and incubated for 30 min in blocking solution (2% goat serum, 2% serum albumin, and 0.2% triton x-100 in pbs). gfap and synapsin antibodies were from abcam (rabbit polyclonal reference 7260) and cell signaling usa (rabbit polyclonal reference 2312), respectively. samples were subsequently washed three times in pbs and incubated for 30 min in pbs solution containing the appropriate fluorescence - conjugated secondary antibodies (all from molecular probes) and were then washed five more times with pbs buffer and mounted using mowiol. statistical analyses were performed using graphpad prism software (graphpad software inc., san diego, ca, usa). a two - way anova with tukey 's multiple comparison test was performed to detect differences in mobile fraction between neurons, cultures, and distance from somas. a one - way anova was employed to study differences among neurons or between individual neurons and the whole population. a kolmogorov - smirnov test was performed to compare cumulative frequency distributions for spine head areas between ast high and ast low conditions. a mann - whitney test was used to test for differences between mf in both culture conditions (ast high and ast low) or between the culture (ast high) and slices. the significance level was set at p < 0.05. a better model of tau distribution was determined by comparing a single gaussian model versus a sum of two, employing an extra sum - of - squares f test in graphpad prism. for all our experiments in ast high conditions, a minimum of 10 neurons from around 5 independent cultures and approximately 212 spines were analyzed. for the ast low condition, we studied a minimum of 30 neurons from 10 independent cultures with more than 100 spines analyzed. culture hippocampal neurons produce dendritic protrusions with distinct stages of morphological progression [33, 34 ]. dendritic filopodia could be observed as early as 6 div and became abundant around 9 div. by div 14, the dominant dendritic protrusions were thin spines, characterized by a relatively long neck and a small head. several studies have reported that transfected neurons accumulate gfp - actin at dendritic spines, making them clearly visible without affecting synaptic transmission [15, 35, 36 ]. we transfected cultured hippocampal neurons with a plasmid encoding gfp - actin under the control of a neuronal pdgf (platelet - derived growth factor) promoter to avoid overproduction and toxicity of gfp - actin (figure 1(a)) [28, 29 ]. in these neurons, growing for more than 18 div, staining with rhodamine phalloidin largely colocalizes with gfp - actin positive dendritic spines (90% of colocalization, data not shown). this result is consistent with the reported enrichment of actin filaments in the spines [37, 38 ]. to characterize actin dynamics at the spine, our approximation is based on the work of star. and koskinen.. basically, we are assuming that (1) actin monomers are free to move in and out of the spine compartment and (2) most of the actins in the spine are in filamentous form, and these are in a dynamic equilibrium, continuously poly- and depolymerizing. therefore, the net recovery of fluorescence at the steady - state (the so - called mobile fraction, mf) includes the free diffusion of actin monomers, plus the proportion of filaments in dynamic equilibrium. assuming that actin monomer diffusion is constant, a low proportion of stable filaments should render high values of mobile fraction, and, conversely, a high proportion of stable actin filaments would produce lower mobile fraction values (figure 1(b)). finally, the fluorescence recovery rate is proportional to the velocity of actin monomer incorporation to the plus ends of filament, making frap a suitable technique to measure actin treadmilling (figure 1(b)). in agreement with the previously reported studies, the recovery curve has two clearly distinguished components, each adjusted to a single exponential curve. the fast (initial) component showed a mean time constant of 0.61 0.09 s. similar time constant diffusion was obtained when spines from monomeric gfp - transfected (mgfp) neurons were analyzed (0.53 0.093 s), supporting the idea that this fast component was driven by pure diffusion (figure 1(b) insert and figure 3(e)). the first component was only uncovered when a fly mode acquisition was employed and was ignored in most of the experiments because it does not provide any information about actin cytoskeleton dynamics. the second component was mostly driven by actin polymerization ; consistently, cytochalasin d (5 m) treatment, a barbed - end capping drug, reduced the mobile fraction to 0.30 0.13 and slowed recovery fluorescence time as previously described (figure 1(b) ;). jasplakinolide 1 m treatment, a membrane permeable actin