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dystrophic and aseptic inflammatory disease of intraabdominal adipose tissue is a rare entity.1,2 this disorder mainly involves the mesentery of the small bowel, especially at its root, and occasionally the mesocolon ; however, it can also occur at any other sites in the abdomen, including the pelvis, the peripancreatic area, and the omentum.3 isolated omental panniculitis means the intraabdominal panniculitis that has no evidence of pancreatitis, inflammatory bowel disease, or extraabdominal fat necrosis, and involves the omentum only.1,4 there are only three cases of intraabominal panniculitis with isolated omental involvement that have been reported in the medical literature ; these prior cases were diagnosed by the exploratory laparotomy.1,4,5 we report a case of isolated omental panniculitis diagnosed by abdominal computed tomography (ct) and confirmed by percutaneous ct - guided biopsy. a 61-year - old man presented with a one week history of left upper quadrant noncramping pain and nausea. ultrasonography on his abdomen which was performed at another hospital revealed two hyperechoic masses in left upper quadrant area around the splenic flexure of the colon and the possibility of colonic mass was suggested. the physical examination showed a low grade fever (37.5) and tenderness at the left upper quadrant of the abdomen. laboratory testing revealed a mild leukocytosis (11,100/mm), elevated fasting serum glucose (142 mg / dl), elevated esr (47 mm / h), elevated high sensitivity crp (10.19 mg / dl), and normal amylase (50 iu / l). ct of the abdomen showed two soft tissue masses (4.43.1 cm and 3.12.2 cm) in the omentum around the transverse colon (fig. we performed a percutaneous ct - guided biopsy instead of a surgical biopsy based on the ct findings which was highly suggestive of a benign condition. the histology revealed aggregates of foamy macrophages and chronic inflammatory process including plasma cells and lymphocytes (fig. the diagnosis of isolated omental panniculitis was made according to the clinical symptom, the ct finding and the histology, and the patient was treated with low dosage of prednisolone. a follow - up ct scan two months later showed the regression of the two omental masses (21 cm and 0.50.5 cm) (fig. 3). the patient continued to be well, and completely asymptomatic for 6 months follow - up. the following criteria are considered necessary for the diagnosis of intraabdominal (mainly mesenteric) panniculitis : (i) diffuse, single, or multiple masslike fatty lesions in the mesentery, retroperitoneum, omentum, and/or pelvis ; (ii) histological confirmation of fat necrosis with inflammatory infiltrates and/or infiltration with foamy lipid - laden macrophages ; and (iii) no evidence of pancreatitis, inflammatory bowel disease, or extraabdominal fat necrosis (weber - christian disease).1 this disease is progressive and has three pathological presentations : degeneration of mesenteric fat (mesenteric lipodystrophy), followed by an inflammatory reaction (mesenteric panniculitis), finally leading to fibrosis of the adipose tissue (retractile mesenteritis).2,6 sclerosing mesenteritis seems the most appropriate diagnostic term because of the overall presence of some degree of fibrosis.7 most cases remain idiopathic.1 at the mesenteric lipodystrophy stage, the disease is usually asymptomatic. at the mesenteric panniculitis stage, abdominal pain, fever, and malaise formation of an abdominal mass and possibly obstructive symptoms occurr.2 on ct, the hallmark of mesenteric panniculitis is increased density of the mesenteric fat tissue compared to the attenuation values of normal retroperitoneal or subcutaneous fat.8 this hyperattenuating fat surrounds the mesenteric vessels, but does not displace them.8 the mesenteric lesion, however, may show some regional mass - effect by locally displacing small bowel loops. several additional ct features have been reported that may provide valuable clues for the diagnosis of mesenteric panniculitis : the fat - ring sign, a tumoral pseudocapsule, soft tissue nodules, and a left - sided orientation of mass maximum transverse diameter.3,8,9 these features are not seen in other mesenteric diseases such as lipoma, liposarcoma, lymphoma, or mesenteric carcinomatosis, and their presence may lead to a more confident ct diagnosis of mesenteric panniculitis.3,9 the fat - ring sign and a tumoral pseudocapsule disappear when the mesenteric panniculitis " evolves " to the retractile mesenteritis.3 the clinical symptoms and the ct findings suggested that the main pathological process present in this patient was inflammation rather than fibrosis. a definitive diagnosis of mesenteric panniculitis can be made only by pathological analysis. before the advent of modern diagnostic imaging, the surgical biopsy was done to verify the high density fatty mass on ct in the majority cases of mesenteric panniculitis or sclerosing mesenteritis.1 - 3,10 however, the incidental benign and often asymptomatic nature of mesenteric panniculitis usually does not justify biopsy.8 wat.11 suggested recently that in the absence of a known malignancy, the diagnosis of sclerosing mesenteritis can be made based on the following ct findings without biopsy : (i) hyperattenuating mesenteric fat (especially at the root of the small bowel mesentery). this typically encases undisplaced vessels whilst displacing adjacent bowel loops ; (ii) a tumoral pseudocapsule ; and (iii) well - defined soft tissue nodules less than 5 mm in diameter surrounded by a fatty halo. if however there are features on ct that are not entirely typical for sclerosing mesenteritis such as soft tissue nodules larger than 10 mm in diameter, retroperitoneal extension, displacement of vasculature, invasion of bowel or increase in size of the nodules on follow - up imaging, then a biopsy is needed to exclude the possibility of a malignancy.11 and omental involvement of the panniculitis may mimic carcinomatosis, tuberculosis, and primary mesenteric mesothelioma,11 multiple biopsies are essential for diagnosis.2 pet - ct may have a future role in improving diagnostic accuracy.12 in the prior reports of the isolated omental panniculitis,1,4,5 described ct finding was only the high density fatty mass without other characteristic ct features, and the diagnoses were made by the exploratory laparotomy because the patients revealed extending abdominal tenderness,5 or other diseases (such as the lipoma, liposarcoma or a malignant omental tumor) were suspected.1,4 wedge resection of the mass,1 subtotal omentectomy,5 and partial colorectomy with a resection of greater omental mass and the excision of the lesser omental mass,4 were performed respectively. in this case, the diagnosis of panniculitis could be made based on the characteristic ct features, but the location of lesions was unusual, then percutaneous ct - guided biopsy was performed. the indolent forms of the disease do not usually require specific treatment.3 the guidelines for the treatment of the symptomatic forms are not well established.3 it would appear that active subacute mesenteric panniculitis, as evidenced by continuing fever, a high erythrocyte sedimentation rate and predominance of inflammatory cells, with only minimal fibrosis on histological section, is likely to respond favorably to steroid treatment.13 once the condition has progressed to established fibrosis, steroid treatment is probably ineffectual.13 in this case, prednisolone therapy likely prevented progression of the disease to a more serious degree of fibrosis. surgical treatment is usually not indicated for mesenteric panniculitis except for those patients with a extrinsic bowel obstruction.2 in summary, we report the case of a patient with idiopathic isolated omental panniculitis that was diagnosed by abdominal ct and confirmed by percutaneous ct - guided biopsy. despite its rarity, it is now well established that the diagnosis of mesenteric panniculitis can be made based on characteristic radiological features alone.11,14 but in the case with unusual location including omental involvement, the percutaneous ct - guided biopsy is recommended for confirmation. knowledge of the ct imaging features of this rare condition may prevent unwarranted aggressive procedure for the diagnosis and the treatment. | the preoperative diagnosis of intraabdominal panniculitis is difficult due to its rarity. however, the increased use of abdominal computed tomography (ct) for a variety of indications has increased the diagnosis of intraabdominal panniculitis, including omental panniculitis. the characteristic ct features of intraabdominal panniculitis are increased attenuation of the adipose tissue, the fat - ring sign, a tumoral pseudocapsule, soft - tissue nodules, and a left - sided orientation of mass maximum transverse diameter. recognition of these features is valuable in the diagnosis of panniculitis, and hence percutaneous ct - guided biopsy to determine their presence may prevent unwarranted surgery. we report the case of a 61-year - old man found to have an idiopathic isolated omental panniculitis that was diagnosed by abdominal ct and percutaneous ct - guided biopsy. |
peripheral odontogenic tumors demonstrate histological characteristics of their intraosseous counterparts, but occur solely in the soft tissue covering the tooth - bearing portion of the mandible and maxilla. these lesions are also known as extraosseous odontogenic tumors, soft - tissue odontogenic tumors, or odontogenic tumors of the gingiva. odontogenic myxoma (om) is a rare, benign odontogenic mesenchymal tumor arising from the embryonic connective tissue associated with the tooth - forming apparatus. it was first described by rudolf virchow as myxofibroma in 1863 and was later renamed as odontogenic myxoma by thoma and goldman in 1947. the evidence for its odontogenic origin arises from its almost exclusive location in the tooth - bearing areas of the jaws, its occasional association with missing or unerupted teeth, and the presence of odontogenic epithelium. it is a slow - growing but locally invasive neoplasm, usually occurring in the second or third decade of life. the occurrence of om in the maxilla is rare as compared to the mandible and most reports show a slight predilection for females. oms are central lesions occurring in the jaw bones, commonly present as a painless swelling. pain, displacement of teeth, and paresthesia are uncommon, thus the lesion can reach considerable size before the patient becomes aware of its presence and seeks treatment. histologically, the tumor closely resembles the mesenchymal portion of a developing tooth. the tumor is not encapsulated and is composed of haphazardly arranged stellate, spindle - shaped, and round cells in an abundant loose myxoid stroma that contains only a few collagen fibrils. the treatment of this lesion involves surgical enucleation and curettage, which are the favored techniques. because of its infiltrative character, this lesion is difficult to be curetted and this explains its high recurrence rate. cryotherapy as an adjunct procedure to curettage can be used to minimize the risk of recurrence. intraoral soft - tissue myxoma is an extremely rare lesion and only few reports are available in the literature. in this article, a rare case of peripheral myxoma of anterior maxilla in a 41-year - old female patient is reported with emphasis on review of relevant literature. a 41-year - old female patient was referred to our department with history of a slowly enlarging gingival mass in the anterior maxilla of 6 months duration. intraoral examination revealed a solitary, non - tender swelling on the labial gingiva of the left maxilla in relation to 21 and 22, measuring about 1 2 cm [figure 1 ]. the lesion was firm in consistency and the mucosa covering the lesion was normal in color ; no mobility of the regional teeth was seen. no radiographic changes were seen associated with the lesion [figure 2 ]. based on the overall findings, provisional diagnosis of fibroma was made. intraoral photograph showing the lesion on labial gingiva of left maxilla radiograph showing no bony changes the lesion was surgically excised under local anesthesia and subjected to histological examination. microscopic examination of hematoxylin and eosin stained section demonstrated atrophic, parakeratinized stratified squamous surface epithelium [figure 3 ]. the tumor was composed of loosely arranged spindle- and stellate - shaped fibroblasts with small round nuclei suspended in a delicate network of collagen fibrils [figures 4 and 5 ]. hematoxylin and eosin stained section showing atrophic, parakeratinized stratified squamous surface epithelium (10) loosely arranged spindle - and stellate - shaped fibroblasts with small round nuclei (20) stellate - shaped fibroblasts in loose myxoid stroma (40) om is a mesenchymal lesion that mimics microscopically the dental pulp or follicular connective tissue. the origin of om is believed to be from the mesenchyme of a developing tooth or the periodontal ligament. some authors had previously associated its origin with a myxomatous change of an odontogenic fibroma or residual foci of embryonic tissue. based on the information gathered from the available literature, however, its histological similarity to the stellate reticulum of a developing tooth, its exclusive occurrence in close proximity to the tooth - bearing parts of the jaws, the occasional association with a missing or an unerupted tooth, the presence of odontogenic epithelium in a minority of cases, and the fact that it rarely appears in other parts of the skeleton offer support to an odontogenic origin. most of the patients reported were young adults with average age ranging from 22.7 to 36.9 years. it is rarely seen in patients younger than 10 years of age and in adults above the age of 50. mandible appears to be more affected than the maxilla, in the ratio of 2.5:1. in both jaws, oms commonly occur centrally within the bone of the jaws, whereas our case was located peripherally on the gingiva. buchner. analyzed one of the largest series of odontogenic tumors reported from one source and not even a single case of peripheral myxoma of maxillary gingiva was reported by them. cases of maxillary peripheral om of maxillary gingiva have been reported in the literature by perrotti. out of these six cases, only two cases were reported in the anterior maxillary gingiva. om is commonly reported as a slow - growing tumor that is generally asymptomatic, although some patients experience progressive pain in lesions involving maxilla and maxillary sinus, with eventual neurological disturbances. central myxomas of the jaw have a tendency for extensive bone destruction, invasion into surrounding structures, and a relatively high recurrence rate ; however, metastasis is rare. our case of soft - tissue myxoma was slow growing and localized in nature, with no evidence of recurrence or metastasis. these tumors usually show variable radiographic features ranging from small unilocular lesions to large multilocular lesions, which often displace the teeth or resorb the roots. soap bubble, tennis racket, wispy, and spider - web appearance. since our case was peripherally located in the gingiva, it showed no radiographic changes. the lesion is not encapsulated and is characterized by a proliferation of few rounded cells, fusiform cells, and star cells in an abundant myxomatous stroma with few collagen fibers. small islands of odontogenic epithelial tissue can occasionally be found scattered in such a stroma. om has to be differentiated from other jaw tumors showing myxoid degeneration, such as myxoid neurofibroma, myxoid liposarcoma, and chondromyxoid fibroma. chondromyxoid fibroma shows areas of cartilaginous differentiation, whereas myxoid neurofibromas tend to have scattered lesional cells that are s-100 positive. immunohistochemically, cells of oms stain positively for transferrin, ferritin, alpha - l - antichymotrypsin, alpha - l - antitrypsin, s-100 protein, and vimentin ; however, neuron - specific enolase, s-100 alpha and beta subunits, factor viii - related antigen, and cytokeratin are negative. the lack of capsule and its infiltrative growth pattern are responsible for high rate of recurrence when conservative enucleation and curettage are performed. oms show a locally aggressive behavior and high recurrence and, hence, call for radical resection. however, soft - tissue myxoma is not reported to cause recurrence or metastasis ; hence, carefully planned conservative treatment or semi - radical approach is justified in these cases. in the present case, the tumor was completely removed by local surgical excision and there was no recurrence 6 months after surgery. | odontogenic myxoma comprises 3 - 6% of all odontogenic tumors. odontogenic myxomas are relatively rare benign mesenchymal tumors found exclusively in the tooth - bearing areas of the jaw and are usually located centrally in the mandible. soft - tissue localization is rarely seen and is classified as peripheral myxoma. peripheral myxoma is slow growing and less aggressive, as compared to the central myxoma. it has a low recurrence rate. till date, only few cases of maxillary gingival myxomas are reported in the literature. here, we present an unusual case of primary peripheral odontogenic myxoma occurring in the gingiva of anterior maxilla in a 41-year - old female patient. |
avaliar a aplicabilidade da escala london chest activity of daily living (lcadl), em pacientes em lista de transplante pulmonar. estudo transversal com 26 pacientes em lista de espera para transplante de pulmo, de ambos os sexos, entre maio e setembro de 2010 tratados no programa de reabilitao pulmonar, complexo hospitalar santa casa de misericrdia de porto alegre, em porto alegre, rs. todos os pacientes foram submetidos ao teste de caminhada de seis minutos (tc6) e a teste de funo pulmonar e foram obtidos os escores das escalas lcadl e de borg modificada para dispneia e fadiga das pernas. o teste alfa de cronbach foi utilizado para verificar a consistncia interna da escala lcadl. a anlise de regresso linear foi utilizada para identificar associaes entre o escore total em porcentagem da escala lcadl e as variveis estudadas. segundo os resultados da lcadl, 69% dos pacientes indicaram que suas atividades de vida diria so muito comprometidas pela dispnia. houve associaes negativas estatisticamente significativas entre o escore total da escala lcadl e distncia percorrida no tc6 (= 0,087 ; p < 0,001) e trabalho realizado no tc6 (= 0,285 ; p < 0,001), quando os dados foram ajustados por idade e vef1. esses achados sugerem que a escala lcadl um instrumento til para avaliar o desempenho funcional dos pacientes em listas de transplante pulmonar. the prevalence of respiratory diseases has increased substantially in recent years, and respiratory diseases have come to play a major role in the morbidity and mortality profiles of the population. a public health problem, copd ranked fourth among the leading causes of death worldwide in 2006, and by 2020, it is estimated that it will be the third leading cause of mortality worldwide. in chronic lung disease patients who have severe progressive impairment and a high level of inactivity, lung transplantation is recommended as an essential therapeutic intervention, being an effective treatment option. the most common indications for this intervention are emphysema (in 36%), idiopathic pulmonary fibrosis (in 20%), and cystic fibrosis (in 16%). in transplantation candidates, assessment of their level of physical activity is an important tool for quantifying the impact of the disease on their activities of daily living (adl). the six - minute walk distance (6mwd) is also considered a good marker of functional capacity in adl, and the use of the six - minute walk test (6mwt) is recommended in the assessment of patients with exercise - induced lung disease or cardiovascular disease, given that it provides an overall analysis of the respiratory, cardiac, and metabolic systems. another way of assessing the functional status of a patient is to use scales or questionnaires. the use of questionnaires or scales has shown the impact that the disease has on the level of physical activity and the quality of life of chronic disease patients, indirectly reflecting the degree to which the disease interferes with their adl. currently, there are few validated scales to investigate the impairment of functional capacity in chronic lung disease patients, especially in severely impaired patients who are candidates for lung transplantation. the london chest activity of daily living (lcadl) scale is a tool aimed at assessing the level of dyspnea during adl. it has four domains : self - care ; household activities ; physical activity ; and leisure activities. the lcadl scale, which is considered an inexpensive and user - friendly instrument, can be a feasible clinical tool for assessment and monitoring of dyspnea - related functional impairment in chronic lung disease patients, as well as for pre- and post - intervention assessment. although this instrument has been validated for use in copd patients, there are few studies evaluating the use of this scale in lung disease patients already on the waiting list for lung transplantation. the objective of the present study was to evaluate the applicability of the lcadl scale in patients on the waiting list for lung transplantation by assessing the psychometric properties of the scale and the relationship between the scale scores and the 6mwt results. this was a cross - sectional study involving patients treated at the pulmonary rehabilitation program in the pereira filho ward of the complexo hospitalar santa casa de misericrdia de porto alegre, located in the city of porto alegre, brazil. all male and female patients who were on the waiting list for lung transplantation between may and september of 2010 and who gave written informed consent were included in the study. patients who had cognitive or motor limitations, as well as those who had had respiratory infection in the three weeks before the assessment, were excluded from the study. the version of the lcadl scale used in the present study has been validated for use in brazil. the lcadl scale consists of 15 questions divided into four domains : self - care ; household activities ; physical activity ; and leisure activities. each question in each domain is scored by patients on a 0 - 5 scale, with 5 representing the greatest dyspnea - related impairment in adl. the total score can range from 0 to 75 points, with higher values translating to greater limitation in adl. in addition, the lcadl scale has a multiple - choice question, i.e., question 16, which refers to specific information about dyspnea - related impairment in adl in any situation and to which patients are required to answer " quite a bit ", " slightly ", or " not at all ". the lcadl scale, which was administered as an interview by the same researcher, was interpreted on the basis of its total score (expressed as a percentage). the 6mwt was performed in accordance with the american thoracic society recommendations, considering reference equations for healthy adults. the six - minute walk work (6mww) was calculated by multiplying distance walked by body weight (in kg). before and after the 6mwt, patients were assessed for their perception of dyspnea and leg fatigue with the modified borg scale. all pulmonary function tests were carried out in accordance with the technical standards and the acceptability and reproducibility criteria of the american thoracic society / european respiratory society. flow - volume curves were obtained with a koko spirometer (ferraris respiratory, louisville, co, usa). continuous variables are expressed as means and 95% cis, and categorical variables are expressed as absolute and relative frequencies. bivariate and multivariate linear regression analyses were performed to identify possible associations between the lcadl scale results and the covariates of interest (6mwd, 6mww, age, fev1, and the borg scale scores). initially, all covariates that had a p < 0.10 in the univariate analysis were included in the multivariate model. the next step was to exclude the covariates that were found to have critical p values (values that were not significant) one by one. this step was repeated until all variables remaining in the model had a p < 0.05. the internal consistency of the lcadl scale was measured with cronbach 's alpha coefficient, which investigates specific correlations between total and domain scores. for the purpose of calculating this coefficient, values of p 0.70 are considered adequate. in addition, the proportion of patients with minimum and maximum scores (floor / ceiling effect) was calculated. the present study included 26 subjects, 10 (38%) of whom were male. table 1characteristics of the sample of patients on the waiting list for lung transplantation. variableresultbmi, kg / m 23.8 30.2fev1, % of predicted35.1 17.4fvc, % of predicted43. 8 14.9fvc / fev1 0.67 0.236mwd, m331.4 118.76mww, m / kg101.9 30.2bmi : body mass index;6mwd : six - minute walk distance;6mww : six - minute walk work.. in the sample as a whole, 14 (54%) and 12 (46%) of the patients had obstructive and restrictive lung disease, respectively. the patients included in the present study had the following diagnoses : pulmonary fibrosis, 13 (50%) ; copd, 4 (15%) ; cystic fibrosis, 2 (8%) ; bronchiectasis, 3 (12%) ; and others, 4 (15%). the mean total lcadl score (expressed as a percentage) was 36%. in the sample as whole, 16 patients achieved a total lcadl score above 50%. in 69% of the cases, the answer to the qualitative question on the lcadl scale, i.e., question 16, which refers to the level of dyspnea - related impairment in adl, was " quite a bit ". the overall alpha value was 0.89, and the alpha values for each domain ranged from 0.72 to 0.94. there was no floor or ceiling effect. in the univariate analysis, the 6mwd, the modified borg scale scores, and the 6mww showed significant associations with the total lcadl score (expressed as a percentage ; table 2). table 2univariate analysis of the total score on the london chest activity of daily living scale (expressed as a percentage).variable95% cipage, years0.226 0.222 to 0.6740.323diagnosis copd0.000 pulmonary fibrosis 16.325 34.000 to 1.3510.070 cystic fibrosis 8.244 26.362 to 9.8740.372 bronchiectasis 9.658 30.574 to 11.2590.365 others 10.746 27.024 to 5.5320.196fev1, % of predicted0.373 0.001 to 0.7410.0056mwd, m 0.079 0.132 to 0.0270.002modified borg scale score 0.056 2.461 to 2.3490.9646mww, m / kg 0.348 0.563 to 0.1330.0026mwd : six - minute walk distance6mww : six - minute walk work : six - minute walk distance : six - minute walk work in the multivariate analysis, the 6mwd (figure 1) and the 6mww showed a significant inverse association with the total lcadl score (expressed as a percentage ; table 3). figure 1relationship between the total score on the london chest activity of daily living scale (expressed as a percentage) and the six - minute walk distance (6mwd). (= 0.103 ; p < 0.001). table 3multivariate analysis of the total score on the london chest activity of daily living scale (expressed as a percentage).variable95% cip6mwd, m0.087 0.128 to 0.046 < 0.0016mww, m / kg0.285 0.448 to 0.123 < 0.0016mwd : six - minute walk distance6mww : six - minute walk work : six - minute walk distance : six - minute walk work the objective of the present study was to evaluate the applicability of the lcadl scale in patients on the waiting list for lung transplantation. this instrument was found to have good reliability and to be associated with the 6mwd and the 6mww. our data showed that the total lcadl score was inversely associated with these parameters in the study population.. showed that the lcadl scale correlated with the 6mwd in copd patients. when we compared the lcadl scores with the different diagnoses of patients, we found no significant differences. however, we observed that copd patients had higher scores on the lcadl scale than did those with other diagnoses. the lack of significance in the differences among the different diagnoses might be due to a lack of power for this analysis. recent studies have reported a negative correlation between movement intensity during adl in copd patients in the self - care domain and the total lcadl score. the reported results suggest that lower movement intensity during adl translates to a higher total score on the lcadl scale, indicating greater functional impairment in adl. when we evaluated the association between the 6mww and the lcadl scores, we also found it to be significant. one group of authors proposed that functional performance during exercise would be better expressed by the product of 6mwd and body weight than by the analysis of 6mwd alone, because subjects perform specific work when walking, given that they transport their own body over a given distance over a given time. in the present study, the lcadl scale was found to have high internal consistency (alpha), with the overall alpha value being 0.90 and the alpha values for each domain ranging from 0.72 to 0.94. previous studies have demonstrated that the lcadl scale has high internal consistency when administered to copd patients. one study reported an alpha value of 0.98, which demonstrates that the lcadl scale is a valid measure of dyspnea during adl. another study showed that the brazilian portuguese - language version of the lcadl scale is reproducible, having an alpha value of 0.86. in the present study, 69% of the patients in the sample achieved a total lcadl score (expressed as a percentage) above 50%, i.e., they showed severe dyspnea - related limitation in their adl. a recent study has suggested the use of a cut - off point of 50%, with a score equal to or greater than 50% indicating severe dyspnea - related limitation in adl. in addition, we should take into account that most of the study sample reported that dyspnea significantly affects their adl, which leads us to believe that dyspnea can impose major restrictions on these patients. the presence of dyspnea has been considered a severe limitation to physical and social activities, having a direct impact on the quality of life of patients with chronic obstructive or restrictive respiratory disease and leading to a marked increase in morbidity and mortality. in the univariate analysis, the modified borg scale score (dyspnea) showed no significant association with the total lcadl score (expressed as a percentage). this finding might be associated with the fact that the borg scale has poor reproducibility, which has been reported in other studies, given the difficulty that patients have in classifying their level of dyspnea because of desensitization. in this sense, the lcadl scale can be a useful and user - friendly tool for assessing patients on the waiting list for lung transplantation, especially those experiencing significant impairment in adl, in whom dyspnea is an incapacitating symptom, even for routine activities. the fev1 was not significantly associated with the total lcadl score (expressed as a percentage). one explanation for this finding is that, although fev1 provides a useful description of the severity of the disease - related pulmonary changes, it does not systematically assess the impact that the disease has on the patients ' ability to perform their adl because of dyspnea. these findings can be attributed to the fact that functional classification scales are more strongly related to patient symptoms and clinical status, neither of which are directly related to age. one explanation for the lack of association between the lcadl scale results and patient age in the present study is the fact that the sample did not include older patients. one group of authors found a strong negative correlation between age and functional capacity, which is evidence that physical aspects are more impaired by advancing age than are mental aspects. however, despite the limited number of patients, it was possible to determine that the lcadl scale behaves appropriately with regard to psychometric properties, as well as being strongly associated with the 6mwd and the 6mww, which are objective measures. in conclusion, the lcadl scale proved to have instrumental reliability, as well as being correlated with conventional objective measures of the level of physical activity. the fact that the lcadl scale performed well in patients on the waiting list for lung transplantation suggests that this instrument can be a valid and feasible tool to assess dyspnea during adl. | objective : to evaluate the applicability of the london chest activity of daily living (lcadl) scale in patients on the waiting list for lung transplantation. methods : this was a cross - sectional study, conducted between may and september of 2010, involving 26 male and female patients on the waiting list for lung transplantation and treated at the pulmonary rehabilitation program in the complexo hospitalar santa casa de misericrdia de porto alegre, located in the city of porto alegre, brazil. we evaluated the patients using the six - minute walk test (6mwt) and pulmonary function tests. we also obtained the lcadl scores, as well as the modified borg scale scores for sensation of dyspnea and leg fatigue. cronbach 's alpha coefficient was used to determine the internal consistency of the lcadl scale. linear regression analysis was used in order to identify associations between the total lcadl score (expressed as a percentage) and the variables studied. results : according to the lcadl scale results, 69% of the patients reported that the performance of their activities of daily living was significantly impaired by their dyspnea. the internal consistency of the lcadl scale was 0.89. after adjusting for age and fev1, we found that the total lcadl scale score showed statistically significant negative associations with the six - minute walk distance (= 0.087 ; p < 0.001) and the six - minute walk work (= 0.285 ; p < 0.001). conclusions : our findings suggest that the lcadl scale is a useful tool for assessing patients on the waiting list for lung transplantation. |
according to the european association of urology guidelines on urinary incontinence, concerning the treatment of female stress urinary incontinence (sui), the retropubic insertion of a midurethral synthetic sling (mus) gives equivalent patient - reported cure of sui at 12 months, when compared to colposuspension. these guidelines also report that midurethral synthetic sling inserted by either the transobturator (to) or retropubic (rp) route gives equivalent patient - reported outcome at 12 months. with an obvious trending towards less and less invasive surgical options, single - incision vaginal slings (sis) have emerged. they require very limited intracorporeal dissection, proposing to further increase safety of suburethral slings, without jeopardizing the success rates reported by conventional rp and to access. these sis outcomes are comparable with conventional mus at short - term follow - up [35 ]. although sparse, two - year follow - up studies are available and seem to maintain steady success rates over this time [6, 7 ]. longer follow - up time reports are needed, to ensure that, in the long run, these sis offer constant success rates. the objective of this study is to describe the outcome of women treated with mini - arc at a mean follow - up of 45 months, based on a baseline population which has already been reported in a short - term paper, after adequate long - term follow - up evaluation. previously considered cured and improved patients were evaluated to access if their condition remains stable, as reflected in a subjective satisfaction evaluation. this is a single - centre prospective evaluation of women with urodynamic stress urinary incontinence, which were submitted to mini - arc (american medical systems, minnetonka, mn, usa) placement as a primary surgical treatment. surgical technique, inclusion and exclusion criteria, baseline population characteristics, and short - term outcome and complications have already been described in a previous paper. on this report, on 105 women with a mean follow - up of 15 months (and a minimum follow - up of 6 months), 84 patients (80%) now, with a mean follow - up of 45 months, cured / improved patients were reassessed by telephone interview and completed patient global impression of improvement (pgi - i), to access treatment response, patient global impression of severity (pgi - s), to access current sui condition, rated their improvement in a 0100 scale, and answered if they would recommend the procedure. this study was approved by the institutions ' ethics committees and each participant provided written informed consent prior to enrollment. at 15-month mean follow - up (initial population of 105 patients), 84 patients were cured and 12 improved. seventy - seven patients could be contacted (80% of the initial population) and have a current mean follow - up of 45 months (median 43.5 months). three were submitted to other forms of sui treatment during the period of follow - up. so, from a total of 77 responders, 70 (91%) maintained the initial cure / improvement situation (figure 1). fifty - three of the cured patients (84%) rated the improvement of sui by the pgi - i as very much better or much better and 4 (6%) considered it to be a little better. four patients (6%) answered no change and two (3%) a little worse the mean rate of improvement in a 0100 scale was 81 15, 52 patients (83%) rating improvement > 70. fifty - four patients (86%) reported their urinary tract condition (utc) to be normal on pgi - s (figure 3). when analyzing improved patients (n = 7), 2 (29%) considered their pgi - i as very much better or much better, 1 (14%) a little better, and 4 (57%) no change (figure 2). only 3 patients (43%) rated their improvement to be equal or superior to 70% in a 0100 score or five patients (71%) answered moderate on pgi - s, with only two patients (29%) considering their utc to be female urinary incontinence is a very common condition, which can affect around 35% of women ; sui is the most prevalent type, but the consultation and treatment rates are very low. the conservative management is the first treatment option and it usually includes pelvic floor muscle training, which can be very successful in around a fourth of the patients, especially in younger patients with mild forms of the condition. obese women can adopt a program of weight reduction associated with physical exercise, which can offer a 25% cure rate, since they stay firmly devoted to the program over time and are willing to wait for the improvements. as a result, surgery is the most common form of sui treatment worldwide. during the last 2 decades we have observed the development of promising sui surgical techniques and the introduction of suburethral, tension - free slings. tvt (gynecare, ethicon, somerville, new jersey, usa) was the first device of this kind to be introduced in clinical practice, in 1996 by ulmsten.. according to the european association of urology guidelines on urinary incontinence, the rp insertion of a mus gives equivalent patient - reported cure of sui at 12 months, when compared to colposuspension. nonetheless, tvt shows low invasiveness, short hospital stay, reduced risk of prolonged catheterization, and low risk of causing future pelvic organ prolapsed. all together, these characteristics were responsible for the swift replacement of burch colposuspension as the preferred surgical approach to female sui. tvt has become the gold standard in the surgical treatment of sui with high cure rates that subsist at long time follow - up. the blind passage of needles through the rp space was associated with severe complications, such as bladder and bowel perforations and life - threatening vascular injuries [16, 17 ]. these concerns led to the development of the to route in 2000, a relatively avascular space for the passage of trocars. however, to tapes have been associated with prolonged and limitative pain referred to the groin and upper thigh, due to the obturator foramen violation and vaginal perforations due to a more horizontal trajectory of the needle passage [16, 17, 19 ]. to our knowledge, this is the longest follow - up prospective report on mini - arc single - incision sling. at roughly four - year follow - up, the majority of patients cured or improved at short - term evaluation maintain a high degree of satisfaction at long term. short- and midterm reports on mini - arc have, on the majority, been consistent with the initial results of this series [6, 7 ] and comparable to conventional mus [20, 21 ], with a low morbidity profile [20, 21 ]. the number of patients available for this evaluation, with 80% responders at almost 4-year mean follow - up, permits having an adequate idea of the long - term outcomes of the initial population, in a reliable way. patient global impression of improvement questionnaire addresses the sui treatment outcomes when compared with baseline condition and the results among the cured patients describe a 90% (57 patients) positive result, with 84% of the patients considering their actual condition to be very much better or much better, which is usually assumed to be equal to a cured situation. these numbers are certainly reliable, as the 0100 improvement scale results mean score is over 80%, with over than 4/5 of the cured patients rating this improvement > 70%. on the other way, the actual urinary tract condition, addressed by pgi - s, is considered normal by 86% of the cured women. the improved - patient population is very small (n = 7), and interpreting their results would not prompt solid conclusions. these reports on long - term evaluation are very important to assure that sis are a valid technique, with fair and comparable results at short- and middle - term evaluations, and that over time the results are maintained stable. this study shows that the majority of patients cured / improved after mini - arc placement maintain a high degree of satisfaction at a long - term evaluation. | single - incision slings were introduced in the surgical treatment of female stress urinary incontinence (sui) to lessen the morbidity associated with traditional midurethral slings. however, long - term reports on patient satisfaction are still scarce. this study describes the outcome of women treated with mini - arc at a mean follow - up of 45 months. in a previous report on 105 women with 15-month mean follow - up, 84 (80%) were found cured and 12 (11%) improved. now, with a mean follow - up of 45 months, cured / improved patients were reassessed by telephone and completed patient global impression of improvement (pgi - i), patient global impression of severity (pgi - s), rated their improvement in a 0100 scale, and answered if they would recommend the procedure. at 45-month follow - up, 73 women cured / improved were available for evaluation. over 80% of the cured patients rated the improvement of sui by the pgi - i as very much better or much better, reported their urinary tract condition to be normal on pgi - s, and described their improvement > 70%. ninety percent would recommend this procedure to a friend. the improved - patient population is very small (n = 7). this study shows that the majority of patients cured / improved after mini - arc placement maintain a high degree of satisfaction at a long - term evaluation. |
the perivascular epithelioid cell tumors (pecomas) are a family of related mesenchymal neoplasms that include angiomyolipoma, lymphangiomyomatosis, and clear cell ' sugar ' tumor of the lung (1). in 1991, investigations of hmb-45 (gp100) immunoreactivity and the presence of premelanosomes in both clear cell ' sugar ' tumors (ccsts) of the lung and the epithelioid clear cell component of angiomyolipoma (aml) of the kidney and liver were published (2 - 6). first proposed a cellular link among these unusual mesenchymal lesions and lymphangiomyomatosis (lam) (7). soon after, the same group suggested the descriptive term ' perivascular epithelioid cell ' (pec) for the distinctive cell type found in these three lesions, and hypothesized that pecs may originate from the walls of blood vessels based on the observation that these cells are frequently and intimately related to such structures (8). most of the patients with lesions of the pecoma family are female, and some patients also show coexistence of the tuberous sclerosis complex (tsc) (9). although there are strong associations between the tsc, aml, and lam, this association is much less clear for the rare pecomas (10). malignant pecomas are extremely rare, and they have been reported to occur in the uterus, broad ligament, prostate, small intestine, rectum, skull base, and heart (11 - 16). a 30-yr - old korean man visited to hospital for the evaluation of a growing palpable abdominal mass, which he had felt growing over 6 months. a computed tomography (ct) scan of the abdomen showed an intraabdominal mass with a size of about 7.5 cm that was supposed to originate from the falciform ligament. the solid portion was yellow - brown in color and revealed foci of hemorrhage and necrotic changes (fig. histological examination showed sheets of spindle - to - epithelioid cells with clear - to - eosinophilic cytoplasm (fig. they were also positive for the s-100, a marker of neurogenic and melanocytic tumors, and were negative for the cd117 (c - kit), which typically shows positivity in classic gastrointestinal stromal tumors (gists). the tumor cells were also negative for factor viii, cd34, cd31, ck, and desmin. however, the tumor showed high cellularity and necrotic foci. these features suggest that the tumor had aggressive potential and a high probability of local recurrence or metastasis. the ki-67 proliferative index was approximatively 15 percent of cells. at 6 days after surgical excision, because the biological behavior of this tumor is still unclear, with descriptions in the literature of pecomas with metastases, we had planned follow - up investigation in the form of ct scans of the abdomen and the thorax. until now the " perivascular epithelioid cell " (pec) was first proposed by bonetti and colleagues in 1992 as an explanation for the presence of hmb-45 (gp100) immunoreactivity and premelanosomes in both clear cell " sugar " tumors of the lung and angiomyolipomas (8). the pecs are characterized by epithelioid cells with a clear to faintly eosinophilic cytoplasm, a perivascular location, and the unique co - expression of smooth muscle actin isoforms and melanocytic markers such as the gp100 protein recognized by hmb-45 (8). hmb-45 and melan - a are the most sensitive melanocytic markers in pecoma. on our case, tumor cells were positive for hmb-45, sma, and s-100. at the time of the final diagnosis, no metastatic lesions were present. a significant association was noted between a tumor size of 8 cm (median size), mitotic activity greater than 1/50 hpf, and necrosis with aggressive behavior (1). patient age (median age : 46 yr), infiltrative growth pattern, gynecologic primary site, high cellularity, high nuclear grade, atypical mitotic figures, vascular invasion, and the growth pattern of the tumor (spindled or epithelioid) were not associated with aggressive behavior (1). in our case, the tumor size was 8.0 cm. these features suggested that the tumor had aggressive potential and a high probability of local recurrence or metastasis. the ki-67 proliferative index was approximately 15 percent of cells. although it has been suggested that pecomas do not express s-100 protein, thereby allowing them to be distinguished from melanoma and clear cell sarcoma, this was not the case. a series of seven very young patients (age range, 3 - 29, median, 11 yr) pecoma of the falciform ligament / ligamentum teres was reported in 2000. therapy consisted of surgical resectioning. during the mean follow - up of 18 months (range, 6 months to 5 yr) in six of seven cases, five patients remained healthy, and relatively few malignant pecomas have been reported, firm criteria for malignancy have yet to be established. special attention should be paid to the lungs and liver because of potential metastasis (18). a pecoma situated elsewhere has to be excluded because the tumor described here could have been a primary tumor or a metastasis. because pecomas can behave in an aggressive manner, careful follow - up is warranted. in addition, to perform a pathological diagnosis, recognition of the clinicopathological, histological, and immunohistochemical (hmb-45 positive) features of pecoma seems to be very important. | we present a case of perivascular epithelioid cell tumors (pecomas) in the abdominal cavity at the falciform ligament. a 30-yr - old korean man visited to hospital for the evaluation of a growing, palpable abdominal mass. he had felt the mass growing over 6 months. there was no family or personal history of tuberous sclerosis. the resected specimen showed a mass of 8.07.05.5 cm in size. histological examination showed sheets of spindle - to - epithelioid cells with clear - to - eosinophilic cytoplasm. immunohistochemically, tumor cells were positive for hmb-4 (gp100) and smooth muscle actin. they were also positive for the s-100, which is a marker of neurogenic and melanocytic tumors. patient was treated with radical resection of tumor without any adjuvant therapy. he is well and on follow - up visits without tumor recurrence. |
over the past few months, the health sciences community has engaged in a fierce debate regarding pathways to achieve the goals of open science.1,2,3 there is a significant divide between the potential for the rapid, reproducible health research that is critical to advancing health science, on the one hand, and the current infrastructure and incentives in place that are designed to keep information private and unavailable to others who may benefit from using health data, on the other. all the communities we work with as part of the electronic data methods (edm) forum health services research, clinical research informatics, medicine, behavioral health, and numerous others are grappling with these issues to better assess the feasibility, cost, and ultimate benefits of a substantial culture change toward greater openness. indeed, as the president s cancer moonshot demonstrates,4 there is a new imperative to accelerate collaboration in implementation science, learning health systems, and precision medicine.5 writ large, open science includes a suite of approaches to facilitate greater access to both data and the information produced by a process of scientific inquiry (fig. 1).6,7 open access (oa) publishing is the most well - known of these concepts. as currently defined, oa publishing is designed to enable the free access, use, modification, and sharing of published research for any purpose.8 increasingly in physics and astronomy,9 and more recently in biological health, open data and open source code10,11 accompany oa manuscripts to accelerate the scientific process by contributing research materials and findings in a computable format. the goal of these open scientific efforts is to facilitate more rapid sharing and, therefore, learning across the field, and to promote reproducibility of programs committed to research, discovery, and continuous improvement. certainly, there are legitimate concerns about the need to ensure adequate security and privacy of individual, protected health information (phi) in an era of open science. however, there are also substantial prevailing interests arguing against open sharing that do not necessarily represent the public s interest. these include the potential value of intellectual property to investigators who withhold data sets in order to preserve the ability to conduct new analyses over time, and even concerns that more reproducible research enables competing researchers to identify flaws in the original work if the original data is reanalyzed. these issues can be heightened in the clinical health domain given its close relationship with a trillion dollar marketplace for health services that is increasingly dependent on data and analytic tools. both examples highlight incentives not to share and not to collaborate that, if not entirely wholesome, are nonetheless entirely human. there are many reasons to believe oa and open science are crucial to enabling a national learning health system. as demonstrated by arxiv in the physics, mathematics, engineering, and astrophysics communities,12 a key value proposition for open science is the ability to spread promising scientific approaches rapidly in a more open and transparent environment both to accelerate innovation and to reduce redundancy. note, however, that as we move beyond the current period in which unprecedented support has been provided for health it infrastructure and research, it is imperative that we consider ways to provide open source tools that can reduce disparities between wealthier systems and regions on the one hand and those that may be more resource constrained, including safety net and public health systems, on the other. open science offers tools to minimize the digital and analytic divide between disparate systems while promoting continuous learning and improvement. the result of these debates over oa can be extremely polarizing because open science is, at its core, a philosophy. while formal requirements to instantiate open science principles as part of the scientific workflow may be on their way, for now we re relying on the goodwill of data liberators13 and individual actors to demonstrate the benefits of openness. those in health care who more freely share data and code believe that the public good resulting from sharing their work is of greater value than the rent they may be able to derive from their work at a later date in applied health research, findings and approaches to improving health care delivery are sufficiently proximal to commercial health care in that they are perceived to have more commercial value. this has historically led to a phenomenon wherein the performance of new predictive algorithms and other tools are published, while the full code or a reference data set needed to replicate the findings are not. with support from the agency for healthcare research and quality, the edm forum s natural experiment to facilitate a culture of open science started in 2010, with the creation of a repository to facilitate contributions from the community, including an opportunity to crowdsource comments and review. fairly quickly however, we perceived a need for an independent oa journal that would ensure good peer review for cutting edge work pertaining to electronic health data. as a result, the edm forum launched generating evidence & methods to improve patient outcomes (egems) in 2013. in an early editorial, i commented on our philosophy for the journal,14 recognizing that egems was an experiment in understanding the culture of oa publishing and the willingness to embrace manuscripts more focused on methods, data, and the implementation issues affecting our ability to build learning health systems.15,16 egems has kept to this commitment. our inaugural efforts published articles discussing the funding from the american recovery and reinvestment act (arra)17 and the health information technology for economic and clinical health (hitech) act. subsequently, an infusion of support from the affordable care act for patient - centered outcomes research supported by the patient - centered outcome research institute (pcori)18 and the center for medicare & medicaid innovation (cmmi) has led to new insights into data, methods, and implementation science. new opportunities to reinvest in paying for quality care via the medicare access and chip reauthorization act (macra)19 and the new regulation implementing payment reform, the merit - based incentive payment system (mips),20 will no doubt generate further opportunities for learning in practice. i am pleased to report that as of june 1, 2016, egems has published its 139 article, and has passed a personal milestone for readership of 100,000 paper downloads. a great boon to reaching this goal has been the journal s inclusion in pubmedcentral (pmc), which ensures egems is fully searchable via keywords in pubmed21 and makes the full text of all articles available in html. the number of citations of egems papers is also growing rapidly in step with increasing readership. while figure 2 demonstrates the rapid rate of increase in downloads year after year, what is not observable is an important change we refer to as the pubmed effect. since march of 2015, when egems papers were made available in pubmed, access to our manuscripts has undergone a rapid shift from accessing egems papers on our own website (www.egems.org) to downloads in pmc as of may 2016 access to manuscripts via pmc now comprises 67 percent of all egems downloads. this transition is informative for our team and the field because it demonstrates the important public good provided by the national library of medicine s catalog as an international marketplace for health research that includes credible oa journals. it also demonstrates the extraordinary power of semantic search to ensure that new ideas are found within the massive catalog available through pmc. interestingly, many of the most highly downloaded papers from pmc were different from the most downloaded papers on the egems website, highlighting that the two sources are accessed for different purposes and perhaps by different types of researchers. an ongoing effort for our team is to understand differences in search and citation patterns due to the portals used to access egems papers bibliometric and sociometric research of interest on its own. our global readership, according to statistics for our website (excluding downloads from pubmed), shows significant international readership (figure 3). since the journal s launch, roughly 60 percent of downloads can be traced to countries outside of north america, demonstrating a strong international readership. at the same time, we have struggled with many of the same issues as other journals, such as swift recruitment of peer reviewers and increasing the rapidity of the peer review process, and are continuously improving our timeliness while maintaining the quality of peer review. a key strategic decision arrived at with leadership from the egems editorial board was to dedicate time to making sure we get things right. at this early stage in the development of science with electronic health data (ehd), it is most important to publish the most useful stories about the journey toward timely and relevant science at the nexus of precision medicine, delivery system science, and implementation science. and it is nt always a simple or quick process to tease out the most salient lessons from ongoing efforts in this space. in what may be the world s greatest understatement ! one critical element of developing a culture of greater experimentation is to promote transparency of the work in an open commons of data, code, and scientific processes. notable examples of communities that have demonstrated effective use of platforms to show and share data include github for software engineering and an increasing number of disciplines, and the open science framework, which supports many projects in biomedicine and other fields. to achieve a similar success in our collaborations with learning health systems interested in improving health and health care, our team has been working with colleagues at the ohio state university to build the third pillar of open resources needed to support a culture of open science in a commons for sharing data and code in applied health science. however, the most salient concept is the translation of the acronym as the spanish word for sky, since the type of sharing we envision is a blue sky concept. cielo is designed to include the version control framework, git, developed by linus torvalds.22 however, unlike github, which does not enable efficient semantic search for work in key areas of health science, cielo enables the community to develop new tags (a folksonomy) that will evolve to create the most salient tags or taxonomy. the goal is for cielo, over time, to develop a robust knowledge - management structure that has strong links to egems keywords and pubmed medical subject headings (mesh) so that items can be found easily and unambiguously cited using digital object identifiers (dois). if these goals can be achieved we ll be pleased to contribute this test bed to the community to facilitate a culture of more rapid adoption and adaptation of new tools techniques. if health care is to join the ranks of other fields that have embraced open science, we must address several issues that we already know are critical to preserving the public s trust in scientific publishing : - the ability to unambiguously cite all published scientific material.ideally, this will occur at the levels of both the object such as dois, as well as that for individual researchers such as open researcher and contributor i d (orcid).23- infrastructure and standards to support metadata (data about the data).this is to ensure unambiguous interpretation at the level of individual data elements, which is key to executing programs in new environments.- seamless links between formal peer - reviewed publications and the materials that support the research.executable papers are perhaps the most advanced concept in this arena24 and propose a promising direction for our work to show not tell.- rigorous quality control of peer review.increasingly, standards for reviewers are being developed,25 in addition to changes in the academic marketplace that promote and reward higher quality reviews.- review for plagiarism.this is to ensure that standards are met, with respect both to the uncredited use of others work, and to self - plagiarism.- credentialing and strong management of journals to ensure predatory practices do not prevail. - the ability to unambiguously cite all published scientific material. ideally, this will occur at the levels of both the object such as dois, as well as that for individual researchers such as open researcher and contributor i d (orcid).23 - infrastructure and standards to support metadata (data about the data). this is to ensure unambiguous interpretation at the level of individual data elements, which is key to executing programs in new environments. - seamless links between formal peer - reviewed publications and the materials that support the research. executable papers are perhaps the most advanced concept in this arena24 and propose a promising direction for our work to show not tell. - rigorous quality control of peer review. increasingly, standards for reviewers are being developed,25 in addition to changes in the academic marketplace that promote and reward higher quality reviews. this is to ensure that standards are met, with respect both to the uncredited use of others work, and to self - plagiarism. resources such as retraction watch26 and beall s list of predatory journals for 201627 are examples of efforts to promote credibility and best business practices among scientific publishers. egems has readily participated in a review of our editorial practices to demonstrate and certify that we manage our journal with the highest standards of scientific credibility and openness, even as these standards evolve to be increasingly rigorous.28 having enumerated this substantial wish list for open science in health, i should acknowledge that the person - hours to achieve these aims are significant and will require strong community engagement and support. we must define a strong value proposition for researchers and other analysts who produce ideas and evidence as their livelihood. likewise, the costs of producing and maintaining repositories of code or data are not well appreciated, and must be understood in order to achieve a rational and sustainable approach. even with cielo, we acknowledge it will be an ongoing effort to achieve our idealized components for a health repository, or repo,29 yet i am optimistic we will resolve these issues by working closely with the scientific communities engaged in the edm forum. health researchers have adapted to great change in the past five years, and we are just now beginning to get our bearings on ways to make the most effective use of the enabling health it infrastructure at our disposal. i anticipate tremendous work from egems authors and readers in the years to come and am pleased to provide resources to help promote the journey toward open science in health research. | open science includes a variety of approaches to facilitate greater access to data and the information produced by processes of scientific inquiry. recently, the health sciences community has been grappling with the issue of potential pathways and models to achieve the goals of open science namely, to create and rapidly share reproducible health research. egems continued dedication to and milestones regarding the publication of innovative, useful, and timely research to help contribute to the push towards open science is discussed, as well as the edm forum s new data sharing platform, cielo. although strides have been made, there is still more work to be done to help health sciences community truly embrace open science. |
the viruses used in this study were isolated from homogenates of the brains of bats by direct intracranial (i.c.) although isolate b58 was obtained in 1989, no further identification or characterization was conducted until this study. was conducted following procedures approved by the animal ethics committee of the institute for viral disease control and prevention, china. mice were observed 2 per day after inoculation, and every 2 hours after the onset of clinical signs. after the appearance of cytopathic effect (cpe), supernatant was harvested and passaged 3 more times. neurovirulence of the 2 bat jev strains and of a mosquito - derived jev strain, m10 (9), were determined. all viruses were consecutively passed 3 times in mice, and virus suspension (defined as the 10 stock) was prepared from the third passage. for determination of a 50% lethal dose (ld50), suckling mice (5 per group) were inoculated i.c. with dilutions from 10 to 10. animals were monitored daily for survival, and the ld50 values were calculated by using a standard method (10). viral rna was isolated by using the viral rna mini kit (qiagen, hilden, germany). first strand cdna was synthesized using the ready - to - go kit (amersham pharmacia biotech, uppsala, sweden). flavivirus - specific primers (11) and primers designed from the sequence of jev beijing-1 (l48916) were used for pcr and sequencing. sequence assembly was conducted by using the atgc software package, version 4.0 (genetyx corp., homology and alignment analysis were conducted by using clustalx version 1.8 (www.clustal.org/download/1.x/ftp-igbmc.u-strasbg.fr/pub/clustalx) and megalign (dnastar, madison, wi, usa). the 2 groups (n = 8 for each) inoculated with b58 and gb30, respectively, displayed clinical signs after 42 h postinoculation (hpi), whereas the control group with buffer only (n = 8) displayed no clinical signs. clinical signs included refusing sucking, no interest in grouping, neck rigidity, tremors and muscular spasms, ataxia, and hind - limb paralysis. the supernatant of brain homogenate was used to inoculate c6/36 cells, and cpe was visible at 96 hpi for the first passage and at 72 hpi for second and third passages. the identity of b58 as jev was confirmed by pcr sequencing. the complete genome sequence of both isolates the 2 genomes have identical size at 10,977 nt with a 95-nt 5 nontranslated region (ntr) and a 583-nt 3 ntr. the genomes have similar guanine - cytosine content (51.44% for b58 and 51.39% for gb30). the 2 bat jev isolates have an overall sequence identity of 99.9% both at nt and aa levels. when compared to 55 known jev isolates of known complete genome sequences, the nt sequence identity varies from 88.6% to 99.3%, and aa sequence identity from 97.0% to 99.3%. analysis of the ntr sequences showed that the bat jev isolates have the same 5 ntr as do the others, but the 3 ntrs of the bat jevs have a g insertion at nt 307, the same as that observed in the nakayama strain (genbank accession no. phylogenetic trees derived from nucleotide sequences of the complete genome or the most variable envelope protein gene of selected jev strains (table 1) indicated that both jev isolates from bats are members of genotype iii as defined by solomon. (3). a more detailed analysis indicated that the bat jevs are most closely related to human isolate liyujie and mosquito isolate bn19 within cluster 6 (figure). similar phylogenetic trees were obtained based on other gene sequences, such as prm (data not shown). phylogenetic tree based on the envelope (e) protein gene of selected japanese encephalitis virus strains. murray valley encephalitis virus (mev) e gene (in boldface) was used as an outgroup. see table 1 for more details of the strains used in this analysis and their genbank accession numbers. neurovirulence of the 2 bat jev isolates was determined as described above, and the ld50 for suckling mice was 8.0 log10/0.02 ml, compared with 3.5 log10/0.02 ml for the mosquito strain m10. neurovirulence of these 2 bat isolates also was predicted from aa sequence comparison to those known to have high neurovirulence (13,14). as shown in table 2, all residues important for virulence and neurotropism were conserved between the bat jev isolates and the nakayama strain. these 8 aa residues of the envelope protein are shown to play a key role in neurovirulence. they vary substantially between the attenuated vaccine strain (sa14 - 14 - 2) and the virulent strain (nakayama). in this study, we analyzed the complete genome sequences of 2 bat jev isolates. although previous serologic studies (15) have indicated the occurrence of jev in a leschenault s rousette, we demonstrate jev infection of marina aurata bats, confirming that the same jev genotype can infect bats of many species. the bats tend to roost in trees, caves, and roofs of residential properties in close proximity to rice paddocks and pig pens, providing ample opportunity for cross - species transmission among bats and between bats and other animals. notably, the 2 jev strains most closely related to the bat viruses were all isolated from yunnan province, liyujie from a human in 1979 and bn19 from mosquitoes in 1982 (figure). our study indicates that the same virus is circulating in hosts of at least 4 different species (human, mosquito, and 2 different bat species), and likely in birds as well, highlighting the broad host range of jevs in this area. we emphasize that the 4 closely related strains (b58, gb30, liyujie, and bn19) were isolated over 2 decades, which suggests that the virus was stably maintained in the region, perhaps by circulating in disparate hosts. the findings from our current study highlight the potential importance of bats in human je outbreaks in the region. needed additional studies of jev in bats should include the determination of viremia in bats of different species and potential seasonal variation of viral loads among different bats at different geographic locations. | genome sequencing and virulence studies of 2 japanese encephalitis viruses (jevs) from bats in yunnan, china, showed a close relationship with jevs isolated from mosquitoes and humans in the same region over 2 decades. these results indicate that bats may play a role in human japanese encephalitis outbreaks in this region. |
plants have been used for therapeutic applications ever since man has been concerned about his health. for centuries, the world has depended on the useful possessions of plants as a source of medicines. ethnobotanical investigations done in the last few decades had discovered the analgesic properties of plants mentioned in the traditional information. the exploration for new analgesic compounds from the enormous arrays of medicinal plant resources is growing. this is because such information may hold guarantee for the finding of new therapeutic agents capable of inhibiting, decreasing, or relieving pain. plants characterize a huge natural supply of valuable compounds that might achieve as lead for the expansion of novel drugs. the exploration of the effectiveness of plant - based drugs used in the traditional medicine has been given great considerations because they are cheap and have little side effects, and, according to world health association (who), about 80% of the world population still relies chiefly on plant - based drugs. one of these medicinal plant species that has dramatic pharmaceutical values is leonurus cardiaca l.. leonurus cardiaca, commonly known as motherwort, is a member of the lamiaceae family that has been consumed in asian countries as a traditional remedy against nervous and functional cardiac disorders [6, 7 ]. it is a perennial herb prevalent in europe, usually found in country areas throughout the plains and hills, as well as in east asia to the himalayas and eastern siberia, northern africa, and north america. motherwort consists of the aerial portions of leonurus cardiaca collected through the flowering time and dried at 35c and, according to european pharmacopoeia 7th edition, must encompass a minimum of 0.2% flavonoids expressed as hyperoside. in the aerial parts of leonurus cardiaca, there are ingredients belonging to the group of terpenes, including monoterpenes such as iridoids, diterpenes of clerodane, furanolabdane, and labdane types, triterpenes including ursolic and oleanolic acids [11, 12 ], nitrogen - containing compounds such as leonurine and stachydrine, and phenylpropanoids such as lavandulifolioside, as well as flavonoids, phenolic acids, volatile oils, sterols, and tannins. pharmacological reports have established antimicrobial [10, 19 ], antioxidant [15, 16 ], and anti - inflammatory effects, as well as the effects of the herb on the heart and circulatory system. sedative and hypotensive properties since pharmacological studies are limited, the use of motherwort is mainly based on traditional suggestions. analgesic drugs are one of the most products that are used in numerous diseases for alleviating the pain. most analgesic drugs, accessible in the market, exhibit an extensive range of adverse effects including gastrointestinal disorders, kidney problems, and other unwanted effects. this situation highlights the need for advent of safe, novel, and effective analgesic compounds. the aim of the present study was to investigate the analgesic activities of aerial part of leonurus cardiaca using three analgesic tests in mice. the animals were handled in accordance with the criteria outlined in the guide for the care and use of laboratory animals (nih us publication 86 - 23 revised 1985). 68 weeks of age, were kept in a controlled environment (22 2c, 50 5% humidity) under a 12-hour light / dark cycle (light on 08:0020:00) and had free access to a standard pellet chow and tap water throughout the study. all experiments were conducted at tehran university of medical sciences according to the recommendations of the ethics committee for animal welfare and have been approved by institutional animal care and utilization committee. one hundred grams of aerial parts was placed into a flask ; one liter of 96% ethanol was then added. after 24 hours, when the solution had become clear, this was transferred into another flask. one liter of 70% ethanol was then added to the solid residue and after 12 hours the supernatant was again decanted into another flask. both solutions were then combined and concentrated by vacuum distillation at a temperature of 50c and 70 rpm rotation speed, until the volume decreased to 1/3 of the initial volume. the solution was then poured into a petri dish and dried in the autoclave at temperatures below 50c in sterile conditions. the formalin test is an acceptable and reliable model of nociception that generates two separate phases of increased licking activity that can be recognized to different nociceptive mechanisms. the initial licking stage lasts for the first five minutes and a late phase takes from 15 to 45 minutes after the injection of formalin. as previously defined, formalin (20 l of a 2.5% solution) was injected subcutaneously into the dorsal surface of the right hind paw. the animals were then placed under a glass funnel on a glass surface ; a mirror was angled at 45 degrees. the pain response time (licking time) was calculated in two periods : 0 to 5 min, the first phase (caused by direct motivation of the nociceptors), and 15 to 45 min, the second phase (inflammatory pain produced by release of inflammatory mediators). morphine (10 mg / kg, temad co., iran) was injected to animals in the positive control group. the other groups received different doses of leonurus cardiaca extract (125, 250, and 500 mg / kg). the tail flick examination was used to calculate analgesic activity by the method defined by d'amour and smith 1941, with minor alterations in the procedure. basal reaction time of animals to radiant heat was recorded by locating the tip (last 1 - 2 cm) of the tail on radiant heat source. the tail removal from the radiant warmth mice were treated with morphine (10 mg / kg), normal saline, and leonurus cardiaca (125, 250, and 500 mg / kg). the latent period of the tail - flick response was determined at 30, 45, 60, 75, and 90 minutes after the administration of drugs. the hot plate test was used to calculate analgesic activity by the method explained by eddy and leimbach with minor modifications. mice were retained on a hot plate having a stable temperature of 55 1c. the time taken for either paw licking or jumping each mouse was individually placed on the hot plate in order to find the animal 's reaction to electrical heat - induced pain (licking of the forepaws and eventually jumping). the latency until mice showed first signs of discomfort (hind paw lifting, hind paw licking, or jumping) was recorded, before (baseline), and response was determined at 30, 45, 60,75, and 90 min after the administration of normal saline, leonurus cardiaca (125, 250, and 500 mg / kg), and morphine (10 mg / kg). one - way analysis of variance (anova) test was used to ascertain the significance of variations between the times of licking in formalin test. two - way repeated measure anova test used to assay the differences in reaction time in tail flick and hot plate tests. the treatment of mice with leonurus cardiaca resulted in an inhibition of the formalin - induced licking in the early phase and inflammatory pain (late phase) of the formalin test. in the early phase, the maximum analgesia was observed at dose of 500 mg / kg of leonurus cardiaca extract (p < 0.01 compared to control group) and was equal to morphine in dose of 10 mg / kg. in the late phase, the dose of 250 mg / kg of leonurus cardiaca extract showed the most analgesic effects (p < 0.05 compared to control group) and it was comparable to morphine in dose of 10 mg / kg. pretreatment with leonurus cardiaca (250 or 500 mg / kg) demonstrated a significant and dose - dependent antinociceptive activity in the tail flick test (figure 2). the 500 mg / kg of leonurus cardiaca increased an antinociceptive activity in 30 (p < 0.01), 45, 60, and 75 (p < 0.01) minutes after injection that were comparable to the normal saline. this effect was significant in time 45 and 60 minutes after injection for doses 125 and 250 mg / kg of leonurus cardiaca. under similar conditions, treatment with morphine significantly increased latency to thermal stimulation 30 min after administration and the antinociceptive effect was maintained during the entire period of evaluation. the results in figure 3 show that the treatment of mice with morphine (10 mg / kg i.p.) increased the latency response in the hot plate test from 30 to 120 minutes after treatment. on the other hand, leonurus cardiaca significantly influence the reaction time of the animals to the hot plate at doses of 500 mg / kg in 45 and 60 minutes (p < 0.05) and 75 and 90 minutes (p < 0.01) after treatment. in this study, we pointed to investigate the potential antinociceptive effect of aerial part ethanol extract from leonurus cardiaca by chemical and thermal models of nociception. the first test to assess the antinociceptive property leonurus cardiaca was the formalin test. the formalin test initially described by dubuisson and dennis and categorized by the first phase, which is induced by direct formalin stimulation of the sensorial c - fibers followed by substance p release, and the second phase (inflammatory) principally due to a following inflammation reaction in the peripheral tissue mediated by the release of various inflammatory mediators has been related with the augmented level of pg, stimulation of cox, and release of nitric oxide (no) [25, 26 ]. the biphasic nature of pain response in this test, which presents diverse pathological procedures, can be used to clarify the probable mechanism involved in analgesia. centrally acting drugs, such as opioids, prevent both phases of pain, whereas peripheral - acting drugs such as acetylsalicylic acid, which inhibit cox activity, only inhibit the second phase [24, 28 ]. our results showed that the leonurus cardiaca extract at dose of 500 mg / kg and 250 mg / kg was more effective in the first and second phases, respectively, as well as morphine 10 mg / kg, suggesting possible peripheral and central antinociceptive mechanism. this extract was possibly able to inhibit this inflammation, so it can be deduced that peripheral mechanisms might also be involved in antinociceptive effects. it is generally believed that centrally acting analgesics possess effects on both phases, while peripherally acting analgesics affect only the first phase. this is because the injected formalin into the paw results in release of several neurotransmitters including glutamate and aspartate in dorsal horn of spinal cord. so the early phase of the formalin test represents the transmission of nociceptive impulses, while late phase is associated with the development of a delayed inflammatory response. opioid analgesics suppress both of early and late phases of formalin test, while none steroidal anti - inflammatory drugs (nsaids) mainly act on the late phase. as shown in figure 1, morphine could alleviate both of phases, whereas leonurus cardiaca at dose of 250 mg / kg diminished only the formalin - induced pain of the late phase. this effect maybe contributed to presence of some anti - inflammatory compounds which may act like nsaids. demonstrating in figure 1, leonurus cardiaca extract only at the maximum administrated dose (500 mg / kg) was capable of reducing the formalin - induced pain in the early phase, and other doses (125 and 250 mg / kg) did not show such effect. a pharmacokinetic involvement may partly clarify this finding that describes that leonurus cardiaca extract at the maximum dose (500 mg / kg) could pass through blood - brain barrier and show its analgesic properties in early phase, which is mainly central, while lower doses did not display such effect perhaps due to poor passing via this barrier. aiming to study the spinal antinociceptive action, we performed the tail flick test. this model, like the hot plate test [22, 29 ], measures animal nociceptive response latencies to thermal stimulus but tail flick is principally a spinal response and hot plate is predominantly supraspinal [3032 ]. treating the animals with leonurus cardiaca, at dose 500 mg / kg, alters mouse latency to painful thermal stimulus in tail flick and hot plate tests. these findings suggest that central and peripheral mechanisms are involved in the antinociceptive activity of the extract. demonstrating in figure 2, leonurus cardiaca extract only at the maximum dose (500 mg / kg) could alleviate the pain in all times of tail flick test, whereas the lower doses (125 and 250 mg / kg) reduced only late pain (times 60 and 75 minutes). this finding is similar to results of formalin test ; in both of them, leonurus cardiaca extract only at dose of 500 mg / kg was capable of diminishing the early pain, which is mainly central, and, as aforementioned, this event can be contributed to pharmacokinetics. in this study, it was shown that the administration of ethanolic aerial part extract of leonurus cardiaca possesses antinociceptive effects in tail flick, hot plate, and both phases of formalin test. the results obtained in this study indicate that the extract possesses analgesic properties, which are mediated through peripheral and central inhibitory mechanisms. this could provide a rationale for the use of this plant in pain and inflammatory disorders in folk medicine. further studies should be performed for the isolation of new chemical constituents of the plant as well as for a better understanding of the mechanism of antinociceptive activity presented by the extract. | leonurus cardiaca, commonly known as motherwort, is a member of the lamiaceae family. it has a number of interesting biological activities, for example, sedative and hypotensive, antioxidant, anti - inflammatory, and antimicrobial activities. the aim of the present study was to investigate the effect of alcoholic extract of aerial part of leonurus cardiaca on nociceptive response using formalin, tail flick, and hot plate tests in mice. the acute treatment of mice with an ethanolic extract at doses of 500 and 250 mg / kg by intraperitoneal administration produced a significant antinociceptive in the first and second phases of formalin test, respectively. the hot plate and tail flick tests showed an increase in the antinociceptive effect at dose 500 mg / kg. these results suggest that leonurus cardiaca possesses central and peripheral antinociceptive actions. |
chromosomal instability phosphorylated histone h2ax kirsten rat sarcoma viral oncogene homolog loss of heterozygosity a peptide sequence motif containing leucine - any amino acid - cysteine - any amino acid - glutamate phosphatase and tensin homolog retinoblastoma protein retinoblastoma susceptibility gene murine retinoblastoma susceptibility gene homolog gene encoding tumor protein p53 heterozygous loss of tumor suppressor genes has emerged as functionally relevant in tumor initiation and progression. haploinsufficient because the remaining wild - type allele is insufficient to maintain normal cellular function. while the repertoire of haploinsufficient tumor suppressor genes increases, the paradigm of knudson 's 2-hit hypothesis suggests that loss of both copies remains critical for eliminating retinoblastoma susceptibility (rb1) gene function. our recent work challenges this model by offering a number of lines of evidence from mouse models, cancer genomic data, and analysis of non - malignant rb1 cells from hereditary retinoblastoma patients to argue that loss of one copy of rb1 is functionally relevant. our study suggests conditional haploinsufficiency of rb1, akin to other paradigms of haploinsufficiency in cancer such as phosphatase and tensin homolog (pten) and tumor protein p53 (best known as p53). in this commentary, our investigation of haploinsufficiency of rb1 follows a number of studies that detail abnormalities in heterozygous tissues or cells from gene targeted mice. most notably, zheng. published an elegant study in which embryonic stem cells heterozygous for the mouse rb1 gene demonstrate chromosomal instability. in our study, we offer a number of independent approaches that implicate rb1 heterozygosity in the maintenance of genome stability. using fibroblast cells carrying null or partial loss of functional alleles of mouse rb1, we compare wild type, heterozygous, and homozygous mutant states so that quantitative assessments can be made about the magnitude of rb1 function. we observe that loss of one functional rb1 allele recapitulates the dna replication defects found with loss of both alleles. in particular, loss of one rb1 allele results in replication stress with accumulation of phosphorylated histone h2ax (h2ax) at pericentromeres and mitotic errors. these observations regarding rb1 heterozygosity in mouse cells lastly, using a partial loss - of - function allele of mouse rb1 (rb1) that is viable but defective for suppressing replication stress, we compare differences in rb1 gene dosage in a transformation - related protein p53 (trp53) knockout background. heterozygous and homozygous rb1 mutants display similar degrees of cancer susceptibility and pathology, presenting tumors with similar levels of aneuploidy. therefore, by a number of measures, loss of one copy of mouse rb1 is equivalent to loss of both copies in preventing replication stress and aneuploidy in cancer, establishing a paradigm where retinoblastoma protein (prb) function is haploinsufficient. as suggested by berger and colleagues in their proposal of a continuum model of tumor suppressor gene function, evidence for haploinsufficiency influencing cancer initiation and progression we report that rb1, a point mutation that specifically interferes with prb binding to chromatin regulators containing a leucine - any amino acid - cysteine - any amino acid - glutamate (lxcxe) peptide motif, accelerates cancer in a trp53 background regardless of whether mice are heterozygous or homozygous for rb1. importantly, heterozygous tumors retain the wild - type rb1 allele, indicating there is no loss of heterozygosity. this is context dependent as rb1 mice are not cancer prone, indicating that p53 status is critical for this phenotype. as with studies of pten mutant mouse models of cancer, the ability of the rb1 mutation to enhance cancer phenotypes is context dependent since the rb1 genotype actually inhibits the ability of the kirsten rat sarcoma viral oncogene homolog (kras) to induce lung tumors. from this perspective our data offer interesting similarities with other haploinsufficient tumor suppressors such that mouse rb1 haploinsufficiency requires key accompanying mutations and for this reason exhibits conditional effects. in addition, rb1-dependent effects on cancer pathogenesis are asymmetric as they largely enhance trp53 inactivation - dependent characteristics rather than displaying their own. importantly it is possible that these similarities with pten may form the basis of simple rules for classifying haploinsufficient tumor suppressors in the future. why tumor suppression is conditionally haploinsufficient in these scenarios is a key question that remains to be investigated. in summary, a model in which rb1 function is eliminated by loss of both copies has been widely known for some time and contributes to retinoblastoma formation among other disease sites (fig. our work suggests an additional pathway in which loss of one copy of rb1 contributes to cancer pathogenesis and is most relevant when accompanied by elimination of p53 function (fig. examination of rb1 loss and its correlation with genome instability in cancer cell lines suggests that mutation of tp53 is a common event that accompanies rb1 elimination. it will be interesting to determine whether this is also true for rb1 cancers. we suggest that the paradigm where this new pathway of partial loss of rb1 may be most relevant is in mesenchymal cancers. it will be interesting to see whether sequencing studies of osteosarcomas, particularly in retinoblastoma survivors, demonstrate rb1 heterozygosity and tp53 loss together. since prb status correlates with differential therapeutic responses for certain cancers, the identification of rb1 heterozygosity as functionally relevant suggests that gene dosage may be an important parameter to include in future diagnostic tests. (a) loss of heterozygosity (loh) model in which the remaining wild - type retinoblastoma susceptibility gene (rb1) allele is eliminated and the nullizygous state contributes to malignancy. the disease sites in humans and mice where this paradigm is most relevant (b) conditional haploinsufficiency model in which loss of one rb1 allele contributes to chromosomal instability (cin). further loss of tumor protein p53 function is conducive to tumorigenesis in this paradigm and the cancer types consistent with this pathway are indicated. (a) loss of heterozygosity (loh) model in which the remaining wild - type retinoblastoma susceptibility gene (rb1) allele is eliminated and the nullizygous state contributes to malignancy. the disease sites in humans and mice where this paradigm is most relevant are depicted at the bottom. (b) conditional haploinsufficiency model in which loss of one rb1 allele contributes to chromosomal instability (cin). further loss of tumor protein p53 function is conducive to tumorigenesis in this paradigm and the cancer types consistent with this pathway are indicated. the authors wish to thank many colleagues for encouragement during the course of this controversial study. fad is the wolfe senior fellow in tumor suppressor genes at western university and the canadian institutes of health research supported investigation of rb1 haploinsufficiency. | recent work demonstrates that retention of a single functional retinoblastoma susceptibility (rb1) allele is insufficient to maintain genome stability. haploinsufficiency of rb1 accelerates cancer pathogenesis in concert with inactivation of tumor protein p53. collectively, multiple lines of evidence suggest revision of the 2-hit model to include conditional haploinsufficiency of rb1. |
primary aortoenteric fistula is a rare but potentially lethal complication of an abdominal aortic aneurysm. a 55-year - old woman was referred to the emergency room for syncope associated with hematemesis, melena and anemia. the patient 's history revealed a heavy smoking habit, chronic alcohol - related liver disease and congenital hiatal hernia. she reported a similar episode of hematemesis five days prior but at that time refused further investigation. on arrival in the emergency room, she was hemodynamically stable, orientated and alert. a gastro - intestinal endoscopy was performed to better understand the possible cause of bleeding. the exam revealed many coagulated blood spots in the stomach and a clot tenaciously adhered to the duodenal wall without active bleeding signs or esophageal varices. a computed tomography (ct) scan showed a saccular abdominal aortic aneurysm (max diameter : 44 mm), a few centimeters downstream from the origin of the right renal artery, the presence of multiple gas bubbles in the thrombus, and the presence of a strong adherence between the aneurysm and the third portion of the duodenum (fig. the patient was unaware of this pathology. according to these clinical signs and images she was urgently taken to the operating room with the diagnosis of primary aorto - enteric fistula (paef). the saccular aneurysm and the duodenum were isolated by a transperitoneal approach after accurate debridement of some adherences between the two structures. we decided to clamp the aorta above the right renal artery because of the proximity of the aneurysm and the origin of the vessel. we used a dacron silver graft to perform an aorto - aortic substitution sutured with a synthetic nonabsorbabable monofilament prolene. proximal clamping lasted 12 minutes and during that time the right kidney perfusion was guaranteed by the infusion of crystalloid fluids at 5 at a flow rate of 1.5 ml / g / min (custodiol benshein, germany). due to a limited extension of the duodenal tear, the intestinal wall was directly sutured and a portion of omentum was interposed between the graft and the bowel (fig. 2). the patient required the transfusion of four units of packed red blood cells. as a result, a broad - spectrum antibiotic prophylaxis with metronidazol, teicoplanin and fluconazol was administered. a ct scan and gastrografin (bracco diagnostic inc., quebec, canada) intestinal transit tests were finally performed showing the success of the repair. on the sixteenth day after the surgery primary aorto - enteric fistula is defined as a direct communication between the aorta and the gastro - intestinal lumen and represents a rare and severe complication of the abdominal aortic aneurysm (aaa). the primary form, less common with an incidence of 0.04 to 0.07%, is due to the erosion of the intestinal wall by an aortic aneurysm.1)2) approximately 350 cases of paef have been reported in the literature since its first description about 200 years ago.3) on the other hand, secondary aorto - enteric fistula has an incidence of 0.4 - 1.6% and may represent the complication of surgical aortic replacement.2) mortality is extremely high : around 100% of untreated patients or patients with active bleeding, and around 30 - 40% of patients treated with surgery4) epidemiological data reveal that the average age of diagnosed paef is 64-years - old and that it mainly affects males (3:1 ratio).5) about 83% of paef is associated with a fusiform aaa (mean diameter 6.2 cm).6)7) other causes of paef are related to infectious diseases such as syphilis, tuberculosis, mycotic infections, and collagen diseases.8) moreover, gastric pathologies (peptic ulcer, intestinal tumors, pancreatitis), history of radiation exposure and neoplastic diseases appear to increase the risk of paef.8)9) the intestinal tracts more frequently involved in paef are the second and third portion of the duodenum and the duodeno - jejunal angle (54%).7) the clinical presentation of paef is often insidious unless doctors suspect gastro - intestinal bleeding (80%)10) with episodes of hematemesis and melena (64%), abdominal pain (32%) and the palpation of an abdominal pulsating mass (25%).11) the onset of paef is rarely related to signs of sepsis (fever, leukocytosis) or with hemorrhagic shock with hypotension, tachycardia, and loss of conciousness.12) the triad of abdominal pain, pulsatile mass and gastrointestinal bleeding is described in only 11% of patients.6) despite the absence of a pathognomonic sign, there is a characteristic presentation of paef defined as " herald bleeding"13)14) it consists of an episode of gastro - intestinal bleeding which is usually self - limiting by formation of a blood clot in the aorto - enteric fistula. however, after infusion therapy, increasing blood pressure or endoscopic procedures, the clot can be removed leading to severe bleeding, with a tendency of hemodynamic instability.2) gastrointestinal endoscopy is the first - step in detecting gastrointestinal bleeding of unknown origin but the sensitivity for detecting paef is < 25%.6) many authors agree that this examination requires experienced operators because of the necessity to evaluate the duodenum in its entire length and to avoid reckless maneuvers leading to the removal of the clot.7) the diagnosis of paef is usually obtained through a ct scan showing air bubbles in the aortic wall and the presence of contrast medium in the intestinal lumen. the sensitivity of this technique varies from 30 to 61%.15) the gold standard in the treatment of paef is surgery. in our case study, we performed an aorto - aortic bypass with a dacron silver prosthesis, which is the preferred choice in a septic or potentially septic area. the advantages of this method are the relative speed of execution, the good long - term patency, and the preservation of the anatomic course of the vessels. other treatment options proposed in the literature include extra - anatomic by - pass, the substitution of the aortic tract with a venous prosthesis (neoaortoiliac system, nais) and the cryopreserved arterial homografts.15)16)17) the endovascular aneurysm repair (evar) of the paef, as reported in the literature, could be applied in specific situations as a " bridge " treatment in patients with haemodynamic instability, where immediate surgery is contra - indicated and has to be delayed.18)19) this solution is still controversial, especially for the persistence of the infective site and the risk of evar 's infection.20) in conclusion, a saccular aneurysm, too small to require elective surgical treatment, is a possible cause of early onset paef. it is mandatory to consider paef in differential diagnoses even in patients without a history of aneurysm but with other risk factors, such as smoking and gastro - intestinal inflammatory diseases, in order to provide prompt surgical treatment. | primary aortoenteric fistula is a direct communication between the aorta and intestinal lumen and it represents a rare but potentially lethal complication of an abdominal aortic aneurysm. however, it may occur less frequently in a naive non - aneurysmatic aorta. diagnosis is often difficult and delayed in most cases, unless there is a high level of clinical awareness. urgent surgery is still the recommended treatment. we describe the case of primary aortoenteric fistula of a saccular aneurysm. a 55-year - old woman was referred to our center with hematemesis, melena, and severe anemia who was dignosed previously with unknown saccular abdominal aneurysm. |
approximately 40% of all non - hodgkin lymphomas (nhls) occur in extranodal locations. primary nhls of the gastrointestinal tract are rare, accounting for only 14% of malignancies arising in that area. primary gastric lymphoma (pgl) accounted for 6875% of patients with primary gastrointestinal tract nhl [4, 5 ]. there was a slight predominance of males with pgl (male : and female, 1.1 : 1). pgl diagnoses reached their peak incidence in individuals between the ages of 50 to 60 years. the most common presenting symptoms included epigastric pain, epigastric discomfort, anorexia, weight loss, nausea, vomiting, and gastrointestinal bleeding. systemic b symptoms occurred in 12% of pgl patients [4, 5 ]. extranodal marginal zone b - cell lymphoma of the mucosa associated lymphoid tissue (malt) type accounted for 4048% of pgl cases [4, 5 ]. according to pathological classification, currently, high - grade stage i / ii pgl can be treated with chemotherapy followed by radiotherapy, and advanced stage pgl is usually treated with chemotherapy only [6, 7 ]. low - grade stage i / ii helicobacter pylori (+) malt lymphomas can be treated with antibiotic therapy. treatment failure with h. pylori eradication can be managed with radiation therapy. in this study, we aimed to determine the clinical characteristics of patients with pgl who were referred to our clinic over the last 12 years. we also aimed to evaluate patient survival and prognostic factors affecting survival and the effect of combined surgical and conservative treatment. we retrospectively reviewed and analyzed the data of patients treated for pgl in our clinic (istanbul university, cerrahpasa medical faculty, department of internal medicine, division of medical oncology) from 1998 through 2010. all patients with malt lymphoma who did not respond to antibiotic therapy for h. pylori eradication or who demonstrated relapse during followup were included in the study. approval from the local ethics committee and informed consent of the patients or their next of kin were obtained prior to the study. the following characteristics or results were recorded for each patient : medical history, physical examination, biochemistry, computed tomography (ct) of the thorax and abdomen, multiple gastroscopic biopsies of the upper and lower gastrointestinal tract, examination of waldeyer 's ring, and bone marrow biopsy. poor performance status was defined using the karnofsky scale (60 years were significant prognostic factors for os in multivariate analyses. pgls represent more than half of all primary gastrointestinal lymphomas, accounting for 5% of all malignant tumors of the stomach [912 ]. according to histological type, pgls are divided into low - grade and high - grade. in this retrospective study, the rate of malt lymphomas (21%) was lower than previously shown [4, 5 ]. about 40% of pgls are low - grade lesions thought to arise in the mucosa from the defined malt lymphoma, and 60% are histologically considered high - grade [4, 6, 10 ]. epigastric pain, epigastric discomfort, anorexia, weight loss, nausea, vomiting, and gastrointestinal bleeding were the most common symptoms in the present study, as reported in other series [4, 1315 ]. in the current report, there was a slight male predominance [4, 13, 14, 16 ], while other studies reported female prominence among cases of pgl [14, 16 ]. in pgl, there are multiple factors that affect survival. in previous studies, advanced stage, poor performance status, age > 60 years, and elevated ldh at presentation of the disease were associated with poor outcome ; female sex, low - grade histology, good performance status, and surgical resection for local disease have been reported to be associated with high os and dfs rates [4, 1719 ]. in our study, the five - year os and dfs rates were 83.1% and 78.6%, respectively. in patients with stage i / ii or iie disease at presentation, the five - year os and dfs rates were 91.2% and 83.9%, respectively. the five - year os and dfs rates of those with stage iv disease at presentation were 70.6% and 65.5%, respectively. in prior studies, the survival rate of patients with pgl was 7892%, according to stage and treatment modality [6, 1922 ]. based on our results, variables associated with poor dfs were advance stage, high ldh level, and poor performance status. in a multivariate analysis, variables associated with poor dfs were also advanced stage, high ldh level, and poor performance status. regarding os, a univariate analysis revealed that advanced stage, age > 60 years, and poor performance status were associated with a poor prognosis. in the multivariate analysis, advanced stage, poor performance status, and age there are no standard therapeutic guidelines for patients in whom antibiotic therapy has failed. in two retrospective studies of patients with malt lymphoma, no significant differences in survival were found between different treatment modalities [23, 24 ]. in our study, patients with malt lymphoma were those in whom antibiotic therapy failed. no significant differences in dfs or os were demonstrated among pgl patients who had nonsurgical or surgical treatment of the local disease (stage i / ii or iie) of malt or non - malt lymphoma. controversy remains over the optimal treatment for early stages of pgl, particularly regarding the role of surgery. recently, it has become evident that there is no difference in survival rates for those who were treated with surgery compared to a conservative modality. surgery is no longer accepted as the cornerstone treatment of pgl and is reserved for when nonsurgical treatment is not possible. surgery should be considered following nonsurgical treatment and when disease complications, such as hemorrhage, obstruction, or perforation, have occurred. however, it is well known that surgical approaches have a number of potential disadvantages. additionally, significant morbidity is associated with gastrectomy, as follows : 17% of patients developed malabsorption syndromes, 38% reported weight loss, and 13% developed dumping syndrome. on the other hand, of the patients who received chemotherapy, only 5% developed acute complications, such as gastric perforation and gastrointestinal hemorrhage [20, 2730 ]. in the present study, there was no difference between conservative and surgical treatment approaches with regard to dfs and os. the main limitations of this study were its retrospective design and the lack of data on patient quality of life and toxicities in patients treated with a conservative approach or surgery. in conclusion, poor performance status, advanced stage of disease, high ldh level, and advanced age were bad prognostic factors for patients with pgl. surgery provides no advantage for survival over conservative treatment ; thus, conservative treatment modalities should be preferred initially at early stages of pgl. further prospective, large - scale, controlled studies are needed to determine the impact of different treatment modalities on the outcome of patients with pgl. | objectives. the aim of this study was to evaluate clinical characteristics, prognostic factors, survival rates, and treatment modalities in patients with primary gastric lymphoma (pgl). methods. we retrospectively reviewed and analyzed data from patients treated for pgl in our clinic from 1998 through 2010. staging was performed using the lugano staging system. overall and disease - free survival (os and dfs) were calculated from the date of diagnosis. results. we identified 79 patients. thirty - seven patients (47%) were male. the median age at presentation was 57 (1885) years. the median follow - up time was 41 (952) months. thirty patients (38%) underwent surgery, 74 (92%) received chemotherapy, and 18 (23%) received radiotherapy. the five - year os and dfs rates were 91.2% and 83.9%, respectively, in patients with stage i / ii or iie disease and 70.6% and 65.5%, respectively, in patients with stage iv disease (p = 0.02 for both rates). treatment modality (surgical or conservative) had no impact on os or dfs in early stages. in a multivariate analysis, poor performance status, advanced stage, and high ldh levels were significant bad prognostic factors for dfs, while advanced stage, poor performance status, and age > 60 years were significant bad prognostic factors for os. conclusion. surgery provides no advantage for survival over conservative treatment ; thus, conservative treatment modalities should be preferred initially at early stages of pgl. |
targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. such genetic manipulations can be achieved using site - specific nucleases. here, we engineered transcription activation - like effector nucleases (talens) for five distinct genomic loci. at all loci tested we obtained hesc and ipsc single - cell - derived clones carrying transgenic cassettes solely at the talen - specified location. thus, talens mediate site - specific genome modifications in human pluripotent cells with comparable efficiency and precision as zinc finger nucleases (zfns). |
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determinar a rugosidade de superficies submetidas a diferentes tratamentos e correlacionar rugosidade com velocidade de escoamento de sistemas adesivos lminas de vidro foram utilizadas como substrato para avaliar a velocidade de escoamento dos sistemas adesivos single bond and prime & bond nt. seis diferentes tratamentos de superfcie foram comparados : 1- sem tratamento ; 2 silanizao (sl) ; 3 jateamento (j) ; 4 j + sl ; 5 condicionamento com cido fluordrico a 10% (hf) ; 6 hf + sl. antes e aps os tratamentos, foi mensurada a rugosidade das superficies (ra, m). gotas de 10 l de adesivo foram despositadas sobre as superficies e as velocidades de escoamento foram observadas significncias estatsticas entre grupos foram analisadas pelos testes anova (um e dois critrios) e snk. houve diferenas significantes na velocidade de escoamento entre materiais (p 0.05). linear regression did not indicate any interaction between spreading velocity and roughness (r = 0.173 ; rsqr = 0.0299). the selection of glass slides as substrates for the measurement of both adhesive spreading velocity and surface roughness was due to their low cost, compared to dental ceramics, and also by they vitreous composition (sio2), similar to that found in feldspathic porcelains13. glass has been already used to study silane adhesion10. according to the adhesion principles, adherend surface free energy and roughness as well adhesive surface tension and viscosity although wettability can not be directly correlated to spreading velocity, it is supposed that higher spreading velocities yield better wettabilities. even though adhesive resins are the most clinically relevant agents to measure contact angles (considering adhesive restorations), studies have been frequently using water for this purpose instead of them22. however, resins yield higher mean contact angles than water, and therefore do not wet ceramics as well as water22. according to the results found here, the mean spreading velocity found for single bond was significantly lower than that of prime & bond nt, regardless of the surface treatment used. although we did not measure the viscosity of the adhesives employed in this study, we can assume, based on the composition of each material, that single bond presents a higher viscosity than prime & bond nt12,27. single bond contains 60 - 70 wt% of bis - gma in its composition (table 1), which can significantly increase its viscosity. bis - gma is a very viscous substance because it has a high molecular weight, it has a large rigid section, and it is capable of hydrogen bonding to its neighbors due to the presence of the hydroxyl group and carbonyl oxygen11. more viscous adhesives do not wet well a substrate as do less viscous ones17. also, the substrate chemical composition and the adhesive complex monomeric mixture influence respectively surface free energy and surface tension16, consequently affecting wettability and the time required to spreading24. once the substrates had the same origin and composition, i.e. glass slides, for all groups, it can be inferred that the surfaces tensions of the adhesive systems used are different, providing more or less wettability, in compliance with other results5. surface treatments can modify both surface free energy and roughness of a solid16 increasing or decreasing adhesives wettability over it. theoretically, different surface treatments create different surface free energies and roughness, and therefore promote distinct adhesive wettabilities. in this study we found that the untreated, sandblasted or etched surfaces allowed adhesives to spread at similar velocities, being the spreading velocity affected only when these surfaces were silanated. indeed, silane application decreased the mean spreading velocity of the adhesives tested (p 0.05), suggesting that all of them have similar wettabilities. moreover, it has been suggested that if a surface is too much rough it can avoid an adequate adhesive spreading16. nevertheless, grooves and porosities found in sandblasted ceramics surfaces can not be considered the only factors responsible to worsen ceramic 's flexural resistance, as this property is highly dependent on the specific composition and microstructure of each material1. hence, sandblasting should not be recommended to increase wettability and adhesion, at least from the standpoint of an adherend / adhesive surface interaction.. the lower roughness of the hf etched surfaces can be due to the formation of insoluble silica salts on the glass slide surface similar to what happens in ceramics6,9. the use of an ultrasonic bath for 1 minute probably did not remove these insoluble precipitates. differences in ceramics microstructure and composition can affect the micromechanical retention created by etchant solutions13,15,19, and this may explain the restrict roughness reached by hydrofluoric acid. acid etching has a limited influence on the surface free energy of ceramics and it is necessary to silanate these etched surfaces to get adhesion improved20. silanization increases the adhesive strength of ceramics to dental tissues, regardless of the material composition or acid etching15. therefore, due to the inadequate surface characterization reached by hf in this study due to a similar roughness found in etched and unetched surfaces, from the standpoint of adhesive strength, it is suggested that silanization alone should be enough to improve adhesion8,14,19,26. silanization statistically decreased the roughness of sandblasted surfaces, but the same effect was not found in other groups. we suggest that silane penetrated into the more pronounced irregularities created by sandblasting, minimizing their magnitude. however, silanization was not enough to reduce the roughness of sandblasted surfaces to the levels found in the non - sandblasted ones, confirming that sandblasting can create too much roughness on a glass surface. nevertheless, even this not so pronounced " smoothing effect " of silanization can be capable to reinforce ceramics structure. most researches on ceramic area have studied tensile strength21 or flexural strength after treatments and silanization. the explanation for the improvement in these mechanical properties after silanization can be related to two aspects, the possibility of silane to fill the irregularities created by surface treatments9,21, and by its chemical adhesion with the substrate, i.e. adhesive / resinous cement / ceramic29. in accordance with the hypotheses tested previously to this study and based on the results found here, we reject the null hypotheses 1, 2 and 3, i.e., there was a significant difference on the surface roughness obtained from the different surface treatments ; there was a significant difference in the spreading velocity of the adhesives tested and there was a significant difference in the spreading velocity of both adhesives over the surfaces submitted to different treatments. we accept the null hypothesis 4 since no correlation between spreading velocity and surface roughness was found. these findings suggest that more research is needed to understand the role of surface treatments on adhesion as well on the stability of dental porcelain, when current adhesive systems are employed. within the limitations of this study, the real need to treat a surface (e.g., sandblasting, hf) of a glass restorative substrate (ceramic) prior to adhesive procedures may be questioned, at least from the standpoint of wetting. although surface treatments yielded different roughness, they did not provide differences in the spreading velocity of the simplified bonding systems studied. silanization decreased bonding systems ' spreading velocities, so it may raise difficulties for an adhesive to properly wet a substrate, thus adversely affecting bonding. | objectives : to determine the roughness of glass surfaces submitted to different treatments and to correlate it with the spreading velocity of two adhesive systems.materials and methods : glass slides were used as substrates to evaluate the spreading velocity of single bond and prime & bond nt adhesive systems. six different surface treatments were compared : 1) no treatment ; 2) silanization (sl) ; 3) sandblasting (sb) ; 4) sb + sl ; 5) 10% hydrofluoric acid treatment (hf) ; 6) hf + sl. before and after treatments, surface roughness was measured by a profilometer (ra, m). drop volumes (10 l) of the adhesive systems were deposited onto substrates with a micropipette to observe materials spreading during 30s. data were expressed in mm / s as spreading velocity. statistical significances among groups were analyzed using one - way and two - way - anova designs and the snk test.results:significant differences in spreading velocity were found between materials (p < 0.001) and among treatments (p < 0.001). silanization decreased the spreading velocity for both adhesives in comparison to groups where it was not performed (p < 0.05). differences in roughness were found only for sb surfaces that were rougher than the others (p < 0.05). silanization decreased the roughness of sb surfaces (p < 0.05). linear regression did not indicate any correlation between spreading velocity and roughness (r = 0.173).conclusions : although surface treatments yielded different roughness, they did not provide differences in the spreading velocity of the simplified bonding systems studied. silanization decreased bonding systems ' spreading velocities. |
a 56-year - old female was referred to oculoplastics at the king khaled eye hospital (kkesh) due to complaints of continuous pain and discharge in the right socket that had begun 4 months prior to presentation. the patient had a history of perforating trauma followed by phthisis bulbi in the right eye when she was 3 years old, and she underwent enucleation without placement of an orbital implant a few years later. when she was 35 years old, she underwent surgery for the placement of an rtv silicone subperiosteal implant in the right orbit to improve the socket volume. ptosis repair was carried out at the age of 37 years, dermis fat graft at the age of 47 years, and a mucous membrane graft and deepening sutures to improve the cul - de - sac at the age of 52 years. on examination, she had a bulging dystopic external prosthesis and the eyelids did not close properly. the socket was inflamed, grade 3, with mild redness and copious discharge (fig. 1 ; upper row). the patient was treated with neomycin, bacitracin and dexamethasone drops (maxitrol ; allergan inc., a ct scan of the orbit showed dense material along the extraconal space of the right orbital cavity adjacent to the right orbital roof and lateral wall with rarefaction along the inner layer and minimal hyperostosis (fig. 1 ; lower row). the patient underwent surgical excisional biopsy of the foreign body through a crease approach. during the surgery, we found that the material was surrounded by a hard pseudocapsule with the consistency of bone and inflammatory tissue. a 48-year - old male was referred to the kkesh due to complaints of pain, copious discharge, redness, contracted cavity and poorly fitting prosthesis in the left eye. the patient had been involved in a road traffic accident when he was 28 years old. he underwent enucleation of the left eye without placement of a spherical implant in the left socket. subsequently, multiple surgeries were performed for socket reconstruction followed by mucous membrane graft, deepening sutures, implantation of a secondary polymethyl methacrylate implant and, later, removal thereof due to dehiscence and exposure, as well as pyogenic granuloma excision. on examination, examination of the left eye and ocular adnexa indicated an anophthalmic cavity with a grade 4 contracted socket, inflammation, with significant discharge and redness. the patient was treated with corticosteroids resulting in temporary improvement with recurrence after tapering the steroid. ct scan and magnetic resonance imaging (mri) indicated an irregular foreign body located in the left orbital roof, associated with adjacent inflammatory changes involving the extraocular muscles (fig. a rubber - like foreign body was identified and removed after removal of the surrounding bony layer (fig. a 56-year - old female was referred to oculoplastics at the king khaled eye hospital (kkesh) due to complaints of continuous pain and discharge in the right socket that had begun 4 months prior to presentation. the patient had a history of perforating trauma followed by phthisis bulbi in the right eye when she was 3 years old, and she underwent enucleation without placement of an orbital implant a few years later. when she was 35 years old, she underwent surgery for the placement of an rtv silicone subperiosteal implant in the right orbit to improve the socket volume. ptosis repair was carried out at the age of 37 years, dermis fat graft at the age of 47 years, and a mucous membrane graft and deepening sutures to improve the cul - de - sac at the age of 52 years. on examination, she had a bulging dystopic external prosthesis and the eyelids did not close properly. the socket was inflamed, grade 3, with mild redness and copious discharge (fig. 1 ; upper row). the patient was treated with neomycin, bacitracin and dexamethasone drops (maxitrol ; allergan inc., a ct scan of the orbit showed dense material along the extraconal space of the right orbital cavity adjacent to the right orbital roof and lateral wall with rarefaction along the inner layer and minimal hyperostosis (fig. 1 ; lower row). the patient underwent surgical excisional biopsy of the foreign body through a crease approach. during the surgery, we found that the material was surrounded by a hard pseudocapsule with the consistency of bone and inflammatory tissue. a 48-year - old male was referred to the kkesh due to complaints of pain, copious discharge, redness, contracted cavity and poorly fitting prosthesis in the left eye. the patient had been involved in a road traffic accident when he was 28 years old. he underwent enucleation of the left eye without placement of a spherical implant in the left socket. subsequently, multiple surgeries were performed for socket reconstruction followed by mucous membrane graft, deepening sutures, implantation of a secondary polymethyl methacrylate implant and, later, removal thereof due to dehiscence and exposure, as well as pyogenic granuloma excision. on examination, examination of the left eye and ocular adnexa indicated an anophthalmic cavity with a grade 4 contracted socket, inflammation, with significant discharge and redness. the patient was treated with corticosteroids resulting in temporary improvement with recurrence after tapering the steroid. ct scan and magnetic resonance imaging (mri) indicated an irregular foreign body located in the left orbital roof, associated with adjacent inflammatory changes involving the extraocular muscles (fig. a rubber - like foreign body was identified and removed after removal of the surrounding bony layer (fig. the clinical symptoms of both our cases at presentation were pain, copious discharge, intense inflammation, redness and a contracted socket. also, both patients had a history of multiple surgeries to correct the chronic problems in the anophthalmic socket. the chronic discharge in both patients was related to an intense chronic inflammation in the orbital tissues. this is a very common symptom in patient with an anophthalmic cavity likely due to various causes including dry socket (common in sockets because of the absent corneal reflex), incomplete blink, meibomian gland inflammation, giant papillary conjunctivitis (mechanical damage from the external prosthesis to the conjunctiva and hypersensitivity to proteins deposited over the prosthesis), implant exposition or pyogenic granuloma (secondary to suture or implant exposure). both patients complained about pain, which may be due to chronic inflammation in the socket. alternatively, pain may be due to phantom eye syndrome that can occur in one third of anophthalmic cavity carriers as a result of the interruption of the sensory nerves impulse, due to the reorganization of the neurosensory cortex, or due to the regenerative sprouting of the axons resulting in a neuroma. in the current study, both patients presented with contracted cavities with chronic inflammation of the socket related to progressive contraction of the anophthalmic cavity, often associated with the presence of myofibroblast, a modified fibroblast linked to the contracted sockets [5, 6 ]. one of the primary goals of anophthalmic socket treatment is the restoration of an adequate orbital volume through the use of appropriately sized spherical alloplastic implants. complications of all types of implants can result in discharge, implant exposure, conjunctival thinning, pyogenic granuloma formation, implant infection, with persistent pain or discomfort. notably, the two cases reported here did not receive a spherical implant in the anophthalmic socket, but a silastice rtv silicone rubber base for volume replacement in the anophthalmic socket. this is a biomaterial which can be used in the orbital floor, lateral wall and roof. the rtv silicone rubber base is a brand injectable silicone rubber elastomer, considered to be a biologically inert, nontoxic, nonreactive thermally stable elastomer, which is resistant to oxidation and vulcanizes at room temperature. rtv silicone with a catalyst was placed in a dissected pocket subperiosteally along orbital walls to correct the volume deficit in both cases, vulcanizing the implant in situ into soft silicone rubber and conforming the implant to the orbital wall [9, 10 ]. in the current cases, ct studies imaged the implant as a foreign body, which was confirmed by mri in 1 patient. the rtv implant was placed in both patients a few years after the enucleation, and they developed inflamed and contracted sockets with poor cosmetic results. however, good outcomes without complications have been reported with this implant [11, 12, 13 ]. the most important difference between our cases and the literature is the duration of follow - up. our cases had the implant inserted 21 years (case 1) and 20 years (case 2) ago, which represents the longest follow - up for rtv implants. hence, long - term follow - up is fundamental to understand the biocompatibility of the implants, as complications such as rejection and subsequent infection, cellulitis, or migration of the material could occur with any type of implants over the lifetime of the patient. we do not know why the previous surgeons opted to use subperiosteal instead of spherical implants to replace socket volume in both patients. both patients had fat atrophy, deep socket, superior sulcus atrophy and poor cosmesis with the prosthesis due to the sunken cavity. other common complications related to silicone implants include hemorrhage, infection, periorbital cellulitis, migration, extrusion, skin fistulae, and sino - orbital fistulae. the index of suspicion should be high when there is persistent conjunctival inflammation despite topical antimicrobial drops, discomfort on palpation of the implant, and pyogenic granuloma. some have reported that locally all the silicone implants (injectable, sheets or blocks) induce a limited foreign body reaction, which may result in a fibrous pseudocapsule around the implant with the possibility of contraction, leading to migration of the implant [3, 12 ], with the possibility of chronic inflammation secondary to the presence of the silicone orbital implant. in the current cases, the chronic inflammation resolved in both patients after the removal of the implants. if, at some point, an implant was placed, chronic inflammation indicates that implant removal is warranted and lifetime follow - up is required [14, 15 ]. in conclusion, our cases presented with chronic inflammation in an anophthalmic socket due to a foreign body reaction against the injectable silicone rtv implant. anophthalmic cavity carriers presenting with recurrent inflammation have to be investigated according to their previous orbital surgeries and the type of implants. orbital imaging is helpful in localizing the cause and determining treatment, such as implant removal. the authors received no financial support for this study and have no financial or proprietary interest in the materials presented in this study. | two case reports are used to illustrate the signs and symptoms, complications and treatments of chronic socket inflammation due to intraorbital implants. the ophthalmic examination, surgeries and treatments are documented. two anophthalmic cases that underwent enucleation and multiple orbital surgeries to enhance the anophthalmic socket volume developed pain, intense discharge and contracted cavities with chronic inflammation in the socket which was nonresponsive to medical therapy. computed tomography indicated a hypodense foreign body in both cases causing an intense inflammatory reaction. the implants were removed by excisional surgery and a room temperature vulcanized silicone implant was retrieved in both cases. socket inflammation resolved in both cases after implant removal. an intraorbital inflammatory reaction against an intraorbital implant can cause chronic socket inflammation in patients with a history of multiple surgeries. diagnosis requires imaging and the definitive treatment is implant removal. |
free vascularised fibula grafting is well described in limb salvage surgery after resection of bone tumours. nonunion at the osteotomy site and fracture of the graft are recognised complications, which may be related to poor blood flow within the graft. graft viability is usually judged by evidence of bony bridging and graft hypertrophy on plain radiographs, but this is problematic and delayed union and infection can not be reliably predicted. the early identification of graft failure would improve patient management, and a reliable assessment of graft viability would also help identify characteristics associated with successful grafts. functional imaging techniques, such as positron emission tomography (pet), can visualise subtle metabolic changes and have the potential for assessing graft viability. the pet tracer f - fluoride has been used to evaluate regional bone metabolism and skeletal kinetics [15 ] as well as monitoring therapeutic response. the dynamic behaviour of complex biological systems can be described by suitable kinetic models in functional imaging. rate constants and macroparameters of kinetic models are related to biological processes ; hence, these parameters could be used to aid clinical evaluation. kinetic modelling of pet imaging usually involves obtaining a series of blood samples for the input function (if), and a tissue time activity curve (ttac), derived from dynamic imaging data as the output function. rate constants are determined by parameter estimation methods, which fit the ttac according to an underlying kinetic model. traditionally, manually defined regions of interest (roi) are used to derive average, but reduced noise, ttacs for the selected regions by deriving the mean activities for the defined roi. alternatively, parameter estimation can be conducted voxel by voxel to form a three - dimensional parametric image volume whose voxel values represent quantitative functional parameters. the parametric image approach reduces operator - dependant bias in the manual delineation of roi and eliminates the need to know the spatial distribution of newly developed tracers. furthermore, the parametric image approach readily visualizes the spatial distribution of quantitative functional parameters. the nonlinear least square (nls) analysis, a standard parameter estimation method, provides estimates with optimum statistical accuracy in kinetic analysis by iteratively adjusting the kinetic parameters of the nonlinear model equations. there is intrinsic noise in functional imaging data, thus an appropriate weight function is usually chosen to derive the objective function in nls fitting. thus, nls is often referred to as the weighted nonlinear least square (wnls). however, nls / wnls are not suitable for the construction of parametric images due to their high computational cost, proneness to be trapped in local minima, and a requirement for appropriate initial parameters. graphical analysis (ga) methods, such as the patlak (pga) and logan approaches, transform kinetic model equations into a simple linear plot of selected variables. the slope and intercept of ga methods are usually related to parameters of interest. computational efficiency and reliable parameter estimates make ga methods suitable for deriving parametric images. a number of investigators reported on the kinetic analysis of f - fluoride in the evaluation of vascularisation of allogenic bone grafts [1116 ]. different kinetic quantitative approaches are also reported [5, 12, 15 ]. however, these comparisons were only limited to the ttacs derived by manually placing roi on the images, which is time consuming and prone to subjective bias. thus, the performance of these techniques for generating three - dimensional parametric images has not been fully evaluated. in addition, there is no consensus on the optimum time period in defining the linear portion of the pga plot, which may also introduce bias in pga plots. our aim was to systematically investigate the performance of parametric images derived by the pga method and evaluate the optimum linear portion of pga using dynamic f - fluoride pet imaging. eight patients with limb salvage surgery and bone grafting were included with volume of interest (voi) defined on bone grafts and normal bone tissues. quantitative rate constants and net influx rates were also derived for voi - derived ttacs with nls and pga analysis for comparison. a three - compartment and four - parameter model has been used in the kinetic analysis for fluoride bone metabolism (figure 1). rate constants describe the transport of f - fluoride between the compartments with k1 representing the unidirectional clearance of fluoride from plasma to bone tissue, k2 the reverse transport of fluoride from bone tissue to plasma, k3 reflecting the incorporation, and k4 the release of fluoride from the bone mineral compartment. the unit of k1 is mlminml, while k2, k3, and k4 have units of min. c p(t) represents the plasma concentration of f - fluoride, while ce(t) and cm(t) are f - fluoride concentrations in extracellular fluid and bone. ct(t) is the sum of ce(t) and cm(t) and represents total concentration of f - fluoride in bone tissue. through kinetic modelling technique, the output function, ct(t), can be represented by the input function, cp(t), in the following : (1)ct(t)=k121[(k41)e1t+(2k4)e2 t ] cp(t)+k1k321(e1te2t)cp(t), where 1,2=(k2+k3+k4(k2+k3+k4)2 - 4k2k4)/2 and denotes the mathematical convolution operator. for the roi - based approach, a fifth parameter of fractional blood volume (fbv) is often included in parameter estimation to address spillover from capillary blood activity within the tissue regions as shown in (2)ct(t)=(1fbv)k121 [(k41)e1t+(2k4)e2 t ] cp(t)+k1k321(e1te2 t) cp(t)+fbvcp(t). the influx rate macroparameter, ki, represents net influx or uptake of plasma fluoride activity by the bone matrix and has units of mlminml and is given by (3)ki = k1k3k2+k3. k i is an important macroparameter for describing the level of osteoblastic activity and is a measure of bone metabolism. the studies and protocols were approved by the ethics committee of the central sydney area health service. eight patients with previous limb salvage surgery and bone grafting were studied ; there were 3 men and 5 women with age range of 2053 years. the time between the bone graft surgery and pet scans was ranging from 1.3 to 4.9 years. all imaging studies were carried out on an ecat 951r whole body pet scanner (siemens / cti, knoxville, tenn, usa) in royal prince alfred hospital. the scanner acquired 31 planes simultaneously with a slice separation of 3.375 mm and axial field of view (fov) of 10.8 cm. 206.2 18.1 mbq f - sodium fluoride was infused at a constant rate over a 3-minute period. simultaneously with the beginning of tracer infusion, a 60-minute dynamic sequence of 29 emission scans was started with twelve 10-second, six 30-second, and eleven 5-minute frames. 12 arterialised - venous blood samples [8, 18 ] were obtained every 30 s for the first 6 min after injection, followed by 4 samples at 1-minute, 2 samples at 5-minute, and 4 samples at 10-minute intervals. plasma samples were counted in a -well - counter cross - calibrated with the pet scanner. transmission images were reconstructed by an ordered - subset median - root - prior (os - mrp) iterative reconstruction algorithm with 2 iterations and 16 subsets, which were then segmented into lung, bone, and soft tissue. the dynamic emission images were reconstructed into 128 128 matrices by an ordered - subset expectation - maximization (os - em) iterative reconstruction technique with 1 iteration and 16 subsets. attenuation correction was implemented in the os - em reconstruction with the attenuation coefficients derived from the segmented reconstructed transmission images. the images from the last six frames, ranging from 30 to 60 min, were averaged to aid definition of voi, which were defined using the pmod package (version 3.1, pmod technologies ltd., zurich, switzerland) by manually delineating rois on voxels with similar values across a sequence of image planes shown in figure 2. vois were defined on the bone graft and also on normal bone in the ilium and lumbar vertebra. mean value and standard deviation (sd) value were then derived for each voi on each frame of reconstructed images to form ttac and sd curve. figure 3 plots typical ttacs and plasma time activity curve (ptac) of one patient in figure 3(a) and sd curves over time in figure 3(b). the patlak graphical analysis (pga) assumes a three - compartment and three - parameter model, where the release of fluoride from the bony matrix is considered negligible, that is, k4 = 0. this leads to (4) to describe the relations between the input and output function in figure 1 : (4)ct(t)cp(t)=k1k3k2+k30tcp()dcp(t)+k2k2+k3ce(t)cp(t). when equilibrium has been reached after a sufficient time (t > t), (4) can be simplified to (5), where const represents a constant value : (5)ct(t)cp(t)ki0tcp()dcp(t)+const, t > t, k i can then be readily derived from the slope of the linear plot of 0cp()d/cp(t) versus ct(t)/cp(t). pga was used to derive the parametric images of ki according to (5) by an in - house package implemented in the idl language (version 6.0, itt visual information solutions, boulder, colo, usa). four linear periods of the pga plot reported in the literature were used to derive ki voxel by voxel for 4 to 60 min, 10 to 50 min [12, 15 ], 20 to 50 min, and 20 to 60 min [13, 14 ]. the value of a voxel was set to zero if the derived ki was negative. for comparison, the derived vois in section 2.3 were used to derive mean and sd values of ki from the parametric images, respectively, for the same patient. pga was also applied to derive ki for voi - derived ttacs with the same linear periods investigated as in the parametric images. the weights used in nls analysis were defined by the inverse of the voi - derived sd curve. the range of parameter variation was set from 0 to 1 for fbv, k1, k2, and k3 and 0 to 0.1 for k4, while the initial parameters were 0.1 mlml, 0.1 mlminml, 0.15 min, 0.1 min, and 0.01 min for fbv, k1, k2, k3, and k4 which were specified. the generated ttacs were fitted by using the marquardt - levenberg algorithm to adjust the rate constant according to (2) using the pmod package. for the equivalent comparison with the pga method, nls analysis was also applied to fit the voi - derived ttac with fbv and k4 predefined to be 0. figure 4 plots the net influx rates derived from voi - based ttacs versus those from the parametric images by pga with 10 to 50 min selected as the linear fitting period. a significant, high correlation (r > 0.999) was observed between the values of parametric images and values of voi - based ttac for the four pga approaches. comparably, low values of sd were observed in all the regions for parametric image of ki (table 1). this indicated that pga provided reliable and consistent estimates of ki for region averaged ttacs as well as voxel - by - voxel ttacs. figure 5 shows the parametric images derived by pga approach for the investigated linear fitting periods. more detail is achieved by the pga with the linear period of 2050 min (figure 5(c)) and 2060 min (figure 5(d)). however, this comes at the expense of increased noise and slightly less reliable parameter estimation reliability. table 2 lists the rate constants derived by nls for the three - compartment and five - parameter model as well as the relevant influx rate (ki5p). the mean values of parametric images of ki derived by pga are also listed for the comparison. the influx rate for three - compartment and three - parameter model with fbv = 0 and k4 = 0 is also given in the table and is referred to as ki3p. some unsuccessful fits were observed by nls analysis such as when k2 was close to zero or one. this is not unexpected because the success of physiological meaningful nls analysis is dependent on not only appropriate initial parameters but also proper noise model and underlying kinetic model. insufficient data quality in ttac may give rise to such unsuccessful fit for the three - compartment and five - parameter kinetic model. interestingly, most of estimated fbv is observed to be zero, which implies that the spillover contribution from surrounding vascular system can be ignored. table 3 lists the mean values and sd for all the vois with unsuccessful fits excluded. the lowest value of k1 was observed for the bone grafts, which implies reduced blood flow. similar values of k2, k3, and k4 were observed in the graft, ilium, and lumbar vertebra. the lower k1 of bone graft in contrast to normal bone suggests that reduced osteoblastic activity, indicated by reduced ki, of the bone graft was mainly caused by poor vascularisation and reduced blood flow. linear regression analysis was applied to compare the accuracy of the net influx rate derived by pga compared with nls for the three - compartment and five - parameter model with unsuccessful fits excluded. table 4 lists the results of linear regression for the studied four pga linear periods. one example of linear regression analysis is plotted in figure 6 for the pga with the linear period of 20 to 60 min. a high linear correlation was observed for ki derived from pga parametric images compared with nls with the linear correlation r 0.961. however, pga tends to underestimate ki with the slope of linear analysis ranging from 0.787 to 0.890 as compared with ki5p derived by nls. the values of ki3p in table 2 are almost identical to the values of original ki5p when fitted k4 is 0, while ki3p is lower than ki5p when fitted k4 is > 0. the same underlying model is assumed in deriving ki3p for nls and ki for pga. ignoring the contribution of k4 is likely to be the main reason for the underestimation of the influx rate with pga. the potential for underestimating ki with pga should be recognised when interpreting the kinetic analysis of bone metabolism. however, the simplicity of pga and the benefit of parametric images make pga a more attractive approach for the quantitative analysis of bone metabolism. overall, taking reliability, accuracy, and linear regression analysis into account, the pga analysis with the linear period of 10 to 50 min should provide best parametric images of ki in the quantitative analysis of bone metabolism. f - fluoride pet with quantitative kinetic analysis has allowed the quantitative assessment of regional lesions and treatment response in metabolic bone disease and also enabled early identification of bone viability and discriminated uneventful and impaired healing processes of fractures, bone grafts, and osteonecrosis. despite its desirable characteristics of high and rapid bone uptake with very rapid blood clearance, f - fluoride pet scanning has yet to become part of routine clinical practice. the gaussian noise model is usually assumed in the nls fitting with the weights defined according to the activity of ttac and the duration of imaging frame. in this study, the weight is defined by the inverse of the voi - derived standard deviation instead, which provides an approximate estimation of noise distribution for the voi - derived ttac. however, due to the low signal - to - noise ratio in early, short imaging frames, the iterative reconstruction caused the values of voxels with low activity to be estimated to be zero and explains why the values of sd are shown to be zero for the early frames in figure 3(b). thus, the extraordinary high weights assigned for the early zero points would give rise to unsuccessful nls fits, especially for the lower f - fluoride uptake in the grafts. with the first sixteen data points (first 4 min) excluded, nls analysis was found to achieve successful fitting for voi - based ttacs as summarised in table 2. 7, graft region in figure 3) are 0.000 for fbv, 0.025 for k1, 0.179, 0.173, and 0.006, respectively, for k2, k3, and k4 while ki5p is 0.012, which is identical to the mean value of influx rate derived from the corresponding parametric images voi. the estimation of k1 in nls analysis was sensitive to data points in early imaging time. the obtained zero points in the first 4-minute period may lead to biased estimation of k1. however, the parameter of interest, ki, was insensitive to the early imaging time. the nls analysis was expected to provide reasonable estimate of ki as the reference in the evaluation of pga method. the late time period inherently used for the pga fit avoids the problem of the low count early frames for the pga method. we observed high reliability for the studied pga with four linear periods in the generated parametric images of ki, which are highly correlated with nls - derived ki values. interestingly, the underestimation of pga - derived ki tends to be higher with later starting times of the linear period. thus, the inclusion of early staring time in pga analysis is necessary to reduce the underestimation of ki due to the simplifying assumptions of the underlying kinetic model. hybrid pet / ct scanner has now become widespread with improved image quality and shorter imaging time with coregistered ct images. combining with the detailed anatomical information readily available from ct images, the state - of - the - art pet / ct imaging may lead to more accurate assessment of graft viability. this requires in - depth investigation on improving the accuracy of ct - based attenuation correction for pet scans, especially for bone and graft tissues, to take advantage of the benefits offered by the hybrid scanners. dynamic f - fluoride pet imaging studies in patients with limb salvage surgery and fibula bone grafts were analysed using nls and pga methods to derive ttacs and parametric images. 39 regions were analysed respectively for the bone graft, ilium, and lumbar vertebrae. the results show that parametric images derived by pga are consistent with voi - based parameter estimation by pga with high reliability. the pga approach tended to underestimate influx rate because the assumption that k4 = 0 was not valid in all cases. the linear portion for pga analysis this quantitative approach using dynamic f - fluoride pet imaging allows the assessment of bone metabolism, and additional clinical studies will clarify its role in identifying bone graft viability. | the early identification of graft failure would improve patient management. 18f - fluoride is a suitable tracer for quantifying bone metabolism. performance of parametric images constructed by patlak graphical analysis (pga) with various time periods was evaluated in the analysis of dynamic 18f - fluoride pet studies of eight patients with fibula bone grafts after limb salvage surgery. the pga parametric image approach tended to underestimate influx rate. the linear portion of pga analysis was found to be from 10 to 50 min. it shows promise in providing a quantitative assessment of the viability of bone grafts. |
gonadotropin - releasing hormone (gnrh) controls the production of gonadotropins, there - by having an orchestrating effect on the reproductive hormone cascade and spermatogenesis (1). active immunization against gnrh has successfully suppressed the secretion of gonadotropins and decreased sperm production, follicular development, ovulation and conception in male and female mammals (2, 3). therefore, it can be used as an alternative for castration and fertility control in farm animals, companion animals and wildlife species (46). application of gnrh vaccination in humans has been suggested for controlling fertility - related endocrine disorders and gonadal steroid - dependent diseases (7). active immunization of adult animals against gnrh causes the loss of synthesis and secretion of gonadotropins and cessation of gonadal function as long as the antibody titers remain elevated (8). it has been reported that cow urine contains all beneficial elements such as chemical properties, potentialities and constituents that are capable of removing all the ill effects and imbalances of body caused by various infectious agents and toxicants. in this way, it ensures a protection against various ailments including the most dreaded diseases like cancer, diabetes, hepatitis etc. kamdhenu ark (distilled cow urine) has been reported as a strong immunomodulator and bioenhancer by various researchers (10, 11). experimental studies of rangasamy and kaliappan revealed the protective effects of cow urine on haematological, serum biochemical parameters and immune status of broilers (12). the present study attempts to evaluate the effects of gnrh - bsa immunization on gonado - somatic indices (gsi) and sperm parameters in male mice and to examine the modulatory role of kamdhenu ark following the immunization. sixty adult male mice, mus musculus, of parke 's strain (p), weighing 305 g were used in the study. the mice in group i served as the controls, receiving intraperitoneal phosphate buffered saline (pbs) injections (100 l) on the 1, 30, 60 and 90 days, while the mice in group ii were immunized by gnrh - bsa conjugate (50/0.2/35 g / ml / g bw) (sigma - aldrich, usa) dissolved in 100 l of phosphate buffered solution (0.01 n) emulsified with an equal volume (100 l) of freund 's adjuvant (sigma aldrich, usa). gnrh - bsa injections were given intra - peritoneally at different intervals, i.e. on days 1, 30, 60 and 90. however, the mice in group iii were supplemented with daily kamdhenu ark (100 ppm) (gaytri shakti peeth, india) orally along with the intraperitoneal injections of gnrh - bsa. five animals from each group were sacrificed in monthly intervals, i.e. on days 30, 60, 90 and 120 and their testes and epididymides were quickly dissected. the testes were weighed for observing gonadosomatic indices [gonad weight/100 g bw ], while the epididymides were processed for semen analysis, i.e. sperm motility, sperm count and morphology by prasad method (13). cauda epididymides were dissected out to release sperms in normal saline (100 mg tissue/2 ml n.s.) for sperm suspension. for studying sperm morphology, leishmans stain was used and the slides were finally observed at 400 magnification (14). the collected data were analyzed through one way anova and post - hoc methods using ezanova software. p - values < 0.05 or < 0.01 were considered significant while values < 0.001 were considered as highly significant. the collected data were analyzed through one way anova and post - hoc methods using ezanova software. p - values < 0.05 or < 0.01 were considered significant while values < 0.001 were considered as highly significant. however, more significant decrease in gsi was observed in the group treated by kamdhenu ark along with gnrh - bsa, especially in the later part of the experiment (p<0.01) (table 1). moreover, sperm motility and sperm count significantly decreased throughout the investigation in all the treated groups compared to the control group (p<0.01) (table 2). however, some mice immunized by gnrh - bsa + kamdhenu ark also showed decreased values for sperm motility and count than the gnrh - bsa immunized groups (p<0.05). the percentage of morphologically normal sperm decreased significantly with increased percentage of abnormal forms of sperms, i.e. pin head, large head, oval head, double head, head less, bent neck, looping mid piece, coiled - tail, double - tailed, tailless in all the experimental groups as compared to the control group (p<0.01) (table 3, figure 1). moreover, some significant alterations in normal sperm morphology, such as large head, headless and pin head sperm were also observed in gnrh - bsa + kamdhenu ark treated group when compared with gnrh - bsa, especially in the later part of the experiment (p<0.01). normal morphological sperm forms in the controls (a) and morphologically abnormal sperms after gnrh - bsa and kamdhenu ark along with gnrh - bsa administration (bi) in male mus musculus. gonadosomatic indices (gsi) in the experimental and control groups of male mice, mus musculus, after different intervals mean sem of five animals (accuracy of calculation up to two decimal digits) significant difference with the controls in the same column (p < 0.01) significant difference with gnrh - bsa groups in the same column (p < 0.01) significant differences (p < 0.05) sperm motility and sperm count in the experimental and control groups of male mice, mus musculus, after different intervals mean sem of five animals (accuracy of calculation up to two decimal digits) significant difference with the controls in the same column (p<0.01) significant difference with the gnrh - bsa groups in the same column (p<0.01) significant differences (p<0.05) percentage of normal and abnormal sperm morphology in the experimental and control groups of male mice, mus musculus, after different intervals mean sem of five animals (accuracy of calculation up to two decimal digits) significant difference with the controls in the same column (p < 0.01) significant difference with gnrh - bsa groups in the same column (p < 0.01) significant differences (p < 0.05) the endocrine effects of active immunization against gnrh have been studied in a variety of young adult male and female animals (1517). experimental studies have demonstrated decreases in gonadotropins, sperm production, follicular development, ovulation and conception after immunization against gnrh, chemically conjugated to a carrier protein. gnrh immuneization affected sperm motility and sperm counts in ram lambs, boars and colts (18, 19). several other experimental studies have revealed the deleterious effects of immunization against gnrh on different sperm parameters in rats, bulls, stallions, cats and dogs (2024). cow urine has been tested for its immuno - modulatory properties that enhance both cellular and humoral immune responses (25, 26). kamdhenu ark (distilled cow urine) has been reported to increase the humoral immunity in rats (27). chauhan., (2004) observed that kamdhenu ark may modulate the immune responses because it increases the secretion of interleukin-1 and 2 (28). recently, ganaie and shrivastava reported the modulatory effects of kamdhenu ark on gnrh - bsa immunization in female mice (29). in corroboration to above studies, our study also revealed that gnrh - bsa immunization significantly decreased the values of gsi, sperm motility, count and morphology in male mus musculus. the aforesaid parameters diminished more significantly in the group supplemented with kamdhenu ark along with gnrh - bsa immunization. all these changes in gsi and sperm parameters suggested that gnrh - bsa immunization could have directly suppressed the activities of gonadotropins and testosterone through hypothalamo - hypophysial - gonadal axis or might have indirectly affected the testicular tissue. however, more significant decreases in the parameters after kamdhenu ark supplementation may be because of its modulatory and bioenhancing properties. | backgroundactive immunization against gnrh decreases the secretion of gonadotropins and causes cessation of gonadal function, thereby, inducing infertility. based on the immunoenhancing activity of kamdhenu ark (distilled cow urine), this study was performed to evaluate its effects on the gonadosomatic indices (gsi) and sperm parameters in male mice receiving a gnrh contraceptive vaccine.methodssixty adult male mice of parke 's strain were divided into three groups of twenty. group i served as the controls, while group ii was immunized by gnrh - bsa conjugate (50/0.2/35 g / ml / g bw) by four intraperitoneal injections at different intervals on days 1, 30, 60 and 90. however, group iii was supplemented daily by oral kamdhenu ark (100 ppm) along with gnrh - bsa immunizations. the animals were sacrificed after 30, 60, 90 and 120 days and their testis and epididymis were dissected out weighed and semen analysis was performed.resultsgsi values, sperm motility, sperm count and sperm morphology in male mus musculus were decreased significantly in all the experimental groups as compared to the control group (p<0.01). kamdhenu ark significantly enhanced the effect of gnrh vaccine on the aforesaid parameters especially in 90 and 120 days treated groups (p<0.05).conclusionthe changes witnessed in sperm parameters suggested that the gnrh - bsa immunization suppressed the activities of gonadotropins and testosterone directly through hypothalamo - hypophysial - gonadal axis and indirectly by acting on the testes which may modulate the sperm morphology, sperm count and motility. however, kamdhenu ark seems to have enhanced these effects because of its immune - modulatory properties too. |
a fundamental barrier to fulfilling the emerging health policy mandate in the united states for monitoring the quality and outcomes of pac is the absence of standardized, patient - centered outcome data that can provide policy officials and managers with outcome data across different diagnostic categories, over time, and across different settings where pac services are provided (wilkerson and johnston, 1997). recently, the national committee on vital and health statistics (ncvhs) (2002) made recommendations on the potential for standardizing data collection and reporting for the purposes of quality assurance as well as for setting future research and health policy priorities in the u.s. the ncvhs (2002) was unanimous in stressing two major goals : to put functional status solidly on the radar screens of those responsible for health information policy, and to begin laying the groundwork for greater use of functional status information in and beyond clinical care the ncvhs project used the term functional status very broadly to cover both the individual 's ability to carry out activities of daily living (adls) and the individual 's participation in various life situations and society. within pac, functional outcome instruments have been developed and are widely used for various applications and for use in specific settings. examples include the functional independence measure (fim) for acute medical rehabilitation (guide for the uniform data set for medical rehabilitation, 1997 ; hamilton, granger, and sherwin, 1987), the minimum data set (mds) for skilled nursing and subacute rehabilitation programs (morris, murphy, and nonemaker, 1995), the outcome and assessment information set for home health care (oasis) (shaughnessy, crisler, and schlenker, 1997) and the short form-36 (sf-36) for ambulatory care programs (ware and kosiniski, 2001). if one looks carefully at the content of these instruments, it becomes apparent that substantial variations exist in item definitions, scoring, metrics, and content coverage, resulting in fragmentation in outcome data available for use across different pac settings (haley and langmuir, 2000). differences in conceptual frameworks used to construct each instrument, the inability to translate scores from one instrument to another, and the lack of outcome coverage and precision to detect meaningful functional changes across settings, severely limit the field 's ability to measure and analyze recovery through the period of pac service provision. if the pac field is to achieve the goal of comprehensive functional outcome assessment and quality monitoring for different patient diagnostic groups across different pac settings, efforts are needed to develop functional outcome assessments that are applicable across a continuum of post - acute services and settings. to our knowledge, no studies exist that have directly compared the content and operating characteristics of functional outcome instruments commonly used in pac to examine their relative merits for monitoring outcomes across care settings. in this article, therefore, we report the results of a direct empirical comparison of the fim, oasis, mds, and the physical function scale (pf-10) of the sf-36, focusing on three aspects of each : instrument content, range of coverage, and measurement precision. the objective of this comparative analysis is to evaluate the commonly held assumption that there exist fundamental deficiencies in the current armamentarium of setting - specific outcome instruments that prevents their applicability for more comprehensive patient - centered functional outcome assessment across diagnoses, over time, and across different settings where pac is provided. in response to identified deficiencies in existing instruments, we also discuss the potential utility of contemporary measurement techniques, such as item response theory (irt) methods and computerized adaptive technology (cat), to yield functional outcome instruments better suited for outcome monitoring across pac settings. these analyses use data from a sample of 485 pac patients drawn from six health provider networks in the greater boston area. all patients enrolled in the study were recruited by study coordinators within their own health care facility and completed informed consent prior to participating in the study. patients were recruited from inpatient (199 from acute inpatient rehabilitation and 90 from transitional care units) ; and community settings (90 from ambulatory services ; and 106 from home care). eligibility criteria included : (1) adults, age 18 or over, (2) recipients of skilled rehabilitation services (physical, occupational, or speech therapy), and (3) english speaking. the sample was stratified by impairment group to include approximately an equal number of subjects within three major patient groups : (1) 33.2 percent neurological (e.g., stroke, multiple sclerosis, parkinson 's disease, brain injury, spinal cord injury, neuropathy) ; (2) 28.4 percent musculoskeletal (e.g., fractures, joint replacements, orthopedic surgery, joint or muscular pain) ; and (3) 38.4 percent medically complex (e.g., debility resulting from illness, cardiopulmonary conditions, or post - surgical recovery). to assure good representation of levels of functional severity, the sampling design was stratified to yield a wide distribution of subjects representing three distinct severity levels : slight (35.9 percent), moderate (44.1 percent), and severe (20.0 percent) based on scores from an adapted modified rankin scale (vanswieten., 1988). the sample contained a wide age range (19 - 100 years ; mean age = 62.7 years.) the majority of the subjects were female (58.8 percent), white (81.6 percent), and non - married (61.1 percent). more than one - half (51.3 percent) had beyond a high school education. the wide range of onset from initial injury / illness (2.0 - 3.9 years) characterizes different stages of recovery within both inpatient and community settings. the sf-8 health survey (ware., 1999) data indicated that physical functioning of the overall sample (mean=40.3, standard deviation (s.d.)=9.9) was below the u.s. population norms (mean=50, s.d.=10), although mental functioning (mean=50.2, s.d.=10.3) was consistent with u.s. population norms (mean=50, s.d.=10). our overall data collection strategy was to assess items from existing functional outcome tools used in pac so that they could be combined for analysis into one common scale. due to practical data collection considerations and potentially high patient response burden rather, we linked items together by administering to the entire sample a core set of 58 activity items applicable for patients in both home and community service settings. this method has been referred to as a common - item test equating design, in which a core set of items serve as a scale anchor from which unbiased parameters can be estimated on items with missing data (mchorney and cohen, 2000). we collected activity items, when available, from standardized instruments administered and recorded in the medical record. these included the 18-item fim for persons in inpatient rehabilitation settings (n=108), 19 mds items (physical functioning and selected cognitive items) for persons in skilled nursing home settings (n=91), and 19 adl / individual adl items from the oasis or persons receiving home care (n=103). since there were no consistent data available from charts in the outpatient setting, we administered the 10 physical functioning items of the sf-36 to individuals receiving outpatient services (n=82). we applied specific rules for handling missing item data within two of the standardized instruments. in accordance with fim scoring rules, items that were not administered we converted these codes to the lowest score for that item, namely total dependence. we reasoned that the most likely explanation for the activity not occurring was that the item could not be performed, an assumption that others have made when comparing functional instruments. the 58 core activity items collected on all 485 subjects were used as scale anchors. in order to compare items from instruments across pac settings, the common - item test equating design was used so that every subject had a similar core set of items. rasch (1980) models (wright and masters, 1982) can be conducted with missing data if a core set of items is used to link the setting - specific instruments into a common scale. core items included : 15 physical functioning, 14 self - care, 12 daily routine, 11 communication, and 6 interpersonal interaction items. activity questions asked, how much difficulty do you currently have (without help from another person or device) with the following activities ? a polytomous response choice included not at all, a little, somewhat, a lot, and can not do. we framed the activity questions in a general fashion without specific attribution to health, medical conditions, or disabling factors. for some individuals in the community settings, we also collected 52 additional functional items (n=196) and added those to the common linking solution, however the results of these items are not reported here. data on items from three of the existing instruments (e.g., fim, oasis, and mds) were recorded from retrospective chart review. data on the pf-10 items (outpatient programs) and core and community items were collected via subject or proxy interviews. each interview (approximately 45 - 60 minutes) was conducted in a quiet atmosphere in an inpatient setting, an outpatient facility, or participant 's home. the ability of a subject to take part in the interview self - report was assessed by a treating clinician who determined if the respondent could : (1) understand the interview questions, (2) sustain attention during an interview, and (3) reliably respond to the questions. if the answer to any of these screening questions was no, then the interview was completed by either a clinician or family member proxy participant. a proxy participant interviews that included information on core items were timed to coincide with administration of standard outcome instruments. for example, fim is administered in most facilities within 3 days of admission and prior to discharge in the inpatient rehabilitation setting. thus, the subject interview was arranged to take place within 3 days of admission or discharge such that fim information was collected close to the time of the interview. likewise, interviews of subjects in transitional care units were scheduled to coincide with the mds assessment. subjects receiving home care therapy were interviewed either near to admission or discharge such that the oasis assessment was performed in close proximity to the subject interview. the interview was fully scripted, with standard instructions and an answer card to help subjects identify the desired response choice. the order of presentation of test sections was randomly assigned to mitigate the possibility of large portions of missing data in any one section due to respondent fatigue. interviewers, who were experienced rehabilitation clinicians (mean years of clinical experience=5.7 years), received training and quality assurance from : (1) an initial 3-hour training session, (2) a protocol manual, (3) supervision by the research project staff on all first interviews, and (4) acceptable completion of a procedural checklist and inter - rater reliability on a subsequent interview. a project staff member accompanied interviewers on approximately every 10th interview to check on adherence to study protocol and to assure overall quality control. all subjects, regardless of setting, were administered the sf-8 items to establish a normative description of the sample. first, we conducted one - parameter rasch (1980) partial credit analyses for the entire item pool (instruments and core / community items) to develop an overall functional ability scale (wright and masters, 1982). for example, the fim scale has a seven - point rating scale, while the oasis incorporates multiple response categories that differ per item. this overall scale was used to estimate parameters for all items in each of the four comparison instruments. we used a method of concurrent calibration, which involves estimating item and ability parameters in the overall subject and item pool simultaneously. this treats items not taken by subjects as missing, as the rasch program uses information available to estimate person and item parameters. our goal was to develop a general scale for functional abilities, and specifically to see the location of item content across the four existing instruments. we calculated internal consistency estimates cronbach alpha (1951)for each of the individual instruments and for the overall functional scale to determine the levels of consistency of the items within the overall functional scale. we also calculated goodness of fit tests for all items with the overall functional scale using the standardized infit statistic (+ /-2.0) to determine the number of items with poor fit within the overall scale. second, we examined the relative amount and range of activity content covered by each of the four existing instruments and made comparisons across instruments. to do this, we used the extreme (highest and lowest) item threshold estimate for each instrument. a threshold represents the point of separation between adjacent item response categories for each item. the rasch partial credit model estimates thresholds for each item, and the minimum and maximum threshold values per instrument were used to establish the range of content within the overall functional ability scale. third, we calculated item and test information functions (lord, 1980 ; dodd and koch, 1987 ; murnki, 1993) to estimate the location of optimal measurement precision of each instrument. the item information function is an index of the degree and location of information that a particular functional item provides for estimating a score along a functional ability scale. item information functions are related to the location and shape of the item characteristic curve (icc), which describe the probabilities of responding to particular response options of an item. the steeper the slope of the icc, the more information about functional ability provided by an item in the scale, thus the greater level of item discrimination and precision associated with estimates of the score at that point along the scale. iccs that have a broad range of coverage along the scale provide information functions across a wide range of the scale. therefore, the information function is the relationship of the amount of information of an item at a particular scale level and is described by the ratio of the slope of the icc and the expected measurement error. test information functions were calculated by summing the item information functions to obtain an estimate of the measurement precision of the entire test at different levels of the functional ability continuum. we compared the test information function with the ability levels of the sample in each major setting (inpatient, community). we calculated a summary score converted to 0 - 100 metric for each person based on the overall item pool. these analyses use data from a sample of 485 pac patients drawn from six health provider networks in the greater boston area. all patients enrolled in the study were recruited by study coordinators within their own health care facility and completed informed consent prior to participating in the study. patients were recruited from inpatient (199 from acute inpatient rehabilitation and 90 from transitional care units) ; and community settings (90 from ambulatory services ; and 106 from home care). eligibility criteria included : (1) adults, age 18 or over, (2) recipients of skilled rehabilitation services (physical, occupational, or speech therapy), and (3) english speaking. the sample was stratified by impairment group to include approximately an equal number of subjects within three major patient groups : (1) 33.2 percent neurological (e.g., stroke, multiple sclerosis, parkinson 's disease, brain injury, spinal cord injury, neuropathy) ; (2) 28.4 percent musculoskeletal (e.g., fractures, joint replacements, orthopedic surgery, joint or muscular pain) ; and (3) 38.4 percent medically complex (e.g., debility resulting from illness, cardiopulmonary conditions, or post - surgical recovery). to assure good representation of levels of functional severity, the sampling design was stratified to yield a wide distribution of subjects representing three distinct severity levels : slight (35.9 percent), moderate (44.1 percent), and severe (20.0 percent) based on scores from an adapted modified rankin scale (vanswieten., 1988). the sample contained a wide age range (19 - 100 years ; mean age = 62.7 years.) the majority of the subjects were female (58.8 percent), white (81.6 percent), and non - married (61.1 percent). more than one - half (51.3 percent) had beyond a high school education. the wide range of onset from initial injury / illness (2.0 - 3.9 years) characterizes different stages of recovery within both inpatient and community settings. the sf-8 health survey (ware., 1999) data indicated that physical functioning of the overall sample (mean=40.3, standard deviation (s.d.)=9.9) was below the u.s. population norms (mean=50, s.d.=10), although mental functioning (mean=50.2, s.d.=10.3) was consistent with u.s. our overall data collection strategy was to assess items from existing functional outcome tools used in pac so that they could be combined for analysis into one common scale. due to practical data collection considerations and potentially high patient response burden, rather, we linked items together by administering to the entire sample a core set of 58 activity items applicable for patients in both home and community service settings. this method has been referred to as a common - item test equating design, in which a core set of items serve as a scale anchor from which unbiased parameters can be estimated on items with missing data (mchorney and cohen, 2000). we collected activity items, when available, from standardized instruments administered and recorded in the medical record. these included the 18-item fim for persons in inpatient rehabilitation settings (n=108), 19 mds items (physical functioning and selected cognitive items) for persons in skilled nursing home settings (n=91), and 19 adl / individual adl items from the oasis or persons receiving home care (n=103). since there were no consistent data available from charts in the outpatient setting, we administered the 10 physical functioning items of the sf-36 to individuals receiving outpatient services (n=82). we applied specific rules for handling missing item data within two of the standardized instruments. in accordance with fim scoring rules, items that were not administered we converted these codes to the lowest score for that item, namely total dependence. we reasoned that the most likely explanation for the activity not occurring was that the item could not be performed, an assumption that others have made when comparing functional instruments. the 58 core activity items collected on all 485 subjects were used as scale anchors. in order to compare items from instruments across pac settings, the common - item test equating design was used so that every subject had a similar core set of items. rasch (1980) models (wright and masters, 1982) can be conducted with missing data if a core set of items is used to link the setting - specific instruments into a common scale. core items included : 15 physical functioning, 14 self - care, 12 daily routine, 11 communication, and 6 interpersonal interaction items. activity questions asked, how much difficulty do you currently have (without help from another person or device) with the following activities ? a polytomous response choice included not at all, a little, somewhat, a lot, and can not do. we framed the activity questions in a general fashion without specific attribution to health, medical conditions, or disabling factors. for some individuals in the community settings, we also collected 52 additional functional items (n=196) and added those to the common linking solution, however the results of these items are not reported here. data on items from three of the existing instruments (e.g., fim, oasis, and mds) were recorded from retrospective chart review. data on the pf-10 items (outpatient programs) and core and community items were collected via subject or proxy interviews. each interview (approximately 45 - 60 minutes) was conducted in a quiet atmosphere in an inpatient setting, an outpatient facility, or participant 's home. the ability of a subject to take part in the interview self - report was assessed by a treating clinician who determined if the respondent could : (1) understand the interview questions, (2) sustain attention during an interview, and (3) reliably respond to the questions. if the answer to any of these screening questions was no, then the interview was completed by either a clinician or family member proxy participant. interviews that included information on core items were timed to coincide with administration of standard outcome instruments. for example, fim is administered in most facilities within 3 days of admission and prior to discharge in the inpatient rehabilitation setting. thus, the subject interview was arranged to take place within 3 days of admission or discharge such that fim information was collected close to the time of the interview. likewise, interviews of subjects in transitional care units were scheduled to coincide with the mds assessment. subjects receiving home care therapy were interviewed either near to admission or discharge such that the oasis assessment was performed in close proximity to the subject interview. the interview was fully scripted, with standard instructions and an answer card to help subjects identify the desired response choice. the order of presentation of test sections was randomly assigned to mitigate the possibility of large portions of missing data in any one section due to respondent fatigue. interviewers, who were experienced rehabilitation clinicians (mean years of clinical experience=5.7 years), received training and quality assurance from : (1) an initial 3-hour training session, (2) a protocol manual, (3) supervision by the research project staff on all first interviews, and (4) acceptable completion of a procedural checklist and inter - rater reliability on a subsequent interview. a project staff member accompanied interviewers on approximately every 10th interview to check on adherence to study protocol and to assure overall quality control. all subjects, regardless of setting, were administered the sf-8 items to establish a normative description of the sample. first, we conducted one - parameter rasch (1980) partial credit analyses for the entire item pool (instruments and core / community items) to develop an overall functional ability scale (wright and masters, 1982). for example, the fim scale has a seven - point rating scale, while the oasis incorporates multiple response categories that differ per item. this overall scale was used to estimate parameters for all items in each of the four comparison instruments. we used a method of concurrent calibration, which involves estimating item and ability parameters in the overall subject and item pool simultaneously. this treats items not taken by subjects as missing, as the rasch program uses information available to estimate person and item parameters. our goal was to develop a general scale for functional abilities, and specifically to see the location of item content across the four existing instruments. we calculated internal consistency estimates cronbach alpha (1951)for each of the individual instruments and for the overall functional scale to determine the levels of consistency of the items within the overall functional scale. we also calculated goodness of fit tests for all items with the overall functional scale using the standardized infit statistic (+ /-2.0) to determine the number of items with poor fit within the overall scale. second, we examined the relative amount and range of activity content covered by each of the four existing instruments and made comparisons across instruments. to do this, we used the extreme (highest and lowest) item threshold estimate for each instrument. a threshold represents the point of separation between adjacent item response categories for each item. the rasch partial credit model estimates thresholds for each item, and the minimum and maximum threshold values per instrument were used to establish the range of content within the overall functional ability scale. third, we calculated item and test information functions (lord, 1980 ; dodd and koch, 1987 ; murnki, 1993) to estimate the location of optimal measurement precision of each instrument. the item information function is an index of the degree and location of information that a particular functional item provides for estimating a score along a functional ability scale. item information functions are related to the location and shape of the item characteristic curve (icc), which describe the probabilities of responding to particular response options of an item. the steeper the slope of the icc, the more information about functional ability provided by an item in the scale, thus the greater level of item discrimination and precision associated with estimates of the score at that point along the scale. iccs that have a broad range of coverage along the scale provide information functions across a wide range of the scale. therefore, the information function is the relationship of the amount of information of an item at a particular scale level and is described by the ratio of the slope of the icc and the expected measurement error. test information functions were calculated by summing the item information functions to obtain an estimate of the measurement precision of the entire test at different levels of the functional ability continuum. we compared the test information function with the ability levels of the sample in each major setting (inpatient, community). we calculated a summary score converted to 0 - 100 metric for each person based on the overall item pool. the internal consistency values of the four functional ability instruments were as follows : mds=0.97, oasis=0.99, fim= 0.99, and the pf-10=0.99. only five of the items within the existing instruments (7.2 percent) exceeded the goodness of fit values. thus, we felt it was acceptable to combine the items from each of the four functional outcome instruments into an overall functional ability scale for the purposes of directly comparing their range of functional content, breadth of coverage, and measurement precision. figure 1 illustrates and compares the location of items in each of the four functional outcome instruments along the broad content dimension of functional ability. these locations are derived as estimates of the average functional ability parameter generated for items in each instrument included in the analysis. across all four instruments it can be seen that cognitive, communication, and bowel and bladder continence function items achieved the lowest functional ability estimates, indicating that those items were usually less difficult for persons in the sample to perform compared with other items contained in these instruments. in general, the pf-10 scale contained items with the highest item functional ability calibrations compared with the other three. a substantial number of items in the fim, oasis, and mds instruments achieved average functional ability calibrations in the mid - point of the functional ability continuum. the actual range of functional ability that is covered by the items contained in each of the four functional outcome instruments is presented in figure 2. the content coverage is calculated by the high and low step estimates for each item 's response categories instead of average estimates, which were the basis of the information presented in figure 1. consistent with the general spread of the functional ability parameters illustrated in figure 1, the range of coverage shown in figure 2 appears greatest for both the mds and the oasis instruments as compared with either the fim or pf-10 scales. because of the high step estimate for one of the transportation items in the oasis, the actual range of coverage of the oasis is nearly the entire range of all four instruments. figure 3 displays the measurement precision of each instrument as depicted by the test information function curves. these curves are superimposed with the average score on the functional ability continuum and 2 standard deviation for the inpatients and community samples. note the location of the peak amount of precision for each instrument in relationship to each sample. although the oasis items contain a broad range of content, as was seen in figure 2, the oasis items provide a high degree of measurement precision at only the very low end of the functional ability dimension for both the inpatient and community samples. the precision of mds items is also greatest at the lower functional ability dimension levels, although the mds items have a greater span of functional ability in which they provide some levels of precision. in contrast, the information function of the pf-10 items peaks at the high end of the community outpatient sample with very poor precision for the inpatient sample. the results of these analyses of instrument content, coverage, and measurement precision provide direct evidence of what many have argued are the major limitations of existing functional outcome instruments currently in use within pac. while each of the four instruments compared in this analysis appears well suited for its primary application, none of them appears well - equipped for the current policy mandate for monitoring the quality and outcome of pac provided over time and across different pac settings. if one looks at the results for the fim, the most widely used outcome instrument in pac, one can see that the fim items cover a very small portion of the functional ability continuum within a narrow range of functional content. the fim is most precise and relevant for pac inpatients whose function is at the low end of the continuum. all of these characteristics of the fim are acceptable when one considers its primary application is for evaluation of inpatient rehabilitation services. these fim characteristics become severe limitations, however, if the intended application is assessment of outcomes or quality across pac settings. an instrument such as the pf-10 suffers from a similar type of limitation, as does the fim. the pf-10 covers a narrow range of functional content, although, unlike the fim, the content covered by the pf-10 is at the higher end of the functional activity continuum. the pf-10 appears most precise for community dwelling outpatients, but much less so for inpatients such as those seen in many rehabilitation facilities. when used with high functioning community patients, the pf-10 covers appropriate content ; its content and range is severely limited in application to patients within institutions. the mds and oasis instruments, in comparison with the fim and pf-10, cover content from the mid portion of the functional ability continuum with less content covering the low or high ends. patients functioning at the very high or very low end of the functional continuum would not be as well served by the oasis and mds. concerns about existing instruments used in pac, such as the four examined in this analysis, have stimulated the development of more comprehensive functional outcome instruments developed specifically for application across diagnostic groups, and across pac settings. an example of this type of work is found in the activity measure for pac (am - pac), developed by the rehabilitation research & training center for outcomes based at boston university (haley and jette, 2000). in constructing the am - pac, its developers used the strategy of combining functional ability items found in existing instruments and from a variety of other sources into quantitative scales that can be employed to assess a wide range of functional content needed to assess quality and outcomes of patients seen across pac settings. although instruments such as the am - pac are promising, the continued use of traditional, fixed - form measurement methodology for constructing functional outcome instruments presents the researcher with two common problems that limit their utility in clinical outcomes assessment. one fundamental problem encountered with fixed - form instruments of modest length is floor and ceiling effects where large numbers of individuals who complete these outcome instruments score at either the top or the bottom of the range of possible scores. these ceiling and floor effects severely reduce measurement precision and thus, restrict the utility of the instruments (andresen., 1999 ;, 1996 ; difabio., 1997 ; rubenstein., 1998). in response to concerns over inadequate measurement precision and inadequate coverage of important outcome domains, some researchers develop more comprehensive and lengthy outcome instruments. lengthy instruments lead to the frustration and fatigue faced by many subjects and busy clinicians overwhelmed by large and burdensome batteries of instruments (meyers, 1999). one promising solution offered to measurement problems faced by traditional fixed form instruments is offered through the combined application of irt methodology and cat techniques (ware., 1999 ; ware, bjorner, and kosinski, 2000 ; weiss, 1982 ; hambleton, 2000). these techniques of test construction are currently being applied to the development of a new generation of functional assessment instruments designed for use in pac settings (haley and jette, 2000). cat methodology uses a computer interface for the patient (or a computerized interview / clinician report) that is tailored to the unique functional ability level of the patient. the basic notion of an adapted test is to mimic what an experienced clinician would do. a clinician learns most when he / she directs questions at the patient 's approximate level of proficiency. administering outcome items that are either too easy or too hard provides little information. an adaptive test first asks questions in the middle of the ability range, and then directs questions to the level based on the patient 's responses, without asking unnecessary questions. this allows for fewer items to be administered while gaining precise information regarding an individual 's placement along a continuum of functional ability. cat applications require a large set of items in any one functional area (item pools), items that consistently scale along a dimension of low to high functional ability, and rules guiding starting, stopping, and scoring procedures. large item sets such as the am - pac can be readily adapted to a cat format in future work. methods like irt that make it possible to calibrate questionnaire items on a standard metric (ruler) also yield the algorithms necessary to run the engine that powers cat assessments. these statistical models estimate how likely a person at each level of function is to choose each response to each survey question. this logic is reversed to estimate the probability of each health score from a particular pattern of item responses. the resulting likelihood function makes it possible to estimate each person 's score, along with a person - specific confidence interval. in principle, one can derive an unbiased estimate of an outcome, i.e., an estimate without systematic error, from any subset of items that fits the model. the number of items administered can be increased to achieve the desired level of precision. most statistical models for estimating such item parameters can be traced to the work of rasch (1980) or on a second tradition both models assume unidimensionality ; i.e., that the items included on a particular scale measure only one concept. whether these techniques, if applied to functional outcome assessment, will solve the problems presented by traditional, fixed form methodology, needs to be carefully evaluated in future research. there are several limitations in the analyses reprinted in this article. to achieve a direct comparison of these four instruments, we used a liberal interpretation of unidimensionality to combine items from all of the four comparison instruments into a single scale. this simplified the presentation so that an instrument - based rather than a content - based comparison could be made. a more detailed examination of common functional dimensions that underlie the item set was beyond the scope of this article. we also point out that, for practical reasons, we combined data from medical records and from interviews to develop the item calibrations for the instrument comparisons. although not ideal, we do find only small amounts of error from clinician and self - report interview modes of testing within these functional items (andres, haley, and ni, forthcoming). more work in this area of combining data across respondents and modes of data collection will be needed as the field advances in assessing functional ability across post - acute core settings. results of this analysis illustrate some of the inherent limitations in the range of content, breadth of coverage, and measurement precision found in functional outcome instruments currently in use within pac. limitations in existing functional outcome methodology has stimulated the ongoing development of more comprehensive outcome assessment systems specifically for monitoring the quality of services provided for patients with different across diagnoses and across pac settings. the careful use of irt - based measurement methods coupled with cat outcome assessment techniques may hold future promise for making outcomes assessment briefer and less burdensome to patients, and thus more acceptable for use in busy clinical settings. what is needed is functional outcome data that is applicable to patients treated across different clinical settings and applications, more efficient and less costly to administer, and, sufficiently precise to detect clinically meaningful changes in functional outcomes. contemporary measurement methodology may hold considerable promise as a vehicle for advancing pac outcomes evaluation, thus avoiding the pitfalls of traditional assessment methodology. | there is a growing health policy mandate for comprehensive monitoring of functional outcomes across post - acute care (pac) settings. this article presents an empirical comparison of four functional outcome instruments used in pac with respect to their content, breadth of coverage, and measurement precision. results illustrate limitations in the range of content, breadth of coverage, and measurement precision in each outcome instrument. none appears well - equipped to meet the challenge of monitoring quality and functional outcomes across settings where pac is provided. limitations in existing assessment methodology has stimulated the development of more comprehensive outcome assessment systems specifically for monitoring the quality of services provided to pac patients. |
the puerto rican crested toad, peltophryne lemur, is the only native toad species of puerto rico and has become a subject of conservation concern due to its small population size, limited breeding sites, and small geographic range [13 ]. although several new populations have been established in puerto rico through captive - breeding and release programs, reproduction of the naturally wild population of this toad species is considered limited to a small region of coastline located in guanica, puerto rico [48 ]. based on direct observation of breeding events during heavy rainfall, the number of observed mature individuals declined from 1984 to 2003, with only 80 mature individuals recorded in 2003 [1, 9 ]. the majority of breeding for the naturally wild population is thought to occur at three distinct breeding sites within a several kilometer radius, and each of these three breeding sites contains at least one ephemeral pool that fills with water under adequate rainfall conditions. the volume, surface area, depth, and duration of each of these temporary pools is dependent on the amount and frequency of rainfall that the region receives. a portion of the largest and most significant site for toad reproduction, the tamarindo site, is accessible for vehicular parking by members of the public when the breeding pools are dry. vehicular traffic is associated with a wide variety of contaminants including polycyclic aromatic hydrocarbons (pahs) from incomplete combustion, exhaust, oil leaks, tire abrasion, asphalt, and other lubricants [1014 ]. pahs have been linked with many undesirable health consequences in humans and animals including carcinogenic, immunotoxic, mutagenic, and teratogenic effects [1518 ]. environmental exposure of amphibians to pahs may cause such broad effects as increased mortality, genotoxicity, larval deformities, histological changes to the integument, slowed development, and larval hyperactivity [11, 1925 ]. a more thorough review and discussion of the effect of pahs and other contaminants on amphibian populations may be found elsewhere [17, 26, 27 ]. passive sampling has been used for several decades as a method to assess contaminant levels within the air and aquatic environments. passive sampling devices (psds) allow time - weighted average concentrations of contaminants to be quantified giving a better understanding of the exposure than random discrete water testing. because the devices accumulate contaminants over a deployment period, passive sampling technology is often able to detect trace levels of contaminants in an aquatic environment better than other methods. the semipermeable membrane device (spmd) is a common configuration, which is usually composed of low - density polyethylene (ldpe) tubing containing a neutral lipophilic triolein core. the ldpe tubing forms transient thermal pores to a size that closely approximates those of aquatic organisms [32, 33 ]. when such spmds are deployed in an environment containing lipophilic contaminants, such as pahs, these analytes diffuse through the membrane to become sequestered within the core or membrane itself. this ensures that only the bioavailable form of the analyte is accumulated within the device and mimics the uptake of contaminants from the environment by live organisms. following a deployment period, these contaminants can be extracted from the device using a nonpolar solvent and analyzed using gas chromatography - mass spectrometry (gc / ms) to identify and quantify the contaminants present within the device. modeling can then be used to determine the concentrations of these contaminants within the aquatic environment [32, 3437 ]. current efforts to conserve the puerto rican crested toad are being made through captive breeding, reintroduction programs, habitat conservation, and public education conducted by the puerto rican crested toad species survival plan [4, 5, 8, 9 ]. habitat preservation may play a critical role in the survival of the remaining naturally wild population of toads. contaminant levels at high enough concentrations could pose a significant risk to the development and survival of offspring within the breeding pools utilized by this species. this study was conducted to measure and perform a risk analysis of the bioavailable concentrations of pahs present within breeding pools utilized by puerto rican crested toads at sites used for vehicular parking compared to those that are not. the psds used in this study were composed of a single strip of dichloromethane - extracted ldpe tubing with the dimensions of 25 cm 7.5 cm, an effective surface area of 375 cm, and an average weight of 4.3 g. during deployment, these strips were housed between two rectangular fenestrated aluminum plates with the dimensions of 30 cm 9 cm, which were curved along the longitudinal axis to form a cylindrical housing for the device. six small slices were made along the edges of each ldpe strip to allow conventional plastic ties to hold the strip in place within the aluminum plates. these plates allowed the strips to be positioned at their respective deployment sites, provided shade, and elevated the strips above the sediment and within the water column. small plastic placards were placed on each device and secured with plastic ties to deter theft during the deployment period. the components of the devices were stored wrapped in baked aluminum foil at 20c until the time of the deployment. deployment of the devices was scheduled for the first adequate rainfall of the rainy season, which occurred on november 8, 2009. recovery of the devices was performed 14 days following the initial deployment of each device to ensure that analytes remained in the linear uptake phase for appropriate modeling purposes. devices were deployed into the ephemeral pools located at the three breeding sites : tamarindo, aroma, and atolladora. device placement was based on the specific pool configuration at each site favoring deep depressions to ensure that devices were not exposed to air as the pools evaporated during the deployment period. 15 devices were deployed at the tamarindo site and were divided between its two major pools. 8 of these devices were deployed at the tamarindo south pool, and 7 devices were deployed at the tamarindo north pool. 6 devices were deployed at the aroma site, and 8 devices were deployed at the atolladora site. following the deployment period, the ldpe strips were removed from their aluminum plates and rinsed in the water at their respective deployment locations to remove any organic debris. each strip was wrapped individually in aluminum foil and placed in a sealed plastic bag inside a cooler containing ice packs for several hours until the devices could be stored below 20c, which has been validated as an appropriate storage technique for spmds for periods up to 6 months. the ldpe strips remained under these storage conditions until analysis, which was performed several weeks following recovery of the devices. the pool located at the aroma site sustained significant natural volume loss during the deployment period, causing all of the devices at this site to become exposed to air prior to recovery. the devices from this site were not analyzed and were removed from the remaining aspects of this study. nitrile gloves were worn during deployment and recovery of the devices and were changed between the handling of each device. to ensure quality control, a separate field blank for each site was exposed to the air during deployment and recovery of the devices at each respective site. these field blanks were stored identically to the other devices prior to and after the deployment period and were stored wrapped in aluminum foil within a sealed plastic bag at 20c throughout the deployment period. to prepare the samples for analysis following recovery, each ldpe strip was allowed to thaw and then scrubbed under deionized water to remove any residual algae and organic matter. each strip was cut into pieces of 1 cm by 4 cm with solvent - rinsed scissors. these pieces were placed into solvent - rinsed and labeled containers and were extracted twice over 24 hours using a total of 45 ml of dichloromethane. gel permeation chromatography was used to fractionate the waxes and analytes of these samples using the technique previously described. a procedural blank was prepared and extracted concurrently with the samples to ensure quality control. between 4 and 7 samples for each site were analyzed using an agilent 6890 gas chromatograph with 5973n mass selective detector. the column used was a varian vf-5ms 30 m 0.25 mm i d (0.25 m film thickness), with 10 m integrated guard column. the initial temperature used was 40c for 1 minute and then increased 25c per minute until a temperature of 100c. following this, a temperature program of 5c per minute to a final temperature of 310c was used with a hold time of 15 minutes. the inlet temperature was held at 300c, and a pulsed splitless injection technique was used. the inlet pressure was increased to 30 psi for 0.9 minutes and then reduced to maintain a constant column flow of 1 ml / min. the transfer line of the mass selective detector was held at 300c and was operated in selected ion monitoring mode for analysis. perdeuterated surrogate recoveries for naphthalene d-8 from each of the devices ranged from 39% to 69% with the procedural control having a recovery of 80%. recovery of acenaphthene d-10 ranged from 42% to 74% with the procedural control having a recovery of 74%. the recovery for chrysene d-12 ranged from 85% to 99% with the procedural control having a recovery of 103%. the recovery for perylene d-12 ranged from 65% to 71% with the procedural control having a recovery of 82%. the linear uptake model was used to derive water concentrations for each pool using the measured amount of analyte recovered from a specific device, field data, and a laboratory - derived sampling rate for that analyte. the following equation was used to calculate the water concentrations of specific analytes in this study, and a full description of the model and the derivation of this equation are described elsewhere [32, 3437 ] (1)cw = npsdrst. in this equation, cw is the concentration of the contaminant or analyte in the water (ng / l), npsd is the amount of analyte sorbed by the device (ng), rs is the laboratory derived sampling rate (l / d), and t is the length of the deployment (d). in order to account for differences between the configuration of the devices used in this study compared to those used for the laboratory derived sampling rates, these rates were adjusted by a factor of 0.833. to assess vehicular traffic density at each site, gps locations over the engine compartment of every car parked in the vicinity of the atolladora, aroma, and tamarindo sites were taken three times per day with data collection not occurring at an interval shorter than two hours from the commencement of the previous data collection. the number of cars parked within a 100 meter buffer zone of the estimated center of each site was measured. the estimated center of each buffer zone was based upon the central location of the largest breeding pool present at each site. the gps unit used to collect data for assessment of parking density was the garmin gpsmap 60csx model. arcgis software was used to perform the analysis of all traffic data collected. to assess the risk associated with the analytes measured at each site, risk quotients were created for each analyte by calculating a ratio of the average water concentration at each site to the published water quality criteria. water quality criteria were obtained from several sources including the epa 's national recommended water quality criteria, canadian water quality guidelines for the protection of aquatic life, british columbia water quality guidelines, toxicological benchmarks for aquatic biota from the us, department of energy, and toxicity studies from the primary literature. in the event that multiple water quality criteria were published for a single analyte the traffic analysis revealed that the average number of cars parked within a 100-meter buffer zone from the center of the tamarindo south site was 17.56 cars. this is almost double the number of cars recorded for the tamarindo north and atolladora sites, which averaged at 9.29 and 9.13 cars, respectively. it is important to note that the tamarindo south site and location of its breeding pool was directly accessible and frequently used for vehicular traffic and parking. vehicular access was not permitted at the tamarindo north and atolladora sites ; however, vehicles did use adjacent parking and roadways. the average derived water concentrations for analytes present within the ephemeral pools of each breeding site biphenyl, c1-naphthalenes, fluorene, c1-c3 dibenzothiophenes, phenanthrene, anthracene, 1-methylphenanthrene, c1-c3 phenanthrenes / anthracenes, fluoranthene, pyrene, c1-fluoranthenes / pyrenes, retene, benz[a]anthracene, chrysene, c1-c3 chrysenes, and benzo[e]pyrene were all found to be in higher concentrations than combined field blank and procedural blank concentrations for the tamarindo south site. naphthalene, biphenyl, 2,3,5-trimethylnaphthalene, c2 naphthalene, c1 - 2 phenanthrenes / anthracenes, fluoranthene, pyrene, and retene were all found in higher concentrations than background levels at the tamarindo north site. phenanthrene, 1-methylphenanthrene, c1 - 2 phenanthrenes / anthracenes, fluoranthene, pyrene, c1-fluoranthenes / pyrenes, and retene were found to be above background levels at the atolladora site. the tamarindo south site had a more diverse population of pah analytes present within its breeding pool. 22 out of the 48 pah analytes examined were found to be above background levels at the tamarindo south site. only 9 and 8 of the pah analytes were found to be above background levels at the tamarindo north and atlladora sites, respectively. the tamarindo south site also had higher concentrations of almost every analyte measured above background levels compared to the other two sites with the exclusion of naphthalene, biphenyl, c-2 naphthalenes, and 2,3,4-trimethylnaphthalene. these 4 analytes were found in slightly higher concentrations at the tamarindo north site. the overall risk as indicated by the risk quotients depicted in table 2 is negligible for most of the analytes measured. chrysene, benz[a]anthracene, and pyrene at the tamarindo south site represent those analytes with the highest risk with quotients of 0.226, 0.022, and 0.021, respectively. as indicated by the calculated quotients, the risk associated with the current concentrations of pah analytes present in the breeding pools appears to be very low based on published water quality criteria. several of the published water quality criteria are based on standards for human consumption and do not necessarily reflect the potential exposure that submerged toad embryos or tadpoles may encounter. criteria designed for human consumption may not reflect the unique physiologic adaptations specific to amphibians that could alter the sensitivity, metabolism, and ultimate effect of analytes in this class of animal. additionally, several analytes present within these breeding pools had no published water quality criteria data, and risk quotients could not be calculated for these analytes. this study was conducted to assess the bioavailable concentrations of pah analytes present within three breeding pools utilized by puerto rican crested toads at sites used for direct vehicular parking compared to those that are not. a more diverse population of pah analytes were found in higher concentrations within the tamarindo south breeding pool, which is associated with higher levels of vehicular activity than the other two sites. risk analysis for each site indicated low - risk quotients for the current concentrations of pah analytes found at all three sites based on published water quality criteria. interpretation of the risk analysis is confounded by the use of several water quality criteria based on human consumption standards as well as a lack of published criteria for some of the analytes found within the breeding pools. this study was limited to the assessment of pah analyte concentrations and does not preclude the possibility that other organic or inorganic contaminants may be affecting toad reproduction at these sites. | habitat preservation and management may play an important role in the conservation of the puerto rican crested toad, peltophryne lemur, due to this species ' small geographic range and declining native wild population. bioavailable water concentrations of polycyclic aromatic hydrocarbon (pah) contaminants within breeding pools at 3 sites were established using passive sampling devices (psds) and gas chromatography - mass spectrometry (gc / ms). a more diverse population of pah analytes were found in higher concentrations at the breeding site that allowed direct vehicular access, but calculated risk quotients indicated low risk to toad reproduction associated with the current pah analyte levels. |
over the past two decades, it has become increasingly common for people to travel overseas in order to access medical procedures and services, including fertility treatment. people travel to receive fertility services for a broad range of push and pull reasons. some travel to receive procedures that are illegal or unavailable to them at home ; others are in search of better care, shorter waiting times, greater privacy or lower costs. the united states, for example, is a destination for some reproductive travellers, including europeans and australians seeking surrogacy or sex selection. at the same time, us citizens have travelled to india and mexico in order to access cheaper surrogacy arrangements. diasporic travellers might travel to their country of origin in order to have their cultural or religious needs met, or to access ethnically matched donors. for example, thailand banned foreigners from accessing surrogacy in 2015, and india restricted surrogacy to heterosexual married couples in 2013 and is in the process of implementing a ban on all foreigners, except those of indian descent. nepal and mexico initially appeared to be alternative low - cost destinations, before restrictions upon foreigners access to surrogacy were enacted in 2015 and 2016, respectively. it is impossible to tell how many people travel in order to access fertility services. in 2010, shenfield estimated that there were about 2430,000 cycles of cross border fertility treatment within europe each year, involving 1114,000 patients. there is, however, no systematic collection of data, so the prevalence and outcomes of reproductive travel are unknown. while the picture of cross - border reproduction (cbr) is simultaneously incomplete and extraordinarily complicated, one certainty is that states can no longer assume that their citizens will seek fertility treatment and services in local, easily - regulated clinics. every country therefore faces the question of what the appropriate regulatory response, if any, should be to this dynamic and widespread (but ultimately unquantifiable) bypassing of domestic healthcare services and regulations. our focus in this article is on domestic responses to cbr, but it is also worth noting that there is increasing interest in the development of cross - national minimum standards. these might take the form of a hague convention on surrogacy, in order both to protect the surrogate s welfare and to avoid the creation of stateless and parentless children, or it might simply involve collaboration among members of the international federation of fertility societies in order to develop uniform clinical and safety standards. research emerging from the uk and australia suggests a number of common threads, which make them useful comparators when developing responses to cbr. in both the uk and australia, people who travel abroad for reproductive treatment may have multiple reasons for doing so, but as both countries provide high quality ivf treatment (with varying degrees of state subsidy) and can broadly be characterised as liberal in their access, our residents may be less likely to be motivated primarily by treatment exclusions or cost considerations. in the uk, in their qualitative study of 51 interviewees in 200910, culley found that those utilising cross - border treatment were motivated by avoiding long waiting lists ; the prospect of higher success rates abroad ; the hope of receiving treatment in a less stressful environment ; and by dissatisfaction with the treatment that they had received in the uk. in australia and new zealand, a survey study in 2014 of 137 respondents (105 from australia and 32 from new zealand) by rodino identified unavailability of treatment, treatment not permitted, long waiting lists for donor gametes and limited choice of donors as the most common motivations. in this article, we contribute to an emerging body of research into the experiences of reproductive travellers as part of our qualitative study examining cbr. in this project we are interviewing reproductive travellers from australia, as well as regulators, agencies and clinicians both within and outside australia, in order to better understand the motivations for, and experiences of cbr. at the time of writing, mid - way through a four year project, we had undertaken 54 interviews with 55 interviewees, of whom 28 had utilised cross border reproductive processes, as well as 11 facilitators and four medical practitioners engaged in cross border treatment. we are publishing these early findings in order to contribute to the development of a more nuanced analysis of cbr that explores both risks and benefits, attending to the perspectives of those who undertake it. although the research is not yet finalised, the themes that we discuss here are significant and have the potential to contribute to legal and policy developments currently being debated in australia, the uk, and elsewhere. the premise of this qualitative study, and of our argument here, is that regulators, clinicians, policymakers, and law - reformers can and should learn from the lived experiences of those who cross borders in search of reproductive treatments and services. we suggest that patients subjective experiences of risk, care, and legality differ markedly from the assumptions about patient behaviour that have tended to inform regulation in this area. we tease out this claim, and its implications for responsive regulation, across four preliminary observations. further, this division of practices is not experienced as meaningful by many participants in cbr, and is openly rejected by some. secondly, the status of the law in cbr is profoundly equivocal. even countries with extra - territorial criminal prohibitions against commercial surrogacy in practice often facilitate these prohibited arrangements through citizenship and parentage provisions. this ambiguity builds an experience of law as both there and not - there for reproductive travellers. in our interviews we have noticed that some people accessing cbr simultaneously acknowledge the presence of legal provisions (such as prohibitions in the criminal law ; rules regarding legal parentage, or the likely unenforceability of surrogacy or egg donation contracts), at the same time as regarding these as abstract technicalities. time and again our participants downplayed the significance of law, as compared with real life, or a well - trodden path, in which others before them, and they themselves, have returned home with children despite the existence of restrictive legal provisions, in australia and the country of treatment. thirdly, self - sourced information, from the internet and more specifically social media such as facebook, is now the principal source of information and peer support for reproductive travellers. clinics and agencies have their own facebook pages, and there are facebook groups for different cohorts of reproductive travellers : gay dads, egg donors, egg recipients, surrogate mothers, and intended parents. personal recommendations and anecdotal evidence about the quality of care appear to matter more to patients than doctors or regulators might expect. given that many patients self - refer with the help of internet forums and facebook, reproductive travellers may be bypassing what has conventionally been a crucial source of information and support when undergoing complicated, stressful and invasive medical treatment, namely local healthcare professionals. drawing on these observations, we suggest that finding a way to ensure that as many reproductive travellers as possible access accurate, balanced information before they depart in order to make an informed assessment of overseas (and domestic) treatment options should be an important, but by no means straightforward, regulatory objective. we need to learn more about why some citizens of countries that pride themselves upon having among the safest and the best regulated fertility services in the world are actively bypassing those services, and seeking out treatment in apparently less well - regulated environments, often on the basis of facebook recommendations. consumer choice in cbr, or that a reform agenda should be shaped to the expectations of those who utilise cbr, especially if there is clear evidence of harmful practices or outcomes. however, we do argue that the experiences of those who travel for reproduction offer important insights that complicate common assumptions about cbr. for instance, as we shall show below, some women found the idea of altruistic surrogacy or egg donation to be more morally problematic than compensated surrogacy and egg donation. commercial involvement in assisted reproduction. to inform a more nuanced approach to the provision and regulation of fertility treatment, we must attend to the subjective experience of risk, quality, and care in cbr, especially when this involves what angela campbell calls morally ambiguous or even ostensibly self - injurious choices. in this article, we suggest that it is impossible to properly evaluate the role of law in cbr without attending to its impact upon participants lived experiences, and that, in the light of a dramatic mismatch between law s goals and reproductive travellers experiences of law, there may be grounds for some form of realignment. in many countries, including the uk and australia, a sharp distinction between altruistic and commercial arrangements has shaped the legal response to surrogacy and assisted reproduction for the past 30 years. in australia, commercial surrogacy is a criminal offence for all participants, as is trading in human gametes. in the uk, professional involvement in commercial surrogacy, egg donors in the uk have been able to receive up to 750 per cycle of donation, to include all expenses, an amount which is not intended to incentivize donation, but which is instead, in the words of the then hfea chair, lisa jardine, a level of compensation which will not deter those interested in donation but will retain donors already in the system, without attracting those who are merely financially motivated. an assumption underpinning much of the reaction to cbr in the uk and australia is that travellers go to commercial jurisdictions in order to avoid, directly or by implication, the constraints of altruistic regimes ; that is to access a more immediate or wider range of reproductive contributors who are more plentiful (and who may also be less powerful negotiators) because they are motivated primarily or solely by financial gain. we suggest that this flat characterisation fails to take account of the realities of payment to whom and for what, in both domestic and cross - border arrangements. in contrast, a common theme among our interviewees is a rejection of the assumption that altruistic surrogacy is morally superior to commercial surrogacy because there are fewer financial incentives. indeed, it was noted that there are aspects of altruistic surrogacy that might be described as coercive. for example, beth had undergone a radical hysterectomy as part of her treatment for cervical cancer and, after an unsuccessful surrogacy arrangement in australia, she travelled to california for an arrangement involving an egg donor and a surrogate mother. beth found the assistance of the agency in california to be vital to the whole process. she was strongly critical of the australian system and did not accept that altruistic surrogacy was less coercive than the commercial arrangements available in california : yes, but you d have a bit of hinting going on, would nt you ? if it s altruistic here and it s in - family there s a lot of hinting ; aunties are talking to sisters, friends are saying would you do it for them, what about them, why do nt you help them, or whatever. in america she s just receiving a whole stack of applications, there s no previous connection. undoubtedly the role of the agency. their role is paramount, is vital, because not only does their reputation rest on this, they have to protect the surrogate before they protect the intended parents, because if the surrogate does nt have a good experience, the surrogate s going to tell other people. there are going to be other surrogates that are wishing to go through the process to be selected. even though you think in america they re paying for them, surely they ve got loads of women, oh no, it s only a certain breed of lady that does this. so it s not just a whole line of ladies waiting around the block, it s just a very small amount of certain special ladies. yes, but you d have a bit of hinting going on, would nt you ? if it s altruistic here and it s in - family there s a lot of hinting ; aunties are talking to sisters, friends are saying would you do it for them, what about them, why do nt you help them, or whatever. in america she s just receiving a whole stack of applications, there s no previous connection. undoubtedly the role of the agency. their role is paramount, is vital, because not only does their reputation rest on this, they have to protect the surrogate before they protect the intended parents, because if the surrogate does nt have a good experience, the surrogate s going to tell other people. there are going to be other surrogates that are wishing to go through the process to be selected. even though you think in america they re paying for them, surely they ve got loads of women, oh no, it s only a certain breed of lady that does this. so it s not just a whole line of ladies waiting around the block, it s just a very small amount of certain special ladies. the distinction between regimes, and practices, characterised as altruistic and those characterised as commercial is contestable both within and across different jurisdictions. altruistic, because under canadian law the surrogate can not be paid more than her expenses. nevertheless, australians travel to canada for surrogacy (and not, it appears, the other way around), and they do so in order to access paid brokering services, even though such services would be criminalised as commercial if they were operating within australia. incoherence is also present in australia s approach to the payment of egg donors, brokers involved in egg procurement and agencies who run egg so for example, eggs imported into australia from us - based services such as the world egg bank involve a cost of $ 20,000 usd to the patient for six eggs, comprising a payment of approximately $ 3000 usd to the egg donor and $ 17,000 in fees to intermediaries (described as administrative and transport costs). in comparison, one us - based broker we interviewed, who matched donors and recipients and organised treatment in eight overseas destinations, reported that the egg providers in her service were paid $ 1500 usd as a price, with $ 100 spending money and $ 50 per day meal allowance on top of their travel expenses, while an additional administrative fee of $ 4500 usd was charged by her to patients (leading to a total cost to patients of less than $ 10,000). bizarrely, the first of these examples is regarded as altruistic donation under australian law, while the latter is not. there is also, as will be discussed below, an illogical and widening gulf between the active domestic medical facilitation of overseas (compensated) egg donation and the prohibition of domestic medical involvement in overseas (commercial) surrogacy. in our cbr study, we have found that the historical stigma attached to the commercialisation of reproductive contributors is not shared by intended parents. for many participants, the lack of payment to the surrogate or egg donor in domestic arrangements was believed to be unfair to her, as she was then, effectively, the only volunteer surrounded by a number of professional participants including doctors, counsellors and lawyers all of whom were acting for profit. some altruistic surrogacy arrangements ended up with an overall cost to the parents that was roughly similar to the cost of a commercial arrangement overseas. so, for example, lachlan, an interviewee with two children born through surrogacy in australia, noted that the cost of their arrangements had been about $ 80,000 for the first child and $ 50,000 for the second child, all of which went to the medical and legal professions rather than to the women who had helped them : the whole debate in terms of commercial surrogacy arrangements, if it s ever spoken about as advocates against it, it s always the quotation from someone in the legal or medical profession because they re getting sizeable rents. you almost expect to pay double in terms of the process as well it would be cheaper for us to go to india, for argument s sake, than going here in australia everybody gets paid in this, apart from the women. yes and [our surrogate ] veronica had an old fridge and the seal was nt working, i just wanted to go out and buy a new fridge for the family. there was a decision then of okay, would that be considered a material item and will that be considered [commercial payment when we are ] going to the courts ? so we made a decision well no, we wo nt say anything.so that s a cause of frustration. the whole debate in terms of commercial surrogacy arrangements, if it s ever spoken about as advocates against it, it s always the quotation from someone in the legal or medical profession because they re getting sizeable rents. you almost expect to pay double in terms of the process as well it would be cheaper for us to go to india, for argument s sake, than going here in australia everybody gets paid in this, apart from the women. yes and [our surrogate ] veronica had an old fridge and the seal was nt working, i just wanted to go out and buy a new fridge for the family. there was a decision then of okay, would that be considered a material item and will that be considered [commercial payment when we are ] going to the courts ? like lachlan, lauren, another interviewee involved in a surrogacy arrangement in australia, expressed a desire to pay her surrogate, and an anxiety about the fuzzy definition of expenses in australia : i think also a con of the altruistic system in general is that a really sort of fuzzy line of what can and ca nt be considered a surrogacy expense. so you re always sort of worrying like oh am i breaking the law by reimbursing this. there s no real sort of set list of what you can and ca nt pay for and i think that causes anxiety for surrogates as well. i think also a con of the altruistic system in general is that a really sort of fuzzy line of what can and ca nt be considered a surrogacy expense. so you re always sort of worrying like oh am i breaking the law by reimbursing this. there s no real sort of set list of what you can and ca nt pay for and i think that causes anxiety for surrogates as well. lauren also expressed a real sense of discontent about not being able to compensate her surrogate, saying:[t]here s such an inequality for giving. i find that for someone to give their body for the sake of creating another family is just i ca nt think of any greater gesture and so to not be able to return that in some way sort of makes me feel, what s the word, inadequate in a way i guess. i ll find other ways to be giving, giving with my heart, giving with my friendship and giving with my love. but if i could i d just i d give everything but yep not allowed. i would nt want to see it turn into a commercial operation but i would love a token amount just to sort of absorb some of those costs that surrogates are nt comfortable with sharing with their intended parents but also just to lighten the load on them a little bit. maybe $ 10,000 or $ 20,000 like not a huge amount of money just not as an incentive to do it but just to kind of sorry my brain s just gone dead. not to attract people for commercial reasons but more just to lighten the load on the families a little bit. so i d really love to see that happen [t]here s such an inequality for giving. i find that for someone to give their body for the sake of creating another family is just i ca nt think of any greater gesture and so to not be able to return that in some way sort of makes me feel, what s the word, inadequate in a way i guess. i ll find other ways to be giving, giving with my heart, giving with my friendship and giving with my love. but if i could i d just i d give everything but yep not allowed. i would nt want to see it turn into a commercial operation but i would love a token amount just to sort of absorb some of those costs that surrogates are nt comfortable with sharing with their intended parents but also just to lighten the load on them a little bit. maybe $ 10,000 or $ 20,000 like not a huge amount of money just not as an incentive to do it but just to kind of sorry my brain s just gone dead. not to attract people for commercial reasons but more just to lighten the load on the families a little bit. so i d really love to see that happen lauren s preference for modest compensation, which would not be enough to attract people for commercial reasons is consistent with the findings of a recent uk survey of surrogates and intended parents, which found that the mean average of 10,00015,000 represents compensation, not payment. indeed, there is evidence that this level of compensatory payment is simply waved through in the magistrates courts in the uk (which deal with parental orders for uk surrogacy), without any need for it to refer to itemized expenses. for many of our participants, being able to pay surrogates modest compensation was fairer and hence more morally satisfactory for them than asking another woman to carry a pregnancy without receiving any compensation for her time and inconvenience. for example, cybil s three - year - old son was born with the help of traditional surrogacy in western australia. cybil explained that she would have preferred to be able to pay her child s surrogate. her central rationale was that payment would create clearer boundaries, in contrast to the current system in which the definition of reasonable expenses is unclear : yeah, it would be much clearer for everyone what the boundaries are. i know that because you ca nt offer not only compensation but even gifts, like technically you ca nt even give them a bunch of flowers. i know that because you ca nt offer not only compensation but even gifts, like technically you ca nt even give them a bunch of flowers. some participants were also clear that they valued the service provision of commercial providers, not only to themselves, but also to the surrogate or egg donor. gerry, who had used a surrogacy agency in canada said : i think i just really liked the way i think we have covered this off before as well, but the agency is very respectful to the surrogate in what they call the fourth trimester, meaning, dealing with her effectively and caring in a caring way about the fact that how s she s going to feel post separation after the birth. i think for us, it s really important to have a sense that we re doing the right thing and that we re not exploiting anyone. i think i just really liked the way i think we have covered this off before as well, but the agency is very respectful to the surrogate in what they call the fourth trimester, meaning, dealing with her effectively and caring in a caring way about the fact that how s she s going to feel post separation after the birth. i think for us, it s really important to have a sense that we re doing the right thing and that we re not exploiting anyone. one of the canadian agencies we spoke to assigned a full - time support worker to each surrogate and a different employee to support each set of intended parents. the agent, sally, explained that these workers played an invaluable role in resolving issues and ensuring that disputes did not arise in the course of the relationship between the parties. in contrast, some of the intended parents who undertook unpaid surrogacy within australia felt that, after the clinic s initial counselling session, they were left on their own. for example, lachlan describes the limited service provided by the australian clinic that he and his wife attended : canberra was a funny situation where the hospital that runs the clinic, there was one lady there involved and she only dealt with surrogacy on tuesday. so if you called up on monday she would nt respond until the tuesday. if you sent an email on the wednesday you had to wait the whole week until the tuesday. as a result, it was very hard to get hold of her because there were a number of parents obviously wanting to get stuff and only tuesday. so that became in itself but you say to yourself okay, i need to call her tomorrow, type thing. canberra was a funny situation where the hospital that runs the clinic, there was one lady there involved and she only dealt with surrogacy on tuesday. so if you called up on monday she would nt respond until the tuesday. if you sent an email on the wednesday you had to wait the whole week until the tuesday. as a result, it was very hard to get hold of her because there were a number of parents obviously wanting to get stuff and only tuesday. so that became in itself but you say to yourself okay, i need to call her tomorrow, type thing. while of course not all offshore providers necessarily commit much, or any, of their commercial fee into the level of service provision offered by sally s canadian agency (and many of our participants undertook egg donation and surrogacy abroad with no preparatory or follow up counselling), we draw on this contrast between a canadian agency and an australian clinic to illustrate that it is not the fee itself which determines whether practices are fair and non - exploitative yet, the legality of surrogacy arrangements are determined solely by reference to this factor. a more responsive approach for law would be to ask : what practices are beneficial and how might they be facilitated (and perhaps also paid for) by a regime that seeks to avoid improper inducement or impaired consent ? in the uk s altruistic only system, it is an offence for anyone other than the surrogate and the intended parents to negotiate a surrogacy arrangement on a commercial basis, and it is a criminal offence for intended parents, surrogates and agencies to advertise their willingness to participate in or facilitate surrogacy. as a result, as mcfarlane j explained in re g (surrogacy : foreign domicile), the role of facilitating surrogacy arrangements has traditionally been left to groups of well - meaning amateurs. if the mischief to which the ban on commercial involvement is directed is the prevention of exploitation, the evidence is by no means clear that this is best achieved by discouraging professional agencies involvement in surrogacy. on the contrary, as natalie gamble has explained : the offer of payment does not necessarily preclude an informed choice ; and nor does the absence of payment ensure it. a much more sophisticated approach is to require regulated intermediaries to ensure that surrogates and parents are given good quality information about the risks and offered counselling to reflect on the long term commitment involved before they proceed. the offer of payment does not necessarily preclude an informed choice ; and nor does the absence of payment ensure it. a much more sophisticated approach is to require regulated intermediaries to ensure that surrogates and parents are given good quality information about the risks and offered counselling to reflect on the long term commitment involved before they proceed. if prohibitions on commercial surrogacy can be readily avoided by buying an airline ticket, with, in practice, few or no penalties for doing so and active facilitation of parenthood by the courts and immigration services, what is the status of those prohibitions ? in the uk, the law has prevented the development of commercial surrogacy brokers, but it does not treat intended parents who engage in commercial surrogacy as criminals. australia s prohibitions on the payment of gamete providers and surrogates are stricter, and in addition three australian states and territories criminalise participation in commercial surrogacy even if it occurs elsewhere. the national health and medical research council s guidelines, which apply to all fertility practitioners in australia, further state