filament stabilizer, greatly impairs fluorescence recovery (mf : around 5% ; statistically nonsignificant), further confirming that the slower component depends of f - actin polymerization. the population of mobile fraction values follows a continuous distribution (values range from 0.2 to 1.1 ; recovery values higher than 110% were discarded), with a mean value of 0.78 0.01 (figure 1(c)). when several spines from the same neuron were analyzed, we observed a large variability of mf values within a single neuron (see figure 1(d) as an example). therefore, our first question concerned the origin of this variability. was the mobile fraction regulated by the age of the culture or by the proximity of the spine to neuronal soma thus, spine mobile fractions were analyzed for a period of five days (18, 20, and 22 days in culture) and mf values were averaged and segregated, according to their dendritic origin (primary, secondary, and tertiary dendrite) and age of the culture. the results (figure 1(e)) indicate that neither age of the culture nor the distance from the cell body affects spine mobile fraction (similar mean values were obtained when spines were segregated in 20 m intervals, data not shown). despite variability, no differences were found when average mobile fraction values were compared among neurons or between single neurons and the whole population of mfs (figure 1(f)). in summary, considering these results as a whole, we assume that mobile fraction variability can be attributed to the individual spines themselves, and not to neurons or culture age. from a spurious observation therefore, to evaluate the role of astrocytes modulating actin dynamics at the spine level, we performed frap experiments with two types of cultures : regular cultures growing over an astrocyte monolayer (condition : ast high) and in the partial / total absence of astrocytes (condition : ast low) (figures 2(a) and 2(b)). both types of cultures developed spines after 16 days in vitro, and recordings were made between 18 and 22 days in vitro. despite the fact that neurons exhibit a normal growth in the absence of astrocytes, we observed a consistent reduction in basal fluorescence levels at the spines. to test whether differences arise from spine size, we analyzed the spine head area in both experimental conditions. no significant differences were found in average head area between the two conditions (ast high : 0.73 0.03 ; ast low : 0.78 0.04 m), although the cumulative frequency distribution indicates that small spine head areas were more abundant in ast low conditions (figure 2(c)). conditions had a mean time value of 0.65 0.04 s, similar to that obtained in ast high conditions, implying that the diffusion rate is unaffected by the presence / absence of astrocytes in the culture. in contrast, different results were observed when the mobile fraction was quantified. in conditions, the mf was drastically reduced (figures 2(d) and 2(g), red open circles). although some variability is present, the frequency distribution clearly indicates that neurons growing in these conditions have lower mobile fractions, with a range between 0.1 and 0.8 and a mean value of 0.54 0.02 (figure 2(d)). to confirm the presence of astrocytes nearby or in close proximity to the spine, in a small number of experiments, fm4 - 64 (a lipophilic dye) was included in the culture media during the recording conditions. in these conditions, and without stimulated endocytosis, fm4 - 64 adheres to all extracellular membranes, allowing easy identification of the presence of membranes around the spine. as indicated in figure 2(e), in fm4 - 64 staining showed a sandwich - like distribution, enfolding dendritic spines (see linear scanning in insert, figure 2(e)(a, b, and c)), indicative of a membrane around the spine. this spine showed a mobile fraction value close to 80% (figure 2(g), black closed circles). in clear contrast, the spine from the ast low conditions was almost devoid of red fluorescence around the spine (see insert in figure 2(f)(a, b and c)). in this case, fluorescence recovery was close to 30% (figure 2(g), red open circles). in addition to the physical contact between the dendritic spines and astrocytes, it is possible that glial cells release soluble factors into the medium that affect actin dynamics. in order to test possible contributions of soluble factors, we studied some ast low neurons treated with astrocyte - conditioned medium, but no significant effects were seen in the actin mobile fraction. due to this absence of significant effects and the fact that the medium composition may depend on many, highly variable factors (age of astrocytes, frequency of medium replacement, degradation, etc.), we chose not to pursue these experiments any