that it is ethically unacceptable to undertake or facilitate surrogate pregnancy for commercial purposes. yet prosecution, disciplinary or licensing action in response to breach or evasion of these prohibitions has been extremely rare, and uniformly unsuccessful. some of our interviewees from australian jurisdictions where criminal prohibitions on commercial surrogacy have extra - territorial effect took a calculated gamble. they understood that they were breaking the law but believed that, because so many other families had not been punished or detected, that they too would be unaffected : one is i knew that technically by the law of new south wales, we were breaking that law. [another parent ] kind of put my feelings in that regard at ease in saying " well, if they arrest you for it, they re going to arrest hundreds of other people who have done exactly the same thing that you re thinking of doing ", which made me feel better about being more open about it. one is i knew that technically by the law of new south wales, we were breaking that law. [another parent ] kind of put my feelings in that regard at ease in saying " well, if they arrest you for it, they re going to arrest hundreds of other people who have done exactly the same thing that you re thinking of doing ", which made me feel better about being more open about it. others who were more concerned about breaking the law undertook a variety of evasion strategies : some sought out surrogacy in canada on the basis that it too was seen as an altruistic jurisdiction ; others moved to a different australian state, like victoria, from where it is not illegal to travel for commercial surrogacy ; some simply falsified documentation in order to appear that they had done so. dian was born without a uterus and, after a negative experience in india that we come back to later, had had a child through a commercial surrogacy arrangement in the usa. she and her husband wayne were very concerned about the new south wales ban on overseas commercial surrogacy when they returned from the usa with their daughter. at the time of the interview a year later wayne was still worried that they had acted against the law, but dian was becoming less anxious : well, we did nt want to do something that broke the law. both [our sets of our ] parents did nt know anything about it. [we ] kept them from that ; because of that reason as well, we did nt want to worry them. wayne s brother i do nt know whether he s currently a lawyer, but he was a lawyer at one stage, and he knew what we were doing. he was extremely worried for us, but supportive, like he did nt say do nt do it. i guess i did nt want to implicate i did nt want to implicate other people. [we ] kept them from that ; because of that reason as well, we did nt want to worry them. wayne s brother i do nt know whether he s currently a lawyer, but he was a lawyer at one stage, and he knew what we were doing. he was extremely worried for us, but supportive, like he did nt say do nt do it. i guess i did nt want to implicate i did nt want to implicate other people. participants also experienced the law of the treating country as highly ambiguous. in some respects, this is understandable, as some of the jurisdictions in which international commercial surrogacy have flourished in recent years are precisely those with a lack of clear regulation. however, we argue that this went further, to actually imbue the whole cbr process as one of law and not - law, often involving an active process of double - think. harry, a gay man who went to thailand with his partner and undertook two surrogacy arrangements simultaneously with embryos created from the same egg donor and each man s sperm, said : i mean thailand did nt have specific laws at the time. we were certainly aware that there [were ] draft laws on the table, but at the time they did nt have those laws. however through just common practice, we actually felt that the laws were quite protective of us, of doing surrogacy. we were certainly aware that there [were ] draft laws on the table, but at the time they did nt have those laws. however through just common practice, we actually felt that the laws were quite protective of us, of doing surrogacy. here, harry is speaking of then - current thai laws, in which the surrogate (and her husband, if any) were the legal parents of any child born ; a male genetic parent not married to the mother could generally not apply for custody before the child was seven years old, and any form of payment for surrogacy was unlawful. quite protective of him as an intended parent, who had no genetic relationship to one of the children, in comparison with the draft laws, passed in haste in 2015, which expressly criminalised all paid surrogacy, and limited unpaid surrogacy to domestic arrangements involving relatives of the surrogate. protected by contracts which provided that surrogates were obligated to surrender babies to them, in contrast to domestic arrangements where she might change her mind and decide to keep it. yet, when pressed, they would acknowledge that such a contract was not likely to be enforceable. as harry explained : no, we never had a lawyer take us through contracts. so the contracts were, you know, we went through the contracts with the agent and they were in english and in thai but again, i do nt think there would be much to be gained by going through that with a lawyer because i m not sure we were happy with what was actually written in the contract, but some of it i think would nt actually be legally binding if it was actually tested in court and things like that, because of the fact that it was quite a legal grey area i think you needed to i think the important thing for intended parents is to understand what environment they were operating in, but i think because there was nt really explicit laws to refer to, that s why i think engaging with lawyers either here or there was less useful. so the contracts were, you know, we went through the contracts with the agent and they were in english and in thai but again, i do nt think there would be much to be gained by going through that with a lawyer because i m not sure we were happy with what was actually written in the contract, but some of it i think would nt actually be legally binding if it was actually tested in court and things like that, because of the fact that it was quite a legal grey area i think you needed to i think the important thing for intended parents is to understand what environment they were operating in, but i think because there was nt really explicit laws to refer to, that s why i think engaging with lawyers either here or there was less useful. likewise tom who undertook surrogacy as a single gay man in india said of the contract he signed : look, i was following what people were saying about contracts and to get the contract looked at i think some of the advice that people have shared was it costs a lot of money to have your contracts looked at and it s not actually legal or legally viable in australia anyway. so i did nt actually then i decided not to seek legal advice because i just thought it was almost pointless. look, i was following what people were saying about contracts and to get the contract looked at i think some of the advice that people have shared was it costs a lot of money to have your contracts looked at and it s not actually legal or legally viable in australia anyway. so i did nt actually then i decided not to seek legal advice because i just thought it was almost pointless. essentially then, legal advice is almost pointless for an unviable contract, which nevertheless is capable of going some way to protect the rights and liabilities of the parties throughout the arrangement. one of the preconditions for the granting of a parental order to intended parents through surrogacy in both the uk and australia is that no payment should have been made other than expenses reasonably incurred. in the uk, the courts have the power to retrospectively authorise payments made in excess of reasonable expenses. because the child s welfare is the paramount consideration when deciding whether to make a parental order, the uk courts are effectively presented with a fait accompli : if the child s settled home is with the intended parents, a parental order will invariably be in his or her best interests. thus, uk courts routinely authorise payments made to the surrogate mother, even when they vastly exceed any plausible expenses. at the same time, the statutory prohibition on payments may discourage some intended parents, concerned about having their financial arrangements scrutinised, from applying for parental orders. the legislation therefore fails to stop payments, while failing to provide legal certainty about what is and is not allowed, and also potentially deterring the acquisition of legal parenthood. in contrast, in australia there is no discretion in the surrogacy parentage transfer regimes controlled by state and territory courts. thus intended parents in commercial arrangements are not legal parents, and can never have parentage transferred. excluded from this process, parents can nevertheless approach the federal family court seeking parental responsibility orders (a lesser form of recognition than legal parentage) and, in a handful of these cases, the court has also perversely, this means that for some male genetic parents in commercial surrogacy their legal parentage has been secured more readily than it has been for intended parents in domestic unpaid arrangements. to add to the confusion, other judges of the court have refuted this approach as a misreading of the legislation, leading to an as yet unresolved judicial lottery. in a similar vein, in both the uk and australia, as with canada and many other altruistic jurisdictions, the state has actively acquiesced in facilitating the acquisition of citizenship for children born as a result of overseas commercial surrogacy arrangements. this is the case in australia even when the intended parents are resident in a state in which they are subject to extra - territorial criminalisation. in a parallel vein, potentially unlawful payments by australian parents to egg providers abroad are not examined as part of state processes granting citizenship or parental responsibility in surrogacy arrangements. gabriel, a gay man who undertook surrogacy in mexico said : it s obvious the australian government s allowing it to happen. if they started doing that then they re really saying, no you re not allowed to do it at all because it s illegal. but the government s not doing that, they re letting it happen. if they started doing that then they re really saying, no you re not allowed to do it at all because it s illegal. but the government s not doing that, they re letting it happen. in this way, as discussed later, intended parents understood themselves as both legal parents and not - legal parents under australian law : their children were granted passports and in practice they could use their overseas birth certificates, at the same time as those birth certificates do not record parentage for the purposes of australian law and their parental relationship had not been legally formalised. if it is possible to establish a working parental relationship in the absence of a parental order, many struggle to see the point of going through yet more expensive red - tape. indeed, some australian facilitators, such as alec, actively discouraged parents from doing so because, unlike the uk, the result was not guaranteed:if we had a system here where it was a guaranteed process and a simple process, of course i 'd be recommending everybody get them, but 95 per cent [do nt ] get them, because we get by without it. why would we want to spend a year and a half in the court system and spend $ 30,000 on this stuff ? if we had a system here where it was a guaranteed process and a simple process, of course i 'd be recommending everybody get them, but 95 per cent [do nt ] get them, because we get by without it. why would we want to spend a year and a half in the court system and spend $ 30,000 on this stuff ? the practical consequence of people bypassing the formal transfer of parenthood may be that the courts have to step in at a later point, perhaps following parental separation or death, in order to resolve some of the difficulties that may arise when a child s social parents are not also her legal parents. in practice, de facto tolerance of the evasion of reproductive travellers may be inevitable ; as one of culley s respondents put it : what are you going to do, confiscate their passports? but whether characterised as a pluralistic safety value or as out - and - out hypocrisy, there is at the very least a mixed message being sent about the status of the extra - territorial prohibition of international commercial surrogacy. there is considerable evidence that the internet is by far the most important source of information for reproductive travellers, and that healthcare professionals in their home country are very rarely people s primary source of information and advice. in the uk, hanefeld at al interviewed 77 outbound patients and found that most had identified a specific clinic or provider through facilitators in the uk or online forums. in australia, hammarberg s study found that those travelling abroad for surrogacy sourced most of their information online and from other parents through surrogacy. our preliminary findings strongly reflect this trend, with patients actively amassing information from a variety of internet sources. the quality and accuracy of online material is decidedly variable, and much of it is unverifiable, yet our participants regularly referred to this process as research. potential travellers visit clinics websites, but more significantly, seek advice and information from online forums, social media, and several layers of intermediaries variously described as facilitators, brokers, consultants and agencies. many of these are run by people who have previously undergone fertility treatment abroad, who go on to set - up businesses through which they can share, and make a living from, their personal experience and knowledge of overseas fertility services. in our study, potential reproductive travellers had sought peer - to - peer information, advice and support from fellow members of internet forums and facebook groups. this often involves users requesting information about other people s experiences at specific overseas clinics or agencies, which fellow forum - users will answer. as tom they were really popular in terms of people communicating and providing stories and getting updated information and asking questions, in mostly a respectful manner most of the time so it was really good well, now i guess the facebook groups have taken over the yahoo groups. some of the facebook groups are country - specific, so there s one or two for nepal, or there used to be one for thailand they were really popular in terms of people communicating and providing stories and getting updated information and asking questions, in mostly a respectful manner most of the time so it was really good well, now i guess the facebook groups have taken over the yahoo groups. some of the facebook groups are country - specific, so there s one or two for nepal, or there used to be one for thailand once people have decided to travel abroad for treatment, they often rely upon personal recommendations in order to select a particular clinic. in our cbr study, olivia chose a us - based clinic for egg donation based upon the recommendation of a real life friend : because obviously going to another country i had absolutely no idea. you do nt know whether the websites are actually legitimate, i mean there is so much fraud online and the amount of money that you re talking about with doing ivf overseas. even having a skype conversation or a telephone conversation you ve got no guarantee. so i felt i would not have gone overseas unless i knew someone who had been at that clinic. you do nt know whether the websites are actually legitimate, i mean there is so much fraud online and the amount of money that you re talking about with doing ivf overseas. even having a skype conversation or a telephone conversation you ve got no guarantee. so i felt i would not have gone overseas unless i knew someone who had been at that clinic. in one respect, olivia s experience is unusual : while all of our interviewees had, like olivia, relied upon personal recommendations, most of these had been from cyber friends. cheryl, who travelled to india for surrogacy noted that all of these clinics, american, indian, greek, they ve all got slick websites and you they all sound amazing. cheryl said that what tipped the balance for her was a very honest forum, even though it was hosted by the clinic itself : i did an awful lot of research. you get a very, very honest account of the experiences that lots and lots and lots of people had had using this clinic. you could send them private messages, so being able to message them and say i m from australia as well and i ve got a question about this that was something that made me well it tipped my decision in their favour. you get a very, very honest account of the experiences that lots and lots and lots of people had had using this clinic. you could send them private messages, so being able to message them and say i m from australia as well and i ve got a question about this that was something that made me the central role played by informal online networks and patient testimonies highlights, as hanefeld put it, the importance of hard information to patients. the information that patients are seeking online thus appears to be qualitatively different from what doctors and regulators might expect. success rates and professional accreditations matter, but patients are often more interested in what treatment will feel like : the attitude of staff, the clinic environment, the gut feelings other patients had had about their treatment. whether it feels safe to be treated somewhere is not the result of evaluating technical data from the clinic or local regulator, but comes instead from personal recommendations and first person narratives, largely gathered online. you get on the websites and you get on the blogs and all that sort of stuff and that provides you with far more of an education, be it right information or not far more education and reassurance than any doctor s given me ; because you re talking to other women that have been there, done that [y]ou do your own research. you get on the websites and you get on the blogs and all that sort of stuff and that provides you with far more of an education, be it right information or not far more education and reassurance than any doctor s given me ; because you re talking to other women that have been there, done that although the internet is the primary source of soft information about the experience of fertility treatment, it is also worth noting the growing popularity of fertility and surrogacy fairs or roadshows, which are huge exhibitions in which visitors can gather information from regulated clinics and the local regulator, while also meeting people from overseas clinics and alternative therapists. discussions about one s need for fertility services have emerged from the strict confidentiality of the doctor - patient relationship to become instead a marketing opportunity for private providers. but while these fairs provide clinics with the chance to sell their services, potential patients also value the opportunity to find out if they feel a connection with the clinic staff and their approach. social media websites like facebook also play an ambiguous role as sources of information and support. facebook friends with an overseas agent or clinic, and gain considerable reassurance from the posts of other patients or clients. the distinction between peer support and viral marketing from commercial brokers in such a setting is not always clear. in the context of travel for cosmetic surgery, holliday have pointed out that, to agents, facebook is an important marketing tool, whereas patients did not recognise (or refused) the marketing definition of agents facebook pages and saw them instead as open forums for discussion. the familiarity of facebook allows it to be both a source of peer support and authentic advice (from the point of view of patients), and a staggeringly successful (because invisible) advertising mechanism for agents. in our study, there were also instances where facebook was used by intended parents to breach the privacy of egg donors or surrogates, for example to identify and approach these women privately, or to seek and store information about them without permission. umar and gabriel were at the beginning of a surrogacy and egg donor arrangement in mexico. although egg donation is anonymous in mexico, umar had found their egg donor s facebook profile because the clinic had given them the egg donor s name, without her knowledge. gabriel said : yeah, umar has already cut out the pictures and cropped them. like i said we ll probably inform the egg donor or just message her on facebook to say thank you and this is what you ve done for us. like i said we ll probably inform the egg donor or just message her on facebook to say thank you and this is what you ve done for us. one response to the issues raised by overseas travel is to try to educate people about the implications of undertaking cbr, by providing information about clinical standards of care and the legal status of children born from such arrangements. counsellors and patient support group representatives interviewed by culley in the uk, for example, thought the only feasible response to reproductive travel is to educate people, and ensure that they go into it with their eyes open and fully aware of the implications. yet, it is hard to intervene in order to provide high quality information when people self - refer to overseas clinics, on the basis of facebook recommendations. in the uk, considering fertility treatment abroad : issues and risks. this politely suggests a number of issues that people should proven record on quality and standards. there is, of course, no guarantee that anyone contemplating treatment outside the uk will read this page, or follow its advice. in the australian system with seven jurisdictions and only two official regulators, varta and the wa reproductive technology council, official information is even less widely available than in the uk, leaving australians even less able to access accurate and reliable advice. there is also a mismatch between the view that healthcare professionals have a key role to play in educating people about possible risks, and the fact that healthcare professionals are seldom the first port of call for information. if people seek out information via google, facebook, and internet chatrooms, there may be little opportunity for clinicians to educate them about risks and potential pitfalls, a problem which is, as we see in the following section, exacerbated by domestic laws which criminalise cbr, and hence deter patients from incriminating themselves in front of healthcare professionals. the reordering of sources of information about fertility treatment from the medical profession to the internet is significant. it suggests that patients are increasingly willing to bypass local healthcare professionals in order to take matters into their own hands. even if doctors are still trusted sources of information, there are other factors which make the internet an attractive source of information. when people are seeking out information about treatments that may be unlawful at home, or which are stigmatised restrictive legal provisions then actively contribute to the bypassing of medical professionals, concerned about their professional registration, as a source of advice and support. shenfield note in the eshre good practice guide to cross border reproductive care that, [c]ollaboration between the home practitioner and the receiving center offers the best chance of optimal care for the cross border patient, but add that this may pose a problem where it is forbidden for doctors to give information about alternatives that are not legal in the country of residence of the patient. indeed, laws which inhibit doctors from offering advice and assistance to patients who are contemplating fertility treatment overseas create a professional conflict of interest in which doctors must choose between making the care of their patient their first concern, which would militate in favour of providing advice and support, and not being seen to endorse or support illegal behaviour, which might instead prompt them to leave patients to their own devices in hammarberg s survey study of australians travelling abroad for surrogacy, fewer than half of the 249 intended parents who responded had sought information from australian ivf professionals and of those who did, around one - third reported a negative reaction. cheryl said : the main ivf doctor that i saw here in sydney was very against offshore surrogacy. oh that s terrible. these are women that are terribly exploited and you ll go over there and you ll get a disease and you ll be in some terrible baby factory and what not. anyway, she said those things and then i just shut down that dialogue with her. the main ivf doctor that i saw here in sydney was very against offshore surrogacy. these are women that are terribly exploited and you ll go over there and you ll get a disease and you ll be in some terrible baby factory and what not. anyway, she said those things and then i just shut down that dialogue with her. some reported that fertility doctors were unwilling to provide any form of information at all. dian, for instance, said : then [my partner ] wayne mentioned the word the phrase it s illegal, its basically, the door was shut at that point here in australia. facilitating commercial surrogacy has prevented fertility experts in australia from engaging in even basic information - giving to their patients, such as what is involved in safe egg stimulation and embryo transfer protocols, or the risks of departing from these protocols. it has also prevented the provision of basic fertility testing or preparatory care (such as checking hormone levels, ovarian reserve, or sperm counts) for patients who are planning to undertake treatment abroad. for dian this meant that she had travelled to india twice and undergone egg retrieval, despite the fact that a simple hormone test could have told her in advance that ivf using her own eggs would be unlikely to work.so i got there by myself. the second or third day, while i maybe the second day after arriving in new delhi i had a consultation with dr i (india). she said that my [hormone ] level was too high ; too high or too low, i ca nt remember, but as far as she s concerned it would just be a complete waste of time to do an ivf treatment on me.so i was quite devastated, but then she said that we should just go ahead with an egg donor. at this stage [wayne ] was nt there, and she wanted me to make the decision right there and then. so i got there by myself. the second or third day, while i maybe the second day after arriving in new delhi she said that my [hormone ] level was too high ; too high or too low, i ca nt remember, but as far as she s concerned it would just be a complete waste of time to do an ivf treatment on me. so i was quite devastated, but then she said that we should just go ahead with an egg donor. at this stage [wayne ] was nt there, and she wanted me to make the decision right there and then. both clinically and emotionally, we suggest that this was an adverse experience that could have been avoided if dian had undergone basic preliminary investigations and preparatory care at home, before travelling to india. professionals within australia have also expressed the concern that media coverage of cross border surrogacy, coupled with difficulties in accessing frank advice from domestic healthcare professionals once commercial surrogacy is mentioned, has meant that some women are travelling abroad for surrogacy as a fertility cure, when they are, in fact, capable of carrying a pregnancy. in contrast to surrogacy, the australian prohibitions on commercial trading in gametes are far more specifically worded, criminalising only the giving and receipt of valuable consideration, rather than potentially implicating anyone involved in facilitating the practice. this has meant that some fertility doctors are willing to recommend overseas egg donation and we found that some even facilitate shared care, with the provision of scans and tests domestically before the woman travels, as well as follow up care. thus, if dian had told her fertility doctor that she was travelling to receive paid egg donation rather than to pursue commercial surrogacy, she would be likely to have received domestic medical assistance and advice. in a parallel vein, general practitioners (who are not covered by the same ethical guidelines as fertility specialists) were reported by our interviewees to be assisting patients with blood and semen tests in preparation for overseas surrogacy, as well as with prescription medications and blood tests in advance and pregnancy tests post - travel for those receiving egg donation abroad. after years of unsuccessful ivf in australia, leah travelled to greece to undergo ivf and egg donation. she explained that her gp in australia was helping her : so how we coordinate it is that he tells me what i need. so i just have to make sure that he s written down for me the correct spelling of the medication that i need, what it s for. then i just tell him a little bit about the background to why we re doing it and then he ll write me an australian script for it, so that s how we re coordinating it at the moment. so i just have to make sure that he s written down for me the correct spelling of the medication that i need, what it s for. then i just tell him a little bit about the background to why we re doing it and then he ll write me an australian script for it, so that s how we re coordinating it at the moment. the implications of this finding need further exploration, but at a minimum suggest that access to local medical care and advice for reproductive travellers is often filtered through general practitioners rather than ivf clinicians, and is more effectively obtained for australian women who are seeking egg donation compared to those seeking surrogacy. it would be possible to regard reproductive travel as an aberration, relied upon in extremis by people who are prevented, either by law or de facto, from accessing reproductive services at home. in response to the increasing numbers of people travelling for reproductive purposes, enabling more people to access local fertility services but while we would support measures to improve access to services, not least because these might also meet the needs of those who can not afford to travel, we would like to suggest that we should also be interested in what local fertility providers and regulators can learn from the experiences of reproductive travellers. as discussed in this article, our preliminary fieldwork has thrown up four themes : that the legal distinction between altruistic and commercial gamete donation and surrogacy is increasingly unsustainable ; that role of the law in cbr is profoundly equivocal ; that facebook is now the principal source of information and peer support for reproductive travellers and lastly, that domestic reproductive service providers are often sidestepped. each theme suggests that the cross - border reproductive traveller does not conform to regulators assumptions about patient behaviour. implicit here is the premise that the law will best protect patients through discouraging international travel. here, the patient experience diverges. it is clear that patients paying for treatment overseas feel as though they are more in control of their treatment, and that, in contrast to their experience of domestic fertility services, they do not have to be grateful for what they receive. indeed, in many of our interviews patients have praised the standard of care they received overseas, considering it superior to that available at home. opting out of local, regulated services is not necessarily always an unwelcome last resort then, but may have positive advantages for some patients. if this is the case, we should be interested in listening to what patients say is overseas clinics may offer more support and more contact time with clinicians and nursing staff, as well as a greater choice of donors or surrogates. overseas clinics also make excellent use of social media to contact patients and to facilitate a high quality care experience. nicky hudson and lorraine culley, for example, found that treatment overseas gave their interviewees active involvement in deciding on treatment protocols, choice about donors, control over the timing of treatment and good access to the clinician leading their care. we should be interested in this apparent disjunction between what matters most to would - be parents and what matters most to doctors, regulators and legislators. in van hoof s study of internet forums, in which dutch patients shared their experiences of having received ivf treatment in belgium, respect for the person behind the patient was identified as the main reason for the patients belief that the quality of care was higher in belgium. van hoof comment that patient centred care is generally seen as a dimension of care that has nothing to do with effectiveness and efficiency, whereas forum users considered that the central position of the patient [was ] key for every dimension of good quality of care. this is a revealing illustration of the gap between the normal markers for success, as judged objectively, and the marker of high - quality care for patients, in which patient - centred care is not just a desirable extra, but is central to every aspect of what matters to them. for example, rosalind, who travelled to greece to use donated eggs observed : weve spent nearly 60,000 dollars in australia and not one of my doctors have called and said, hey how are you or let s do this now or anything like that.so that s why we chose him because he just took this real personal approach with us and it was him that was in contact with us. we ve spent nearly 60,000 dollars in australia and not one of my doctors have called and said, hey how are you or let s do this now or anything like that.so that s why we chose him because he just took this real personal approach with us and it was him that was in contact with us. clinicians, regulators and politicians have tended to assume that patients choose clinics on the basis of their success rates and the costs of treatment. like to be treated there with scepticism because, by definition, anecdotal evidence is not evidence at all. but while it may not be statistically significant, in practice, the anecdotal clearly matters to patients. patients want to receive treatment from caring clinicians, and they want to feel a connection with their donor or surrogate. in our interviews, we note how frequently the language of intimate relationships is invoked in relation to donors and surrogates. clinicians might assume that patients are principally interested in the health and screening results of potential donors and/or surrogates, whereas patients may be looking for an emotional bond with them. the most striking example of this from our cbr study was an intended mother who explained that she and her partner had fallen in love with an egg donor, and that, for them, this trumped the discovery that she was a carrier of the tay sachs gene. a gap between what matters to experts and what matters to patients is also evident in relation to the law, including but not limited to criminal prohibitions, citizenship and the rules of legal parentage. for example, in our study, it is striking how few intended parents of children born through surrogacy had sought legal formalisation of their relationship. tom, who had undertaken surrogacy in india said : im not going to the family court [to seek parental responsibility ]. i m fine with [the indian birth certificate ] as being a formal document. because my name is on it i feel that gives me the parental rights that i need even though legally i know i m not actually the parent in australia. that s the weird thing is that in india i m the legal parent and the surrogate has no parental rights and i come back to australia and i do nt have any parental rights but the surrogate has got all the parental rights, and she s not even actually living in the country. (emphasis added) i m not going to the family court [to seek parental responsibility ]. i m fine with [the indian birth certificate ] as being a formal document. because my name is on it i feel that gives me the parental rights that i need even though legally i know i m not actually the parent in australia. that s the weird thing is that in india i m the legal parent and the surrogate has no parental rights and i come back to australia and i do nt have any parental rights but the surrogate has got all the parental rights, and she s not even actually living in the country. (emphasis added) many interviewees appeared unconcerned about, or actively avoided knowing about the risks to which their family was exposed as a result of their lack of legal parentage. what mattered instead was the lived experience of the practicalities of new parenthood : being able to get back into australia, obtain a medicare card to access medical treatment for the child, and later to enrol him or her in school. this was particularly the case for those whose names appeared on the foreign - issued birth certificate, who had encountered a no - questions - asked response from authorities. charlotte, a parent through surrogacy and egg donation in the us, described her decision not to seek legal advice as i just assumed everything would be fine because i m on their birth certificate. in our interviews, it was common for genetic and non - genetic parents to take such a view, relying upon a document that they acknowledge, when pushed, would not be likely to stand up to challenge. like the unenforced or ambiguous laws of the treating country, and the blind - eye approach of domestic regulators, the birth certificate both is, and is not, legal protection. we suggest that any attempt to further reform or refine laws relating to cbr must attend to the subjective and lived experience of law, which like the personal experience of treatment, may stand in contradiction to policy maker s understandings of participants motivations or of law s effect upon behaviour. these experiences can usefully be explored to complicate, for example, the dominant assumption that payment in reproductive endeavours is exploitative and inevitably impairs informed consent. our interviews explore both cross - border compensated surrogacy and egg donation and domestic uncompensated arrangements. though we are still at the beginning of our work, it is already clear that the picture is far more complex than might have been expected, both morally and legally. indeed, as this preliminary snapshot from our interviews has shown, some participants chose compensated arrangements precisely because they considered an uncompensated arrangement to be ethically questionable. perhaps more importantly, our research has begun to demonstrate that the incentive of legal parentage under australian law, or threat of its denial, will not drive intended parents away from paid surrogacy or egg donation. the law, as it currently stands, works in obstructive and confounding ways that push people outside its reach or encourage them to ignore its limits and this is ultimately counter - productive and likely to cause harm to the participants and their future children. there is a still a great deal to learn about cross - border reproductive treatment. large - scale bypassing of domestic fertility services is a relatively new phenomenon and we have no reliable way of recording its outcomes. in order to ensure that the law is capable of minimising the risk of harm to all participants, we argue that it is necessary to learn from the experience of those undertaking cross - border reproductive treatment, at and outside the limits of the law. | abstractdrawing upon the preliminary findings of an australian empirical project on cross - border reproduction (cbr), this article argues that regulators and policymakers could learn from the experiences of those who travel overseas in order to access fertility treatment and surrogacy. it makes four principal observations. first, the distinction between so - called altruistic and commercial gamete donation and surrogacy is increasingly unsustainable and is not experienced as meaningful by many participants in cbr. secondly, the status of the law in cbr is profoundly equivocal ; for participants it is often there and not there at the same time. thirdly, self - sourced information, from the internet and more specifically social media such as facebook, is now the principal source of information and peer support for reproductive travellers. fourthly, and relatedly, domestic reproductive services providers are often sidestepped. if one of the goals of regulation is to minimise the risk of harm to participants, it is not clear that it is currently achieving this aim, and this article argues that any reforms will only work if they are more responsive to the reality of cbr. |
bacterial parapneumonic pleural effusion (ppe) accounts for 40% of community - acquired pneumonia cases and has high morbidity and mortality (1). a study in the uk showed that the mortality rate of bacterial ppe was 15% and 20% of patients needed to be hospitalized for several months (2). an early identification of the causative pathogen cultured from pleural samples can improve the treatment outcomes by indicating the appropriate antibiotics as well as treatment interventions such as surgical drainage (4, 5). the use of blood culture bottles for ascites fluid or joint fluid gave a better yield of causative agents than did the standard culture bottle (6, 7). similar results were obtained in a study to detect bacteria in pleural fluid in the uk. the blood culture bottle method increased the pathogen identification rate by 20.8% compared with sterile culture bottles in 53 bacterial ppes (8). pathogens of pneumonia, causes of ppe, may be different between asian and the western countries (9, 10). the northeastern parts of thailand and australia have a higher incidence of melioidosis or burkholderia pseudomallei infection (11). there are limited data of using blood culture bottles to yield the identification of bacterial pathogens in asian patients with bacterial ppes. the aim of this study was to compare the culture - positive rate by the blood culture bottles and the standard culture bottles in bacterial ppes in different settings and larger study populations from the original study (8). patients diagnosed with bacterial ppe in khon kaen hospital, khon kaen, thailand, were enrolled consecutively and prospectively. the study period was from june first, 2012 to december 31st, 2013. the inclusion criteria were patients aged > 18 years and with exudative, neutrophilic ppe. the patients were excluded if causes other than bacterial pleural effusion were suspected including the presence of lymphocytes in the pleural fluid at levels of more than 50%, the presence of abnormal cells, lung abscesses, or bloody pleural effusions (13). pleural fluids of all the eligible patients were collected in blood culture bottles in addition to standard culture bottles to compare the organism identifications in both bottles. in this practice, a standard culture bottle is a sterile bottle without any media ; 5 ml pleural fluid was put in both culture bottles. blood culture bottles were bact / alert fa for aerobic organisms, containing soybean - casein digestive broth, sodium polyanethol sulfonate, pyridoxal hcl, menadione, hemin, activated charcoal, l - cysteine, carbohydrate, and amino acids (biomerieux, inc., blood culture bottles were used for aerobic organisms because aerobic organisms are the common pathogens in bacterial ppe (8), and patients with pleural effusion from anaerobic pathogens were excluded. the blood culture bottles were incubated in bact / alert system at 35c for seven days (15, 16), and the procedures for bact / alert system were followed (16), while pleural fluids in the standard culture bottles or sterile tubes were cultured using blood, macconkey, and chocolate media. baseline patient characteristics, laboratory results, and culture results were collected and compared with the disease severity. the culture results using blood culture bottles and standard culture bottles were compared using descriptive statistics. the disease severity was classified into the most severe empyema thoracis, complicated ppe, and simple ppe as the least severity (17). in brief, empyema thoracis is diagnosed if the pleural fluid is purulent, while complicated ppe is warranted if one of the following features is met ; positive pleural fluid smears for bacterial pathogens ; pleural fluid glucose 0.05). the results of the previous study (8) wherein the blood culture bottle, specifically bact / alert fa increased the bacterial identification rates in ppe, were confirmed. this study was different from the original study in terms of the study location (the uk vs an asian tropical country), the sample size (53 vs 129), plus different possible organisms in the effusion. the overall bacterial identification rate in bacterial ppe by blood culture bottle in this study was 24.0% and even higher at 33.8% in the empyema group. previous antibiotic use in this study population (79.1%), as shown in table 1 (18), and delayed transport process to the culture lab (19) might also be considered as factors. the culture - positive rate by blood culture bottle in the study, however, was still higher than a previous report by ferrer at 15.0% (20). better bacterial culture yield for pleural effusion using blood culture bottles may be due to using charcoal - containing medium, while the standard culture bottles do not have such medium (14). charcoal may increase the oxygenation, resulting in a higher rate of organism recovery. in this study, the most common pathogen in the pleural fluid was streptococcus sp. burkholderia pseudomallei, the causative agent of melioidosis, was found in five patients (31 patients, 16.1% in our study). reechaipichitkul (12) reported pleural effusion in 12.2% and 15.3% of cases with acute and subacute / chronic pulmonary melioidosis, respectively. s. pneumoniae is usually the most common pathogen causing community - acquired pneumonia, but was not found in this study. this may also be explained by the high rate of previous antibiotic use (79.1%). the main limitation of this study was that the size of the study population was somewhat lower than the calculated power sample size (126 vs. 135 subjects). the results, however, showed statistically significant advantages of the blood culture bottle method over the standard culture bottle method for the detection of bacteria in pleural effusion. the relatively low bacterial detection rate for the pleural fluid may be due to the high rate of previous antibiotic use. the results of this study strongly supported the previously report of menzies. using a relatively larger sample size (8). blood culture bottle method should be used in routine clinical practices for pathogen identification in pleural fluids of patients suspected of bacterial ppe. in conclusion, the blood culture bottle method was more effective than the standard culture bottle method for the detection of bacterial pathogens in ppe. | background : bacterial parapneumonic pleural effusions (ppes) have high morbidity. the accurate identification of pathogens is vital for initiating the appropriate treatment. a previous study suggested that the use of blood culture bottles might improve the bacterial yield in ppes.objectives:the aim of this study was to compare the culture positivity rate by the blood culture bottles and the standard culture bottles in bacterial ppes.patients and methods : patients diagnosed with ppes at the khon kaen hospital, khon kaen, thailand, which is an endemic area of melioidosis, were enrolled consecutively and prospectively. the study period was from june first, 2012 to december 31st, 2013. the inclusion criteria were adult patients aged > 18 years, with exudative, neutrophilic parapneumonic effusion. of the pleural fluid samples, 5 ml from all the eligible patients were collected in both blood culture bottles and the standard culture bottles. patient baseline characteristics, laboratory results, and culture results were collected and analyzed.results:during the study period, 129 patients met the study criteria. the bacteria - positive rate of pleural fluid culture using the standard culture bottle was 14.0%, whereas the positive rate using blood culture bottles was 24.0% (p < 0.001).conclusions : the blood culture bottle method is more effective than the standard culture bottle method for the detection of bacterial pathogens in ppe. |
metabolic syndrome (metsy) is defined as a combination of adverse cardiovascular disease (cvd) and metabolic risk factors including abdominal obesity, dyslipidemia, hyperglycemia, and hypertension. a growing body of evidence documented that the metsy is a risk factor for atherosclerotic cvd and type 2 diabetes mellitus (t2 dm) incidence and mortality. three definitions have been suggested by various organizations. the revised national cholesterol education program third adult treatment panel (rncep : atpiii) definition requires three or more of the following components : waist circumference ([wc ] 102 cm for men and 88 cm for women);increased triglycerides ([tg ] 150 mg / dl or being under treatment);low, high - density lipoprotein cholesterol (hdl - c < 40 mg / dl for men and < 50 mg / dl for women or being under treatment);elevated blood pressure systolic blood pressure ([sbp ] 130 mmhg, or diastolic blood pressure [dbp ] 85 mmhg or receiving anti - hypertensive medications);increased fasting plasma glucose ([fpg ] 100 mg / dl or treatment for hyperglycemia). waist circumference ([wc ] 102 cm for men and 88 cm for women) ; increased triglycerides ([tg ] 150 mg / dl or being under treatment) ; low, high - density lipoprotein cholesterol (hdl - c < 40 mg / dl for men and < 50 mg / dl for women or being under treatment) ; elevated blood pressure systolic blood pressure ([sbp ] 130 mmhg, or diastolic blood pressure [dbp ] 85 mmhg or receiving anti - hypertensive medications) ; increased fasting plasma glucose ([fpg ] 100 mg / dl or treatment for hyperglycemia). in the international diabetes federation definition, central obesity is necessary as a prerequisite (wc 94 cm for men and 80 cm for women) and in addition at least two of the raised tg, low hdl - c, elevated blood pressure (bp) and fpg. the recent joint interim statement definition requires the presence of three out of the five above mentioned components, but with considering national or regional - specific adoption for measure of the central obesity mainly wc. the prevalence and clinical usefulness of the metsy are age, gender, and culture - dependent, augmenting with increasing age and more useful in males. in the middle east, the prevalence of the metsy is more than western countries, with higher frequency in females than males. due to the high burden of the cvds and t2 dm, identifying individuals who are at higher risk for these disorders would be useful. as mentioned above, the metsy is a risk factor for cvd and t2 dm but during recent years using a continuous metsy risk score (cmetsys) is recommended instead of a yes / no definition. recently, hsiao., using data from a middle - aged cohort, published the chinese metsy risk score. they have used binary logistic regression and receiver operation characteristic (roc) curve to construct their cmetsys. during their study follow - up 30 of the 352 participants developed metsy. they indicated tg and dbp have highest and lowest area under curve (auc). they concluded that tg and wc are the most important variables, and their model could be useful for clinical screening for the metsy. the aim of this study was to construct a cmetsys in an iranian adult population and to provide an evaluation of the predictive performance of the cmetsys to identify individuals with metsy. the isfahan healthy heart program (ihhp) data were used in this cross - sectional analysis. ihhp is a comprehensive, community - based healthy lifestyle program with a reference area ; the details are published elsewhere. briefly, data were collected from three communities located in the central area of iran including : isfahan, najaf - abad, and arak. five to 10% of households were selected using a random two - stage clustered sampling method. during survey, data from 8313 individuals aged 19 years (one random selected adult within each household) was collected in isfahan, najafabad, and arak. the participants were interviewed to complete validated questionnaires containing questions on demography, socioeconomic status, smoking behavior, physical activity, nutritional habits, and other behavior regarding cvd. in clinics, participants were asked to fast for 12 h prior to the examinations. high - sensitivity c - reactive protein (hs - crp) for 7087 participants, fpg, total cholesterol (t - chol), hdl - c, low - density lipoprotein cholesterol (ldl - c), and tg were assessed. all blood samples were examined in the central laboratory of the isfahan cardiovascular research center (a collaborating center of the world health organization), with adherence to external national and international quality control. in this study, although binary metsy was defined according to the rncep : atpiii and its criteria for the components of the metsy, but after comparing the auc from models including each components as a categorical or continuous independent variable, we decided to use continuous values of the components of the metsy during model building. for approaching elevated bp and for reducing the number of model parameters, mean arterial pressure (map) was used. it was calculated using the following equation : map = ([sbpdbp]/3) + dbp. then, we compared model including only map with which one consists of map and sbp using likelihood ratio test (lrt) (p = 0.041) and also auc using delong method (p = 0.05). therefore, we used map to consider bp during modeling. using logistic regression with a binary outcome, based on modified national cholesterol education program (ncep) : atpiii metsy definition for asian populations, including gender and age and also one of the map, wc, t - chol, hdl - c, tg, hip circumstance (hc), wc to hc ratio (waist to hip circumferences), body mass index (bmi, fpg, and 2 hpg as independent variables then auc and its binomial 95% confidence interval (ci) were calculated for each model. considering the value of auc and clinical practicability, more useful predictors were selected and modeled in different approaches. finally, we construct four different models as following list : model 1 : including informative blood glucose measurement (fbs).model 2 : including powerful nonlaboratory measurements (wc, bmi, and map).model 3 : including main serum lipids measurements (tg, hdl - c, and ldl - c).model 4 (full model) : including all components of metsy (fpg, hdl - c, wc, map, and tg). model 2 : including powerful nonlaboratory measurements (wc, bmi, and map). model 3 : including main serum lipids measurements (tg, hdl - c, and ldl - c). model 4 (full model) : including all components of metsy (fpg, hdl - c, wc, map, and tg). to model building and validation, we conducted user stepwise selection (using lrt) and leave - one - out cross - validation methods (using crossval macro for stata statistical software) (release : 11.2 ; statacorp, tx, usa). for each model, the auc and its 95% ci was calculated and compared (delong method) with its sub - models which were included less independent variables and also a full model. we calculated the probability of the metsy for each person using following formula only for model 4 : pr = 1/(1 + e) where x represent the suggested cmetsys. after constructing the cmetsys, by using multiple linear regression, we assessed linear relationship of the cmetsys with smoking status, percentiles of the crp value (hs - crp), global dietary index, total, leisure time, homework, and workplace physical activity scores, and bmi. we compared determinants of the metsy as a binary outcome with its determinants when it was defined as a continuous risk score. to do this, we fitted a logistic regression with statistically significant determinants of the cmetsys as its independent variables. all statistical analyses were conducted using stata statistical software (release : 11.2 ; statacorp, tx, usa). mean and standard deviation of age for men were 38.5 (15.9) and for women were 39.3 (15.3). total prevalence of the metsy was 21.9%, 29.0% and 14.7% in females and males, respectively. percentages of abnormality of each component of the metsy were estimated based on ncep : atpiii criteria. the metabolic description of study participants gender - divided percentages of abnormality of the components of the metsy (according to ncep : atpiii) among study participants percentages of subjects with 0, 1, 2, 3, 4 or 5 abnormal component were calculated by gender. these percentages were 14.7, 29.3, 27.0, 19.7, 7.7, and 1.5 for females and 28.8, 30.9, 25.7, 10.6, 3.7, and 0.5 for males, respectively. the auc of the models including both gender and age and one or more independent variables and its binomial 95% ci are presented in table 3. it also illustrates the results of the comparison of each auc with the next one based on delong method. the statistical significance level of lr test to assessing the goodness of fit of each model comparing with the same, but without underlined variable in the first column presented in the last column in this table. auc and its 95% binomial ci for each model and statistical significance of their auc and goodness of fit table 4 shows coefficient, odds ratio, and statistical significance level of each variable in the best - fitted logistic models [which been bold in table 3 ] to predict the probability of presence of the metsy in iranian adults. the cross - validated (leave - one - out) estimates of auc for models 1 to 4 were 0.78, 0.88, 0.90, and 0.95, respectively. best - fitted logistic models to predict presence of the metsy in iranian adults, sorted based on the auc from lowest to highest although four separate models were fitted, but as an example we calculated the x (where probability of presence of the metsy is pr = 1/(1 + e^x).) using the best one (model 4). for calculations, we used following equation where all except gender are continuous variables with units mentioned in table 1, gender is a binary variable with one and two codes representing female and male, respectively. x = 14.43465 + (0.13hdl - c) + (0.01tg) + (0.03fps) + (0.11wc) + (0.09 map) + (0.02age) + (2.83 gender). best cut point of x for classification of the metsy (yes / no) was 1.15 with 89% sensitivity and 87.9% specificity and 88.2% correct classification. the auc and roc curve for each of four models mentioned in table 4 are illustrated in figure 1. the receiver operation characteristic curve and area under curve for each of suggested models to construct continuous metabolic syndrome risk score for iranian adults. model 2 : including powerful nonlaboratory measurements (waist circumference [wc ], body mass index and mean arterial pressure [map ]). model 3 : including main serum lipids measurements (triglycerides, high - density lipoprotein cholesterol [hdl - c ] and low - density lipoprotein cholesterol). model 4 : including all components of the metsy (fasting plasma glucose, hdl - c, wc, map, and tg) we also assessed the probable determinants of the cmetsys constructed by model 4. table 5 shows the determinants of the cmetsys (x) determined by linear regression and their coefficient, and also p - values provided by logistic regression. in this study, we constructed and validated four cmetsys to identify the metsy in iranian adults. this is the first study of its kind in a middle - eastern adult population. considering some circumstances in which clinicians or epidemiologists do not have values on some components of metsy, first, when we have only age, gender and fpg, the model had lower discriminative power but still plausible auc (78.1 [95% ci : 77.1 - 79.1 ]). although 2 hpg added to this model, but it had no statistically significant added value on auc or goodness of fit. the second ones provide cmetsys for conditions in which we have nonlaboratory variables, e.g., bmi, wc, and map. the next model was provided for circumstances in which only serum lipid profile and tg were considered. if we have values of each five components of metsy, the model 4 will be the best one. our study shows that tg and wc are the number one and two contributors in presence of the metsy. hdl - c and map are in third and fourth order, and the last one is fpg. hsiao., found tg and wc as more important components of metsy. based on our findings, hdl - c has the same rank as in hsiao. but fpg and bp do not have the same importance as in their study. in our study, the predictive performance of map had no statistically significant difference with sbp. however, one probable reason for this distinction may be because of considering the bp as map in our study and as sbp and dbp in the study of hsiao. despite ranks for importance of bp as well as other components, the auc for bp in our study was 81.3 (80.4 - 0.82.2) and 59.5 (50.8 - 68.3) in their study. considering the follow - up nature of data that used in hsiao. (provide ability to predict metsy for healthy people in the future) and cross - sectional nature of data which we used (provide ability to identifying peoples with metsy at this time), this difference could provide probable evidence on more predictive performance of bp and other components for discriminate the present metsy than for its future incidence. considering our full model, this study provides approval evidence on independent predictive performance of tg and wc to discriminate the metsy., our best model demonstrates independency of influence of other components of the metsy to estimate the risk of presence of the metsy. one possible reason for this discrepancy may be a large difference between the sample size of two studies as well as the possible effects of regional and ethnical differences. in concordance with our findings, there are some evidences on the presence of gradient relationship between the cmetsys and number of abnormal components of metsy. although univariate study results showed statistically significant differences between those with metsy and healthy participants in terms of all type of physical activity, the cmetsys had significant relationships with leisure time and workplace physical activity, but the corresponding figure was not significant for homework physical activity and total physical activity score. against univariate results, logistic regression showed no statistical association between the metsy and all types of physical activities. these findings demonstrate two key points : first, the importance of leisure time and workplace physical activities and second, according to more analysis (not shown), probable confounding effect of gender on those effects when using yes / no definition for the metsy. the significant association of the cmetsys with leisure time physical (ltphya) was shown earlier in american children. hence, we could conclude cmetsys in adults or children has a probable relationship with ltphya activity. linear regression also showed a significant association between the cmetsys and bmi or hs - crp percentiles. this finding is in concordance with some other studies. although we do not have any confirmation on the association of metsy with hs - crp if accept present evidence from other studies, comparing results of logistic regression and linear regression could provide some evidence on using the cmetsys instead of yes / no definition.. showed there is also a relationship between obesity and hs - crp level and other authors revealed the relationship of hs - crp and the metsy. these findings are in agreement with studies on risk factors of cvds. in this study, our limitation was cross - sectional context of the study although most risk score studies are cross - sectional, but the goal of risk score studies is prediction of future disorder and this need cohort designs. however, considering the concordance of our findings with which in other studies with cohort designs, it can be suggested that this limitation had no or a very weak effect on our study conclusion. four generalizable continuous risk score models with plausible predictive performance to identify the metsy in iranian adults were generated. the best cmetsys provided in this study had a significant relation with hs - crp, bmi, leisure time, and workplace physical activity as well as age and gender. although we provided a practicable cmetsys to predict the metsy in iranian adults but for better predictions follow - up studies are needed. smh contributed in the design of the study, revising the draft and approval of the final version of the manuscript, and agreed for all aspects of the work. rk contributed in the design of the study, revising the draft and approval of the final version of the manuscript, and agreed for all aspects of the work. hmv contributed in the design of the study, data analysis, writhing and revising the draft and approval of the final version of the manuscript, and agreed for all aspects of the work. mm contributed in the data analysis, revising the draft and approval of the final version of the manuscript, and agreed for all aspects of the work. ns contributed in the design of the main study, revising the draft and approval of the final version of the manuscript, and agreed for all aspects of the work. sa contributed in the design of the main study, revising the draft and approval of the final version of the manuscript, and agreed for all aspects of the work. | background : metabolic syndrome (metsy), an important predisposing factor for the most of noncommunicable diseases, has become a global pandemic. given different definitions used for the metsy, recently using a score termed continuous metsy risk score (cmetsys) is recommended. the aim of this study was to provide a cmetsys in a population - based sample of iranian adults and to assess its determinants.materials and methods : we used the data of the baseline survey of a community trial entitled the isfahan health heart program. the metsy was defined according to the revised national cholesterol education program third adult treatment panel. all probable predictive models and their predictive performance were provided using leave - one - out cross - validated logistic regression and the receiver operation characteristic curve methods. multiple linear regression was performed to assess factors associated with the cmetsys.results:the study population consisted of 8313 persons (49.9% male, mean age 38.54 15.86 years). the metsy was documented in 1539 persons (21.86%). triglycerides and waist circumference were the best predictive components, and fasting plasma glucose had the lowest area under curve (auc). the auc for our best model was 95.36 (94.83 - 95.83%). the best predictive cutoff for this risk score was 1.151 with 89% sensitivity and 87.93% specificity.conclusion:we provided four population - based leave - one - out cross - validated risk score models, with moderate to perfect predictive performance to identify the metsy in iranian adults. the cmetsys had significant associations with high sensitive c - reactive protein, body mass index, leisure time, and workplace physical activity as well as age and gender. |
neuralgic amyotrophy (brachial plexus neuropathy, brachial plexus neuritis, or parsonage - turner syndrome) is an uncommon inflammatory condition typically characterized by acute and severe shoulder pain followed by paresis with muscle weakness and atrophy of the upper limb or shoulder girdle. we report an unusual clinical manifestation of neuralgic amyotrophy, namely bilateral phrenic nerve palsy with concomitant laryngeal paresis. a 55-year - old male presented with orthopnea and aphonia after an episode of bilateral shoulder pain preceded by an upper respiratory tract infection. spirometry, chest x - ray and videolaryngoscopy revealed bilateral and simultaneous paresis of the diaphragm and the vocal cords. clinical examination at admission and at the 2-month follow - up did not show upper limb weakness or atrophy, except for a mild atrophy of the right supraspinatus muscle. an electromyography of the upper limb muscles and nerve conduction studies did not reveal signs of denervation. after treatment with prednisolone, vocal cord function markedly improved within 8 weeks, whereas paresis of the diaphragm persisted. shoulder pain followed by diaphragmatic paralysis with dyspnea and hoarseness may be a manifestation of neuralgic amyotrophy even if upper limb or shoulder girdle palsies are absent. neuralgic amyotrophy (na) is an uncommon neuropathy first described in 1948 by parsonage and turner. typically, the earliest manifestation of this disease is a sudden onset of severe and sharp pain in the shoulder lasting for 24 weeks, mostly followed by a sudden weakness and gradual atrophy of the muscles innervated by the c56 or other nerve roots in the vicinity. rare manifestations include isolated or concomitant affection of other peripheral motor nerves derived from the cervicobrachial plexus, such as the phrenic or laryngeal nerves [2, 3, 4, 5 ]. very rarely, bilateral pareses of the diaphragm or laryngeal palsies had been observed [6, 7, 8 ]. although the pathogenesis of na is unknown, an idiopathic autoimmune process triggered by an unspecific infection is presumed. infections of the respiratory tract, trauma or surgery of the shoulder often precede the onset of na. besides the idiopathic form, a hereditary autosomal dominant form caused by a mutation in the sept9 gene on chromosome 17q25 has recently been described. in the majority of patients, the course of na is benign, with recovery of muscle function within weeks to months. however, 25% of the patients are not able to return to work after 3 years. the diagnosis of na relies on a characteristic history of severe pain in the shoulder, followed by pain relief and subsequent brachial palsies. chest x - ray and cerebrospinal fluid analysis should be obtained to rule out a direct viral or bacterial infection, or a mechanical compression of the brachial plexus. plexus mri usually reveals no pathological findings, although t2 hyperintensities may occasionally be observed. corticosteroids seem to accelerate the recovery of na patients and are recommended in the early stages of the disease ; however, there are no randomized controlled treatment studies of na conducted to date. we report an unusual case of na presenting with simultaneous, bilateral phrenic nerve palsies and vocal cord paresis as the predominant symptoms, with partial gradual recovery after 2 months. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a 55-year - old male patient with an unrevealing medical history except for nicotine abuse (50 pack years) presented with an acute onset of dyspnea and aphonia. about 7 weeks before referral to our tertiary neurological center, the patient had suffered a viral infection of the upper airways, which was followed by a distinct sharp pain in both shoulders lasting for several days. the pain subsided ; however, the patient developed increasing shortness of breath and orthopnea. a chest ct was normal and spirometry showed a reduced vital capacity and a reduced forced expiratory vital capacity per second. the working diagnosis of chronic obstructive lung disease was made, and the patient was discharged on inhaled fenoterol and ipratropium bromide. five days later, the patient suffered from progressive hoarseness, leading to complete aphonia within 48 h. he complained of the impossibility to cough and to clear his throat. the initial neurologic examination at our department showed no weakness or atrophy of the upper limbs and normal tendon reflexes. the patient was short of breath with paradoxical abdominal movement, extensive use of the accessory respiratory muscles and a slight inspiratory stridor which prevented him to sustain a supine position. a chest ct and a whole - body positron emission tomography - ct showed no evidence of malignancy or inflammation. an x - ray cinematography of the chest revealed an elevated diaphragm with only marginal movement during inspiration (fig. videolaryngoscopic examination showed signs of partial paralysis of both branches of the vagus nerves, namely both the recurrent laryngeal nerves and the superior laryngeal nerves. the excavated vocal folds were in an intermediate position, with minimal non - intended adduction movements. 2). a needle electromyography (emg) of the right and left m. biceps brachii, the right m. infraspinatus and m. extensor pollicis longus and the left m. deltoideus, m. trapezius and paravertebral muscles was normal, without pathological spontaneous activity. motor and sensory nerve conduction velocities including phrenic nerve conduction were normal, as were the f - waves. spirometry revealed a reduction of the forced expiratory vital capacity per second (66% of reference) and of the vital capacity (75% of reference). the maximum inspiratory pressure was severely reduced (34.3% of reference), whereas the maximum expiratory pressure was not decreased (table 1). because of severe dyspnea when lying down, lung function testing could not be performed in the supine position. the diagnosis of bilateral na was made, and the patient was treated with intravenous corticosteroids (500 mg methylprednisolone for 5 days). at reassessment after 2 months, phonation had markedly improved, but hoarseness was still present. further neurological examination remained unchanged, except for a mild atrophy of the right supraspinatus muscle. during videolaryngoscopy, the excavated vocal cords were mobile ; however, during phonation, glottal closure was still insufficient over the whole length of the glottis. while the gap between the processus vocales was closely to 1 mm, it was approximately 2 mm between the excavated vocal folds in the mid of the glottis (fig. results of the emg, phrenic nerve conduction velocity, lumbar puncture and spirometry were unaltered. a chest x - ray revealed an unchanged elevation of the diaphragm, with restricted mobility during inspiration. the typical course of na is initiated by a distinct pain in the shoulder, followed by muscle weakness and atrophy. however, na involving other peripheral nerves or isolated affection of single motor nerves can be difficult to diagnose. in our case, acute - onset dyspnea and orthopnea were the predominant symptoms suggestive of a cardiac etiology or bronchial asthma. the rapidly progressive hoarseness, leading to aphonia within 48 h and worsening of dyspnea when lying flat, was indicative for an atypical na [3, 7, 12 ]. a decrease in the forced vital capacity of more than 40% in the standing compared to the supine position is suspicious of diaphragmatic paresis ; therefore, the forced vital capacity should be assessed in both positions, if possible. phrenic nerve conduction velocity can be prolonged or, as in our case, normal. the emg of the diaphragm could have helped to confirm the diagnosis of diaphragmatic paralysis [3, 13 ], but was not performed in our case due to the associated relatively high risk of pneumothorax. the chest x - ray revealed an elevated diaphragm on both sides with nearly no movement during inspiration, and the maximum inspiratory pressure was severely reduced. laryngeal paresis is a rare feature of na [2, 6 ]. to and traquina reported a case of a child with bilateral vocal cord paresis due to na. since both superior laryngeal nerves were additionally affected, glottic stenosis was incomplete and the patient was still able to breathe spontaneously, although a slight inspiratory stridor was noticed on auscultation. recovery of respiratory muscle weakness can take months to years, and only about 50% of the patients experience an improvement of respiratory function, whereas improvement of laryngeal paresis usually occurs faster [2, 14 ]. in our case, the diagnosis of na in this case was based on the affection of multiple motor nerves, the typical onset of the disease, the reported shoulder pain and the negative results of additional diagnostics. isolated phrenic nerve paralysis without weakness of the upper limbs despite normal emg and nerve conduction velocities has been described to occur in na [3, 5, 8, 12 ]. however, to the best of our knowledge, bilateral and simultaneous vocal cord paresis with concomitant bilateral palsy of the diaphragm without involvement of the proximal upper limb muscles or weakness of the shoulder girdle has not been reported in na so far. therefore, physicians should be alerted to a preceding history of an upper airway infection and shoulder pain even if pareses of the upper limbs and the shoulder girdle are absent and the emg and nerve velocity conduction studies are unrevealing. ringelstein has received travel expenses and honorariums from boehringer ingelheim, sygnis, neurobiological technologies, novartis, novo - nordisc, sanofi - aventis, solvay, bayer vital, m 's science, sevier, ucb, and trommsdorff for serving as a member of steering committees, safety committees in clinical trials, and as a speaker and consultant. | backgroundneuralgic amyotrophy (brachial plexus neuropathy, brachial plexus neuritis, or parsonage - turner syndrome) is an uncommon inflammatory condition typically characterized by acute and severe shoulder pain followed by paresis with muscle weakness and atrophy of the upper limb or shoulder girdle. we report an unusual clinical manifestation of neuralgic amyotrophy, namely bilateral phrenic nerve palsy with concomitant laryngeal paresis.case reporta 55-year - old male presented with orthopnea and aphonia after an episode of bilateral shoulder pain preceded by an upper respiratory tract infection. spirometry, chest x - ray and videolaryngoscopy revealed bilateral and simultaneous paresis of the diaphragm and the vocal cords. clinical examination at admission and at the 2-month follow - up did not show upper limb weakness or atrophy, except for a mild atrophy of the right supraspinatus muscle. an electromyography of the upper limb muscles and nerve conduction studies did not reveal signs of denervation. analysis of the cerebrospinal fluid and an mri of the neuraxis were unremarkable. after treatment with prednisolone, vocal cord function markedly improved within 8 weeks, whereas paresis of the diaphragm persisted.conclusionshoulder pain followed by diaphragmatic paralysis with dyspnea and hoarseness may be a manifestation of neuralgic amyotrophy even if upper limb or shoulder girdle palsies are absent. |
in recent years, great emphasis has been placed on the role of arterial stiffness and central blood pressure (bp) as independent predictors of the development of cardiovascular (cv) diseases [13 ]. consequently, the assessment of arterial stiffness and central hemodynamics is recommended as additional tests for the clinical evaluation of hypertensive patients (based on history, physical examination, and findings from routine laboratory tests), particularly for those at risk of cv complications. regional and local arterial stiffness may be measured directly and noninvasively, at various sites along the arterial tree, by assessing pulse wave velocity (pwv) and augmentation index (ai). the most widely employed methods for evaluating pulse waveforms are those based on applanation tonometry and transfer functions, although recently oscillometric ambulatory blood pressure monitoring (abpm) devices using specific algorithms for pulse wave analyses have been proposed for assessing arterial stiffness [69 ]. at present, oscillometry is an affordable technique and may allow a comfortable, accurate, repeated, and prolonged estimation of arterial stiffness and central hemodynamics over the 24 hours in daily life conditions. the most recent studies seem to indicate reliability and feasibility of ambulatory arterial stiffness evaluation based on analysis of brachial oscillograms [10, 11 ]. in the present study we aimed at assessing the feasibility of determining central bp and various indices of arterial stiffness over the 24 hours by a noninvasive, clinically validated technology of pulse wave analysis based on oscillometric bp measurements, integrated in an ambulatory bp (abp) monitor [10, 12 ]. potential differences in arterial hemodynamics and stiffness were sought between healthy normotensive volunteers and hypertensive patients evaluated in a real - life context. treated or untreated hypertensive outpatients and untreated healthy volunteers, aged 18 years or more, were included in the study. healthy individuals were eligible for inclusion into the study in absence of arterial hypertension (office systolic, sbp < 140 mmhg, and office diastolic, dbp < 90 mmhg plus 24-hour average sbp < 130 mmhg and dbp < 80 mmhg), blood test abnormalities (including impaired fasting glucose, impaired glucose tolerance, or dyslipidemia), obesity, and other major cardiovascular risk factors. both healthy subjects and hypertensive patients were excluded in case of previous or current cardiovascular disease or any other concomitant significant systemic condition. all individuals were submitted to an abpm, preceded by an office automatic bp measurement with the same device used for ambulatory monitoring. office bp was measured in the sitting position after 5-minute rest : three measurements were obtained at 2 min intervals and the average of the three measurements was taken as the reference for office bp. hypertensive patients were recruited among consecutive patients with a known history of high bp presenting at the outpatient clinic of the cardiology research complex, moscow. healthy volunteers were recruited among the staff and personnel at the russian railroad and at the russian navy. the study was conducted according to good clinical practice guidelines and the declaration of helsinki, and the protocol was approved by the ethics committees of the centers involved. written informed consent was obtained from all patients and controls prior to their inclusion into the study. abpm was performed noninvasively over the 24 hours by the bplab electronic, oscillometric, automated bp monitor (bplab gmbh, germany). the device accuracy in measuring additionally, the device has passed validation also for estimation of vascular indices against the most commonly noninvasive device, recommended as reference standard, the sphygmocor [12, 14 ], in accordance with the artery guidelines. the optimal adult cuff was wrapped around the nondominant arm and the patient was asked to keep her / his arm still during the automatic bp measurements. the device was programmed to measure bp every 1530 min during daytime (from 06:00 to 22:00) and every 3060 min during nighttime (from 22:00 to 06:00). each recording started in the morning and was preceded by verification of the accuracy of oscillometric bp measurements against auscultatory technique in every subject. after fitting the device, patients were sent home and asked to resume normal life and to come back 24 hours later for removal of the instrumentation. the bplab bp monitor makes use of brachial oscillometric bp waves for a noninvasive estimation of central bp and arterial stiffness [10, 1214 ]. the vasotens principle of oscillometric pulse wave analysis is based on plethysmography and on recording the pulsatile pressure in the brachial artery. during bp measurement, the pressure waveforms in the cuff are digitalized and stored in the device memory while performing step - by - step deflation. thereafter, signal processing is performed using a special mathematical algorithm, which is based on a specially developed transfer function that utilizes a modification in a certain frequency range within the acquired pulse signal to derive the aortic pressure wave. the modulus and phase characteristics of the vasotens transfer function have been published previously. the difference in time between the first wave and the second wave (i.e., the reflected wave) correlates to the distance, according to the manufacturer 's instructions, and allows calculation of the pulse wave velocity (pwv). a detailed description of the vasotens methodology may be found in previous publications [10, 12, 17 ]. the reflected wave transit time (rwtt) represents the transit time of pulse wave along a corresponding artery and is the reciprocal of pwv. in case of a stiff artery, the method used to estimate this parameter is based on the identification of the reflected wave on the pulse curve in sphygmogram records by original vasotens algorithm. since measures of arterial stiffness depend on bp and heart rate (hr) values, rwtt is usually normalized to a sbp of 100 mmhg and a hr of 60 bpm by a regression analysis of 24-hour rwtt to 24-hour sbp and 24-hour hr in each individual. pwv indicates the pulse wave speed in the arterial tree : if the artery is stiff, the speed is increased. pwv was also normalized by bp and hr, applying the same methodology used for rwtt. the augmentation index (ai) is defined as the percentage ratio of the pressure increment caused by the reflected wave to the direct wave. normally, the reflected component in peripheral waves is always smaller than the direct component and ai is negative. in case of high arterial stiffness, the addition of the reflected component caused by different timing may exceed the direct component and the index becomes positive. ai is strongly dependent on hr, so the index is corrected for a hr of 75 bpm as described above. finally, we calculated the ambulatory arterial stiffness index (aasi), as one minus the slope of regression of dbp relative to sbp. the analysis of 24-hour bp recordings was preceded by removal of artifacts according to the previously described editing criteria. recordings were considered valid when at least 70% of expected measurements were available, as recommended by current guidelines [16, 19 ]. all the bp and arterial stiffness indices estimated in each single bp measurement were averaged for any given subject in order to obtain the 24-hour mean value. additionally, the daytime and nighttime subperiods were defined according to sleeping times reported in the individual patient 's diary cards : average measures for such awake and asleep periods were then computed. mean values obtained in each individual subject were averaged for the whole study population, separately for the healthy normotensive and hypertensive group. differences in hemodynamic indices were assessed by analysis of variance, without adjustment (crude estimate) and after accounting for age, gender, body mass index (bmi), antihypertensive drug treatment, and mean abp (adjusted estimate). adjustment for mean abp was not applied to normalized rwtt and normalized pwv, because these measures were already normalized to a sbp of 100 mmhg and to central bp. comparison of categorical variables was made by a chi - square test. to check the relation between the studied parameters, pearson 's correlation coefficient (r) data are shown as mean sd or as mean and 95% confidence interval for continuous variables and as absolute (n) and relative (%) frequencies for discrete variables. overall, 916 people were recruited and performed an abp recording, of which 182 were healthy volunteers and 734 were hypertensive patients. in the control group, 40 subjects were not considered eligible for inclusion in the analysis because either of abp recordings did not meet quality criteria (n = 10) or office or abp were elevated. in the hypertension group thus, in summary, 803 subjects were included in the analysis, of which 142 were controls and 661 were patients with arterial hypertension. the average percentage of valid readings obtained over the 24 hours was 93.9% in the healthy control and 93.1% in the hypertensive group. the average number of valid readings available during the day was 32.9 9.4 in the control group and 26.7 5.6 in the hypertension group, while the corresponding figure for the nighttime period was 7.6 2.6 and 8.2 2.6. comparison of the baseline clinical characteristics of the two groups showed that hypertensive patients were older, were more often females, were thinner, had higher office sbp values, and had higher abp than healthy controls (table 1). a quarter (24.1%) of the hypertensive patients were regularly taking bp lowering medications. both 24-hour central sbp and dbp were significantly higher in hypertensives than in healthy controls (table 2). rwtt was significantly lower, whereas aortic pwv and peripheral and aortic ais were significantly higher, in the hypertension group (table 2). after adjustment for confounding factors, a statistically significant between - group difference was still observed for central bp, rwtt, and ai, only. when indices were assessed separately for the awake and asleep periods, before adjustment, all of them were significantly different between the two groups for both the daytime and nighttime periods, with the exception of nighttime pwv (figure 1(a)). after adjustment for confounding factors, only rwtt and central bp resulted systematically different between healthy subjects and hypertensive patients for both daytime and nighttime (figure 1(b)). to note, all estimates of vascular health displayed a typical circadian rhythm : during night sleep rwtt and ai increased, while pwv and central bp decreased. such a pattern was lost after correcting rwtt and aortic pwv by sbp and hr and peripheral ai by hr (normalized indices). as shown in table 3, for both healthy subjects and hypertensive patients the relation between age and bp or between age and the different indices of arterial stiffness was statistically significant : the only exception was peripheral sbp and aasi in controls. the magnitude of the correlation coefficient was the highest for ai, with no differences between controls and hypertensives. conversely, a weak relationship was observed between brachial bp and the different indices of arterial stiffness, though in some cases such a correlation was statistically significant, particularly in the hypertensive group (table 3). the different measures of arterial stiffness were variably correlated with each other (table 4). regarding central bp, the best correlation was found between peripheral or aortic ai and aortic sbp, with higher values in the hypertensive group. aortic ai and brachial ai were highly correlated with each other, as was the case with rwtt versus pwv. a poor correlation was observed between pwv and ai and between all arterial stiffness indices and aasi, the only exception being represented by ai in hypertensive patients. in almost all cases correlation coefficients were better in healthy controls. in this study we report on the absolute levels and circadian pattern of arterial stiffness indices and central hemodynamics evaluated in dynamic conditions over the 24 hours in a large cohort of healthy volunteers and hypertensive subjects. estimation was based on brachial pulse wave analysis of oscillograms obtained noninvasively by a validated cuff - based bp measuring device. we documented higher peripheral and central bp, higher pwv and ai, and lower rwtt in hypertensive than in normotensive subjects, suggesting that arterial indices derived from oscillometric ambulatory bp measures may help to detect differences in arterial function and to investigate vascular impairment in hypertension. when crude estimates were corrected by confounding factors (age, gender, bmi, and antihypertensive treatment 24-hour average bp levels), statistically significant between - group differences were still observed for central bp, rwtt, and ai, only. this suggests that these indices may be unaffected by intrinsic subjects ' characteristics and/or bp levels and may thus represent a more sensitive index for evaluating arterial function, at least in ambulatory conditions. the different indices displayed a typical circadian pattern, regardless of the normotensive or hypertensive status. in particular, central bp followed a diurnal course similar to that of peripheral brachial bp and thus decreased during nocturnal sleep. conversely, ai increased overnight, likely because this index is inversely related to heart rate, which decreases at night, and it is strongly affected by the body posture, with absolute values increasing during recumbency [20, 21 ] and decreasing from supine to upright position, irrespective of age, due to a decrease in arterial wave reflection [22, 23 ]. standardization of the ai to heart rate removed the diurnal profile, in case of being peripheral but not aortic ai. rwtt and pwv both depend on bp and were, respectively, higher and lower at night than at day. as in the case of peripheral ai, circadian pattern of rwtt and pwv both disappeared after correction for sbp and hr, suggesting that standardized parameters may be more robust as compared to uncorrected ones. all these findings are in line with and confirm those obtained in healthy volunteers or hypertensive patients with other oscillometric devices ; although such studies were based on a different technology, they were carried out in smaller groups of subjects and the majority of them evaluated central bp only [11, 2426 ]. our study also adds data to existing evidence collected in normotensive volunteers with the same technology. interestingly, regardless of the awake or asleep period and of the presence or absence of a circadian rhythm and with the only exception of nighttime pwv, average transit time was lower and pwv, ai, and central bp were higher in hypertensive than normotensive individuals. we also examined the correlation between the different central and peripheral hemodynamic and arterial stiffness indices. a close relation was found between age and 24-hour arterial stiffness, in both healthy individuals and hypertensive patients, confirming previous evidence collected in resting conditions [28, 29 ]. abp was weakly correlated with arterial stiffness indices suggesting that normalized pulse wave analysis may provide an estimate of arterial function involvement independently of bp levels. however, further studies are required in this sense. though limited in size, the statistically significant correlation between the two main measures of arterial stiffness (pwv and ai) and between peripheral and central ai, found in our study, is consistent with results of published reports [28, 3037 ]. however, our study is the first documenting such a relation in ambulatory conditions and in either apparently healthy subjects or hypertensive patients. finally, pwv was poorly related to aasi, a finding which is in contrast with the results of a recent systematic review and meta - analysis of 51 cross - sectional and longitudinal studies in adults, which reported a good correlation between pwv and aasi. however, unlike our study, in all the studies included in the meta - analysis, pwv was measured in resting and not in ambulatory conditions. conversely, our results support the evidence of another study which explored the relative importance of the different determinants of the aasi through a previously validated one - dimensional computer model of the arterial circulation applied to 10,000 abpm simulations. outcomes of such study suggest that the aasi may not accurately reflect arterial stiffness in ambulatory conditions. the results of the present study must be interpreted also in the context of its limitations. first of all, we assessed arterial parameters noninvasively by applying transfer function analysis to an oscillometric reconstructed waveform rather than via a direct measurement. indeed, several authors have questioned the goodness of the principle of one - site central bp, pwv, and ai measurements by oscillometry [7, 40, 41 ]. however, at present, oscillometry is a method that can be easily and conveniently employed for 24-hour monitoring of central hemodynamics, allowing obtaining repeated measurements in daily life conditions with hardly any discomfort to the patient. additionally, the device used in our study has been properly validated versus alternative algorithms for computing arterial stiffness indexes and central hemodynamics, according to commonly accepted and standardized protocols. all these studies documented a good agreement between the oscillometric cuff - based estimates of central bp, pwv, and ai measured by the bplab and the established radial tonometry methods [12, 14 ]. we must acknowledge that all these validation studies were conducted in resting laboratory conditions and not in ambulant subjects. thus, we can not exclude that values collected in dynamic conditions might be, at least in part, unrelated to those collected with other devices at rest. we can only rely on studies documenting that the feasibility and reproducibility of noninvasive assessment not only of bp but also of vascular biomarkers derived from the pulse wave analysis of oscillograms by the vasotens technology are acceptable [10, 14 ]. second, absolute data collected in this study may be useful as a reference for indices collected with the same device but may not be used for other ambulatory devices, which are based on different algorithms. third, our hypertensive subjects were characterized by office dbp values that were on average slightly lower than those observed in healthy individuals, while sbp values were only marginally higher. fourth, though based on a large sample of subjects, information provided by our study needs to be corroborated by data collected in future large cohort studies. we need to specifically address arterial stiffness indices and central hemodynamics in extended age ranges, in high - risk hypertensive patients, and in subjects with established target organ damage or comorbidities, such as diabetes. in this regard, a large database of patients evaluated at different centers has been recently established (vasotens registry). our results suggest that noninvasive assessment of ambulatory arterial stiffness and central hemodynamics may be feasible and help in assessing the degree of impairment of the arterial tree in hypertensive subjects in daily life dynamic conditions. such an approach may help unraveling subclinical organ damage or functional changes, for instance, due to an increased sympathetic tone, which are typically associated with hypertension. however, further observations in diverse populations are required before ambulatory assessment of the central hemodynamic variables can make it to the clinical practice. future studies should validate whether the assessment of noninvasive 24-hour central hemodynamics can provide further information regarding cv risk stratification and target organ damage beyond the 24-hour brachial bp. additionally, reference values specifically obtained by the bplab monitor, ideally in prospective studies, are needed. nevertheless, our data may be used as preliminary diagnostic values of bplab abpm additional indices in adult healthy normotensive and hypertensive subjects. | objective. central blood pressure (bp) and vascular indices estimated noninvasively over the 24 hours were compared between normotensive volunteers and hypertensive patients by a pulse wave analysis of ambulatory blood pressure recordings. methods. digitalized waveforms obtained during each brachial oscillometric bp measurement were stored in the device memory and analyzed by the validated vasotens technology. averages for the 24 hours and for the awake and asleep subperiods were computed. results. 142 normotensives and 661 hypertensives were evaluated. 24-hour central bp, pulse wave velocity (pwv), and augmentation index (ai) were significantly higher in the hypertensive group than in the normotensive group (119.3 versus 105.6 mmhg for systolic bp, 75.6 versus 72.3 mmhg for diastolic bp, 10.3 versus 10.0 m / sec for aortic pwv, 9.7 versus 40.7% for peripheral ai, and 24.7 versus 11.0% for aortic ai), whereas reflected wave transit time (rwtt) was significantly lower in hypertensive patients (126.6 versus 139.0 ms). after adjusting for confounding factors a statistically significant between - group difference was still observed for central bp, rwtt, and peripheral ai. all estimates displayed a typical circadian rhythm. conclusions. noninvasive assessment of 24-hour arterial stiffness and central hemodynamics in daily life dynamic conditions may help in assessing the arterial function impairment in hypertensive patients. |
polycystic ovary syndrome (pcos), one of the most common endocrine disorders occurring during reproductive age, is characterized by ovulatory dysfunction, biochemical or clinical hyperandrogenism, and polycystic ovaries. its prevalence ranges from 5% to 20% depending on the diagnostic criteria used [2, 3 ]. pcos is currently considered a syndrome with metabolic consequences that could affect women 's health during different stages of reproductive age. several studies have highlighted that the risk for maternal, neonatal, and obstetric complications may be increased in women with pcos [57 ]. gestational diabetes mellitus (gdm) is the most commonly reported pregnancy complication in women with pcos. pregnant women with pcos have been reported to develop insulin resistance and impaired -cell function. this pathogenic mechanism may be associated with glucose intolerance, resulting in a greater incidence of gdm in women with pcos. observational studies have revealed an association between pcos and gdm, hypertension during pregnancy, and preterm birth. however, these studies are limited by significant heterogeneity, which indicates that the reliability of the finding of increased risk of pregnancy and adverse birth outcomes in women with pcos could be compromised [9, 10 ]. therefore, properly designed studies should be performed before formulating recommendations for pregnant women with pcos. determining the risk for gdm and adverse birth outcomes in women with pcos is important for preventive intervention through screening in the early stage of pregnancy. we conducted a large historic cohort study of pregnant women, including those who conceived spontaneously and through assisted reproductive technology, to assess the risk for gdm and adverse birth outcomes among chinese women with pcos. this historic cohort study was performed at guangzhou women and children 's medical center (gwcmc), china, between january 1, 2011, and december 31, 2014. the inclusion criteria were singleton pregnancies, 35 days or < 10 periods / year. all pregnant women at the antenatal clinic in gwcmc underwent a routine 75 g oral glucose tolerance test between 24 and 28 weeks of gestation. gdm was diagnosed according to the modified international association of diabetes and pregnancy study groups (iadpsg) criteria when one or more of the following glucose levels were elevated : fasting plasma glucose level 5.1 mmol / l, 1 h plasma glucose level 10.0 mmol / l, and 2 h plasma glucose level 8.5 mmol / l. preterm birth was defined as birth at < 37 weeks of gestation, classified as moderately (32 to 36 weeks) and very preterm birth (< 32 weeks). sga was defined by a fetal growth less than the 10th percentile at each completed week of gestation, and lga was defined by a fetal growth greater than the 90th percentile, according to the report on chinese infants born between 28 and 44 weeks of gestation in 2014. we defined sga and lga at less than 28 weeks of gestation based on the united states national reference, because of the lack of a reference for the chinese population. low birth weight (lbw) was defined as a birth weight < 2500 g. macrosomia was defined as a birth weight 4000 g. potential confounders included characteristics with a possible association with gdm, preterm birth, and fetal growth, including maternal age, maternal education, parity, prepregnancy body mass index (bmi), use of assisted reproductive technology, gestational age at delivery, and newborn sex. the institutional review board of guangzhou women and children 's medical center approved the study. we compared women with pcos and those without pcos group using the chi - squared test for categorical variables and t - test for continuous variables. multivariable logistic regression models were used to examine the association of the risk for gdm and adverse birth outcomes with pcos after adjusting for confounders. the crude and adjusted odds ratios (ors) with 95% confidence intervals (95% cis) were computed to estimate the degree of association. statistical analysis was performed using the statistical package spss, version 20 (spss inc., demographic characteristics of mothers and newborns according to pcos status are presented in table 1. women with pcos before early pregnancy were more likely to be older, had higher prepregnancy bmi, and used assisted reproductive technology compared with women without pcos. table 2 presents multivariate associations of pcos during pregnancy with gdm and birth outcomes. in the adjusted analysis, women with a previous diagnosis of pcos had a higher risk for gdm than women with no such diagnosis (adjusted or 1.55, 95% ci : 1.142.09). there was also a strong association between pcos and preterm birth (adjusted or 1.69, 95% ci : 1.082.67). in the stratified analysis using multivariable logistic regression, the adjusted or for gdm among women with pcos undergoing assisted reproductive technology was 1.44 (95% ci : 1.031.92) and among women with pcos who conceived spontaneously was 1.60 (1.182.15) (figure 1). also, the risk of preterm birth was increased in women with pcos regardless of use of assisted reproductive technology. the present study, in which maternal age at birth, parity, education, prepregnancy body mass index, and use of assisted reproductive technology were controlled for, indicated that a previous diagnosis of pcos increases the risk of gdm and preterm birth, although no increased risk for other adverse birth outcomes was observed. a 1.5-fold increased risk for gdm in women with pcos in early pregnancy was seen in our study. this finding is consistent with that of another study, which reported the incidence of gdm to be 19.2% in jewish women, 44.4% in iranian women, 40.9% in american women, and 26.9% in mexican women with pcos compared with 3%, 7.3%, 9.4%, and 9.6% in women without pcos, respectively [3, 1416 ]. these findings show that pcos has a different impact on the risk of gdm depending on ethnicity. there are three possible explanations : (1) pcos has different characteristics and clinical impact in different ethnic groups, (2) increased insulin resistance in pcos has different clinical effects depending on the insulin metabolism characteristic of different ethnicities and environments, and (3) the prevalence of gdm in women with pcos is affected by the different diagnosis criteria (the who or the modified iadpsg criteria) [3, 17 ]. in addition, pcos is a major cause of infertility in women, and these women might require assisted reproductive technology to become pregnant. some studies have suggested that assisted reproductive technology is associated with an increased risk of gdm [1921 ], which indicated that women with pregnancies that were conceived while undergoing assisted reproductive technology have impaired glucose tolerance compared with those who conceived spontaneously. of note, most prior studies have not examined pcos status independently of assisted reproductive technology. in this setting, we did a stratified analysis in two separate groups, one restricted to women undergoing assisted reproductive technology versus those who were not. after regression analysis of the two separate groups, we showed that women with pcos had a greater risk of gdm during pregnancy regardless of assisted reproductive technology. women with pcos were associated with an increased risk of gdm that could not be attributed to the increased use of assisted reproductive technology. this may explain our findings that pcos, an inherent insulin resistant condition, is independently associated with gdm. similarly, a large population based cohort study found that pcos was strongly associated with very preterm birth, and a systematic review showed that pcos increased the risk of preterm birth by at least 2-fold. however, the pathophysiological mechanisms underlying the association between pcos and preterm birth are not completely understood. various etiologies have been suggested, including the increased incidence of multiple pregnancies and nulliparity, the associated increased estrone levels, hyperinsulinemia, and the subsequent diabetic and hypertensive predispositions [5, 24, 25 ]. in previous reports, women with pcos often required assisted reproductive technology to become pregnant, increasing the risk of multiple births and hypertensive disease, which are associated with preterm birth [2628 ]. the association between pcos and preterm birth may thus be an interaction with assisted reproductive technology. in our stratified analysis, the results did not support the statement that adverse pregnancy outcomes among women with pcos were mediated by assisted reproductive technology. there was no significant association between the interaction of pcos with assisted reproductive technology and preterm birth. this finding is supported by two studies from northern europe [2, 29 ], which reported that preterm birth associated with assisted reproductive technology could be explained by factors that lead to infertility, rather than the assisted reproductive technology. in the current study, we could not identify the pathophysiological mechanisms behind the increased risk of preterm birth among women with pcos, a potential relationship that should be addressed in future studies. a major strength of this study was the relatively large sample size and the fact that potential confounding variables that were not controlled for in most previous studies were controlled for in this study. we also included women who conceived spontaneously and those who conceived by assisted reproductive technology to study pregnancy outcomes. however, certain limitations must be noted. the sample size was based on the primary outcome and is therefore not suitable for estimating the risk for all perinatal outcomes such as stillbirth and neonatal death. because the study was a retrospective cohort study, hormone levels were not measured to examine individual pcos status, and data on medical therapy for pcos were not available. in summary, our results suggest that women with pcos were more likely to develop gdm and experience preterm birth. future longitudinal studies are needed to better determine the underlying processes of pcos during gestation and to develop efficient preventive strategies to preclude the adverse effects on both the mother and child. | objective. to examine the association of polycystic ovary syndrome (pcos) in early pregnancy with gestational diabetes mellitus (gdm) and adverse birth outcomes. methods. in this retrospective cohort study including 2389 pregnant women, the medical records of 352 women diagnosed with pcos were evaluated. outcomes included gdm, preterm birth, low birth weight, macrosomia, and being small and large for gestational age. multivariable logistic regression models were used to examine the association of the risk for gdm and adverse birth outcomes with pcos after adjusting for confounders. results. women previously diagnosed with pcos had a higher risk of gdm (adjusted odds ratio [or ] 1.55, 95% confidence interval [ci ] : 1.142.09). a strong association was seen between pcos and preterm birth (adjusted or 1.69, 95% ci : 1.082.67). on stratified analysis, the adjusted or for gdm among women with pcos undergoing assisted reproductive technology was 1.44 (95% ci : 1.031.92) and among women with pcos who conceived spontaneously was 1.60 (1.182.15). no increased risk for other adverse birth outcomes was observed. conclusions. women with pcos were more likely to experience gdm and preterm birth. |