further. nevertheless, we can not completely rule out that some undetermined soluble factors could affect actin dynamics. as mentioned earlier, the recovery rate of gfp - actin fluorescence is proportional to actin polymerization velocity. to study the variability of this parameter in our neuronal population, we analyzed the rate of recovery by fitting the second component of the recovery curve to an exponential growth described by a tau value (). recovery time values show a high degree of variability, ranging from 1.1 to 46.8 seconds. the frequency distribution graph suggests the existence of two populations of spines, determined according to their recovery time (figure 3(a)). the frequency distribution was fitted to a double gaussian distribution, with two average values of 6.02 2.70 and 14.87 6.32 seconds, respectively. the kinetics of recovery were also affected by the lack of astrocytes. in these culture conditions, constant times presented a double gaussian distribution, with two mean values of 13.16 5.84 and 33.22 3.55 (figure 3(b), red bars). we then proceeded to evaluate whether there was any relation between recovery times and mf values (in ast high condition). as shown in figure 3(c), the relation between mf and tau values reinforces the existence of two populations of spines : one characterized by a faster recovery (up to 10 s) and lower mf values and a second population characterized by slower recovery times and higher levels of mf (figure 3(c)). a similar distribution can be observed when mobile fraction values are plotted versus spine head size. two populations became apparent in this graph : one with a smaller size and lower mobile fractions and a second one with larger areas and higher mobile fractions (figure 3(d)). therefore, it follows that tau and spine size area are also related, with smaller spines displaying faster recovery times and larger spines being more prone to showing slower recovery times (figure 3(e), closed dots, left axis). it can be argued that if diffusion is the main driver, the recovery time constant and photobleached area will follow a linear regression that is simply the effect of increasing the bleached area. to test this hypothesis, recovery rates were analyzed for a simple diffusion process employing egfp transfected neurons, and recovery time values were plotted against spine head areas (figure 3(e), open circles, right axis). as figure 3(e) indicates, recovery times are ten times slower when employing gfp - actin, which rules out diffusion as a main driver controlling actin velocity recovery. to confirm whether this distribution of actin recovery times was a general characteristic of the spines or a peculiarity of the hippocampal cultures, we performed a similar experiment employing hippocampal organotypic slices transfected with gfp - actin (figure 3(f)). in this condition, the estimated mean mf was 0.84 0.02, which was not statistically different from the mf obtained from the cultures. the differences between these two models emerged when recovery times were analyzed. as figure 3(f) shows, the frequency distribution of tau values from the organotypic slices indicates the presence of a single population of spines with a mean value of 25.06 1.9 seconds (blue bars). this result was confirmed when the spine area was plotted against mf. as figure 3(d) indicates, all values from slices are segregated into a population of spines with larger areas and higher mf values (figure 3(d), blue circles). previous studies using photoactivated actin in combination with high - resolution techniques suggested the existence of polymerization hot spots along spine head structure. to evaluate this point, we devised a simple experimental protocol employing conventional confocal microscopy. to this end, a line scanning mode (x, t mode) was used to perform frap. employing this acquisition mode, only a narrow longitudinal area was scanned (close to 300 nm wide). this allowed us to reduce time sampling values to 1 - 2 ms (figures 4(a)-4(b)). using this acquisition mode, we were able to differentiate between the recovery rate of the distal part of the spine (cortical head area) and the proximal area (closer to the neck of the spine) (highlighted as a dashed yellow box in figure 4(b)). when fluorescence recovery curves from each section were independently analyzed, transient changes in the slope of recovery were visible (figure 4(c)). the changes in one area were accompanied by an equivalent alteration, but in the opposite direction, in the other areas of the spine (figure 4(c), comparison of recovery between distal and proximal areas). a similar phenomenon was observed in 52% of the spines studied (13 of a total of 25 spines). oscillations in the slope of recovery were observed in either the distal or