growth hormone deficiency (ghd) is one of the most important endocrine and treatable causes of short stature (ss). children with a height that is at least two standard deviations (sds) below average or approximately the third percentile for that age and gender are deemed to have ss. in the us alone, 90,000 children have height below the second percentile and are classified as having ss. considering a cut - off of fifth percentile that is often used in clinical practice, about 150,000 children a year would be referred as having ss. in a developing nation like india, perception of height as a marker of general health is less pronounced as compared to weight, due to the social health system being more focused on common causes of malnutrition, rather than on normal growth and development. this factor might lead to underdiagnosis of pathologic causes of ss and other conditions potentially associated with poor growth. further, there is insufficient awareness of the need to measure height, height measurement techniques, and thresholds for referral among primary care physicians. it is often up to families to recognize that their child is not gaining proper height and seek advice from a primary care physician. in the majority of cases, the families wait until adolescence and late puberty, when chances to improve final height are limited. the prevalence of ghd in children with ss ranges from 2.8% to 69% [36 ]. ghd is also estimated to be prevalent in more than 80% patients undergoing postneurosurgical procedure [7, 8 ]. in india, a study by colaco. assessed the profile of ghd in 430 children in bombay and found that 31% of the children had familial ghd and about 17% of children had idiopathic ghd. these studies are biased by patterns of referral to tertiary centres of care. however, there is no pan - india study on the prevalence of ghd. the studies that have been conducted so far are few and regional [9, 10 ]. the lack of larger prevalence studies hinders accurate estimation of the magnitude of ghd problem. knowing the prevalence would greatly help in streamlining the screening of children with ghd. diagnosis of ghd is based on a combination of auxology, biochemical analyses such as growth hormone (gh) stimulation tests and insulin - like growth factor 1 (igf-1), skeletal age, magnetic resonance imaging (mri), and exclusion of other systemic diseases which can have a similar presentation [1618 ] (table 1). diagnosis of ghd is more challenging in resource constrained countries like india for various reasons. recombinant human gh therapy was first approved for children with ghd in 1985 and later for the treatment of various conditions like idiopathic short stature (iss), turner syndrome (ts), noonan syndrome (ns), prader - willi syndrome (pws), chronic renal failure (crf), and small for gestational age (sga) [1921 ]. in india, diagnosis and treatment of ghd are hurdled with various challenges restricting the availability of gh therapy to only a very limited segment of the children. this review will look at the challenges in diagnosis and treatment of ghd in india. in march 2015, merck serono convened discussion forums in six indian cities (delhi, bangalore, kolkata, hyderabad, chennai, and kochi) to identify the challenges in diagnosis and management of gh deficiency in the country. leading experts in endocrinology discussed their views on overcoming these challenges. challenges on diagnosis and management of the growth disorders were formulated based on a literature search conducted in databases including the us national institutes of health (pubmed), medline, scopus, and google scholar and the findings were discussed at the forums of experts. the literature search identified gaps in diagnosis of ghd (suboptimal referral of children with growth disorders, poor recognition, and lack of proper training and education of physicians regarding growth disorders and lack of easy availability of latest diagnostic technologies with accuracy and reproducibility) and optimizing growth response and issues with patient adherence to therapy as the key challenges in the recognition and treatment of ghd. suboptimal referral of children with growth disorders to the endocrinologist was agreed to be the single most important obstacle for diagnosis and management of ghd in india. most pediatricians tend to focus on measuring weight rather than height despite the availability of indian growth charts [31, 34 ] and guidance from the indian academy of pediatrics (iap). advantages and disadvantages of indian growth charts are presented in table 2. for children under the age of 5 years, the indian government and iap have accepted the new standards for growth monitoring from world health organization (who) released in 2006. and, for the children in age group 518 years old, the revised iap growth charts are recommended. several endocrinologists mentioned that insufficient medical and family history does not allow them to make accurate growth predictions. others pointed out the central board of secondary education (cbse) electronic health record data that has been collected but never used for referrals. it would be reasonable to select appropriate cut - offs (e.g., 3rd and 97th centiles) to get both short and tall children being referred to primary or secondary care with further investigations, for example, height sd score (hsds) being calculated by nurses or pediatric doctors and a decision made for further referral to endocrinologists. in studies from india, the mean age of patients diagnosed with ghd for various trials range from 8.6 to 14 years (table 1). however, in general clinical practice, outside of clinical trial settings, most physicians participating in the advisory boards were commenting on late referral for the first consultancy of children with growth disorders. to the best of our knowledge, no literature is available on late referral of children with growth disorders in india. however, it is acknowledged that patients with hypothyroidism have been referred late as well. this suggests the need for a threshold for referral in order to identify growth abnormalities in children. the sd score (sds) used in the detection of ss is helpful in distinguishing between normal and ss due to ghd. the dutch consensus guidelines (dcg) interpreted the cut - off value for referral as sds < 1.3 sd in order to identify the risk groups that need further evaluation. in addition, the guidelines provide several other referral criteria including clinical symptoms, persistent ss after being born sga, height standard deviation score (hsds), and growth deflection [37, 38 ]. a study by grote. compared the referral criteria of the dcg with those of the uk consensus guideline (ukcg) and the who global database on child growth and malnutrition cut - off values. the study concluded that too many children aged < 18 years (nearly 80%) would be referred if we use dcg, whereas use of the ukcg leads to only 0.3% referrals and the who criteria to approximately 10%. in a consensus document by the iap, clear guidelines are given on growth monitoring, plotting on growth charts, and criteria for referral (figures 1 and 2). however, implementation of these guidelines is not satisfactory. the who has also developed growth monitoring charts ; and a training course is available to help in assessing child growth. suggested that, for a good differential diagnosis, it is essential to get medical history, family history, physical examination, analysis of the growth curve, and weight - for - height measurements. a review by nwosu and lee on evaluation of short and tall stature in children suggested that firstly a thorough history and physical examination should be conducted and the laboratory investigations should then be based on the finding of these examinations. in india, children are followed up for immunization and minor illness by general practitioners and pediatricians in most areas. weight recording to identify protein - energy malnutrition (pem) has been ingrained in pediatric practice, as pem is still a major public health problem in india. height measurement is also critical to assess wasting (weight for height) and stunting (height for age). this is justified by the study of growth retardation by nath. where pem and chronic anemia resulted in more than 60% of cases of ss. in various endocrine causes of growth failure like ghd including hypopituitarism, primary hypothyroidism, precocious puberty, and other rare congenital genetic disorders (e.g., ns, ss homeobox - containing gene deficiency (shox - d), and ts), there is a failure to gain height but the weight continues to be within normal centiles [43, 44 ]. the other important factor that contributes to delay in diagnosis of growth disorders is poor recognition and understanding of growth disorders by pediatricians. this was closely associated with delayed societal alertness, whereby families only start to worry about the short height of their child at the late adolescent age. at this stage, the growth potential is greatly diminished and the efficacy of gh treatment interventions is limited. although several diagnostic tools are available for the diagnosis of growth disorders, none of them can be completely relied upon to confirm the diagnosis [4547 ]. tests used for the diagnosis of ghd include auxology, measurement of igf-1 and igf binding protein 3 (igfbp-3), radiographic assessment of bone age, cranial mri, gh provocation testing, and genetic testing [4547 ]. igf-1 and igfbp-3 are other commonly suggested tools for screening (or confirmation) of ghd. igf-1 measurement is limited because of the sensitivity of the assay and the results may be inaccurate because igf-1 circulates as a complex with acid labile subunit (als) or with igfbp-3 or other igfbps. juul and skakkebaek conducted a study to assess the outcome of igf-1 and igfbp-3 in screening children for ghd. in children < 10 years, the sensitivity of igf-1 and igfbp-3 was reported to be 53.3% and 60%, respectively. another study by cianfarani. also showed that igf-1 and igfbp-3 possessed a sensitivity of 73% and 30%, respectively. these studies have inferred that both these tests possess good specificity but lack sensitivity. however, in india there is a lack of availability of these assays in semiurban and rural areas. although normative igf-1 data for indian children has been derived, it is seldom used in commercial laboratory reports. gh provocation testing is one of the other methods of diagnosing gh deficiency. despite their limitations in terms of types of provocation stimuli, the need for sex steroid priming, cut - off levels for ghd diagnosis, assay related problems, and lack of normative data, gh provocation testing is commonly used by most practitioners for decision - making on initiating treatment. in india, clonidine is the most commonly used gh provocation stimulus. the insulin tolerance test is labour intensive and most advisors felt that it is impractical outside an academic setup. rarely, glucagon is used as gh provocation stimulus. other stimuli, such as gh releasing peptide-2 (ghrp-2), arginine, and l - dopa, are not available in india routinely. however, physicians should not underestimate clinical clues that increase the likelihood of abnormal mri findings and congenital pituitary hormone deficiencies, particularly with regard to facial dysmorphology and more common clinical syndromes. outside india, gh1 and gh releasing hormone receptor (ghrhr) mutations have been identified in several clinical studies to be associated with ghd and familial cases of ss. genes, for example, hesx1, prop1, pou1f1, lhx4, and lhx3, could also be considered based on the probability of identifying a mutational lesion that may be responsible for phenotype. in india, desai. in their study of 31 patients with ghd reported that 22 (71%) of the patients had a homozygous g to t transversion in exon 3. the majority of the patients (71%) had an e72x mutation in the ghrhr gene. other genes known to be associated with ss include ptpn11, sos1 (ns), fgfr3 (achondroplasia and hypochondroplasia), shox (shox - d), npr2, aggrecan, and pappa2 [5456 ]. shox deficiency is the first indication being approved for gh treatment that requires genetic testing. the availability of genetic testing for ss syndrome is limited outside metropolitan cities and is expensive. the growth genetics consortium (ggc), an international collaborative effort, has created a public database and website which includes information on molecular defects of the gh - insulin growth factor (gh - igf) axis. this database can provide guidance to healthcare professionals for identification, evaluation, and management of patients with defects of the gh - igf axis. somatotropin, a recombinant gh, is used for treatment of several conditions including ghd, ts, iss, sga, pws, crf, and ns. however, inappropriate growth response to gh treatment has been observed in clinical studies [59, 60 ]. multiple factors result in an inappropriate response to gh therapy including significantly late initiation of the therapy and dosage limitations imposed by regulatory authorities. variability in response to treatment from person to person may be due to several characteristics including diagnosis, body composition, age, and several other exogenous and endogenous factors. thus, to analyze or predict the probable amount of growth that can be expected during treatment, researchers have developed prediction models. ranke. developed and validated a gh treatment prediction model for patients born with sga using the data of children from the kigs (pharmacia international growth database) and/or those who participated in previous clinical trials. this model inferred that gh dose is the crucial factor for response prediction. despite possessing several advantages associated with the use of prediction models, their use in clinical practice is still limited. the unavailability of user - friendly software systems and the lack of prediction models for the indian population deter physicians from considering the use of these prediction models. the second important challenge which limits the effectiveness of gh therapy is patient adherence. a literature search has found several studies which identified that poor adherence is the major factor that reduces the effectiveness of gh therapy [63, 64 ]. aydin. conducted a multicentric study on 217 gh - nave patients to assess adherence to gh therapy. the study found poor adherence to the therapy and determined it to be the underlying factor responsible for suboptimal growth during therapy. a systematic review by fisher and acerini also observed that adherence to gh therapy is suboptimal. it could not identify the cause of nonadherence and recommended further research to be conducted. several factors are known to be associated with nonadherence to the therapy such as type of delivery system, discomfort with injections, cost of treatment, socioeconomic status, lack of communication / training from healthcare providers, poor understanding of disease and consequences of missed doses, requirement for long - term treatment, and lack of immediate clinical improvement and peer or psychosocial pressure (e.g., during adolescence) [57, 65 ]. poor response to gh therapy can be identified and managed as presented in figure 3. bozzola. suggested that regularly interviewing ghd patients could be a useful approach to improve adherence and also mentioned that communication with patients and their parents should be in a nonaggressive manner. muller. conducted a randomized crossover study comparing a liquid formulation of gh with the older freeze - dried product and found that an overall preference was given to the liquid product, with 98% patients rating it as easier to use. in another study by iyoda. optimization of the preservative and buffer content of a liquid gh formulation may reduce injection pain and, hence, improve patient compliance. an observational study assessed treatment adherence with the easypod in children and their views regarding its use. a total of 87.5% patients showed adherence to the therapy during the 3-month period of the treatment. more than 80% of children reported it to be easy to use, speedy, and comfortable. this device is unique as it helps in tracking the daily injections of the medication and thus can help in assessing whether it has been taken as prescribed. a study on the use of recombinant somatotropin (r - hgh) as a long - term therapy (11 years) to treat ghd reported positive catch - up growth response and bone age acceleration in accordance with age versus height age. the median hsds improved significantly from 3.8 at baseline to 3.3 (p < 0.001) during the first year of r - hgh therapy and improved further to 1.5 after 7 years of the therapy. this review has summarized the challenges associated with the timely diagnosis of ghd and treatment. experts at the advisory meetings also presented their suggestions to improve early diagnosis of ghd, optimization of gh therapy, and patient adherence. basic education to the public on the awareness of growth monitoring will aid in improving referrals of the children. suggestions to improve referral were based around early enough awareness of height delay, with one or two critical time points during infancy, in children 4 - 5 years of age and before the onset of puberty, when early intervention would aim to improve adult height. several schools have initiated a regular height and weight measurement activity, the data for which are collected and stored in the database owned by cbse. however, these data are underutilized and not transferred to healthcare institutions. simple tools such as electronic excel databases which allow data to be collected and make a simple analysis by selecting subgroups of children with ss (e.g., below 3rd or 5th centile of height for chronological age) will be useful. these tools will improve referral of children with ss to secondary care and eventually improve late diagnosis of disorders associated with ss beyond ghd. advisors suggested having further collaboration with the endocrine society of india (esi) to implement joint efforts to improve referral of children with ss related disorders. they also opined that screening guidance needs to be revisited and should endorse collaboration with iap, the esi, indian society for pediatric and adolescent endocrinology (ispae), and cbse electronic health record data. at advisory board meetings, questions on awareness about the best practice of using stadiometers were also raised (e.g., selection of the right stadiometer, measurement technique, and stadiometers that do not pass quality control). a stadiometer, which consists of a vertical ruler with a sliding horizontal rod, is a tool to measure height accurately. since accurate measurement of height is the key to diagnose growth disorders in a child and is usually done by nurses and primary care physicians, it is of great importance that these healthcare providers are trained in the minimal standards of good quality stadiometers and height measurement techniques. plotting of the growth chart helps in assessing a child 's growth pattern over time. have developed charts that can help in monitoring the growth from birth up to the age of 18 years, unlike the who charts that can be used to monitor growth only up to the age of 5 years [34, 72 ]. these charts are recognized to be the best charts for growth monitoring in indian children. the iap growth chart committee recommends the use of revised growth charts for height, weight, and body mass index (bmi) for children in the age group of 518 years as suggested by khadilkar. and who standards for growth assessment in children below 5 years of age. the panel also emphasized the need to create awareness among parents to remain cautious if the child is growing well till the age of 4, suggesting that parents should initiate monitoring if they then observe that the child is not growing well after the age of 4 years. a visit to a specialist is recommended at least 1 year before the start of puberty (i.e., 8 years in girls and 9 years in boys). it was suggested by the panel members that it should be the social responsibility and joint efforts of all stakeholders (government, schools, and healthcare institutions) to implement an effective screening system that includes height as one of the measurements. this screening should include measurements of height in addition to weight, eye sight, and dental care. the use of dcg criteria for referral suggested that a large percentage of the referrals were mainly due to the deflection of length during the first 3 years of life. nevertheless, availability of growth charts, awareness, and training of medical personnel to measure growth properly and plotting data on the growth chart was agreed to be the cornerstone of success towards early diagnosis / detection of growth disorders [31, 34 ]. although several diagnostic tests are available for the diagnosis of ghd, none of them can be relied upon independently. in one of the advisory board meetings it was suggested that diagnosis of ghd should be based on multiple diagnostic criteria, since there is no standard diagnostic test. experts also mentioned the need to improve availability and standardization of gh, igf-1, and igfbp-3 assays for the diagnosis of ghd. in addition, the results are also affected by the pattern of gh secretion prior to the administration of the stimulus. short - term nutrition is another important factor that affects the plasma concentration of igf-1, which is reduced by undernutrition even with high gh secretion. thus, there is a need for other tests to accurately assess ghd [45, 73 ]. a review by clemmons reported obstacles in gh and igf-1 standardization which include use of different calibrator materials, varying results with the assays because of the different antibody types used that bind to different forms of gh and igf-1 and the effect of matrix component. binding proteins including gh - binding protein (ghbp) and igfbps also interfere with the assay findings. thus, to improve the standardization of the different assays, these obstacles need to be overcome. patient compliance is of critical importance to ensure benefits of the treatment. in the past, today, we have entered an era of virtually unlimited supply of gh although the cost still remains a limiting factor. the advisory board suggested the following : a therapy device that is easy to use and appealing to children would greatly help in improving patient adherencereduce the cost of gh therapy and support the use of gh treatment by the governmentdesigning robust patient support programs will help to achieve good adherenceorganize educational activities (educational camps) for patients and parentssocial media can be a good mode to spread awareness about the early diagnosis and treatment of ghdpatients should be asked to use calendars to improve adherence to the therapy and doctors can take the initiative to send reminders to the patients via text messages on their phones for their upcoming appointmentsincreased family and social support to the patient can also help in improving adherence a therapy device that is easy to use and appealing to children would greatly help in improving patient adherence reduce the cost of gh therapy and support the use of gh treatment by the government designing robust patient support programs will help to achieve good adherence organize educational activities (educational camps) for patients and parents social media can be a good mode to spread awareness about the early diagnosis and treatment of ghd patients should be asked to use calendars to improve adherence to the therapy and doctors can take the initiative to send reminders to the patients via text messages on their phones for their upcoming appointments increased family and social support to the patient can also help in improving adherence to date no single test has been developed that can be considered to be definitive in diagnosing ghd. therefore, more research needs to be conducted to develop a robust diagnostic criterion for ghd. further, there is a need for database and registries for monitoring various possible ethnicity - specific growth responses and adverse effects (aes). india - specific databases or registries may help provide epidemiological data for ghd in an indian context. education and awareness about growth disorders among parents would help improve the diagnosis and treatment of children with ghd. the first point of contact in a patient 's journey is that physician / pediatrician needs to be well equipped to identify cases of ghd. encouraging the use of prediction models by pediatricians / endocrinologists will help in optimizing the treatment. physicians in india should be given regular training and more emphasis should be put on using the indian growth charts for growth monitoring. further, compliance to treatment is one of the major obstacles in poor growth response. the availability of an easy - to - use delivery system would be beneficial in improving adherence and achieving satisfactory or optimal outcomes. | in clinical practice, every year approximately 150,000 children are referred with short stature (ss) based on a cut - off of fifth percentile. the most important endocrine and treatable cause of ss is growth hormone deficiency (ghd). the lack of reliable data on the prevalence of ghd in india limits estimation of the magnitude of this problem. the diagnosis and treatment of ghd are hurdled with various challenges, restricting the availability of growth hormone (gh) therapy to only a very limited segment of the children in india. this review will firstly summarize the gaps and challenges in diagnosis and treatment of ghd based on literature analysis. subsequently, it presents suggestions from the members at advisory board meetings to overcome these challenges. the advisory board suggested that early initiation of the therapy could better the chances of achieving final adult height within the normal range for the population. education and awareness about growth disorders among parents, regular training for physicians, and more emphasis on using the indian growth charts for growth monitoring would help improve the diagnosis and treatment of children with ghd. availability of an easy - to - use therapy delivery system could also be beneficial in improving adherence and achieving satisfactory outcomes. |
there is an increased incidence of major and minor congenital abnormalities in infants born to epileptic mothers (6 - 7% compared with 2c3% in the general population). sodium valproate is a popular drug because of its broad range of anticonvulsant effects and relative freedom from sedative and behavioral effects. exposure to valproic acid during first trimester can result in the constellation of minor craniofacial anomalies and major organ malformations in human fetuses. here, we report a case of a 3-month - old baby with facial dysmorphism, as a case of fetal valproate syndrome (fvs) based on the phenotype and maternal use of valproic acid during the antenatal period. a 3-month - old male child admitted with complaints of breathlessness since 3 days with fever. it was a full - term normal delivery with birth weight of 2.5 kg without any postnatal complications. mother was known case of epilepsy and was controlled on sodium valproate 800 mg / day since 3 years. before delivering this child mother had three spontaneous abortions within first trimester during those 3 years. on admission, the child was in congestive cardiac failure with heart rate-168/min, respiratory rate - 64/min. child had severe failure to thrive with current weight 3.8 kg at 3 months of age despite on exclusive breastfeeding. features of facial dysmorphism [figure 1 ] such as prominent metopic sutures, trigonocephaly, tall forehead, epicanthal folds, infraorbital groove, and medial deficiency of eyebrows, shallow philtrum, anteverted nares, and broad root of nose, low set ears, thin upper lip, and small mouth were present. broad hands and feet, loose skin [figure 2 ], and hypospadias were other features. based on facial dysmorphism, congenital heart disease and hypospadias in the setting of maternal valproic acid consumption during antenatal period, diagnosis of fvs prominent metopic suture fetal valproate syndrome on auscultation, pansystolic murmur with loud pulmonary component of second heart sound were present. two - dimensional echocardiography showed moderate atrial septal defect, ventricular septal defect with tiny patent ductus arteriosus with moderate pulmonary hypertension. ophthal evaluation, ultrasonography abdomen, and x - ray spine were within normal limits. valproic acid crosses the placenta and is present in a higher concentration in the fetus than in the mother. complications of epilepsy and antiepileptic drug treatment, include stillbirths, prematurity, low birth weight, major and minor malformations, and cognitive delay later in life. following several other case reports of the teratogenic effects of vpa, all of which documented similar major and minor anomalies. the facial features seen in fvs are trigonocephaly, tall forehead with bifrontal narrowing, epicanthic folds, infraorbital groove, medial deficiency of eyebrows, flat nasal bridge, broad nasal root, antiverted nares, shallow philtrum, long upper lip and thin vermillion borders, thick lower lip, small downturned mouth. our patient had almost all facial features of fvs that has previously been described in literature. the timing of exposure and the dose of the drug are important in influencing the outcome of pregnancy. first - trimester exposures are more likely to result in malformations as this is the main period of structural development in the fetus. the efficacy of valproic acid as an antiepileptic drug can not be disputed, but the extent of its teratogenic effects can not be under - estimated either. hence, the balance between the therapeutic effects of this drug and its teratogenic effects is critical in the management of women with epilepsy. high - dose folic acid (4 mg / day) is recommended during pregnancy, starting at least 6 weeks preconception and continuing through the first trimester. | antenatal use of anticonvulsant valproic acid can result in a well - recognized cluster of facial dysmorphism, congenital anomalies and neurodevelopmental retardation. in this report, we describe a case with typical features of fetal valproate syndrome (fvs). a 26-year - old female with epilepsy controlled on sodium valproate 800 mg / day since 3 years, gave birth to a male child with characteristic features of fvs. she also had 3 spontaneous first - trimester abortions during those 3 years. sodium valproate, a widely used anticonvulsant and mood regulator, is a well - recognized teratogen that can result in facial dysmorphism, craniosynostosis, neural tube defects, and neurodevelopmental retardation. therefore, we strongly recommend avoidance of valproic acid and supplementation of folic acid during pregnancy. |
stakeholders in public health research are researchers, funding agencies, organizations hosting research activities, policymakers, health managers, professionals in the health care system, patients and the community as well as the healthcare industry. it is important to understand the perceptions and views of various stakeholders on priorities in public health research to maximize the benefits of research1. involvement of various stakeholders apart from researchers is crucial to align the research initiatives with policies and public health programmes2. it is particularly important in view of low public health research funding and low public health research output3. the need for better collaboration between researchers and policymakers to enhance the use of research has been reported14. in uk, these efforts were successful in addressing needs of various stakeholders1. in canada, the study that evaluated the interactions between researchers and decision makers identified three models of decision maker involvement namely formal supporter, responsive audience, and integral partner4. there is a lack of data regarding involvement of policymakers in the public health research prioritization in india. health is a state subject in india, therefore, public health programmes are delivered by the state health systems. state level stakeholders who are actively involved in the implementation of public health programmes are one of the most important stakeholders in identifying public health research agenda that would lead to direct programmatic benefits. in this context, it is important to understand the public health priorities as perceived by these stakeholders. in india, department of health research (dhr) was constituted in 2007 with the mandate to serve as an apex department for medical, allied basic sciences, clinical and public health research in the country. the dhr mandate is to translate the innovations into products/ processes and to introduce these innovations into public health service through health systems research, and to strengthen the coordination between various stakeholders to increase the use of research findings in practice of public health5. a two day consultative meeting was organized by the dhr in september 2011 to bring the state government officials and public health researchers together on the same platform. a survey was conducted prior to the two day consultation in august, 2011 to obtain perspectives of state government health system stakeholders regarding public health research priorities in various indian states. the survey also covered public health researchers from leading public health organizations in india to understand their views. an exploratory study was conducted to generate information that could serve as a basis for discussion in the meeting. the major objective of the meeting was to identify the key public health research priorities as perceived by state officials and researchers. a cross - sectional survey was conducted by national institute of epidemiology (nie), chennai, india in june - july, 2011 among stakeholders of various state government and national level programme stakeholders as well as researchers from public health research organizations and academic organizations. the state officials included national rural health mission (nrhm) directors, directors of health services or state level national programme managers. public health researchers from leading national public health research and academic organizations in various states were also approached. sample size and procedure : at least two officials from all 35 states were contacted. these officials were informed that they could take the feedback from state level programme managers for the survey. nearly 30 leading public health research institutions in india were listed and at least one renowned public health researcher was contacted from each of these institutions. data collection : a self - administered semi - structured questionnaire was used for data collection. the study questionnaire included structured questions to capture demographic profile and work experience of the respondents and open ended questions requiring them to write five leading public health research priorities. additionally, the participants were requested to rank the six pre - identified public health research domains namely reproductive / maternal health including family planning, child health problems for under - five age group, adolescent health, undernutrition including micronutrient deficiencies, infectious diseases and non - communicable diseases, from one to six. the questionnaires were sent to the individuals by post and email and follow up was done by telephone and email. data collection was facilitated by graduates of epidemiology and public health training programmes of nie working in various state governments. they contacted the respective state officials, explained the background of the survey, handed the questionnaires, and made the required follow up to encourage the state officials to complete the survey. the study was approved by the nie ethics committee, and informed consent was obtained from all the participants. data analysis : the proportions for the quantitative data were computed. the data were coded from the open ended questions regarding five leading public health research priorities. overall 35 state officials from 15 indian states and 17 public health researchers responded to our request and participated in the study (figure). in response to an open ended query regarding public health research priorities, 153 responses (multiple responses from each respondent) were obtained from 35 state officials and 64 from 17 public health researchers. five leading priorities that were identified included maternal and child health (24%), non - communicable diseases (22%), vector borne diseases (6%), tuberculosis (6%) and hiv / aids / sti (5%) (table i). participating indian states (highlighted in blue) represented by state officials in the survey, 2011. public health research areas listed by experts and state officials in response to the open ended question regarding five leading research priorities for the state, 2011 in addition to open ended query, a list of five broad areas was separately ranked for urban and rural areas by the participants. reproductive/ maternal health including family planning was ranked one or two by 23 (66%) state officials and 12 (71%) public health researchers in rural areas. child health problems were ranked one or two by 18 (51%) state officials and 12 (71%) public health researchers. infectious diseases were ranked one or two by higher proportion of state officials as compared to public health researchers [12 (34%) vs. 5 (29%) ]. in contrast, undernutrition was ranked one or two by higher proportion of public health researchers as compared to state officials in rural areas (table ii). public health research priorities ranked 1 or 2 for urban and rural areas by experts and state officials, 2011 in urban areas, higher proportion of state officials ranked reproductive / maternal health at one or two as compared to public health researchers [22 (63%) vs. 4 (24%) ]. child health problems for under - five age group were ranked higher by nearly half of the state officials and public health researchers. at least one fourth of the state officials and public health researchers ranked adolescent health as one or two for urban areas. non - communicable diseases were given higher ranking by 10 (59%) public health researchers and only 9 (26%) state officials for urban areas (table ii). in addition to disease specific priorities, health systems research, community involvement and environmental issues were also identified among the leading ten priority areas (table i). various other broad research areas such as impact assessment / translational research, adolescent health, economic impact of public health programmes, technology, human resources, immunization and vaccine preventable diseases, gender empowerment, urban health, de - centralized planning and new emerging infectious diseases were among the other identified priorities. this survey was an effort to include the state level policymakers and public health managers in the process of planning relevant public health research. maternal and child health research continued to remain the leading priority ; however, public health researchers gave more emphasis on need for research in the emerging public health challenges such as non - communicable diseases and adolescent health. involvement of non researchers in the process of research to define the problems better can increase the use of research by practitioners and society. traditionally it is believed that researchers should be able to determine the research agendas on their own, however, the need for collaborative research with other stakeholders involvement in setting the agenda has also been emphasized7. several countries initiated structured interventions in 1990s to increase the collaborations between researchers and policymakers1. national health system research and development strategy - uk, agency for health care policy and research in us and prime minister 's national forum on health in canada are some of the examples of such initiatives7. in india, the dhr proposes to establish special linkages with central and state public health services to fulfill the mandate of translating the research findings to public health programmes and policies5. this survey provided an insight into the perceived priorities of important stakeholders based on the experiences in the respective states. it needs to be emphasized that these are initial steps and the survey has to be expanded and also extended to other states for getting a comprehensive picture. maternal and child health was perceived as the leading priority by researchers as well as state officials. however, in context of the need to achieve millennium development goals, this should continue to be the focus of research. this is also consistent with the health research agenda of 12 plan and goals of national rural health mission, an initiative of the central / federal government to strengthen the primary care and achieve the millennium development goals for maternal and child health68. non - communicable diseases related research was reported higher in the priority, more so for urban areas. this is consistent with rising burden of ncds in india and is one among the ten health research priorities identified in the 12 plan69. lower priority given to research related to under - nutrition by the state officials was not consistent with the data that indicated high rates of malnutrition including micronutrient deficiencies in most of the states10. this may be because nutrition is perceived beyond the health system functions and the interventions involve programmes and initiatives from other social sector departments of the government as well. however, 12 plan calls for need for convergent action on nutrition and need for monitoring the health impact of programmes and policies of non - health sector6. health systems research was identified as an important priority area only by a small proportion of participants probably due to low exposure to health systems research. health systems research is cross - cutting and may help in strengthening the health systems and implementation of various programmes. among the ten key priorities for health research in 12 plan, there are three priority areas of health systems research namely health financing, health information systems, and public health systems strengthening6. there is a need for sensitization of the public health professionals regarding scope and need for health systems research. it is possible that some respondents could not have clearly differentiated between public health priorities from public health research priorities though it had been clarified in the invitation letters. another limitation of the study was that we received the responses from 15 (42%) states even after repeated efforts to contact using various modes of communication. for every state at a particular level of progress and development which was not represented in our survey, there was a comparable state with similar geographic location which was represented in the survey. hence it is appropriate to conclude that in spite of non - representation of some states, overall national views have been captured. however, it is important to explore the views and options of the health programme managers and policymakers from the states not represented in the survey at some later time point for consensus building. special emphasis will have to be given to consider the viewpoints of stakeholders in the less developed states to consider their unique problems. strength of our study was bringing together the views of both researchers and non - researchers. in conclusion, structured initiatives are needed to promote interactions between policymakers and researchers at all stages of research starting from defining problems to the use of research to achieve the health goals as envisaged in the 12 plan over next five years. there is a need to identify specific research questions in the priority research areas that are most relevant and useful to the policymakers. special focus to promote health systems research will be required to address the cross - cutting challenges in the implementation of public health programmes. | public health research has several stakeholders that should be involved in identifying public health research agenda. a survey was conducted prior to a national consultation organized by the department of health research with the objective to identify the key public health research priorities as perceived by the state health officials and public health researchers. a cross - sectional survey was done for the state health officials involved in public health programmes and public health researchers in various states of india. a self - administered semi - structured questionnaire was used for data collection. overall, 35 state officials from 15 states and 17 public health researchers participated in the study. five leading public health research priorities identified in the open ended query were maternal and child health (24%), non - communicable diseases (22%), vector borne diseases (6%), tuberculosis (6%) and hiv / aids / sti (5%). maternal and child health research was the leading priority ; however, researchers also gave emphasis on the need for research in the emerging public health challenges such as non - communicable diseases. structured initiatives are needed to promote interactions between policymakers and researchers at all stages of research starting from defining problems to the use of research to achieve the health goals as envisaged in the 12th plan over next five years. |
in recent years, the efficacy of endovascular treatment after the administration of intravenous recombinant tissue plasminogen activator (rt - pa) has been demonstrated in several studies (1 - 5). in fact, the number of patients undergoing endovascular treatment after intravenous rt - pa administration has been increasing. in addition, carotid artery stent placement during endovascular treatment is performed in selected patients in clinical practice. however, strong evidence that supports the effectiveness of emergent stenting treatment is unavailable. patients who undergo emergency stent placement are recommended to receive antiplatelet therapy to prevent in - stent thrombosis (6), but only a few studies have reported the use of these drugs after carotid artery stenting and intravenous rt - pa administration (7,8). we herein report our experience with acute antiplatelet therapy in three stroke patients who underwent emergency carotid artery stent placement after intravenous rt - pa administration. a 71-year - old man was brought to our hospital with the chief complaint of acute left hemiplegia. he had a history of hypertension and non - valvular atrial fibrillation (nvaf) and had been receiving oral administration of rivaroxaban (15 mg / day). on arrival, the patient 's national institute of health stroke scale (nihss) score was 14. head magnetic resonance imaging (mri) revealed acute cerebral infarction in the right basal ganglia extending to the corona radiata [diffusion - weighted imaging (dwi)-alberta stroke program early computed tomography score (aspects) : 9 ] (figure a). magnetic resonance angiography (mra) revealed a right internal carotid artery occlusion (figure b). at 110 minutes after the onset of stroke, rt - pa (time from the last administration of rivaroxaban to rt - pa : 590 minutes) and cerebral endovascular treatment were administered. the thrombus was aspirated using the penumbra system (penumbra, alameda, usa). although balloon percutaneous transluminal angioplasty (pta) was performed for the residual right internal carotid artery stenotic lesion, elastic recoil occurred. therefore, clopidogrel and aspirin were administered at loading doses of 300 and 200 mg, respectively, followed by emergency carotid artery stenting (carotid wallstent, boston scientific, natick, usa) (figure c). at 300 minutes after onset (time from the last administration of rivaroxaban to recanalization : 780 minutes), complete recanalization was achieved. after treatment, antiplatelet therapy (clopidogrel 75 mg + aspirin 100 mg) was continued. the patient 's systolic blood pressure (sbp) was maintained at 100 to 130 mmhg by a continuous intravenous infusion of nicardipine. during the follow - up period, the patient remained free of concurrent symptomatic intracranial hemorrhaging (sich) (figure d). although no neurologic changes were noted, follow - up mra performed on hospital day 4 showed reocclusion, likely resulting from in - stent thrombosis (figure e). on hospital day 35, the patient 's modified rankin scale (mrs) score was 2, and he was transferred to another hospital. a : diffusion - weighted imaging (dwi) on arrival showed acute cerebral infarction in the right basal ganglia extending to the corona radiata, b : magnetic resonance angiography (mra) on arrival showed an occlusion of the right internal carotid artery, c : before and after stent placement, d : computed tomography (ct) at 24hours after t - pa showed no hemorrhagic change, e : mra on the 4th hospital day showed reocclusion due to in - stent thrombosis. f : dwi on arrival showed acute cerebral infarction in the right frontal temporal lobe and corona radiata, g : mra on arrival showed a bilateral occlusion of the internal carotid artery, h : before and after stent placement, i : ct at 24 hours after t - pa showed a hemorrhagic change in the right basal ganglia, j : mra on the 5th hospital day. k : dwi on arrival showed acute cerebral infarction in the left basal ganglia extending to the corona radiata, l : mra on arrival showed an occlusion of the left internal carotid artery, m : before and after stent placement, n : ct at 24 hours after t - pa showed no hemorrhagic change, o : mra on the 14th hospital day. the carotid stent was patent. an 85-year - old man was brought to our hospital with the chief complaints of left hemiplegia and dysarthria. he had a history of hypertension, old cerebral infarction, and nvaf and had been receiving oral administration of aspirin (100 mg / day). on arrival, head mri revealed acute cerebral infarction in the right frontal temporal lobe and corona radiata (dwi - aspects : 6) (figure f). mra revealed a bilateral internal carotid artery occlusion (figure g). at 240 minutes after onset, rt - pa was administered, and cerebral endovascular treatment was additionally administered. because stenosis at the origin of the right internal carotid artery was so severe that a therapeutic catheter could not be crossed over the lesion, clopidogrel was administered at a loading dose of 300 mg, followed by emergency carotid artery stenting (wallstent) (figure h). then, aspiration with the penumbra system was performed for a right middle cerebral artery occlusion (m1 segment), and complete recanalization was achieved at 323 minutes after onset. after treatment, antiplatelet therapy (clopidogrel 50 mg + aspirin 100 mg) was continued. sbp was maintained at 100 to 130 mmhg by a continuous intravenous infusion of nicardipine. head computed tomography performed 24 hours later revealed asymptomatic hemorrhagic changes (figure i). the course of rehabilitation therapy was uneventful, however, aortic dissection occurred on hospital day 38. the patient was therefore transferred to the department of cardiovascular medicine. a 72-year - old man with aphasia and a history of diabetes mellitus and dyslipidemia head mri revealed acute cerebral infarction in the left basal ganglia extending to the corona radiata (dwi - aspects : 9) (figure k). after the administration of rt - pa at 130 minutes after onset, he was transferred to our hospital, where cerebral endovascular treatment was added. to remove an occlusion at the origin of the left middle cerebral artery, the thrombus was retrieved using the penumbra system and solitaire fr (covidien, irvine, usa). although pta for residual stenosis in the left internal carotid artery was performed, elastic recoil occurred. therefore, clopidogrel and aspirin were administered at loading doses of 300 and 200 mg, respectively, followed by emergency carotid artery stenting (wallstent) (figure m). the patient 's sbp was maintained at 100 to 130 mmhg by a continuous intravenous infusion of nicardipine. during the follow - up period, he remained free of concurrent sich (figure n). on hospital day 25, the patient 's mrs score was 2, and he was transferred to another hospital. the use of antiplatelet drugs within 24 hours after intravenous rt - pa administration for patients undergoing emergency stent placement was approved by the ethical committee of our hospital (application no. a 71-year - old man was brought to our hospital with the chief complaint of acute left hemiplegia. he had a history of hypertension and non - valvular atrial fibrillation (nvaf) and had been receiving oral administration of rivaroxaban (15 mg / day). on arrival, the patient 's national institute of health stroke scale (nihss) score was 14. head magnetic resonance imaging (mri) revealed acute cerebral infarction in the right basal ganglia extending to the corona radiata [diffusion - weighted imaging (dwi)-alberta stroke program early computed tomography score (aspects) : 9 ] (figure a). magnetic resonance angiography (mra) revealed a right internal carotid artery occlusion (figure b). at 110 minutes after the onset of stroke, rt - pa (time from the last administration of rivaroxaban to rt - pa : 590 minutes) and cerebral endovascular treatment were administered. the thrombus was aspirated using the penumbra system (penumbra, alameda, usa). although balloon percutaneous transluminal angioplasty (pta) was performed for the residual right internal carotid artery stenotic lesion, elastic recoil occurred. therefore, clopidogrel and aspirin were administered at loading doses of 300 and 200 mg, respectively, followed by emergency carotid artery stenting (carotid wallstent, boston scientific, natick, usa) (figure c). at 300 minutes after onset (time from the last administration of rivaroxaban to recanalization : 780 minutes), complete recanalization was achieved. after treatment, antiplatelet therapy (clopidogrel 75 mg + aspirin 100 mg) was continued. the patient 's systolic blood pressure (sbp) was maintained at 100 to 130 mmhg by a continuous intravenous infusion of nicardipine. during the follow - up period, the patient remained free of concurrent symptomatic intracranial hemorrhaging (sich) (figure d). although no neurologic changes were noted, follow - up mra performed on hospital day 4 showed reocclusion, likely resulting from in - stent thrombosis (figure e). on hospital day 35, the patient 's modified rankin scale (mrs) score was 2, and he was transferred to another hospital. a : diffusion - weighted imaging (dwi) on arrival showed acute cerebral infarction in the right basal ganglia extending to the corona radiata, b : magnetic resonance angiography (mra) on arrival showed an occlusion of the right internal carotid artery, c : before and after stent placement, d : computed tomography (ct) at 24hours after t - pa showed no hemorrhagic change, e : mra on the 4th hospital day showed reocclusion due to in - stent thrombosis. f : dwi on arrival showed acute cerebral infarction in the right frontal temporal lobe and corona radiata, g : mra on arrival showed a bilateral occlusion of the internal carotid artery, h : before and after stent placement, i : ct at 24 hours after t - pa showed a hemorrhagic change in the right basal ganglia, j : mra on the 5th hospital day. k : dwi on arrival showed acute cerebral infarction in the left basal ganglia extending to the corona radiata, l : mra on arrival showed an occlusion of the left internal carotid artery, m : before and after stent placement, n : ct at 24 hours after t - pa showed no hemorrhagic change, o : mra on the 14th hospital day. the carotid stent was patent. an 85-year - old man was brought to our hospital with the chief complaints of left hemiplegia and dysarthria. he had a history of hypertension, old cerebral infarction, and nvaf and had been receiving oral administration of aspirin (100 mg / day). on arrival, head mri revealed acute cerebral infarction in the right frontal temporal lobe and corona radiata (dwi - aspects : 6) (figure f). mra revealed a bilateral internal carotid artery occlusion (figure g). at 240 minutes after onset, rt - pa was administered, and cerebral endovascular treatment was additionally administered. because stenosis at the origin of the right internal carotid artery was so severe that a therapeutic catheter could not be crossed over the lesion, clopidogrel was administered at a loading dose of 300 mg, followed by emergency carotid artery stenting (wallstent) (figure h). then, aspiration with the penumbra system was performed for a right middle cerebral artery occlusion (m1 segment), and complete recanalization after treatment, antiplatelet therapy (clopidogrel 50 mg + aspirin 100 mg) was continued. sbp was maintained at 100 to 130 mmhg by a continuous intravenous infusion of nicardipine. head computed tomography performed 24 hours later revealed asymptomatic hemorrhagic changes (figure i). the course of rehabilitation therapy was uneventful, however, aortic dissection occurred on hospital day 38. a 72-year - old man with aphasia and a history of diabetes mellitus and dyslipidemia was brought to a previous hospital with a chief complaint of right hemiplegia. head mri revealed acute cerebral infarction in the left basal ganglia extending to the corona radiata (dwi - aspects : 9) (figure k). after the administration of rt - pa at 130 minutes after onset, he was transferred to our hospital, where cerebral endovascular treatment was added. to remove an occlusion at the origin of the left middle cerebral artery, the thrombus was retrieved using the penumbra system and solitaire fr (covidien, irvine, usa). although pta for residual stenosis in the left internal carotid artery was performed, elastic recoil occurred. therefore, clopidogrel and aspirin were administered at loading doses of 300 and 200 mg, respectively, followed by emergency carotid artery stenting (wallstent) (figure m). the patient 's sbp was maintained at 100 to 130 mmhg by a continuous intravenous infusion of nicardipine. during the follow - up period, he remained free of concurrent sich (figure n). on hospital day 25, the patient 's mrs score was 2, and he was transferred to another hospital. the use of antiplatelet drugs within 24 hours after intravenous rt - pa administration for patients undergoing emergency stent placement was approved by the ethical committee of our hospital (application no. carotid artery stent placement during endovascular treatment for patients who are unresponsive to intravenous rt - pa administration has become a popular treatment in clinical practice. the multicenter randomized clinical trial of endovascular treatment for acute ischemic stroke in the netherlands reported that acute carotid artery stent placement was performed in 12.9% of patients (1). we have performed immediate endovascular treatment after intravenous rt - pa administration for 29 patients in our hospital since september 2012 ; 23 of these patients had a stroke in the area of anterior circulation. in particular, carotid artery stent placement was performed in three (13%) of 23 patients. the leading complication of carotid artery stent placement after intravenous rt - pa administration is considered to be sich. in the present study, all three patients received loading doses of antiplatelet drugs during surgery and subsequently received dual antiplatelet therapy, without showing sich. in our hospital, the imaging indications for endovascular treatment after intravenous rt - pa administration regard, dwi - aspects 6 and a major artery occlusion [internal carotid artery or middle cerebral artery (m1 segment) ] were considered to indicate mra - dwi mismatch and therefore provided support for the use of additional endovascular treatment. selecting patients according to their mri findings (e.g., mra - dwi mismatch) appears to be important to prevent sich. in our department, the goal of bp control in patients undergoing carotid artery stenting after intravenous rt - pa therapy is set at sbp 130 mmhg. in addition, patients with a vulnerable plaque are at an increased risk for artery - to - artery embolism and acute in - stent thrombosis, as shown in case 1. in this study, one of the patients experienced thrombus formation in the placed stent without any worsening of the neurological symptoms. symptomatic in - stent thrombosis is rare and is reported to be between 0.04 - 2% (10,11). however, because in - stent thrombosis can result in serious consequences, emergency surgical treatment (e.g., pta, stent - in - stent placement, or thrombectomy) should be considered for such cases. there is no evidence of emergency stent placement for acute cerebral infarction and there are no clear guidelines for specific medications or loading dosage of antiplatelet agents at the time of stent placement (6). in each of the presented cases, the antiplatelet drug types and doses for both pre - stent loading and after therapy were decided by the attending physician according to the patient 's background variables, including age and pre - onset history of antiplatelet drug use. therapeutic strategies for the use of antiplatelet drugs in patients undergoing emergency stent placement should be established, with a particular focus on the type and dosing of antiplatelet drugs for patients that have received intravenous rt - pa therapy. | we herein report three ischemic stroke patients who underwent emergency carotid artery stenting after receiving intravenous tissue plasminogen activator (t - pa) treatment. all patients received antiplatelet medications immediately before stent placement for loading as well as dual antiplatelet therapy after stenting. under high - dose and dual antiplatelet therapy, none of the three patients showed symptomatic intracranial hemorrhaging. however, one case showed reocclusion of the placed stent after acute thrombosis. as a result, new treatment strategies for the use of antiplatelet agents during emergency stent placement must be developed, particularly for patients who have received intravenous t - pa therapy. |
pd is a chronic progressive brain disorder caused by a loss of dopamine - producing brain cells. pd leads to shaking (tremors), rigidity, difficulty with walking, slowness of movement, and impaired balance and coordination. in the later stages of illness, there can be cognitive problems, and in the advanced stages of illness, dementia is not uncommon. at the present time, there is no cure for pd. the goal of treatment is symptom control, mainly involving the use of levodopa (a drug that converts in the brain into natural dopamine) and dopamine agonists. when the prescribed drug therapy is no longer effective in controlling the tremors and/or paucity of movement, and the drug induced dyskinesias are significant, another treatment option for some parkinsonian patients is dbs. dbs can be effective in reducing tremors, slowness of movements, and gait problems, but there can be serious unwanted side - effects. the preferred surgical targets for pd are the subthalamic nucleus (stn) and the globus pallidus interna (gpi)two brain areas involved in motor control. for patients with tremor - dominant pd the electrodes are connected to a small battery - operated neuro - stimulator that is surgically implanted under the skin, usually in the upper part of the chest below the collarbone. the stimulator, which looks like a cardiac pacemaker and is sometimes referred to as a brain pacemaker, is externally programmed to deliver continuous electrical signals to the targeted brain area. the stimulation parameters the amplitude, the frequency, and the pulse width are set by the physician. with some devices, at the discretion of the programmer, the patient may control the amplitude (the voltage) within certain parameters (e.g., 2.43.2 v). the potential benefits of dbs for the treatment of patients with advanced pd are documented in narrative form in testimonials available on medtronics1 websites and in case reports. consider, for example, richard s narrative on medtronics australian website:when you are an actor, your body is a tool. if your tool is broken, you ca nt work. my left arm would nt cooperate, so i d put it in my pocket, or hold it. i was always going to another doctor, going to another specialist, having another test or procedure. it felt like all i did was live in waiting rooms and walk down endless corridors without ever getting anywhere. eventually, [richard s ] his research led him to deep brain stimulation and he decided to go ahead. i d made up my mind ; done all the tests, had the haircut and passed the audition. the day of the operation was nt easy then when they turned the stimulator on the stiffness disappeared. now i stand up with ease, where once it had been an almighty struggle. i cross the room like any normal person going for a stroll. when you are an actor, your body is a tool. my left arm would nt cooperate, so i d put it in my pocket, or hold it. i was always going to another doctor, going to another specialist, having another test or procedure. it felt like all i did was live in waiting rooms and walk down endless corridors without ever getting anywhere. eventually, [richard s ] his research led him to deep brain stimulation and he decided to go ahead. i d made up my mind ; done all the tests, had the haircut and passed the audition. the day of the operation was nt easy then when they turned the stimulator on the stiffness disappeared. now i stand up with ease, where once it had been an almighty struggle. below is an excerpt from chris story : chris noticed something wrong when he was out walking with one of his three daughters. my arm swing on my right side just quit, he recalled. i had no idea what was happening. within a year of being diagnosed he lost the use of his right hand, which meant he could nt write. my life basically became just work and sleep. on his first morning back at work loading trucks [after the surgery ], chris felt like a new person. i could tell that i was nt going through the normal problems in the morning, he says. it usually would take an hour and a half of cramping and walking weirdly. chris noticed something wrong when he was out walking with one of his three daughters. my arm swing on my right side just quit, he recalled. i had no idea what was happening. within a year of being diagnosed he lost the use of his right hand, which meant he could nt write. my life basically became just work and sleep. on his first morning back at work loading trucks [after the surgery ], chris felt like a new person. i could tell that i was nt going through the normal problems in the morning, he says. it usually would take an hour and a half of cramping and walking weirdly. less well documented, but no less relevant, are the common physical and psychological side - effects of dbs experienced by patients with pd. for example, there is the potential problem of weight gain, as well as the potential for serious disruptive changes in behaviour, mood and cognition. these changes can include speech dysfunction (impaired fluency and vocabulary), reduced working memory and processing speed, acute depression, pathological crying, mania, fear and alienation. if we return to richard s story (excerpted above), it is not all happiness and light. richard experiences depression and slurred speech : while the operation was a success, a few weeks later, richard went through a period of depression the ordeal of the operation was so profound that a sense of anticlimax began to hover over me. back home in the suburbs you re a normal bloke, but you feel like you ve been to another planet. richard is having to write now rather than act, because he s suffering from a side - effect of the operation known as dysarthria or slurred speech. he finds that his speech therapy is like going back to acting classes think loud and speak slow. while the operation was a success, a few weeks later, richard went through a period of depression the ordeal of the operation was so profound that a sense of anticlimax began to hover over me. back home in the suburbs you re a normal bloke, but you feel like you ve been to another planet. richard is having to write now rather than act, because he s suffering from a side - effect of the operation known as dysarthria or slurred speech. he finds that his speech therapy is like going back to acting classes think loud and speak slow. a more dramatic case of untoward consequences, originally reported by leentjens and colleagues, and subsequently referred to by many others, involves a 62-year - old male patient with pd who is admitted to a psychiatric hospital for a manic state resulting from dbs. as reported by walter glannon : a mood stabiliser failed to control his symptoms, which included megalomania and chaotic behaviour that resulted in serious financial debts. adjustment of the stimulator resolved the mania and restored his cognitive capacity for insight and rational judgment. yet this resulted in a return of his motor symptoms, which were so severe that the patient became bedridden. this left the patient and his healthcare providers with a choice between two mutually exclusive options : to admit the patient to a nursing home because of a serious physical disability, despite intact cognitive and affective capacities ; or to admit the patient to a chronic psychiatric ward because of a manic state, despite restoration of good motor function (, p. 290).while competent (that is, off dbs), the patient was presented with these options. he chose dbs to improve his motor function, understanding that the resulting mania would result in his institutionalization in a chronic psychiatric ward. a mood stabiliser failed to control his symptoms, which included megalomania and chaotic behaviour that resulted in serious financial debts. adjustment of the stimulator resolved the mania and restored his cognitive capacity for insight and rational judgment. yet this resulted in a return of his motor symptoms, which were so severe that the patient became bedridden. this left the patient and his healthcare providers with a choice between two mutually exclusive options : to admit the patient to a nursing home because of a serious physical disability, despite intact cognitive and affective capacities ; or to admit the patient to a chronic psychiatric ward because of a manic state, despite restoration of good motor function (, p. 290). clearly, patients with pd who undergo dbs can (and do) experience significant benefits. these same patients, however, also can (and do) experience significant unwanted side - effects including cognitive and psychiatric disturbances (such as major depression and mania). whether the effects of dbs are positive or negative, physical or psychological, transient or permanent, they can have a profound impact on personality, on familial, marital, social, and professional relations, as well as occupational functioning. for those who embrace a static view of personal identity as comprised of core inclinations and character traits, changes in these domains arguably represent a serious threat to personal identity. for others, who embrace a dynamic view of personal identity and consider the notion of an authentic self given by nature and unchanged by time outdated, changes in these domains do not represent a threat to personal identity, but rather are constitutive of personal identity. below, i introduce a philosophical account of relational personal identity as a dynamic interpersonal activity based in narrative. on this view, personal identity is not tied to an individual s core inclinations or character traits, but reflects an individual s lived experience (and perhaps some measure of introspection) as integrated into her autobiographical narrative. personal identity is a concept we rely on in attributing moral responsibility to one person rather than another, and in ensuring that persons get their just desserts. marya schechtman makes this point succinctly when she writes of personal identity that it serves as a minimum condition in the assessment of responsibility, obligation, and certain sorts of entitlement. our practices of promising, contracting and assessing praise or blame depend on this notion in sharp contrast, our practices of promising, contracting and assessing praise or blame in no way depend upon personality. for example, if i thought you were a kind and considerate person when i made you a promise, i ca nt simply disregard my promise to you because, in my estimation, you are no longer the kind and considerate person i once thought you were. similarly, if i contract with you to pay you an hourly wage to build me a fence, i ca nt simply default on any payment owed to you because you have become apathetic since your partner ran off with a younger man. if you are an imprisoned murderer who has experienced a religious conversion so that you are now a pacifist who abhors violence, you are not thereby absolved of responsibility for your prior criminal behavior. in all of these hypothetical situations your personality may have changed, and the change may be sufficiently radical as to prompt others to say of you that you are a different person. generally, it would be understood that, notwithstanding any change(s) in personality, you are nonetheless the person to whom a promise, a wage or a punishment is owed. by the same token, barring specific terms suggesting otherwise, neither the promise, the wage nor the punishment is owed to another in your stead. in brief, a change in personality, whether gradual or abrupt, subtle or radical, temporary or permanent, does not in and of itself make for a change in personal identity.2 the two concepts personality and personal identity are not synonymous. a highly influential answer to this question can be found in schechtman s book the constitution of selves. in this book, schechtman helpfully draws our attention to the pivotal identity question which she dubs the characterization question which is what actions, experiences, beliefs, values, desires, character traits and so on (hereafter abbreviated (, p. 73) ? in exploring this question, schechtman argues that a person creates his identity by forming an autobiographical narrative a story of his life (, p. 93). according to schechtman:[i]ndividuals constitute themselves as persons by coming to think of themselves as persisting subjects who have had experience in the past and will continue to have experience in the future, taking certain experiences as theirs a person s identity is constituted by the content of her self - narrative, and the traits, actions, and experiences included in it are, by virtue of that inclusion, hers (, p. 94). [i]ndividuals constitute themselves as persons by coming to think of themselves as persisting subjects who have had experience in the past and will continue to have experience in the future, taking certain experiences as theirs a person s identity is constituted by the content of her self - narrative, and the traits, actions, and experiences included in it are, by virtue of that inclusion, hers (, p. 94). some such narratives are at most personal myths. according to schechtman, for a self - narrative to be identity - constituting, an identity - constituting self - narrative must be capable of local articulation (that is, the person must be able to provide some account of her history, her life situation, and her motivations). as schechtman insists, the narrator should be able to explain why he does what he does, believes what he believes, and feels what he feels (, p. 114). a person need not be able to narrate her whole life in a self - conscious way, but she must be able to narrate parts of it (, p.105). that is, she must be able to render her self - narrative intelligible. as regards the reality constraint, schechtman insists that the self - narrative must cohere with basic observational facts about the world. that is, an identity - constituting self - narrative must cohere with reality : this is not to say that a narrative must be totally accurate in every regard or contain no trivial mistakes, but it should exhibit a fundamental grasp of what the world is like (, p. 83). from another perspective, laurence thomas argues that identity is an interpersonal social construct. he writes:[w]e are constituted through others, the way in which we conceive of ourselves, at least in part, owes, much to how others conceive [of ] us, and this is necessarily so. the way in which we think of ourselves is inextricably tied to the way in which others think of us (, p. 365).this view is consonant with the views of those who espouse a feminist relational understanding of autonomy. as susan sherwin reminds us:[n]o one is fully independent the view of individuals as isolated social units is not only false but impoverished : much of who we are and what we value is rooted in our relationships and affinities with others [w]e are constituted through others, the way in which we conceive of ourselves, at least in part, owes, much to how others conceive [of ] us, and this is necessarily so. the way in which we think of ourselves is inextricably tied to the way in which others think of us (, p. 365). [n]o one is fully independent the view of individuals as isolated social units is not only false but impoverished : much of who we are and what we value is rooted in our relationships and affinities with others finally, it is also important to recognize the ways in which the body can and does influence who we are and how we can be in the world. race, class, gender, ethnicity, age, sexual orientation, and ability are features of the self that others read off the body in the context of complex networks of social norms, institutions, practices, conventions, expectations, and attitudes agents identities are formed within the context of social relationships and shaped by a complex of intersecting social determinants, such as race, class, gender and ethnicity reasoning along similar lines, linda martn alcoff writes more specifically about how race and gender affect our relations in the world, which in turn affects our interior life, that is, our lived experience or subjectivity (, p. 92). taken together, these discrete perspectives on identity inform my philosophical account of relational identity as a dynamic, socially, culturally, politically, and historically situated communicative activity (based in narrative and performance) that is informed by the interests, perspectives, and creative intentions of close and distant others (, p. 110). this account of relational identity takes seriously the claim that a person creates his identity by forming an autobiographical narrative a story of his life (, p. 93). it also embraces the claim that we are constituted / constructed in and through personal (intimate) relationships and public (impersonal social and political) interactions. finally, it accepts the claim that we are embodied selves situated in particular social, cultural, political and historical contexts. in this way, relational personal identity recognizes that the desires, beliefs, values, emotions, intentions, memories, actions and experiences that make up a person s self - narrative are shaped in and through relationships. this relational account of personal identity is distinct from both the somatic (or biological) account of personal identity, as well as the psychological account of personal identity. my identity is not in my body or in my brain, but in the negotiated spaces between my body and brain, and the bodies and brains of others:[i]dentities are created by relational beings mutually engaged in the never - ending project of constituting themselves in and through personal relationships and public interactions in order to answer such personal questions as : who am i ? where am i from ? where have i been ? where am i going ? indeed, it is through our (more or less conscious) interpretations of our values, memories, actions, experiences, and so on as well as the (more or less conscious) interpretations of these same characteristics by others that we come to embody answers to these pivotal questions, thereby instantiating our place in the world as we continually strive for balance between how we see and understand ourselves and how others see and understand us (, p.117). [i]dentities are created by relational beings mutually engaged in the never - ending project of constituting themselves in and through personal relationships and public interactions in order to answer such personal questions as : who am i ? where am i from ? where have i been ? where am i going ? what do i stand for ? who do i want to be ? who am i becoming ? indeed, it is through our (more or less conscious) interpretations of our values, memories, actions, experiences, and so on as well as the (more or less conscious) interpretations of these same characteristics by others that we come to embody answers to these pivotal questions, thereby instantiating our place in the world as we continually strive for balance between how we see and understand ourselves and how others see and understand us (, p.117). relational identity is a dialectical process aimed at achieving equilibrium some kind of temporary and temporizing balance between self - ascription and ascription by others (i.e., others who are a part of one s familial, social, cultural, and political clusters of meaning and belonging). this view is consonant with the work of lorraine code who suggests that uniqueness, creativity, and moral accountability grow out of interdependence and continually turn back to it for affirmation and continuation the identity - constituting narrative is the narrative that effectively balances how a person sees and understands herself, with how others see and understand her. that is, the identity - constituting narrative is the narrative that satisfies the equilibrium constraint. the equilibrium constraint requires minimal endorsement or uptake by others of one s projected self - narrative. when there is no uptake, the cyclical and iterative process of identity formation that involves combining and resolving projected and perceived self - narratives into a coherent, identity - constituting narrative begins (again). to be clear, equilibrium is different from stability, insofar as the latter is presumed to be more enduring. equilibrium, on the other hand, is a temporary state of affairs during which time the projected self - narratives and perceived self - narratives are in balance. at such time, the self is able to take notice of her place in the world and choose to embrace, refine or revise her projected self - narrative as part of the ongoing, never - ending process of identity formation. a projected self - narrative can be a preferred self - narrative or performed self - narrative and most likely is some combination of both. the preferred self - narrative is the story of who the person wants to be. if we think back to the cases described at the outset, richard s preferred self - narrative might be that of the brilliant actor struck down in his prime by illness. if others perceive richard in this way then they will endorse his story, and for a time this will be his identity - constituting narrative. if this is not how others perceive richard, however, then the equilibrium constraint will not be satisfied. others, for example, might consider richard an average actor, but an excellent writer who might never have discovered his talent for writing, but for his illness. in tandem with the preferred self - narrative the performed self - narrative is the story of who the person can be, given the ways in which his life is constrained by self and others. richard s performed self - narrative may be that of the self - assured curmudgeon who writes for a living, because that is what brilliant actors do when they retire. but maybe richard fears his illness and chooses to mask his fear with cocky, surly behaviour. if we look for an intelligible self - narrative articulated by richard and facts that cohere with reality, as schechtman would have us do, richard is a man who once worked as an actor, and whose acting career has come to an end because of pd. for example, can we say that richard was a brilliant actor ; is an acerbic character ? on a relational account of personal identity, richard is the person at the intersection of who he wants to be, and who others will minimally let him be. from a more technical standpoint, richard s identity - constituting narrative is the self - narrative that satisfies the equilibrium constraint. that is, richard s identity - constituting narrative is the narrative that he is able to construct and maintain through complex (conscious or unconscious) intimate and public interactions involving the fact that he worked as an actor, may be sufficient minimal uptake of his self - narrative to satisfy the equilibrium constraint. then again, richard may need further endorsement of additional features of his self - narrative to effectively balance how he sees and understands himself with how others see and understand him. if there are no such external endorsements, however, and instead richard is faced with an alternative narrative of average actor, a number of strategies are available to richard in an effort to establish equilibrium. for example, richard could modify his self-perception and self-projection to better accommodate the perceptions and reactions of others in his community of belonging. for example, richard may come to see and understand himself as others see and understand him a talented writer who has found his calling. as a result, richard may (consciously or unconsciously) come to have less care about his previous career as an actor and may have less vested in the persona of brilliant actor. as a result of this change in perception, richard may change his projected self - narrative and in so doing garner sufficient uptake of his revised narrative for there to be equilibrium. on the other hand, if neither the original self - narrative nor any revised self - narrative garners minimal support / endorsement, then another option for restoring equilibrium would be for richard to change his community of belonging. then again, richard could attempt to achieve equilibrium by simply dismissing any discordance between his projected narrative and the narrative perceived by others, as their error. with the equilibrium constraint, what matters for richard s identity - constituting narrative is not what others objectively believe to be true (as these others could be mistaken, bigoted or hostile (, p. 92)), nor what richard asserts to be true (as he may be ignorant, mistaken, self - deceived, or mendacious with respect to his self - narrative (, p. 116)), but what balance can be achieved between who richard says he is, and who others will minimally let him be. on a relational account of personal identity, richard s identity - constituting narrative is what emerges in the negotiated space between richard and others. richard s identity is a balance between self - ascription and ascription by others (self-perception, other-reaction) as he (consciously or unconsciously) engages in the never - ending dialectical project of tailoring his identity (or shifting his community of belonging) in response to events, experiences and perceptions. i met helmut dubiel, philosopher, university professor, and author of deep in the brain : living with parkinson s disease in april 2009. we were both invited speakers at a conference in freiburg, germany entitled pimp your brain ! i do nt understand or speak german and so after his talk we met for coffee. when i met him he had been living with pd for close to 16 years and had been fitted with his brain pacemaker for about three years. as a side - effect of dbs, when the amplitude was high, his voice was low and his articulation was slurred. in his words, his speech was soft and washed out there were difficulties writing on the chalk board, speaking clearly and commanding the respect of students. as i listened to dubiel, i reflected on how important it is for those of us who lecture in front of small and large classes to have control of our mobility and our speech. dubiel also alluded to his academic successes and how he had imagined (hoped) his writings would be remembered as important contributions to the literature. while he was pleased with the critical acclaim for his autobiography, he also bemoaned the fact that he likely would be remembered more for this work than his academic writing. when i asked dubiel more pointed questions about identity, he spoke about the importance of erotic touching and how with pd it had become more difficult for him to see himself, and for others to see him, in the role of lover. of the early days following diagnosis, dubiel reports that [e]rotic touching and encounters grew all the more important the thicker the glass wall became between me and my social environment this prompted me to ask him if he was self - conscious doing this in a public place, and whether it would be better if the neuro - stimulator could be placed under the skin near the wrist instead of in the upper part of the chest and if it could be activated by applying gentle pressure. on this point dubiel was adamantno, then i would feel like a machine. at the end of our conversation, dubiel told me that his autobiography would soon be available in english. in reading this book, i learned more about dubiel the professor, the lover, the father, the son and the friend. in each of these roles, dubiel of particular note were the ways in which the diagnosis of pd and the treatments available to him (including dbs) shaped his sense of self in and through his relationships with others. early in his autobiography, dubiel reports on his diagnosis of pd : the first and simultaneously most lasting feeling that the diagnosis of parkinson s triggered was a sense of narcissistic injury, one that no other injury inflicted by a human being has ever surpassed. at a single blow, i felt excluded from the fellowship of those who simply had their bodies at their disposal, who experienced no friction losses between an impulse to act and the action itself (, pp. the first and simultaneously most lasting feeling that the diagnosis of parkinson s triggered was a sense of narcissistic injury, one that no other injury inflicted by a human being has ever surpassed. at a single blow, i felt excluded from the fellowship of those who simply had their bodies at their disposal, who experienced no friction losses between an impulse to act and the action itself (, pp. this is a clear statement in explicitly relational terms about how pd had a profound impact on dubiel s personal identity in excluding him from the fellowship of able - bodied individuals. so it is that dubiel s body is a constitutive element of his self ; it influences who he is and how he can be in the world. how he sees and understands himself, and how others see and understand him is mediated through his body and, more particularly, his limited ability to control his body. they do not allow him to see himself as he would like to be : virile and in full control of my life, a man who lifts his eyes to the stars but has both feet on the ground (, p. 38). in the few photos of dubiel taken during his first years with pd, dubiel notices a mask - like rigidity, while in contrast i find my eyes to be larger, sadder, and more expressive than before i fell ill at the time of diagnosis, dubiel is, from his perspective, at the top of his game : i had just been appointed a full professor, while retaining my position as deputy director of a small but prestigious research institute. at the time, my ambition and energy level were ample for two jobs. i was publishing books and monographs on a regular basis, and they found readers i was the crown prince of the institute (, p. 50).once i received the diagnosis, however, a gradual process of alienation arose between the institute and me, which would conclude three years later with a humiliating dismissal (, p. 50). i had just been appointed a full professor, while retaining my position as deputy director of a small but prestigious research institute. at the time, i was publishing books and monographs on a regular basis, and they found readers once i received the diagnosis, however, a gradual process of alienation arose between the institute and me, which would conclude three years later with a humiliating dismissal (, p. 50). here the theme of alienation is even more explicit and continues to be so for dubiel as his illness progresses. in the year it was surely not a transformation caused by verifiable changes in brain physiology, but rather a psychological adjustment to a biorhythm that had becomes utterly unpredictable due to the illness and medication (, p.50) i became estranged from many colleagues and friends whom i had known for ages, (p. 52).in further contemplating his illness, dubiel writes:[w]hen it [parkinson s ] is full - blown, [it ] robs a person of the ability to continually reinvent himself. one s life becomes similar to that of a plant : silent, without transcendence and autonomy. in a sense, it s not even a life anymore just existence in an atrophied form (, p. 60). it was surely not a transformation caused by verifiable changes in brain physiology, but rather a psychological adjustment to a biorhythm that had becomes utterly unpredictable due to the illness and medication (, p.50) i became estranged from many colleagues and friends whom i had known for ages, (p. 52). [w]hen it [parkinson s ] is full - blown, [it ] robs a person of the ability to continually reinvent himself. one s life becomes similar to that of a plant : silent, without transcendence and autonomy. in a sense, it s not even a life anymore just existence in an atrophied form (, p. 60). at the time of writing dubiel dubiel s experience is one of fragmentation of living at least two lives a private life in which he partially acknowledges his illness to himself and a close circle of friends, and a public life in which the illness is a well - guarded secret. eventually, the drug therapy is no longer effective in counteracting the symptoms of dubiel s disease and the dyskenesias caused by over - medication have become torturous. at this time, dubiel opts for dbs in the hope of improving his motor functions. post - surgery he reports that from a medical perspective all went well : according to the surgeons criteria, the operation was a full success. my medication intake could be drastically reduced (, p. 84). according to the surgeons criteria, the operation was a full success. off conditions were simply gone. [the ] medication intake had dropped by twenty - five percent, [and ] overall mobility and endurance were markedly better than before (, p. 94). nonetheless, similar to the experience of other patients with pd who have undergone dbs, dubiel is dissatisfied with the overall outcome of the procedure (, p. 93) : in my case the operation had merely replaced the plague with cholera. i simply had the impression that during the entire first year the upshot of this major and extremely expensive surgery had been to replace one set of grave symptoms with another (, p. 93). i simply had the impression that during the entire first year the upshot of this major and extremely expensive surgery had been to replace one set of grave symptoms with another (, dubiel experiences profound depression and a range of physical problems including a speech disturbance, shortness of breath when bending, inability to write, small - stepped gait and a tendency to fall on stairs and no sense of smell and taste. dubiel identifies his speech problem as the worst of these side - effects : my worst post - operative symptom, which remains unchanged to this day, is a speech disturbance : my volume is too low, and my articulation is poor, slurred. often i ca nt even command twenty percent of my normal speech volume (, p. 94). my worst post - operative symptom, which remains unchanged to this day, is a speech disturbance : my volume is too low, and my articulation is poor, slurred. often i ca nt even command twenty percent of my normal speech volume (, p. 94). while dubiel describes his worst side - effect as reduced speech volume, in relational terms, the volume of this voice (and more particularly the risk of making unintelligible, poorly articulated comments in a feeble voice (, p. 123)) mark him in public and set him apart from his peers. another source of stigmatization is his movement patterns : now i walk through the street and count the oncoming pedestrians who look at my asymmetrical shuffling gait with curiosity or alienation (, p. 109). these physical limitations negatively affect dubiel s relationships with close and distant others. in time, dubiel learns to deal with the unwanted side - effects by adjusting the pacemaker amplitudes. if he wants to enunciate clearly, he sets the amplitude very low knowing this will lead to relative immobility and depression if he wants to walk any distance, he sets the amplitude much higher knowing that his speech will become inaudible. while having to choose between talking and walking is less than ideal, dubiel s restored ability (within limited parameters) to control his body in social situations restores his pride and professional authority. in turn, this allows him to conceive of a continued place for himself within the academy. meanwhile, he dreams about the things he would like to be able to do including, walk through large crowds of people without fear, dance, talk with strangers in noisy train stations, stroll along the path that leads from harlem down to battery park on a sunny september day in new york dubiel is the person at the intersection of who he wants to be, and who others will minimally let him be. his identity - constituting narrative is the self - narrative that satisfies the equilibrium constraint. it is the narrative that he is able to construct and maintain through complex (conscious or unconscious) intimate and public interactions involving s autobiographical narrative we know that at least during one period of equilibrium following dbs, he was a person alienated from colleagues and loved ones. this question can be answered in the affirmative or the negative depending upon the theory of personal identity that informs the answer. for example, applying the narrative self - constitution view of identity, schechtman concludes that dbs for pd can be a threat to personal identity. garrison, a 61-year - old american with pd who consents to dbs to treat his tremors and severe apathy. following surgery, garrison experiences significant improvement in his motor symptoms and dramatic changes in personality. where once he was shy and introverted, he is now outgoing and gregarious where once he was enthusiastic about his work, he has now quit his job to promote various social, political and charitable causes. schechtman believes that since narrative is a dynamic notion, continuity of narrative is thoroughly compatible with even quite radical change nonetheless, she concludes that dbs can be a threat to personal identity. in her discussion of mr. garrison s values, motivations and actions are the result of dbs is, according to schechtman, at odds with the articulation constraint on identity - constituting narratives according to which the narrator should be able to explain why he does what he does, believes what he believes, and feels what he feels (, p. 114). plans, projects, intentions, beliefs, and desires because they do nt ; his current passions and interests the things he takes as reasons were caused by manipulation of his brain garrison s personal identity is threatened insofar as he runs afoul of the articulation constraint. from another perspective garrison were to suggest that his newfound passions and interests were the result of personal development and not dbs, then he would run afoul of the reality constraint, according to which the self - narrative must cohere with basic observational facts about the world. schechtman s affirmative answer to the question is dbs a threat to personal identity ? to appreciate the limitations of this affirmative response, however, we need only reflect briefly on the use of dbs to treat psychological problems instead of motor symptoms. consider, for example, the use of dbs for the treatment of severe, treatment - refractory depression or obsessive compulsive disorder. imagine that following dbs the depressed patient is happy or the patient with obsessive compulsive disorder does nt experience the same impulses. would we find the conclusion that dbs is threatening to personal identity as intuitively appealing in these instances ? or, would we be more inclined to endorse the view that even quite radical change can be successfully integrated into a patient s autobiographical narrative (i.e., her life story) ? further, if we return to schechtman s original description of the articulation constraint on identity - constituting narratives that the person be able to provide some account of her history, her life situation, and her motivations ; that she be able to narrate parts of her life in a self - conscious way ; that she be able to render her self - narrative intelligible it is unclear why a patient could not satisfy the articulation constraint by including a description of consent to dbs in her self - narrative. in my view, schechtman s intuitively appealing affirmative answer to the question is dbs for pd a threat to personal identity ? is unsatisfactory. below, applying a relational account of personal identity, i offer alternative responses to the question is dbs for pd a threat to personal identity ? a first plausible response to this question affirms that dbs for pd is not (and never could be) a threat to personal identity because personal identity is a dynamic concept:[t]here is no true self, only a dynamic socially, culturally, and politically constituted self that is historically situated and that at any one point in time can be more or less stable. indeed, it is only in interaction with others and through their instantiation of, or resistance to, a storied and projected self that a person can experience either affirmation or disruption, both of which are relevant to the project of stabilizing one s self - narrative in an effort to achieve a period of equilibrium (, p. 123).a patient with pd who undergoes dbs may experience a unique form of biographical disruption (, p. 1850). it does not follow, however, that the patient s personal identity is under threat. if the self is a dynamic, socially, culturally and politically constituted self that is shaped over time through experience (and perhaps introspection), it makes no sense to describe any particular event or experience (including dbs) as threatening. there is no pre - set autobiographical narrative (i.e., life story) that has been thwarted by some unfortunate event or experience. there just is the life story as it unfolds. on this view, what matters is whether (and, if so, to what extent) an event or experience is integrated (consciously or unconsciously) into an identity - constituting narrative. [t]here is no true self, only a dynamic socially, culturally, and politically constituted self that is historically situated and that at any one point in time can be more or less stable. indeed, it is only in interaction with others and through their instantiation of, or resistance to, a storied and projected self that a person can experience either affirmation or disruption, both of which are relevant to the project of stabilizing one s self - narrative in an effort to achieve a period of equilibrium (, p. 123). this brings us to a second plausible response to the question is dbs for pd a threat to personal identity ? this response involves looking at the question from a moral perspective, and seeing that an identity - constituting narrative can be damaging when it is the result of oppression, as when an individual in certain contexts and circumstances is forced to live for periods of time within the confines of another s ideas about what makes for an appropriate self - narrative : an identity - constituting narrative is not, in and of itself, a good thing much depends on the extent to which the identity is asserted or assigned when a person is able to fashion and project an identity - constituting narrative that she values (hopefully a self - narrative that fosters her talents and dreams) and is able to motivate appropriate uptake of the self - narrative she embraces, there may be evidence of autonomy on the other hand, when a person is unable to contribute effectively to a satisfying self - narrative and finds herself forced to live within constraints set by others who have fixed ideas about who she is and who she can be, there may be evidence of oppression (, pp. an identity - constituting narrative is not, in and of itself, a good thing much depends on the extent to which the identity is asserted or assigned when a person is able to fashion and project an identity - constituting narrative that she values (hopefully a self - narrative that fosters her talents and dreams) and is able to motivate appropriate uptake of the self - narrative she embraces, there may be evidence of autonomy on the other hand, when a person is unable to contribute effectively to a satisfying self - narrative and finds herself forced to live within constraints set by others who have fixed ideas about who she is and who she can be, there may be evidence of oppression (, pp. when considered from a moral perspective, a person must be able to contribute actively to the authoring of her life in a manner that is consistent with her broader interests, values and commitments. when this is not the case, as when others have a disproportionate hand in writing stories for [others ] that are limiting, cruel, oppressive or alienating (, p. 127), a serious moral problem arises because of the ways in which agency and autonomy are constrained. consider, for example, the scope of possible identity - constituting narratives available to persons with pd (with or without dbs) in a society that is not welcoming of persons with physical and psychological disabilities. in such a society, a person s experiences will be significantly affected by stories others have constructed to restrict the range of narratives that can be appropriated and successfully enacted. in such a society, discriminatory attitudes towards persons with disabilities, not dbs for pd, would be a serious potential threat to personal identity. to be clear on this point, the threat to personal identity experienced by persons with disabilities is analogous to the threat experienced by women in a patriarchal society, by coloured people in a racist white society, and by gay people in a homophobic society. the threat, such as it is, are the beliefs and attitudes of others that result in stigmatization and alienation, which in turn may result in negative experiences and feelings being integrated into one s identity - constituting narrative. a third response to the question is dbs for pd a threat to personal identity ? is similar in orientation to that offered by schechtman. on a relational account of personal identity, but for the dbs the identity - constituting narrative would be quite different (perhaps even radically different). on this view, dbs for pd distorts the dialectical process of identity formation and, for this reason, is a threat to personal identity. dbs for pd limits how a person sees and understands herself ; as such dbs limits what the protagonist of an autobiographical narrative can project for minimal endorsement by others. a person with pd who has been treated with dbs can not successfully project a self - narrative that is impervious to the fact of dbs. this constraint on what can be projected, acts as a constraint on what can be perceived. in turn, the interplay between constrained projected and perceived self - narratives gives rise to a different identity - constituting narrative. a problem with this perspective on dbs for pd as a threat to personal identity, however, is that it renders any and all life events and experiences (whether initially considered positive or negative) potential threats to identity. if dbs for pd is a threat to personal identity because it constrains how a person sees and understands herself, which in turn constrains the dialectical process of identity formation, then so too pd is a threat to personal identity, and so too is potentially every other life event or experience integrated into an identity - constituting narrative including graduation, promotion, job loss, marriage, birth of a child, tsunami, divorce, death of a loved one, earthquake and so on. as we live our lives (that is, as our story unfolds), we experience countless events that irrevocably constrain what narrative(s) we can project. for example, marriage following a brief passionate love affair does away with the identity of bachelor / bachelorette. is this event / experience to be understood as a threat to personal identity ? does the answer to this question change if the partner turns out to be physically or psychologically abusive, or unfaithful, or obsequious ? but for the marriage, the spouse might have more self - confidence, more independence, more friends, better health, and so on. for some this will be a devastating occurrence, for others this may prove to be the making of the man. in either case the narrative flow will be disrupted. michael j. fox was a prominent canadian actor in the 1980s and 90s, well - known for his roles in the television series family ties, and spin city and in the back to the future movie franchise. in 1991, he was diagnosed with early onset pd. in 2000, a few years after having publicly announced his illness, he launched the michael j. fox foundation for parkinson s research. at this time, he also turned his attention to writing and has authored several bestsellers lucky man (2002), always looking up : the adventures of an incurable optimist (2009), and a funny thing happened on the way to the future, (2010). michael j. fox s identity - constituting narrative is complex and many of the narrative details are subject to multiple interpretations. at minimum, he is an accomplished actor, a published author, a person living with pd and effective advocate for others living with pd. to be sure, michael j. fox s life trajectory would have been very different but for his illness. moreover, without this prior successful career, he might not have become a successful advocate for people living with pd. and, without both of these life experiences he might not have been awarded the order of canada in 2011 for a lifetime of outstanding achievement, dedication to the community and service to the nation. all of these events and experiences are part of his identity - constituting narrative ; none of these events can properly be described as a threat to his identity. to insist on this point, the actor, famous for his role in the movie superman (and sequels), was paralyzed as a result of an equestrian accident. along with his wife dana, he founded the christopher and dana reeve foundation to support spinal cord injury research and to improve the quality of life of persons living with spinal cord injury. christopher reeves identity is that of the actor - turned - advocate for spinal cord injury research. his accident was no more a threat to his personal identity (his autobiographical narrative) than his career as an actor. both of these facets of his life were an integral part of his personal identity his life story. to recap, a major life event or experience that dramatically disrupts the narrative flow undeniably constrains the dialectical process of identity formation (and thereby alters a planned or anticipated narrative), but this biographical disruption does not, in itself, constitute a threat to identity. a fourth plausible response to the question is dbs for pd a threat to personal identity ? suggests that dbs is such a threat but only insofar as it is a threat to agency the ability to make informed and rational choices as when a person s actions do not flow from her intentions or beliefs but rather are the result of direct brain manipulation. here it is worth noting that following dbs patients not only report i do nt feel like myself anymore, and i feel like a robot, and i feel like an electric doll (, p. 1813). emerging research on the effects of dbs on patients with pd suggests that there is cause for concern with respect to the ways in which direct brain stimulation potentially affects agency. for example, pathological gambling is now recognized as a potential side - effect of dbs [2527 ]. this means that some patients with pd who have been treated with dbs and who spend excessive amounts of time in front of slot machines do not do so as a matter of choice, but as a consequence of brain manipulation. when direct brain manipulation explains a belief or behavior there is reason to think of this as a serious threat to agency, which in some instances may give rise to a threat to identity. for illustrative purposes, this article has focused narrowly on dbs for pd. the conclusions that follow, however, apply to dbs in general. dbs can be a uniquely disruptive experience resulting in dramatic changes in behaviour, mood and cognition. such changes can have a major impact on personality and, according to some, can also have a major impact on personal identity. if personal identity is understood in static terms, it may be reasonable to worry about how changes in personality potentially threaten personal identity. but, if personal identity is understood in dynamic, narrative and relational terms (and if personality and personal identity are understood not to be synonymous), then the claim that dbs is a threat to personal identity is problematic. from one perspective, the claim is false because it misunderstands the dynamic nature of personal identity. from a second perspective, the claim is misdirected because it is not dbs for pd but rather the discriminatory attitudes of others towards persons with disability that are threatening to personal identity. from yet another perspective, the claim is trivially true as all dramatic events and experiences integrated into an autobiographical narrative, not only dbs for pd, are then potentially threatening to personal identity there is one sense, however, in which it may be accurate and not trivially true to describe dbs as a threat to personal identity. this is when dbs undermines agency to such an extent that the person is no longer able to meaningfully contribute to the authoring of her own life (i.e., to contribute to the cyclical and iterative process of projecting, defending and revising a self - narrative). without the ability to contribute to the process of identity formation | this article explores the notion of the dislocated self following deep brain stimulation (dbs) and concludes that when personal identity is understood in dynamic, narrative, and relational terms, the claim that dbs is a threat to personal identity is deeply problematic. while dbs may result in profound changes in behaviour, mood and cognition (characteristics closely linked to personality), it is not helpful to characterize dbs as threatening to personal identity insofar as this claim is either false, misdirected or trivially true. the claim is false insofar as it misunderstands the dynamic nature of identity formation. the claim is misdirected at dbs insofar as the real threat to personal identity is the discriminatory attitudes of others towards persons with motor and other disabilities. the claim is trivially true insofar as any dramatic event or experience integrated into one s identity - constituting narrative could then potentially be described as threatening. from the perspective of relational personal identity, when dbs dramatically disrupts the narrative flow, this disruption is best examined through the lens of agency. for illustrative purposes, the focus is on dbs for the treatment of parkinson s disease. |
immunization categories, experimental groups, and influenza virus strains used are shown in the table. twenty - seven pigs were intranasally (in) inoculated with 7.0 log10 50% egg infectious doses of swine / belgium/1/98 (avian - like swine influenza [h1n1 ]), swine / gent/7625/99 (reassortant influenza [h1n2 ] with human - lineage ha), swine / flanders/1/98 (reassortant influenza [h3n2 ] with human - lineage ha), or some combination of the three two groups received a single inoculation with either siv (h1n1) or siv (h1n2). three groups received dual, consecutive inoculations with 2 siv subtypes at a 4-week interval. twenty - four pigs received 2 intramuscular (i m) doses of commercial, inactivated siv vaccine, at a 4-week interval. one vaccine contained a classical swine - lineage subtype h1n1 virus, a / new jersey/8/76 ; the remaining 3 contained various avian - like subtype h1n1 sivs (table). phylogenetic lineage of the hemagglutinin gene : c, classical swine ; hu, human ; av, avian. infection with swine / belgium/1/98 (h1n1), swine / gent/7625/99 (h1n2), or swine / flanders/1/98 (h3n2). the first 3 vaccines are bivalent (subtypes h1n1, h3n2) ; the fourth is trivalent (subtypes h1n1, h3n2, h1n2). # infection with swine / belgium/1/98 (h1n1). each individual pig was hyperimmunized with a different european siv isolate : subtype h1n1 viruses were swine / finistre/2899/82, swine / belgium/1/98, and swine / gent/132/2005 ; subtype h1n2 viruses were swine / scotland/410440/94, swine / gent/7625/99, and swine / gent/177/2002 ; subtype h3n2 viruses were swine / gent/1/84, swine / flanders/1/98, and swine / gent/131/2005. six pigs were first inoculated in with swine / belgium/1/98 (h1n1) followed by a single i m administration of new jersey/8/76-based siv vaccine 5 weeks later. nine pigs were hyperimmunized against various european sivs (table) by in inoculation, followed by an i m innoculation with the same virus in combination with freund s complete adjuvant 4 weeks later. all serum samples were examined in hemagglutination - inhibition (hi) assays against the european subtype h1n1, h1n2, and h3n2 viruses listed above ; 3 north american sivs with a classical h1 ; and a / california/04/2009, a prototype pandemic (h1n1) 2009 virus (7). the north american sivs included swine / iowa / h04ys2/2004 (triple reassortant influenza [h1n1 ]), swine / ontario/11112/04 (reassortant influenza [h1n1 ]), and swine / indiana/9k035/99 (triple reassortant influenza [h1n2 ]). low amino acid homology was present in the ha1 region of the ha gene between the european h1 sivs used for infection of pigs and the north american h1 sivs (range 70%75%) or the pandemic (h1n1) 2009 virus (69%72%). when compared with avian - like influenza (h1n1) strains in european vaccines, the new jersey/8/76 vaccine strain was more closely related to north american sivs (77%81% vs. 92%94%) and pandemic (h1n1) 2009 virus (72%75% vs. 90%). hi antibodies induced by a single infection with european subtype h1n1 or h1n2 sivs did not cross - react with north american h1 sivs or with pandemic (h1n1) 2009 virus. in contrast, consecutive infections with 2 european subtypes frequently induced cross - reactive antibodies, even though european viruses do not contain a classical swine h1 ha. three european vaccines also induced cross - reactive antibodies to north american h1 sivs, and 2 induced antibody titers > 20 to pandemic (h1n1) 2009 virus in most pigs. hi titers against pandemic virus were lower with a / new jersey/8/76-based vaccine than with vaccine containing an avian - like siv (h1n1). a single vaccination with a / new jersey/8/76-based vaccine induced only minimal hi antibody titers in influenza - naive pigs, but existing antibody titers (data not shown) to european avian - like subtype h1n1 siv in pigs previously infected with this virus dramatically increased, even though these viruses contain ha proteins of the avian and classical swine lineages respectively. all pigs had antibodies to the viruses with a classical swine h1 ha, including pandemic (h1n1) 2009 virus. finally, hyperimmunization with european sivs of subtype h1n1, h3n2, or h1n2 resulted in high titers to the homologous viruses. cross - reactions with viruses with a classical h1 were consistently observed with serum samples from pigs hyperimmune to avian - like european influenza (h1n1) viruses, but they were rare or absent after hyperimmunization with subtype h1n2 or h3n2 strains. a large antigenic and genetic distance exists between european h1 sivs and viruses with a classical h1 ha (8,9). nevertheless, pigs infected or vaccinated with european sivs frequently have cross - reactive hi antibodies to pandemic (h1n1) 2009 virus and related north american sivs. two factors predispose for serologic cross - reactivity : 1) elevated antibody titers to european avian - like subtype h1n1 sivs, as in hyperimmune swine serum and some postvaccination serum ; and 2) infection with 2 european siv subtypes. consecutive experimental infection of pigs with the antigenically distinct sivs of subtypes h1n1 and h1n2 causes a strong boost of already existing hi antibody titers to the first infecting virus, as shown by longitudinal investigations (6). these pigs may even develop low levels of cross - subtype - reactive hi antibodies to siv (h3n2). in humans and in mice, sequential infections with influenza virus variants seemingly lead to a predominant antibody response against cross - reactive epitopes on the ha, which are shared with the first infecting virus, whereas the response to strain - specific epitopes may be lower (10). such cross - reactive antibodies may cause cross - recognition of viruses with a classical h1 ha in our dually infection immune pigs, but further studies with monoclonal antibody escape mutants are needed to understand cross - reactivity at the molecular level. multiple siv infections have become common in swine - dense regions of europe since the introduction of the h1n2 subtype in the late 1990s (11). the hi test will clearly fail to differentiate here between established sivs and pandemic (h1n1) 2009 virus. this conclusion is further supported by preliminary hi tests on sera collected from finishing pigs in belgium during 2007. of 172 serum samples, 35% and 36%, respectively, were positive against swine / iowa/2004 (h1n1) and swine / indiana/99 (h1n2) ; 95% of these positives had antibodies to > 1 european siv subtype. besides serum antibody to the ha, an infection with live influenza virus also stimulates mucosal immunity and cellular immune responses to highly conserved viral epitopes (12). these responses mean partial protection against a subsequent infection with an antigenically unrelated strain may occur in the absence of cross - reactive antibodies (13). in contrast, protection offered by inactivated influenza vaccines is almost entirely dependent on serum hi antibody titers. two of the 4 vaccines used induced hi antibody titers against pandemic (h1n1) 2009 virus that are considered protective. unexpectedly, avian influenza (h1n1) siv - based vaccine gave higher antibody titers than the a / new jersey/76-based vaccine. the amino acid homology between vaccine and test strains is thus a less reliable predictor of serologic cross - reactivity than one would assume. our data suggest that preexisting immunity to established siv strains may partially protect pigs in europe against pandemic (h1n1) 2009 virus, but the extent of such protection needs to be assessed in well - controlled challenge experiments. pandemic (h1n1) 2009 infection in pigs in europe has so far been limited to countries where sivs are absent or circulating at low levels. whether the virus will become established in countries where sivs are widespread remains to be seen. the divergence between h1 of contemporary seasonal influenza (h1n1) viruses of humans and pandemic (h1n1) 2009 virus is approximately the same as that between sivs in europe and north america. however, cross - reactive hi antibody responses to pandemic (h1n1) 2009 virus have been almost exclusively detected in humans born before 1950 (14). these persons have been exposed to older variants of seasonal influenza (h1n1) viruses that are more closely related to classic swine influenza (h1n1) viruses and pandemic (h1n1) 2009 virus. two antigenically distinct influenza (h1n1) viruses seasonal and the 2009 virus could cocirculate in humans in the future. this cocirculation will likely broaden serologic responses and protection against influenza but may also complicate interpretation of hi test results. | we tested serum samples from pigs infected or vaccinated with european swine influenza viruses (sivs) in hemagglutination - inhibition assays against pandemic (h1n1) 2009 virus and related north american sivs. we found more serologic cross - reaction than expected. data suggest pigs in europe may have partial immunity to pandemic (h1n1) 2009 virus. |
diabetes mellitus (dm) is one of the major public health problems around the world. according to the compiled data of the world health organization (who), approximately 150 million people have diabetes mellitus worldwide, and this number of diabetic patients may be doubled by the year 2025. alongside established lifestyle factors, such as smoking, adiposity, and diet, ethanol is a very commonly used chemical substance and also has dose - related effect on cardiac events. possible cardioprotective effects of ethanol consumption continue to be hotly debated in the medical literatures and popular media. systematic reviews and meta - analyses have addressed that there is a curvilinear relationship between ethanol consumption and cardiovascular disease, with a protective effect of moderate ethanol consumption and a detrimental effect of large amounts intake [37 ]. importantly, epidemiological studies suggest that light to moderate alcohol consumption decreases the risk of cardiovascular events, that is, 1 - 2 drinks per d or 39 drinks per wk (one beer, one glass of wine, or one glass of spirit was approximated to one standard drink defined as 1.5 cl or 12 g of pure ethanol). moderate drinking can activate metabolism of ethanol by acetaldehyde dehydrogenase-2 generation to reduce the incidence of diabetic cardiomyopathy [8, 9 ]. diabetic cardiomyopathy is defined as the ventricular dysfunction that occurs in diabetic patients independent of another cause, such as coronary artery disease or hypertension. diabetic cardiomyopathy is a common complication of diabetes which has become a major cause of diabetes - related morbidity and mortality. mitochondrial acetaldehyde dehydrogenase-2 (aldh2) is a key enzyme which plays an important role in the metabolism of acetaldehyde and other toxic aldehydes. furthermore, in transgenic mice, overexpression of aldh2 has been shown to be protective against streptozotocin - induced diabetic cardiomyopathy. our previous results had indicated that aldh2 expression was further decreased accompanying the development of diabetes. meanwhile, we investigated the effects of low - dose ethanol feeding in diabetic rats which could promote myocardial protection through activation of aldh2 expression. and more, we also found that upregulation of aldh2 played a protective effect in myocardial ischemia and reperfusion injury and diabetes cardiomyopathy. aldh2 may be an endogenous cardiac protective factor in myocardial injury [2, 8, 14 ]. several biological mechanisms have been proposed to explain that myocardial fibrosis is one of the main pathological changes of diabetic cardiomyopathy. studies have found c - jun n - terminal kinase (jnk) signaling pathway played a key role in the process of myocardial fibrosis [1517 ]. evidence had shown that activation of the jnk pathway was involved in the progression of diabetes induced myocardial fibrosis and that such a pathway could be a therapeutic target for diabetic heart injury and cardiomyopathy [18, 19 ]. however, whether the jnk pathway is involved in the cardioprotective effect of low - dose ethanol on diabetic rats has not been fully elucidated. so in this study, we mimic diabetes model by intraperitoneal injection of streptozotocin (stz) in combination with low - dose ethanol ; the purpose of the present study is as follows : (1) to investigate the mechanism of low - dose ethanol which alleviates myocardial fibrosis in diabetic cardiomyopathy ; (2) to clarify whether low - dose ethanol mediated protection is associated with downregulating the jnk signaling pathway. this study might shed some light on low - dose ethanol as an effective therapeutic in the treatment of diabetic cardiomyopathy. adult male sprague - dawley rats (200 to 250 g) were obtained from bengbu medical college animal administration center. all animal studies were approved by the animal ethics committee of bengbu medical college and performed in accordance with the ethical standards. trizol was purchased from invitrogen (usa) ; hydroxyproline (hp) was purchased from nanjing jiancheng bioengineering institute (china). ethanol (etoh) was purchased from bengbu new chemical reagent factory (china). -actin antibodies were purchased from santa cruz biotechnology (usa), rabbit c - jun n - terminal kinase (jnk) and phosphorylated jnk (p - jnk) were purchased from anbo biotechnology (usa). chemiluminescence reaction (ecl) all primers were purchased from shanghai sangon biotech (china). as previously described by our laboratory, stz at 55 mg / kg freshly dissolved in 0.1 mol / l sodium citrate buffer (ph 4.5) was injected intraperitoneally to induce diabetic models in overnight fasted rats. all rats were randomly divided into four groups : normal control group (con), diabetes at 4 weeks group (dm4w), diabetes at 8 weeks group (dm8w), and ethanol + diabetes at 8 weeks group (etoh + dm8w), respectively (n = 6). in con group, rats were fed with standard rat chow and received an intraperitoneal injection of the same volume of citrate buffer for 8 weeks. in etoh + dm group, dm rats were fed with 2.5% etoh in their drinking water for one week to initiate drinking then, it was changed to 5% etoh continuous access through the remaining 7 weeks. male sd rats were anaesthetized by use of chloral hydrate (100 mg / kg) through intraperitoneal injection. throughout the experiment, systolic pressure (sp), diastolic pressure (dp), mean arterial pressure (map), and heart rate (hr) were determined by invasive hemodynamic evaluation methods for 30 min. at the end of the experimental period, hearts were excised rapidly, placed in ice - cold krebs - henseleit (k - h) buffer, and weighed and the ratio of heart weight / body weight (h / b) was calculated. heart tissue (100 mg) the supernatant was collected after centrifugation for 20 min (2000 rpm). left ventricular tissue obtained from all groups was stained with masson 's trichrome for the quantification of collagen. the histological sections were taken in 10% neutral formalin - fixed, dehydrated, paraffin embedded sections. then, the samples underwent the dehydration of gradient ethanol, neutral resin embedding, and masson trichrome staining. myocardial collagen was quantified at a final magnification of 200x with a polarized microscope connected to a video camera. myocardial collagen volume fraction (cvf) was analyzed using image pro analysis software, expressed as the mean percentage of collagen area to the total area of each microscopic field. five visions under microscope of each sample were randomly chosen and the average of them was taken for analysis. total rna was extracted from the left anterior myocardium using trizol according to the manufacturer 's instructions. total rna (3 g) was reversely transcribed to cdna, and pcr was performed by a routine method. the primers used were as follows : for aldh2, forward : 5-gtg ttc gga gac gtc aaa ga-3 and reverse : 5-gca gag ctt ggg aca ggt aa-3 and the product size was 187 bp ; for -actin, forward : 5-gag acc ttc aac acc cca gcc-3 and reverse : 5-ggc cat ctc ttg ctc gaa gtc-3 and the product size was 312 bp. the pcr condition was as follows : predenaturing at 95c for 3 min and then 40 cycles (50 s denaturation at 95c, 50 s annealing at 62.5c, and 60 s extension at 72c), followed by a final step at 72c for 10 min. myocardium tissues (100 mg) from each group were collected and homogenized in a lysis buffer. homogenates were sonicated and centrifuged at 12,000 g for 30 min at 4c. the protein concentration was determined using the bicinchoninic acid (bca) protein assay kit. total protein (80 g) was separated by sds - polyacrylamide gel electrophoresis (page) and transferred electrophoretically to a polyvinylidene difluoride (pvdf) filter membrane. the membranes were blocked with 5% nonfat milk in tris - buffered saline tween (tbst) for 2 h, and then they were incubated at 4c overnight with the corresponding primary rabbit jnk antibody (1 : 1000), rabbit p - jnk antibody (1 : 1000), and mouse -actin antibody (1 : 500). all membranes were incubated for 1 h with corresponding secondary antibody hrp - linked anti - mouse igg or hrp - linked anti - rabbit igg. one - way analysis of variance (anova) followed by student - newman - keuls (snk) was used for multiple comparisons. in contrast to con group, there was no statistical difference about hemodynamic parameters in dm4w groups, but systolic pressure (sp), diastolic pressure (dp), mean arterial pressure (map), and heart rate (hr) were decreased significantly in dm8w groups. compared with dm8w group, sp, dp, map, and hr were rather increased significantly in etoh + dm8w group. (table 1) compared with con group, there was no significant difference about the ratio of heart weight to body weight (h / b) and hydroxyproline (hp) content in myocardial tissue in diabetic rat in dm4w group ; however, with extended duration, h / b ratio and myocardial hp content were increased in dm8w group, and the difference was statistically significant (table 2). compared with dm8w group, h / b ratio and myocardial hp content were significantly deceased in etoh + dm8w group (p < 0.05). in masson trichrome staining, however, collagen tissue was increased markedly and disrupted in some area in diabetic group. in dm4w group, myocardial cells were approximately arranged well, collagen fibers were sparsely distributed, and interstitial collagen was dyed a little blue. compared with control group, myocardial cells were in a disordered arrangement, the interstitial collagen were edematous, and collagen fibers were unevenly distributed and increased markedly in dm8w group. compared with dm8w group, myocardial cells were arranged neatly, and collagen fibers were significantly reduced in etoh + dm8w group. quantitative analysis results. as displayed in table 2, compared to control group, the contents of collagen volume fraction (cvf) were significantly increased in dm4w group (p < 0.05), and with the development of diabetes, cvf were further increased in dm8w groups (p < 0.01). but in contrast to dm8w group, the contents of cvf were markedly decreased in etoh + dm8w group (p < 0.01, figure 1, table 2). real - time pcr revealed that, compared with control group, the expression of aldh2 mrna level was reduced in dm4w (p < 0.05). with the development of diabetes, aldh2 mrna in contrast to dm8w group, the expression of aldh2 mrna was increased in etoh + dm8w group (p < 0.01, table 3). on western blot analysis, compared with control group, the ratio of p - jnk / jnk protein expression was increased in dm4w (p < 0.05), and with the development of diabetes, the ratio of p - jnk / jnk was further increased in dm8w groups (p < 0.01). compared with dm8w group, the ratio in etoh + dm8w group was decreased (p < 0.01, figure 2, table 3). diabetes mellitus is becoming an epidemic health threat and represents one of the most prevalent chronic noncommunicable disorders. cardiovascular disease is a serious complication of diabetes and is responsible for 80% of the deaths among diabetics. diabetic cardiomyopathy is one of the most common complications of diabetes ; its major pathological characteristics are hypertrophy or hyperplasia of cardiac myocytes. excessive deposition of myocardial interstitial collagen and myocardial fibrosis often leads to cardiac hypertrophy and decrease of heart function, which play a vital role in the occurrence and development of diabetic cardiomyopathy. in the present study, our results demonstrated that, with the progression of diabetes, systolic pressure, diastolic pressure, mean arterial pressure, and heart rate were declined significantly in dm8w group compared with con group, but the ratio of heart weight / body weight, hydroxyproline content, and cvf were markedly increased. meanwhile, the mrna expression of aldh2 was decreased and the ratio of p - jnk / jnk was increased. when the dm rats were treated with ethanol at low concentration, hemodynamic parameters were improved and hydroxyproline content and cvf were decreased, accompanied with the increase of myocardial aldh2 mrna expression and decrease of p - jnk / jnk protein expression. the results suggested that with low - dose ethanol intervention, enhanced aldh2 expression can antagonize the happening of myocardial fibrosis in diabetic cardiomyopathy, which may be relevant with downregulating the jnk pathway. mitochondrial aldehyde dehydrogenase 2 (aldh2) is a kind of aldehyde oxidase that is involved in ethanol metabolism, which is closely related to human alcohol drinking behavior. heavier alcohol consumption is associated with increased risk of developing diabetes mellitus, hypertension, and cardiovascular and cerebrovascular disease. nevertheless, there are increasing reports showing a critical role for light to moderate alcohol ingestion could protect against myocardial injuries. researches indicated a u - shaped relationship between alcohol consumption and cardiovascular disease mortality [6, 26 ]. statistics manifested the mortality of drinking alcohol up to or over 6 cups (355 ml / cup) per day is 1.6 times as high as control, while light - to - moderate drinking of 1~2 cups alcohol per day [fewer than 2 cups or 1 ounce (28.3495 g) ] reduced mortality from cardiovascular disease. in addition, clinical data showed that moderate drinking can protect metabolic syndrome (cardiovascular disease and diabetes) via influencing various factors to reduce the diabetes prevalence, such as glycosylated hemoglobin, high density lipoprotein cholesterol, and fibrinogen. after continuously feeding c57bl/6 mice with 18% of ethanol for 12 weeks, the myocardial mechanics were restored and the expression and activation of pkc and akt were increased, which established that etoh feeding causes cardiac expression of activated pkc-. sun. observed that ischemic injury could result in downregulation of mitochondrial aldh2 in mice hearts inducing an elevation of aldehyde 4-hydroxy-2-nonenal (4-hne), leading to cardiomyocyte apoptosis through downregulation of hsp70 and activation of jnk and p53. aldh2 detoxifies 4-hne, a mediator of programmed cell death events, by transmitting a mitochondrial aldh2 signal to elicit a cytosolic response through the jnk / p53 pathway. however, using transgene technology to increase the expression of aldh2 can contribute to antagonizing heart failure and decreasing heart function. furthermore, our previous research found that there was a close relationship between aldh2 and oxidative damage. with the progression of diabetes, the myocardial antioxidant ability of diabetic rats was decreased, which exacerbated the progression of myocardial fibrosis [8, 13, 31 ]. in this study, we found that the ratio of heart weight / body weight, hydroxyproline content, and cvf were markedly increased in diabetic 4 and 8 weeks ' rats, which suggested diabetic induced myocardial fibrosis. when the diabetic rat was treated with low doses of ethanol to induce aldh2 activity, the hemodynamic parameters were increased and hydroxyproline content and cvf were decreased, which indicated that increasing aldh2 expression can attenuate the happening of myocardial fibrosis and the destroying of myocardial injuries. mitogen - activated protein kinases (mapks) play an important role in the signal transduction pathways from the membrane to intracellular compartments including the nucleus. they regulate the functions of many gene products and therefore affect cell growth, differentiation, and apoptosis. oxidative stress, inflammation, endoplasmic reticulum stress, and autophagy defect can activate mapks signaling pathway in the progression of diabetes complications. li. observed that production of a large number of ros was thought to be an important contributing factor, concomitant with activation of jnk, p38 mapk, and tgf- in the development and the progression of diabetic cardiomyopathy. c - jun nh2-terminal kinase (jnk) is one of the major members of mapks, and jnk activation is also implicated in cardiac fibrosis [33, 34 ]. jnk signaling pathway plays a key role in the growth of cardiac fibroblasts induced by high glucose. studies of diabetic rats had demonstrated that myocardial fibrosis was developed ; meanwhile, jnk mrna expression level and activity were upregulated. furthermore, disruption of the jnk protein kinase decreased the occurrence and development of diabetic myocardial fibrosis [18, 35 ]. it is worthwhile to note that, in our experiment, we found that, with the progression of diabetes, the expression of myocardial aldh2 at mrna level was decreased and the ratio of p - jnk / jnk at protein level was increased, which suggested aldh2 and jnk signaling pathway both participated in the occurrence and development of diabetic cardiomyopathy. moreover, further enhancing activation of aldh2 expression of low doses of ethanol for 8 weeks in diabetic rats, accompanied with the high - expression of aldh2, p - jnk / jnk were decreased in contrast to diabetes rats, which suggested activation of aldh2 expression might be associated with downregulating the jnk signaling pathway to relieve myocardial fibrosis and myocardial injuries. we only observed that the protection of low - dose ethanol was relevant with downregulating the jnk pathway in diabetic cardiomyopathy. to better understand the mechanisms involved, we will adopt the activation or inhibition of jnk to investigate the downstream signaling molecules in the follow - up experiments. to address this issue, we will use jnk inhibitor to observe whether inhibiting jnk pathway can play the cardiovascular role in diabetes rats. moreover, we further explore whether enhanced activation of jnk signaling pathway can antagonize the cardioprotective effect of activation of aldh2 by low - dose ethanol. through these experiments, we want to verify low - dose ethanol could attenuate myocardial fibrosis via downregulating the jnk pathway in diabetic cardiomyopathy. in summary, our results demonstrate that with the progression of diabetes, aldh2 expression was decreased accompanied with the happening of myocardium fibrosis. treatment with ethanol at low concentration can protect the heart by upregulating aldh2 and downregulating the jnk signaling pathway against myocardial fibrosis in diabetic cardiomyopathy. | objective. to investigate the effects of low dose ethanol feeding in diabetic rats and analyze its underlying mechanisms. methods. male sprague - dawley rats were divided into 4 groups : control (con), diabetes at 4 weeks (dm4w), diabetes at 8 weeks (dm8w), and etoh + dm8w. after 8 weeks, hemodynamic parameters were recorded and heart weight / body weight (h / b) and hydroxyproline (hp) content in myocardium were measured. morphology of collagen in myocardial tissue was observed with masson 's trichrome staining method and collagen volume fraction (cvf) was analysed. the mrna expression of aldh2 was assessed with real - time pcr. the protein expressions of p - jnk and jnk were evaluated using western blot. results. in contrast to con group, there was no difference in hemodynamic parameters in dm4w group, but mean arterial pressure and heart rate were decreased in dm8w group, and the ratios of h / b, hp, and cvf were markedly increased. aldh2 mrna expression was decreased, while the ratio of p - jnk / jnk were increased. compared with dm8w group, the above indexes were improved in etoh + dm8w group. conclusion. with low dose ethanol intervention, enhanced aldh2 expression can antagonize the happening of myocardial fibrosis in diabetic rats, which may be relevant with downregulating the jnk pathway. |
children brought for medical treatment are often found to be suffering from more than one morbid condition, making a single diagnosis impossible. the effective management of those conditions is more dependent on adopting a holistic approach using cheap, universally available and accessible strategies rather than sophisticated and expensive technology. according to the world bank report 1993, for situations where laboratory support and clinical resources are limited, such an approach is more realistic and cost - effective, and therefore, has the potential to make the greatest impact on the global burden of disease. during the year 1992, the world health organization (who), who, in collaboration with united nations integrated children 's emergency fund (unicef), and some other agencies, institutions and individuals, responded to the challenge by adopting a strategy known as integrated management of childhood illness (imci)., priority has not been given to diagnosis of individual disease, rather the classification of the diseases and assessment of severity according to the common signs and symptoms were approached. along with curative care for common childhood illnesses like acute respiratory infection, diarrhea, measles, malaria and malnutrition, the strategy also addressed aspects of nutrition, immunization, and other important elements of disease prevention and health promotion. imci addressed the age group between 2 weeks and 59 months, but in india it was found that neonatal mortality constitutes 64% of under - five mortality, all neonates are included in the strategy, starting from the day of birth and it is adapted in the indian version as imnci. in the adapted version, the entire age group of 0 to 59 months (as against 2 weeks to 59 months in imci) was included to address the neonatal mortality challenge. in the imnci version, the age group was divided in two groups as 0 - 2 months and 2 months to 59 months. in both the age groups, through the combination of signs and symptoms classification of the condition is made. this classification indicates the severity of conditions and call for specific actions like red indicating urgent referral, yellow indicating management at existing health facility and green indicating management at home. the imci algorithm in both 7 days to 2 months and 2 months to 5 years age group was validated in india and other developing countries by several studies. as imnci is the adapted version of imci, followed only in india, there is paucity of published study testing the validity of imnci. however, two studies were done at department of pediatrics, kalawati saran children 's hospital and lady hardinge medical college testing the validity of imnci algorithm during 2002 - 2003 by goswami v, singh v, dutta ak and by kaur s, singh v, dutta ak, chandra j during the year 2005 - 2006. in west bengal imnci algorithm was operational in purulia district since the year 2008, but there is hardly any published study testing its validity. under this background, the present study has been done with the objective to assess the validity and reliability of the algorithm with provisional diagnosis of senior pediatricians for each imnci classifications. this observational, cross sectional study was done at department of pediatrics, in a tertiary care hospital of kolkata during january to march 2009 with the young infants between 0 - 2 months. all the young infants presented during this study period with a fresh episode of illness were included in the study with o an informed consent from their parents. altogether 117 mothers were interviewed with a pre tested, semi structured interview schedule and the young infants were assessed, classified and categorized for treatment using physician 's chart booklet for imnci along with the assessment form used for imnci. these cases were then sent to the pediatricians for further assessment without any mention of the classifications made by the researchers using the imnci algorithm so as to reduce observer bias. pediatricians examined the young infants as they did in any tertiary care hospital and recorded the presenting symptoms, clinical features and provisional diagnosis in the opd or emergency examination tickets. the imnci classifications were compared with those provisional diagnoses. the provisional diagnosis which could be compared with a particular imnci classification was determined after discussion with senior faculty members of department of pediatrics, medical college, kolkata. validity characteristics like sensitivity, specificity, positive predictive value, negative predictive value, and reliability characteristics like percent agreement, and kappa were assessed for individual imnci classifications against pediatrician 's provisional diagnosis. the kappa test is used to exclude the extent of percent agreement which was due to chance. out of the 117 young infants, 25.64% were within 7 days of age and 2.56% were presented within 24 hours. the young infants were presented with one or multiple presenting symptoms and among them cough and cold (33.33%) was the main symptom. next were fever (12.82%), loose stool (11.11%) and respiratory distress (11.11%). other important presenting symptoms were convulsion (7.69%), vomiting (7.69%), yellow discoloration of skin (5.98%), inability to suck (5.98%), unsatisfactory feeding (5.13%), and no cry after birth (2.56%). the common classifications were severe malnutrition (23.08%), feeding problem (22.22%), low weight for age (21.37%), possible serious bacterial infection (19.66%), possible serious bacterial infection, not able to feed (18.80%), severe jaundice (1.71%) etc [table 1 ]. distribution of study subjects according to the imnci classification (multiple classification) n = 117 when pediatricians assessed the cases, a single provisional diagnosis was made for each study subject based on initial clinical evaluation. the important provisional diagnoses as found in the opd or emergency tickets were common cold (22.20%), followed by septicemia (15.40%), jaundice (7.70%), breast fed loose stool (6.80%), birth asphyxia (6.80%), upper respiratory tract infection (6.80%), local infection (6%), lower respiratory tract infection (4.30%), low birth weight baby (3.50%), thrush (1.70%) [table 2 ]. distribution of study subjects according to provisional diagnosis by pediatricians n = 117 in eight cases (6.80%), the pediatricians found no abnormality and the initial diagnoses were healthy baby. for fourteen cases (12%), there were some other single provisional diagnoses like spina bifida occulta, anorectal malformation, cephalhematoma, congenital hypothyroidism, down 's phenotype, abdominal colic, congenital leukemia, stenosis of gut, hemorrhagic disease of newborn, meconium aspiration syndrome, excessive jitteriness, prune belly syndrome, congenital cyanotic heart disease [table 2 ]. the classifications made by imnci algorithm were compared with the provisional diagnoses of the pediatricians. as per the opinions of senior faculty members of pediatrics department, possible serious bacterial infection was comparable with septicemia ; local bacterial infection with cellulites, boil ; whereas for jaundice, low body temperature, and diarrhea, the comparisons should be with similar conditions. there was no diagnosis as severe jaundice, severe dehydration, severe malnutrition, low weight for age, feeding problem and no feeding problem by the pediatricians and so comparison could not be made for those conditions. both imnci algorithm and pediatrician 's provisional diagnosis did not find any case of some dehydration, severe persistent diarrhea or severe dysentery and obviously no comparison could be done for those cases. in case of possible serious bacterial infection, in the present study, sensitivity was found as 88.89%, positive predictive value was 36.39%, negative predictive value was 97.26% and specificity was 71.72% [table 3 ]. along with validity, the reliability of the classification was also assessed by percent agreement, by exclusion of chance by kappa test. but it was evident that most of the agreement was due to chance ; so when chance agreement was excluded by kappa test, the kappa value was only 0.38 [table 3 ], which indicates only minimal agreement. distribution of validity and reliability characteristics of imnci classification, against pediatrician s provisional diagnosis n = 117 in case of local bacterial infection, sensitivity was only 14.29% whereas the specificity was 99.09% [table 3 ]. the percent agreement of local bacterial infection was 94% with the kappa value 0.20, indicating only minimal agreement [table 3 ]. in cases of jaundice, sensitivity was 66.67% whereas the specificity was 99.07% [table 3 ]. the percent agreement was 97% with kappa value 0.73, indicating good agreement [table 3 ]. for no dehydration classification in algorithm, the sensitivity was 25% whereas the specificity was 94.50% [table 3 ]. the percent agreement was 90%, the kappa value was only 0.19, which indicates only negligible agreement [table 3 ]. as per the algorithm, there were 44 cases of possible serious bacterial infection and among those, there were 21 cases, which had no ability to feed or suck. though in the algorithm, those cases were classified separately, pediatricians did not consider them as separate entity and diagnosed as septicemia as a whole. when chance agreement was excluded by kappa test, the kappa value was only 0.29, indicating only minimal agreement [table 3 ]. integrated management of neonatal and childhood illness (imnci) is already operational at the field level in india, but there is paucity of published study testing its validity and reliability. in the present study, it was found that young infants were presented with one or multiple symptoms and among them cough and cold (33.33%) was the main symptom. the other important presenting symptoms were fever (12.82%), loose stool (11.11%) and respiratory distress (11.11%). in the study done by sachdev. the most common presenting symptom was cough (65.3%), followed by fever (53.8%), running nose (40.3%), diarrhea (17.3%), respiratory distress (9.6%). the young infants were assessed and classified according to the imnci algorithm and it was found that majority (54.70%) had single classification, 38.47% had two classifications, 5.98% had three and 0.85% had four classifications. the mean number of classifications was 1.53 with standard deviation 0.65. in the study done by sachdev, the mean number of classification by imnci algorithm was 1.8 with standard deviation 0.8. in the study done by kaur, singh, dutta, chandra, the mean number of morbidities was 1.75. in the present study, the important provisional diagnoses as found in the opd or emergency tickets were common cold (22.20%), followed by septicemia (15.40%). in the study done by sachdev. the most common diagnosis of the pediatricians was low birth weight (75.20%), followed by diarrhea (27.9%), upper respiratory tract infection (26.3%) and septicemia (21.7%). in case of possible serious bacterial infection, in the present study, sensitivity was found as 88.89%, positive predictive value was 36.39% and specificity was 71.72%, whereas in the study done by sachdev, the sensitivity was 96.5% and specificity was 51.8%. in the study done by kaur, singh, dutta, chandra, the sensitivity of algorithm to identify bacterial infection was 88.5% while the specificity was relatively low (57.4%). the reasons for this low specificity and positive predictive value might be that the predictors for possible serious bacterial infection as mentioned in the algorithm could predict other conditions also as convulsion or bulging fontanelle might occur in other cns disorders like hypoxic - ischemic encephalopathy, birth asphyxia, intracranial hemorrhage, meningitis, hypoglycemia, hypocalcaemia or any other causes of subdural effusion apart from septicemia. similarly increased respiratory rate, severe chest indrawing or nasal flaring could be the manifestation of hyaline membrane disease, transient tachypnea of newborn, meconium aspiration syndrome and other causes of respiratory insufficiency. dehydration fever, which is very much common particularly in an overheated nursery, might be an important cause of over diagnosis of possible serious bacterial infection. in the study by goswami, singh, dutta, algorithm tends to over diagnose serious bacterial infection by 8 - 20% (in three age groups). in the study done by kaur, singh, dutta, chandra, also, it was found that out of 80 cases classified by the algorithm as possible serious bacterial infection with difference in diagnosis with the pediatricians, 31 (38.7%) had birth asphyxia with hypoxic - ischaemic encephalopathy, 16 (20%) had hypocalcemic seizures, 11 (13.7%) had meconium aspiration syndrome, and 7 (8.8%) had hemorrhagic disease of newborn. other conditions included respiratory distress syndrome (9 cases), transient tachypnea of newborn (4 cases), and neonatal seizures (2 cases). pediatricians diagnosed one big boil and more than 10 pustules as boil or cellulites, comparable with local bacterial infection whereas for the imnci algorithm, it was considered as possible serious bacterial infection. this difference in detection might be the cause of low sensitivity. in cases of jaundice, sensitivity was 66.67% whereas the specificity was 99.07. pediatricians diagnosed all relevant cases as jaundice ; clinically they did not categorize any case as severe jaundice. however, in the algorithm when the jaundice appears in the 1 day of life or persists for more than 14 days and extends in the palms and soles, it was classified as severe jaundice. in the study done by kaur, singh, dutta, chandra, the algorithm under - diagnosed the severity of jaundice in few subjects (12/131) and over - diagnosed (8/131) the severity in few subjects. for no dehydration classification in algorithm, probability of low sensitivity might be due to some cases like lactose intolerance, breast - fed loose stool and not consideration of urination status by the algorithm. in the study done by kaur, singh, dutta, chandra, of the 76 cases identified as diarrhea by the algorithm, 22 (29%) had breast - fed stools. in the algorithm, some conditions like birth asphyxia, down 's phenotype, abdominal colic, stenosis of gut, hemorrhagic disease of newborn, meconium aspiration syndrome, excessive jitteriness, prune belly syndrome, congenital cyanotic heart disease, anorectal malformation, cephalhematoma, congenital leukemia, breast - fed loose stool or lactose intolerance, spina bifida occulta, and congenital hypothyroidism were not covered. in the study done by kaur, singh, dutta, chandra, also it was seen that the algorithm under diagnosed some surgical conditions and congenital anomalies. therefore, in conclusion, it could be mentioned that imnci is a quite sensitive strategy and could identify the severe illnesses of the young infants requiring referral to higher facility. presence of other diagnosis with similar symptoms might result in false positive errors and low specificity. however, as this study was done in a tertiary care setting, further study particularly in primary health care setting is required. | background : integrated management of childhood illness (imnci) is already operational in many states of india, but there are only limited studies in indian scenario comparing its validity and reliability with the decisions of pediatricians. aims andobjectives : to assess the validity and reliability of the imnci algorithm with provisional diagnosis of senior pediatricians for each imnci classifications.materials and methods : the present study is done with all the young infants between 0 - 2 months presented during the study period with a fresh episode of illness to test the validity and reliability of the algorithm in comparison to provisional diagnoses of senior pediatricians. the study was done in a tertiary care hospital. validity characteristics such as sensitivity, specificity, positive predictive value, negative predictive value, and reliability characteristics such as percent agreement and kappa were assessed for individual imnci classifications.results:the sensitivity of possible serious bacterial infection, local bacterial infection, jaundice, no dehydration and possible serious bacterial infection, not able to feed were 88.89, 14.29, 66.67, 25 and 44.44% respectively. the specificities for the same conditions were 71.72, 99.09, 99.07, 94.50 and 86.87%. percent agreements for similar conditions were 74, 94, 97, 90 and 80% respectively and the kappa ratios were 0.38, 0.20, 0.73, 0.19 and 0.29 respectively.conclusion:it could be concluded that imnci is quite a sensitive strategy and could identify severe illnesses of young infants requiring referral to higher facility. further studies, particularly in primary health care setting, are required. |
cardiopulmonary resuscitation (cpr) is an emergency procedure which is performed on a person in cardiac arrest in an effort to reverse the otherwise inevitable progression to death. however, success is not always possible and not infrequently this procedure is associated with a high level of morbidity. it is for these conditions when cpr is not envisaged to be viable that the do not resuscitate (dnr) order was conceived.[25 ] this determination had almost exclusively been a physician - led decision until the 1980s when a shift in attitudes began to take shape. dnr orders began to veer toward patient - led determinations and were seen as a means for patients to reclaim the right to self - determination in the face of medical paternalism. even if the doctor was ultimately responsible for the decision, there was growing empirical evidence to suggest that most patients would like to play an active role in the determination of their resuscitation status. yet within asian cultures, family - centric rather than patient - centric determinations prevail, sometimes limiting such assertions of autonomy.[912 ] family members, invested with the responsibility of safeguarding the patient from psychological distress which in turn was believed to hasten clinical deterioration, often influenced how much the patient knew and consequently how involved the patient was in deciding about treatments. while in western cultures, the disclosure of diagnosis and prognosis to patients has been associated with a better quality of life, this is not necessarily so in asian cultures. showed that in korea, patients who were aware of their illness and who actively participated in the decision - making process did not score higher than others on outcome measures of quality of life and quality of death. in fact, those who were not fully involved in decision making scored better in some domains, some of which were statistically significant. singapore is a society that holds fast to asian values but is also increasingly influenced by western biomedical ethical frameworks. for physicians and nurses, the increasing onus placed on patient autonomy and a growing tendency to limit the influence of the family in the decision - making process do seem at odds with traditional mores of the familial determination fed mainly by a beneficent wish to protect patients from distress and also to maintain hope. as a result, local physicians have been found to maintain this status quo and prefer to discuss treatment options with the family rather than the patients themselves, particularly when dnr statuses are being determined. nonetheless, there is increasing evidence that patient perception and involvement in the decision - making process might be changing. it is from within this context that this study sets out to explore attitudes of oncology and palliative care doctors and nurses faced with sometimes conflicting cultural intuitions and professional opinions when deliberating dnr discussions. additionally, this paper also attempts to elucidate the understanding of the implications of a dnr order among physicians and nurses of the various oncology specialities and palliative medicine as well as their perceptions of patient care following a dnr order being implemented. the study took place in a tertiary specialist cancer center and an inpatient oncology ward located within the same campus. participants were doctors and nurses who worked in oncology or palliative medicine. to minimize disruption to service provision, the questionnaire was explained and distributed at department meetings for doctors and at staff handover periods for nurses. each participant completed a consent form and an anonymized once - off questionnaire comprising questions relating to discussing and deciding on dnr orders. the completed forms were placed within a box by each participant and collected by the investigators at the end of the day to ensure anonymity. over a 1-month period between march 1 and 31, 2011, 187 questionnaires were distributed and 146 questionnaires were returned (response rate 78.1%), out of which 37 (25.3%) were completed by doctors and 109 (74.7%) by nurses. the age of participants ranged from 19 to 68 years (mean 32.0, median 29) and years of experience in oncology or palliative care ranged from 0 to 40 years (mean 5.77 years, median 3.75). the responses to the questions are shown in table 2. where questions were left unanswered or more than one option was ticked, these are accounted for under void. demographic details of participants (n=146) responses to the questionnaire survey there was a variation in what participants thought a dnr order involved. although most agreed that therapies administered should be limited to those that can be provided in a general ward, negating any transfer to an intensive care unit, there was a significant discrepancy as to what this would in fact encompass. nearly half the participants (48%) felt that this determination would not entail the utilization of aggressive fluid replacements in a crisis or the implementation of antibiotics should the need arise despite the fact that both could be carried out in a general medical ward. conversely, 43% of respondents felt otherwise and would prevail upon the use of these measures should the need arise. when asked who a dnr decision should be discussed with, 78.8% of all responders felt that such determinations ought to include the patients themselves while 78.1% of all responders felt that the next of kin must play a part in such a process. thirty - six (24.7%) responders thought that a wider involvement of the family beyond simply the next of kin was called for in such deliberations ; 21 (14.3%) participants felt conversely that neither the patient nor the next of kin should be involved in dnr discussions. participants were then asked as to who should ultimately be responsible for determining a dnr order. responses appeared spread across the various options offered ranging from always the doctor to always the patient / family. there was a statistically significant difference (p<0.001 for pearson 's chi - square test) between the responses from nurses and those from the doctors. physician respondents tended to favor the physician being given the final say while nurses, the patient / family [figure 1 ]. responses of participants to the question of who should ultimately decide on a dnr order on the question of primacy between patient and family, responses were varied with 23.3% believing that priority was situation dependent. a small majority of 81 (55.4%) participants were found in favor of the patient rather than the family. twenty - seven (18.5%) participants however felt that the family ought to be afforded precedence. a striking finding of this study is the presence of variance in the understanding of the do not resuscitate while there was an acceptance that a dnr order signified a change of tact from treatments focused upon life extension to one of maximizing the quality of life and comfort measures, there were differing views on how patients would be managed when they deteriorated. the use of antibiotics and fluids in sepsis may extend life expectancy, but may also facilitate symptom management without being detrimental to the patients, which may in turn improve their quality of life. even if the goal of treatment has shifted away from life extension, the small amount of time granted by such interventions may prove to be providential where there are goals to be met or families awaited. hence, in patients with a dnr order, the clarification of how aggressive medical management should be is helpful particularly to the out - of - hours on - call team. it is remarkable that within such diversity in the comprehension of a dnr status, nearly half the participants (49.3%) had no concerns that a dnr order would lead to the provision of substandard care. perhaps this is related to an increasing tendency for decisions to be regularly reviewed and a feeling that these patients would be referred on to the palliative care teams for better symptom control and holistic management. likewise patients at the end of life who upon declaration of a dnr order would become eligible for the care for the dying pathway, which is increasingly being employed on campus and evidenced to be an effective tool in the early identification and therefore treatment of symptoms.[2224 ] with regard to the question of who should be involved in the determination of a dnr order, this study shows that patients and family should play a more important role than other healthcare professionals, reflecting the increasing importance of patient autonomy within the medical decision - making process. although a large majority (78.8%) of participants thought that dnr orders should be discussed with patients, this does not seem to be consistent with local practice. in many cases, patients tend to be too ill to discuss such plans and frequently it is the family that occupies the main decision - making position. it is possible that while healthcare professionals think that they should have dnr discussions with patients, in reality they are reluctant to broach this subject due to lack of time, fear of patient 's response, a sense of inadequacy in ability to handle such conversations, or as a result of the local perception that such discussions about death are in fact taboo. it is thus not altogether surprising that 78.1% of healthcare professionals felt that a dnr decision should be discussed with the next of kin. while helpful in trying to ascertain goals, values, and preferences of the patient, the next of kin may harbor vested interests and may not act in the patient 's best interests, in which case a discussion with the wider family unit may clear the air. it is perhaps for this reason that 24.7% respondents suggested such a wider familial involvement in decision making. why is there a great discrepancy between the involvement of next of kin and as many family members as possible ? it is possible that doctors involve only the next of kin mainly for pragmatic purposes, as a decision - making process involving many family members can be difficult and inefficient. communicating to the next of kin as a surrogate decision maker may also be seen as easier than discussing a dnr order directly with the patient., evidence exists that would suggest that patients may have a better quality of life if they are not actively involved in decision making. it appears that within the regnant family - centric asian ethical framework, acting in patients best interest may mean denying them of their right to self - determination. the acceptability and viability of such a posit however still requires further elucidation.[2830 ] questions regarding whether familial determinations without patient involvement are really made in the patient 's best interests need to be balanced against the necessity of family input to ascertain the many psychosocial, financial, physical, clinical, and spiritual issues to be considered so as to formulate a care plan that best provides for the needs of the patient.[3133 ] despite the importance of patient and family involvement, a significant minority of 14.3% of participants thought that dnr decisions should not be discussed with the patient or family. this may reflect the view that the dnr order is a medical decision regarding a treatment with medical indications that do not have to be discussed with the patient or family if the treatment is thought to be inappropriate for the patient.[53436 ] in keeping with local guidelines, the scottish integrated policy on decision making and communication of dnr orders states that where cpr is unlikely to have a medically successful outcome, it should not be attempted and it is an unnecessary and cruel burden to ask patients and relevant others to decide about cpr when it is not a treatment option. dnr orders are highly emotive and may be associated with negative connotations of failure ; hence, patients and their families may struggle to make a decision that is truly in patient 's best interest. simultaneously, discussions on a futile treatment in the vain hope of family acquiescence for this venture may in fact be in breach of the ethical principle of nonmaleficience. furthermore, if the patient or family decides against a dnr order, the provision of a futile treatment may in fact be in breach of the physician 's overriding duty of care to the patient and their family. exchanging patient autonomy for a treatment that will be more harmful than beneficial to the patient would also be a contravention of a physician 's primary obligation to act in the patient 's best interests. even so, should not patients right to self - determination be valued above all, even if their choice is perceived by the physician to cause more harm than good ? should it be the patient and/or family because it is about the patient 's life in the end, with input from healthcare professionals so that they can make an informed decision ? or should it be the doctor because it is ultimately a medical intervention with medical indications, with input from the patient and/or family so that the decision can be in line with the patient 's values and preferences ? results from this study appeared to veer toward a decisive response by either of the key personnal within the deliberations rather than the safety there was almost an equal preference for the doctor (41.8%) as there was for the patient and/or family (41.1%) to fulfill this fundamental role. indeed, only 16.4% opted for a response that would depend on the situation. in keeping with legal and practice guidelines, it was the physicians who opted for the physician holding the decisive position while in keeping with other studies, most nurses felt that it ought to be the patient who ought to wield the final say.[3941 ] existing studies in the asian context suggest that between the patient and family, it is the family that presides over key decisions, particularly in the end - of - life issues.[2527 ] the rationale it would seem is so that the patient is relieved of the burden of knowledge and weight of such a major decision.[9124243 ] certainly, received knowledge and current practice suggest that the involvement of the patient in the process is seen as unimportant and possibly even detrimental to general well - being and health outcomes.[25274445 ] however, this norm is challenged by the results of this study. when asked whether the patient or the family should be more involved in dnr discussions and decisions, a majority of 55.4% of participants thought that patients should be more involved than the family, suggesting that even in end - of - life decision making, the asian culture does not preclude the involvement of patients. indeed the right approach to dnr discussions and decisions varies even within a particular culture, and needs to be individualized. yet, even when it has been determined how and with whom to have a dnr discussion with, it is difficult to know when is the most appropriate time to have that discussion, as shown by the wide range of responses to the final question. a discussion too early may be unnecessarily distressing for the patient or family and may be misinterpreted as the doctor giving up on the patient. on the other hand, a discussion that occurs too late may mean that the patient is not physically able to participate and the family may be too preoccupied with the patient 's imminent death to be able to engage in it. this study is limited by the small sample size which prevented further statistical analysis of factors associated with various responses to the questions. further research employing qualitative methodology will also be valuable in helping to better understand the results obtained from this questionnaire study. additionally, the study could be expanded to include nurses, physicians, and allied heathcare from other specialities to facilitate a wider grasp of the situation. in summary, the determination of dnr statuses is difficult and practices vary depending on country, culture, and even healthcare professionals working in different specialties. in this study, even though all participants were oncology or palliative care healthcare professionals working within a single institution, there was a wide range of responses to questions on various aspects of dnr discussions and decisions, indicating that views differed even within this narrow profile. this was also present across all questions in the subgroup of palliative care professionals, who may be expected to deal with dnr discussions and decisions in a more consistent manner. an institutional guideline may facilitate a standardized approach to this complex issue, but given the myriad factors involved would serve more as a guide to a thought process rather than a protocol to be followed. from the viewpoint of local oncology and palliative care healthcare professionals, patients should be involved in discussions pertaining to their own resuscitation status even though when and to what extent this should occur remains unclear. moreover, questions persist as to whether such practical and perceptual changes can and will be translated to clinical practice. efforts to confront issues of collusion and improve direct patient involvement remain unstudied as do efforts to change perceptions ; however, these efforts do continue as further work is still required in this field. it is hoped that more research on this matter will further illuminate this process toward a patient - centered care framework balanced upon the specifics of an individual case scenario. | background:do not resuscitate (dnr) orders are put in place where cardiopulmonary resuscitation is inappropriate. however, it is unclear who should be involved in discussions and decisions around dnr orders.aim:the aim was to determine the views of oncology and palliative care doctors and nurses on dnr orders.materials and methods : a questionnaire survey was conducted on 146 doctors and nurses in oncology and palliative care working within a tertiary specialist cancer center in singapore.results:perceived care differences as a result of dnr determinations led to 50.7% of respondents reporting concerns that a dnr order would mean that the patient received a substandard level of care. on the matter of dnr discussions, majority thought that patients (78.8%) and the next of kin (78.1%) should be involved though with whom the ultimate decision lay differed. there was also a wide range of views on the most appropriate time to have a dnr discussion.conclusions:from the viewpoint of oncology and palliative care healthcare professionals, patients should be involved at least in discussing if not in the determination of dnr orders, challenging the norm of familial determination in the asian context. the varied responses highlight the complexity of decision making on issues relating to the end of life. thus, it is important to take into account the innumerable bio - psychosocial, practical, and ethical factors that are involved within such deliberations. |
hemophilia a, a deficiency in the activity of coagulation factor (f) viii, is an x - linked bleeding disorder with an approximate incidence of one in 5,000 male infants.1,2 absence or reduction of the fviii protein affects secondary hemostasis, which manifests as induced or spontaneous bleeding depending on the severity of the disease. the severity of hemophilia is classified depending on the patient s baseline plasma level of fviii. coagulation factor levels are often expressed as a percentage of factor activity or as international units. one international unit (iu) one hundred percent (100 iu / dl) is the average amount of activity for a person without hemophilia. severe hemophilia is characterized by a fviii level of < 1% (1 iu / dl). iu / dl) result in moderate hemophilia, and levels between 5% and 40% (540 while one - half of all hemophilia cases have severe deficiency, moderate and mild factor deficiencies correspond to 10% and 40% of cases, respectively.3,4 the correlation between factor levels and the severity of bleeding is not perfect, but in general, clinical phenotype corresponds to the factor level. the hallmark clinical presentation of severe hemophilia a is spontaneous, traumatic, and excessive soft tissue, muscle, body cavity, and joint bleeding. annual treatment cost of severe fviii deficiency is several hundred thousand us dollars and bleeding - related complications often result in greater severity of disease, poor quality of life, surgical interventions for severe joint destruction, and shortened life span.5 from the late 1950s and through the 1960s, fresh frozen plasma was the main treatment modality for hemophilia. each unit of fresh frozen plasma contains only a small amount of fviii, thus large volumes of intravenously administered fresh frozen plasma were needed to stop bleeding episodes, and patients were usually hospitalized for treatment of joint bleeding. due to reluctance to be hospitalized, delayed treatment in many adolescents and young adults led to the development of chronic joint disease with deformities. over the past several decades, plasma - derived fviii concentrates and recombinant preparations have become the center of successful management of hemophilia a.2,3 modern management began in the early 1970s with the discovery of plasma - derived fviii concentrates. this home - based replacement therapy allowed early control of spontaneous bleeding and reduced the degree of musculoskeletal damage. this single, life - altering advancement in hemophilia care became a paragon of the successful management and secondary prevention of the most common hereditary coagulation disorder. however, a major setback for the entire hemophilia community and plasma - derived concentrate replacement therapy came with the discovery of the transmission of potentially deadly blood - borne pathogens through the products. by the early 1980s, human blood, plasma, and these plasma - derived products manufactured from the pooled plasma donated by thousands of people were discovered to be transmitting viruses, including hepatitis b and c, and hiv. after this discovery, and by the late 1980s, advanced screening methods were developed and purification steps were incorporated into the production of donor - derived concentrates. safer plasma concentrates of coagulation factors became available, but, unfortunately, by this time, many patients had already been infected and had succumbed to the epidemic.2,3,6 another major breakthrough for hemophilia treatment following on the heels of this tragedy came with the cloning of the fviii gene in 1984. after extensive clinical trials and by the 1990s, genetically engineered recombinant factors became the mainstay of hemophilia treatment.2,6,7 infusion of fviii concentrates either with newer purified plasma - derived factors or these recombinant preparations have led to successful management of hemophilia.2 with the availability of plasma - derived and recombinant fviii products, the benefits of primary prophylaxis were demonstrated and they became the standard of care for patients with severe factor deficiencies.8,9 the widespread use of prophylactic regimens has improved joint outcome and increased quality of life,9,10 but it has also been associated with the development of neutralizing antibodies, or inhibitors, in ~25.9%32% of these patients.11,12 additional factors contribute to the risk of inhibitor development, including genetic mutation, age, race, and intensity of exposure, and these are confounding factors to the problem. the development of inhibitors is generally considered the most frequent serious adverse event in these patients. without an effective rise in factor activity following an infusion, patients continue to bleed frequently and severely, and the benefit of prophylaxis is eliminated. low levels of inhibitors may be overcome by utilizing higher doses for infusion, but a different strategy is necessary when the inhibitor levels are high. patients who develop high levels of inhibitors may be managed effectively with bypass agents, such as activated prothrombin complex concentrate (such as feiba [factor eight inhibitor bypassing activity ]) or activated fvii. these replacement products are extremely effective in the management of acute bleeds and, for some, in the long - term management of bleeds in patients with inhibitors. however, breakthrough bleeding is still common and leads to early joint damage, hemophilic arthropathy, and long - term disability. most patients who develop inhibitors will therefore be tried on immune tolerance induction (iti) therapy. they are exposed to high doses of a fviii product on a frequent basis. in patients who respond, the repeated exposure allows the immune system to tolerize itself to fviii and recovery of fviii activity occurs. once this is achieved, the frequency of exposure is reduced until a return to a standard prophylactic schedule is achieved. approximately 70% of patients will be able to tolerize after going through iti, and the time that is required for tolerization may be several years.13 the cost of high doses of factor can also be prohibitive for some patients.14 the human fviii protein was purified in 1982 by fay,15 and was cloned independently and simultaneously by three different groups in 1984. toole isolated clones corresponding to the fviii gene from a human complementary dna library. gitschier and wood also cloned and expressed the fviii gene. with a 186 kb size, it is still one of the largest described genes. in 1986, tantravahi concluded that the fviii gene is positioned in the most distal band (xq28) of the long arm of the x chromosome. the fviii gene comprises of 26 exons, which encode for a polypeptide chain of 2,351 amino acids. once the 19 peptide secretory leader sequence is removed, fviii has a mature sequence of 2,332 amino acids with the domain structure a1-a1-a2-a2-b - a3-a3-c1-c2. von willebrand factor (vwf), which noncovalently binds with plasma fviii, acts as a carrier and protects fviii from proteolysis and rapid clearance. the domain structure of fviii is very similar to that of coagulation fv.1518 the in vivo expression site of fviii has not been definitively determined. based on mouse studies, fviii is believed to be produced in sinusoidal endothelial cells, predominantly in the liver.18 however, a variety of other tissues are also capable of producing it, including the spleen, lymph nodes, and kidneys. after posttranslational modifications, the coagulation factor is released into the circulation as a set of heterodimeric proteins. when the coagulation cascade is triggered, the fviii heterodimer becomes subject to multiple proteolytic cleavages by thrombin to form activated fviii (fviiia). fviiia acts as an essential cofactor for activated fix in the contact factor pathway (intrinsic coagulation cascade). after conversion into its active form and participation in this fx activating complex, fviiia loses its activity quickly. inactivation and loss of procoagulant function occur through either spontaneous dissociation of a2-a2 or proteolysis of fviiia by activated protein c.1522 fviii is highly sensitive to proteolytic processing after secretion, and only a small fraction of circulating fviii is in the single - chain form. the majority consists of heavy chains of variable length (consisting of the a1 and a2 domains together with variable lengths of b domain) linked noncovalently to light chains consisting of the a3, c1, and c2 domains. this proves that the b domain is not required for activation of the protein of procoagulant function.1518 factor replacement therapy is highly effective in controlling bleeding, but has its limitations. therefore, the therapy frequently requires repeat dosing, and the development of anti - fviii alloantibodies that inhibit the function of plasma - derived and recombinant fviii occurs in a significant number of patients. the development of these inhibitory anti - fviii antibodies is a complex and multifactorial process. our understanding of the pathophysiological mechanisms leading to alloantibodies development has improved over the past few decades. inhibitors are defined and controlled by both genetic and environmental factors, including immune regulatory cells (eg, dendritic cells, macrophages, and cd4 t cells), cytokines, and other immune regulatory molecules. the understanding of alloantibody development has improved, but it is still not possible to confidently predict the immune response of an individual patient to a given factor or the risk of developing inhibitory alloantibody at the onset of fviii replacement therapy.2,2224 current hemophilia research is focusing on the creation of new factor replacement therapies with longer half - lives, accessing alternative mechanisms to achieve desired hemostasis and to enhance bypassing activity, and on limiting the immunogenicity of the protein. the availability of longer - acting factor concentrates would reduce the number of infusions during acute bleeding episodes and prolong the time between prophylactic infusions. among these newer techniques, covalent attachment of fviii to polyethylene glycol (peg ; pegylation), fusion to the fragmented crystallizable (fc) portion of immunoglobulin g1 (igg1) molecule, and fusion to recombinant albumin are most likely to gain approval.25 pegylation involves the covalent attachment of peg to a protein, peptide, or a small molecule drug. peg effectively increases the molecular weight and size of the protein by creating a hydrophilic cloud around the molecule. this molecular change may reduce the susceptibility of the molecule to proteolytic activity and degradation. it is also believed that pegylation changes the surface charge of the protein that ultimately interferes with some receptor - mediated clearance processes. the pharmacokinetic studies of peg - fviii conducted in hemophilic mouse models demonstrated that the half - life of pegylated factor is more than doubled (4.9 hours) when compared to non - pegylated full - length recombinant fviii (1.9 hours).2225 human igg - based therapeutics is another approach for the treatment of a variety of conditions, including hemophilia. fusion of a biologically active protein to the fc domain of human igg facilitates binding of the protein to the neonatal fc receptor (fc rn). once the fc - fusion protein binds to fc rn, it becomes protected from lysosomal degradation. when fc - fusion proteins and igg are taken up from circulation into cells by nonspecific endocytosis, they interact with fc rn endosomes and are redirected back to plasma. this naturally occurring recycle pathway prolongs the half - lives of various biologics, including recombinant fviii. although biologically unrelated, both these molecules have an extended plasma half - life exceeding 20 days, which makes them a subject of common interest in fusion protein technologies. it has been shown that in animal models of hemophilia b, genetically fusing recombinant fix to recombinant albumin factor prolongs the half - life, with increased in vivo recovery and equivalent activity when compared to recombinant fix only.2225 these provide an alternative to the extension of factor half - life that is shown with peg. in fact, efmoroctocogalfa (eloctate ; biogen, cambridge, ma, usa), fviii - fc fusio protein, was the first approved recombinant factor specifically designed to extend the half - life of conventional fviii. studies have shown that the mean half - life (t) is 19.0 hours, which is 1.51.7 longer than conventional factor. one schedule utilizes dosing every 35 days based on individual pharmokinetics results, and the other utilizes dosing with alternative fixed dose (65 iu / kg) weekly administration. the fc - fusion protein product shows superior results with an annualized bleeding rate (abr) of 1.6 bleeding episodes with individualized prophylaxis and 3.6 bleeding episodes with alternative weekly dosing schedule, compared to an on - demand dosing result of 33.6 bleeding episodes. in addition, 45.3% of patients on individualized prophylaxis and 17.4% of patients on alternative weekly prophylaxis had no bleeding at all during the study period compared to 0% in the group receiving on - demand treatment. during the course of the trials, there were 757 episodes of bleeding. of these, 87.3% resolved after one dose of treatment and 97.8% were treated with only one or two doses.26 there is no albumin fusion fviii replacement product currently in widespread development. it is a single - chain b - domain deleted fviii that is activated upon thrombin cleavage. in animal models, of significant note is that it appears to have increased affinity for vwf, and there are some indications that it might have decreased immunogenicity.27 several peg factor products are currently approved or are in clinical trials (nct01736475, nct01458106, nct01480180, nct01580293) in addition to the non - peg products, and these have or will likely gain approval in the near future (table 1). rurioctocog alfa pegol (bax 855, adynovate ; baxalta, bannockburn, il, usa) is a full - length recombinant fviii with a 20 kda branched peg covalently bound to the b - domain region and has recently been approved for use in the us. studies on this product report a half - life that is 1.4 longer than the half - life of a comparable conventional factor. there is an abr of 1.9 bleeds on prophylaxis, which is a 90% reduction compared to on - demand therapy. of the patients receiving prophylaxis bleeds were treated with either one or two doses 95.9% of the time, and a median dose of 30.87 iu / kg was used with 97% of patients, reporting either good or excellent results. there were no reports of any inhibitors after exposure to bax 855.28 turoctocog alfa pegol (n8-gp) is a glycol - pegylated product. it is a b - domain truncated fviii with a 40 kda peg bound by a unique o - linked glycan on the residual 21 amino acid b - domain region with a half - life of 1.6 that of a full - length fviii product (19 hours). efficacy data reports an abr of 1.3 on prophylaxis compared to an on - demand dosing rate of 30.9. treatment of bleeding events while on clinical trials showed n8-gp was equally effective compared to a standard half - life factor. no patients developed inhibitors while on the studies.29 bay 94 - 9027 is another b - domain truncated fviii that has a 60 kda peg linked by a modified cysteine region on the a3 domain. the half - life of this product there have been two hypersensitivity reactions reported but no reports of any patients with inhibitors. there are multiple foreseeable benefits of using a longer - acting pegylated product compared to those currently available. with the product s longer half - life prophylactic dosing decreases from three times a week or every other day to twice a week or every fifth day. acute bleeds may be managed with one or two doses of factor replacement rather than continuing the treatment over multiple days or weeks, as is now frequently required. currently, many patients require hospitalization, not only for the threat to life and limb during a bleeding episode but also due to the frequency of the dosing regimens. as a result of these longer - acting products this in turn may lead to reduced costs associated with hospitalization and absence from work and school, as well as increased productivity when recovering from bleeds.31,32 the realized promise of a longer half - life in the pegylated products results in fewer required doses for prophylaxis, potentially decreasing the burden of infusion and increasing compliance to these regimens.27 many patients who have been reluctant to start prophylactic therapy because of the frequent dosing of these regimens may now be more inclined to consider it and move away from a purely on - demand regimen, which is associated with increased bleeding rates and worsened joint outcomes.9 fewer factor infusions in the pediatric population in particular may also decrease the need for central venous line (cvl) access due to better preservation of patient veins. this in turn, even when a cvl is required, reduces both the burden of infectious complications from a cvl and the risk of venous thrombotic events. patient noncompliance with a prophylactic regimen is a challenging factor in hemophilia treatment, especially in teenagers and young adults when compared to younger children. because prophylaxis is the standard of care in the us and other developed nations, a number of patients may have never experienced a significant bleeding episode. convincing these patients of the continuing benefits of prophylaxis in the absence of a first - hand bleeding episode is often difficult and almost impossible at times. this noncompliance leads to increased complications seen in adulthood.33 a prophylactic regimen that uses fewer needle sticks may help in reducing the number of patients who choose to stop their prophylaxis. fviii - fc fusion protein was the first extended half - life product to gain regulatory approval and was met with cautious enthusiasm. it has now been available in the us for more than a year, but not all patients have consistently observed the gain of a prolonged half - life and its benefits ; in fact, some patients have not seen any benefit from this product over standard therapy due to its variability. the introduction of one or more pegylated products may be an alternative for patients who do not benefit from fviii - fc fusion protein products. inversely, patients who may not respond to peg factor products may still have any alternative with fviii - fc fusion product. the extended half - life fviii replacement products are not a cure for hemophilia a, but are tools that can be used to manage and control the disease. each new treatment option that becomes available gives rise to the question of how to best use the product along with the other tools available to provide the best possible outcome for each patient. with the greater interpatient variability of dosing, a one - size - fits - all model to prophylactic dosing no longer appears to be appropriate. the variation in a product s half - life for each patient results in dosing regimens that range from every 3 days to once a week. this dosing challenge has resulted in the need for hemophilia treatment centers to develop a new model for individualized dosing. thus far, the primary focus has been on the potential for these products in patients with severe deficiency, but patients with mild and moderate deficiencies could also see benefits. due to the infrequency of their bleeds, the proficiency with self - intravenous infusion is not as high. management of these bleeds can be achieved more simply and with fewer doses in these cases. some patients with moderate deficiency, and even some patients with mild deficiency, can have regular bleeding. in these patients, a prophylactic regimen, perhaps less dose intensive, might help to prevent long - term joint damage and disability much in the same way that prophylaxis has helped patients with severe deficiency. with limited information available, the questions arise about when and how to start fviii products in patients not previously exposed to any fviii products. the sippet trial was designed to demonstrate the difference in inhibitor development when an untreated patient is first exposed to a plasma - derived factor when compared to first exposure with a recombinant product.34 although the study has been completed, the data has not been published yet. however, results of the study suggested that there might be some risk of inhibitor development with the use of recombinant therapy in previously untreated patients when compared to plasma - derived factor products.35 this data might argue for the use of a plasma - derived product when initiating prophylaxis, especially in those who have additional high risk features, but might not a pegylated product be equally effective in reducing the risk of inhibitor formation ? could pegylation prevent exposure of the antigenic sites of the protein and the prolonged exposure result in early tolerization ? how does a pegylated factor affect inhibitor formation in patients with the highest risk of inhibitor development such as those with large mutations or a family history of inhibitors ? does the size of the peg moiety, which varies between the products, play a role in altering inhibitor development ? so far, there have been no reports of any peg factor replacement products causing inhibitor formation in the initial clinical trials ; however, study results from previously untreated patients are yet to be reported. by decreasing the possibility of a patient developing inhibitors, primary prophylaxis may be warranted earlier in the treatment plan of patients with mild - to - moderate hemophilia rather than as secondary prophylaxis. after the development of inhibitors, is there a role for the use of extended half - life factor for the induction of immune tolerance ? some feel that a plasma - derived product that contains some amount of vwf and other proteins might modulate the immune system to better protect and prolong exposure to fviii and improve the induction of immune tolerance.36 it is reported that tolerance induction with fviii - fc protein does occur in hemophilia a mice.37 in addition, groomes provide the first report of a patient who was able to reduce his factor inhibitor titers in response to an iti regimen using fviii - fc fusion protein product, although full tolerization was not reported. might not the protection of fviii by pegylation also allow a prolonged exposure and possibly improve the likelihood of iti ? in fact, work is underway to explore this potential, but no results from these studies have been published to date. we do not know the unintended consequences of the incremental improvement. a poorly measured, but much hoped for, benefit of an extended half - life product is increased patient satisfaction with health care and an increase in the quality of patients lives. we expect to see fewer missed days from school and work when bleeds and injuries are more easily and better controlled. do fewer doses result in the inability or delay in pediatric patients learning how to self - infuse ? what, if any, is the effect of the frequent and continuous exposure to peg and the potential buildup in the tissues ? so far, there is no evidence of tissue accumulation or harm, but what will be the experience after years and decades of use of these products ? the dawn of a new era in the care of hemophilia patients is upon us with the release of recombinant fviii products with extended half - lives, and products with even more extended half - life will become available in the very near future. with all the promise of these new agents, many questions still remain. over the decades, we have learned much about the treatment of hemophilia a. the collective experience gathered, as the care of hemophilia has improved, provides caution and optimism. we will come to learn how to best use these tools and the answers to our questions | hemophilia a, a deficiency in the activity of coagulation factor (f) viii, is an x - linked bleeding disorder with an approximate incidence of one in 5,000 male infants. bleeding - related complications often result in greater severity of disease, poor quality of life, surgical interventions for severe joint destruction, and shortened life span. with the availability of plasma - derived and recombinant fviii products, the benefits of primary prophylaxis were demonstrated and is now the standard of care for patients with severe factor deficiencies. current hemophilia research is focusing on the creation of new factor replacement therapies with longer half - lives ; accessing alternative mechanisms to achieve desired hemostasis and enhance bypassing activity ; and limiting the immunogenicity of the protein. pegylation involves the covalent attachment of polyethylene glycol (peg) to a protein, peptide, or a small molecule drug. peg effectively increases the molecular weight and size of the protein by creating a hydrophilic cloud around the molecule. this molecular change may reduce susceptibility of the molecule to proteolytic activity and degradation. it is also believed that pegylation changes the surface charge of the protein that ultimately interferes with some receptor - mediated clearance processes. the half - life of pegylated factor is more prolonged when compared to non - pegylated full - length recombinant fviii. the dawn of a new era in the care of hemophilia patients is upon us with the release of recombinant fviii products with extended half - lives, and products with even more extended half - life will become available in a very short time. with all the promise of these new agents, many questions still remain. |
calcifications are present almost solely in relation to atherosclerosis in the coronary vessels except for individuals with chronic kidney disease.1) therefore, coronary artery calcium score (cacs) has been used as a useful surrogate marker for coronary atherosclerosis burden. furthermore, cacs provides prognostic information in asymptomatic population.2 - 5) from these data, the use of cacs for risk stratification in asymptomatic population is advocated by professional guidelines.6 - 8) coronary computed tomographic angiography (ccta) has been introduced as a noninvasive imaging modality to directly assess the degree of coronary artery stenosis.9)10) the outstanding diagnostic accuracy of ccta has been demonstrated by prospective multicenter studies using coronary angiography (ca) as the reference standard.11)12) widespread use of ccta revealed a non - negligible prevalence of non - calcified plaque (ncp) in subjects with cacs of zero, who have very low probability of future cardiac adverse events.13 - 15) in symptomatic patients, ncps are predictive of myocardial hypoperfusion and future coronary events.16 - 18) however, the clinical predictors and the prognosis of ncp in asymptomatic subjects with zero cacs have not been fully evaluated.19) therefore, we evaluated the association of standard risk factors with ncp, and the prognosis of ncp in middle aged asymptomatic subjects with cacs of zero using ccta. between december 2005 and january 2008, we enrolled 5632 consecutive subjects who underwent ccta using 64-slice multi - detector row computed tomography (mdct) at seoul national university bundang hospital (snubh). we included asymptomatic individuals who had ccta as part of general health evaluation in a health promotion center. we excluded subjects as follows : 1) insufficient medical records or inadequate imaging for analyses (n=24) ; 2) age less than 35 or more than 75 years (n=195) ; 3) subjects who had cacs > 0 (n=922) for current analyses. as a result, 4491 middle - aged individuals with cacs of zero were finally enrolled. the study protocol was approved by the institutional review board and all patients provided written informed consent. data acquisition and image post - processing for ccta and cacs were done in accordance with the society of cardiovascular computed tomography guidelines,20) and detailed methods were described in previous manuscript.21) coronary artery calcium scores were measured using the scoring system previously suggested by agatston.22) the ccta was performed on a 64-slice mdct scanner (brilliance 64, philips medical systems, best, the netherlands). co., ltd., seoul, korea) was used to achieve a heart - rate of 1 mm that existed either within the coronary artery lumen or adjacent to the coronary artery lumen, which could be clearly discriminated from surrounding pericardial tissue or the vessel lumen itself. each lesion was identified with a multiplanar reconstruction technique and maximum intensity projection of short - axis, 2-chamber, and 4-chamber views. each identified lesion was examined using maximum - intensity - projection and multi - planar reconstruction techniques along multiple longitudinal axes and in the transverse plane. we analyzed plaque characteristics on a per - segment basis according to a 16-segment coronary artery tree model (left main ; proximal, mid and distal left anterior descending artery (lad) ; first and second diagonal branches of the lad ; proximal and distal left circumflex artery ; first and second obtuse marginal branches of the left circumflex artery ; proximal, mid and distal right coronary artery ; posterior descending artery ; and left and right posterolateral branch).20)23) coronary artery stenosis was defined as presence of any plaque. obstructive stenosis was defined when coronary artery segments exhibited plaque a luminal diameter stenosis 50%, and non - obstructive stenosis was defined when coronary artery segments exhibited plaque with a luminal diameter stenosis 20% }, moderate - risk (more than 2 risk factors and 10-year risk > 20%), and low - risk group (0 to 1 risk factor).24) to assess prognosis of individuals with ncp, we selected age, gender, ccta date (7 days)-matched controls among the subjects without ncp. if there are more subjects who meet the matching criteria, the matched - control subject was randomly selected among the qualified subjects. after the matching group was selected, follow - up for events was performed by a dedicated physician and/or research nurse blinded to the ccta results. the primary end - point was all - cause death and the secondary outcome was the composite outcome of cardiac death, non - fatal myocardial infarction (mi), unstable angina (ua) requiring hospitalization, and revascularization after 90 days of index ccta. we only considered revascularizations more than 90 days after ccta as outcome events to exclude downstream revascularization procedures related to index ccta.25 - 27) continuous variables are expressed as means1 standard deviation, whereas categorical variables are presented as frequencies. differences between continuous variables were analyzed by student 's unpaired t - test and those between categorical variables by the chi - square test or fisher 's exact test, as appropriate. in addition, to compare differences of continuous variables and categorical variables between matched controls and patients, paired t - test and mcnemar test were performed. univariable and multivariable binary logistic regression analyses were performed to determine association of standard risk factors and ncp. in the analysis, we selected standard risk factors as follows : age, gender, current cigarette smoking status, body mass index (bmi), disease status of hypertension, diabetes mellitus. the covariates - adjusted odds ratios (or) and their 95% confidence intervals (ci) were derived in multiple logistic regression models. null hypotheses of no difference were rejected if p were less than 0.05 or, equivalently, if the 95% cis of or estimates excluded 1. all analyses were performed with statistical package for the social sciences (spss) 13.0 statistical package (spss inc., chicago, il, usa). between december 2005 and january 2008, we enrolled 5632 consecutive subjects who underwent ccta using 64-slice multi - detector row computed tomography (mdct) at seoul national university bundang hospital (snubh). we included asymptomatic individuals who had ccta as part of general health evaluation in a health promotion center. we excluded subjects as follows : 1) insufficient medical records or inadequate imaging for analyses (n=24) ; 2) age less than 35 or more than 75 years (n=195) ; 3) subjects who had cacs > 0 (n=922) for current analyses. as a result, 4491 middle - aged individuals with cacs of zero were finally enrolled. the study protocol was approved by the institutional review board and all patients provided written informed consent. data acquisition and image post - processing for ccta and cacs were done in accordance with the society of cardiovascular computed tomography guidelines,20) and detailed methods were described in previous manuscript.21) coronary artery calcium scores were measured using the scoring system previously suggested by agatston.22) the ccta was performed on a 64-slice mdct scanner (brilliance 64, philips medical systems, best, the netherlands). co., ltd., seoul, korea) was used to achieve a heart - rate of 1 mm that existed either within the coronary artery lumen or adjacent to the coronary artery lumen, which could be clearly discriminated from surrounding pericardial tissue or the vessel lumen itself. each lesion was identified with a multiplanar reconstruction technique and maximum intensity projection of short - axis, 2-chamber, and 4-chamber views. each identified lesion was examined using maximum - intensity - projection and multi - planar reconstruction techniques along multiple longitudinal axes and in the transverse plane. we analyzed plaque characteristics on a per - segment basis according to a 16-segment coronary artery tree model (left main ; proximal, mid and distal left anterior descending artery (lad) ; first and second diagonal branches of the lad ; proximal and distal left circumflex artery ; first and second obtuse marginal branches of the left circumflex artery ; proximal, mid and distal right coronary artery ; posterior descending artery ; and left and right posterolateral branch).20)23) coronary artery stenosis was defined as presence of any plaque. obstructive stenosis was defined when coronary artery segments exhibited plaque a luminal diameter stenosis 50%, and non - obstructive stenosis was defined when coronary artery segments exhibited plaque with a luminal diameter stenosis 20% }, moderate - risk (more than 2 risk factors and 10-year risk > 20%), and low - risk group (0 to 1 risk factor).24) to assess prognosis of individuals with ncp, we selected age, gender, ccta date (7 days)-matched controls among the subjects without ncp. if there are more subjects who meet the matching criteria, the matched - control subject was randomly selected among the qualified subjects. after the matching group was selected, follow - up for events was performed by a dedicated physician and/or research nurse blinded to the ccta results. the primary end - point was all - cause death and the secondary outcome was the composite outcome of cardiac death, non - fatal myocardial infarction (mi), unstable angina (ua) requiring hospitalization, and revascularization after 90 days of index ccta. we only considered revascularizations more than 90 days after ccta as outcome events to exclude downstream revascularization procedures related to index ccta.25 - 27) continuous variables are expressed as means1 standard deviation, whereas categorical variables are presented as frequencies. differences between continuous variables were analyzed by student 's unpaired t - test and those between categorical variables by the chi - square test or fisher 's exact test, as appropriate. in addition, to compare differences of continuous variables and categorical variables between matched controls and patients, paired t - test and mcnemar test were performed. univariable and multivariable binary logistic regression analyses were performed to determine association of standard risk factors and ncp. in the analysis, we selected standard risk factors as follows : age, gender, current cigarette smoking status, body mass index (bmi), disease status of hypertension, diabetes mellitus. the covariates - adjusted odds ratios (or) and their 95% confidence intervals (ci) were derived in multiple logistic regression models. null hypotheses of no difference were rejected if p were less than 0.05 or, equivalently, if the 95% cis of or estimates excluded 1. all analyses were performed with statistical package for the social sciences (spss) 13.0 statistical package (spss inc., chicago, il, usa). the distribution of individuals according to plaque severity was described in table 1. among 4491 of overall individuals without cac, 313 subjects (7%) had coronary plaques : 279 patients (6%) with non - obstructive plaque and 34 (1%) with obstructive plaque. among the subjects with obstructive plaque, 30 subjects (88%) were 1-vessel diseases (vd), 1 subject (3%) was 2-vd, and 3 subjects (9%) were left main disease. the main clinical characteristics of study subjects without cac according to plaque characteristics were listed in table 2. overall, the study population consisted of 4491 individuals without cac : 57% were male with a mean age was 488 years. subjects with ncp were generally older, male, and with a higher prevalence of hypertension, diabetes, and dyslipidemia (all p 0%) was 6.2% and obstructive ncp (50%) was 0.7% in subjects with zero cacs. cheng.28) showed prevalence of detectable ncp was 6.5% (27/416) (6.0%, non - obstructive ; 0.5%, obstructive) in low to intermediate risk patients (80% of patients had chest pain or dyspnea). akram.13) reported that 8.2% (4/49) had obstructive ncp in symptomatic patients, but no asymptomatic subjects (0/76) had obstructive ncp. however, ergn.14) reported higher prevalence of ncp compared to the current study : 17% (54/320) had ncp and 3% (10/320) had obstructive ncp in asymptomatic subjects. rubenstein.15) also found 14% (25/125) of symptomatic patients with zero calcium had ncp. the discrepancy of the prevalence of ncp might originate from the difference in pretest probability of coronary artery disease (cad) and ethnicity of study population. we believe that the accuracy study of ccta using ica as a standard test by cademartiri. provided a good reference of the prevalence of ncp. the prevalence of obstructive ncp in zero calcium demonstrated at cag was 1.4% (1/71) in asymptomatic subjects which was similar to current study (0.7%). to our knowledge, there is no study that evaluated risk factors for ncp in an asymptomatic population with zero cacs. multivariable logistic regression analyses identified old age, male gender, diabetes, hypertension, and dyslipidemia as independent risk factors for ncp in asymptomatic population with zero calcium in current study. therefore, it should be noted that individuals with old age, male gender, diabetes, hypertension, and dyslipidemia have a higher risk of ncp although they have zero cacs in clinical practice. in patients with suspected cad van werkhoven.18) found that ncp was independent predictor with an incremental prognostic value to cacs. motoyama.17) also showed that low - attenuation plaque on ccta was a risk factor for future acute coronary syndrome in patients with suspected or known cad. however, there is lack of study that analyzed the prognosis of ncp in asymptomatic population with zero cacs. the current study clearly demonstrated a negligible risk of exclusive ncp in subjects with zero cacs. during the median follow - up duration of 22 months (interquartile percentile : 18 to 28 months), there was no cardiac event in the ncp group nor even in the control group without ncp. this indicates that a cacs of zero successfully discriminates the future risk of cardiac events in asymptomatic population. recently, the results from international multicenter ccta registry showed that ccta barely added prognostic value over cacs in population without chest pain syndrome.29) the results of the current study, which showed fairly good prognosis in an asymptomatic population with zero cacs, would be able to explain why ccta failed to show significant added prognostic value over cacs. another important reason why ccta failed to stratify future risk of ncp in asymptomatic subjects with cacs of zero is that the pretest probability of cad in the current study population is low. although 4491 subjects with no cac were included in current analyses, the subjects with ncp were only 7% (313 subjects). moreover, a follow - up duration of 22 months might not be enough to discriminate future risk of ncp in the current asymptomatic population. therefore, future studies with longer follow - up and a larger population might prove the prognostic value of ncp identified by ccta for risk prediction in this low - risk population with zero cacs. however, ccta has potential hazards including radiation exposure and intravenous use of contrast agent. therefore, for present, the application of ccta to detect ncp for risk stratification purpose in an asymptomatic population with cacs of zero should not be justified. we retrospectively looked at a population who underwent ccta in a health promotion center on a self - referral basis. in addition, the influence of post - test medical treatments, coronary intervention within 90 days, or risk factor control was not considered. further, we used a 90 day window period to exclude downstream revascularization procedures related to ccta. thus, a revascularization procedure which was performed within 90 days from ccta, but not a downstream procedure might be excluded from the clinical outcome. moreover, the topographic distribution of coronary plaques, and various plaque assessment approaches including area stenosis, minimal lumen diameter, and minimal lumen area were not included in current analyses. in the largest series of asymptomatic subjects with cacs of zero undergoing ccta, we find a negligible risk of exclusively ncp. our findings suggest that the application of ccta to detect ncp for risk stratification purpose in asymptomatic population with cacs of zero is not justified. the prevalence of any ncp (> 0%) was 6.2% and obstructive ncp (50%) was 0.7% in subjects with zero cacs. cheng.28) showed prevalence of detectable ncp was 6.5% (27/416) (6.0%, non - obstructive ; 0.5%, obstructive) in low to intermediate risk patients (80% of patients had chest pain or dyspnea). akram.13) reported that 8.2% (4/49) had obstructive ncp in symptomatic patients, but no asymptomatic subjects (0/76) had obstructive ncp. however, ergn.14) reported higher prevalence of ncp compared to the current study : 17% (54/320) had ncp and 3% (10/320) had obstructive ncp in asymptomatic subjects. rubenstein.15) also found 14% (25/125) of symptomatic patients with zero calcium had ncp. the discrepancy of the prevalence of ncp might originate from the difference in pretest probability of coronary artery disease (cad) and ethnicity of study population. we believe that the accuracy study of ccta using ica as a standard test by cademartiri. provided a good reference of the prevalence of ncp. the prevalence of obstructive ncp in zero calcium demonstrated at cag was 1.4% (1/71) in asymptomatic subjects which was similar to current study (0.7%). to our knowledge, there is no study that evaluated risk factors for ncp in an asymptomatic population with zero cacs. multivariable logistic regression analyses identified old age, male gender, diabetes, hypertension, and dyslipidemia as independent risk factors for ncp in asymptomatic population with zero calcium in current study. therefore, it should be noted that individuals with old age, male gender, diabetes, hypertension, and dyslipidemia have a higher risk of ncp although they have zero cacs in clinical practice. in patients with suspected cad van werkhoven.18) found that ncp was independent predictor with an incremental prognostic value to cacs. motoyama.17) also showed that low - attenuation plaque on ccta was a risk factor for future acute coronary syndrome in patients with suspected or known cad. however, there is lack of study that analyzed the prognosis of ncp in asymptomatic population with zero cacs. the current study clearly demonstrated a negligible risk of exclusive ncp in subjects with zero cacs. during the median follow - up duration of 22 months (interquartile percentile : 18 to 28 months), there was no cardiac event in the ncp group nor even in the control group without ncp. this indicates that a cacs of zero successfully discriminates the future risk of cardiac events in asymptomatic population. recently, the results from international multicenter ccta registry showed that ccta barely added prognostic value over cacs in population without chest pain syndrome.29) the results of the current study, which showed fairly good prognosis in an asymptomatic population with zero cacs, would be able to explain why ccta failed to show significant added prognostic value over cacs. another important reason why ccta failed to stratify future risk of ncp in asymptomatic subjects with cacs of zero is that the pretest probability of cad in the current study population is low. although 4491 subjects with no cac were included in current analyses, the subjects with ncp were only 7% (313 subjects). moreover, a follow - up duration of 22 months might not be enough to discriminate future risk of ncp in the current asymptomatic population. therefore, future studies with longer follow - up and a larger population might prove the prognostic value of ncp identified by ccta for risk prediction in this low - risk population with zero cacs. however, ccta has potential hazards including radiation exposure and intravenous use of contrast agent. therefore, for present, the application of ccta to detect ncp for risk stratification purpose in an asymptomatic population with cacs of zero should not be justified. we retrospectively looked at a population who underwent ccta in a health promotion center on a self - referral basis. in addition, the influence of post - test medical treatments, coronary intervention within 90 days, or risk factor control was not considered. further, we used a 90 day window period to exclude downstream revascularization procedures related to ccta. thus, a revascularization procedure which was performed within 90 days from ccta, but not a downstream procedure might be excluded from the clinical outcome. moreover, the topographic distribution of coronary plaques, and various plaque assessment approaches including area stenosis, minimal lumen diameter, and minimal lumen area were not included in current analyses. in the largest series of asymptomatic subjects with cacs of zero undergoing ccta, we find a negligible risk of exclusively ncp. our findings suggest that the application of ccta to detect ncp for risk stratification purpose in asymptomatic population with cacs of zero is not justified. | background and objectivesnon - calcified plaque (ncp) identified by coronary ct angiography (ccta) has been reported in up to 10% of individuals with coronary artery calcium score (cacs) of zero. however, clinical risk factors and the prognostic value of ncp in asymptomatic subjects with cacs of zero are unknown.subjects and methodsthe study population consisted of consecutive asymptomatic subjects (488 years, 57% men) who underwent ccta from december 2005 to january 2008 as part of a general health evaluation.resultsamong 4491 of overall asymptomatic individuals with cacs of zero, 313 subjects (7%) had ncp : 279 patients (6%) with non - obstructive and 34 (1%) with obstructive. in multivariable analyses, age, male gender, diabetes, hypertension, and dyslipidemia were significantly associated with presence of ncp (all p<0.05). during the median follow - up duration of 22 months (interquartile percentile : 18 to 28 months) of subjects with ncp (n=313) and age, gender, and ccta date matched individuals without ncp (n=313), there was no clinical event including all - cause death nor composite outcome of cardiac death, myocardial infarct, unstable angina requiring hospitalization, and revascularization after 90 days from index ccta in both groups.conclusionin the largest series of asymptomatic individuals with cacs of zero undergoing ccta, age, male gender, diabetes, hypertension, and dyslipidemia were independently associated with ncp. however, a future risk of exclusive ncp in asymptomatic subjects with cacs of zero was negligible. |
traditionally, clinical microbiologists handle and read routine bacterial culture plates on the open laboratory bench. at the beginning of each shift, stacks of inverted plates are removed from incubators and set on the benchtop. throughout the day this work includes collecting plates from different incubators, inspecting media for bacterial growth, examining colony morphology, isolating pure cultures, performing biochemical testing on isolates, preparing media for antimicrobial susceptibility testing (ast), interpreting ast, and discarding or archiving old culture plates. this work also includes reviewing notes from the previous days interpretations of any growth and deciding the next step in analysis and/or reporting for these cultures. the plates are eventually returned to their incubator(s), and the same process recommences the following day. with the advent of digital imaging, digital technology has recently been applied in the clinical microbiology laboratory to perform digital plate reading (dpr) in some laboratory settings. the dpr approach is similar to the aforementioned manual reading process, but is highly modified. with dpr, technologists still read plates, but they can now do so virtually without physically touching the culture plate. dpr today in the clinical laboratory consists of a digital camera juxtaposed or attached to an incubator which, through automation, moves the culture plate to the camera for the image to be captured. the incubator and dpr combination allow for continuous incubation of cultures and scheduled digital image capture of those plates. dpr is typically combined with a middleware system that resides between the image capture system and the laboratory information system. these middleware systems have functionality that can present to the technologist images of the cultures plates not only from a single culture but other cultures where there is perhaps growth from other sources., the technologist can perform the work that has historically been relegated to the open bench ; dpr and middleware solutions allow images to be captured, colonies to be circled, zones to be measured, annotations to be made, and next steps to be planned. with dpr, the time required for collecting and collating plates can be reduced, and software enables stored images to be referenced when working up a culture. the duration at which the cultures are at suboptimal incubation temperature (i.e., physically on the counter) is reduced, and the productivity of the technologist is improved. with dpr the ability to incorporate computer aided tools and image analysis to support this review aims to discuss the advantages and challenges associated with dpr and explores some of the existing platforms that incorporate this technology into an overall solution for total laboratory automation (tla) within the clinical laboratory space. perhaps the earliest dpr system used in clinical microbiology was the biogram (giles scientific, new york, ny) system, which was only partially digital. the biogram system employed electronic calipers, which a technologist could use to measure the inhibition zone size on a culture plate created from an antimicrobial disk. the measurement from the calipers was automatically transferred to a computer, which converted the measurement to a minimal inhibitory concentration value. an updated platform, biomic video (giles scientific, new york, ny, usa), was subsequently developed and demonstrated to be reliable and feasible for clinical use in ast using disk diffusion testing. one study demonstrated that it was more cost effective than a more automated ast alternative. the latest generation of this system, biomic v3 (giles scientific ; santa barbara, california), incorporates a color analysis software tool that is designed to facilitate the interpretation of microbial growth on chromogenic media (i.e. chromagar) (http://youtu.be/kou9h8iooyy). the biomic v3 is also capable of counting colonies (http://youtu.be/ct-0rzgyk_w) and analyzing certain ancillary tests such as etest assays (http://youtu.be/b2fxjs37vzi). the biomic v3 requires manual loading and unloading of one culture plate at a time, and it is not designed or marketed for comprehensive routine analysis of primary culture plates. other semi - automated ast dpr systems that are similar to the biomic v3 have been developed. for example, i2a (montpellier, france) developed the sirscan 2000 which has been reported to have similar accuracy and better precision than manual measurement of ast zone sizes. therefore, it appears that dpr may reduce inter - operator variability, at least when measuring ast zone sizes. some early clinical microbiology imaging systems were custom - built and used for comprehensive telemicrobiology, which included off - site dpr. these initial systems used extremely manual techniques and custom system designs that would not be feasible for the daily workflow of a contemporary, high - volume clinical microbiology laboratory. as stated above, newer automated systems are comprised of an incubator that incorporates an automated imaging component that interfaces with dpr middleware for viewing and analyzing images. currently, at least four companies are offering or developing off the shelf automated dpr technologies : bd kiestra (drachten, netherlands), biomeriux (marcy - ltoile, france), copan (murrieta, california), and i2a. bd kiestra 's systems are currently the most widely implemented dpr systems with dozens of installations in europe. its systems include tla and work cell automation platform lines, which use kiestra 's reada browser software for dpr analysis. copan 's tla system is the wasplab, and it uses a web - based interface. the i2a system is the maestro, and this is still in development. currently available systems have been registered with the united states food and drug administration as class i devices. giles scientific 's biomic is registered as a microbiology automated zone reader, " and bd kiestra 's reada browser is registered as a microbiology manual colony counter. copan 's wasplab image acquisition station, interface software, and computer hardware as additional functionality is added to these dpr systems, it remains to be seen how the regulatory landscape may evolve. the intent of this paper is not to compare the specific features of each of these systems, but rather to review advantages and disadvantages of digital microbiology in general, using examples that may be common to all of these systems or unique to only one of them. given that the technology incorporated into dpr systems and software is still emerging, it should be borne in mind that some of the systems details may be subject to modifications and updates. currently, dpr enables the capture of and analysis of images from traditional bacterial culture plates. the systems can also be used for yeasts, but they are not currently designed for use with mycobacteria or fungi. the incubators have at least three components : a holding area for inoculated plates, a robotic handling mechanism for the plates, and an image capture station [figure 1 ]. these incubators allow for automated image capture at user - defined intervals and on demand while being incubated continuously. about half a minute per plate is needed for image capturing because the plates have to be robotically moved to the image capture station, and then numerous lighting strategies are used to capture multiple images of each plate [figure 2 ]. as is the case with manual plate reading, different lighting conditions highlight different aspects of the culture plate and its colonies. for example, backlighting is used to highlight hemolysis, and tangential lighting is used to enhance colony texture. captured images are compiled into a composite image, which incorporates the advantages of each original primary image [figure 3 ]. a composite image helps to consolidate key visual data from each original image, so that the technologist viewing the composite image can quickly interpret the relevant visual information contained in multiple original images. another advantage is that a composite image may appear more similar to what the plate might actually appear as if viewed manually. copan 's wasplab image capture station (left) and a close up picture of the imaging stage (right) are pictured. the front end operation of digital plate reading is an automated system that retrieves a stored culture plate at user - defined intervals, moves the plate to the stage for imaging, removes the lid of the plate, illuminates the plate in multiple ways during the photographic process, and returns the plate to its storage location. images are courtesy of copan a diagram of the image capture station in a biomeriux incubator is depicted. digital image capturing stations feature the capability to use different illumination wavelengths, lighting angles, lighting directions, light diffusion patterns, and backgrounds (without a background color, with a white background, or with a black background). for example, backlighting can help reveal hemolysis patterns but may make it difficult to discern details of colony texture. figure is courtesy of biomeriux three captured images and one composite image of eikenella corrodens are shown. the blood agar plate was inoculated with biomeriux 's previ isola instrument and then incubated. primary images were collected using top annular illumination, (a) bottom annular illumination, (b) and in high contrast black and white. (c) the myla software then created a composite image, (d) which incorporates features of each primary image. full resolution images are available at http://goo.gl/kbbeuw or by using this qr code although the time required to photograph an individual culture plate is relatively short, the time required to photograph every plate in an incubator can be substantial. hence, the frequency of photographing culture plates may be limited by the number of specimens in an incubator. for example, a full incubator may require 8 h of constant imaging to complete a photograph cycle and capture images of each plate. it is important to consider that a delay between image capture by the system and image analysis by a technologist could lead to challenges. for example, it may be appropriate to photograph specimens that are of the greatest clinical importance more frequently (e.g., spinal fluids every 4 h, compared to urine cultures that may only need to be photographed every 12 h). software and middleware tools are used with dpr to facilitate and expedite the analysis of the cultured specimens [figures 4 and 5 ]. these tools can include a contact sheet of all the plates associated with a single specimen, side - by - side temporal comparison of a culture plate, side - by - side primary specimen gram stain and primary culture plate (in development), pop - up magnification of an area of interest, and automatic zone measurement. these software tools facilitate plate reading for technologists and potentially enhance their ability to interpret cultures beyond what is possible when performing manual plate reading. the software interface (top - left) and images of culture plates from copan 's wasplab digital plate reading system are pictured. a culture of streptococcus pneumoniae after 18 h of incubation on sheep blood agar is shown in this screenshot of the software. other plates set up from the specimen, which include chocolate and macconkey, are visible at the bottom of the screen. although no growth is present on the macconkey plate, the growth on the blood agar is visible at 18 h (top - right) and 48 h (mid - right), and the growth on the chocolate agar is visible at 18 h (bottom - left) and 48 h (bottom - right). full resolution images are available at http://goo.gl/kbbeuw or by using this qr code examples of bd kiestra 's digital plate reading software tools are demonstrated. a plate of interest can be examined at multiple time points (top pane), which enables the microbiologist to more objectively analyze the change in a culture 's appearance over time. an area of interest can be viewed at a greater magnification without the need to manually adjust the lighting, nor use a physical magnifying lens (middle pane). zones of inhibition can be measured in silico without ever removing the plate from the incubator (bottom pane). digital plate reading has advantages (some potential and some realized) over traditional (manual) plate reading [table 1 ]. culture plates remain in incubation during routine dpr examination, so cultures have decreased time to the detection of growth. the modified incubators used in dpr do not alter the appearance of colonies and culture plates, so the cultures appear the same as cultures that are incubated in traditional incubators. dpr systems enable a reduction in time spent by skilled staff in transporting, sorting, and retrieving culture plates, which in turn allows for increased time spent actually analyzing cultures, and these changes enable technologists to be more efficient. the use of a modern dpr system can enhance technologist efficiency and decrease time to organism detection, which leads to decreased turnaround times. when using dpr, plates are physically handled less often. this automated labeling reduces the need to manually apply printed stickers or to manually write on culture plates, which can increase the efficiency of the process and decrease errors made in the laboratory when reading plates. these barcodes enable the laboratory to identify and track culture plates while in the dpr incubator, while being manipulated on the bench, and when returning the plates to the incubator. because culture plates need to be handled less often, workstations can be configured to optimize ergonomics and minimize risk of repetitive motion injuries. also, the decreased physical exposure to pathogens provides a potential decrease in the risk of laboratory acquired infections for laboratory workers. the current advantages and challenges of using digital plate reading for bacterial cultures in the clinical microbiology laboratory software tools associated with dpr provide unique advantages. these analyses include identifying no growth or enumerating colonies in cultures, measuring zone sizes on ast plates [figure 5 ], and identifying a colony color, which can be used to identify organisms growing on chromogenic media [figure 6 ]. software can interpret simple culture results, such as identifying no growth plates, so these results can be released quickly. additionally, dpr software allows skilled technologists to annotate plates and delegate additional work - up to support staff or an automated colony picking instrument. these software tools help to maximize the amount of time workers spend performing tasks at the top of their skill levels. a bi - plate of bbl chromagar orientation medium and columbia cna agar is shown that was inoculated with the bd kiestra inoqula and imaged with bd kiestra digital plate reading (dpr) system. colonies suggestive of escherichia coli (pink), enterococcus (blue), and staphylococcus epidermidis (white) are visible on the chromogenic agar, and only the gram positive organisms are evident on the cna medium. dpr software can be used to quantify colony color and qualitatively interpret colony types by color. full resolution images are available at http://goo.gl/kbbeuw or by using this qr code the use of preserved digital images is an advantage of dpr because it enables integrative interpretive analysis, rapid consultation, archiving important teaching cases, and sharing of visual information. archived images of culture plates at user - defined time points facilitates the analysis of specimens. the development of software tools to enable simultaneous viewing of digital plates and digital gram stains will allow for a more integrative analysis of microbiology testing. similarly, simultaneous analysis of multiple specimens from a variety of sources all obtained from a single patient (including archived images from previous specimens) facilitates a patient - centric strategy of analysis instead of the source - centric microbiological analysis that is commonly employed. difficult to interpret cultures that demonstrate unusual or discordant findings can be shared electronically with the laboratory director for rapid consultation, which can streamline and expedite analysis of difficult cases. work is being done to integrate microbiology images into the hospital information system, so that clinicians can view images of finalized cultures and specimen gram stains. retrospective quality review of plate interpretations and work - up is also possible, which can be used as a means to monitor or measure the proficiency and competency of a technologist. in summary, the advantages of dpr can be attributed to one or more of the following : image capturing is designed to occur within an incubator, so cultures can undergo routine analysis while maintaining continuous incubation ; culture plates require less manual manipulation, which can save time and improve safety ; software can be used to analyze and annotate digital images, which can increase objectivity and efficiency ; storing images electronically enables increased flexibility in analysis and sharing of information. the two most common and significant challenges to adopting and implementing dpr are likely the capital investment required to obtain the equipment and workflow changes required to implement dpr [table 1 ]. as with the training and implementation associated with any new technology the capital investment for a dpr system is significant, but the return on investment may result in an eventual net cost - savings due to improved efficiency. however, only preliminary studies describing the increased efficiency associated with dpr and tla are available, so extrapolation and best - guessing is currently required when estimating return on investment. the willingness of technologists and technicians on the bench to change processes needs to be considered, and their expectations as well as concerns need to be heard. additionally, techs that do not feel confident or comfortable using computers may feel anxious and apprehensive when considering moving from a manual analysis of culture plates to dpr. medical leadership and administration need to agree on the goals of changing to a dpr system. ideally, representatives from all stakeholder groups will be involved in all stages of planning, implementation, and process revision. open communication of goals is important in order to foster a unified vision of change. digital plate reading is an emerging technology that is only now entering the early adoption stage in the united states, so the unknowns associated with dpr are largely unexplored. additionally, emerging technologies can undergo a period of rapid evolution during which the cost of the technology can rapidly decline and during which the quality of the output can rapidly improve. it is unknown if early adoption of a dpr system will lead to its early obsolescence because of the rapid improvements that may occur in the near future, and it is unknown if current buyers are paying a premium price to be an early adopter of dpr. these concerns may stifle the readiness of financial decision makers to release large amounts of capital to immediately invest in dpr. the concerns associated with adopting this emerging technology can begin to be overcome by performing independent studies that objectively analyze and report quality and efficiency metrics associated with dpr (e.g., accuracy, precision, changes turnaround time, changes in productivity, changes in laboratory space needs, changes in staffing needs) and dpr 's return on investment. such studies will help others in clinical microbiology to make more informed decisions about the value associated with implementing dpr and tla. fulchiron and colleagues identified a novel challenge associated with dpr, which is the loss of colony isolation. this loss can occur because cultures continue to be incubated after their images have been captured, so colonies continue to grow after imaging. therefore, if the images are not examined in a timely matter (i.e., < 2 h after image capture), then colonies that appeared to be isolated may have time to collide with others on the agar plate. this occurrence would require reanalysis of the sample, which could impair workflow and throughput. additionally, dpr workstation design needs to be carefully considered and implemented because the potential exists for increased risk of repetitive motion injuries if design is suboptimal. nuances such as this, which are unique to dpr, need to be identified empirically, studied formally, and published in the peer - reviewed literature. in 2010 and 2011 ; farrington., described the process of tla implementation and some of the changes they encountered after implementing dpr as part of tla in a laboratory that processes approximately 500,000 samples annually. in total, eight dpr workstations were installed. their site preparation caused significant disruption within the laboratory for weeks leading up to the installation of the equipment. each technologist underwent approximately 25 h of training before performing independent dpr, but full confidence with using the system was not achieved after the initial training. during the first 6 months of dpr use, they surveyed 6 weeks of work and reported their findings. during those 6 weeks, 23,630 samples were processed from which 46,725 plates were inoculated (1.98 plates per sample) and 149,762 images were obtained (3.21 images per plate). of those 46,725 plates ; 9,552 (20.4%) errors in the tla system were most frequently identified immediately following system changes, such as following initial implementation of the system or following the addition of another incubator. although these authors describe their system as being generally consistently reliable, they did note a significant breakdown that lasted 5 h caused by a software error. the authors report that these errors gave them the opportunity to improve fall - back systems that will reduce or abolish the impact of any similar failures in the future. after implementation, the number of staff required for daily operation decreased, but the number of second shift staff needed for optimal operation increased. after implementation, four staff were needed for first shift, and four staff were required for second shift. the full - time equivalents required to operate the laboratory decreased from 26.5 to 18.43 (30% reduction) after the implementation of dpr and tla, even though the laboratory 's sample volume increased. further advancements in dpr systems are anticipated as this technology matures. through the use of dpr and tla, automated colony picking and subculturing work is currently being done to improve the computer 's ability to interpret dpr images. the addition of automated colony analysis to an existing dpr system would most likely only require the validation of software and no additional capital equipment. these advanced dpr informatics tools could include the use of neural networks and artificial intelligence, which would leverage a computer 's ability to more accurately and reproducibly quantify image components that humans typically analyze qualitatively (e.g., color, size, speed of growth). copan is supporting an effort (microbia.org) that is working toward this type of advancement. the addition of such advanced computer - assisted analysis for dpr would facilitate specimen work - up and streamline analyses. the continued development and implementation of tools that could reduce the number of plates that need to be physically manipulated by individuals would help to increase the efficiencies associated with dpr. although dpr will initially be performed locally within the laboratory, it has been proposed that it may even be feasible that dpr could be performed remotely by telepathology. how dpr might fit into the routine analysis of more complex microbial culturing techniques, such as mold and mycobacteria cultures, is yet to be explored. the development and adoption of dpr is so recent that no significant body of formal studies or peer - reviewed literature is currently available. the real - world challenges associated with dpr need to be better identified and reported to the clinical microbiology and clinical informatics communities. successful (and unsuccessful) downtime strategies, downtime frequency, and downtime performance reports would be useful additions to the literature. guidelines for validation and verification of quality measures as well as proficiency testing have not been established. consensus recommendations as to the minimum specifications for image capture quality and quantity, as well as digital display quality have yet to be broached. image archiving requires increased digital storage space, and guidelines as to the appropriate duration and integrity of culture image storage are needed. moreover, practical recommendations as to whether or not these images should be part of the discoverable medical record are also needed. digital plate reading is a novel tool that should be added to the growing applications made possible by introducing digital imaging technology into the pathology laboratory. laboratorians will begin to derive ensuing benefits from improved efficiency of laboratory workflow, expedited generation of results, and enhanced characterization of microbial isolates as the clinical microbiology laboratory becomes more automated, more digital, and more reliant on informatics tools. initial studies and anecdotal evidence suggest that dpr can improve the clinical microbiology laboratory 's efficiency while improving turnaround times. improved turnaround times could in turn allow patients to more quickly receive optimal antimicrobial management. unfortunately, rigorous studies of dpr have yet to be reported in the peer - reviewed literature, and guidance regarding implementation, management, and monitoring of dpr systems is currently lacking. | digital plate reading (dpr) is increasingly being adopted as a means to facilitate the analysis and improve the quality and efficiency within the clinical microbiology laboratory. this review discusses the role of dpr in the context of total laboratory automation and explores some of the platforms currently available or in development for digital image capturing of microbial growth on media. the review focuses on the advantages and challenges of dpr. peer - reviewed studies describing the utility and quality of these novel dpr systems are largely lacking, and professional guidelines for dpr implementation and quality management are needed. further development and more widespread adoption of dpr is anticipated. |