proximal areas of the spine in a similar proportion, with no differences between large or small spines. similar phenomena in the fluorescence profile were also evident during basal recording, even though the reduction of fluorescence after bleaching facilitated the discrimination (data not shown). to confirm that actin polymerization was the primary cause of these oscillations in fluorescence recovery rate, a series of experiments were performed, adding 200 nm of latrunculin a (lata) to the extracellular solution. lata, an organic compound with a high affinity for monomeric actin, prevents actin polymerization by sequestering actin monomers. despite a reduction in the recovery rate in the presence of lata, the recovery profile was similar between the two areas of the spine head (figure 4(d), n = 16). similar results were obtained when jasplakinolide 1 m was added to the culture media (data not shown). it has been proposed that a highly dynamic actin cytoskeleton in dendritic spines is necessary to support and regulate spine morphology, as well as synaptic transmission and plasticity. in the present report, we have confirmed the plastic nature of this actin cytoskeleton. spine actin mobile fraction values were not homogenous in either slices or cultures. on the contrary, we found a large degree of variability, with values between 20 and 100%, although the majority of spine mf values were concentrated close to 80%. similar to the results of the pioneering work of star and colleagues, we found that nearly all spines contained a large amount of dynamic actin. other authors have previously reported a progressive reduction in mobile fraction associated with culture aging. however, our results show a large and stable mf mean value that is independent of the age of the culture or even the distance to the soma, a result that is in agreement with the lack of changes in hippocampal cultures reported by star and colleagues. similar to this work, in our study, large spines that theoretically must bear large postsynaptic densities were associated with large mobile fractions and relatively slow actin recoveries. confirming these findings, in hippocampal organotypic slices from 7-day - old animals, however, it must be recognized that different age and culture conditions, or even frap protocols, among laboratories would certainly induce different spine actin turnovers that could contribute to explaining the discrepancies in the reported results. the main finding of our paper is the presence of two spine populations (faster and slower recovery) in culture conditions based on their polymerization rate, and, notably, only one population in organotypic slices (slower). spine heads typically contain a major dense network of short cross - linked and branched filaments. since fluorescence recovery after photobleaching quantifies the incorporation of new fluorescent monomers, recovery time constants express, or must be proportional to, the polymerization rate. attending to the double gaussian distribution observed, we have classified the spines into slow polymerization (tau values between 10 and 25 seconds) and fast polymerization (between 2 and 10 seconds) groups. interestingly, analysis of the spine head areas demonstrated that large spines were associated with slower recovery rates, while small spines displayed a faster recovery. a large set of actin binding proteins, such profilin ii, gelsolin, debrin, and arp2/3, have been found to be associated with the spine cytoskeleton (for a review, see cingolani and goda). among them, cofilin 1/adf has been recognized as a key regulator controlling f - actin assembly and disassembly. binding of adf / cofilin to actin is controlled via phosphorylation (inactivation) and dephosphorylation (activation) by lim kinases (limk) and slingshot phosphatases, respectively, both of which are known to exert powerful control over spine morphology and synaptic plasticity. overexpression of an inactive form of cofilin results in more mature spines through an ampa receptor traffic - dependent mechanism. inactive cofilin mutants increase f - actin contents and reduce the actin dynamics measured by frap. on the other hand, cofilin 1 promotes f - actin assembly during ltp ; conversely, it is required for f - actin disassembly and spine shrinkage during ltd. such a dual function of cofilin 1 thus suggests that it may be responsible, at least in part, for the observed variability among turnovers and actin mobile fractions. however, based on the complexity of the signal cascades that control actin dynamics, it is very likely that additional molecular pathways are also involved. an accurate proportion and compartmentalization of the actin binding proteins inside the spine would be crucial to ensuring proper spine