primitive neuroectodermal tumors (pnets) are highly malignant embryonal neoplasms of bone and soft tissues accounting for 4%17% of all pediatric soft tissue tumors. metastasis at presentation is quite common, with central pnets metastasizing to cranium and leptomeninges while peripheral pnets (ppnets) disseminating to lungs, bone, liver, and lymph nodes. primary pulmonary pnet is known to metastasize to pancreas, adrenal gland and ovaries while it metastasizing to brain is exceedingly rare. a 29-year - old female presented with a cough and right - sided chest pain of 1-month duration. computed tomography scan of chest [figure 1 ] showed an 11.3 cm 11 cm 10 cm soft tissue mass lesion in right perihilar region with the loss of fat planes with esophagus, aorta, and diaphragm without chest wall or pleural involvement. biopsy showed atypical round to oval cells with scanty cytoplasm intermixed with respiratory epithelium [figure 2 ]. immunohistochemistry (ihc) was positive for neuron - specific enolase (nse) [figure 3 ], synaptophysin, chromogranin, cd 99, and vimentin. smooth muscle actin (sma), epithelial membrane antigen (ema), cytokeratin (ck), leukocyte common antigen (lca), cd 20, cd 45, thyroid transcription factor (ttf-1) and desmin were negative, confirming the diagnosis of pnet lung. computed tomography scan (axial section) of thorax showing a large heterogeneously enhancing soft tissue mass lesion in the right perihilar region involving right lower lobe extending into the mediastinum with loss of fat planes with esophagus, aorta, and right crus of the diaphragm. there is no chest wall or pleural involvement biopsy from lung lesion showing small round cells with scanty cytoplasm with condensed chromatin and inconspicuous nucleoli (h and e, 200) immunohistochemistry picture from lung lesion showing tumor cells positive for neuron specific enolase (200) magnetic resonance imaging (mri) brain and positron emission tomography scan showed localized disease. received chemotherapy vincristine, adriamycin, and cyclophosphamide alternating with ifosfamide plus etoposide (vac / ie) resulting in partial response (pr) to therapy. conformal radiotherapy (rt) was given to postchemotherapy residual volume to a dose of 60 (gray) gy in 30 fractions. post - rt patient experienced significant symptomatic relief with improved performance status though with a static disease. the patient was on follow up for 9 months when she presented with a headache, vomiting, seizures and left - sided hemiparesis. mri brain [figure 4 ] showed an 8.6 cm 7.5 cm solitary lesion with areas of altered signal intensity at right periventricular and periatrial parietal lobe with significant perilesional edema suggestive of hemorrhagic metastatic deposit. she underwent right fronto - temporo - parietal craniotomy with excision of tumor and evacuation of hematoma. ihc was positive for synaptophysin [figure 5a ], chromogranin [figure 5b ], vimentin [figure 5c ], and cd 99 [figure 5d ] with ki67 of 90% while negative for ck, ttf-1, and melan - a which suggested metastasis from primary pnet lung. we treated her with whole brain rt (wbrt) to a dose of 30 gy in 10 fractions followed by oral pazopanib 800 mg once a day as a palliative intent. the patient is under follow up for over 1 year with a karnofsky performance status of 70%. magnetic resonance imaging brain (a = coronal section and b = axial section) showing a solitary lesion with areas of altered signal intensity at right periventricular and periatrial parietal lobe with significant perilesional edema suggestive of hemorrhagic metastatic deposit immunohistochemistry picture from brain metastasis showing tumor cells positive for (a) synaptophysin (100), (b) chromogranin (200), (c) vimentin (100), (d) cd 99 (200) thoracic pnets are the most aggressive form of ppnets arising from chest wall or underlying lung pleura invading bone, lung or mediastinum in children and young adults. they are extremely aggressive with a dismal prognosis as patients may present with an upfront metastatic disease to contralateral lung, bone, bone marrow, lymph nodes, liver, pancreas, adrenals, and ovaries. primary lung pnet can often be misdiagnosed with a metastatic lung lesion, mucinous adenocarcinoma or squamous cell lung carcinoma. since pnets and other small round cell tumors share the same histological picture, ihc plays a vital role in their differentiation. cd 99, vimentin and s100 are nonspecific while nse, synaptophysin and chromogranin confirm the diagnosis of pnet. ttf-1 is found in pulmonary adenocarcinomas and thyroid malignancies, ck for carcinoma, desmin for rhabdomyosarcoma, sma and ema for soft tissue sarcoma while lca, bcl-2, cd 20, cd 45 are for acute lymphoblastic lymphoma and leukemia. however, to confirm the diagnosis, amplification of ewing sarcoma breakpoint region-1 by situ hybridization techniques is required. according to javery. and previous other series on extraskeletal ewing sarcoma (ees), lung is the most common site of metastasis followed by bone comprising 80% and 40% cases, respectively. huh. reported lymph nodes as the most frequent metastatic site (75.9%) followed by bone, lung, peritoneum, pleura while least being brain (3.4%). the most prevalent primary sites were extremities, abdomen, pelvis, thorax, paravertebral space followed by head and neck current treatment recommendations for lung pnet and ppnets / ewing 's are same with vac / ie. rt concurrent with chemotherapy is preferred for the limited stage while sequential rt is beneficial for extensive lesions. we have treated two more cases of adult primary lung with a radiological pr but a significant symptomatic response. both patients are on follow - up for more than a year now without any evidence of disease progression or metastasis. for the management of brain metastasis from pnet lung, at present wbrt to a dose of 30 gy was exhibited as it is the standard of care in patients with brain metastasis which has shown superiority in preventing neurologic morbidity and mortality. pazopanib is an oral multikinase inhibitor of angiogenesis indicated mainly for progressive or chemotherapy refractory soft tissue sarcomas. although pazopanib is not the standard therapeutic option in ewing sarcoma, it has demonstrated its efficacy in refractory and ees metastatic cases. since ewing 's tumors and ppnets share similar genetic characteristics with reciprocal translocations of chromosomes 11 and 22 at ewsr1, differing only in degree of neural differentiation with the former being more differentiated than latter, we, therefore, exhibited pazopanib to our patient with a palliative intent as she had progressed after standard chemotherapeutic regimen. the effectiveness of pazopanib by children 's oncology group (cog) nct01956669 phase-2 study and another multi - kinase inhibitor regorafenib by nct02048371 and nct02389244 phase-2 studies for refractory ewing sarcoma are underway, and the results are awaited. newer prognostic markers should be developed to identify primary pnet lung at risk of developing brain or distant metastases. a better understanding and interpretation of the molecular and biological mechanisms of this entity we recommend craniotomy and excision followed by wbrt for brain metastasis and oral pazopanib to check further dissemination as an incisive treatment guideline, though more prospective data favoring this recommendation is required. | primitive neuroectodermal tumors (pnets) are highly malignant neoplasms of embryonal origin manifesting in children and adolescents, rarely seen in adults. carcinoma lung with hemorrhagic metastasis to the brain is very common, but primary lung pnet with hemorrhagic brain metastasis is extremely uncommon. we hereby report a 29-year - old female diagnosed as pnet lung was treated with vincristine, adriamycin, and cyclophosphamide alternating with ifosfamide plus etoposide followed by radiotherapy (rt). after 9 months, she developed hemorrhagic brain metastasis from pnet lung confirmed from tissue immunohistology postcraniotomy. received palliative whole brain rt followed by oral pazopanib resulting in significant improvement in performance status. a thorough review of literature reveals that our case may be the second case of primary lung pnet with hemorrhagic brain metastasis and also the first to be exhibited oral pazopanib resulting in a significant therapeutic effect to be reported in world literature till date. |
levels of the polybrominated diphenyl ethers (pbdes), a class of widely used flame retardants, appear to be rising rapidly in human tissues, as evidenced by studies of human breast milk. the case of the pbdes illustrates the value of breast - milk monitoring programs in identifying important emerging pollutants, and highlights why such monitoring programs are needed in the united states. a review of the use, occurrence, and toxicity of pbdes indicates many parallels between some pbdes, pcbs, and other polyhalogenated persistent organic pollutants, and suggests that the pbdes may be a significant environmental challenge in the future.imagesfigure 1figure 2 |
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the international community is rapidly approaching the deadline for achieving the 2015 millennium development goals. given that we have the tools at our disposal to significantly bend aids - related morbidity and mortality curves and reduce hiv incidence, it is essential to redouble efforts to reach the goal of placing 15 million people on art by 2015. in reaching this milestone, we can write a new chapter in the history of global health, demonstrating that a robust, multidimensional response can succeed against a complex pandemic that presents as many social and political challenges as it does medical ones. this milestone is also critical to advance our ultimate goal to end aids, translating into a world in which aids - related deaths and new hiv infections are exceedingly rare. in at least 13 high - prevalence countries where hiv treatment has been brought to scale, the annual increase in the number of people receiving art now exceeds the number of people newly infected with hiv. a recent study in kwazulu - natal, south these achievements indicate that the aids response is starting to outpace the epidemic, which should reinforce our efforts to achieve similar successes around the globe. as the who 's 2013 consolidated guidelines emphasize, early art initiation is essential to leverage the full range of benefits that art offers. fully implementing these guidelines would prevent an additional 3 million aids - related deaths and avert 19 million new hiv infections between 2013 and 2025. yet again, time is of the essence, as experience demonstrates that rapid art scale - up achieves far greater health gains [9, 11 ]. although art has reshaped the aids response, the hiv treatment revolution has yet to reach many around the world. only modest gains in art coverage have been reported in eastern europe and central asia as well as in the middle east and north africa, and art - eligible individuals in west and central africa are notably less likely to obtain art than those in eastern and southern africa. treatment coverage is > 20% lower among men than among women, and globally the pace of art scale - up for children 15 pills a day that was routinely prescribed just a few years ago, the majority of patients today are initiated on art regimens consisting of 1 pill per day in a fixed - dose combination. the evolution has benefited many, particularly with respect to the challenge of achieving optimal art adherence ; however, innovation must continue to be leveraged to further simplify hiv treatment, such as the ongoing research around longer - acting antiretroviral drugs. this will further reduce art - related side effects and toxicities, enhance adherence, achieve maximum effectiveness, and significantly minimize the risk of costly drug resistance. there have been dramatic reductions in the cost of antiretroviral drugs from us$15 000 per person per year in 2001 to < us$100 today for first - line art regimens in some countries. further reducing art and related costs (eg, diagnostics) should remain a priority, particularly for people in low- and middle - income countries. further, cost avoidance can also be derived from strengthening art programs, thus preventing avoidable treatment failure and consequent drug resistance, which require more expensive second- and third - line art regimens. urgent attention is also required to bring services to populations that experience unique barriers to access. expanding earlier art access for newly diagnosed infants requires countries to ensure 100% active case - finding for all children born of mothers living with hiv, and universal access to hiv treatment for infants testing positive for hiv. similarly expanded art access should be our goal with respect to pregnant women living with hiv, in line with the who 's recommendation to offer lifelong art to all hiv - positive pregnant women regardless of their cd4 count (option b+). tailored efforts are needed to encourage women and men to seek hiv testing and treatment services, to develop services that are accessible to and user - friendly for young people as well as an aging population of hiv - positive individuals, and to implement community - driven, rights - based outreach and service channels for key marginalized populations, such as men who have sex with men, injecting drug users, and sex workers. punitive laws and other structural impediments that deter access to hiv testing and treatment services should immediately be removed, both because they represent violations of human rights and because they undermine efforts to control hiv. to mobilize the financial resources that will be needed to achieve and sustain universal access to hiv treatment, all stakeholders in the hiv response, including countries and international donors, must continue to share responsibility for the global aids response. unaids recommends that countries develop national investment cases that define the most strategic use of resources, identify opportunities for efficiency gains, and describe clear plans for long - term financing, with an emphasis on innovative domestic financing sources and the rapid scale - up of hiv treatment. at least 14 countries have begun aligning national hiv responses with investment principles that include treatment as a keystone activity, with an additional 30 countries planning to develop a national hiv investment case over the next 2 years. investing wisely requires that resource allocation reflects the mix of approaches that is calculated to maximize the health returns for available funding. rational resource allocations also need to be focused on the geographic settings and populations with the greatest need. kenya, for example, has pledged to prioritize hiv investments in the 9 counties that account for 54% of new hiv infections, while nigeria is reallocating resources towards the 12 states and the federal capital territory that collectively represent 70% of the country 's hiv burden. strategically designing national or regional antiretroviral procurement tenders and improving drug forecasting and distribution systems can help further reduce drug costs and avert stock - outs. all countries should actively explore innovative financing options to ensure a robust, sustainable response. a growing number of countries are exploring various financing strategies, such as a dedicated hiv tax levy or the creation of a national hiv or health trust fund, and transitioning towards national self - financing of the hiv response. even with such innovations, low - income countries and those with especially heavy hiv burdens will continue to require external assistance to close the hiv treatment gap, highlighting the urgent importance of strengthened and sustained support from the international community, particularly from the global fund to fight aids, tuberculosis and malaria and the us president 's emergency plan for aids relief (pepfar). to drive success and ensure accountability, countries should take steps to develop new, ambitious national treatment targets beyond 2015 that aim to achieve the end of aids. in addition to coverage targets, countries should identify specific goals with respect to knowledge of hiv status, linkage to care, and long - term retention in care. treatment targets should take into account the who 's 2013 consolidated guidelines, which nearly doubled the number of people clinically eligible for art. with so much at stake, and with the tools at our disposal to envision the end of aids, there is no room for complacency. building on the decades - long history of the global response to aids, we must do what must be done now : get people tested. link people who test hiv positive but who are not clinically eligible for art by current criteria to comprehensive, holistic care. treat people who need art so that they may stay alive, healthy, and productive members of society. leverage the full potential of hiv treatment to prevent not just aids - related morbidity and mortality, but also hiv transmission, thus avoiding the inevitable cost of inaction. we must hold nothing back as we work to transform the end of aids from a promise into reality. | we have the tools at our disposal to significantly bend aids - related morbidity and mortality curves and reduce human immunodeficiency virus (hiv) incidence. it is thus essential to redouble our efforts to reach the goal of placing 15 million people on life - saving and -enhancing antiretroviral therapy (art) by 2015. in reaching this milestone, we can write a new chapter in the history of global health, demonstrating that a robust, multidimensional response can succeed against a complex pandemic that presents as many social and political challenges as it does medical ones. this milestone is also critical to advance our ultimate goal of ending aids by maximizing the therapeutic and preventive effects of art, which translates into a world in which aids - related deaths and new hiv infections are exceedingly rare. |
both the psychomotor and emotional development can be significantly impaired when the child has hearing problems. lack of information and acoustical stimuli in the first years of life restricts or even disables the development of speech. earlier diagnosis of hearing problems in an infant or child is enhances the possibility of choosing the proper diagnostic and therapeutic path. timely intervention is an important component of any early hearing detection and intervention (ehdi) screening program. the first neonatal screening programs (nhs) in poland were designed in the 1980s and subsequent studies took place in the mid-1990s. the results of the polish nhs / edhi program suggest that the incidence of congenital hearing disorders ranges from 2 to 7 per 1000 births, estimates which have been verified by other studies in the literature [611 ]. a child might present a hearing loss caused by acquired hearing or genetic disorders, usually in the form progressive hearing loss. data in the literature suggest that any undetected impairment, even of a mild degree, can significantly affect the language development and social and emotional development of children, as well as their educational achievements [13,13 ]. for example, undetected mild unilateral hearing loss can cause difficulties in speech understanding and problems with sound - source location, significantly affecting the learning process. these children do not achieve the same progress in school as their peers with good hearing, and 40% of school - age children with unilateral hearing loss fail final - year exams and must repeat a class. studies in various populations show that 7695% of all examined children had this disease at least once during early childhood. there is a growing incidence of high - frequency hearing loss in young children caused by listening to loud music through headphones [1618 ]. other causes of hearing loss in school - age children include infectious diseases (e.g., mumps, measles, and meningitis), mechanical injuries, and congenital cholesteatoma. in 1999, the institute of physiology and pathology of hearing (warsaw, poland), conducted hearing screening tests in different regions of poland, assessing a group of more than 6200 school - age children. data from this study suggested that 1 in every 5 children aged 618 years has hearing problems, with the most common locus in the middle ear. in 2008 a team from the institute of physiology and pathology (warsaw, poland) carried out numerous mass - screening programs for a period of 4 years. to ensure credible results, unique devices and proprietary solutions were developed. in a period spanning 4 years, more than 300 000 school - age children were assessed. the important innovation of this program was in choosing primary schools located not in the big cities, but in small schools in urban peripheries. during this period, specific computerized tools were developed (e.g., the sense examination - platform szok, which is a telemedicine model) that made possible the organization and performance of hearing tests at relatively low costs. in addition, medical specialists were able to review, via the platform, remotely collected data and to identify children requiring additional care. the data from this study showed that 714% of the assessed children had peripheral hearing loss. for 70% of those children it was the first hearing screening test in their life and for 60% of these children these large - scale screening experiences called for a european scientific consensus agreement, which was defined and signed during the european federation of audiology societies (efas) meeting in june 2011, at warsaw, poland. as a result of the consensus agreement and under the auspices of the institute of physiology and pathology of hearing, a number of pilot hearing screening programs were started in various countries [2224 ], promoting hearing - loss detection and treatment of communication disorders in young school - age children. the medical community was exposed to information regarding the screening equipment developed in poland and various organizational solutions (szok) that allow data collection on a larger scale. the project was organized in cooperation with the mz - rt national medical center (dushanbe, tajikistan), the trade and investment promotion section of the polish embassy in tashkent, and the department of otolaryngology of abuali ibni sino tajik state medical university in dushanbe. the objectives of this pilot project were : (i) to perform audiometry screening tests in schools and to classify the results via a telemedicine model ; (ii) to provide, if possible, medical consultation ; and (iii) to raise awareness, among both medical specialists and parents in tajikistan, about the potential causes of hearing loss and the possibilities for prophylaxis, diagnosis, treatment, and rehabilitation. due to significant differences in the socia - economic levels, these 2 public schools were randomly chosen, excluding elite private schools. in one of the schools the pilot study assessed a total of 143 (286 ears) students aged 78 years. testing was conducted by a team from the institute and from the abuali ibni sino tajik state medical university (medical doctors and other specialists). the final results were given to the directors of the schools, who contacted the parents of the children who did not pass the screening, for further clinical assessments and information on the observed hearing deficits. the model used remote client devices and software developed during the 2008 polish screening program. additional details on the model / platform are presented in sections a2 and a3 in the appendix. tests were conducted in quiet rooms in order to determine hearing threshold in the 5008000 hz range (see additional details in section a1 of the appendix), with the same paradigms as in a previous study. according to already published criteria, cases presenting threshold levels > 25 db at 1 or more frequencies or in 1 ear were marked with a hearing loss status. prior to testing, the children s parents were informed of the testing procedures and provided their written consent. they had the option to receive additional information by completing a questionnaire with data on the potential causes of the child s hearing problems, medical history, possible presence of tinnitus, and any presence of learning difficulties. in total, the results of the individual tests (pure tone averages and questionnaires) were sent via an internet connection to the szok platform via encrypted lines. test results presenting a hearing loss status were divided into 2 classes : subjects with unilateral or bilateral hearing losses. the subjects in each class were further assigned into 3 groups according to the pattern of their corresponding audiograms : low - frequency hearing loss (lfhl) the value of the hearing threshold for the frequencies of 500 and/or 1000 hz was at least 25 db hl, while the hearing threshold for the frequencies of 2000, 4000, and 8000 hz did not exceed 20 db hl ; high - frequency hearing loss (hfhl) the value of the hearing threshold for the frequencies of 4000 and/or 8000 hz was at least 25 db hl, and for the frequencies of 500, 1000, and 2000 hz it did not exceed 20 db hl ; other the hearing threshold exceeded 20 db hl for at least 2 non - adjacent frequencies. this category was initially called mixed (i.e., low- and high - frequency losses), but the name improperly suggested the existence of a specific middle and inner ear pathology and it was abandoned for a more generic term. based on the hearing test results and the questionnaire outcomes, and in accordance with the screening criteria developed during the programs implemented in poland, medical specialists from the institute of physiology and pathology of hearing (ifps) classified children with hearing losses into 2 groups : (i) a group requiring further clinical care ; and (ii) a group requiring continuous monitoring over time, according to standard hearing prophylaxis procedures. the parents of the latter group obtained additional information on hearing disorders and the various factors causing the deterioration of hearing. analyses of variance (anova) was conducted with spss version 16.0 at a significance level of p 0.05. the testing variables were defined as follows : school id / language ; gender ; screening outcome (pass / fail) ; unilateral hearing impairment ; bilateral hearing impairment ; and lateralization (right / left impaired ear). the age variable was not tested because it was found to be homogenous in the assessed sample (i.e., the children had the same age). the use of the remote client devices (pcs) and the transmission of data via the szok platform did not present any particular technical complications. on occasion, due to poor internet connections (transmission rates below 40 kb / s), the transmission of data took twice the time necessary, but no corrupted data were reported. the anova analyses suggested that the language (i.e., the site of assessment) was a significant factor in every tested variable, but this was caused by the asymmetric sample sizes taken from the 2 schools (see numbers of participants in table 1). no gender effects were observed on the screening outcome or on the incidence of unilateral / bilateral hearing impairment. in terms of screening, 109 children passed the set criteria, while 34 children (24%) presented audiometric levels above the 25-db criterion and were considered as refer cases. in both schools, the proportion of bilateral to unilateral hearing impairment was found to be very similar, at close to 1:1. based on the screening results of the 34 refer cases and the data gathered from the questionnaires, audiologists and otolaryngologists from the ifps referred 24 students to further specialist care. the parents of the remaining 10 students were provided with information on hearing loss prophylactic procedures. the data are summarized in table 1. in terms of ears presenting hearing losses, the total number was 51, derived from 17 unilateral and 17 bilateral cases. among these, 31.4% (16 ears) presented low - frequency hearing loss (lfhl), 25.5% (13 ears) high - frequency hearing loss (hfhl), and the remaining 43.1% (22 ears) presented hearing losses involving all tested frequencies. for the low and high - frequency loss - groups, anova analyses showed a significant lateralization effect favoring the right ear. similarly, for the cases classified in the other category, anova analyses showed a lateralization effect favoring the left ear. due to the small numbers of ears in these groups, these observations may be slightly biased. the questionnaires showed that only 9 (37%) of the 24 children needing additional health care had previously undergone hearing tests. children with refer screening results had experienced otitis media twice as often as the other normal children and had a runny nose during the hearing tests at school. the objective of this pilot hearing screening program in tajikistan was to validate a telemedicine model with methods and procedures already tested in other programs, in a region where hearing screening is rare. data were collected from first - graders and the hearing screening outcomes were enriched with additional information from questionnaires. the latter proved to be an adequate tool in assessing non - specific information related to screening, such as otitis incidence, tinnitus incidence, and runny nose incidence. unfortunately, questionnaires require time and smooth collaboration between parents and the audiology staff and these factors in many instances prevent the proper collection of information. the data suggest that almost 1 in 4 students (i.e., 34/143=23.7%) has a hearing impairment. data from previous hearing screening tests conducted by the institute of physiology and pathology of hearing in 1999 (5, 15) showed a lower incidence, suggesting that 1 in 5 (20%) students has a hearing problem. although these studies assessed different sample sizes and children of different ages, the hearing deficit estimates successfully describe the variability that commonly characterizes hearing screening outcomes in school - age children. thirty - four percent of the tested children in tajikistan were diagnosed with a low - frequency hearing loss. similar data have been reported in a study in nigeria, where 167 out of 500 examined children (33.4%) presented lfhl in their right ear and 39 in their left ear. very different data were reported from an american population, where the percentage of children with lfhl was reported as 7.1%. the differences in the results between prior studies and the tajikistan data may be caused by the fact that the research in tajikistan was carried out in the autumn, when there is an increased incidence of upper respiratory tract infections (urti). research conducted at the pediatric clinic of otolaryngology, audiology, and phoniatrics of the medical university of lodz, poland showed a correlation between urti and hearing loss. the data from the tajikistan project showed that the low- and high - frequency hearing loss occurs more likely in the right ear than in the left. similar results were observed in studies in nigeria, where there is a higher incidence of hearing loss involving high frequencies in the right ear. in these tests, high - frequency hearing loss occurred almost 3 times more often in the right ear as in the left. data from this study suggest that for subjects presenting hearing losses at multiple frequencies, there is a strong lateralization effect towards the left ear. these findings confirm the data trends found in a previous study by the same authors in a pilot study in poland. nevertheless, the findings in both studies refer to a finite sample size ; therefore, data from a larger pool of subjects should be considered prior to forming any definite conclusions. according to kuppler, the most common hearing disorder is the unilateral hearing loss, which is found in approximately 3% of school - aged children. reported values of unilateral hearing loss incidence as high as 88% (59/67) in a group of 296 assessed children, of which 67 presented hearing deficits. conducted screening tests in 6166 american children aged 619 years ; almost 15% of the children presented a low- or high - frequency hearing loss with 82% unilateral losses. data from an iranian children population report an incidence of unilateral hearing loss of 75% of all reported hearing losses. in the present study the incidence of unilateral hearing disorders was 50% (17/34 cases), an estimate which is much lower than the estimates reported in the above - mentioned studies. a number of hypotheses (or their combinations) might explain this outcome, such as : (i) the population of dushanbe has this particular characteristic ; (ii) the assessed sample of 143 students did not accurately represent the characteristics of the general population ; and (iii) previous studies considered a wider age interval, which could have led to different unilateral / bilateral hearing deficit ratios. the findings of the present study suggest that many factors play important roles in the data reported from child screening programs. the age - range and the sample size of the assessed population are important, but one has to also consider other factors such as the hearing assessment protocols and the instrumentation errors during the data assessment. the proposed screening procedures allow the detection not only of children with hearing loss, but also those with other hearing disorders, such as tinnitus. according to data in the literature, data from the current study show that 9% of the normal hearing group who passed the screening has tinnitus. for the cases classified to a group requiring further clinical care, additional data from screening in poland have reported an even larger percentage, showing that nearly 32% of children with normal hearing have tinnitus [3234 ]. the successful use of the szok software platform in a population with a different language is a positive step towards a model of telemedicine / telehealth [3537 ] serving rural areas where specialist medical care is not available. the proposed platform can be used not only for screening children but also for starting edhi programs. the platform was not evaluated in terms of quality of data or transmission efficiency because these tasks were performed in previous studies. despite intenet a novel aspect of using this telemedicine model in the future could be the education of professionals and families on issues of hearing deficits and intervention policies. the results of this pilot study confirm that, even in developed countries, the awareness of hearing disorders is low. many school - age children have hearing loss, but often the problem stays unnoticed by parents and teachers. the data from the present study suggest that : it is possible to apply a telemedicine model to assess the hearing status of children and to provide long - distance expert assistance. the reported data suggest that in dushanbe, tajikistan, 1 in every 4 young children has a hearing deficit. the incidence of unilateral hearing loss was very similar to the incidence of bilateral losses. this estimate is much lower than the figures presented in previous reports in the literature. a number of factors (e.g., age, sample size, and protocol used), can significantly influence these values ; therefore, direct comparison of screening outcomes from various studies can be difficult. audiometry testing was conducted during lecture hours in quite rooms (noise level=4550 db a). the testing audiometers could also measure ambient noise and testing was stopped when the noise level before and during testing exceeded 50 db a. in a free - field audiometry testing, it is very difficult to obtain low frequency (125 and 250 hz) threshold estimates without many repetitions. the low audiometric frequencies were excluded from the tajikistan testing protocol. since the main deliverable of the project was to identify the pros and cons of future screening guidelines, this procedural compromise was considered acceptable. the sense examination platform (szok) is a telemedicine model for screening and testing : (i) hearing and sight ; and (ii) speech in children, young adults, and, in general, of those with special needs. the system was developed by the institute of sensory organs in collaboration with the institute of physiology and pathology of hearing (warsaw, poland). the sense examination platform is equipped with a variety of screening tests enabling it to detect a number of dysfunctions in the field of hearing, sight, and speech. in addition, the platform was designed to collect data for surveys and general epidemiological research. the platform is built around an internet network solution, interfacing a central computer system and a series of portable computers (remote client devices) equipped with audiometric headphones and a response - button interface. the platform allows the user to conduct the following tests : audiometric testing (module audiogram 2009) : this feature allows the user to perform air conduction audiometric testing for each ear separately, in a tone frequency range from 250 to 8000 hz and for hearing threshold levels not exceeding 80 db hl. hearing screening test (module i can hear 2009) : this feature enables the implementation of hearing screening tests for tones at the frequencies 1000, 2000, and 4000 hz (in various stimulus intensities) and the assessment of speech intelligibility in noise. speech screening test (module i can speak 2009) : the speech test is carried out to obtain reliable information on : (i) the quality of verbal behavior of the child and (ii) the degree of speech development (or of any potential delays) and any pathological linguistic phenomena occurring in the speech of the child. sight screening test (module i can see 2009) : the sight screening test is based on the contrast differentiation test, the color vision test, and the stereoscopic vision test. audiological survey (module survey 2009) : this module allows the user to conduct a general survey regarding the hearing, sight, and speech of a patient. the surveys were developed by specialists based on years of experience in specific areas and they provide reliable information on the tested person. test module ddt 2009 : this is a dichotic listening test. during the test, pairs of sounds are presented to each ear and the task of the tested subjects is to repeat what they heard in one or both ears. test module gdt 2009 : this test allows assessment of the potential of perception of gaps in noise. during the test | backgroundaccording to the guidelines of the european scientific consensus on hearing (european federation of audiology societies efas congress, june 2011, warsaw, poland), the detection and treatment of communication disorders in early school - age children is of the highest importance. this objective was adopted by the polish president of the efas council from the second half of 2011 ; as a result, pilot programs on children s hearing screening were initiated in various european countries. this paper reports data from a pilot program in dushanbe, tajikistan.material/methodswe randomly selected 143 children from 2 primary schools. each child was assessed by pure tone audiometry and 2 questionnaires (dedicated to parents and children). the study allowed the validation of : (i) hearing screening procedures in young children, and (ii) data collection via a telemedicine model.resultshearing impairments were identified in 34 cases (23.7%) with a 50% ratio between unilateral and bilateral losses. we found a higher incidence of hearing impairment in children than that reported in previous polish studies.conclusionsthe data from the present study suggest that it is possible to use a telemedicine model to assess the hearing status of children and to provide a long - distance expert assistance. the latter is very important for rural areas without specialized medical services. |
piroxicam is a non - selective prostaglandin g / h synthase (better known as cyclooxygenase or cox) inhibitor that acts on both prostaglandin g / h synthase 1 and 2 (cox-1 and -2). cox catalyzes the conversion of arachidonic acid to a number of prostaglandins involved in fever, pain, swelling, inflammation, and platelet aggregation. piroxicam antagonizes cox by binding to the upper portion of the active site and preventing its substrate, arachidonic acid, from entering the active site. the analgesic, antipyretic and anti - inflammatory effects of piroxicam occur as a result of decreased prostaglandin synthesis. piroxicam also inhibits the migration of leukocytes into sites of inflammation and prevents the production of thromboxane a2, an aggregating agent, by platelets (1, 2, 5, 6 and 7). industrial powder of piroxicam is an amorphous white powder but piroxicam also has two different needle and cubic polymorphs crystalline shapes (1, 5 and 6). different solvents, different technological procedures (melting, rapid and slow cooling) and some other operating parameters such as ph, agitation speed and agitator paddle shape affect the particles shapes and formation of polymorphous piroxicam (1 - 3 and 17). in the current study using taguchi experimental design approach, the influences of some main physicochemical properties, possibilities of their formation and their avoidance were examined and then the dissolution rate of piroxicam powder was optimized. experimental design the conventional one - at - a - time approach to evaluate the influences of process parameters on product quality or quantity requires numerous experimental runs (particularly when a lot of variables are to be investigated at different levels) to fully explore the entire parameter space. in this respect, the taguchi experimental design method can reduce the number of experiments while retaining data collection quality. the quantified and comparative analysis of the factor effects is the second advantage of this approach (9, 10). the first important step in design of experiment is the proper selection of factors and their levels. in this study, five operating factors include the type of solvent, type of cooling (rapid and slow cooling), type and shape of mixture paddle, ph, and agitator speed were considered in three levels (table 1). the factors and their levels have been chosen according to a literature review on previous publications as well as industrial recipes for production and optimization of piroxicam powder (1, 4 - 8). for design of experiments with five factors and three levels for each factor, a standard l18 orthogonal array was employed (table 2). each row of the matrix represents one run at specified condition. in order to avoid the systematic bias, the statistical analysis of the results was carried out using qualitek-4 (nutek inc.) software (9, 10). selected factors and their levels taguchi orthogonal array for experiments based on coded levels c) placed inside the reactor, and was controlled by a ben marry set. at first step of recrystallization, the reactor was first charged with 0.5 litter of solvent according to the experimental design table (table 2??1, 2, 11 - 14). yield determination where m1 and m2 are the amount (grams) of final dried recrystallized piroxicam and initial samples, respectively (1, 9). particle size and shape measurement a 1 mg.ml?qudix-scatheroscope particle size analyzer. this method was applied identically to all 18 samples to keep a similar condition for samples (1, 2 and 15 - 17). dissolution rate test for determination of dissolution rate, 100 mg of the crystals was added to 250 ml of distilled water at 25 c in a dissolution testing device according to references (dissolution tester erweka - dt800, japan). rotation speed was 100 rpm. at regular intervals (15 minutes ; 0, 15, 30, 60), concentrations were determined using the uv - spectrophotometer at 350 nm (uv-160a spectrophotometer, shimadzu, japan) (1, 15 - 17). the results of all measurements are summarized in table 3. the measured responses for each run in taguchi method the results are statistically analyzed using analysis of variance (anova) to determine the partial contribution of each operating factor on the response. the strategy of anova calculation is to extract from the results that how much each factor is effective on response or responses of experiments (9, 10). there are many statistical terms in anova table, among them few are more meaningful. the f - ratio is a criterion for distinguishing the important factors from those with less significance. it should be emphasized that the interpretation of anova table is valid just in the range of levels considered for each factor. the main effects of factors are determined using average values of response at each level (9, 10). effects of process parameters on particle size table 4 shows the analysis of variance for particle size. as it is observed, the predominant factor for controlling the particle size in recrystallization process is the type of cooling and ph is the second major effective factor. the type of mixture paddle and type of solvent show low effects on the particle size formation. it is implied from data in anova table that the agitation speed has no significant influence on particle size. these statistical results have been confirmed by reported literatures (11 - 15, 17). analysis of variance (anova) for particle size figure 1 indicates the effect of type of cooling on particle size. it shows that slow cooling (level 2) results in formation of the smallest particle versus other levels and figure 2 shows the second level (ph=7) is the best ph for formation of the smallest particles. influence of type of cooling on particle size influence of ph on particle size effects of process parameters on dissolution rate table 5 shows the anova table (analysis of variance) of dissolution rate of piroxicam samples. as it is observed the predominant factors for controlling the dissolution rate in samples are the type of solvent and then type of cooling respectively. the ph and agitation speed during crystallization process have slight affects on the dissolution rate of sample powders and type of agitator paddle has no significant effect on the response. analysis of variance (anova) for dissolution rate figure 3 indicates that the effect of type of solvent on dissolution rate. it shows that ethanol solvent causes to produce better particles versus other levels at dissolving rate. figure 4 shows after type of solvent factor, the first level (fast cooling) is the best versus other levels for formation of faster dissolution rate particles. influence of type of solvent on dissolution rate influence of type of cooling on dissolution rate table 5 shows the optimum conditions for the best response (fast dissolution rate particles) that qualitek4 software suggests of particle size data. optimum conditions for producing fast dissolution rate particles table 6 shows the software suggests optimum conditions for crystallization process to produce the best powder by increased dissolution rate. the optimum condition for attaining the best powder by increased dissolution rate is reported in table 6. the best level of each factor for improvement of response is given in this table. optimum conditions for dissolution rate these conditions were tested again and the powder was extracted and purified by current last methods that used for other samples. the properties of prepared powder were reported in table 7. properties of optimum powder dissolution rate of prepared powder was tested again and reported in figure 5. dissolution rate of optimum powder based on these results it is revealed that an optimum particle size between 120 - 169 m and needle crystals are the best style for dissolution rate of piroxicam powder. run 6, 13, 16 response shows particles with cubic shape and optimum size (120 - 160 m) have no effective dissolution rate but particles with needle shape demonstrate better dissolution rate so it is confirmed the conditions for the best dissolution rate are an optimum particle size in range of 120 - 160 m and needle shape crystals. using taguchi experimental design the influence of five operating variables on particle size and dissolution rate of industrial piroxicam powder in recrystallization process was statistically analyzed. the main conclusions are summarized below : 1) type of cooling and ph of recrystallization process show maximum effects on the particle size with respect to the selected levels in this study. 2) slow cooling and ph=7 result in producing small particles but acidic ph and fast cooling result in producing larger particles. 3) type of solvent and type of cooling in recrystallization process demonstrate the maximum effects on dissolution rate of prepared particles. 4) ethanol solvent and fast cooling result in producing the particles with best dissolution rate. 4) the optimized conditions to prepare the powder with the best dissolution rate are summarized below : type of cooling = fast agitation speed = 200 type of mixture paddle : turbine the optimum produced powder has 27.6% faster releasing versus industrial sample in first 30 minutes. our obtained results reveal that an optimum particle size between 120 - 169 m and needle shape crystals are the best style for dissolution rate of piroxicam powder. | piroxicam has two different crystalline forms (known as needle and cubic forms), that they are different in physicochemical properties such as biological solubility. in the current research, using taguchi experimental design approach the influences of five operating variables on formation of the piroxicam polymorph shapes in recrystallization were studied. the variables include type of solvent, cooling methods, type of mixture paddle, ph, and agitator speed. statistical analysis of results revealed the significance order of factors affecting the product quality and quantity. at first using the taguchi experimental method, the influence of process factors on the yield, particle size and dissolution rate of piroxicam powder was statistically investigated. the optimum conditions to achieve the best dissolution rate of piroxicam were determined experimentally. the results were analyzed using qualitek4 software and it was revealed that the type of solvent and method of cooling respectively are the most important factors that affect the dissolution rate. it was also experimentally achieved that some factors such as type of agitator paddle, ph and agitation rate have no significant effects on dissolution rate. |
skills such as social smiling, crawling, the first walking steps, grasping, and the first spoken word are known as developmental milestones. important developmental milestones that are commonly studied include gross motor, fine motor, language, and social skills. children who do not achieve developmental milestones at the expected ages are said to have delayed developmental milestones. globally, 200 million children do not reach their developmental potential in their first five years. in india,. the achievement of gross motor milestones is frequently delayed, and these children exhibit divergent outcomes. a number of children with gross motor development delays achieve typical milestones at later ages. some children have permanent motor disabilities, such as cerebral palsy or others conditions, which generally become more evident as the child reaches the age of 5 years and older. the early identification of delays in achieving milestones is critical for a child 's development. the early identification of motor developmental delays aids timely referral for diagnosis, interventions, and treatment. developmental status is determined by complex interactions between internal constitutional factors and the external environmental factor. various factors that are known to be associated with delays in achieving milestones including early gestational age, twin status, nutrient intake, and low socioeconomic status. the world health organization (who) has developed normal age ranges for the achievements of motor milestones by healthy children. the who multicentre growth reference study (mgrs) generated new growth curves for assessing growth and the achievement windows of six gross motor development milestones by infants and young children around the world. this study aimed to assess the delay in gross motor developmental achievements and associated factors among 418-month - old children in rural india. a community - based cross - sectional study was conducted in the intensive field practice area of the comprehensive rural health services project (crhsp) of ballabgarh, northern india. the crhsp, ballabgarh caters to 28 villages, one secondary - level hospital in ballabgarh, two primary health centers and 12 subcenters in the ballabgarh block of the faridabad district of haryana in north india. this project was established as collaboration between the all indian institute of medical sciences, new delhi, and the state government of haryana in 1961 to develop a model for rural healthcare practice in india. the crhsp, ballabgarh caters to a population of nearly 90,000 as of december 31, 2011. in addition to community - based research, this project provides preventive, promotive, and curative services to its population. the required sample size was calculated to be 213 based on a prevalence of developmental delay of 14%, with an alpha error of 0.05, an absolute precision of 5%, and a nonresponse rate of 10%. very sick children and those who could not be examined were excluded from the study. pretesting was performed with 30 children to examine the feasibility of the survey and the questionnaire, and appropriate modifications were made. three visits were made to each eligible child in a village / cluster before any child was excluded from the study. the mother / caregivers were given an information sheet and explanations of the study, its objectives and procedure and the rights of the participants. if the mother / caregiver agreed to participate in the study after reviewing the information sheet, written consent was collected, and the mother / caregiver was interviewed. after obtaining preliminary information about the child, each child was subjected to anthropometric measurements. the child 's weight was measured via tared weighing with minimum clothing over the child 's body and without shoes using an omron weight scale hn 283., the child was laid on an infantometer with its head positioned firmly against the fixed hardboard and eyes looking forward. the knees were extended via the application of firm pressure, and the feet were flexed at right angles to the lower legs on the board. each anthropometric index was measured twice, and the average of the two readings was calculated. the highest milestone achieved was assessed by observing the child and verbally confirming with the mother / caregiver that the child could complete a particular activity. six who gross motor milestones described in the who motor development study were determined. for this analysis, the world health organization (who) anthro software (version 3.2.2, january 2011) was used to compute the children 's weight - for - age, weight - for - length, and length - for - age z scores as per the who standards. further analyses of the data were completed using spss version 17.0 (spss inc., the median age at the highest observed milestone was calculated and compared with the who reported medians for each of the six milestones. for the logistic regressions of each gross motor milestone, the number of children who were within the windows of achievement the potential explanatory variables that were studied included the nutritional status of the child, sex, infant and child feeding practices, immunization, and others factors. step - wise selection was employed to obtain the variables that were significant at the level of p < 0.05 and were entered into the final multivariate logistic regression models of the six motor milestones. ethical clearance was obtained from the ethics committee of the all india institute of medical sciences, new delhi, india. appropriate management and referrals to more advanced health facilities were provided for all children who required referrals. a total of 221 children ranging in age from 4 to 18 months were studied. the mean age of the children was 11.5 months (standard deviation : 4.1). more than half of the mothers and fathers reported having a middle school education or higher. the details of the children, i.e., birth order position of 3 or lower, childbirth practices, infant feeding practices and immunization statuses, are presented in table 1. the prevalence of underweight, stunting, and wasting was 20.4%, 42.1%, and 10.4%, respectively [table 1 ]. characteristics of the study subjects (children aged 4 - 18-months) and their parents (n=221) the median motor development age exhibited a 0.12.1-month delay compared with the median age reported by the who [table 2 ]. the prevalences of the achievements of each of the gross motor milestones ranged from 91.6% to 98.4% table 3. univariate analyses revealed that complete immunization for age, normal nutritional status, greater paternal education level, timely weaning, and low birth order position were significantly associated with achievement of gross motor milestones. the birth order positions of one and two were found to be significantly associated with the timely achievement of gross motor milestones in the multivariate logistic regression [odds ratio (confidence interval), p value : walking with assistance 2.1 (0.254.1), 0.02 ; standing alone 6.5 (0.53.2), < 0.001 ; and walking alone 5.2 (0.42.9), 0.01 ; table 4 ]. gender, nutritional status, infant and child feeding practices, immunization status, institutional delivery and maternal and paternal education levels were not significantly associated with the achievements of any of the motor milestones in the multivariate analyses [data not shown ]. comparison of median ages (months) of motor milestone achievement between the world health organization, the published literature, and the present study proportions of children who achieved age - appropriate motor milestones per the world health organization windows of achievement in the present study (n=221) odds ratios (95% confidence interval) from univariate logistic regressions of the factors associated with achieving motor milestones during the world health organization windows of achievement in the present study to the best of our knowledge, this study is the first of its type to assess delays in the achievement of six gross motor milestones using the who mgrs windows of achievement scale for these milestones among children in a rural community in india. in addition, there is a lack of published literature about the rural community of india in the delayed achievement of six gross motor milestones based on the who mgrs windows of achievement scale. in this study, developmental delays in the achievement of gross motor milestones were present in 6.3% of children. similarly, other population - based studies have reported that the prevalence of any developmental delay ranges from 3.5% to 10%. according to the who, approximately 5% of children below the age of 14 years exhibit a developmental delay or disability. in india, the prevalence of developmental delay among those under the age of 2 years is approximately, 2%. the higher prevalence of delayed developmental milestones observed in this study might be due to the small sample size or differences in the study instrument used. the prevalence of delayed milestones was lower than that reported in another clinic - based study conducted in bhopal, india, among children below the age of two years. the authors of this study found that the prevalence of any developmental delay was 9.5% using the trivandrum developmental screening chart. in addition to motor milestones, this study tool also assesses the achievement of language and social milestones. the prevalence of delayed milestones was nearly 50% in a study conducted using the ages and stages questionnaire followed by the developmental assessment scale for indian infants in a tertiary care setting in north india. the median age of motor development exhibited a 0.12.1-month delay relative to the median age reported by the who. the primary reason for this delay might be that the present study was a cross - sectional study, whereas the mgrs was a longitudinal study. similar delays have also been found in other cross - sectional studies from nepal, indonesia, vietnam, and zanzibar. birth order was the only variable that was found to be significantly associated with the achievement of gross motor milestones in a study conducted in a tertiary care setting in north india. the major limitation of this study is that we were not able to assess all of the developmental milestones for logistic reasons. the history of development was assessed based on the report from the informant ; therefore, some degree of recall bias can not be ruled out. moreover, the past histories of the children, for example, feeding history, breastfeeding, and weaning, could not be confirmed based on documentation. although low, the prevalence of delayed gross motor milestone achievements supports the need for a health facility - based awareness campaign to promote the timely identification of and interventions for children with delayed milestones. the apparently healthy children in the rural area of haryana achieved the gross motor milestones with some delays as compared to the who windows of achievement. a further evaluation of the problem of the delay in achieving milestones needs to be performed and should utilize a mixed methods approach to facilitate understanding of issues related to both identification and management. | background : nearly 14% of children worldwide do not reach their developmental potential in early childhood. the early identification of delays in achieving milestones is critical. the world health organization (who) has developed normal age ranges for the achievement of motor milestones by healthy children. this study aimed to assess the gross motor developmental achievements and associated factors among children in rural india.materials and methods : a cross - sectional study was conducted with rural children in north india. a pretested questionnaire was used to collect the data. the median age at the time of the highest observed milestone was calculated and compared with the who windows of achievement.results:overall, 221 children aged 418 months were included in the study. the median age of motor development exhibited a 0.12.1-month delay compared to the who median age of motor milestone achievement. the prevalence of the gross motor milestone achievements for each of the six milestones ranged from 91.6% to 98.4%. developmental delay was observed in 6.3% of the children. after adjusting for different variables, children with birth order of second or more were found to be significantly associated with the timely achievement of gross motor milestones.conclusion:the apparently healthy children of the rural area of haryana achieved gross motor milestones with some delay with respect to the who windows of achievement. although the median value of this delay was low, awareness campaigns should be implemented to promote timely identification of children with development delays. |
the prevention of diet - related diseases, such as coronary heart disease and obesity, is a key public health priority within europe and internationally. nutrition labels are a potentially valuable tool in assisting consumers to make informed decisions about their food choice. voluntary use of front - of - pack (fop) labelling is relatively new and seeks to provide consumers with simplified at - a - glance information to supplement that provided on back of pack (bop) to help them make healthier choices. there are many types of fop labels currently used in the eu and internationally that vary both in format and the type of information that they convey. these range from logos, such as the dutch choices logo, which provide summary information on the overall healthiness of a food, through to more detailed information on the amounts of individual nutrients contained in a specified portion size, which are supplemented with information such as percentage of guideline daily amount (gda) and/or traffic light (tl) colour coding (red, amber and green). many food manufacturers and retailers within europe have taken up these various schemes and multiple schemes now co - exist within many countries and also within individual food retail chains. an audit assessing the penetration of nutrition information on food labels in five product categories in the eu-27 plus turkey found on average, 85% of the products contained bop nutrition information and 48% contained fop nutrition information, with the lowest penetration in turkey (24%) and the highest in the uk (82%). discussions on a proposal for a new eu food information to consumers regulation are drawing to a close and while the nutrition declaration will become mandatory (bop), provisions for fop nutrition remain voluntary. member states will also have the ability to recommend additional forms of expression for fop labels subject to meeting certain criteria. there are a range of views and little consensus on the form that fop labels should take. there is an extensive and growing body of research on nutrition labels that has been recently reviewed. these reviews show that while there has been much work investigating consumer use and understanding of different types of nutrition labels and logos, most of this has focused on reported use and understanding with relatively little work examining how consumers actually use labels and process the information they contain in real - life contexts. further, while a number of studies have examined consumer understanding of individual fop labels, these have all examined comprehension of a single label format and not when they are used in combination, for instance, to compare two products. none of the recent reviews identifies any research on the impact of multiple label formats on comprehension or use, but campos. do note that barriers to consumer understanding need to be identified and addressed to promote appropriate label use. given the number of different fop schemes currently in use across europe, there is a need to understand the consequences of multiple label formats in the market place on consumer use and comprehension and this is the policy relevant question addressed here. the data presented came from a study commissioned by the uk food standards agency in 2008. the study was conducted by tns - bmrb in association with the food, consumer behaviour and health research centre, university of surrey. the study addressed two initial research questions : how do consumers use fop labels in real - life contexts (stage 1) and how well do individual fop schemes and their elements enable consumers to correctly interpret levels of key nutrients (stage 2). a third stage was included to examine whether the co - existence of different fop formats affects accurate interpretation by consumers, following a strong suggestion from early findings that the co - existence of different fop schemes in the market place caused problems for consumers. the qualitative work in stage 3 of the overall study was designed to explore whether there are difficulties for shoppers when using the different fop label formats currently in the uk market place (gdas, tl colour coding and combinations of these) in making product comparisons and, if so, to uncover the sources of difficulty and their effects. fifty in - depth interviews were conducted in which participants were presented with pairs of different fop labels (for either breakfast cereals or ready meals) and asked to make a healthier choice. this task was used, as earlier development work had identified product comparison as a common use of fop. a structured topic guide was used that focused on three areas that had emerged as salient : how people make comparisons when deciding which of two products is healthier, the decision - making process that they go through when making comparisons and what is important to people when making comparisons and decisions. participants were asked using the information on these two labels, which of these two products do you think is healthier? for a pair of similar products labelled with different fop labels. talk aloud technique used by higginson., enabling any difficulties to emerge and to be used as the starting point to discuss their nature and source. four different fop labels were used to represent those used by the main supermarkets in the uk (figure 1) : a % gda only label ; a tl only label ; a label containing % gda, tl and text ; and a label with % gda and non - tl colour, where colour is a design feature, rather than indicative of nutrient levels (as in tl schemes). this label was used to explore problems with the interpretation of colour, when it was not part of a tl format, as this was identified as a source of confusion in stage 1 of the study. as many consumers shop in a variety of supermarkets purchasing a mixture of brand and supermarket own - brand products, they encounter the same or similar product categories with differing fop formats. therefore, shopping decisions may involve comparisons both across and within product categories using different fop formats. the task presented to participants thus corresponds to a real - life purchasing decision when comparing two products. however, because packaging, claims, other labelling information and endorsements can influence purchasing decisions, the task involved comparison of label pairs in a test situation with no actual products presented. all other elements of packaging and presentation were absent as shown in figure 1, as the goal was to isolate and explore in more depth the difficulties described by consumers in the earlier real - life research of stage 1. participants were presented with four pairs from a series of 24 label pairs and asked to decide which label represented the healthier product. the label pairs included some where there was no obvious answer as to which was the healthier product (for instance where some nutrients were higher on one label, but others higher on the second) to explore participants thought processes when the task was more complex. figure 1the array of labels used with the topic guide the array of labels used with the topic guide participants were purposively recruited to ensure a spread across geographical areas (brighton, london, nottingham and swansea), users of the four main uk retailers (sainsburys, morrisons, tesco and asda), users and non - users of fop labels, gender, age, household type, ethnicity and socio - economic status. both users and non - users were included to allow examination of whether there is less confusion among label users and whether there were any specific issues that affected comprehension among non - users. while other studies have shown some degree of over - reporting of label use, this was explored further in the interview to ensure accuracy. there is no objective measure of familiarity, but again this was further examined in the interview. participants were recruited using free find methods, with target quotas to find individuals who met the inclusion criteria. free find recruitment entails quotas being set for recruiters who then find eligible participants in a particular area. this produces a purposive sample that reflects the diversity of the relevant population as is usual practice in qualitative research. interviews were conducted in participants homes january february 2009 and lasted 1 h. participants were given 25 for taking part. the data were synthesized into a framework based on a priori and emerging themes ; the matrix was used to search for themes, similarities and differences which were then mapped out for further examination. this structured approach allows identification and mapping of key themes and issues, as they occurred across individual accounts, and the development of typologies and explanations. table 1 shows the characteristics of the sample. table 1number of subjects by characteristics of the sample (26 label users, 24 non - label users)ngeographic location brighton12 london12 nottingham12 swansea14main retailer used sainsburys12 morrisons10 tesco16 asda12age group (years) 163014 315014 516413 658household type with children < 16 years at home19 without children < 16 years at home15 living as a couple9 not living as a couple7ethnicity white30 asian9 black11socio - economic status ab7 c1/c224 de19a : socio - economic status is a household - based proxy measure of social class based on the normal occupation of the chief income earner in the household categorized as ab : professional, managerial and technical ; c1 : skilled non - manual ; c2 : skilled manual ; d : partly skilled and unskilled ; e : dependent on state and casual workers number of subjects by characteristics of the sample (26 label users, 24 non - label users) a : socio - economic status is a household - based proxy measure of social class based on the normal occupation of the chief income earner in the household categorized as ab : professional, managerial and technical ; c1 : skilled non - manual ; c2 : skilled manual ; d : partly skilled and unskilled ; e : dependent on state and casual workers the qualitative interviews revealed the nature of the decision - making process when judging the relative healthiness of products using different fop labels, the nature of problems encountered and whether participants were likely to complete similar tasks in a real - life situation. some information is common to all fop labels, (i.e. weight of nutrient in grams) and some label types share common elements with other label types (i.e. text, tl or % gda). the label containing text, tl and % gda did not present problems for participants in making comparisons with either tl or % gda labels, because they were able to use the common element to make the comparison. participants were also able to make decisions on which product was healthier in some pairs simply by glancing at the fop labels and, if unfamiliar with the scheme, they tended to resort to gram weights to make comparisons although some were unable to see this consistency. when asked how they were making the decision they were able to articulate clearly that the nutrient levels (weight in grams) were higher in one of the fop labels, home in on one nutrient, such as salt, and then use the gram weights of this to make a decision. when participants were unfamiliar with any type of fop scheme they were unsure where to start. participants experienced difficulties, however, in comparing some pairs of fop label types, especially where there was no common element beyond the gram amount. for instance, when attempting to compare a label with tl colour but no % gda (label 2) and with a label with % gda but no tl colour (label 3) : it 's like speaking different languages. i 'm trying to compare french with german with english why do n't we just have everything in english, and then there 's a direct comparison ? but where we 've got different details, it 's pretty confusing. i 'm trying to compare french with german with english why do n't we just have everything in english, and then there 's a direct comparison ? but where we 've got different details, it 's pretty confusing. further difficulties were encountered with complex comparisons where some nutrients were high on one label and other nutrients high on the other label. in these situations, where a correct answer was not immediately obvious many participants had an internal dialogue about the relative healthiness of individual nutrients, e.g. whether it was better to have higher sugar and fat or higher salt and saturates. some participants considered one or two particular nutrients that they needed to keep low, with the choice depending on their personal circumstances and health status. however, the most usual way of deciding was to choose one or two nutrients as proxies for healthiness and make a decision on those alone : i 'm looking at the fat and the salt. i would n't be looking into saturates because i do n't think it 's all that important i would n't be looking into saturates because i do n't think it 's all that important participants indicated that while they were prepared to persevere with this relatively demanding decision - making process in an interview situation, while shopping they were unlikely to have done so and would have given up much sooner. it was not unusual for participants to comment that they would have become frustrated by the effort required. to put it literally that 's what i 'd do.i would get annoyed because it should be easy. to put it literally some participants said that they would have used other factors to make a decision, including attractiveness of the packaging or other labelling information, packaging health claims, nutrition claims, brand information or product familiarity. some participants saw an advantage in being able to compare gda percentages in % gda labels and text, tl and % gda labels, because the common % gda element allowed them to understand whether the differences in the levels of nutrients were big enough to matter or not. however, it was more usual that participants did not understand % gdas and looked to other elements of the labels to make comparisons. one common misunderstanding was that the figure of % gda represented the proportion contained in the whole product rather than the proportion of the gda contained in a serving. in the example below, the participant believed that half of the meal represented by the label consisted of salt : because 45% [salt on % gda label ] that 's, like, nearly half of the whole meal ! because 45% [salt on % gda label ] that 's, like, nearly half of the whole meal ! further complications arose because shoppers did not understand how information in gram weight could be shown to be 0% of gda : i ca n't grasp that one. different uses of colour on fop schemes also caused difficulties ; in particular, the non - tl colours on % gda labels caused confusion when making comparisons with labels using tl colour. some participants thought that the colours on % gda labels (both the pastel colours used in nutrient - specific schemes and monochrome colours) provided an indication of the level of nutrients present in a product, in the same way that tl colours provide this information. these were usually participants who were familiar with, and often understood, the tl colour scheme. i 'm confused with this one, as i said, red is for danger, but that 's a cooler colour [non - tl coloured % gda label ], but yet it 's got 68% in here [tl label ] there is only 56%. i 'm confused with this one, as i said, red is for danger, but that 's a cooler colour [non - tl coloured % gda label ], but yet it 's got 68% in here [tl label ] there is only 56%. some participants were uncomfortable working with any numerical information (gram weights or % gda) and relied on tl colours and text (high, medium and low) elements when they were present. of all the label elements, text alone caused participants no difficulties in understanding : i do like the high and medium [text labels ] because for people like me that want to go on a quick easy shop, that you want it basically told to you, rather than you trying to work something else out. i do like the high and medium [text labels ] because for people like me that want to go on a quick easy shop, that you want it basically told to you, rather than you trying to work something else out. those with more confidence in their abilities to complete the tasks found the comparisons less daunting, although ultimately no less difficult. familiarity with particular fop label schemes did affect participants confidence and willingness to engage with the various fop labels. pause while they stopped trying to make the comparison and tried to work out what the differences between the labels were. this pause is of interest because it is the point where participants reported that they would be likely to give up trying in a real - life situation, abandoning the comparison due to frustration and falling back on other factors (e.g. other information on packaging). at this point, some people questioned why the labels were different, and why the food industry did not use a consistent labelling scheme. i think it would be a lot easier if they were all just the same, cos i do n't know why they 'd need to be different and you would maybe think why has that got that on, and that one got that on why are they them colours, and they are their colours if they all had [label with % gda and tl ], that would be really straightforward, you know what the colours are, you got the grams there if you want them, and the percentages, you can compare between. i think it would be a lot easier if they were all just the same, cos i do n't know why they 'd need to be different and you would maybe think why has that got that on, and that one got that on why are they them colours, and they are their colours if they all had [label with % gda and tl ], that would be really straightforward, you know what the colours are, you got the grams there if you want them, and the percentages, you can compare between. some information is common to all fop labels, (i.e. weight of nutrient in grams) and some label types share common elements with other label types (i.e. text, tl or % gda). the label containing text, tl and % gda did not present problems for participants in making comparisons with either tl or % gda labels, because they were able to use the common element to make the comparison. participants were also able to make decisions on which product was healthier in some pairs simply by glancing at the fop labels and, if unfamiliar with the scheme, they tended to resort to gram weights to make comparisons although some were unable to see this consistency. when asked how they were making the decision they were able to articulate clearly that the nutrient levels (weight in grams) were higher in one of the fop labels, home in on one nutrient, such as salt, and then use the gram weights of this to make a decision. when participants were unfamiliar with any type of fop scheme they were unsure where to start. participants experienced difficulties, however, in comparing some pairs of fop label types, especially where there was no common element beyond the gram amount. for instance, when attempting to compare a label with tl colour but no % gda (label 2) and with a label with % gda but no tl colour (label 3) : it 's like speaking different languages. i 'm trying to compare french with german with english why do n't we just have everything in english, and then there 's a direct comparison ? but where we 've got different details, it 's pretty confusing. i 'm trying to compare french with german with english why do n't we just have everything in english, and then there 's a direct comparison ? but where we 've got different details, it 's pretty confusing. further difficulties were encountered with complex comparisons where some nutrients were high on one label and other nutrients high on the other label. in these situations, where a correct answer was not immediately obvious many participants had an internal dialogue about the relative healthiness of individual nutrients, e.g. whether it was better to have higher sugar and fat or higher salt and saturates. some participants considered one or two particular nutrients that they needed to keep low, with the choice depending on their personal circumstances and health status. however, the most usual way of deciding was to choose one or two nutrients as proxies for healthiness and make a decision on those alone : i 'm looking at the fat and the salt. i would n't be looking into saturates because i do n't think it 's all that important i would n't be looking into saturates because i do n't think it 's all that important participants indicated that while they were prepared to persevere with this relatively demanding decision - making process in an interview situation, while shopping they were unlikely to have done so and would have given up much sooner. it was not unusual for participants to comment that they would have become frustrated by the effort required. to put it literally, it gives me a headache, and i just put it down. that 's what i 'd do.i would get annoyed because it should be easy. to put it literally some participants said that they would have used other factors to make a decision, including attractiveness of the packaging or other labelling information, packaging health claims, nutrition claims, brand information or product familiarity. some participants saw an advantage in being able to compare gda percentages in % gda labels and text, tl and % gda labels, because the common % gda element allowed them to understand whether the differences in the levels of nutrients were big enough to matter or not. however, it was more usual that participants did not understand % gdas and looked to other elements of the labels to make comparisons. one common misunderstanding was that the figure of % gda represented the proportion contained in the whole product rather than the proportion of the gda contained in a serving. in the example below, the participant believed that half of the meal represented by the label consisted of salt : because 45% [salt on % gda label ] that 's, like, nearly half of the whole meal ! because 45% [salt on % gda label ] that 's, like, nearly half of the whole meal ! further complications arose because shoppers did not understand how information in gram weight could be shown to be 0% of gda : i ca n't grasp that one. different uses of colour on fop schemes also caused difficulties ; in particular, the non - tl colours on % gda labels caused confusion when making comparisons with labels using tl colour. some participants thought that the colours on % gda labels (both the pastel colours used in nutrient - specific schemes and monochrome colours) provided an indication of the level of nutrients present in a product, in the same way that tl colours provide this information. these were usually participants who were familiar with, and often understood, the tl colour scheme. i 'm confused with this one, as i said, red is for danger, but that 's a cooler colour [non - tl coloured % gda label ], but yet it 's got 68% in here [tl label ] there is only 56%. i 'm confused with this one, as i said, red is for danger, but that 's a cooler colour [non - tl coloured % gda label ], but yet it 's got 68% in here [tl label ] there is only 56%. some participants were uncomfortable working with any numerical information (gram weights or % gda) and relied on tl colours and text (high, medium and low) elements when they were present. of all the label elements, text alone caused participants no difficulties in understanding : i do like the high and medium [text labels ] because for people like me that want to go on a quick easy shop, that you want it basically told to you, rather than you trying to work something else out. i do like the high and medium [text labels ] because for people like me that want to go on a quick easy shop, that you want it basically told to you, rather than you trying to work something else out. those with more confidence in their abilities to complete the tasks found the comparisons less daunting, although ultimately no less difficult. familiarity with particular fop label schemes did affect participants confidence and willingness to engage with the various fop labels. pause while they stopped trying to make the comparison and tried to work out what the differences between the labels were. this pause is of interest because it is the point where participants reported that they would be likely to give up trying in a real - life situation, abandoning the comparison due to frustration and falling back on other factors (e.g. other information on packaging). at this point, some people questioned why the labels were different, and why the food industry did not use a consistent labelling scheme. i think it would be a lot easier if they were all just the same, cos i do n't know why they 'd need to be different and you would maybe think why has that got that on, and that one got that on why are they them colours, and they are their colours if they all had [label with % gda and tl ], that would be really straightforward, you know what the colours are, you got the grams there if you want them, and the percentages, you can compare between. i think it would be a lot easier if they were all just the same, cos i do n't know why they 'd need to be different and you would maybe think why has that got that on, and that one got that on why are they them colours, and they are their colours if they all had [label with % gda and tl ], that would be really straightforward, you know what the colours are, you got the grams there if you want them, and the percentages, you can compare between. these data reveal that making comparisons using multiple fop label formats poses problems for consumers, particularly, when there is no interpretive element in common ; participants coped better when there was a common element. the kinds of problems that participants encountered correspond to the problems uncovered by mitchell. in their proposed model of consumer confusion. firstly, consistency was wrongly assumed across different label formats and notably non - tl colour coding was assumed to signpost the level of a nutrient by those already familiar with the tl scheme. this suggests that people are transferring meaning from a scheme they are familiar with to other fop schemes, and sometimes inappropriately. the second source of confusion arose when consistent elements were obscured (gram weight of nutrient) by other differences in label format. these problems meant that considerable perseverance was required by shoppers to make comparisons of product healthiness using different fop labels and beyond the effort most are likely to commit in real life. the label containing text, % gda and tl colour overcame many of the problems that participants encountered by providing consistency and also allowing participants to use those elements that they were already familiar with. there was no evidence that the inclusion of all elements caused participants problems in identifying the relevant information. as noted earlier, there are no other equivalent studies that have included a comparative assessment of the understanding of different fop label formats to contextualize these findings, although participants in a recent citizens forum on fop labelling conducted in the uk wanted a standardized scheme, feeling it would be more user - friendly and hence easier and more convenient to use. similarly, kelly. found that australian respondents reported finding multiple fop label formats confusing and overwhelmingly wanted a single scheme and feunekes. also report that in their study conducted in germany, uk, italy and the netherlands participants expect one labelling format across food products. a limitation is that this study was conducted in a test situation and not during real - life shopping in a retail environment, but the task used corresponds to a common real - life shopping decision of product comparison. the exclusion of other product information, such as packaging and brand information, means that other factors that influence purchasing decisions were removed and that difficulties encountered by participants can be attributed the challenge of making a product comparison using different fop formats. the data support the conclusion that making product comparisons using different label formats presents consumers with considerable challenges and also takes them longer. feunekes. examined comprehension of single label types and found that the time taken to make an evaluation increases with the complexity of the fop label. there are many other factors that influence label use and food choice, and price is particularly important to those on a low income. however, as campos. note the relationship between use of nutrition labels and healthier diets is probably bi - directional with those whose diets are already healthier diets being more likely to use them, but also that appropriate labelling can promote healthier eating. their review also shows that to maximize use and comprehension, particularly, among certain population groups (those who are older, of lower socio - economic status and from some ethnic minorities), it is vital to ensure that information on nutrition labels is both accessible and understandable. while these qualitative data were not analysed by socio - demographic difference, these groups were all found to have lower levels of comprehension in the main stage survey indicating the importance of minimizing any possible sources of misunderstanding and confusion such as that caused by multiple formats. the findings of this study thus indicate that, if fop labels are to achieve their potential in both informing food choice and encouraging healthy eating, they should be standardized to one format. the study was funded exclusively by the uk food standards agency, contract pau 217. key pointsthis is the first study that has comprehensively examined how people actually use different fop labels to make comparisons and judgements about the healthiness of food products using different label formats.lack of standardization in fop labels, for instance, in the use of colour across schemes, causes confusion and can lead to incorrect inferences being made.making comparisons across label formats was frustrating and time - consuming deterring usage in a real - life situation.label usage and impact in promoting healthy food choices may be enhanced by achieving greater consistency in the fop format. this is the first study that has comprehensively examined how people actually use different fop labels to make comparisons and judgements about the healthiness of food products using different label formats.lack of standardization in fop labels, for instance, in the use of colour across schemes, causes confusion and can lead to incorrect inferences being made.making comparisons across label formats was frustrating and time - consuming deterring usage in a real - life situation.label usage and impact in promoting healthy food choices may be enhanced by achieving greater consistency in the fop format. this is the first study that has comprehensively examined how people actually use different fop labels to make comparisons and judgements about the healthiness of food products using different label formats. lack of standardization in fop labels, for instance, in the use of colour across schemes, causes confusion and can lead to incorrect inferences being made. making comparisons across label formats was frustrating and time - consuming deterring usage in a real - life situation. label usage and impact in promoting healthy food choices may be enhanced by achieving greater consistency in the fop format. | background : nutrition labels are a potentially valuable tool to assist consumers in making healthy food choices. front - of - pack labels are a relatively new format and are now widely used across many european countries, but it is unclear which of the many formats in use are best understood by consumers. it is also unclear whether the existence of multiple formats impedes understanding and use. this article addresses this question with findings from a study commissioned by the uk food standards agency to provide evidence to inform policy decisions in this area. methods : in - depth qualitative interviews were used to explore consumers decision - making processes when using two different front - of - pack label formats to judge the relative healthiness of a pair of products. participants were presented with product pairs differently labelled and a series of structured prompts were used to access their internal dialogues and to identify any difficulties encountered. results : the interviews revealed that making product comparisons using different label formats was challenging for participants and particularly for those product pairs where there was not an obvious answer. when the label formats on the product pairs lacked a common element, such as text, this also caused difficulties and misinterpretation. the comparisons also took time and effort that would be a deterrent in real - life situations. conclusions : these findings indicate that the existence of multiple front - of - pack label formats in the marketplace may impede consumer comprehension and discourage use. they suggest that a single format may encourage consumers to use front - of - pack labels in making healthy food choices. |
the disease is caused by 3 serotypes of poliovirus (poliovirus types 1, 2, and 3), belonging to the genera enterovirus and picornaviridae family. recently, the virus has been reclassified as enterovirus c spp. in the enterovirus genus. the virus is transmitted through contaminated food and water and multiplies in the intestine, from where it can invade the nervous system. many infected individuals may be asymptomatic but do excrete the virus in their faeces, hence transmitting infection to others. in about 1% of affected individuals, the virus enters the central nervous system and replicates in anterior horn cells, that is, motor neurons of the spinal cord. the typical neurological manifestation of paralytic poliomyelitis is acute flaccid paralysis (afp) of limbs, predominantly lower limbs, usually asymmetric and with intact sensation. in rare cases, viral destruction of bulbar cells results in respiratory paralysis and the world health assembly in 1988 resolved to eradicate poliomyelitis from the world and marked the launch of the global polio eradication initiative (gpei). the gpei is a partnership led by national governments and spearheaded by the world health organization (who), rotary international, the us centers for disease control and prevention (cdc), and the united nations children 's fund (unicef). since then, there has been a decline in global polio incidence, from an estimated 350,000 cases in 1988 to under 3,500 in the year 2000. in 2012, a total of 223 polio cases were reported from five countries : afghanistan, chad, niger, nigeria, and pakistan. out of these cases, 97% (217 out of the 223) were reported from the three remaining endemic countries : afghanistan, nigeria, and pakistan. polio eradication strategies rest on two main activities : immunization coverage and surveillance of acute flaccid paralysis (afp) cases. oral polio vaccine (opv) has been the choice for routine immunization in over 120 countries that have eliminated poliomyelitis. in malaysia, opv was licensed and introduced into the childhood immunization program in 1972. since then, the incidence of poliomyelitis declined with no reported cases between 1986 and 1991. in 1992, malaysia experienced a small outbreak with 3 cases of paralytic poliomyelitis caused by importation of wild poliovirus that originated from the indian subcontinent. various studies from developing countries suggest that, after 3 doses of opv, the mean proportion of infants with detectable serum neutralizing antibodies level was only 73% (3699%) for type 1, 90% (71100%) for type 2, and 70% (4099%) for type 3 poliovirus. this suboptimal seroconversion was related to many factors including interference with other enteroviruses, inhibition of type 1 and type 3 by type 2 in the opv, diarrheal illnesses, and presence of maternal antibodies. although opv is a very safe vaccine, on rare occasions, vaccine - associated paralytic poliomyelitis (vapp) may occur following ingestion of opv. the mechanism of vapp is believed to be a mutation or reversion of the vaccine virus to a more neurotropic form. vaccine - associated paralytic poliomyelitis is defined by who as paralytic poliomyelitis occurring in a vaccinated individual between the 7th and 30th day after receiving a dose or in a close contact of the vaccinated recipient between the 7th and 60th day after a dose was taken. sabin viruses can replicate in populations with low opv coverage, acquire the neurovirulence and transmissibility characteristics of wpv, and cause circulating vaccine - derived poliovirus (cvdpv) cases and outbreaks [11, 12 ]. the ways in which cvdpvs are generated and the conditions that promote them are still not clear. it is believed that these sequences are acquired by reassortment inside the intestinal tract of an infected person with the opv virus and the enterovirus ; the progression of the infection remains unknown. in september 2003, the who informal consultation on identification and management of vaccine - derived polioviruses concluded that, after eradication of wild poliovirus, continued use of opv would compromise the goal of a polio - free world. ipv could be useful preeradication for prevention of paralysis due to wild or vaccine - derived polioviruses. most industrialized countries had already decided that, in their specific settings (i.e., geographical distance from endemic countries, very high immunization coverage, temperate climates, and high sanitation and hygiene), the risks of cvdpvs and of vapp due to continued use of opv are greater than those due to wild poliovirus importations. consequently, some of these countries have adopted routine vaccination schedules that rely either exclusively on inactivated polio vaccine (ipv) or on a sequential ipv / opv schedule [14, 15 ]. in october 2008, the ministry of health, malaysia, approved a vaccine policy change from opv to ipv. the switch to ipv in malaysia into the expanded programme of immunization was rolled out in 8 states : selangor, federal territory, sabah (including labuan) sarawak, perak, pahang, kelantan, and terengganu. in january 2010, all states used ipv in the epi. the institute for medical research (imr), malaysia, was designated the national poliovirus laboratory (npl) in 1992. since then, our polio laboratory has collaborated actively with the disease control division, ministry of health (moh), and who towards achieving polio eradication. the polio laboratory plays an important role in surveillance with the virological investigation of afp cases. the objectives of this paper are to review isolation of sabin polioviruses and non - polioviruses before and after switch of opv to ipv in the childhood immunization schedule and share experiences in the performance of the national reference laboratory. since 1992, when the virology unit, imr, was designated as the npl, till december 2012, the npl received 3360 stool specimens from 1930 reported afp cases sent from hospitals throughout malaysia. another 619 specimens, comprised of throat swabs, and cerebrospinal fluid were also received from these cases. the specimens were accompanied by an afp notification form with details of patient personal and clinical history. till 2008, the methods used for virus isolation and microneutralization for identification of positive isolates were as described in who polio laboratory manual 2004 and, from 2008 onwards, the supplemental manual of 2006 for the new algorithm technique was used for poliovirus isolation. all stool specimens were processed with chloroform before inoculation into rd and l20b cell lines from our laboratory stock held in liquid nitrogen at low passage. inoculated cell cultures were examined daily for cytopathological effect (cpe) and confirmed by microneutralization assay using standard who antisera. poliovirus isolates were sent to the victorian infectious disease reference laboratory (vidrl) in melbourne, australia, for further identification and intratypic differentiation (it d), till august 2010. between 2008 and 2010, following a new who standard algorithm, all positive cultures in l20b were sent to the vidrl in australia for it d and microneutralization assay was not done. the method for it d of poliovirus isolates by rrt - pcr was as recommended by who using kit developed by usa centers for disease control and prevention. the principles of the rrt - pcr technique involved the conversion of viral rna (vrna) to complementary dna (cdna) using reverse transcriptase. the cdna was amplified in a pcr reaction using taq polymerase and simultaneously the pcr products were detected by specific taqman probes. both the cdna synthesis and the pcr reaction used multiple sets of oligonucleotide 6 primers (pv serotype 1, pv serotype 2, and pv serotype 3, pan - poliovirus, pan - enterovirus, and sabin multiplex) and probes. this combination of primers and probes resulted in serotype identification and intratypic differentiation of poliovirus isolates. specimens that were diagnosed as sabin - like were further screened for vdpv using a vdpv diagnostic rrt - pcr kit. the primers and probes used consisted of sabin 1 vdpv, sabin 2 vdpv, and sabin 3 vdpv. specimens that were diagnosed as group - specific for enterovirus (pan - enterovirus - positive) but negative for polioviruses were further sequenced for non - polio enterovirus differentiation. this activity was monitored by the surveillance unit, disease control division of the ministry of health, by communication with clinicians attending to the afp cases and from monthly notification data obtained from state health departments. clinical assessment of all reported afp cases was also reviewed at the expert polio review meetings. one hundred and seventy - six enteroviruses were isolated from the 3360 stool specimens investigated between 1992 and december 2012 (table 1). 55 out of 176 virus isolates were polioviruses (pv) and the remaining were non - polio enteroviruses (npev). since 2009, after the sequential introduction of ipv in the childhood immunization programme, no sabin polioviruses were isolated. out of 55 polioviruses isolated, 3 were wild type isolated in 1992, caused by importation of wild polioviruses that originated from the indian subcontinent. the wild - type polioviruses were confirmed by intratypic differentiation by centers for disease control, atlanta, usa. the remaining were vaccine - related sabin - like strains. out of these, 21 were sabin type 3 viruses, 15 were sabin type 2, 7 were a mixture of sabin 2 and sabin 3, and 9 were sabin type 1. the non - polio enteroviruses (npev) included coxsackie a viruses, coxsackie b viruses, echoviruses, and enterovirus 71. the primary goal of the afp surveillance for poliovirus eradication is to promptly detect possible areas of circulating wild poliovirus and circulating vaccine - derived poliovirus to implement immediate control measures. it remains essential that surveillance is maintained until global eradication is achieved because of the risk of wild virus importation from endemic regions. in malaysia, the last major outbreak of poliomyelitis occurred in 1977 with 121 cases including 4 deaths. the number of poliomyelitis cases decreased dramatically from 1978 following an effective national oral polio vaccine immunization programme introduced in 1972. however, three cases of poliomyelitis were reported in 1992 due to importation of wild poliovirus. since 1993, no wild poliovirus has been identified. malaysia together with other countries in the western pacific region (wpr) was certified as polio - free in 2000. since then, considering the reduced risk of wild - type poliovirus indigenous or imported, many countries including malaysia had started using ipv either completely or sequentially, driven by concerns to avoid vapp. the national committee on immunisation policy and practice (ncipp), ministry of health, reviewed the safety, immunogenicity, herd immunity, cost benefit, and practicality such as implementation issues and sustainability. the transition policy in malaysia from opv to ipv began in 2008 and by january 2010 was implemented in all states. for overall public health benefit, and based on recommendations by the who, a sequential vaccination schedule of 4 doses of ipv is given to infants at the second, third, and fifth months of age followed by a booster dose at 18 months. the challenge in the use of ipv is the vigilance of wpv importation from wild polio - endemic areas. as long as wpv transmission has not been interrupted everywhere, all polio - free countries and areas remain at risk of reimportation of wpv, particularly from the remaining polio - endemic countries. from 2003 to 2009, who has recorded 133 wpv importation events in 29 previously polio - free countries. the risk of importations with subsequent spread was the highest in countries immediately bordering endemic countries and was also higher in countries with low routine immunization coverage. in 2011, wpr experienced a wild poliovirus importation from pakistan into western china, causing a polio outbreak of 21 cases in young children and adults in xinjiang uyghur autonomous region. the outbreak in china reaffirms the continued risk for any country to be reinfected until such time as all wild poliovirus transmission is interrupted globally. in may 2012, at the world health assembly, the who 's executive board (eb) adopted a landmark resolution, declaring the completion of polio eradication a programmatic emergency for global public health, requiring the full implementation of current and new eradication strategies. this includes maintaining very high population immunity against polioviruses through routine immunization programmes, keeping vigilance for poliovirus importations, and the emergence of cvdpvs, by achieving and sustaining certification - standard surveillance for polioviruses. timely identification of polioviruses and vdpvs that can cause acute flaccid paralysis (afp) is becoming increasingly important because of reported circulating - vdpv outbreaks. in this respect, the npl in imr has continuously strengthened the laboratory afp surveillance with monthly reporting of the virological investigation of afp cases to who and to the ministry of health, afp review committee. the performance of npl is annually reviewed by who - wpro using various quality indicators such as accuracy and timeliness of results. the annual performance review assessment also includes an on - site laboratory inspection by an expert virologist and proficiency testing of panel specimens. the npl has achieved these quality indicators set by who to reach international standards required for certification of poliomyelitis eradication as evidenced by the accreditation status awarded by the who in the global polio laboratory network since 1998. when the new standard who algorithm for poliovirus isolation and identification was introduced in the wpr laboratory network in 2006, our npl introduced the algorithm for virus isolation in 2008 and real - time polymerase chain reaction method was used for poliovirus it d in 2010. this is important for prompt investigation of poliovirus variants isolated for any potential outbreak management. fifty - five polioviruses were isolated during the 20-year period from 1992 till 2012. except for the 3 cases of wild poliovirus importation from the indian subcontinent, the remaining were sabin - like strains. no vdpv has been isolated, attributed possibly to malaysia 's high immunization coverage. since the sequential introduction of ipv in the epi, no sabin polioviruses were isolated from afp cases. however, 26 sabin polioviruses were isolated from non - afp cases in the laboratory between 2009 and 2012. in 2012, thirteen sabin polioviruses were also isolated from sewage in the same year, but no vdpv was detected. with near eradication of poliomyelitis, viruses other than polioviruses have been reported to cause afp. among these viruses are echoviruses, coxsackie viruses, and enterovirus 71 which are also members of the enterovirus genus and associated with a vast range of clinical presentations, from asymptomatic, hand - foot - mouth disease to acute flaccid paralysis resembling polio. the npev isolated belonged to the group coxsackie a viruses, coxsackie b viruses, echoviruses, and enterovirus 71. the laboratory has reviewed and investigated many contributing factors but the isolation rate has been sustained below 3%, possibly attributed to good public health and hygiene. seven out of 121 npevs were enterovirus 71 and the clinical picture for these cases included acute encephalopathy and enteroviral monoplegia. ev 71 has been reported in countries of the asia pacific region causing large outbreaks of hand - foot - mouth disease (hfmd). in malaysia, the first major hfmd outbreak occurred in 1997 and in 2000, where ev71 was reported to cause neurological complications in some children which included acute flaccid paralysis and brain stem encephalitis. since then, several hfmd outbreaks have occurred in cyclic patterns, complicated by fatalities due to severe neurological involvement ; ev71 has been implicated as the major causative agent for these outbreaks. a high level of laboratory surveillance has shown that no outbreaks of wild poliovirus have occurred since 1992. no cases of vdpv were recorded. the npl continues to play an integral role in the afp surveillance and is committed towards who 's goal of polio eradication. | since 1992, surveillance for acute flaccid paralysis (afp) cases was introduced in malaysia along with the establishment of the national poliovirus laboratory at the institute for medical research. in 2008, the ministry of health, malaysia, approved a vaccine policy change from oral polio vaccine to inactivated polio vaccine (ipv). eight states started using ipv in the expanded immunization programme, followed by the remaining states in january 2010. the objective of this study was to determine the viral aetiology of afp cases below 15 years of age, before and after vaccine policy change from oral polio vaccine to inactivated polio vaccine. one hundred and seventy - nine enteroviruses were isolated from the 3394 stool specimens investigated between 1992 and december 2012. fifty - six out of 107 virus isolates were polioviruses and the remaining were non - polio enteroviruses. since 2009 after the sequential introduction of ipv in the childhood immunization programme, no sabin polioviruses were isolated from afp cases. in 2012, the laboratory afp surveillance was supplemented with environmental surveillance with sewage sampling. thirteen sabin polioviruses were also isolated from sewage in the same year, but no vaccine - derived poliovirus was detected during this period. |
worldwide, cervical cancer is the second most common malignant tumor in women and is one of the leading causes of cancer - related death in developing countries. despite the general downward trend in incidence of cervical cancer, it is still a serious public health problem worldwide, especially in developing countries ; in addition, cervical cancer is occurring in younger patients. during the past decades, many distinct advances have been made in the prevention, surgical resection, radiotherapy, and chemotherapy of cervical cancer, but the prognosis of cervical cancer patients remains poor. therefore, it is important that we better understand the molecular events involved in the invasion and metastasis of cervical cancer, and develop novel prognostic markers and therapeutic strategies for cervical cancer. the infection of human papilloma virus (hpv) is considered the major cause of cervical cancer, but viral infection alone is not sufficient for the development of cervical cancer. emerging evidence indicates that the chronic inflammatory response is significantly correlated with various types of cancers. interleukin 1(il-1), as a pleiotropic pro - inflammatory cytokine, plays an important role in tumor growth and metastasis. two of them are active, il-1 and il-1, and the third one is an antagonist, il-1ra. il-1 and il-1 are coded by different genes, but their biological functions are identical. interleukin 6 (il-6), a 23- to 30-kda pleiotropic cytokine, is produced by many types of cells like fibroblasts, immune cells, and some tumor cells. increasing evidence reveals that il-1 and il-6 play crucial roles in cancer initiation, development, and metastasis. however, to the best of our knowledge, the final role of il-1 and il-6 in cervical cancer remains unclear. in the present study, we used tissue microarray and immunohistochemistry methods to detect the expression of il-1 and il-6 in 105 case - samples of human cervical cancer tissues and their paired adjacent tissues. the purpose of this study was to investigate the association of il-1 and il-6 expression with clinicopathological parameters of cervical cancer and evaluate the prognostic value of il-1 and il-6 expression in cervical cancer patients. this study included a total of 105 formalin - fixed, paraffin - embedded cervical cancer tissues and their adjacent non - tumor tissues taken from patients at the time of resection surgery, from january 2002 to november 2013 at east hospital, tongji university. all cancers were confirmed as cervical cancer by the medical examination of hematoxylin and eosin after surgical resection. none of patients had received adjuvant chemotherapy, radiation therapy, or other anti - tumor therapies. important related clinicopathological parameters of the patients, such as age, tumor size, figo stage, lymphatic metastasis, stromal invasion, differentiation, and survival time were obtained from each patient s medical records and are shown in tables 1 and 2. the survival time was calculated from the date of surgery to the date of death, or the last known follow - up. all cervical cancer tissue samples include in this investigation were obtained with patients written informed consent. a standard immunohistochemistry method was used to inspect il-1 and il-6 expression in cervical cancer tissues and their adjacent tissues. briefly, the tumor tissues and adjacent tissues were fixed in 10% formaldehyde and embedded in paraffin ; then cut into 4-m sections. all 4-m tissue sections are dewaxed and rehydrated with xylene and graded alcohol, respectively. we washed the sections with buffer solution for 5 minutes and then added the primary antibody at 4c overnight. afterwards, we washed the sections with phosphate buffered saline (pbs) and stained them with 3, 3-diaminobenzidine (dab) for 5 minutes, then counterstained with hematoxylin. finally, all the sections were assessed under an optical microscope by two independent investigators ; any discrepancy in immunohistochemistry was resolved by consensus. the expression of il-1 and il-6 in cervical cancer and adjacent tissues was compared with paired wilcoxon test. chi - square test and fisher s exact test were used to assess the association between clinical characteristics of cervical cancer patients and il-1 and il-6 expression. the prognostic indicators of cervical cancer and il-1 and il-6 expression were determined using kaplan - meier survival analysis and log - rank test for univariate analysis ; the significant variables resulting from univariate tests were included in the cox multivariate regression analysis. a p value of 0.05). similarly, the expression of il-6 in cervical cancer tissues was significantly correlated with tumor size (=5.695, p=0.017), figo histology grade (=10.239, p=0.001), and tumor differentiation (=5.210, p=0.022). however, there was no statistical correlation found between il-6 expression and patient age, lymph node metastasis, or stromal invasion (p>0.05). therefore, these results demonstrated that higher il-1 and il-6 expression in cervical cancer tissues was positively correlated with tumor metastasis and cancer progression, suggesting that il-1 and il-6 play important roles in tumor progression. in order to further evaluate the relationship between il-1 and il-6 expression and prognosis of cervical cancer, we performed log - rank survival analysis according to il-1 and il-6 expression level and patient survival data. the survival analysis demonstrated that the cervical cancer survival rate of patients with negative il-1 or il-6 expression was significantly better than that of patients with positive expression (p0.05). similarly, the expression of il-6 in cervical cancer tissues was significantly correlated with tumor size (=5.695, p=0.017), figo histology grade (=10.239, p=0.001), and tumor differentiation (=5.210, p=0.022). however, there was no statistical correlation found between il-6 expression and patient age, lymph node metastasis, or stromal invasion (p>0.05). therefore, these results demonstrated that higher il-1 and il-6 expression in cervical cancer tissues was positively correlated with tumor metastasis and cancer progression, suggesting that il-1 and il-6 play important roles in tumor progression. in order to further evaluate the relationship between il-1 and il-6 expression and prognosis of cervical cancer, we performed log - rank survival analysis according to il-1 and il-6 expression level and patient survival data. the survival analysis demonstrated that the cervical cancer survival rate of patients with negative il-1 or il-6 expression was significantly better than that of patients with positive expression (p<0.05, figures 3, 4). furthermore, a multivariate cox regression analysis demonstrated that il-1 expression and lymph node metastasis were independent predictors of overall survival in cervical cancer patients. in the past decade, various studies have provided substantial evidence to support the role of inflammation and inflammation - related pathways in the pathogenesis of numerous human cancers, including cervical cancer [810 ]. it has been shown that the inflammatory microenvironment consists of many important components such as tumor cells, stromal cells, and immune and inflammatory cells. all of these components interact intimately and produce chemokines, growth factors, and adhesion molecules, and further promote the initiation and progression of many cancers. il-1, a key inflammatory signaling cytokine, is secreted by various types of cells such as monocytes, macrophages, neutrophils, smooth muscle cells, fibroblasts, and cervical epithelium. increasing evidence suggests that il-1 within the cancer microenvironment can induce the expression of growth factors, such as il-6, il-8, tumor necrosis factor- (tnf-), vascular endothelial growth factor (vegf) ; metastatic genes such as the matrix metalloproteinases (mmps) ; transforming growth factor- (tgf), and stimulate the production of angiogenic proteins [1416 ]. il-1 plays an important role in the development, invasion, and metastasis of cancer. previous studies have found elevated expression of il-1 in a variety of cancers such as breast cancer, pancreatic cancer, and head and neck cancer. moreover, the enhanced expression of il-1 has been correlated with poorer prognosis of cancer. il-6, a 23- to 30-kda multifunctional cytokine, is produced by numerous kinds of immune and non - immune cells, including fibroblasts, immune cells, and some cancer cells. emerging evidence revealed that il-6 is a master regulator of cell proliferation, immune defense, and hematopoiesis, and plays a crucial role in the development and progression of various cancers [2224 ]. il-6 can regulate cancer cell survival, proliferation, and metastasis by activating jak - stat3 signaling pathway and the ras - mapk signaling pathway. the activation of stat3 has been shown to maintain nf-b activity and enhance the expression of angiogenesis - related genes such as vegf. therefore, angiogenesis oncology signaling pathways within the inflammatory microenvironment maybe a crucial mechanism of il-6 induced cervical cancer. furthermore, il-6 can promote b cell differentiation and proliferation and then induce antibody secretion in b cells. moreover, il-6 can enhance, directly or indirectly, nk cell cytotoxicity and t cell tumoricidal activity. the results of our study demonstrated that the expression of il-1 and il-6 was correlated with invasion and progression of cervical cancer. meanwhile, the cervical cancer survival rate of patients with negative il-1 or il-6 expression was significantly better than that of patients with positive expression. il-1 and il-6 are implicated as important to the cytokine network in the development of cervical cancer through several complicated mechanisms that involve cell proliferation, apoptosis, angiogenesis, inflammatory microenvironment, and numerous complex signaling pathways. it is well known that cancer pathogenesis is a multi - factor, multi - step, complicated process that involves gene - gene and gene - environment interactions. large and well - designed studies are still needed to elucidate the pathogenesis of cervical cancer. our study showed that the expression of il-1 and il-6 was significantly elevated in cervical cancer tissues. kaplan - meier and cox regression analysis demonstrated that il-1 and il-6 was associated with cervical cancer progression and metastasis and they might act as important molecular markers for prognosis, and be potential therapeutic targets for cervical cancer. | backgroundil-1 and il-6 are associated with the prognosis of a wide range of cancers, but their value in cervical cancer remains controversial. the aim of this study was to investigate the expression of il-1 and il-6 in cervical cancer and their significance in clinical prognosis.material/methodsthe expression of il-1 and il-6 in 105 formalin - fixed, paraffin - embedded cervical cancer tissues and adjacent non - tumor tissues was examined by immunohistochemistry. the results were semi - quantitatively scored and analyzed by chi - square test. patient overall survival (os) data was collected by follow - up and analyzed by kaplan - meier analysis.resultsthe expression level of both il-1 and il-6 in cervical cancer tissue was higher than in adjacent non - tumor tissues (p<0.05). il-1 expression was shown to be correlated with tumor size, figo histology grade, lymph node metastasis, stromal invasion, and tumor differentiation (p<0.05). il-6 expression was shown to be correlated with tumor size, figo histology grade, and tumor differentiation (p<0.05). patients with positive expression of il-1 or il-6 tended to have much shorter survival times than patients with negative expression. in addition, a multivariate cox regression analysis demonstrated that il-1 expression and lymph node metastasis were independent predictors of os in cervical cancer patients.conclusionsthe expression of il-1 was significantly associated with tumor size, figo histology grade, lymph node metastasis, stromal invasion, and tumor differentiation. the expression of il-6 was significantly associated with tumor size, figo histology grade, and tumor differentiation. positive il-1 and il-6 expression was significantly correlated with poor prognosis. they may be considered valuable biomarkers for prognosis and potential therapeutic targets for cervical cancer. |
recovery of normal knee range of motion (rom) is among the primary goals of total knee arthroplasty (tka) since knee rom is an important clinical indicator of knee joint function that can be used as a basis for the assessment of the success of tka1234). most daily living activities require 90-120 knee rom24) ; however, kneeling, squatting, and sitting cross legged, which necessitate high flexion of the knee joint over 120, are also required for daily living activities in some cultural, religious, or occupational environment2356). among the various factors influencing postoperative knee joint rom, implant design plays an important role, and the high - flexion tka implant design was developed to enable more than 120 postoperative flexion of the knee joint123678). until recently, several manufacturers have introduced various designs of high - flexion tka implants, and a number of researches regarding the difference between the conventional implant design and high - flexion implant design have been performed. however, the reality is there are as many studies reporting insignificant differences between the implant designs910111213) as those reporting higher flexion after implantation of the high - flexion tka designs14151617). we compared the minimum 5-year follow - up results at our institution with the results of the previous papers. although a number of studies reported good results of high - flexion tka, few reports assessed the results after years of repetitive high - flexion. the purpose of this study is to analyze the survival rate of nexgen lps - flex (zimmer inc., warsaw, in, usa) high - flexion implant at minimum 5-year follow - up based on radiological measurements and revision rates. during the period from february 2007 to february 2008, a single surgeon operated tka using nexgen lps - flex implant on 143 knees of 127 patients at our institution. among them, 118 knees of 80 patients which could be followed for minimum 5 years (range, 5 to 7 years and 1 month ; mean, 5 years and 9 months) were enrolled and their clinical and radiological data were analyzed. patients with more than 15 of preoperative varus alignment or more than 30 of flexion contracture were excluded from the enrollment. there were 8 males (8 knees) and 72 females (110 knees), and their age at the time of tka was a mean of 67.4 years (range, 44 to 77 years). the indication for surgery was osteoarthritis in 73 patents (110 knees) and rheumatoid arthritis in 7 patients (8 knees). unilateral surgery was done in 42 patients (42 knees) and staged bilateral surgery was done in 38 patients (76 knees) (table 1). cruciate ligaments were resected as the implant design directs, and both the femoral and tibial components were fixed with bone cement. bone cement was spread on the cut surface of the tibia and femur, and also on the implant itself. we cemented and implanted tibial implant first with 6 - 7 times off impaction using manufactured impactor and then removed excessive cement. after implantation of the tibial component was completed, the same routine was performed with the femoral implant. then, the trial insert was inserted and maintained in position until the heat from the consolidating cement completely dissipated to secure balanced extension gap and to prevent the implant lift - off by the cement volume increment during the consolidation phase. patellar resurfacing was not done in any case, but osteophyte removal and micro - drilling were done only in the cases with predictable poor patellofemoral tracking due to their severe degeneration. passive and active rom exercises were begun on the 3rd postoperative day after the removal of drain. patients were encouraged to gain their maximum rom until 2 to 3 months after tka ; however, we did not recommend squatting, kneeling, or sitting cross legged. patients were instructed to avoid weight - bearing high - flexion activities as much as possible ; however, they were encouraged to gain the ability to perform high - flexion. on clinical evaluation, knee society knee score (ksks) and knee society function score (ksfs) assessed preoperatively and at the 5th postoperative year follow - up visit were compared. the roentgenographic evaluation and scoring system of the american knee society was used to measure and compare the preoperative radiography and postoperative 5-year radiography. flexion angle of the knee joint was measured by comparing the angle between the longitudinal midline of the femur and tibia in full extension and full flexion positions on the sagittal plane radiograph. as for the change in the implant position, femoral component angle in coronal plane (angle), tibial component angle in coronal plane (angle), femoral component angle in sagittal plane (angle), and tibial component angle in sagittal plane (angle) were measured radiographically. changes in the,,, and angles between the immediate postoperative and 5-year postoperative follow - up were assessed. bone - cement interface was set to 7 zones in coronal plane view of the femoral component and another 7 zones in coronal plane view of the tibial component, and 3 zones in sagittal plane. the sum of the scores in all of the zones was regarded as insignificant if it was less than 5 and highly suspicious of failure if the score was over 9 regardless of clinical symptoms. scores between 5 and 9 were regarded as an indication for serial follow - up as to the progression of implant loosening. gross displacement or tilting of the implant kaplan - meier survival analysis was done based on the 1-year follow - up results. the 1-year maintenance of the implant in its original location and in positions suggesting possibly good maintenance of the implant for a longer period was defined as annual success. patient who were in their 5th postoperative year follow - up period were not included in this analysis, and deaths due to diseases or follow - up loss cases were excluded. independent t - test was performed to compare clinical and radiological results of preoperative and last follow - up assessments using spss ver. 18.0 (spss inc., log - rank test was used to determine the relationship between the survival rate and gender and causative diseases. during the period from february 2007 to february 2008, a single surgeon operated tka using nexgen lps - flex implant on 143 knees of 127 patients at our institution. among them, 118 knees of 80 patients which could be followed for minimum 5 years (range, 5 to 7 years and 1 month ; mean, 5 years and 9 months) were enrolled and their clinical and radiological data were analyzed. patients with more than 15 of preoperative varus alignment or more than 30 of flexion contracture were excluded from the enrollment. there were 8 males (8 knees) and 72 females (110 knees), and their age at the time of tka was a mean of 67.4 years (range, 44 to 77 years). the indication for surgery was osteoarthritis in 73 patents (110 knees) and rheumatoid arthritis in 7 patients (8 knees). unilateral surgery was done in 42 patients (42 knees) and staged bilateral surgery was done in 38 patients (76 knees) (table 1). cruciate ligaments were resected as the implant design directs, and both the femoral and tibial components were fixed with bone cement. bone cement was spread on the cut surface of the tibia and femur, and also on the implant itself. we cemented and implanted tibial implant first with 6 - 7 times off impaction using manufactured impactor and then removed excessive cement. after implantation of the tibial component was completed, the same routine was performed with the femoral implant. then, the trial insert was inserted and maintained in position until the heat from the consolidating cement completely dissipated to secure balanced extension gap and to prevent the implant lift - off by the cement volume increment during the consolidation phase. patellar resurfacing was not done in any case, but osteophyte removal and micro - drilling were done only in the cases with predictable poor patellofemoral tracking due to their severe degeneration. passive and active rom exercises were begun on the 3rd postoperative day after the removal of drain. patients were encouraged to gain their maximum rom until 2 to 3 months after tka ; however, we did not recommend squatting, kneeling, or sitting cross legged. patients were instructed to avoid weight - bearing high - flexion activities as much as possible ; however, they were encouraged to gain the ability to perform high - flexion. on clinical evaluation, knee society knee score (ksks) and knee society function score (ksfs) assessed preoperatively and at the 5th postoperative year follow - up visit were compared. the roentgenographic evaluation and scoring system of the american knee society was used to measure and compare the preoperative radiography and postoperative 5-year radiography. flexion angle of the knee joint was measured by comparing the angle between the longitudinal midline of the femur and tibia in full extension and full flexion positions on the sagittal plane radiograph. as for the change in the implant position, femoral component angle in coronal plane (angle), tibial component angle in coronal plane (angle), femoral component angle in sagittal plane (angle), and tibial component angle in sagittal plane (angle) were measured radiographically. changes in the,,, and angles between the immediate postoperative and 5-year postoperative follow - up were assessed. bone - cement interface was set to 7 zones in coronal plane view of the femoral component and another 7 zones in coronal plane view of the tibial component, and 3 zones in sagittal plane. the sum of the scores in all of the zones was regarded as insignificant if it was less than 5 and highly suspicious of failure if the score was over 9 regardless of clinical symptoms. scores between 5 and 9 were regarded as an indication for serial follow - up as to the progression of implant loosening. gross displacement or tilting of the implant kaplan - meier survival analysis was done based on the 1-year follow - up results. the 1-year maintenance of the implant in its original location and in positions suggesting possibly good maintenance of the implant for a longer period was defined as annual success. patient who were in their 5th postoperative year follow - up period were not included in this analysis, and deaths due to diseases or follow - up loss cases were excluded. independent t - test was performed to compare clinical and radiological results of preoperative and last follow - up assessments using spss ver. 18.0 (spss inc., log - rank test was used to determine the relationship between the survival rate and gender and causative diseases. the mean knee flexion angle increased from a mean of 110.214.5 (range, 60 to 140) preoperatively to a mean of 132.45.2 (range, 90 to 145) at the last follow - up. the mean knee flexion contracture decreased from 7.8 (range, 0 to 25) preoperatively to 1.3 (range, 0 to 10) at the last follow - up. the ksks increased from a mean of 36.96.4 preoperatively to a mean of 94.23.2 at the last follow - up. the ksfs increased from a mean of 30.55.7 preoperatively to a mean of 93.74.12 at the last follow - up (table 2). most patients were satisfied with the improvement in pain : 68 patients (100 knees, 85%) out of 80 patients reported no pain or intermittent slight pain at the last follow - up visit. mechanical axis of the lower limb changed from a mean of 10.22. varus (range, 23 varus to 7 valgus) preoperatively to a mean of 3.20.6 valgus (range, 4 varus to 7 valgus) postoperatively. as for the implant position, the mean values of,,, and angles were 96.21.5 (range, 94 to 99), 92.41.1 (range, 8 to 94), 3.21.2 (range, 0 to 5), and 85.62.4 (range, 80 to 90), respectively, immediately after surgery and 95.71.7 (range, 93 to 9), 92.91.9 (range, 89 to 96), 3.21.2 (range, 0 to 5), and 85.62.4 (range, 80 to 90), respectively, at the last follow - up. there was no significant change in the position and alignment of implant between the immediate postoperative and last follow - up measurements (table 3). radiolucent lines were observed in 13 knees (11%) out of 118 knees during the 5-year follow - up. all of the radiolucent lines were in the femoral component : 4 were in zone 1 (anterior surface) and 9 were in zone 4 (posterior surface) (figs. 1 and 2). the mean score of radiolucent lines in the femoral component was 0.5 (table 4). however, there was no statistically significant relationship between the existence of radiolucent lines and postoperative rom (p=0.901) and ksks (p=0.321). removal of the implant due to loosening or revision operation was done in 1 knee (0.84%). the overall survival rate calculated using the kaplan - meier method was 99.2% (fig. 3), and there was no statistically significant relationship between the survival rate and the gender (p=0.121) and causative diseases (p=0.257). there were 2 cases of delayed wound healing and 1 case of revision due to aseptic loosening of the implant (fig. the 2 knees with delayed wound healing had past history of diabetes mellitus and their wounds were healed 3 weeks after the operation. the mean knee flexion angle increased from a mean of 110.214.5 (range, 60 to 140) preoperatively to a mean of 132.45.2 (range, 90 to 145) at the last follow - up. the mean knee flexion contracture decreased from 7.8 (range, 0 to 25) preoperatively to 1.3 (range, 0 to 10) at the last follow - up. the ksks increased from a mean of 36.96.4 preoperatively to a mean of 94.23.2 at the last follow - up. the ksfs increased from a mean of 30.55.7 preoperatively to a mean of 93.74.12 at the last follow - up (table 2). most patients were satisfied with the improvement in pain : 68 patients (100 knees, 85%) out of 80 patients reported no pain or intermittent slight pain at the last follow - up visit. mechanical axis of the lower limb changed from a mean of 10.22. varus (range, 23 varus to 7 valgus) preoperatively to a mean of 3.20.6 valgus (range, 4 varus to 7 valgus) postoperatively. as for the implant position, the mean values of,,, and angles were 96.21.5 (range, 94 to 99), 92.41.1 (range, 8 to 94), 3.21.2 (range, 0 to 5), and 85.62.4 (range, 80 to 90), respectively, immediately after surgery and 95.71.7 (range, 93 to 9), 92.91.9 (range, 89 to 96), 3.21.2 (range, 0 to 5), and 85.62.4 (range, 80 to 90), respectively, at the last follow - up. there was no significant change in the position and alignment of implant between the immediate postoperative and last follow - up measurements (table 3). radiolucent lines were observed in 13 knees (11%) out of 118 knees during the 5-year follow - up. all of the radiolucent lines were in the femoral component : 4 were in zone 1 (anterior surface) and 9 were in zone 4 (posterior surface) (figs. 1 and 2). the mean score of radiolucent lines in the femoral component was 0.5 (table 4). however, there was no statistically significant relationship between the existence of radiolucent lines and postoperative rom (p=0.901) and ksks (p=0.321). removal of the implant due to loosening or revision operation was done in 1 knee (0.84%). the overall survival rate calculated using the kaplan - meier method was 99.2% (fig. 3), and there was no statistically significant relationship between the survival rate and the gender (p=0.121) and causative diseases (p=0.257). there were 2 cases of delayed wound healing and 1 case of revision due to aseptic loosening of the implant (fig. the 2 knees with delayed wound healing had past history of diabetes mellitus and their wounds were healed 3 weeks after the operation. recently, vigorous research efforts have been made on implant designs, especially high - flexion designs, due to the increasing number of tka even among young patients and the growing expectation on functional outcomes based on the improvement of implant durability. high - flexion after tka is generally defined as greater than 125 of knee joint flexion18). compared with the conventional implant designs that allow maximum 120 knee flexion, high - flexion designs theoretically enable more than 135 knee joint flexion. although the preoperative knee joint rom is among the most important factors influencing high - flexion after tka19), maximum flexion angle could only be achieved with the aid of an improved implant design. there are several design characteristics of high - flexion implant designs for improved dynamic conditions compared to the conventional designs20). increased posterior condylar offset makes the posterior condylar curve similar to the round shape, which enables femoral rollback in the higher flexion angle without impinging of the tibial component162122). trochlear groove of the implant is elongated to avoid patella being entrapped in the intercondylar space with the longer distal contact surface of the patella and femoral components under the high - flexion circumstances. anterior lip of the tibial polyethylene insert is beveled to avoid irritating patella and patellar tendon in the high - flexion state. cam - post mechanism is also modified to increase the jump distance while preventing implant dislocation. such changes in the implant design necessitate several changes in the surgical techniques as well. for example, 2 - 3 mm more resection of the posterior condyle is required to insert an implant with greater posterior offset. sufficient exposure of the posterior joint capsule enhances removal of the posterior osteophytes impinging during high - flexion and enables concise balancing of the flexion gap21). meticulous resection of the tissues in the intercondylar space must be done for the well - operating high cam - post mechanism. individualized implantation of a tibial component with a proper posterior slope and slightly posterior placement of the component to prevent flexion limitation caused by anterior sloping or anterior positioning of the tibial component are also required. joint line elevation should be minimized to avoid patellar baja, which causes flexion limitation23). on the other hand, it is also true that there are studies reporting increased chances of aseptic loosening, polyethylene insert wear, patellar insufficiency, and instability related to the characteristics of high - flexion implant designs35624). during our research on the nexgen lps - flex single implant design, we could find 9 previous literatures regarding the same implant design5812131524252627282930) (table 5). however, only 3 studies followed a relatively large group of more than 100 cases, and only 2 reports were based on more than 5-year follow - up. there was only 1 report which followed over 100 cases for more than 5 years. under such circumstances, our minimum 5-year follow - up research on 118 knees with the nexgen lps - flex high - flexion implant design provides valuable and rare data. our results showed a mean 22.2 increase in the knee joint flexion angle from 110.214.5 (range, 60 to 140) preoperatively to 132.45.2 (range, 90 to 145) at the last follow - up, which was the highest flexion angle compared to that in the abovementioned researches, suggesting excellent improvement. ksks was improved from 36.96.4 preoperatively to 94.23.2 at the last follow - up, and it was similar to the results of conventional literatures ranging from 76.9 to 97.6. in particular, in our study, radiological assessment on implant position and radiolucent lines was carried out to identify the presence of aseptic loosening and osteolysis. there was no significant change in the implant position from preoperative period to the last follow - up. radiolucent lines were observed in 13 knees (11%) and all of which were in the femoral component, but none of them met the criteria for implant failure. in addition, the patients who showed radiolucent lines in the follow - up radiography reported they could perform more squatting and kneeling during daily living activities than they were recommended to do. compared to the study of han.24), which reports high incidence of radiolucent lines (38%) and revision surgery (21%) at a mean of 23-month follow - up in the patients with the same nexgen lps - flex implant, our results showed 11% incidence of radiolucent lines and revision surgery was required in 1 knee due to aseptic loosening. han.24) allowed patients to do weight - bearing high flexion activities as tolerated. on the contrary, we advised patients not to squat or kneel while encouraging them to recover the ability to squat and kneel. we suspect that the postoperative patient education and activity restriction could have a profound influence on the difference of the study results. as for the survival analysis, there are literatures calculating survival rates merely based on the ratio of the total number of cases to the number of revision surgery, ignoring the timing of revision surgery or the influence of drop - outs during the research period. in contrast, the 99.2% survival rate in our study was calculated from the kaplan - meier survival analysis factoring in the influence of drop - out cases as well. until recently, numerous research and a few meta - analysis studies have been carried out regarding tka implants. however, it is difficult to draw a convincing conclusion from a simple comparative study of the results due to the diverse operative techniques of individual surgeons and differences in the measurement methodology. the limitation of our study lies in the fact that it is not a comparative study, simply providing clinical results and the mid - term survival rate of a single implant design. therefore, we believe it is necessary to conduct a long - term comparative study on the conventional implant design vs. high - flexion design to analyze practical influence on the results of tka in future research. high - flexion tka using nexgen lps - flex implant design yielded satisfactory clinical and radiological outcomes with 99.2% implant survival rate after 5 years of protected activities of daily living. | purposethis study is to report clinical and radiological results of high - flexion total knee arthroplasty (tka) using nexgen lps - flex system at a minimum 5-year follow - up, and to analyze the implant survivorship based on the results.materials and methodsa total of 80 patients (118 knees) who underwent patellar preserving tka using nexgen lps - flex implant between february 2007 and february 2008 and could be followed for minimum 5 years were reviewed. the range of motion (rom), hip - knee - ankle angle, knee society knee score (ksks), and knee society function score (ksfs) were assessed preoperatively and at the last follow - up and analyzed. implant position of the femoral and tibial components on the immediate postoperative and last follow - up x - rays were compared.resultsthe mean rom was 110.214.5 (range, 60 to 140) preoperatively and 132.45.2 (range, 90 to 145) at the last follow - up. ksks was 36.96.4 preoperatively and 94.23.2 at the last follow - up. ksfs was 30.55.7 preoperatively and 93.74.1 at the last follow - up. there was no statistically significant change in the implant position measured as,,, and angles at the last follow - up compared to the immediate postoperative values. radiolucent lines were observed in 13 knees (11%) on the last follow - up x - rays. revision tka was performed due to aseptic implant loosening in 1 knee (0.84%), and the survival rate at the 5th postoperative year was 99.2%.conclusionsthe clinical and radiological outcomes of high - flexion tka using nexgen lps - flex implant design were satisfactory with 99.2% implant survival rate after 5 years of protected activities of daily living. |
field emission is a quantum mechanical tunneling phenomenon in which electrons escape from a solid surface into vacuum, as explained theoretically by r. h. fowler and l. nordheim in 1928. field emission is widely used in many kinds of vacuum electronic applications such as flat panel displays, microwave power tubes, electron sources, and electron - beam lithography. over the past decade, research groups worldwide have shown that carbon nanotubes (cnts) are excellent candidates for electron emission. cnts possess advantages in aspect ratios, tip radius of curvature, chemical stability, and mechanical strength. however, issues related to the placement and throughput of cnt arrays has hampered the development of such arrays for commercial applications. graphene is a two - dimensional honeycomb - structured single crystal showing ballistic transport, zero band gap, and electric spin transport characteristics [3 - 5 ]. in previous studies, graphene layers were randomly distributed on cathode electrodes for field emission display applications. however, further field emission studies are required using high - quality, planar graphene structure (e.g. obtained from a highly oriented pyrolyzed graphite (hopg) block). in order to understand the fundamental behavior of graphene field emission and expand its application into vacuum nanoelectronics beyond the field emission display, the characterization and analysis of field emission from an individual graphene sheet is necessary. in this paper such a graphene triode structure can be used as a fundamental unit for vacuum nanoelectronics. the triode has an in - plane graphene tip (emitter) with the other in - plane electrodes used as source, drain, and gate on the substrate. depending on the gate voltage applied, electrons are emitted from the graphene tip creating an electron current that can be modulated on and off. to realize this conceptual device, the field emission characteristics of graphene layers with different thicknesses need to be characterized. conceptual schematic view of a graphene - based triode as a fundamental unit for vacuum nanoelectronics. depending on the gate voltage applied, electrons are emitted from the graphene tip creating an electron current that can be modulated on and off to create the graphene layer for this experimental study, graphene sheets were prepared by mechanical exfoliation and placed on insulating sio2 substrate. a thermo - curable elastomer, polydimethylsiloxane (pdms, sylgard 184, dow corning co.) film was prepared using a standard recipe on an oxidized si wafer (see fig. after peeling the film from the wafer, its polished side was scrubbed on a highly oriented pyrolyzed graphite (hopg) block (see fig. 2b, c), and lifted off, transferring graphene layers to the pdms (fig. the exfoliated graphene layers were transferred onto sio2 thin film by scrubbing the pdms film and subsequently detaching, leaving behind thin graphene layers (see fig. 2e, f). in order to find and evaluate the graphene layers, the thickness of sio2 layer on si was set to 300 nm considering optical interference. the graphene sheets are transferred from hopg block to sio2layer a zyvex nanomanipulator operating inside a scanning electron microscope (sem : xl-40 sem, fei co.) was used to measure field emission from individual graphene sheets (fig. 2). figure 3 shows the schematic view of the experimental setup for measuring a field emission current from graphene sheets. in the sem vacuum chamber, two tungsten tips were located on the graphene sample ; one was contacted directly to the sample and grounded as a cathode, and the other was placed an arbitrary distance, d, apart from the edge of the sample as the anode. the tungsten tips were connected to a keithley semiconductor measurement system via a feed - through in the vacuum chamber to apply and sense the electric signal for field emission. the thickness of the layer was optically measured on 300 nm thick sio2 layer by using the change of color due to optical interference and transparency. 4a, cobalt blue, purple, and light purple stand for 8, 4 and 2 nm thicknesses, respectively. figure 4b shows an sem image of graphene sheets with a pair of tungsten tips controlled by the nanomanipulator. schematic view of the experimental setup using a nanomanipulator graphene sampleaoptical image of graphene sheets on sio2. thecolorof graphene sheets determines thickness of the graphene layer.scale bar : 6 m.bsem image of a graphene sample with tungsten tips controlled by nanomanipulator after adjusting the position of the tips, a positive potential was applied to the second tip. figure 5a shows ie curves of graphene for an arbitrary gap < 1 m. the graphene sheet started to emit electron current around 20 v and increased exponentially up to 170 na following the behavior of the fowler nordheim relationship. the field emission current fluctuated for applied voltages higher than 33 v. figure 5b shows fn curves obtained as a result of field emission from a graphene sheet. 5a, the emission current is increased exponentially, and the fn curve shows linear relationship following the field emission behavior. the estimated turn - on voltages of the tested graphene sheet is 12.1 v, where the slope of fn curve is changed and the linear region (red line) begins as shown in fig. fn parameters were evaluated by linear fit of the red line as shown in the equations.(1)(2)(3) where i : current, e : electric field (v / d), : field - enhancement factor, : work function, a : area, : reduced planck constant, and m : electron mass. assuming the work function of graphene is 5 ev and the gap between the graphene sheet and the nanomanipulator tip is 1 m, the estimated field - enhancement factor,, is 3519. it is found that the measured field - enhancement factor is comparable with previous results of graphene film prepared by electrophoresis, and the field emission efficiency of graphene is twice as high as other carbon nanomaterials such as cnt and diamond film. from the experimental results, it is found that one can further reduce the voltage for electron emission as the fabrication process is refined to create a fine emitter tip from graphene sheets. the field emission properties of graphene need further investigation in terms of the number of graphene layers and crystallographic arrangement of the carbon lattice. in the near future ai eplot for emission current.bfnplot for emission current this field - emitting nanodevice based on the planar form of graphene potentially allows for top - down cmos compatible process flows, an advantage for potential industrial fabrication of electronic devices. for applications where high field emission currents or low turn - on voltages are required, nanodevices based on graphene would inherently provide the necessary alignment based on its crystallographic nature this work has partially been supported by exchange student program by brain korea 21, award no kuk - f1 - 038 - 02 made by king abdullah university of science and technology (kaust) and national science foundation (major research instrumentation program, award no. | this paper describes an experimental study on field emission characteristics of individual graphene layers for vacuum nanoelectronics. graphene layers were prepared by mechanical exfoliation from a highly oriented pyrolyzed graphite block and placed on an insulating substrate, with the resulting field emission behavior investigated using a nanomanipulator operating inside a scanning electron microscope. a pair of tungsten tips controlled by the nanomanipulator enabled electric connection with the graphene layers without postfabrication. the maximum emitted current from the graphene layers was 170 na and the turn - on voltage was 12.1 v. |
in 2002, a baseline survey for this study was performed in the town of hisayama, japan. briefly, of the total of 3,896 residents aged 40 to 79 years, 3,000 consented to participate in the survey (participation rate, 77.0%). among them, 178 participants were not administered a 75-g oral glucose tolerance test (ogtt) : 100 refused the test, 46 had already eaten breakfast, and the other 32 were receiving insulin therapy for diabetes. consequently after further excluding 485 participants who had newly diagnosed or known diabetes and 8 for whom there was no measurement of angptl2, the remaining 2,329 (953 men and 1,376 women) were enrolled in the baseline examination. the baseline participants were followed up prospectively, from 2002 to 2009, by yearly health examinations during which an ogtt was administered. of the baseline participants, 2,164 (865 men and 1,299 women) who underwent reexaminations during the follow - up period were finally selected for this study (follow - up rate, 92.9% ; mean follow - up period, 6.0 years). these participants completed the follow - up examinations an average of 4.9 times, and among them, 861 (39.8% of the follow - up population) underwent all 7 annual ogtts. during the follow - up, t2 dm occurred in 221 participants (115 men and 106 women). in the baseline and follow - up examinations, the study participants underwent the ogtt after an overnight fast of at least 12 h. diabetes was defined by the 2003 american diabetes association criteria (7). serum angptl2 concentrations were measured with the human angptl2 sandwich enzyme - linked immunosorbent assay using two mouse monoclonal antibodies that were confirmed to recognize only angptl2 and not to react with other angptls or angiopoietins (4). angptl2 levels were divided into quartile categories : 2.15, 2.162.71, 2.723.40, and 3.41 the incidence of t2 dm was calculated by the person - year method and adjusted for age and sex by the direct method using 10-year age groupings. the adjusted hazard ratios (hrs) and their 95% cis in 2002, a baseline survey for this study was performed in the town of hisayama, japan. briefly, of the total of 3,896 residents aged 40 to 79 years, 3,000 consented to participate in the survey (participation rate, 77.0%). among them, 178 participants were not administered a 75-g oral glucose tolerance test (ogtt) : 100 refused the test, 46 had already eaten breakfast, and the other 32 were receiving insulin therapy for diabetes. consequently after further excluding 485 participants who had newly diagnosed or known diabetes and 8 for whom there was no measurement of angptl2, the remaining 2,329 (953 men and 1,376 women) were enrolled in the baseline examination. the baseline participants were followed up prospectively, from 2002 to 2009, by yearly health examinations during which an ogtt was administered. of the baseline participants, 2,164 (865 men and 1,299 women) who underwent reexaminations during the follow - up period were finally selected for this study (follow - up rate, 92.9% ; mean follow - up period, 6.0 years). these participants completed the follow - up examinations an average of 4.9 times, and among them, 861 (39.8% of the follow - up population) underwent all 7 annual ogtts. during the follow - up, t2 dm occurred in 221 participants (115 men and 106 women). in the baseline and follow - up examinations, the study participants underwent the ogtt after an overnight fast of at least 12 h. diabetes was defined by the 2003 american diabetes association criteria (7). serum angptl2 concentrations were measured with the human angptl2 sandwich enzyme - linked immunosorbent assay using two mouse monoclonal antibodies that were confirmed to recognize only angptl2 and not to react with other angptls or angiopoietins (4). angptl2 levels were divided into quartile categories : 2.15, 2.162.71, 2.723.40, and 3.41 the incidence of t2 dm was calculated by the person - year method and adjusted for age and sex by the direct method using 10-year age groupings. the adjusted hazard ratios (hrs) and their 95% cis at baseline, the mean age of participants was 58.6 years, and the proportion of men was 40.9%. the age- and sex - adjusted incidences of t2 dm increased significantly with elevating quartiles of angptl2 concentrations, and the risk was significantly higher in the second, third, and fourth quartiles than in the first quartile (table 1, model 1). in the multivariate analysis, this association remained substantially unchanged even after adjustment for age, sex, family history of diabetes, fasting insulin, high - molecular - weight adiponectin, bmi, triglycerides, hdl cholesterol, hypertension, alcohol intake, smoking habits, and regular exercise (model 2). as shown in model 3, after further adjustment for high - sensitivity c - reactive protein (hs - crp) values, the risk of developing t2 dm was significantly higher in the highest angptl2 quartile than in the lowest quartile (hr, 1.80 ; 95% ci, 1.142.85 ; p = 0.01). these findings remained substantially unchanged when waist circumference was used instead of bmi in the adjusted models. in a prospective study of a cohort of the general japanese population, we clearly demonstrated that the risk for the development of t2 dm increased with increasing serum angptl2 levels. this association remained robust even after controlling for other confounding factors, including hs - crp levels. to our knowledge, this is the first report to indicate that serum angptl2 levels are an independent risk factor for developing t2 dm in a general population. the concept that heightened inflammation is important in the pathogenesis of t2 dm (8) is supported by the evidence that inflammation in islets, adipose tissue, liver, and muscle may provoke insulin resistance and -cell dysfunction (9,10) and may therefore antedate the diagnosis of t2 dm. prospective observational studies have demonstrated that several nonspecific indicators of inflammation were found to be predictive of incident t2 dm (1114). among them, c - reactive protein is a nonspecific inflammatory marker and the most commonly measured circulating marker for subclinical inflammation (13,15). the standardized assays for its measurement are widely available (13,15). in this study, the association between serum baseline angptl2 levels and incident t2 dm was found to be independent of the hs - crp levels. nevertheless, further studies would be required to reveal whether the association is truly independent of other established inflammatory markers. this analysis clearly showed that elevated serum angptl2 levels were independently associated with incident t2 dm. further studies are needed to reveal the role of angptl2 in inflammation in human adipose tissue and the development of t2 dm. | objectiveto examine, for the first time, the association between a novel inflammatory cytokine, angiopoietin - like protein (angptl) 2, and the development of type 2 diabetes (t2dm).research design and methodsa total of 2,164 community - dwelling japanese individuals aged 40 to 79 years without diabetes were followed up for 7 years. serum angptl2 levels were divided into quartile categories at baseline : < 2.15, 2.162.71, 2.723.40, and 3.41 ng / ml. during follow - up, 221 participants developed t2dm.resultsin multivariate analyses, after adjusting for comprehensive risk factors and high - sensitivity c - reactive protein (hs - crp) levels, the risk of developing t2 dm was significantly higher in the highest angptl2 quartile than in the lowest quartile (hazard ratio, 1.80 ; 95% ci, 1.142.85 ; p = 0.01).conclusionselevated serum angptl2 levels were positively associated with the development of t2 dm in a general population, independent of other risk factors including hs - crp levels. |
the ability to recognize meaningful information from faces is a crucial aspect of our social life. when we are exposed to an environment that is rich in visual features and stimuli, we can immediately detect a face and easily recognize its individual identity and emotional state. this suggests the presence of an efficient system in the primate brain for the visual analysis of faces. recent functional magnetic resonance imaging (fmri) studies have revealed the existence of several brain areas that participate in the analysis of visual characteristics of faces in both humans and macaque monkeys, thus pointing to multiple functionally specialized prior to these studies, face - responsive neurons were recorded in the macaque temporal cortex (bruce., 1981 ; perrett., 1985), which includes some of these face patches, and their functional properties have been studied from various aspects (desimone., 1984 ; yamane.,, we recorded neuronal activity in the anterior temporal cortex of monkeys while presenting visual stimuli that included monkey and human faces, and studied the temporal modulation of neuronal responses to the faces. we found that the initial transient responses of face - responsive neurons represented information about global categories, namely human vs. monkey vs. simple shapes, and the later portion of the responses represented information about more detailed facial categories, i.e., facial expression or identity (sugase. we identified an important role for temporal firing patterns in the information coding of visual facial features and suggested that the initial transient response and the later response encode different aspects of facial characteristics. recent single - unit studies in non - human primates have provided further evidence supporting the important role of temporal neuronal modulation. in addition, human studies reveal a close correlation between neuronal events and the timing of face recognition. in this review, we will first describe studies using macaque monkeys and then summarize studies using human subjects. the accumulated body of evidence reveals a close correlation between the temporal encoding of facial information by inferior temporal (it) cortex neurons and the temporal dynamics of face recognition. in the ventral visual cortical pathway, visual signals are processed sequentially through areas v1, v2, v4, and the it cortex (areas teo and te ; mishkin., 1983). in the macaque it cortex, neurons show selective responses to complex visual images (baylis., 1987 ; richmond., 1987 ; kobatake and tanaka, 1994 ; tsunoda, 2001 ; brincat and connor, 2004 ; yamane., 2006, 2008), such as faces or animals (gross., 1972 ; bruce., 1981 ; desimone., 1984 ; perrett., 1985 ; hasselmo., 1989 ; nakamura., 1994 ; tanaka, 1996 ; tsao., face - responsive / selective neurons give stronger responses, often twice as strong, to face images as compared with other images such as objects, geometric shapes, or scrambled face images. information about faces is sparsely represented by a population of face - responsive neurons (young and yamane, 1992). the responses of these neurons are sensitive to the configuration of facial parts, such as the height of the forehead from the left eye to the hairline, the distance between the eyes and the mouth, or a combination of these parameters (yamane., 1988 ; freiwald., a possible role for it face - responsive neurons in face recognition has been reported. the responses of the majority of face - responsive neurons are correlated with the animal 's perceptual experiences (sheinberg and logothetis, 1997, 2001). it neuronal responses have been shown to represent the configuration of facial parts that are useful for a categorization task when using monkeys that are well experienced with the categorization task (sigala and logothetis, 2002). in the anterior it cortex, the response latency of neurons encoding view - invariant face - identity information correlates with the monkey 's behavioral response latency during identification of a face (eifuku., 2004). performance in face discrimination tasks based on the configuration of facial parts is affected by cooling of the temporal cortex (horel, 1993). at each stage along the ventral pathway, namely areas v2, v4, and the it cortex, stimulus selectivity of neural responses to complex images becomes sharper during the later portion of the response as compared with selectivity during the initial transient response. hegde and van essen (2004, 2006) studied neuronal responses to gratings (sinusoidal, hyperbolic, and polar gratings) and contour stimuli (bars, crosses, and angles) in areas v2 and v4. they found that the population response of the neurons was better able to categorize the stimuli into broad groups, e.g., gratings vs. contour, during the initial phase of the response (area v2, 4080 ms after stimulus onset ; area v4, 80100 ms), and was able to distinguish between individual stimuli within the stimulus groups after the initial phase (hegde and van essen, 2004, 2006). brincat and connor (2006) examined neuronal responses in the posterior part of the it cortex to simple shape stimuli combining convex, concave, and straight contours. they found that information about individual contour fragments is carried in the initial transient response (90% maximum at 122 ms after stimulus onset) and information about the specific multipart contour configurations emerges gradually (90% maximum at 184 ms after stimulus onset). tamura and tanaka (2001) showed that the stimulus selectivity of neuronal responses to photographs of natural objects and geometric shapes in the anterior part of the it cortex is more selective in the later portion of the response (after 240 ms from stimulus onset) compared to the initial transient response (130 ms from stimulus onset). the importance of temporal firing patterns during information coding of visual stimuli by it neurons was first described by optican, richmond, and colleagues (optican and richmond, 1987 ; richmond and optican, 1987 ; richmond., 1987) and has been confirmed since then. (1993) showed that it neuronal responses represent the greatest information about the test stimuli identity during the first 100200 ms after stimulus onset (tovee., 1993). the robustness of the temporal firing pattern across days and weeks of visual experience was also shown (bondar., 2009). we examined neuronal response in the anterior it gyrus and the anterior part of the superior temporal sulcus in the it cortex to visual stimuli, including geometric shapes and monkey / human faces with various expressions, using a fixation task (recoding location shown in figure 1a ; sugase., 1999). some neurons showed initial transient responses to faces, but not to shapes, and maintained a sustained response to only a particular facial expression or identity, indicating sharper stimulus selectivity in the later portion of the response (e.g., figure 1 of sugase., 1999, and figure 2 of matsumoto., 2005b). using a moving time window, we calculated the time course of the transmitted facial stimuli information with respect to both the global category (human faces vs. monkey faces vs. shapes) and sub- or fine categories within each category member (monkey facial expression, monkey identity, human facial expression, or human identity). we found that the latency and peak of information for the global categories were earlier than those of the fine categories (measured as the middle of the 50-ms analysis window ; latency, 91 and 142 ms after stimulus onset ; peak, 152 and 179 ms, for global and fine categories, respectively ; figure 1b). this result indicates that the temporal firing pattern is important for information processing of faces and suggests that the initial transient response and the later response encode different aspects of facial characteristics, i.e., information about global categories and about fine categories. the result that both the peak of the information for the global categories and the peak of the information for the fine categories were observed within 100200 ms after stimulus onset is consistent with the finding by tovee. (1993) and supports the notion that neuronal responses within this time period represent the greatest information about the stimuli identity. because each neuron displays a different temporal firing pattern (as shown in figure 2 of matsumoto., 2005b), we further analyzed the responses of a population of neurons and found that the separation of human vs. monkey vs. shapes was maximized at the [90, 140]-ms period after the stimulus onset, and that the separation of monkey facial expressions and human facial identities was maximized at the [140, 190]-ms period (figure 1c ; matsumoto. this result indicates that global categorization occurs earlier than finer categorization at the population level. ap0 represents the position of the external auditory meatus ; a14, a19, a22, and a24 represent anterior 14, 19, 22, and 24 mm, respectively. summation of ic curves for global (red, human faces vs. monkey faces vs. shapes) and fine categories (black ; monkey facial expression, monkey identity, human facial expression, or human identity) from 32 neurons aligned at the stimulus onset. ic was calculated over a 50-ms sliding analysis window, and plotted at the middle of the time window. the 32 neurons represent information about both global and fine categories. in the time axis (abscissa), the time of stimulus onset is 0 ms and the duration of stimulus presentation is indicated as a dashed line. (c) population activity vectors in two - dimensional space rearranged using pca in the [90, 140 ] and [140, 190]-ms windows. the horizontal axis represents the first principal component, while the vertical axis represents the second principal component. the points indicate the population activity vectors of 45 face - responsive neurons for the individual stimuli. the colors of the points represent the global category : the vectors for human faces, monkey faces, and shapes are shown in red, blue, and green, respectively. the red, blue, and green ellipses indicate the distributions of the population activity vectors for the fine categories human identity, monkey expression, and shape - form, respectively. recent single - unit studies in non - human primates provide evidence that is consistent with our findings. neurons in the anterior it cortex respond to human faces on average 15 ms earlier (103 ms mean latency) than to non - primate animal faces (118 ms ; kiani., 2005), showing that the initial transient response is modulated with respect to the global category, i.e., human vs. animal. tsao, freiwald, and colleagues made remarkable advances in identifying face - processing systems along the ventral visual pathway, identifying six interconnected cortical regions that consist of face - selective neurons, i.e., face patches (figure 2a ; tsao.. in the middle face patch (ml and mf in figure 2a), which is located within the superior temporal sulcus, categorization (faces, bodies, fruits, hands, gadgets, or scrambled images) performance by the neuronal response (133 ms) precedes identification performance (192 ms ; tsao., 2006), showing an earlier representation of the global category information than of the subcategory image identity information. freiwald and tsao (2010) found that in the anterior medial patch (am in figure 2a), which is located in the anterior part of the it gyrus, information about facial identity across different facial views emerges gradually and peaks at around 300 ms after stimulus onset (figure 2b). the fact that view - invariant facial identity information is represented in the response at a later period (300 ms) indicates that additional information processing is necessary to compute facial identity across different facial views, which may take a considerable amount of time. inflated macaque left hemisphere (dark gray areas mark sulci, light gray dark gray boundaries mark the middle of the bank within a sulcus) showing six regions, i.e., face patches, in the temporal lobe that responded significantly more to faces than to objects in fmri experiments. the six patches are indicated as pl, posterior lateral ; ml, middle lateral ; mf, middle fundus ; af, anterior fundus ; al, anterior lateral ; and am, anterior medial patch. view - invariant identity - selectivity index, computed over a 200-ms sliding response window beginning at the indicated time point, plotted for am, al, and ml / mf (solid curves in magenta, blue, and green, respectively). the dotted curves show the mean view - invariant identity - selectivity index over time computed after the relationships between the face view and the face identity are shuffled. the grayscale traces show the time course of the mean response across the population in each face patch. there is the substantial delay between the peak of the mean response to the stimuli and the peak of the view - invariant identity - selectivity index. this type of coding seems to be a plausible mechanism underlying the temporal dynamics of face recognition, e.g., the process of face detection / categorization followed by facial identification of an individual. the causal relationship between signals from face - responsive neurons in the it cortex and face perception microstimulation within face - selective sites around the time of the initial transient response, i.e., [50, 100 ] ms after stimulus onset, biased the monkey 's decision toward faces during a face / non - face categorization task (afraz., 2006). this result indicates that the initial transient response of face - selective neurons has a causal relationship with face perception, i.e., whether an image contains a face, and the perception of a global category. the causal relationship between the later portion of the response and face recognition, there are at least three different possibilities for the underlying mechanism that governs how initial transient responses and later responses encode different aspects of facial characteristics. one possibility is that the initial transient response encodes achromatic information through the magnocellular pathway, which arrives at the it cortex earlier than chromatic information through the parvocellular pathway. (2003) found that colored images evoked larger responses from it face - responsive neurons than did achromatic images during the earliest part of the response. however, the possibility of the magnocellular and parvocellular pathways playing different roles in encoding fine / coarse facial information remains open for future studies. the initial transient response may encode information about the low spatial frequency components of face images through the magnocellular pathway, and the later portion of the response may encode information about the high spatial frequency components through the parvocellular pathway. this idea is supported by human fmri studies (goffaux., 2010, the relationship between responses at different temporal domains and the high and low spatial frequency components of images remains to be elucidated at the single - unit level in non - human primate studies. the second possibility is that the representation of each facial part is followed by complete representation of multiple facial parts. the third possibility is that a visual stimulus is processed at different scales, from global - to - local, with the passage of time. with respect to the second possibility, individual object parts of abstract shapes are represented during the initial transient responses (peak, 122 ms after stimulus onset), and multipart configurations are represented in the later portion of the responses (peak, 184 ms ; brincat and connor, 2006), in the posterior part of the it cortex (area teo and posterior te), which sends efferent projections to the anterior temporal cortex (saleem., 2000 ; borra., consistent with the suggestion by brincat and connor (2006), neurons in the anterior it cortex may represent a diagnostic area that is useful in detecting facial features, such as the eyes (lewis and edmonds, 2003), during the initial transient responses and represents information about the configuration of facial parts that is useful for recognition of facial identity (tanaka and farah, 1993) during the later portion of the response. the third possibility has been investigated using navon (1977) figures, e.g., a large letter n consisting of small letter h 's (tanaka and fujita, 2000 ; tanaka., 2001 ; sripati and olson, 2009, 2010a, b). the behavioral response time to discriminate the global form is 2030 ms earlier than that to discriminate the local form, for both monkey and human subjects (tanaka and fujita, 2000 ; sripati and olson, 2009). the global form is represented during the initial transient response, and the local form is represented approximately 30 ms later in the it cortex (sripati and olson, 2009). differences in the global local latency of the neuronal response are related to the large small latency difference. these results indicate that the global signal emerges earlier, because shapes at a larger scale elicit discriminative neuronal activity earlier. therefore, characteristics of faces at a larger scale, e.g., the outline of the face, may elicit differential neuronal responses earlier than do facial characteristics at a smaller scale, e.g., face parts. with respect to the cellular mechanisms underlying the representation of global - to - fine category information, we speculated that signals related to fine categories emerge through intra - areal contribution or feedback from other areas (sugase., 1999). the amygdala is one of the candidate areas that send feedback signals and affect the later portion of the responses related to fine category information, since the it cortex receives projections from the amygdala (amaral and price, 1984 ; amaral., 1992) and neurons in the amygdala, 1985 ; nakamura., 1992 ; kuraoka and nakamura, 2006 ; gothard., 2007). with respect to the contribution of intra - areal connections, the fine category information might emerge through their recurrent connections. we tested this possibility using an attractor network model that consisted of excitatory and inhibitory neurons (matsumoto., 2005a). since the model reproduced the temporal dynamics of the responses of the face - responsive neurons, we speculated that the recurrent processing within the it cortex might be sufficient to give rise to the later portion of the response. we proposed two physiological experiments to investigate whether the attractor network model is a plausible mechanism : one using noisy, fine feature degraded images, and the other using weakening connections between excitatory neurons within the anterior it cortex. although inhibition by -aminobutyric acid within the it cortex contributes to neuronal stimulus selectivity (wang., 2000), the relationship between the intra - areal inhibition (contribution of both the recurrent and lateral inhibition) and the temporal firing patterns of it neurons still remains indefinite. in addition, the correlation between the responses of neuronal pairs in the it cortex is higher during the presentation of face - like drawings than during the presentation of non - face - like drawings as early as [100, 300 ] ms after the stimulus onset (hirabayashi and miyashita, 2005). the result indicates that connections between neurons in a local circuit may be strengthened during information processing of face images. there is another line of research, which suggests that the processing of visual stimuli occurs in a rapid feed - forward pass during the early portion of the response, with no role in the basic performance of recurrent / feedback processing during the later portion of the response (although it likely plays a role in top - down effects, such as attentional biases), i.e., the feed - forward hypothesis (riesenhuber and poggio, 1999 ; serre., 2007). as supporting evidence for this hypothesis, (2005) showed that information about object categorization and identification can be read out from an unbiased sample of the it neuronal site within a narrow time window (12.5 ms) at the earliest part of the response (starting from 125 ms after stimulus onset) using a support vector machine classifier. since this study did not focus on face - responsive neurons, the time course to the readout of detailed information specific to human / monkey faces, facial identity and expression, in the ventral visual cortical pathway, visual signals are processed sequentially through areas v1, v2, v4, and the it cortex (areas teo and te ; mishkin., 1983). in the macaque it cortex, neurons show selective responses to complex visual images (baylis., 1987 ; richmond., 1987 ; kobatake and tanaka, 1994 ; tsunoda, 2001 ; brincat and connor, 2004 ; yamane., 2006, 2008), such as faces or animals (gross., 1972 ; bruce., 1981 ; desimone., 1984 ; perrett., 1985 ; hasselmo., 1989 ; nakamura., 1994 ; tanaka, 1996 ; tsao., face - responsive / selective neurons give stronger responses, often twice as strong, to face images as compared with other images such as objects, geometric shapes, or scrambled face images. information about faces is sparsely represented by a population of face - responsive neurons (young and yamane, 1992). the responses of these neurons are sensitive to the configuration of facial parts, such as the height of the forehead from the left eye to the hairline, the distance between the eyes and the mouth, or a combination of these parameters (yamane., 1988 ; freiwald., a possible role for it face - responsive neurons in face recognition has been reported. the responses of the majority of face - responsive neurons are correlated with the animal 's perceptual experiences (sheinberg and logothetis, 1997, 2001). it neuronal responses have been shown to represent the configuration of facial parts that are useful for a categorization task when using monkeys that are well experienced with the categorization task (sigala and logothetis, 2002). in the anterior it cortex, the response latency of neurons encoding view - invariant face - identity information correlates with the monkey 's behavioral response latency during identification of a face (eifuku., 2004). performance in face discrimination tasks based on the configuration of facial parts is affected by cooling of the temporal cortex (horel, 1993). at each stage along the ventral pathway, namely areas v2, v4, and the it cortex, stimulus selectivity of neural responses to complex images becomes sharper during the later portion of the response as compared with selectivity during the initial transient response. hegde and van essen (2004, 2006) studied neuronal responses to gratings (sinusoidal, hyperbolic, and polar gratings) and contour stimuli (bars, crosses, and angles) in areas v2 and v4. they found that the population response of the neurons was better able to categorize the stimuli into broad groups, e.g., gratings vs. contour, during the initial phase of the response (area v2, 4080 ms after stimulus onset ; area v4, 80100 ms), and was able to distinguish between individual stimuli within the stimulus groups after the initial phase (hegde and van essen, 2004, 2006). brincat and connor (2006) examined neuronal responses in the posterior part of the it cortex to simple shape stimuli combining convex, concave, and straight contours. they found that information about individual contour fragments is carried in the initial transient response (90% maximum at 122 ms after stimulus onset) and information about the specific multipart contour configurations emerges gradually (90% maximum at 184 ms after stimulus onset). tamura and tanaka (2001) showed that the stimulus selectivity of neuronal responses to photographs of natural objects and geometric shapes in the anterior part of the it cortex is more selective in the later portion of the response (after 240 ms from stimulus onset) compared to the initial transient response (130 ms from stimulus onset). the importance of temporal firing patterns during information coding of visual stimuli by it neurons was first described by optican, richmond, and colleagues (optican and richmond, 1987 ; richmond and optican, 1987 ; richmond., 1987) and has been confirmed since then. (1993) showed that it neuronal responses represent the greatest information about the test stimuli identity during the first 100200 ms after stimulus onset (tovee., 1993). the robustness of the temporal firing pattern across days and weeks of visual experience was also shown (bondar., 2009). we examined neuronal response in the anterior it gyrus and the anterior part of the superior temporal sulcus in the it cortex to visual stimuli, including geometric shapes and monkey / human faces with various expressions, using a fixation task (recoding location shown in figure 1a ; sugase., 1999). some neurons showed initial transient responses to faces, but not to shapes, and maintained a sustained response to only a particular facial expression or identity, indicating sharper stimulus selectivity in the later portion of the response (e.g., figure 1 of sugase. using a moving time window, we calculated the time course of the transmitted facial stimuli information with respect to both the global category (human faces vs. monkey faces vs. shapes) and sub- or fine categories within each category member (monkey facial expression, monkey identity, human facial expression, or human identity). we found that the latency and peak of information for the global categories were earlier than those of the fine categories (measured as the middle of the 50-ms analysis window ; latency, 91 and 142 ms after stimulus onset ; peak, 152 and 179 ms, for global and fine categories, respectively ; figure 1b). this result indicates that the temporal firing pattern is important for information processing of faces and suggests that the initial transient response and the later response encode different aspects of facial characteristics, i.e., information about global categories and about fine categories. the result that both the peak of the information for the global categories and the peak of the information for the fine categories were observed within 100200 ms after stimulus onset is consistent with the finding by tovee. (1993) and supports the notion that neuronal responses within this time period represent the greatest information about the stimuli identity. because each neuron displays a different temporal firing pattern (as shown in figure 2 of matsumoto., 2005b), we further analyzed the responses of a population of neurons and found that the separation of human vs. monkey vs. shapes was maximized at the [90, 140]-ms period after the stimulus onset, and that the separation of monkey facial expressions and human facial identities was maximized at the [140, 190]-ms period (figure 1c ; matsumoto. this result indicates that global categorization occurs earlier than finer categorization at the population level. ap0 represents the position of the external auditory meatus ; a14, a19, a22, and a24 represent anterior 14, 19, 22, and 24 mm, respectively. summation of ic curves for global (red, human faces vs. monkey faces vs. shapes) and fine categories (black ; monkey facial expression, monkey identity, human facial expression, or human identity) from 32 neurons aligned at the stimulus onset. ic was calculated over a 50-ms sliding analysis window, and plotted at the middle of the time window. the 32 neurons represent information about both global and fine categories. in the time axis (abscissa), the time of stimulus onset is 0 ms and the duration of stimulus presentation is indicated as a dashed line. (c) population activity vectors in two - dimensional space rearranged using pca in the [90, 140 ] and [140, 190]-ms windows. the horizontal axis represents the first principal component, while the vertical axis represents the second principal component. the points indicate the population activity vectors of 45 face - responsive neurons for the individual stimuli. the colors of the points represent the global category : the vectors for human faces, monkey faces, and shapes are shown in red, blue, and green, respectively. the red, blue, and green ellipses indicate the distributions of the population activity vectors for the fine categories human identity, monkey expression, and shape - form, respectively. recent single - unit studies in non - human primates provide evidence that is consistent with our findings. neurons in the anterior it cortex respond to human faces on average 15 ms earlier (103 ms mean latency) than to non - primate animal faces (118 ms ; kiani., 2005), showing that the initial transient response is modulated with respect to the global category, i.e., human vs. animal. tsao, freiwald, and colleagues made remarkable advances in identifying face - processing systems along the ventral visual pathway, identifying six interconnected cortical regions that consist of face - selective neurons, i.e., face patches (figure 2a ; tsao. 2008 ; freiwald., 2009 ; freiwald and tsao, 2010). in the middle face patch (ml and mf in figure 2a), which is located within the superior temporal sulcus, categorization (faces, bodies, fruits, hands, gadgets, or scrambled images) performance by the neuronal response (133 ms) precedes identification performance (192 ms ; tsao., 2006), showing an earlier representation of the global category information than of the subcategory image identity information. freiwald and tsao (2010) found that in the anterior medial patch (am in figure 2a), which is located in the anterior part of the it gyrus, information about facial identity across different facial views emerges gradually and peaks at around 300 ms after stimulus onset (figure 2b). the fact that view - invariant facial identity information is represented in the response at a later period (300 ms) indicates that additional information processing is necessary to compute facial identity across different facial views, which may take a considerable amount of time. dark gray boundaries mark the middle of the bank within a sulcus) showing six regions, i.e., face patches, in the temporal lobe that responded significantly more to faces than to objects in fmri experiments. the six patches are indicated as pl, posterior lateral ; ml, middle lateral ; mf, middle fundus ; af, anterior fundus ; al, anterior lateral ; and am, anterior medial patch. view - invariant identity - selectivity index, computed over a 200-ms sliding response window beginning at the indicated time point, plotted for am, al, and ml / mf (solid curves in magenta, blue, and green, respectively). the dotted curves show the mean view - invariant identity - selectivity index over time computed after the relationships between the face view and the face identity are shuffled. the grayscale traces show the time course of the mean response across the population in each face patch. there is the substantial delay between the peak of the mean response to the stimuli and the peak of the view - invariant identity - selectivity index. this type of coding seems to be a plausible mechanism underlying the temporal dynamics of face recognition, e.g., the process of face detection / categorization followed by facial identification of an individual. the causal relationship between signals from face - responsive neurons in the it cortex and face perception was addressed by afraz. microstimulation within face - selective sites around the time of the initial transient response, i.e., [50, 100 ] ms after stimulus onset, biased the monkey 's decision toward faces during a face / non - face categorization task (afraz., 2006). this result indicates that the initial transient response of face - selective neurons has a causal relationship with face perception, i.e., whether an image contains a face, and the perception of a global category. the causal relationship between the later portion of the response and face recognition, there are at least three different possibilities for the underlying mechanism that governs how initial transient responses and later responses encode different aspects of facial characteristics. one possibility is that the initial transient response encodes achromatic information through the magnocellular pathway, which arrives at the it cortex earlier than chromatic information through the parvocellular pathway. (2003) found that colored images evoked larger responses from it face - responsive neurons than did achromatic images during the earliest part of the response. however, the possibility of the magnocellular and parvocellular pathways playing different roles in encoding fine / coarse facial information remains open for future studies. the initial transient response may encode information about the low spatial frequency components of face images through the magnocellular pathway, and the later portion of the response may encode information about the high spatial frequency components through the parvocellular pathway. this idea is supported by human fmri studies (goffaux., 2010, the relationship between responses at different temporal domains and the high and low spatial frequency components of images remains to be elucidated at the single - unit level in non - human primate studies. the second possibility is that the representation of each facial part is followed by complete representation of multiple facial parts. the third possibility is that a visual stimulus is processed at different scales, from global - to - local, with the passage of time. several studies support the second and third possibilities. with respect to the second possibility, individual object parts of abstract shapes are represented during the initial transient responses (peak, 122 ms after stimulus onset), and multipart configurations are represented in the later portion of the responses (peak, 184 ms ; brincat and connor, 2006), in the posterior part of the it cortex (area teo and posterior te), which sends efferent projections to the anterior temporal cortex (saleem., 2000 ; consistent with the suggestion by brincat and connor (2006), neurons in the anterior it cortex may represent a diagnostic area that is useful in detecting facial features, such as the eyes (lewis and edmonds, 2003), during the initial transient responses and represents information about the configuration of facial parts that is useful for recognition of facial identity (tanaka and farah, 1993) during the later portion of the response. the third possibility has been investigated using navon (1977) figures, e.g., a large letter n consisting of small letter h 's (tanaka and fujita, 2000 ; tanaka., 2001 ; sripati and olson, 2009, 2010a, b). the behavioral response time to discriminate the global form is 2030 ms earlier than that to discriminate the local form, for both monkey and human subjects (tanaka and fujita, 2000 ; sripati and olson, 2009). the global form is represented during the initial transient response, and the local form is represented approximately 30 ms later in the it cortex (sripati and olson, 2009). differences in the global local latency of the neuronal response are related to the large small latency difference. these results indicate that the global signal emerges earlier, because shapes at a larger scale elicit discriminative neuronal activity earlier. therefore, characteristics of faces at a larger scale, e.g., the outline of the face, may elicit differential neuronal responses earlier than do facial characteristics at a smaller scale, e.g., face parts. with respect to the cellular mechanisms underlying the representation of global - to - fine category information, we speculated that signals related to fine categories emerge through intra - areal contribution or feedback from other areas (sugase., 1999). the amygdala is one of the candidate areas that send feedback signals and affect the later portion of the responses related to fine category information, since the it cortex receives projections from the amygdala (amaral and price, 1984 ; amaral., 1992) and neurons in the amygdala respond to specific facial identity or expression (leonard., 1985 ; nakamura., 1992 ; kuraoka and nakamura, 2006 ; gothard., 2007). with respect to the contribution of intra - areal connections, the fine category information might emerge through their recurrent connections. we tested this possibility using an attractor network model that consisted of excitatory and inhibitory neurons (matsumoto., 2005a). since the model reproduced the temporal dynamics of the responses of the face - responsive neurons, we speculated that the recurrent processing within the it cortex might be sufficient to give rise to the later portion of the response. we proposed two physiological experiments to investigate whether the attractor network model is a plausible mechanism : one using noisy, fine feature degraded images, and the other using weakening connections between excitatory neurons within the anterior it cortex. although inhibition by -aminobutyric acid within the it cortex contributes to neuronal stimulus selectivity (wang., 2000), the relationship between the intra - areal inhibition (contribution of both the recurrent and lateral inhibition) and the temporal firing patterns of it neurons still remains indefinite. in addition, the correlation between the responses of neuronal pairs in the it cortex is higher during the presentation of face - like drawings than during the presentation of non - face - like drawings as early as [100, 300 ] ms after the stimulus onset (hirabayashi and miyashita, 2005). the result indicates that connections between neurons in a local circuit may be strengthened during information processing of face images. there is another line of research, which suggests that the processing of visual stimuli occurs in a rapid feed - forward pass during the early portion of the response, with no role in the basic performance of recurrent / feedback processing during the later portion of the response (although it likely plays a role in top - down effects, such as attentional biases), i.e., the feed - forward hypothesis (riesenhuber and poggio, 1999 ; serre., 2007). as supporting evidence for this hypothesis, (2005) showed that information about object categorization and identification can be read out from an unbiased sample of the it neuronal site within a narrow time window (12.5 ms) at the earliest part of the response (starting from 125 ms after stimulus onset) using a support vector machine classifier. since this study did not focus on face - responsive neurons, the time course to the readout of detailed information specific to human / monkey faces, facial identity and expression, at the behavioral level, visual recognition speed has been examined using large sets of images consisting of faces, animals, or objects. thorpe and colleagues have shown that human observers are able to categorize briefly presented natural scenes with a reaction time of less than 400 ms and approximate accuracy of 94% using an animal vs. no - animal go / no - go task with manual responses (thorpe., 1996 ; vanrullen and thorpe, 2001 ; rousselet., 2002). the reaction time of human subjects during the task was reported to be 100180 ms (median reaction time) slower than monkey subjects (fabre - thorpe., 1998). recently, these authors used saccadic eye movement for a choice task, and showed that subjects were especially rapid at detecting human faces. in the task, two images were simultaneously presented and the subjects made a saccade to an image of a particular target category, e.g., human faces or motor vehicles. the reaction time (start time) of the saccade was substantially shorter toward human faces than toward the other category images, e.g., motor vehicles (in their third experiment, human faces, 159 ms mean, 100 ms minimum ; vehicles, 183 ms mean, 170 ms minimum ; crouzet., the reaction time was longer during the choice task than during a simple saccadic detection task, with only one stimulus being presented, both for the human face targets (at least 20 ms longer) and for the vehicle targets (80 ms longer). these results suggest that a lower number of processing steps is required to select faces than to select other objects. it remains to be clarified whether signals in the it cortex, e.g., early global category information (face vs. non - face), are required for the saccadic choice. the face bias during the saccadic choice task is at least partially related to the low - level physical characteristics of the images, as scrambling the orientation contents of the images but not scrambling their relative positions disrupts the face bias (honey., 2008). the detection of an object or categorization of an object at a basic categorical level (bird, car, dog, etc.) is approximately 65 ms faster than within - category identification (grill - spector and kanwisher, 2005). face detection using synthetic faces occurs 24 and 31 ms earlier than viewpoint and face identification, respectively (or and wilson, 2010). this result suggests that face recognition is a process of face detection / categorization followed by viewpoint / face identification and that additional processing is required to obtain the latter information. on the other hand, face identification becomes as fast as face categorization depending on the familiarity of the face. for example, identifying a face such as that of bill clinton is as fast as categorizing the face as human or monkey (tanaka, 2001 ; anaki and bentin, 2009). studies examining whether information about each facial part is analyzed, accumulated, and integrated over time during face recognition remain controversial (singer and sheinberg, 2006 ; anaki. humans and macaque monkeys have similar brain systems for face - processing (tsao., 2008 ; bell., 2009 ; the temporal dynamics of the neural correlates of facial information processing have also been studied in human subjects or patients using non - invasive brain - imaging techniques or invasive techniques. the results for these studies are consistent with studies on non - human primates and human behavioral studies. studies using magnetoencephalography (meg) showed that a face - selective response 100 ms after the stimulus onset (m100) was related to categorization of the stimuli as a face (liu., 2002 ; meeren., 2008) and that the later response at 170 ms after the stimulus onset (m170) was related to both facial categorization and recognition of individual faces (liu., 2002). both m100 and m170 were observed over the occipito - temporal cortex, though m100 was distributed slightly posteriorly. since the existence of facial parts (eyes, nose, and mouth) is important for m100 regardless of their configuration, and the part - configuration is important for m170, liu. (2002) speculated that m100 and m170 responses reflect processing related to the detection of diagnostic facial parts and to processing related to the analysis of the part - configuration, respectively, instead of global - to - local processing. the familiarity of faces affected the m170 response (kloth., 2006) and other later responses (latency around 250400 ms, m400) from the occipito - temporal sensors (harris and aguirre, 2008). event - related potential (erp) studies have shown that the representation of facial categories and face - inversion effects occurs during an early negative component that peaks at around 170 ms after stimulus onset (n170, starting at 130150 ms after stimulus onset ; rousselet., 2008). the strength of face selectivity of the n170 response (calculated from the peak amplitude between 140 and 200 ms) has been shown to be highly correlated across subjects with those of the hemodynamic response in the temporal lobe, i.e., the fusiform face area (ffa) and the posterior part of the right superior temporal sulcus, by a simultaneous erp a later response (e.g., a negativity between 300 and 500 ms from a parietocentral electrode) was shown to be associated with the familiarity of faces, suggesting that the late signals are related to familiar facial identity (eimer, 2000). electrocorticography (ecog) recordings indicate that an enhanced activation in gamma power (3070 hz) and evoked responses that occur 150200 ms after stimulus onset (similar timing as n170) are associated with successful recognition of the stimulus category, i.e., face, house, or object (fisch., 2009). studies using intracranial or subdural electroencephalography (eeg) recordings showed that face - specific erps (face - n200) within the fusiform and it gyri were affected neither by semantic priming and face - name learning / identification nor by selective attention, but that subsequent slow evoked potential was affected (p290 and n700, or 240 ms after stimulus onset, respectively ; puce. these results indicate that the face - n200 is largely insensitive to cognitive task manipulations or attention, unlike the later responses, suggesting that the face - n200 reflects an initial and obligatory neural response that is related to the visual analysis of faces and that the later responses are susceptible to cognitive demands and the top - down control of attention. (2005) suggested an important role for the human medial temporal lobe (mtl ; the hippocampus, amygdala, entorhinal cortex, and parahippocampal cortex) in recognizing facial identity. some neurons in the mtl respond exclusively to images of a particular famous person regardless of different views, e.g., the actress jennifer aniston. the latency of the responses to a famous person are primarily observed during the later period, i.e., between 300 and 600 ms after stimulus onset (quiroga., 2005 ; mormann., the results indicate that the higher selectivity of the mtl neurons was found with longer latencies, in other words, in the later responses. furthermore, these data suggest that interactions between the it cortex and mtl may play an important role in the recognition of facial identity regardless of view variations. a recent fmri study revealed a role for temporal dynamics in face information processing, i.e., coarse - to - fine processing (goffaux., 2010). by presenting face images that preserve either low spatial frequency or high spatial frequency for a 75, 150, or 300-ms duration, goffaux. (2010) found that the coarse structure of a face, which is carried by low spatial frequency, is processed prior to the fine details transmitted by high spatial frequency in the ffa. at a 75-ms exposure, the responses to low spatial frequency face images are greater than the responses to high spatial frequency face images. in contrast, at 150 and 300-ms exposure, the responses to high spatial frequency face images, which contain local fine information useful for identification, are stronger than the responses to low spatial frequency face images. another study suggests that coarse signals about visual stimuli, i.e., achromatic but with luminance - contrast stimuli (magnocellular biased stimuli), are derived rapidly through the dorsal visual pathway via the occipital visual cortex and orbitofrontal cortex to the it cortex (kveraga., 2007). these results support the hypothesis that the initial and later portions of neuronal responses in the it cortex play important roles in the coarse and fine processing of face images, respectively. monkey and human studies suggest at least two temporal processing stages underlying the recognition of faces. the correlation between the two processing stages and neuronal activity has been shown by human meg studies, indicating that m100 is related to face detection / categorization and m170 is related to face identification. the timing of the m100 response (averaged response peak time, 105 ms after stimulus onset, range 84.5130.5 ms) and that of the m170 response (160 ms) overlaps with the peak time of global information (mean sd, 152 57 ms) and that of fine information (179 49 ms) reported by sugase. the m100 response appears slightly earlier compared to the peak time of the global information, most likely due to m100 being distributed slightly more posteriorly than m170, thus including signals from posterior parts of the brain, i.e., the occipital. the n170 response of erp studies and the face - n200 of intracranial or subdural eeg recordings seem to reflect at least processing of the detection / categorization of faces. it is not apparent whether the later response (m400 for meg, 300500 ms for erp, p290, and n700 for eeg) that is correlated with the familiarity of faces parallels the fine information reported by sugase. (1999), or a slowly emerging signal reflecting the view - invariant identity information reported by freiwald and tsao (2010). future studies at the single - unit level using non - human primates should address which temporal domains are influenced by the familiarity of faces. the accumulated body of evidence on non - human primates shows that temporal firing patterns in it cortex neurons are important for information coding of visual features of faces. it has been shown that the initial transient response represents the face category, and that the later response represents facial identity and facial expression. future studies should investigate the role of signals in the later response of face recognition to reveal the causal relationship between the later response and face detection / categorization, recognition of facial identity, or even face familiarization. it should also be examined whether the temporal processing stages are specific to face images or are general properties of the visual system. 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. | in this review, we focus on the role of temporal stages of encoded facial information in the visual system, which might enable the efficient determination of species, identity, and expression. facial recognition is an important function of our brain and is known to be processed in the ventral visual pathway, where visual signals are processed through areas v1, v2, v4, and the inferior temporal (it) cortex. in the it cortex, neurons show selective responses to complex visual images such as faces, and at each stage along the pathway the stimulus selectivity of the neural responses becomes sharper, particularly in the later portion of the responses. in the it cortex of the monkey, facial information is represented by different temporal stages of neural responses, as shown in our previous study : the initial transient response of face - responsive neurons represents information about global categories, i.e., human vs. monkey vs. simple shapes, whilst the later portion of these responses represents information about detailed facial categories, i.e., expression and/or identity. this suggests that the temporal stages of the neuronal firing pattern play an important role in the coding of visual stimuli, including faces. this type of coding may be a plausible mechanism underlying the temporal dynamics of recognition, including the process of detection / categorization followed by the identification of objects. recent single - unit studies in monkeys have also provided evidence consistent with the important role of the temporal stages of encoded facial information. for example, view - invariant facial identity information is represented in the response at a later period within a region of face - selective neurons. consistent with these findings, temporally modulated neural activity has also been observed in human studies. these results suggest a close correlation between the temporal processing stages of facial information by it neurons and the temporal dynamics of face recognition. |
sequence alignment tools are essential to biological research [see, e.g. (1), for a survey of multiple sequence alignment methods ]. in addition to merely the residues / nucleotides, biologists often possess more knowledge regarding function, structure or conserved patterns of the sequences to be analyzed. it is generally desirable to have such information incorporated into an alignment procedure, so that the alignment result can be more biologically meaningful. for example, functionally important sites are generally expected to be aligned together, but a typical alignment tool often fails to achieve this if the sequence similarity is low. imposing constraints representing such information turns out to be an effective manner to incorporate biological knowledge into an alignment tool. (2) formulated the constrained multiple sequence alignment problem, where each constraint is a single residue / nucleotide. they considered alignment of rnase sequences, which are known to have a sequence of conserved residues his (h), lys (k) and his. using h, k, h as constraints, in the resulting constrained alignment each of these three residues can be found aligned together in a column of the alignment, appearing in the order as specified. (3) then proposed an improved algorithm for pairwise alignment and an approximation algorithm for multiple alignment. it is also noted that there have been other formulations regarding alignment with constraints proposed from different perspectives with various approaches (414). conserved sites of a protein / rna / dna family are often of several residues / nucleotides long. for these patterns, 15) proposed a generalized formulation and algorithm, where each constraint is a (usually short) string pattern allowing mismatches. lu and huang (16) then proposed a space efficient algorithm for this formulation. web - based systems, music (15) (available at http://genome.life.nctu.edu.tw/music) and music - me (16) (available at http://genome.life.nctu.edu.tw/musicme), were also developed ; from now on these two systems will be referred to as music jointly. with the aid of music, tsai. (15) and lu and huang (16) successfully identified a fragment in the 3 untranslated region (3-utr) of a sars (severe acute respiratory syndrome) coronavirus sequence that can fold into a pseudoknot, which is potentially responsible for self - replication of the virus. indeed, since its release, music has been found useful in, e.g. detection of functionally and/or structurally important residues / motifs in sequences (17,18), prediction of rna pseudoknotted structures (15,19,20), prediction of protein structures (21) and so on. there are, however, formulations of many biologically significant patterns beyond the capability of music. for example, many function - related protein sites as those collected in the prosite database (22) are expressed in regular expressions, which can not be modeled using the substring - with - mismatch formulation of constraints implemented in music. an example of regular expression patterns is the egf - like domain signature 2 (egf_2, ps01186 in prosite) : c - x - c - x(2)-[gp]-[fyw]-x(4,8)-c, which is related to the initiation of a signal transduction that results in dna synthesis and cell proliferation. the meaning of this pattern is that, the first residue is cys, followed by one residue of any kind, then a cys, followed by two residues of any kind, then a gly or pro, etc. regular expressions are also convenient in describing variable ranges between patterns or between blocks within a pattern, which is necessary for some single patterns themselves, and useful in applications where different patterns are expected to exhibit proximity in their occurrences. in the above example of egf_2, the x(4,8) symbol preceding the last cys indicates a range of length varying from 4 to 8 between a residue of [f, y or w ] (phe, tyr or trp) and that last cys. due to the usefulness of regular expressions in describing biological patterns, an enhanced web server, re - music (multiple sequence alignment with regular expression constraints), capable of handling regular expression constraints, is developed. dialign (8,9,12,13) (http://dialign.gobics.de/) is a well - known web server that can accept user - defined constraints as anchor points. it can be noted that the constraint formulation of dialign and the one of re - music are significantly different. in dialign, a constraint consists of the exact positions of a pair of equal - length segments on two of the sequences, where these two segments are expected to be aligned together. conflicts of constraints, if any, are resolved according to a weight function defined on the segment pairs. each pattern may occur many times in a sequence, where each occurrence needs not have the same length. the occurrences to be aligned together so as to satisfy the constraints will be those that can make the overall alignment optimized. re - music provides an intuitive user interface (figure 1). the user enters or pastes the input sequences (in fasta format) in the largest blank field. the format for the constraints follows the prosite pattern format (please see the help page at http://140.113.239.131/re-music/help.html for details). each constraint is put within quotes, and adjacent constraints are separated by space characters.. the preferred scoring matrix may be chosen, and the gap open / extension penalties can be assigned. the user can also enter an email address so that a hyperlink to the alignment result will be sent via email. the output page shows the constrained alignment with the regions for the satisfactions of the constraints shaded in yellow (figure 2b). on the output page the user can also choose to download the alignment result in fasta format or clustalw format. another field with title regular expression constraint(s) is for the user to enter the regular expression constraints, in a prosite - like format (for details please refer to the help page at http://140.113.239.131/re-music/help.html). in this figure, (b) a partial view of the alignment produced by re - music. a common pattern of these gst sequences, namely [st]-x(2)-[de] (ps00006), as obtained from prosite, is used as constraint. in the resulting alignment, re - music annotated the region for the satisfaction of the constraint with a yellow block. it can be seen that the g - site residues are aligned properly, as desired. for both tools, another field with title regular expression constraint(s) is for the user to enter the regular expression constraints, in a prosite - like format (for details please refer to the help page at http://140.113.239.131/re-music/help.html). in this figure, (b) a partial view of the alignment produced by re - music. a common pattern of these gst sequences, namely [st]-x(2)-[de] (ps00006), as obtained from prosite, is used as constraint. in the resulting alignment, re - music annotated the region for the satisfaction of the constraint with a yellow block. it can be seen that the g - site residues are aligned properly, as desired. sequence alignment tools are essential to biological research [see, e.g. (1), for a survey of multiple sequence alignment methods ]. in addition to merely the residues / nucleotides, biologists often possess more knowledge regarding function, structure or conserved patterns of the sequences to be analyzed. it is generally desirable to have such information incorporated into an alignment procedure, so that the alignment result can be more biologically meaningful. for example, functionally important sites are generally expected to be aligned together, but a typical alignment tool often fails to achieve this if the sequence similarity is low. imposing constraints representing such information turns out to be an effective manner to incorporate biological knowledge into an alignment tool. (2) formulated the constrained multiple sequence alignment problem, where each constraint is a single residue / nucleotide. they considered alignment of rnase sequences, which are known to have a sequence of conserved residues his (h), lys (k) and his. using h, k, h as constraints, in the resulting constrained alignment each of these three residues can be found aligned together in a column of the alignment, appearing in the order as specified. (3) then proposed an improved algorithm for pairwise alignment and an approximation algorithm for multiple alignment. it is also noted that there have been other formulations regarding alignment with constraints proposed from different perspectives with various approaches (414). conserved sites of a protein / rna / dna family are often of several residues / nucleotides long. for these patterns, 15) proposed a generalized formulation and algorithm, where each constraint is a (usually short) string pattern allowing mismatches. lu and huang (16) then proposed a space efficient algorithm for this formulation. web - based systems, music (15) (available at http://genome.life.nctu.edu.tw/music) and music - me (16) (available at http://genome.life.nctu.edu.tw/musicme), were also developed ; from now on these two systems will be referred to as music jointly. with the aid of music, tsai. (15) and lu and huang (16) successfully identified a fragment in the 3 untranslated region (3-utr) of a sars (severe acute respiratory syndrome) coronavirus sequence that can fold into a pseudoknot, which is potentially responsible for self - replication of the virus. indeed, since its release, music has been found useful in, e.g. detection of functionally and/or structurally important residues / motifs in sequences (17,18), prediction of rna pseudoknotted structures (15,19,20), prediction of protein structures (21) and so on. there are, however, formulations of many biologically significant patterns beyond the capability of music. for example, many function - related protein sites as those collected in the prosite database (22) are expressed in regular expressions, which can not be modeled using the substring - with - mismatch formulation of constraints implemented in music. an example of regular expression patterns is the egf - like domain signature 2 (egf_2, ps01186 in prosite) : c - x - c - x(2)-[gp]-[fyw]-x(4,8)-c, which is related to the initiation of a signal transduction that results in dna synthesis and cell proliferation. the meaning of this pattern is that, the first residue is cys, followed by one residue of any kind, then a cys, followed by two residues of any kind, then a gly or pro, etc. regular expressions are also convenient in describing variable ranges between patterns or between blocks within a pattern, which is necessary for some single patterns themselves, and useful in applications where different patterns are expected to exhibit proximity in their occurrences. in the above example of egf_2, the x(4,8) symbol preceding the last cys indicates a range of length varying from 4 to 8 between a residue of [f, y or w ] (phe, tyr or trp) and that last cys. due to the usefulness of regular expressions in describing biological patterns, an enhanced web server, re - music (multiple sequence alignment with regular expression constraints), capable of handling regular expression constraints, is developed. dialign (8,9,12,13) (http://dialign.gobics.de/) is a well - known web server that can accept user - defined constraints as anchor points. it can be noted that the constraint formulation of dialign and the one of re - music are significantly different. in dialign, a constraint consists of the exact positions of a pair of equal - length segments on two of the sequences, where these two segments are expected to be aligned together. conflicts of constraints, if any, are resolved according to a weight function defined on the segment pairs. each pattern may occur many times in a sequence, where each occurrence needs not have the same length. the occurrences to be aligned together so as to satisfy the constraints will be those that can make the overall alignment optimized. re - music provides an intuitive user interface (figure 1). the user enters or pastes the input sequences (in fasta format) in the largest blank field. the format for the constraints follows the prosite pattern format (please see the help page at http://140.113.239.131/re-music/help.html for details). each constraint is put within quotes, and adjacent constraints are separated by space characters.. the preferred scoring matrix may be chosen, and the gap open / extension penalties can be assigned. the user can also enter an email address so that a hyperlink to the alignment result will be sent via email. the output page shows the constrained alignment with the regions for the satisfactions of the constraints shaded in yellow (figure 2b). on the output page the user can also choose to download the alignment result in fasta format or clustalw format. another field with title regular expression constraint(s) is for the user to enter the regular expression constraints, in a prosite - like format (for details please refer to the help page at http://140.113.239.131/re-music/help.html). in this figure, (b) a partial view of the alignment produced by re - music. a common pattern of these gst sequences, namely [st]-x(2)-[de] (ps00006), as obtained from prosite, is used as constraint. in the resulting alignment, re - music annotated the region for the satisfaction of the constraint with a yellow block. it can be seen that the g - site residues are aligned properly, as desired. another field with title regular expression constraint(s) is for the user to enter the regular expression constraints, in a prosite - like format (for details please refer to the help page at http://140.113.239.131/re-music/help.html). in this figure, (b) a partial view of the alignment produced by re - music. a common pattern of these gst sequences, namely [st]-x(2)-[de] (ps00006), as obtained from prosite, is used as constraint. in the resulting alignment, re - music annotated the region for the satisfaction of the constraint with a yellow block. it can be seen that the g - site residues are aligned properly, as desired. for both tools, the regular expression constrained sequence alignment problem was originally formulated by arslan (23). the algorithm proposed in (23) is for pairwise alignment with a single constraint. in (24) arslan extended the algorithm in (23) to support multiple alignment with multiple constraints. unfortunately, the time complexity is extremely high, involving an exponential multiplicative factor in addition to the exponential time complexity for optimal (unconstrained) msa computations. even for pairwise alignment with multiple constraints, its worst case time and space requirements are intensive. in addition, the algorithms in (23,24) can not find in the resulting alignment the regions responsible for the satisfactions of the constraints ; only the alignment score, without the alignment itself, is reported. but being able to report alignments is important for a web server. it is therefore necessary to propose a solution more suitable for practical applications. for pairwise alignment with one regular expression constraint, in a previous study (25) we have proposed an algorithm, which is more efficient both in time and in space than the one in (23). furthermore, the alignment in addition to the score can be reconstructed without worsening the time and space complexity. in this work we extend the algorithm in (25) to support multiple constraints and multiple sequences, as required in re - music. the resulting algorithm is more efficient than the one in (24) for pairwise alignment with multiple constraints. to deal with multiple sequences, a progressive method is implemented, using our improved pairwise algorithm as the kernel. for details of the algorithm the reader the glutathione binding site (g - site) on glutathione s - transferase (gst) had been found to have conserved architectures across species (26). the chemical natures of their residues acting as g - site ligands and interactions facilitated with glutathione are also analogous (26). in a reasonable alignment of gst protein sequences, therefore, the residues for the g - site are expected to be aligned together. a structural superposition of the crystal structures of gst proteins from different species also suggests that most of these g - site residues should be aligned together (26). the sequence identity of those gst proteins from different species, however, is quite low ; for example, it is reported in (26) that the pairwise sequence identity between the a. thaliana gst and each of other six non - plant gsts is no more than 20.2%. in such a case, interfered by the low - similarity regions, it would be difficult for a typical alignment tool to align the important residues well. an experiment is therefore undertaken to examine the performance of a typical alignment tool in this case, as well as to demonstrate how re - music can be used to produce a more reasonable alignment. in this experiment we analyze three gst proteins : (i) atgst : a phi class gst from plant a. thaliana (pdbid : 1gnw) ; (ii) sjgst : an alpha class gst from non - mammalian s. japonicum (flat worm) (pdbid : 1m99) ; (iii) ssgst : a pi class gst from mammalian s. scrofa (pig) (pdbid : 2gsr). the result is shown in figure 2a, where active site residues shared by these gsts are boxed. it can be seen that, part of the g - site residues failed to be aligned together, due to the low sequence similarity among these gst proteins. by querying prosite with the three proteins, it is found that they all share the pattern ps00006 ([st]-x(2)-[de ]). using this pattern as constraint, re - music these suggest that, with some information about common patterns, re - music is more reliable to produce alignments in which biologically important residues can be lined up, which is particularly important when the sequence identity is low. being more reliable in aligning together important residues, re - music may also be applied to align an unknown sequence with other sequences whose relevant residues are known, thus providing a convenient and cheap way for a preliminary prediction of the residues in question on the unknown sequence. note also that, in this experiment, the knowledge about the active site residues are not utilized in constructing the alignment ; the constraints do not involve the active site residues themselves. such a property is useful when the residues to be predicted are not expected to be conserved in the sequence level. there is considerable evidence that suggests phylogenetically conserved pseudoknots found in the 3-utrs of various coronaviruses are involved in rna replication of these viruses (28). in an alignment of the 3-utr sequences of coronaviruses, however, it is often the case that the sequence identity among the coronaviruses from different groups is low. it is not an easy task for a typical alignment tool to align together the conserved pseudoknots. in this experiment four coronaviruses are considered in this experiment (genbank accession numbers in parentheses) : (i) hcov-229e : human 229e coronavirus (af304460), (ii) pedv : porcine epidemic diarrhea virus (af353511), (iii) bcov : bovine coronavirus (af220295) and (iv) mhv : murine hepatitis virus (af201929). the first two are group 1 coronaviruses, while the others belong to group 2. not surprisingly, since the sequence identity is low, the phylogenetically conserved pseudoknots (shaded regions) are not aligned well. in (28), predicted secondary structures of the pseudoknots found in the 3-utr of various coronaviruses are given. a consensus of the pseudoknots is to be taken. since, in general, loops in pseudoknots are less conserved, to enhance flexibility, we exclude loop regions nucleotides from the consensus. then the consensus of the pseudoknots can be described as x(5)-c - u - x(4)-c - x(15,16)-u - g - x(2)-a - x(5,7)-g - x(4)-a - g - x(7,10)-u - x(3)-a - x(5). using this consensus as the constraint, re - music is applied to align these 3-utr sequences again. in figure 3b, the pseudoknot regions on these coronaviruses can be seen to have been aligned properly. this demonstrates that re - music can be used to help locate fragments that are conserved in structure. actually, this property, being a common advantage of the music series, had been utilized to predict the pseudoknot in the 3-utr of the sars - tw1 coronavirus by aligning the 3-utr of sars - tw1 with those of some other coronaviruses whose pseudoknot regions are known (15,16). re - music further makes it possible to provide the flexibility of variable ranges between conserved nucleotides or regions in constraints, which is necessary for describing the whole consensus of the pseudoknot in this experiment. the shaded regions, corresponding to the phylogenetically conserved pseudoknots, are not aligned well. as expected, the regions for the pseudoknots are aligned properly by re - music. for both tools, the shaded regions, corresponding to the phylogenetically conserved pseudoknots, are not aligned well. as expected, the regions for the pseudoknots are aligned properly by re - music. for both tools, the glutathione binding site (g - site) on glutathione s - transferase (gst) had been found to have conserved architectures across species (26). the chemical natures of their residues acting as g - site ligands and interactions facilitated with glutathione are also analogous (26). in a reasonable alignment of gst protein sequences, therefore, the residues for the g - site are expected to be aligned together. a structural superposition of the crystal structures of gst proteins from different species also suggests that most of these g - site residues should be aligned together (26). the sequence identity of those gst proteins from different species, however, is quite low ; for example, it is reported in (26) that the pairwise sequence identity between the a. thaliana gst and each of other six non - plant gsts is no more than 20.2%. in such a case, interfered by the low - similarity regions, it would be difficult for a typical alignment tool to align the important residues well. an experiment is therefore undertaken to examine the performance of a typical alignment tool in this case, as well as to demonstrate how re - music can be used to produce a more reasonable alignment. in this experiment we analyze three gst proteins : (i) atgst : a phi class gst from plant a. thaliana (pdbid : 1gnw) ; (ii) sjgst : an alpha class gst from non - mammalian s. japonicum (flat worm) (pdbid : 1m99) ; (iii) ssgst : a pi class gst from mammalian s. scrofa (pig) (pdbid : 2gsr). the result is shown in figure 2a, where active site residues shared by these gsts are boxed. it can be seen that, part of the g - site residues failed to be aligned together, due to the low sequence similarity among these gst proteins. by querying prosite with the three proteins, it is found that they all share the pattern ps00006 ([st]-x(2)-[de ]). using this pattern as constraint, re - music these suggest that, with some information about common patterns, re - music is more reliable to produce alignments in which biologically important residues can be lined up, which is particularly important when the sequence identity is low. being more reliable in aligning together important residues, re - music may also be applied to align an unknown sequence with other sequences whose relevant residues are known, thus providing a convenient and cheap way for a preliminary prediction of the residues in question on the unknown sequence. note also that, in this experiment, the knowledge about the active site residues are not utilized in constructing the alignment ; the constraints do not involve the active site residues themselves. such a property is useful when the residues to be predicted are not expected to be conserved in the sequence level. there is considerable evidence that suggests phylogenetically conserved pseudoknots found in the 3-utrs of various coronaviruses are involved in rna replication of these viruses (28). in an alignment of the 3-utr sequences of coronaviruses, however, it is often the case that the sequence identity among the coronaviruses from different groups is low. it is not an easy task for a typical alignment tool to align together the conserved pseudoknots. in this experiment four coronaviruses are considered in this experiment (genbank accession numbers in parentheses) : (i) hcov-229e : human 229e coronavirus (af304460), (ii) pedv : porcine epidemic diarrhea virus (af353511), (iii) bcov : bovine coronavirus (af220295) and (iv) mhv : murine hepatitis virus (af201929). the first two are group 1 coronaviruses, while the others belong to group 2. not surprisingly, since the sequence identity is low, the phylogenetically conserved pseudoknots (shaded regions) are not aligned well. in (28), predicted secondary structures of the pseudoknots found in the 3-utr of various coronaviruses are given. since, in general, loops in pseudoknots are less conserved, to enhance flexibility, we exclude loop regions nucleotides from the consensus. then the consensus of the pseudoknots can be described as x(5)-c - u - x(4)-c - x(15,16)-u - g - x(2)-a - x(5,7)-g - x(4)-a - g - x(7,10)-u - x(3)-a - x(5). using this consensus as the constraint, re - music is applied to align these 3-utr sequences again. in figure 3b, the pseudoknot regions on these coronaviruses can be seen to have been aligned properly. this demonstrates that re - music can be used to help locate fragments that are conserved in structure. actually, this property, being a common advantage of the music series, had been utilized to predict the pseudoknot in the 3-utr of the sars - tw1 coronavirus by aligning the 3-utr of sars - tw1 with those of some other coronaviruses whose pseudoknot regions are known (15,16). re - music further makes it possible to provide the flexibility of variable ranges between conserved nucleotides or regions in constraints, which is necessary for describing the whole consensus of the pseudoknot in this experiment. (a) a partial view of the alignment produced by clustalw. the shaded regions, corresponding to the phylogenetically conserved pseudoknots, are not aligned well. as expected, the regions for the pseudoknots are aligned properly by re - music. the shaded regions, corresponding to the phylogenetically conserved pseudoknots, are not aligned well. as expected, the regions for the pseudoknots are aligned properly by re - music. for both tools, re - music adopts regular expressions as its constraint formulation, which is useful in expressing prosite patterns or structural elements that often involve variable ranges between conserved parts. the algorithm underlying re - music represents an improvement over the previously proposed algorithm, and is more appropriate for implementation in a web - server. experiments on gst proteins and on coronaviruses with phylogenetically conserved pseudoknots demonstrate that, with additional knowledge incorporated, re - music is able to produce meaningful alignments in which important residues or structural elements can be aligned properly, even if the similarity among input sequences is low. | re - music is a web - based multiple sequence alignment tool that can incorporate biological knowledge about structure, function, or conserved patterns regarding the sequences of interest. it accepts amino acid or nucleic acid sequences and a set of constraints as inputs. the constraints are pattern descriptions, instead of exact positions of fragments to be aligned together. the output is an alignment where for each pattern (constraint), an occurrence on each sequence can be found aligned together with those on the other sequences, in a manner that the overall alignment is optimized. its predecessor, music, has been found useful by researchers since its release in 2004. however, it is noticed in applications that the pattern formulation adopted in music, namely, plain strings allowing mismatches, is not expressive and flexible enough. the constraint formulation adopted in re - music is therefore enhanced to be regular expressions, which is convenient in expressing many biologically significant patterns like those collected in the prosite database, or structural consensuses that often involve variable ranges between conserved parts. experiments demonstrate that re - music can be used to help predict important residues and locate phylogenetically conserved structural elements. re - music is available on - line at http://140.113.239.131/re-music. |