morphology and function. future experiments quantifying and analyzing the distribution of proteins controlling polymerization within the spine are necessary. in primary cultures, a large proportion of spines were smaller than their counterparts found in slices. we must keep in mind that our measurements are relative, based on an estimation of the spine area from a two - dimensional image. nevertheless, a simple explanation might be the different developmental stage of spines in these two systems. thus, young spines of small size may be more abundant and more easily found in primary cultures, while this category of spines progressively diminishes in slices until its final elimination. further experiments analyzing spine size distribution, comparing primary cultures and slices, would be needed to clarify this point. an unexpected result was the role of astrocytes, which participated in the dynamics of the actin cytoskeleton of the spine. the absence of astrocytes shifted the actin mobile fraction distribution to smaller values and slower recoveries. a substantial series of reports have demonstrated that astrocytes play a critical role in regulating synapse formation and activity in the central nervous system [53, 54 ]. astrocyte presence increases synapse formation, maturation, and stabilization [20, 27, 55, 56 ]. several soluble factors secreted by astrocytes have been already identified, including thrombospondins, cholesterol complexes, and sparc, which are known to be involved in synaptic formation and maturation. moreover, the age of astrocytes in cultures regulates the probability of release and synapse maturity of cocultured neurons. in addition to secreted factors, astrocytes can regulate synaptogenesis through physical interactions, and local contact by astrocytes thus elicited pkc activation by means of integrin receptor activation within the neuron, facilitating glutamatergic synaptogenesis. epha4, a family of tyrosine kinase receptors, is enriched in dendritic spines and its ligand ephrin - a3 is localized at the astrocytic processes. acute inhibition of ephrin / epha4 signaling in hippocampal neuronal cultures produces irregular spines with thinner heads. consistent with a role in neuron - astrocyte signaling, acute application of epha4/fc (which inhibits endogenous interaction of epha4) decreases the contact lifetime between astrocyte processes and spines and reduces astrocyte - dependent stabilization of newly formed dendritic spines in organotypic hippocampal cultures. therefore, synaptic maturation and neuronal activity are among the many forms of astrocytic control. at this point, we can not determine which signaling pathways might mediate the effect of surrounding astrocytes on the actin cytoskeleton within the spine. further experiments will be needed to address this issue. finally, our experiments confirmed the existence of polymerization hot spots along the spine structure, as previously shown by frost and colleagues. in their work employing a combination of palm techniques, the authors demonstrate the existence of discrete and separate foci along the spine head and neck, where actin polymerization velocity was elevated. the authors conclude that some of these hot spots can be associated with areas of receptor endocytosis. our results are based on the use of fast line scans with a low spatial resolution, but a fast acquisition rate (1 - 2 ms). our calculations employing fixed cells established a wide 300-nm range, limiting the measured area and therefore reducing the probabilities of detecting simultaneous hot spots. interestingly, the presence of a polymerization hot spot was accompanied by a similar area of slower polymerization, suggesting a flux of actin monomers within the spine. this net flux of actin monomers would remain undetected when whole spine fluorescence is measured. synaptic changes that support long - term plasticity (i.e., ltp) evolve through consecutive stages, and every stage involves a different set of actin functions (for a review, see rudy). remarkably, these changes are not coupled with changes in nearby spines [6467 ], supporting the functional / biochemical independence of each spine. interestingly, the development of the two - photon glutamate uncaging technique has allowed the stimulation of a single spine while simultaneously imaging its morphology. with this approach, it has been found that, upon stimulation, a single dendritic spine rapidly changes its morphology, enlarging its head for the first few minutes and eventually experiencing a whole - volume change that lasts for hours [67, 68 ] (for a review, see nishiyama and yasuda). we have observed a large degree of actin variability among spines, even on the same dendrite. this finding reinforces the notion that, at the biochemical and structural levels, each spine is self - regulated independently of its neighbors. one can speculate about the reasons for the observed variability among the spines, but an independently regulated actin cytoskeleton would indisputably subserve a large degree of systemic plasticity. in other words, every spine would independently adapt its structure to the ongoing synaptic strength, with the actin cytoskeleton being the main element responsible for these changes. as professor yuste proposed, the electrical and biochemical independence of each spine supports the brain 's ability to form a plastic nonsaturated distributed circuit, where every spine is independently regulated. it goes without saying that we are still far from having a complete understanding of actin dynamic participation in spine morphogenesis and physiology. we believe that future work must be undertaken to understand the different roles of actin binding proteins within the spine and to specifically quantify the participation of actin dynamics in the process of ampa glutamate receptor endocytosis. the main findings of our report are, first, the confirmation of the dynamic nature of the actin cytoskeleton at the spine head level. this dynamic is individually regulated by each spine, independently of neuron age or distance from the cell body. second, we have found that the presence of astrocytes is an important regulator of the actin mobile fraction and polymerization rate. third, according to their polymerization rate, spines can be categorized into two populations in primary cultures, or a single population in organotypic slices. | dendritic spines are mushroom - shaped protrusions of the postsynaptic membrane. spines receive the majority of glutamatergic synaptic inputs. their morphology, dynamics, and density have been related to synaptic plasticity and learning. the main determinant of spine shape is filamentous actin. using frap, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. the most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. according to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. |
folin - ciocalteu 's reagent, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxilic acid), dpph (2,2-diphenyl-1-picylhydrazyl), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2 carboxylic acid), gallic acid, and ascorbic acid were purchased from sigma chemical co. (st. surface color and firmness of the fruits were used as criteria for selecting the samples. fruits were classified in 2 categories depending on plant domestication : cultivated and wild edible fruits. cultivated fruits were obtained from the central market of panama city, panama, and wild edible fruits were harvested from their fruit tree. after the fruits were cleaned with tap water, edible portions were cut and analyzed. the edible part of the each fruit (10 g) was homogenized using a blender for approximately 2 min. homogenized samples were then placed in an erlenmeyer flask and extracted at room temperature with 40 ml of methanol for 1 h by using an orbital shaker. antioxidant activity of the fruit extracts was measured according to the method described by lamaison., namely by measuring the reduction of the colored free radical dpph in the presence of the fruit extract at 517 nm. a calibration curve was computed by using trolox, and the total antioxidant activity was expressed as mg trolox per 100 g of fresh fruit weight (mg teac/100 g fw). total phenolics were estimated from the fruits extracts spectrophotometrically as described by singleton and rossi. developed blue color phenolic content was expressed as gallic acid equivalent per 100 g of fresh weight (mg gae/100 g fw). a tritrimetric method was used for determination of vitamin c content in certain fruit extracts by using the 2,6-dichloroindophenol as indicator. results were expressed as mg ascorbic acid, equivalents per 100 g of fresh weight (mg aa/ 100 g fw). folin - ciocalteu 's reagent, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxilic acid), dpph (2,2-diphenyl-1-picylhydrazyl), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2 carboxylic acid), gallic acid, and ascorbic acid were purchased from sigma chemical co. (st. surface color and firmness of the fruits were used as criteria for selecting the samples. fruits were classified in 2 categories depending on plant domestication : cultivated and wild edible fruits. cultivated fruits were obtained from the central market of panama city, panama, and wild edible fruits were harvested from their fruit tree. after the fruits were cleaned with tap water, edible portions were cut and analyzed. the edible part of the each fruit (10 g) was homogenized using a blender for approximately 2 min. homogenized samples were then placed in an erlenmeyer flask and extracted at room temperature with 40 ml of methanol for 1 h by using an orbital shaker. antioxidant activity of the fruit extracts was measured according to the method described by lamaison., namely by measuring the reduction of the colored free radical dpph in the presence of the fruit extract at 517 nm. a calibration curve was computed by using trolox, and the total antioxidant activity was expressed as mg trolox per 100 g of fresh fruit weight (mg teac/100 g fw). total phenolics were estimated from the fruits extracts spectrophotometrically as described by singleton and rossi. developed blue color phenolic content was expressed as gallic acid equivalent per 100 g of fresh weight (mg gae/100 g fw). a tritrimetric method was used for determination of vitamin c content in certain fruit extracts by using the 2,6-dichloroindophenol as indicator. results were expressed as mg ascorbic acid, equivalents per 100 g of fresh weight (mg aa/ 100 g fw). there have been reports in the scientific literature of a strong relation between a regular intake of fruits and a reduced risk of various pathologies such as cardiovascular diseases, cancer, and neurological impairment.[2123 ] oxidative stress, i.e. disequilibrium between the body antioxidant defense system and the generation of radical species, has been linked largely to these ailments. fruits contain phytochemicals that possess known antioxidant activity such as polyphenols and vitamin c, which can reinforce the body antioxidant defense response by using different mechanisms such as scavenging deleterious free radicals from susceptible cells and tissues. consequently, the antioxidant activity of a fruit is a significant parameter to take into account for establishing its nutritional value. dpph radical - scavenging assay measures the ability of an antioxidant to react with dpph radicals generated in the experimental milieu. this assay was used to assess and compare the scavenging activity of extracts of 39 tropical fresh fruits harvested in panama, 7 of which are not domesticated fruits, i.e. caimito (chrysophyllum cainito), guava (inga edulis), algarrobo (hymenaea coubaril), nance (byrsonima crassifolia), jobo (spondias mombin), jagua (genipa americana), and toreta (anona purpurea). table 1 summarizes the fruits surveyed, their antioxidant activity reported as trolox antioxidant capacity (teac), and the total phenolic content. of the 39 fruits studied, guinda (ziziphus mauritania), soursop (annona muricata), and coffe plum (flacourtia jangomas) exhibited the highest antioxidant capacity (1083.33, 975.00, 928.57, and 928.27 mg teac/100 g fw, respectively). on the other hand, avocado (persea americana) and toreta (anona purpurea) presented very low antioxidant activity (17.00 and 16.00 mg teac/100 g fw). in this research, tree tomato showed a very similar teac as guava, which may be due to the possibility that at their physiological maturity (determined by subjective criteria such as their characteristic aroma, taste, and color), both fruits held compounds that altogether make up similar antioxidant activity. in the present study, undomesticated fruits showed a moderate radical scavenging capacity, yet chrysophyllum cainito showed a remarkable high antioxidant capacity (886.36 mgte c/100 g fw). the total phenolic content and teac of panamanian tropical fruits (based on fresh weight) ikram. studied the antioxidant activity of underutilized malaysian fruits, and found that ziziphus mauritania had a moderate antioxidant activity compared to our results. our results regarding the high antioxidant properties of psidium guajava (780.00 mgteac/100 g fw) edible pulp is consistent with surveys conducted in fruits harvested in mauritius, the united states, and india, all of which confirm this fruit 's substantial nutritional value. polyphenols determine central fruits quality properties such as color, taste, and nutritional value. epidemiological research has established an association between the high consumption of fruit polyphenols and a reduced risk of certain kinds of cancer and cardiovascular ailments. polyphenols can end hazardous free radicals propagation, which causes cell lipid peroxidation by transferring an electron equivalent to radicals. in spite of the role these substances play in health maintenance, they are neglected in most food composition surveys. extensive reports can be found in the literature regarding polyphenols in fruits from non - tropical regions ; nevertheless, there is a lack of a comprehensive investigation on polyphenols contained in fruits from tropical areas of the world, mainly central america, despite the wide variety of exotic fruits that are consumed by their populations. bearing this in mind, we evaluated the total polyphenols contained in fruits harvested and most commonly consumed in panama, by using the folin - ciocalteu 's reagent, and correlated this information with the antioxidant activities of the extracts obtained from each assayed fruit. the total phenolic content of the 39 fruits tested are presented in table 1, ranging from 604.80 to 35.10 mg gae/100 g fw. the largest concentration of polyphenols were found in ziziphus mauritiana, followed in order by hibiscus sabdariffa, annona cherimola, chrysophyllum cainito, flacourtia jangomas, and annona muricata (more than 400 mg gae/100 g fw). on the other hand, genipa americana, musa balbisiana, anona purpurea, persea americana, and bactris gasipaes showed the lowest polyphenol content. most of the fruits analyzed possess a moderate phenolic concentration (between 90 and 400 mg gae/100 g fw). comprehensive studies have been carried out to study the antioxidant capacity and polyphenol content of common and underutilized tropical fruits harvested in asia, showing substantial differences when the same fruits are compared with the panamanian species. this variability may be due to different growing conditions, cultivars, climate, geography, and maturation stage. reported decreasing total phenolic content values for psidium guajava > averrhoa carambola > pouteria sapota > manguifera indica > and carica papaya, in fruits grown in the united states. on the other hand, 7 fresh fruits commonly consumed in brazil were assessed for their polyphenolic content, 3 of which, i.e. psidium guajava > averrhoa carambola > ananas comosus, were included in the present study as well. when considering the total polyphenol content of fruits grown in panama, a similar trend emerges, excepting for manguifera indica. in order to determine the influence of polyphenols contained in fruits grown in panama on the overall antioxidant activity, the correlation between these 2 factors was evaluated [figure 1 ] a strong relationship was found [r(37) = 0.89, p 100 mg ascorbic acid/100 g fw), while the lowest quantity was found in solanum quitoense, manguifera indica, hibiscus sabdariffa, passiflora edulis, ananas comosus, musa sapientum, manilkara zapota, and annona muricata (< 10 mg ascorbic acid/100 g fw). most of the fruits had a moderate concentration of this nutrient (between 100 and 10 mg ascorbic acid/100 g fw). when considering the influence of vitamin c on the antioxidant activity of the fruits, vitamin c has a negligible influence [r(18) = 0.32, n.s. ] upon the overall antioxidant properties of the fruits [figure 2 ], which is consistent with previous observations. vitamin c content of fruits surveyed (based on fresh weight) assessment of the effect of vitamin c content on the antioxidant activity of 20 panamanian tropical fruits the present research provides, for the first time, a comprehensive report on the antioxidant activity and total polyphenol content of 39 fruits that are part of the panamanian diet. most of the fruits contained between a moderate and large antioxidant activity and polyphenol content, with a strong correlation between these 2 parameters, indicating that polyphenols presented in the fruits, are strongly associated with their antioxidant activity. when considering the contribution of fruits in the prevention of cardiovascular diseases, many types of cancer, and neurological disorders, such as alzheimer 's and parkinson disease, the important role that polyphenols play in the antioxidant mechanism of prevention of these ailments should be stressed.our results can be used in epidemiologic surveys regarding the intake of polyphenolic antioxidants that foster healthy living conditions in the panamanian population. | objectives : the present research was undertaken to determine the antioxidant activity and total polyphenol content of cultivated and wild edible fruits consumed in panama.materials and methods:39 cultivated and wild edible fruits antioxidant activity and total polyphenol content was assessed by using the dpph and the folin - ciocalteu assays, respectively.results and discussion : the antioxidant composition of the fruits varied between 1083.33 and 16.22 mg teac/100 g fresh weight. on the other hand, the total phenolic content of the 39 fruits tested ranged from 604.80 to 35.10 mg gae/100 g fw. ziziphus mauritania presented the highest antioxidant activity and the largest phenolic content, whereas most fruits had a moderate teac value.conclusion:fruits polyphenol content was strongly correlated with antioxidant properties, which pointed out the important role of these compounds in the prevention of many types of cancer, neurological ailments, and cardiovascular diseases through diverse antioxidant mechanisms. |
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