the immune system is composed of two major subdivisions the innate immune system and the adaptive immune system. the innate immune system, comprised of cytokines, macrophages, and nk cells is rapidly responsive, while the adaptive system is antigen specific and relatively slow to develop. on the other hand, immunotherapy using activated mononuclear cells is a way to harness the adaptive immune response, which is comprised of the antigen - presenting cells (apcs) including dcs and cd4 and cd8 t cells to fight malignancies. the apcs activate t cells by processing antigens and present them to t - cell receptors (tcrs) in the context of the mhc restriction, while cd4 t cells include both helper and regulatory t cells (treg). humoral immune responses are usually thought to play an important role in inflammation, which is characterized by edema and the recruitment of phagocytic cells. actually, these humoral factors are found in serum in patients with malignancies, or they are formed at the site of tumorigenesis. immunological treatment strategies for cancer fall into two distinctive categories, namely, specific and nonspecific immunotherapy. nonspecific immunotherapy induces inflammation or otherwise amplifies an already present immune response, for example, ifn, il-2, and bacillus calmette - guerin (bcg). for decades bcg in non - muscle - invasive bladder cancer is standard primary therapy, and il-2 in renal cell carcinoma is adjunctive therapy. specific immunotherapy makes use of antigen - specific t lymphocytes or antibodies produced by b lymphocytes. recently, prostate cancer vaccines have attempted to induce cancer - specific systemic immune responses and represent a new class of targeted therapies. several immunotherapeutic strategies effective against prostate, bladder, or renal cancer in animal models are under clinical investigation for their efficacy against human gu malignancies. in addition to existing therapies, novel approaches that attempt to exploit the immune system ability to identify, target, and eradicate gu malignancies are now being developed. this review highlights current immunotherapy strategies that may prove to be successful treatments for gu malignancies. kidney cancer is the tenth leading cause of cancer deaths in males in the united states, and death rates of kidney cancer are the highest among american indians / alaskan natives. in europe approximately 14,000 people die annually of renal tumors with an estimated 27,000 new cases per year. about two - thirds of all patients present with localized disease, which can mostly be cured by radical or partial nephrectomy with a 60% to 70% 5-year survival rate. a third of patients present with metastatic disease and the prognosis in patients with metastatic renal cell carcinoma (mrcc) is poor with approximately 1-year median survival and a 10% to 20% 2-year survival rate [2, 3 ]. this is largely due to the absence of effective chemotherapy agents and the limited usefulness of radiation therapy for mrcc. several risk factors for developing rcc have been reported, including smoking, hypertension, and obesity [4, 5 ]. the association to obesity is widely accepted and has been reported consistently in several studies [68 ]. there is evidence of elevated levels of numerous proinflammatory molecules in the blood of obese [911 ]. also, the association between obesity and kidney disease is described, suggesting that inflammation could play an important role in the pathogenic mechanism of renal injury in obese patients [12, 13 ]. indeed, c - reactive protein (crp) represents a promising prognostic variable in patients with rcc [1418 ]. potential biologic mechanisms that have been hypothesized, including higher levels of estrogen and insulin, higher concentrations of growth factors in adipose tissue, and immune dysfunction reported that levels of leptin, which is produced in adipose tissue and plays a modulatory role between metabolism and immunity, were inversely associated with rcc risk. adipose tissue produces a variety of inflammatory factors, including leptin, adiponectin, as well as cytokines. indeed, plasma leptin levels are strongly associated with total adipose tissue mass [2023 ]. the exact mechanism by which obesity, a state of chronic, slightly systemic inflammation, is a risk factor for developing rcc still remains unknown. not only is plasma leptin increased in obese subjects, but leptin is decreased in adipose tissue mass induced by exercise and anorexia nervosa. interestingly, it is reported that excess risk for developing rcc was observed among patients with low plasma levels of leptin, after adjusting for potential confounding factors, such as central obesity, dm and adiponectin. given the immunogenic nature of rcc and the role of leptin in the regulation of immunocompetence, crosstalk between lymphocytes and adipocytes may contribute to immune regulation in patients with rcc, contributing to tumor development. in addition to drugs already used in clinical practice, novel drugs are already under evaluation in clinical trials (table 1). immunotherapy for mrcc mainly involves the direct administration of effector molecules or cells to a patient and requires no relationship with the host immune system. cytokines can indirectly affect tumor growth by inducing cytolytic t cells or by acting directly on tumor cells. interferon (ifn)-alfa and il-2 are widely studied examples of passive immunotherapy for mrcc. since il-2 has believed to have no direct impact on mrcc cells, the effect of il-2 on mrcc is believed to be its ability to expand t - cell populations with antitumor activity. if, however, the cytokine activate 's the host immune system, these cytokine therapies are considered to be active immunotherapy. evidence from knockout mice suggests that il-2 is crucial for the homeostasis and function of cd4 cd25 regulatory t cells in vivo. although the response rate in patients with mrcc treated with il-2 varies between 10% and 20%, some responses are durable. high - dose il-2 appears to be able to cure a small percentage of highly selected patients. some studies suggest that combination therapies involving il-2 combined with additional cytokines may be more effective than il-2 alone.. investigated the efficacy of combination therapy of low - dose il-2 and ifn - alpha. in the 46 patients evaluated in phase 1 and phase 2, the response rate was 26.1% (12 of 46 patients), being highest in 38.7% (12 of 31 patients) of those who were nephrectomized, and with only lung metastases. conducted phase 3, randomised, multicentre trial of maintenance immunotherapy with low - dose il-2 and ifn - alfa for mrcc patients. maintenance immunotherapy after disease progression was found to be feasible but did not significantly increase os. oblimersen is an 18-base oligodeoxynucleotide encoding antisense to the gene for bcl-2, an antiapoptotic protein that is upregulated in renal and other cancers. margoline. evaluated the combination of oblimersen with ifn - alfa in mrcc. they found that only 1 patient of 23 patients enrolled in the study had a partial response lasting 2.5 months, concluding oblimersen given in the dose and schedule used with ifn - alfa does not appear sufficiently active to warrant further study in mrcc. another cytokine, il-12, induces the differentiation of t cells into a t - helper-1(th1) phenotype. cd8 population of the th1 phenotype is considered to be cytotoxic t lymphocyte (ctl) which is highly cytolytic. in a murine model combination therapy with il-12 and il-2 caused the regression of primary and metastatic disease with significantly better results than solo treatment with either agent. granulocyte - macrophage colony - stimulating factor (gm - csf) has received greater focus from the therapeutic perspective because of its ability to activate monocyte and macrophages, which can directly mediate antitumor activity through activated macrophages to release tnf - alfa. garcia. conducted phase 2 trial of subcutaneous il-2, gm - csf, and ifn - alfa in patients with mrcc. the overall response rate was 20% (one complete response and 11 partial responses of 60 patients), and the median progression - free survival and overall survival were 6.0 and 23.4 months, respectively, suggesting the potent efficacy. flt3 ligand (flt3-l), a member of a growth factors that stimulate the proliferation of hematopoietic stem cells, is being tested for its ability to increase dc numbers. flt3 has also been tested in mrcc patients in clinical trials, and results demonstrated that although flt3-l is capable of inducing the expansion of circulating dcs in patients with mrcc, it lacked relevant clinical activity at the doses and schedules examined [33, 34 ]. active specific immunotherapy includes the vaccination of patients virtually to induce long - lasting, tumor - specific immunity which is capable of rejecting cancer cells as well as inducing safe and protective immunological memory. autologous tumor cell vaccines are being used in an attempt to induce tumor - specific immune responses in mrcc. these vaccines include genetically altered tumor cells to enhance the immunogenicity of the tumor cells, thereby, inducing a tumor - specific t - cell response. schendel. tested the possible application of vaccination with allogeneic tumor cells. in the study, a human rcc cell line was genetically modified by retroviral transduction to express the costimulatory molecule cd80. it was possible to isolate ctl clones that were able to accomplish tumor lyses that expressed all of the corresponding allospecificities, demonstrating that the induction of allospecific responses did not hinder the development of tumor - associated ctls in vitro. in practice, lemoine. conducted phase 2 trial to check whether systemic administration of il-2 or infusion of dcs loaded with tumor extracts could lead to some response rates with concomitant survival improvements. no adverse effect due to the vaccinations was observed in 5 patients enrolled. a specific immune response against autologous tumor cells a transient and massive increase of circulating natural regulatory t - cells was evidenced in 3 patients following il-2 administration. these results may support the use of modified allogeneic tumor cells for the vaccination of partial mhc - matched patients with rcc. newer studies suggest that targeted therapy for mrcc, including sunitinib, sorafenib, temsirolimus, everolimus, or combined bevacizumab and ifn - alfa, has proved efficacious as first- or second - line treatment although they have not yet shown a statistically significant survival benefit except for temsirolimus in the high - risk mrcc subgroup. cho., conducted a retrospective analysis of the tolerability and efficacy of il-2 therapy in patients who had previously received vegf - targeted therapy including tyrosine - kinase inhibitor (tki). no patients achieved a partial or complete response to therapy, and the incidence of severe cardiac toxicities in patients receiving prior tki reached 40%, concluding that the assumption that il-2 therapy can be safely administered after tki therapy may not be valid. tested the activity and tolerability of tki sorafenib administered with ifn - alpha-2b as first- or second - line therapy in mrcc patients. in this phase 2 study, the response rate was 33% (95% ci, 19% to 49% ; 13 of 40 patients), including 28% partial responses (n = 11) and 5% complete responses (n = 2), with the median duration of response 12 months. accumulating data by larger cohort will further the rationale for new drugs based on combination therapy with immunotherapy to enhance the effect of immunomodulators on patient survival. bladder cancer is the 4th most common genitourinary cancer in men and the 7th in women with an incidence of more than 70,000 new cases in the united states in 2010. at presentation up roughly 80% of patients are found to have non - muscle - invasive bladder cancer (nmibc), which is disease confined to the superficial layer of the bladder (mucosa, lamina propria), specifically ta, t1 or carcinoma in situ (cis). currently the gold standard treatment for noninvasive bladder cancer is bcg instillation into the bladder, which is now the most commonly used intravesical treatment for high - risk nmibc endorsed in european association of urology and american urological association practice guidelines [4042 ]. virtually it is believed that there exists a host - immune escape associated with bladder cancer. hence, the generation of a localized immune response in the bladder by the intravesical administration of live bcg may transiently restore impaired immune system in the peritumoral bladder wall, so that bcg can elicit the tumor rejection. some studies were conducted to evaluate the mechanism of binding of bcg within the bladder. in a mouse model, it is known that bcg attaches to the bladder wall only in areas with urothelial damage. studies performed using purified extracellular matrix proteins to identify the proteins responsible for attachment suggest that bcg preferentially attaches to surfaces coated with purified fibronectin (fn) and to a lesser extent to other purified proteins including laminin, collagen, or fibrinogen. immunological aspects of bcg therapy for nmibc are related to the presence of delayed - type hypersensitivity. inflammatory response occurs after bcg instillation in to the bladder, which is characterized by an induction of leukocyte subpopulations, such as granulocytes, cd4 and cd8 t cells, nk cells, b lymphocytes, activated lymphocytes, and dendritic cells. following this cellular recruitment, cytokines characterized as part of the th1 and th2 immune response accumulate. cytokines which are in charge of the antitumor response are essentially those related to th1, including il-2, il-12, tnf - alfa, and ifn - gamma. intravesical bcg, a nonspecific active immunotherapy, has been used in the intravesical treatment of nmibc for about 35 years. despite an initial treatment success patients with cis are more at risk for recurrence or advanced disease. despite bcg treatment 42% to 83% of patients with cis associate with papillary nmibc and 20% to 34% with primary cis experience progression to muscle invasive disease. although bcg is an effective adjuvant treatment for preventing bladder cancer recurrence, it is associated with a high incidence of adverse effects, which include nausea, vomiting, weight loss, anorexia, bladder irritation, dysuria, polyuria, hematuria, cystitis, urinary urgency, urinary tract infections, flu - like syndrome, and so on [46, 47 ]. no consensus has been reached about the optimal dose for bcg therapy nor about how the toxicity of bcg treatment can be reduced. variations in the reported frequency of bcg - associated adverse events could be caused by variations in the dose of bcg used. therefore, dose reductions may be a potential option for the prevention of bcg - associated adverse events, particularly for those patients known to be intolerant to standard - dose bcg. mack and frick reported the results of a phase 2 study with low - dose bcg therapy in high - risk nmibc and concluded that low - dose bcg therapy is an effective treatment in high - risk t1 bladder cancer, especially with maintenance therapy to prevent progression and recurrence. long - term outcome of a low - dose intravesical bcg therapy for cis of the bladder is reported. complete response was achieved in 84% of the patients, in whom the recurrence - free rate was 72.4% after 3 years and 61.9% after 5 years. the median cr duration was 37.5 months, suggesting the efficacy and safety of low - dose bcg therapy for cis of the bladder. a number of unresolved questions surround the bcg host interplay which may be characterized by a number of different strains of bcg. bcg daughter strains are divided into the early strains : japan, birkhaug, russia and brazil, which were brought to each country between 1924 and 1926, and the late strains : pasteur, danish, glaxo and connaught, which were obtained after 1931. although all of these strains are descendants of the original m. bovis isolate, subsequent passage under different conditions has resulted in a variety of strains with unique genetic alterations. dutch south east cooperative urological group evaluated bcg - tice versus bcg - rivm in 469 patients with pta / pt1 carcinoma and found that there were no statistical differences were observed in toxicity between the two strains of bcg. connaught (canada), pasteur (france), armand frappier (canada), and tokyo 172 (japan) were employed and studied for the efficacy in low - dose regimens [5153 ]. lamm reported that there were differences in complication rates with various bcg strains [47, 54 ]. the incidence of cystitis like symptoms, haematuria, and fever in our series was 64%, 40%, and 20%, respectively [47, 54 ]. prospectively evaluated the efficacy and adverse events of low dose (40 mg) tokyo 172 strain. there was no significant difference in tumor recurrence rate between the low dose (40 mg) group and the standard dose (80 mg) group. similarly, takashi. reported that the dose (40 versus 80 mg) of bcg was not a significant determinant for cr in patients with cis of the bladder. these studies suggest that 40 mg would be an adequate dose for tokyo 172 strain, and comparable study is possible with a dose of 40 mg. no prospective studies have been conducted to compare low dose tokyo 172 strain (40 mg) with other bcg substrains. delay or interruption of instillation due to side - effects may actually be detrimental to efficacy. so an important issue is whether a low - dose regimen can reduce toxicity while maintaining efficacy. from a phase iii randomized trial comparing low - dose versus standard - dose bcg (pasteur strain, 75 versus 150 mg), pagano. reported a significant decrease in most of the common side effects (cystitis, fever, haematuria ; p < 0.05), clarifying the relationships between dose and toxicity. studies, however, provided only short - term followup ranging from months to two years and data on the long - term condition of bladder patients originally treated with low - dose bcg is very rare. retrospectively reviewed 70 consecutive patients with primary or secondary carcinoma in situ with or without concomitant solitary or multifocal papillary tumor treated with weekly instillations of low - dose pasteur strain for 6 weeks with median followup of 74 months. mean time was 18 months (range 6 to 69) to treatment failure and 13 months (range 7 to 53) to progression. they conclude that low - dose bcg is similarly effective, with a lower incidence of side effects and long - lasting positive outcome. similarly, kamel. retrospectively evaluated 74 patients with g3, t1 bladder cancer treated by a 6-week course of low - dose pasteur strain with median followup of 61 months. regarding toxicity, irritative symptoms occurred in 24% of patients, fever in 9%, microscopic hematuria in 14%, which appeared to be lower when compared with the rates reported for regular doses of bcg. assessed the effectiveness of low - dose bcg for high - risk t1/g3 bladder cancer patients who had weekly instillations of low - dose pasteur strain for 6-week. with a median followup of 33 months, 28 of 51 patients (54.9%) were disease - free, and the risk of treatment failure was significantly greater for solid than papillary tumors (p = 0.0006), recurrent than primary tumors (p = 0.0052), and coexisting carcinoma in situ (p = 0.124) in multivariate analysis, suggesting that this low - dose pasteur bcg is effective in the treatment of high - risk nmibc, except for some tumor characteristics, such as solid appearance, coexisting carcinoma in situ, history of superficial transitional cell carcinoma, and early relapse after the initial induction course. in contrast, herr concluded that patients with highly malignant bladder cancer would not benefit from a dose reduction. now although, long - term tolerance remains an important issue with maintenance schedule. because complex immunological pathway contributes to the success of bcg therapy, stratification of the patients by their immunological aspects may help physicians to predict the response to bcg. immunotherapy has also been carried out on other gu malignancies, except for mrcc and nmibc, and primarily the prostate cancer has been a particular focus of immunotherapy. despite improvements in surgery or radiotherapy for localized prostate cancer, 30% will develop metastatic disease. once become metastatic, usually to the bone, the disease is no longer curable and is usually treated with androgen depletion therapy (adt). some studies support the evidenced that adt induce an immune response against the prostate, which includes t - cell infiltration of the prostate and induce thymic regeneration. disappointingly, over 50% of these patients treated by adt progress to castration - resistant prostate cancer (crpc) within a median of 18 to 24 months. crpc remains an incurable disease when treated with docetaxel - based chemotherapy, and prednisone currently the only fda - approved chemotherapeutic agent for the treatment of crpc, because docetaxel - prednisone extended median overall survival modestly to 19 months and only 20% patients attained 3-year survival based on the results of two large randomized trials [60, 61 ]. several immunotherapeutic strategies effective against prostate cancer in animal models are under clinical investigation for their efficacy against human crpc. cytokines, including il-2 and ifn - alfa, which are mainly used in mrcc patients may also be useful for treating other gu malignancies. in a pilot study of crpc involving il-2 and ifn - alfa administration, some partial responses and psa serum level decreases were reported. in the last 10 years, many cancer vaccines, to be specific active immunotherapy against specific tumor - associated antigens, were tried in clinical trials. these vaccines include dc, whole tumor cell, peptide, viral vectors, and so on. most recently, us fda approval of 2 immunotherapies including sipuleucel - t (provenge, dendreon corp, wa, usa) and ipilimumab (yervoy, bristol - myers squibb). sipuleucel - t is a novel autologous dendritic cell - based vaccine, and the tissue - specific antigen for immunization is prostatic acid phosphatase (pap), which is expressed in about 95% of prostate tumors, and has highly specific expression for prostatic tissue. pap is linked to gm - csf, so that gm - csf functions to enable efficient gm - csf - receptor - mediated uptake of the pap antigen, resulting in enhancing its antigenicity and dc - stimulating properties, moiety and augments the immune response. in a randomized crossover trial, 127 previously the median survival for sipuleucel - t was significantly better than for placebo (25.9 versus 21.4 months, p = 0.01) and 3-year survival was prolonged with sipuleucel - t (34 versus 11%, p = 0.0046). also, in the double - blind, placebo - controlled, multicenter phase 3 trial, there was a relative reduction of 22% in the risk of death in the sipuleucel - t group as compared with the placebo group (p = 0.03), suggesting that sipuleucel - t prolongs overall survival among men with metastatic crpc. the activation of negative regulatory signals in t cells is required to avert an unduly exuberant immune response, which include cytotoxic t - lymphocyte antigen-4 (ctla-4) which is essential for maintaining tolerance for self - antigens. ipilimumab, ctla-4-inhibiting fully human monoclonal antibodies have demonstrated objective clinical and psa responses in advanced crpc in phase 1 and phase 2 trials. in addition, other novel targets for immunotherapy against crpc are likely to expand the therapeutic venue in the near future. therefore, the proper sequence of immunotherapy and appropriate selection of patients will assume benefit. recent advances in immunotherapy in gu malignancies have provided insight into the complexity of immune manipulation and the promise of improving efficacy and reducing adverse reactions by better understanding of mechanisms of immunotherapy. despite the clinical failures of some immunological treatments, several therapies | most cancer patients are treated with some combination of surgery, radiation, and chemotherapy. despite recent advances in local therapy with curative intent, chemotherapeutic treatments for metastatic disease often remain unsatisfying due to severe side effects and incomplete long - term remission. therefore, the evaluation of novel therapeutic options is of great interest. conventional, along with newer treatment strategies target the immune system that suppresses genitourinary (gu) malignancies. metastatic renal cell carcinoma and non - muscle - invasive bladder caner represent the most immune - responsive types of all human cancer. this review examines the rationale and emerging evidence supporting the anticancer activity of immunotherapy, against gu malignancies. |
the aryl hydrocarbon receptor (ahr) is a transcription factor that belongs to the basic helix - loop - helix /per - arnt - sim family of dna binding proteins. there are two major categories of environmental compounds that activate ahr signaling : halogenated aromatic hydrocarbons (hah), such as 2,3,7,8-tetrachlorodibenzo - p - dioxin (tcdd) and polycyclic aromatic hydrocarbons (pah), such as benzo(a)pyrene. unliganded ahr forms a complex including two copies of 90kd a heat shock protein (hsp90), one x - associated protein (xap), and one p23 molecular chaperone protein in the cytoplasm.[14 ] after being activated by its ligands, cytoplasmic ahr translocates into the nucleus, disassociates from the chaperone complex, dimerizes with the aryl hydrocarbon receptor nuclear translocator (arnt) and transactivates target genes through binding to dioxin response elements (dre) in promoter regions. ahr target genes include phase i and phase ii metabolic enzymes, such as cytochrome p450 1a1 (cyp1a1), cytochrome p450 1b1 (cyp1b1), nad(p)h : quinone oxidoreductase i (nqo1) and aldehyde dehydrogenase 3 (alhd3a1) [figure 1 ]. the induction of xenobiotic metabolizing enzymes following ahr activation is considered, at least in part, an adaptive response of the organism to its environment, which could decrease the potential toxicity of foreign chemicals. on the other hand, activation of ahr aryl hydrocarbon receptor signaling pathway the ahr molecule varies significantly across species in mediating tcdd toxicity, as well as in molecular weight by almost 30kd, which is primarily due to the different positions of the translational termination codon. four murine ahr alleles, ahr, ahr, ahr and ahr, have been found and cloned from different inbred and wild mouse strains.[69 ] the ahr receptor has a lower ligand - binding affinity compared to the ahr and ahr alleles. the ahr allele encodes a protein of 805 amino acids, the ahr and ahr alleles encode proteins of 848 amino acids, and the ahr allele encodes a protein of 883 amino acids. all proteins of the four alleles contain a basic helix - loop - helix motif (bhlh), per - arnt - sim (pas) domain and a transactivation domain (tad), and their varied amino acids exist at the carboxyl end. human ahr is identical to mouse ahr at n - terminus and has 60% identity with mouse ahr at c - terminus. an ala375val375 polymorphism is responsible for the reduced ligand - binding affinity of the ahr receptor compared with the ahr receptor in both rodent and human. aryl hydrocarbon receptor structure through evolution of multicellular organisms, the function of ahr in environmental adaption has also been put to use in important physiological processes. ahr mrna is expressed in multiple human tissues, with the highest expression in the placenta, relatively high expression in the lung, heart, pancreas and liver, and lowest expression in the kidney, brain and skeletal muscle. its mrna has also been detected in multiple vascular beds in human, including pulmonary microvasclature, aortic arch and umbilical vein. in the absence of exogenous ligands, the intrinsic activity of ahr signaling is subject to regulation by either endogenous ligands, including 2-(1h - indole-3-carbonyl)-thiazole- 4-carboxylic acid methyl ester, arachidonic acid metabolites, such as prostaglanding2 and lipoxin4a, and heme metabolites, such as bilirubin ; or nonligand activators, such as shear stress, camp and modified low - density lipoprotein (ldl).[1422 ] although none of these factors have been proved as high - affinity physiological activators of ahr, the endogenous function of ahr signaling, including heart function, vascular development and blood pressure regulation, has been characterized using ahr gene - deficient mice. due to the nature of ahr, a mediator of xenobiotics and a potential target in genetic modification of cardiovascular function, in this review, the function of this receptor in the cardiovascular system is summarized and discussed, which may shed light on the development of a new therapeutic methodology in cardiovascular disease prevention and treatment. in the 1990s, ahr - deficient mice were developed independently in three labs, by either deleting exon 1 or exon 2 of the gene. all three ahr - null mice had a mixed c57bl/6 129 background and displayed a slower growth rate within the first few weeks after birth, tcdd resistance, failure of xenobiotic cyp1a1 and cyp1a2 induction, maintained but decreased fertility and liver pathology. ahr - deficient mice develop cardiac hypertrophy and fibrosis in adulthood with a sophisticated mechanism.[2628 ] early characterization of the enlarged heart in ahr - null mice suggested that enhanced vascular endothelial growth factor (vegf) expression may contribute to the hypertrophy phenotype. in 2003, vasquez., reported increased size of cardiomyocytes and an anatomic remodeling without typical features of molecular remodeling, which was not consistent with hypertrophic growth secondary to pressure or volume overload. this suggested an intrinsic role of ahr in cardiomyocyte size control. in the same year, lund., indicated that cardiac hypertrophy in ahr - null mice was associated with high systemic arterial blood pressure as well as increased circulating angiotensin ii (ang ii) and plasma endothelin-1 (et-1) level. this cardiac hypertrophic phenotype was primarily mediated by elevated circulating et-1, thus treatment with bq-123, an eta receptor antagonist, significantly attenuated the phenotype as well as the mrna expression of cardiac hypertrophy markers, atrial natriuretic factor (anf) and -myosin heavy chain (-mhc). cardiac fibrosis were observed by both groups in ahr - null mice, suggesting a functional remodeling of the heart. a further study on altitude acclimated low blood pressure ahr knockout mice revealed that the hypertrophied heart is more likely a compensatory physiological effect to increase cardiac output in an attempt to increase blood pressure. this is consistent with the absence of pathological cardiac hypertrophy markers reported by vasquez. a recent study on ahr - null mice also indicated cardiac hypertrophy and fibrosis, which might involve vav3, an activator of rho / rac gtpases, regulated by ahr. the authors also demonstrated a thickening of arterial media wall and increased number of vascular smooth muscle cells in arterial walls. all the research data above, although inconsistently, suggest that local ahr signaling contributes to the development of cardiac hypertrophy and fibrosis that reflects a cardiac functional remodeling. the role of endogenous ahr in vascular development is also uncovered from the research on ahr knockout mice, which exhibit a spectrum of hepatic defects, including portal fibrosis and a smaller liver. the mechanism underlying the liver defect seems due to fetal hepatic necrosis caused by compromised perfusion, and partially resulted from a patent ductus venosus in adulthood, which is mediated by loss of ahr in endothelial cells specifically.[3436 ] abnormal vascular structures have also been reported in the liver, kidney and hyaloid of ahr knockout mice. a further investigation showed that the mice carrying the hypomorphic ahr allele also develop patent ductus venosus, which could be rescued by tcdd treatment. in addition, nuclear translocation and dna binding abilities of ahr are both required in the closure of ductus venosus, suggesting a transactivation mechanism in this particular endogenous ahr function. taken together, these two models suggest that the endogenous and exogenous ligand - activated ahr signaling may share the same signal transduction mechanism in mediating vascular development. the role of the ahr agonist, tcdd, in inducing high blood pressure has been demonstrated in both epidemiology studies and research using mouse models, in which ahr - mediated cytochrome p450 overexpression may be involved.[3943 ] due to the similarity between endogenous and exogenous ahr signaling, it is not surprising that endogenous ahr also contributes to blood pressure regulation in addition to the cardiovascular development mentioned above. anesthetized ahr - null mice were first found hypotensive in the absence of a heart rate difference at eight months of age. the authors also reported a decreased cardiac output caused by diminished stroke volume in four - month - old ahr knockout mice. later in the same year, lund., reported high blood pressure in conscious ahr - null mice, associated with elevated circulating ang ii and et-1 levels. in this study, angiotensin converting enzyme blockade by captopril attenuated, but did not normalize elevated arterial blood pressure. subsequently, et-1 was identified as the primary factor causing high arterial blood pressure in those ahr - null mice. treatment with bq-123, an eta receptor blocker, dramatically attenuated mean arterial blood pressure as well as plasma ang ii levels in ahr - null mice, suggesting increased ang ii as a secondary effect of et-1 elevation. another group also reported elevated arterial blood pressure in ahr - null mice, which was normalized by captopril treatment. their model also suggested an increase of vascular -1d adrenoceptor expression that was involved in the hypertensive phenotype. interestingly, both groups reported hypertension in ahr - null mice located at mild high altitude (albuquerque nm, 1620 m ; mexico city, 2240 m). a further investigation of blood pressure in ahr - null mice indicated that loss of endogenous ahr signaling in mice led to hypotension at sea level and hypertension at mild high altitude, which was caused by different atmospheric oxygen levels. a recent study performed by the group in albuquerque comprehensively investigated the role of ahr in blood pressure regulation using ahr heterozygous and null mice. their up - to - date data indicated a very interesting phenotype of ahr - null mice. after living for a few years at high altitude, the ahr null mice have a hypotensive phenotype, which mimics the blood pressure phenotype observed at sea level. additionally, the former proposed mediators of high blood pressure, including high circulating ang ii and et-1 levels, no longer occur in these animals. this suggests that the ahr - null mice in albuquerque have physiologically adapted to the altitude and exhibit a blood pressure phenotype consistent with sea level animals. the hypotensive ahr - null mice exhibit a significantly higher level of endothelial nitric oxide synthase (enos) and enhanced vascular nitric oxide (no) production compared to both wild - type and ahr heterozygous mice, which both have normal blood pressure. however, this is not likely the cause of hypotension in ahr - null mice, since n -nitro - l - arginine (lnna), a non - selective nitric oxide synthase (nos) blocker, failed to normalize the blood pressure. moreover, neither prazosin, an alpha1 adrenoceptor antagonist, nor hexamethonium, a ganglionic blocker treatment, causes any differences in the blood pressure change among ahr wild - type, heterozygous and null mice, suggesting an intact sympathetic activity in the blood pressure regulation of ahr - null mice. however, a research group in spain, salamanca (802 m) compared ahr - null and vav3-null mice, which developed similar cardiovascular remodeling and blood pressure, and suggested that the hypertension of ahr - null mice is mediated by vav3 through a sympathoexcitation mechanism. although the role of ahr in blood pressure regulation remains to be elucidated, there is no doubt that ahr could serve as a target in the treatment of high blood pressure and other no - dependent vascular diseases. most cardiovascular diseases are attributed to long term, repeated functional interruption and deposition of harmful factors in the cardiovascular system. the role of ahr in mediating xenobiotics - induced vascular damage has been well documented. however, the research results on the role of endogenous ahr in vascular homeostasis and blood pressure regulation still remain contradictory. from all the research on ahr gene - deficient mice, there is no doubt that ahr is one of the most important factors in maintaining blood pressure stability in those animals. the mechanism of more than 90% of the cases of human hypertension is unknown and ahr gene polymorphism has been detected in humans. therefore epidemiology research to correlate ahr gene polymorphism, altitude of residence and blood pressure phenotype will provide valuable insight into the role of ahr in human blood pressure control. on the other hand, further understanding of the role of ahr in no generation in vasculature endothelium and vascular remodeling will also contribute to the prevention of vascular diseases, such as atherosclerosis. the nature of cardiovascular diseases suggests a multifactorial etiology and a long - lasting disease development process. the endogenous ahr signaling represents a very promising target for cardiovascular disease prevention and treatment due to its role in heart and vascular physiology, blood pressure regulation and vascular no generation. thus, the ahr function in the cardiovascular system requires careful and comprehensive investigation with employment of true littermate animals with a pure genetic background and well - controlled animal husbandry environment. | the aryl hydrocarbon receptor (ahr) is an orphan nuclear receptor with a primary function of mediating xenobiotic metabolism through transcriptional activation of phase i and phase ii drug - metabolizing enzymes. although no high - affinity physiological activators of ahr have been discovered, the endogenous signaling of the ahr pathway is believed to play an important role in the development and function of the cardiovascular system, based on the observations on ahr gene - deficient mice. the ahr knockout mice develop cardiac hypertrophy, abnormal vascular structure in multiple organs and altered blood pressure depending on their host environment. in this review, the endogenous role of ahr in cardiovascular physiology, including heart function, vascular development and blood pressure regulation has been summarized and discussed. |
the female child is six - years - old and comes from fortaleza in the state of ceara in brazil. initial symptoms began six months ago and included fever, asthenia, and weight loss. tests findings include : cbc : normochromic and very microcytic anemia (hb : 7.1 g), leucopenia (3,300 wbc), and thrombopenia (70,000 platelets / mm) ; esr : 77 mm for first hour ; serology of leishmaniasis : positive (1/1600 using immunofluorescence and 5 archs in electrosyneresis) ; bone marrow biopsy : the medulla is rich, but no leishmania are seen. the patient is given a meglumine antimoniate treatment with increasing doses reaching 60 mg / kg / day after three days. transient hepatic cytolysis emerges with alanine transaminasi (alt) and aspartate aminotransferase (ast) 1.5 times above upper normal limit. according to the world health organization, leishmaniasis is a poverty - related disease whose public health impact has been, until recently, grossly underestimated. the disease is caused by protozoan parasites belonging to the genus leishmania that are transmitted by the bite of a phlebotomine sandfly. there are approximately two million new cases each year 1.5 million of cutaneous leishmaniasis and 500,000 of visceral leishmaniasis or kala - azar. kala - azar is endemic to south america, east africa, the mediterranean basin, the middle east, india, and china. the cardinal sign of kala - azar is an anarchic type of fever resisting all forms of treatment. the disease always yields splenomegaly and is thought by many to produce the largest spleens in parasitology. contrary to adult manifestations, cutaneous signs such as erythematous, hyper- or hypopigmented papules and nodules of various sizes are rare in children. although not performed in the case reported, protein immunoelectrophoresis reveals hypergammaglobulinemia with an igg peak in patients with kala - azar. serological tests, which are useful for the diagnosis of kala - azar, include elisa (enzyme linked immunosorbent assay), direct agglutination test (dat), indirect fluorescent antibody test (ifat), and the rk39 dipstick test. elisa is widely performed, particularly in epidemiological studies, because it is very simple. anemia secondary to kala - azar is aregenerative because of bone medullar parasitic invasion. even without direct evidence of leishmania, this child s poor health condition coupled with the epidemiological, clinical, and biological presentations called for immediate treatment. in brazil, side - effects of this case s treatment drug of choice, meglumine antimoniate, include the following : i) intolerance that usually appears after the first injections and consists of fever, chills, cough, myalgia, and/or skin rash. treatment discontinuation is mandatory if such intolerance occurs ; ii) intoxication resulting in fever, cough, skin rash, polyneuritis, hepatitis, cardiac, and renal signs. alternative treatments for kala - azar include sodium stibogluconate, amphotericin b, liposomal amphotericin b, pentamidine, and paromycin. the cardinal sign of kala - azar is an anarchic type of fever resisting all forms of treatment. the disease always yields splenomegaly and is thought by many to produce the largest spleens in parasitology. contrary to adult manifestations, cutaneous signs such as erythematous, hyper- or hypopigmented papules and nodules of various sizes are rare in children. although not performed in the case reported, protein immunoelectrophoresis reveals hypergammaglobulinemia with an igg peak in patients with kala - azar. serological tests, which are useful for the diagnosis of kala - azar, include elisa (enzyme linked immunosorbent assay), direct agglutination test (dat), indirect fluorescent antibody test (ifat), and the rk39 dipstick test. elisa is widely performed, particularly in epidemiological studies, because it is very simple. anemia secondary to kala - azar is aregenerative because of bone medullar parasitic invasion. even without direct evidence of leishmania, this child s poor health condition coupled with the epidemiological, clinical, and biological presentations called for immediate treatment. in brazil, the etiological agent of kala - azar is leishmania donovani. without treatment, side - effects of this case s treatment drug of choice, meglumine antimoniate, include the following : i) intolerance that usually appears after the first injections and consists of fever, chills, cough, myalgia, and/or skin rash. treatment discontinuation is mandatory if such intolerance occurs ; ii) intoxication resulting in fever, cough, skin rash, polyneuritis, hepatitis, cardiac, and renal signs. alternative treatments for kala - azar include sodium stibogluconate, amphotericin b, liposomal amphotericin b, pentamidine, and paromycin. | we report the case of a six - year - old brazilian girl referred for splenomegaly who first presented with fever, asthenia, and weight loss. geographical location, clinical exam, and blood laboratories suggested kala - azar. serology confirmed kala - azar diagnosis, but direct evidence of the parasites was not made. a treatment by meglumine antimoniate is given under hospital surveillance for two weeks. thereupon, the patient is asymptomatic and all tests are normal. |
one factor contributing to immunological memory is the much higher frequency of lymphocytes specific for the priming antigen in the memory pool than among naive cells. long - term persistence of memory cells is achieved by a combination of two processes : first, the long - term survival of individual cells, and second, periodic cell division to balance attrition through cell death. both processes are dependent on contact with mhc peptide complexes and with cytokines such as il-15 and il-7 (1). current understanding of how memory cell populations persist has been aided by studies investigating the kinetic behavior of t cells in various animal species, including humans, which have shown that those cells with a memory phenotype (cd45r0 in humans) exhibit a considerably higher rate of turnover than naive phenotype t cells and have a relatively short lifespan, on the order of weeks or months (27). however, memory t cells are phenotypically and functionally heterogeneous, and it is not known if rapid turnover applies to all memory cells. a major subdivision among memory t cells is that between central memory (tcm) and effector memory (tem) cells (8). these subpopulations, which are identified among human cd45r0 t cells as being ccr7cd62l or ccr7cd62l respectively, exhibit distinct functional properties. tcm cells enter lns through high endothelial venules and recirculate primarily between blood and lymph, whereas tem cells migrate preferentially from the blood into peripheral tissues such as the lung or intestinal mucosa (911). in addition, tem cells express effector activity (e.g., cytolytic activity or secretion of cytokines such as ifn-, il-4, or il-5) more rapidly than tcm cells upon restimulation with antigen (8, 9, 12), although some studies suggest that this distinction may be less clear for memory cd8 t cells (13). on this basis, tcm and tem cells have been proposed to play complementary roles in the secondary response to infection, with tem cells providing a rapid effector response at sites of pathogen entry and tcm cells serving as an expanded pool of precursors that can proliferate in the secondary lymphoid organs to rapidly generate large numbers of effector cells (8). although both tcm and tem cd4 populations appear to persist long term in humans (8), the kinetic behavior of the cells which comprise these subpopulations is unknown. to measure turnover of human tcm and tem cd4 cells, we used h - glucose, which is incorporated into dna via the de novo nucleotide synthesis pathway, to label dividing cells in vivo (14). the results show a clear difference in turnover rates between these subpopulations, with tem cd4 cells being much shorter lived. six young healthy volunteers (ranging from 19 to 30 yr of age) were given a primed 24-h i.v., their diet was restricted to small, low energy meals. for measurement of plasma glucose h enrichment, for estimation of h enrichment in cell dna, 50 ml heparinized blood samples were taken at 3, 4, 10, and 21 d after commencement of the infusion. all subjects gave written informed consent under protocols approved by the local research ethics committee. pbmcs were isolated from blood by ficoll - paque (amersham biosciences) density gradient centrifugation and stained (10/ml in pbs + 0.2% bsa) in one of three ways. (i) mouse anti human ccr7 (igm) (bd biosciences), goat anti mouse igm f(ab)2-rpe (southern biotechnology associates inc.), anti cd45ra - rpe - cy5 (serotec ltd.) and anti cd4apc (bd biosciences). (ii) anti human ccr7, anti - igm (fab)2-pe, anti - cd45ra - biotin (bd biosciences), streptavidin - percp (bd biosciences), and anti cd4apc. (iii) anti human ccr7, anti - igm (fab)2-pe, anti cd4-biotin (bd biosciences), streptavidin - percp, anti - cd45r0-allophycocyanin (caltag). typical purities and cell yields for the three major subpopulations (naive, tcm, tem) were 8598% and 16 10, respectively ; much smaller numbers of ra7 cells were obtained (4 10), and purity was usually lower than for the other three populations. enrichment of deuterium in dna was assayed essentially as described previously (7, 15). purified deoxyadenosine was converted to its aldononitrile triacetate derivative by reaction with hydroxylamine / pyridine and acetic anhydride. the resulting derivative was analyzed in triplicate by gas chromatography mass spectrometry, monitoring ions m / z 198 and 200 (sim mode in pci, hp-225 column, hp 6890/5973 gcms ; hewlett packard). plasma glucose enrichment was measured using the same derivatization (m / z 328 and 330). for each subject, mean glucose enrichment for the 24-h labeling period was derived from the area under the curve for the enrichment time profile. results were expressed as the fraction of labeled cells (f) present on each day, given by the ratio of the enrichment of label in dna (e) and the precursor enrichment (b, the mean glucose enrichment over 24 h 0.65) (14). the magnitude of the peak value for f represents a crude measure of the cellular proliferation rate. data was modeled as previously described (7) (sigmaplot v8.02 ; spss), to estimate the rate of proliferation during labeling, p, and subsequent rate of loss of labeled cells, d, taking into account cell death between the end of labeling and first sampling on day 3 and assuming no changes in pool sizes, according to the following equations : \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}f \left \left(t\right) \right = \frac{p}{d } \left \left(1-e^{-dt}\right) \right t{\leq}{\tau}{\mathrm{during\;the\;labeling\;period}}\end{equation}\end{document}\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}f \left \left(t\right) \right = \frac{p}{d } \left \left(1-e^{-d{\tau}}\right) \right e^{-d \left \left(t-{\tau}\right) \right } \;t>{\tau}\;{\mathrm{after\;the\;labeling\;period}}\end{equation}\end{document}where t is time and is the length of the labeling period. in this model, no assumption of equality between p and d has been made ; p represents the average proliferation rate of the whole population, whereas d refers only to labeled cells (i.e., cells which divided during the labeling period). for a kinetically heterogeneous population, even one at steady - state, these two rates will not be the same, as discussed elsewhere (7). where the day 3 value fell below the day 4 value for the same lymphocyte pool, it was assumed that the day 3 value lay within the lag phase between division and appearance in the circulation, and it was excluded from modeling. where proliferation is expressed as doubling time (t2) or disappearance as half - life (t1/2), these were calculated as ln2/p and ln2/d, respectively. six young healthy volunteers (ranging from 19 to 30 yr of age) were given a primed 24-h i.v., their diet was restricted to small, low energy meals. for measurement of plasma glucose h enrichment, for estimation of h enrichment in cell dna, 50 ml heparinized blood samples were taken at 3, 4, 10, and 21 d after commencement of the infusion. all subjects gave written informed consent under protocols approved by the local research ethics committee. pbmcs were isolated from blood by ficoll - paque (amersham biosciences) density gradient centrifugation and stained (10/ml in pbs + 0.2% bsa) in one of three ways. (ii) anti human ccr7, anti - igm (fab)2-pe, anti - cd45ra - biotin (bd biosciences), streptavidin - percp (bd biosciences), and anti cd4apc. (iii) anti human ccr7, anti - igm (fab)2-pe, anti cd4-biotin (bd biosciences), streptavidin - percp, anti - cd45r0-allophycocyanin (caltag). typical purities and cell yields for the three major subpopulations (naive, tcm, tem) were 8598% and 16 10, respectively ; much smaller numbers of ra7 cells were obtained (4 10), and purity was usually lower than for the other three populations. enrichment of deuterium in dna was assayed essentially as described previously (7, 15). briefly, cellular dna was extracted and digested enzymatically to deoxynucleosides. purified deoxyadenosine was converted to its aldononitrile triacetate derivative by reaction with hydroxylamine / pyridine and acetic anhydride. the resulting derivative was analyzed in triplicate by gas chromatography mass spectrometry, monitoring ions m / z 198 and 200 (sim mode in pci, hp-225 column, hp 6890/5973 gcms ; hewlett packard). plasma glucose enrichment was measured using the same derivatization (m / z 328 and 330). for each subject, mean glucose enrichment for the 24-h labeling period was derived from the area under the curve for the enrichment time profile. results were expressed as the fraction of labeled cells (f) present on each day, given by the ratio of the enrichment of label in dna (e) and the precursor enrichment (b, the mean glucose enrichment over 24 h 0.65) (14). the magnitude of the peak value for f represents a crude measure of the cellular proliferation rate. data was modeled as previously described (7) (sigmaplot v8.02 ; spss), to estimate the rate of proliferation during labeling, p, and subsequent rate of loss of labeled cells, d, taking into account cell death between the end of labeling and first sampling on day 3 and assuming no changes in pool sizes, according to the following equations : \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}f \left \left(t\right) \right = \frac{p}{d } \left \left(1-e^{-dt}\right) \right t{\leq}{\tau}{\mathrm{during\;the\;labeling\;period}}\end{equation}\end{document}\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}f \left \left(t\right) \right = \frac{p}{d } \left \left(1-e^{-d{\tau}}\right) \right e^{-d \left \left(t-{\tau}\right) \right } \;t>{\tau}\;{\mathrm{after\;the\;labeling\;period}}\end{equation}\end{document}where t is time and is the length of the labeling period. in this model, no assumption of equality between p and d has been made ; p represents the average proliferation rate of the whole population, whereas d refers only to labeled cells (i.e., cells which divided during the labeling period). for a kinetically heterogeneous population, even one at steady - state, these two rates will not be the same, as discussed elsewhere (7). where the day 3 value fell below the day 4 value for the same lymphocyte pool, it was assumed that the day 3 value lay within the lag phase between division and appearance in the circulation, and it was excluded from modeling. where proliferation is expressed as doubling time (t2) or disappearance as half - life (t1/2), these were calculated as ln2/p and ln2/d, respectively. to measure t cell turnover in vivo, healthy volunteers were infused with h - labeled glucose for 24 h, and enrichment of h in the dna of isolated subpopulations of peripheral blood lymphocytes was determined at several time points. day 3 was chosen as the earliest time point since previous work has shown that the appearance of labeled t cells in the blood peaks at 34 d after starting the infusion (7). cd4cd3 cells were subdivided by cell sorting into four populations : cd45r0 (or cd45ra) ccr7 tcm cells, cd45r0 (or cd45ra) ccr7 tem cells, cd45r0 (or cd45ra) ccr7 naive cells and cd45r0 (or cd45ra) ccr7 cells (referred to as ra7 cells). lack of ccr7 expression on the latter subset, which represents a small fraction of total cd45ra cd4 t cells, suggests that ra7 cells are not naive ; antigen - primed cells have been identified previously among cd45ra cd4 cells based on an absence of cd31 expression (16). on average, these subpopulations represented 21% (tcm), 12% (tem), 64% (naive), and 3% (ra7) of total peripheral blood cd4 t cells in the subjects studied. from the measurement of h enrichment in dna, the proportion of cells in each subpopulation that was labeled at the different time points was calculated, and these values are shown in fig. first, labeling of naive cells was extremely low, confirming previous work showing that there is little proliferation within this subpopulation (27). second, labeling of tem cells occurred at a substantially higher rate than for tcm cells. this was observed clearly in five of the six subjects studied. in the sixth subject (c15), similar rates of labeling were observed for tem and tcm cells, which appeared to be due to a higher than average labeling of tcm cells in this individual. third, ra7 cells showed a relatively rapid rate of labeling, consistent with the view that these cells are not naive but rather are antigen - primed ; the exact relationship between these cells and tcm or tem cells remains to be defined. peak height values (the maximum measured fraction of labeled cells) for each subpopulation are summarized in table i. mean values were higher in ccr7 than ccr7 populations for both cd45r0 and cd45ra cells. kinetics of labeling of naive, tcm, tem, and ra7 cd4 t cells after infusion of h - glucose. subjects were infused with h - glucose for 24 h starting from time 0, and the proportion of labeled cells (relative to total cells in each subpopulation) at different time points is shown for cd45ra (left) and cd45r0 cd4 t cells (right). data for ccr7 and ccr7 cells are indicated by filled or open symbols, respectively ; error bars represent the sd of triplicate gcms measurements. lines represent best - fit curves calculated based on the data points and assuming maximal labeling at 24 h. glucose enrichment and peak fraction of labeled cells after 24 h 6,6-d2-glucose from area under curve of enrichment time profile. maximal value for labeling expressed as f (fraction of labeled cells) measured in lymphocyte subpopulations at time points sampled. cells were sorted according to method i, ii, or iii (see materials and methods). to determine the rates of proliferation (p) and disappearance (d) for the various cell subpopulations, curves were fitted to the data based on the fraction of labeled cells detected at each time point (fig. 1). note that in modeling the data it was assumed that maximal labeling was present at 24 h (i.e., at the cessation of h infusion) and that label disappeared exponentially thereafter with a disappearance rate constant, d ; this model takes into account cell death between the end of labeling and first sampling on day three. as shown in table for the five subjects where this was the case, p for tem cells was on average five times of that of p for tcm cells ; tem and tcm cells had roughly equivalent proliferation rates in the sixth subject. overall, the average proliferation rates for tcm and tem cells were 1.5 and 4.7% per day, respectively. from p, the average intermitotic time for cells within that population (t2) can be calculated, yielding values of 48 d for tcm cells and 15 d for tem cells. proliferation and disappearance rates for tcm, tem, naive, and ra7 cd4 t cells values for proliferation (p) and disappearance (d) rates were derived from the best fit curves applied to the data (see materials and methods). t2 and t1/2 represent doubling time or half - life equivalents of p or d, respectively ; mean values for t2 and t1/2 are calculated from the mean values for p and d, respectively. > indicates t2 or t1/2 exceeds maximal value detectable (p or d close to zero). disappearance rates, though more variable, were also higher for tem than tcm cells in five of the six subjects. on average, the disappearance rates were 4.1% per day for tcm cells and 11.3% per day for tem cells (equivalent to half - lives [t1/2 ] for disappearance of the cells of 17 and 6 d, respectively). note that d is a measure of the disappearance rate of labeled cells only, whereas p applies to all cells, explaining why d can exceed p at steady - state (even though d, the disappearance rate of the entire population, must equal p). as discussed previously, several explanations can be proposed for the relatively rapid disappearance of recently divided cells (7). although this was true for both tem and tcm cells in many subjects, it is notable that labeled tcm cells in two individuals exhibited negligible disappearance rates, implying that some tcm cells can differentiate into long - lived cells after division. in contrast, d was higher than p for tem cells in all subjects. for naive cd4 t cells, p was very low, 0.2% per day, which converts to a t2 for the naive population of about a year (table ii). this contrasts with the rapid proliferation observed among ra7 cells, which had an average p of 3.3% per day (t2 = 21 d). the labeling that does occur amongst truly naive cd4 t cells could reflect division of precursors in the thymus and their release into the blood over the time course of the analysis. alternatively, some proliferation of mature naive cells could occur in the secondary lymphoid organs, perhaps in response to contact with homeostatic cytokines (1721). whichever the case, however, it is evident that the contribution of cell division to maintaining the naive cd4 t cell pool in young adults is minimal. in addition, the disappearance rate for the majority of naive (cd45raccr7) cd4 t cells was shown to be extremely slow. in fact, we found that d for cd45raccr7 cells in all subjects was too small to estimate using this method. this contrasts with earlier results showing a relatively rapid disappearance rate for labeled total cd4cd45ra cells of > 7% per day (7). this disparity may be partly explained by the disproportionate contribution of ra7 cells to labeling in the previous study, since labeled cells in this population disappear rapidly. the present data are striking in their implication that human naive cd4 t cells can remain in interphase for extremely long periods of time after their derivation from dividing precursors. overall, the relative quiescence of naive cd4 t cells and rapid turnover of memory phenotype cells reported here are in agreement with the general pattern of kinetic behavior that has been observed for these cells in many different species. our data show that tcm and tem cd4 t cells in humans have distinct rates of turnover, with tem cells typically being replaced at a much faster rate than tcm cells. these results imply that an almost continuous input of new cells is required to maintain the tem cd4 t cell population, which could have important implications for vaccination given that tem may serve to provide a rapid effector response to reinfection. at face value, it could be argued that the shorter lifespan observed for tem cells in the blood is simply a consequence of these cells having an ability to migrate rapidly and irreversibly into tissues. however, if one assumes that the number of tem cells in tissues is constant (rather than increasing) under steady - state conditions, turnover of cells in the blood ultimately reflects what is occurring in the total tem population. therefore, it is evident from the rapid rate of production that there is also a rapid rate of disappearance among tem cells. several factors could contribute to differences in the kinetic behavior between tcm and tem cells. first, their distinct patterns of migration make it likely that the extent to which they contact cell division- and survival - inducing factors will differ (22). second, it has been shown that tcm and tem cells exhibit intrinsic differences in their responsiveness to homeostatic cytokines (23). in particular, tem cells have higher cell surface expression of the il-2/il-15r -chain, allowing them to proliferate in response to lower concentrations of il-15 than tcm cells. third, tem and tcm cells may also differ in their intrinsic death rates or their ability to respond to survival factors. the present data should be considered with respect to the uncertainty that exists in understanding the lineage relationship between tcm and tem cells, specifically whether these cells represent independent subpopulations or merely different activation states along a common differentiation pathway. previous work has shown that phenotypic conversion can take place between tcm and tem cells, suggesting that one subpopulation could serve as a source for the other ; this has been observed for human cd4 cells, where tcm cells can acquire characteristics of tem cells in response to stimulation through the tcr or by cytokines (23), and in the reverse direction for mouse cd8 t cells, where virus - specific tem cells were shown to convert to tcm cells with time after infection (24). in contrast, based on analysis of t cell clonotypes present among tcm and tem cd8 t cells it was concluded that these cells represent independently regulated cell subpopulations in humans (25). although the present kinetic data could indicate that tcm and tem cells are maintained as separate pools with distinct turnover rates, they are also compatible with a model in which interconnection of tcm and tem cells occurs in both directions. according to this latter model, tem cells are maintained primarily through cell division, either by existing tem cells or by tcm cells that acquire a tem phenotype concomitant with division. replacement of tcm cells would occur through more limited cell division among tcm cells that do not change phenotype and by phenotypic reversion of tem cells after these cells have returned to a quiescent state. finally, these results raise the question of whether further heterogeneity exists in the kinetic behavior of human cd4 t cells. previously, evidence for kinetic heterogeneity amongst antigen - primed human cd4 t cells had been suggested by analysis of total memory effector - phenotype cd4 t cells (26). by using ccr7 as a marker, we were able to demonstrate that there are distinct subpopulations with differing turnover rates present in both cd45ra and cd45r0 cd4 t cell populations. in the future, it will be of interest to use other cell surface markers, for example, those associated with recent activation, to characterize the in vivo behavior of additional cell populations. | memory t cells can be divided into central memory (tcm) and effector memory (tem) cells, which differ in their functional properties. although both subpopulations can persist long term, it is not known whether they are maintained by similar mechanisms. we used in vivo labeling with deuterated glucose to measure the turnover of cd4 + t cells in healthy humans. the cd45r0+ccr7 tem subpopulation was shown to have a rapid proliferation rate of 4.7% per day compared with 1.5% per day for cd45r0+ccr7 + tcm cells ; these values are equivalent to average intermitotic (doubling) times of 15 and 48 d, respectively. in contrast, the cd45ra+ccr7 + naive cd4 + t cell population was found to be much longer lived, being labeled at a rate of only 0.2% per day (corresponding to an intermitotic time of approximately 1 yr). these data indicate that human cd4 + tem cells constitute a short - lived cell population that requires continuous replenishment in vivo. |
ascending aortic dissection (aad) is a rare and serious complication of aortic valve replacement. multiple risk factors such as connective tissue disease, aortic wall thinning, aortic diameter, calcification of wall, structural features of aortic wall and associated diseases have been considered as a predisposing factor for the occurrences of aad. preoperative recognition of these variables with proper intra intra operative logic judgment may decrease tehe incidence of this complication. we herein present a huge ascending aorta with dissecting aneurysm (aad) with a largeintra - operative diameter (15 cm) that has not been recorded in the medical literature so far. he presented with dyspnea, chest pain and amazing symptom of superior vena cava syndrome. the patient underwent open heart surgery with resection of ascending aorta aneurysm with classic bentall operation. the post - operative period was associated with uneventful course and the patient was discharged with good condition on 12 post - operative day. a six months ' follow - up revealed abolishment of chest pain and superior vena cava (svc) syndrome and good prosthetic composite graft function with no recurrence of pseudo aneurysm or dissection. aortic dissection is not a rare event in general population but is a very rare and serious complication in post aortic valve replacement. however, the incidence of aortic dissection (type a) varies between 3.5 to 9 per 100,000 persons in general population, but this prevalence is much higher and reaches between 0.6 to 1% in post avr surgery. although avr itself is considered as an independent risk factor for the development of subsequent dissection, the preoperative and the patient 's risk factors also determinate occurrences of post - operative dissection (1,2). these risk factors include aortic insufficiency, systemic hypertension, male gender, thinned or fragile aortic wall and ascending aortic dilatation (3). in this article, we present a successful surgical treatment of a giant dissecting aneurysm of ascending aorta late after aortic valve replacement with large diameter (15 cm). the largest reported dissecting aneurism in post avr surgery (9 cm) related to fukui study was smaller than our case (4). however, svc syndrome was reported in post avr surgery but two unique features of ours case related to size of dissecting aneurism and svc syndrome that rarely has been reported in medical literature. the most common causes of superior venacava syndrome are mediastinal tumors that by external compression or intra luminal invasion of the cava leadd to svc syndrome. among benign causes, a 67-year - old man with cough, chest pain and dyspnea and intermittent symptom of superior vena cava syndrome (svcs) as upper thorax plethora, dilated jugular veins, dark blue change of neck and severe headache from 4 weeks earlier was referred to our center from another hospital. as his dyspnea worsened, he was referred for transe thoracic echocardiography examination after a widening of mediastinum was seen on chest radiography. the patient 's history showed prosthetic aortic valve replacement with carbomedic (23 number of sorin group) that has been performed 10 years earlier. in the previous operation, risk factors such as coarectation, bicuspid aortic valve, aortic wall, calcification or thin aortic wall were not reported. however, patient 's native valve and its ring was calcified, and ascending aorta had a diameter of 41 mm. physical examination on admission showed mild cyanosis and plethora of head and neck and distended jugular veins. arterial blood gases showed mild respiratory acidosis (ph : 7.33) due to elevated pco2 : 48 mmhg. the contrast ct angiography of the chest was remarkable, revealing a large aneurysm of the ascending aorta with a maximum diameter of 15 cm compressing the superior vena cava. transthoracic echocardiography (tee) showed normal functional prosthetic aortic valve and ascending aortic aneurysm involving sinuses of valsalva. there was a discrepancy between reported size of aneurysm in tee and c - t angiography and intra operative inspection. the gross size of aneurysm was much larger than the one reported in tee (figure 3). aneurysm 's transverse diameter was 15 cm with c - t angiography and gross examination. right coronary arteries were found normal by angiography, but lad and lcx were not detected by angiography (figure 4). the cpb was instituted by femoral artery, and right atrial cannulation and midline sternotomy were performed. a huge ascending aorta aneurysm attached to svc by inflammatory reaction and subsequently leading to its narrowing was detected intraoperatively (figure 5). following ascending aorta clamping, aortotomy was performed and direct cold cardioplegia was delivered to both coronaries ostium (figure 6). both right and left coronary ostium bottoms were prepared and released from the surrounding structures. due to aneurysm of valsalva sinuses and despite normal prosthetic aortic valve function, modified bentall operation was not performed. the supra annular space as valsalva sinus had abnormal tissue consistency, and was not appropriate for modified bentall surgery. the prosthetic valve was removed and classic bentall procedure was performed with no : 25 carbomedic valved conduit composite graft of sorine group. the patient was discharged on the 12 day of the postoperative period in well condition. our patient had two unique feature i.e. huge diameter of aneurism (15 cm) and superior vena cava syndrome. c - t angiography shows huge ascending aorta aneurysm shows pressure effect of dissecting aneurism on superior vena cava transe thoracic echocardiography shows large aneurysm angiography shows huge ascending dissecting aneurism with prosthetic valve shows huge size of ascending aorta in intra operative view shows left main coronary ostium (vertical arrow), cardioplegin right angel cannula (transverse arrow) and intimal flap (curve arrow) immediately above the coronary ostium and prosthetic aortic valve (oblique arrow) factors predicting aortic root complication after avr could be classified as preoperative, intra operative and post - operative variables. pre - operative variables include, hypertension, aortic root dilatation, bicuspid aortic valve, connective tissue disorder (marfan syndrome, vascular ehlers - danlos syndrome, turner syndrome, and loeys - dietz syndrome) (5). any concomitant procedure that needs accomplishment of avr such as aortic root canuula for cardioplegin, aortotomy site decalcification of aortic ring or aorticvalves, partialor or complete aortic clamping site, accidental trauma to aortic wall by electrocautery, and use of suture with large needle (57). post - operative factors include hypertension and infection of suture lines. however, these complications are rare which potentially have serious consequence. the most important factor for predicting pos - toperative dissection after avr is aortic root dilatation.when the aorta has a markedly enlarged diameter (> 50 mm) at initial avr, combined ascending aortic replacement is a preferable surgical procedure. however, it is an issue of controversy whether a mildly dilated ascending aorta (4050 mm) should be replaced. in one, study, aortic regurgitation, systemic hypertension, male sex and a thinned or fragile aorta with mild dilation (> 45 mm) at initial avr have been considered as risk factors for late aortic complications. it seems that management of dilated aorta in the avr candidate patients depended on coexisting risk factors observed in the pre and intra operative periods (810). post - operative dissection occurs in all avr surgery that aorta is handled during initial surgery. the dissection 's flap can begin at multiple location, such as aortic cannulation site, cardioplegia cannula site, aortic cross clamping site, aortotomy site and in severe atherosclerosis of aortic wall. it is vital to carefully close the aortotomy site with small needle and sutures close together that have appropriate distance from the aortotomy at the initial surgery. in our case, the intimal tear was found immediately above the left main coronary artery ostium in sinotubular junction ; however, the entry site had enough distance from the previous aortotomy line. it seems that direct cardioplegin perfusion to left coronary ostiummay caused small intimal tear that extended upward to posterior aortic wall in this patient with fragile aortic wall. some surgeons consider an aortic diameter limit of 44 mm as maximum diameter that could be treated by aortoplasty or replacement in initial avr. other surgeon believe that aortic diameter between 45 to 50 mm is an issue of debate (11). most cardiac surgeons refrain to perform replacement or aortoplasty in patients with concomitant risk factors such as low ejection fraction, elderly patients ' renal dysfunction or history of copd or other organ dysfunctions. prolonged cardiopulmonary bypass may lead to further cardiac dysfunction, cerebral complication, respiratory dysfunction, acute renal failure, which can increase post - operative icu stay and increase morbidity and mortality (12). to avoid a prolonged cpb time, more studies are necessary to conclude whether both of these conservative surgeries could be considered as an alternative to aortic replacement in high risk patients to prevent future dissection. if svcs occurred following the pseudo aneurysm of aorta, it usually results from extrinsic compression of vessel (13). etiology of svcs has been changed considerably in recent decades. in mcintire and sykes'sstudy in a case series of svcs 60 years ago, 70% of svcs were caused by benign etiology of which the most common causes were secondary to syphilitic aneurysms (55%) and 15% were secondary to other mediastinal inflammation such as tuberculosis, sarcoidosis or mediastinal fibrosis, whereas 30% were caused by malignant tumors (14). in recent decades, the common etiology of svcs has reversed from infection causes to malignancy (15). in a recent study by baker, malignant and benign diseases accounted for 90% and 10% of cases respectively. today, due to low incidence, benign causes of svcs are often missed in the differential diagnosis (16). however, post - cardiac surgery cause of svcs is easily detected by recent development of imaging modality and differentiated from rare benign mediastinal mass, inflammatory pseudo tumor, autoimmune or granulomatous processes. dissection of a post avr ascending aorta producing huge pseudo aneurysm is an exceedingly rare cause of svcs (17). | backgroundascending aortic dissection (aad) is a rare and serious complication of aortic valve replacement. multiple risk factors such as connective tissue disease, aortic wall thinning, aortic diameter, calcification of wall, structural features of aortic wall and associated diseases have been considered as a predisposing factor for the occurrences of aad. preoperative recognition of these variables with proper intra intra operative logic judgment may decrease tehe incidence of this complication.case detailswe herein present a huge ascending aorta with dissecting aneurysm (aad) with a largeintra - operative diameter (15 cm) that has not been recorded in the medical literature so far. he presented with dyspnea, chest pain and amazing symptom of superior vena cava syndrome. the patient underwent open heart surgery with resection of ascending aorta aneurysm with classic bentall operation. the post - operative period was associated with uneventful course and the patient was discharged with good condition on 12th post - operative day.conclusiona six months ' follow - up revealed abolishment of chest pain and superior vena cava (svc) syndrome and good prosthetic composite graft function with no recurrence of pseudo aneurysm or dissection. |
cardiovascular diseases, such as coronary heart disease (chd), are among the leading causes of death in western countries and in iran (1). these disorders are responsible for more than 25% of all deaths worldwide (2). in the uk, for example, more than 90,000 people die from chd each year (3). according to the world health organization, chronic disease causes 70% of deaths globally, with chd ranking first (4). it is reported that the age at onset of cardiovascular disease in iran is approximately 7 - 10 years earlier than in other countries (5). chd s physical and psychosocial consequences not only increase the mortality rate, but also considerably increase disability rates for a large portion of the country s workers during their best years of productivity, ultimately reducing productivity and increasing the cost of healthcare (6, 7). studies have shown that 30% - 72% of chd patients demonstrate symptoms of depression, and 40% - 65% show symptoms of anxiety (8 - 10). these two problems, in addition to lack of social support, are among the most common psychological responses in patients with cardiovascular disease, and they not only increase the use of healthcare services, but also the risk of disease relapse or exacerbation (11) and the costs of acute and long - term healthcare (12). lifestyle changes are also recommended in order to eradicate or decrease the underlying factors (13). in recent years, alternative therapies, such as music therapy, relaxation, therapeutic massage, and guided imagery, have been used to decrease patients anxiety and distress related to acute and chronic disorders (14). in addition to the measures cited previously, studies indicate reduced distress, anxiety, depression, and certain psychological problems through the use of psychological treatments, such as cognitive - behavioral therapies, psychoeducational programs (peps), and the establishment of support systems. recently, researchers have focused on using peps as a fundamental treatment for patients psychological problems (12). peps are usually aimed at directing the patients learning, providing opportunities for them to express their emotions in a safe environment, creating real hope or strengthening it, offering solutions to enhance the patients self - awareness, and providing opportunities for them to practice their new knowledge (15 - 17). several studies have investigated the effects of peps on mental health and psychological problems in patients with different disorders. luciano. (18) studied the effect of a pep on the general health of patients with fibromyalgia, and reported that the intervention was effective at improving mobility and reducing pain, fatigue, morning tiredness, anxiety, and depression. (19) reported that psychological interventions that provide a supportive environment and cognitive - behavioral therapy can reduce symptoms of depression and anxiety in cancer patients undergoing radiotherapy, improving their health - related quality of life. however, in a meta - analysis of peps, dusseldorp. reported that these methods did not significantly affect anxiety (10 studies) or depression (13 studies) in patients with chd (20). moreover, hartford. investigated the impact of telephone - based psychological training with nurses and reported that the intervention did not significantly affect the patients anxiety after coronary artery bypass grafting (cabg) (21). in another study, johnston. also examined the effects of education and psychological counseling on anxiety, depression, and functional limitations in patients with myocardial infarction ; the results showed that the intervention did not significantly affect depressive symptoms (22). in a recent systematic review of psychological interventions for chd patients and their partners, reid. reported that the effects of psychological interventions for patients with chd were inadequately studied, and that the available studies were mostly out dated and of poor overall quality, including methodological weaknesses, and they showed a non - significant trend (8). therefore, further studies in the field of psychological interventions for chd patients are needed. a majority of the studies on peps have been conducted on patients with psychiatric disorders, while being limitedly implemented in patients with medical disorders, especially cardiovascular disease. however, healthcare providers, including doctors and nurses, have increasingly found that in addition to tending to the physical aspects of patients, it is necessary to pay more attention to their psychosocial needs in order to help them achieve normal lives (23, 24). therefore, indigenous forms of peps may be useful for patients with chronic disease, such as chd, to help them reach maximum functional health (12, 25). this study aimed to examine the effects of a pep intervention on the mental health of chd patients. this randomized controlled trial was conducted on 70 patients with chd who were hospitalized in the coronary care unit (ccu) at shahid beheshti hospital in kashan, iran, in 2014. according to previous studies (12) and based on the formula of = 0.95, 1 - = 0.8, and d = 0.65, the sample size was determined to be 35 patients in each group. the subjects who met the inclusion criteria were randomly allocated into experimental and control groups through a randomized block sampling method. the inclusion criteria were age of 21 - 65 years, willingness to participate in the study, ability to respond to inquiries and attend meetings, no history of angioplasty or cabg, absence of brain disorders (such as alzheimer s, stroke, or transient ischemic attack), and ability to read and write in the persian language. the exclusion criteria were death or migration, the occurrence of any acute or urgent medical or psychological upheaval, and the patient s decision to leave the study. for controlling the confounding variables, patients with substance addictions or known mental and psychological disorders were excluded from the study. the intervention group underwent eight group sessions of pep, two sessions per week at two - day intervals, with each session lasting for 45 - 60 minutes. data on the patients demographics were collected at the start of the study. at the same time, the primary outcome measures were collected for the two groups. in addition to routine medical care, the experimental group received pep intervention, which included discussions and training materials on skills for coping with anxiety, with an emphasis on lifestyle, anger management, problem - solving and muscular relaxation techniques, as presented in box 1 (26 - 28). all pep sessions were facilitated by a trained nurse. at the end of each session, assignments were given to the participants to complete at home. the participants experiences with the assignment were reviewed and discussed at the start of the next session, and then new materials were delivered. during the intervention period, the investigator conducted a 5 - 10 minutes weekly telephone call with each participant in the intervention group to track the home assignments, answer questions, and organize the sessions. the study questionnaire was re - answered by each participant in the intervention group after the eight pep sessions. patients in the control group only received routine medical care, plus a training pamphlet from the american heart association containing information on general cardiac healthcare (29). these participants answered the study questionnaire at the beginning of the recruitment and again after four weeks. the first part contained questions about socio - demographic characteristics (the patient s age, gender, surgical history, and smoking). the second part of the instrument was goldberg s general health questionnaire (ghq). the ghq consists of 28 items that are rated on a three - point likert scale. the scale has four dimensions : somatic symptoms, anxiety, social dysfunction, and depression. the overall score of the scale indicated the individual s level of mental health, with higher scores indicating a lower level of mental health (30). ahmadian (31) confirmed the instrument s content validity and assessed its reliability through the internal consistency method, and cronbach s alpha ranged from 0.77 to 0.90 for the different subscales. in another study, ebrahimi. reported cronbach s alpha for the overall persian scale to be 0.97 (32). permission for this study was obtained from the ethics committee of kashan university of medical sciences. ethical issues in this study involved the assurance of confidentiality and the anonymity of the participants. all participants were informed of the purpose and design of the study, and understood that their participation was voluntary. this study was registered in the iranian registry for clinical trials (irct) with registration code 2014060114086n4. kolmogorov - smirnov was used to examine the normal distribution of variables, and the chi - square test was used to compare the distribution of socio - demographic variables in the two groups. the independent - sample t - test was used to examine the difference between the mean ages of the two groups. the independent - sample t - test was also used to examine the differences between the overall ghq mean scores, as well as the subscale mean scores in the two groups at the start and the end of the study. in addition, the paired - sample t - test was used to compare the changes in the overall ghq mean scores of the individual groups at the start and the end of the study. data on the patients demographics were collected at the start of the study. at the same time, the primary outcome measures were collected for the two groups. the pep was then initiated in the intervention group. in addition to routine medical care, the experimental group received pep intervention, which included discussions and training materials on skills for coping with anxiety, with an emphasis on lifestyle, anger management, problem - solving and muscular relaxation techniques, as presented in box 1 (26 - 28). all pep sessions were facilitated by a trained nurse. at the end of each session, assignments were given to the participants to complete at home. the participants experiences with the assignment were reviewed and discussed at the start of the next session, and then new materials were delivered. during the intervention period, the investigator conducted a 5 - 10 minutes weekly telephone call with each participant in the intervention group to track the home assignments, answer questions, and organize the sessions. the study questionnaire was re - answered by each participant in the intervention group after the eight pep sessions. patients in the control group only received routine medical care, plus a training pamphlet from the american heart association containing information on general cardiac healthcare (29). these participants answered the study questionnaire at the beginning of the recruitment and again after four weeks. the first part contained questions about socio - demographic characteristics (the patient s age, gender, surgical history, and smoking). the second part of the instrument was goldberg s general health questionnaire (ghq). the ghq consists of 28 items that are rated on a three - point likert scale. the scale has four dimensions : somatic symptoms, anxiety, social dysfunction, and depression. the overall score of the scale indicated the individual s level of mental health, with higher scores indicating a lower level of mental health (30). ahmadian (31) confirmed the instrument s content validity and assessed its reliability through the internal consistency method, and cronbach s alpha ranged from 0.77 to 0.90 for the different subscales. in another study, ebrahimi. reported cronbach s alpha for the overall persian scale to be 0.97 (32). permission for this study was obtained from the ethics committee of kashan university of medical sciences. ethical issues in this study involved the assurance of confidentiality and the anonymity of the participants. all participants were informed of the purpose and design of the study, and understood that their participation was voluntary. this study was registered in the iranian registry for clinical trials (irct) with registration code 2014060114086n4. kolmogorov - smirnov was used to examine the normal distribution of variables, and the chi - square test was used to compare the distribution of socio - demographic variables in the two groups. the independent - sample t - test was used to examine the difference between the mean ages of the two groups. the independent - sample t - test was also used to examine the differences between the overall ghq mean scores, as well as the subscale mean scores in the two groups at the start and the end of the study. in addition, the paired - sample t - test was used to compare the changes in the overall ghq mean scores of the individual groups at the start and the end of the study. a p value of 0.05). however, the means of the overall ghq scores and most of the ghq subscales were significantly decreased post - test in the intervention group, so that the differences between the two groups were statistically significant in both the overall ghq scores and in all of the subscales (p 0.07) (table 4). the findings of this study showed that pep improves mental health by decreasing somatic and psychological symptoms, such as anxiety and depression, in patients with chd. this finding is consistent with the results of taylor - rodgers and batterham, who studied the effects of pep intervention on the help - seeking attitudes and intentions among young adults with regard to mental health (12). luciano. (18) and guo. (19) have also reported that psychological training could reduce anxiety and depression in patients with fibromyalgia or cancer. peps combine methods of relaxation with psychological training, such as anger management and problem - solving techniques. several studies have used single methods, such as anger management or relaxation, and reported positive effects. for instance, ahangarzade and ezadi reported that anger management training could improve mental health in nursing students (33). (10) and dehdari. (11) used relaxation and distraction in patients with cardiac disorders and post - cardiac surgery, and reported that these methods were effective in reducing patients anxiety. some studies, such as d'souza.s also used psychological training, and reported that this method was effective not only in reducing the severity of anxiety and depressive symptoms, but also in decreasing the rate of relapse in patients with bipolar disorder (26). it seems that peps have a positive effect on anxiety, depression, and mental health, not only by decreasing muscular tension but also by combining relaxation with improved anger management and problem - solving skills. this could be attributed to differences in the content and techniques used (34), or to the methods and time - intervals. for example, various studies implemented psychological training methods through telephone contact, email, or cds (21, 22). it appears that pep shave a positive impact on mental health, especially if it is applied in a face - to - face approach and is tailored to the individual s culture and lifestyle. in general, it seems that pep changes the patient s mental framework, increases his or her awareness of the present moment, and improves the cognitive and information - processing systems. moreover, group - session peps show more benefits in facilitating and speeding up the treatment process (14, 35, 36), as they allow patients to be gathered in one place to discuss their problems. this intervention reduces patients tensions, relieves their negative emotions, and improves their social relationships (37). therefore, it is recommended that such programs be used in conjunction with other treatments. this study had some limitations, including its non - blind design and the short follow - up period. moreover, the psychological status of the patients while answering the questionnaire, and the level of information or support they received from sources other than the investigators, were not under the researchers control. psychoeducation is a well - known intervention for psychiatric patients, but its use has been limited in other health conditions, such as coronary heart disease. the results of this study show that pep has positive effects on the mental health of patients with chd. further studies are suggested, with greater sample sizes and longer follow - up periods at different time - intervals. studies are also suggested on the barriers to and facilitators of using pep in practice. considering the important psychological needs of patients with coronary heart disease, it is suggested that similar interventions be integrated into routine cardiac care plans, and that pep interventions be added to medical and nursing education curricula. | backgroundpatients with coronary heart disease are at high risk for mental health disorders, such as depression and anxiety. psychoeducation is a well - known intervention for psychiatric patients, but its use has been limited in other health conditions, such as coronary heart disease.objectivesthe aim of this study was to evaluate the effect of psychoeducation on mental health in coronary heart disease patients.patients and methodsthis randomized clinical trial included 70 patients with coronary heart disease at shahid beheshti hospital, in kashan, iran, in 2014. the patients were randomly assigned into two groups : the experimental group, which received eight sessions of psychoeducation, and the control group, which received routine care. data were collected with the goldberg mental health questionnaire (ghq) and were analyzed using independent and paired t - tests performed with spss version 16.resultsthe means of overall ghq scores were significantly decreased post - test in the intervention group, and the differences between the two groups were statistically significant in the overall ghq scores (p = 0.0001). a significant difference was observed between the mean ghq scores of the intervention group prior to and after the psychoeducational program (pep) intervention (30 4.66 vs. 20.50 3.30) (p = 0.0001). no significant changes were observed in the control group pre- and post - test (p = 0.07).conclusionspsychoeducation resulted in improved mental health in patients with coronary heart disease. therefore, it is recommended that this approach be performed as a complementary, effective, non - invasive, low - cost nursing intervention to reduce psychological problems in these patients. |
wild - type, pigmented long - evans (le) rats, obtained from charles river (seattle, wa, usa), were used as controls because although the retina layers thin with age, there is no appreciable cell loss or change in total retinal volume. in addition, le rats have previously been used to back breed pigment in the rcs line and to generate the nondystrophic rcs line. rats were housed in standard conditions under a 12/12-hour light dark cycle (light cycle : 200 lux). all experiments were approved by the institutional animal care and use committee at ohsu and adhered to the arvo statement for the use of animals in ophthalmic and vision research. rcs rats were imaged weekly from p17 to p60 and final images were obtained at p90. both right (od) and left (os) eyes were imaged from 7 to 10 different rcs rats at each time point. both od and os were imaged from four different le rats at each time point. before imaging, pupils were dilated with 1% tropicamide and 2.5% phenylephrine. sedation was induced by ketamine (80 mg / kg)/xylazine (5 mg / kg). spectral - domain oct images were obtained by using the envisu r2200-hr sd - oct device (bioptigen, durham, nc, usa) with the reference arm placed at approximately 1187 mm. single horizontal and vertical linear scans (2-mm preset scan width, 1500 a - scans / b - scan 20 frames / b - scan) were obtained first while centered on the optic nerve, then with the nerve displaced either temporally / nasally or superiorly / inferiorly. to analyze interscan variability, three consecutive od and os temporal, nasal, superior, and inferior scans were obtained in three additional rcs rats at p30. axial and transverse export resolution was 1.175 m / pixel and 3.175 m / pixel, respectively. these files were loaded into imagej (version 1.45 ; provided in the public domain by the national institutes of health, bethesda, md, usa) where they then were registered by using the stackreg plug - in and averaged as a z - stack. manual segmentation and quantification of retinal layer thickness was performed with a custom - designed sd - oct segmentation program built in igor pro (igor pro 6.12 ; wavemetrics, inc., lake oswego, or, usa). segmentation software is sharable with permission of the author (yuquan wen). for segmentation analysis, total retina (tr), inl, outer nuclear layer+ the onl+, the photoreceptor layer from the outer limiting membrane (olm) to the inl / outer plexiform layer (opl) interface, was used to enable consistent measurements across ages as hyperreflectivity in the photoreceptors increased. from p30 to p90, a hyperreflective layer (hrl) was observed and measured in rcs rats. to ensure our segmentation analysis was reliable and repeatable, interscan and interanimal variability were assessed with intraclass correlation coefficients (icc) and 95% confidence intervals by using ibm (armonk, ny, usa) spss statistics version 24 (tables 1, 2). to calculate interscan variability, three repeated scan measurements from three different p30 rcs rats were correlated (table 1). to calculate the variation of od and os scans, measurements from nine different p30 rcs rats were correlated (table 2). reproducibility of first, second, and third scan retinal layer thickness measurements retinal layer thickness variation between rcs od and os scans at p30 total retina, inl, onl+, rpe, and hrl thicknesses (micrometers, y - axis) were measured 40 outward in the inferior (od and os), nasal (os), or temporal (od) direction and + 40 outward in the superior (od and os), nasal (od), or temporal (os) direction from the optic nerve head (x - axis). the resulting waves for tr and onl+ in the superior and inferior retina were averaged and plotted as spider graphs. additionally, points from 40 to 25 and 25 to 40 were averaged to obtain tr, inl, onl+, rpe, and hrl thicknesses for each eye in each quadrant. right and left eye thicknesses were averaged and plotted against age (days) for all of the animals scanned., la jolla, ca, usa) prism 7 software and plotted for each data set. a 2-way anova, multiple comparisons test was used to compare average rcs retinal layer thickness between each quadrant at each time point. once enucleated, eyes were placed immediately in prefer, a glyoxal fixative (anatech ltd, battle creek, mi, usa) and incubated overnight at room temperature. orientated eyes were processed and embedded in paraffin for sectioning (tissue - tek vip 6, tissue - tek tec 5 ; sakura finetek usa, inc., sections were cut with a microtome to a thickness of 4 m, stained with hematoxylin - eosin (h&e), and viewed on a leica dmi3000 b microscope (leica microsystems gmbh, wetzlar, germany). wild - type, pigmented long - evans (le) rats, obtained from charles river (seattle, wa, usa), were used as controls because although the retina layers thin with age, there is no appreciable cell loss or change in total retinal volume. in addition, le rats have previously been used to back breed pigment in the rcs line and to generate the nondystrophic rcs line. rats were housed in standard conditions under a 12/12-hour light dark cycle (light cycle : 200 lux). all experiments were approved by the institutional animal care and use committee at ohsu and adhered to the arvo statement for the use of animals in ophthalmic and vision research. rcs rats were imaged weekly from p17 to p60 and final images were obtained at p90. both right (od) and left (os) eyes were imaged from 7 to 10 different rcs rats at each time point. both od and os were imaged from four different le rats at each time point. before imaging, pupils were dilated with 1% tropicamide and 2.5% phenylephrine. sedation was induced by ketamine (80 mg / kg)/xylazine (5 mg / kg). spectral - domain oct images were obtained by using the envisu r2200-hr sd - oct device (bioptigen, durham, nc, usa) with the reference arm placed at approximately 1187 mm. single horizontal and vertical linear scans (2-mm preset scan width, 1500 a - scans / b - scan 20 frames / b - scan) were obtained first while centered on the optic nerve, then with the nerve displaced either temporally / nasally or superiorly / inferiorly. to analyze interscan variability, three consecutive od and os temporal, nasal, superior, and inferior scans were obtained in three additional rcs rats at p30. axial and transverse export resolution was 1.175 m / pixel and 3.175 m / pixel, respectively. these files were loaded into imagej (version 1.45 ; provided in the public domain by the national institutes of health, bethesda, md, usa) where they then were registered by using the stackreg plug - in and averaged as a z - stack. manual segmentation and quantification of retinal layer thickness was performed with a custom - designed sd - oct segmentation program built in igor pro (igor pro 6.12 ; wavemetrics, inc., lake oswego, or, usa). segmentation software is sharable with permission of the author (yuquan wen). for segmentation analysis, total retina (tr), inl, the onl+, the photoreceptor layer from the outer limiting membrane (olm) to the inl / outer plexiform layer (opl) interface, was used to enable consistent measurements across ages as hyperreflectivity in the photoreceptors increased. from p30 to p90, a hyperreflective layer (hrl) was observed and measured in rcs rats. to ensure our segmentation analysis was reliable and repeatable, interscan and interanimal variability were assessed with intraclass correlation coefficients (icc) and 95% confidence intervals by using ibm (armonk, ny, usa) spss statistics version 24 (tables 1, 2). to calculate interscan variability, three repeated scan measurements from three different p30 rcs rats were correlated (table 1). to calculate the variation of od and os scans, measurements from nine different p30 rcs rats were correlated (table 2). reproducibility of first, second, and third scan retinal layer thickness measurements retinal layer thickness variation between rcs od and os scans at p30 total retina, inl, onl+, rpe, and hrl thicknesses (micrometers, y - axis) were measured 40 outward in the inferior (od and os), nasal (os), or temporal (od) direction and + 40 outward in the superior (od and os), nasal (od), or temporal (os) direction from the optic nerve head (x - axis). the resulting waves for tr and onl+ in the superior and inferior retina were averaged and plotted as spider graphs. additionally, points from 40 to 25 and 25 to 40 were averaged to obtain tr, inl, onl+, rpe, and hrl thicknesses for each eye in each quadrant. right and left eye thicknesses were averaged and plotted against age (days) for all of the animals scanned. best - fit equations were generated by using graphpad (graphpad software, inc., la jolla, ca, usa) prism 7 software and plotted for each data set. a 2-way anova, multiple comparisons test was used to compare average rcs retinal layer thickness between each quadrant at each time point. once enucleated, eyes were placed immediately in prefer, a glyoxal fixative (anatech ltd, battle creek, mi, usa) and incubated overnight at room temperature. eyes were then placed in cassettes and stored in 70% ethanol at room temperature. orientated eyes were processed and embedded in paraffin for sectioning (tissue - tek vip 6, tissue - tek tec 5 ; sakura finetek usa, inc., torrance, ca, usa). sections were cut with a microtome to a thickness of 4 m, stained with hematoxylin - eosin (h&e), and viewed on a leica dmi3000 b microscope (leica microsystems gmbh, wetzlar, germany). retinal layers including the ganglion cell layer (gcl), inner plexiform layer (ipl), inl, opl, onl, olm, photoreceptor inner and outer segments (pr), and rpe were clearly visible in sd - oct images of le rats at all ages examined (fig. the ellipsoid zone (ez), which consists of mitochondria within the ellipsoid layer of the outer portion of the inner segments and represents the inner / outer segment junction, could be identified from p17 to p23, but the outer segments (os) were becoming hyperreflective, which caused them to become isoreflective with the ez. thus, differentiation between the ez and os was less distinct in p17 to p23 rcs retinas than in le controls. by p30, the pr and olm were indistinguishable and isoreflective. thus, from p30 to p90, this area of the retina was observed as a single layer and termed the hyperreflective layer (hrl). from p30 to p60 the onl became progressively hyperreflective and thinned relative to baseline. by p45, it became isoreflective with the opl and became difficult to distinguish, but was not detectable by p90 (fig. images acquired with sd - oct illustrate the longitudinal retinal degeneration in the rcs rat. representative linear sd - oct scans from right eyes of long - evans and rcs rats at differing ages from p17 to p90. images are representative of the same location in both long - evans and rcs retinas, approximately 30 from the optic nerve head in each retinal quadrant. each image is approximately 2. photoreceptors were maintained in long - evans retinas and degenerated over time in rcs retinas. is, inner segments ; onl, outer nuclear layer ; os, outer segments. to characterize the progressive retinal degeneration in the rcs rat, manual segmentation was performed to calculate tr, inl, onl+, hrl, rpe thickness (see schematic for segmentation lines in figure legend ; fig. to validate our segmentation analysis, interscan and interanimal variability were assessed with icc (tables 1, 2). our manual segmentation provided reproducible retinal layer thickness measurements, as most thickness measurements, obtained from three individual scans, were correlative (icc = 0.51 ; table 1). there was also good correlation (icc = 0.6440.944) between retinal layer thickness measurements obtain from right and left eyes of p30 rcs rats, suggesting that degeneration rates were consistent between eyes (table 2). (a) representative linear sd - oct scans of superior retina from right eyes of long - evans and rcs rats. schematic for segmentation lines : tr, bottom purple line to the top red line ; inl, blue line to the green line that surround the inl label ; outer nuclear layer plus outer plexiform layer (onl+), red line to blue line that surround the onl+ label ; rpe, purple line to the blue line (le p30p90 and rcs p17p23) or purple line to the yellow line (rcs p30p90) that surround rpe label ; hrl, yellow line to the red line (rcs p30p60) or yellow line to the blue line (rcs p90) that surround hrl label. (b) right eye and left eye tr, onl+, inl, rpe, and hrl thicknesses from long - evans and rcs retinas in each retinal quadrant were averaged and plotted against age. rcs tr and onl+ degeneration was best fit with an exponential decay function, whereas all other data points were best fit with linear regressions. a 2-way anova, multiple comparisons test was used to compare average rcs retinal layer thickness between each quadrant at each time point. thickness of the superior retina was significantly increased compared to all other quadrants at specified age (p 0.01). thickness of the superior retina was significantly increased compared to temporal and inferior retina at specified age (p 0.01). long - evans : n = 4 animals ; rcs : n = 710 animals. retinal layers and segmentation remained consistent from p30 to p90 in le rats. in rcs rats, segmentation changed at p30 due to the appearance of the hrl and at p90 due to the loss of the onl+ (fig. scatter plots of average tr and onl+ thickness obtained from rcs rats over time demonstrated considerable declines that were best fit, based on r values, with exponential decay functions (fig. in contrast, all other retinal layers quantified in both rcs rats and le controls were best fit with linear functions (fig. 2b ; supplementary table s1). these data demonstrated that total retinal degeneration in the rcs rat was primarily due to the loss of photoreceptors, which is highlighted in figure 3. spider graphs of the superior and inferior retina illustrate that average tr and onl+ thickness decreased consistently over time in the rcs rat, whereas le rats had a narrow range in average tr and onl+ thickness from p30 to p90 (fig. scatter plots in figure 2 also demonstrated the consistency of retinal layer thickness between the different retinal quadrants. superior hrl thickness was significantly increased as compared to nasal, temporal, and inferior hrl thickness from p30 to p56 (fig. 2b, supplementary table s2 ; p 0.01). at p60, superior hrl thickness was still significantly increased as compared to temporal and inferior hrl thickness. by p90, hrl thickness was not significantly different in any retinal quadrant (fig. not surprisingly, average superior tr thickness was affected and was also significantly increased as compared to nasal, temporal, and inferior tr thickness from p30 to p37 (fig. 2b, supplementary table s2 ; p 0.01). interestingly, superior onl+ was thicker than all other retinal quadrants at p37 (fig. 2b, supplementary table s2 ; p 0.01). rcs total retina and onl+ thicknesses obtained from right eye and left eye inferior and superior sd - oct scans were averaged and plotted versus retinal eccentricity from the optic nerve head. gray shading represents long - evans total retina and onl+ range from p30 to p90. rcs onl+ is undetectable by sd - oct at p90 and thus is plotted as zero (). histology of retinal sections from p21, p30, p45, and p60 rcs rats was compared to locally matched sd - oct images (fig. 4). at p21 the gcl, inl, opl, onl, pr, and rpe were clearly observable in both histology and sd - oct images. at this age, os disorganization was only evident at the apical side of the rpe. by p30, os disorganization expanded and the correlation between the pr / debris layer and the hrl was evident, suggesting that both layers are comprised of is, disorganized os, and debris (fig., both modalities demonstrated that this layer is thicker in the superior retina and thins over time (fig. the onl has significantly degenerated and less than half of the onl remained, which was clearly visible in the histology images. in the sd - oct images, the onl became hyperreflective, isoreflective with the opl, and was more difficult to distinguish than in the retinal sections (fig. although the onl resolution declined with disease progression, an onl+ layer was still measurable in sd - oct images at this time point (fig. when comparing the histology and sd - oct images from p21 to p60, both demonstrated an observable decrease in tr and onl thickness (fig. representative histology and sd - oct images of superior and inferior quadrants of the posterior retina from right eyes of rcs rats at p21 (a), p30 (b), p45 (c), and p60 (d). the hrl correlates with the pr / debris layer indicating a composition of is, disorganized os, and debris (arrowheads). retinal layers including the ganglion cell layer (gcl), inner plexiform layer (ipl), inl, opl, onl, olm, photoreceptor inner and outer segments (pr), and rpe were clearly visible in sd - oct images of le rats at all ages examined (fig. the ellipsoid zone (ez), which consists of mitochondria within the ellipsoid layer of the outer portion of the inner segments and represents the inner / outer segment junction, could be identified from p17 to p23, but the outer segments (os) were becoming hyperreflective, which caused them to become isoreflective with the ez. thus, differentiation between the ez and os was less distinct in p17 to p23 rcs retinas than in le controls. by p30, the pr and olm were indistinguishable and isoreflective. thus, from p30 to p90, this area of the retina was observed as a single layer and termed the hyperreflective layer (hrl). from p30 to p60 the onl became progressively hyperreflective and thinned relative to baseline. by p45, it became isoreflective with the opl and became difficult to distinguish, but was not detectable by p90 (fig. images acquired with sd - oct illustrate the longitudinal retinal degeneration in the rcs rat. representative linear sd - oct scans from right eyes of long - evans and rcs rats at differing ages from p17 to p90. images are representative of the same location in both long - evans and rcs retinas, approximately 30 from the optic nerve head in each retinal quadrant. each image is approximately 2. photoreceptors were maintained in long - evans retinas and degenerated over time in rcs retinas. is, inner segments ; onl, outer nuclear layer ; os, outer segments. to characterize the progressive retinal degeneration in the rcs rat, manual segmentation was performed to calculate tr, inl, onl+, hrl, rpe thickness (see schematic for segmentation lines in figure legend ; fig. 2a). to validate our segmentation analysis, interscan and interanimal variability were assessed with icc (tables 1, 2). our manual segmentation provided reproducible retinal layer thickness measurements, as most thickness measurements, obtained from three individual scans, were correlative (icc = 0.51 ; table 1). there was also good correlation (icc = 0.6440.944) between retinal layer thickness measurements obtain from right and left eyes of p30 rcs rats, suggesting that degeneration rates were consistent between eyes (table 2). (a) representative linear sd - oct scans of superior retina from right eyes of long - evans and rcs rats. schematic for segmentation lines : tr, bottom purple line to the top red line ; inl, blue line to the green line that surround the inl label ; outer nuclear layer plus outer plexiform layer (onl+), red line to blue line that surround the onl+ label ; rpe, purple line to the blue line (le p30p90 and rcs p17p23) or purple line to the yellow line (rcs p30p90) that surround rpe label ; hrl, yellow line to the red line (rcs p30p60) or yellow line to the blue line (rcs p90) that surround hrl label. (b) right eye and left eye tr, onl+, inl, rpe, and hrl thicknesses from long - evans and rcs retinas in each retinal quadrant were averaged and plotted against age. rcs tr and onl+ degeneration was best fit with an exponential decay function, whereas all other data points were best fit with linear regressions. a 2-way anova, multiple comparisons test was used to compare average rcs retinal layer thickness between each quadrant at each time point. thickness of the superior retina was significantly increased compared to all other quadrants at specified age (p 0.01). thickness of the superior retina was significantly increased compared to temporal and inferior retina at specified age (p 0.01). long - evans : n = 4 animals ; rcs : n = 710 animals. retinal layers and segmentation remained consistent from p30 to p90 in le rats. in rcs rats, segmentation changed at p30 due to the appearance of the hrl and at p90 due to the loss of the onl+ (fig. scatter plots of average tr and onl+ thickness obtained from rcs rats over time demonstrated considerable declines that were best fit, based on r values, with exponential decay functions (fig. in contrast, all other retinal layers quantified in both rcs rats and le controls were best fit with linear functions (fig. these data demonstrated that total retinal degeneration in the rcs rat was primarily due to the loss of photoreceptors, which is highlighted in figure 3. spider graphs of the superior and inferior retina illustrate that average tr and onl+ thickness decreased consistently over time in the rcs rat, whereas le rats had a narrow range in average tr and onl+ thickness from p30 to p90 (fig. scatter plots in figure 2 also demonstrated the consistency of retinal layer thickness between the different retinal quadrants. superior hrl thickness was significantly increased as compared to nasal, temporal, and inferior hrl thickness from p30 to p56 (fig. 2b, supplementary table s2 ; p 0.01). at p60, superior hrl thickness was still significantly increased as compared to temporal and inferior hrl thickness. by p90, hrl thickness was not significantly different in any retinal quadrant (fig. 2b, supplementary table s2 ; p 0.01). not surprisingly, average superior tr thickness was affected and was also significantly increased as compared to nasal, temporal, and inferior tr thickness from p30 to p37 (fig. 2b, supplementary table s2 ; p 0.01). interestingly, superior onl+ was thicker than all other retinal quadrants at p37 (fig. rcs total retina and onl+ thicknesses obtained from right eye and left eye inferior and superior sd - oct scans were averaged and plotted versus retinal eccentricity from the optic nerve head. gray shading represents long - evans total retina and onl+ range from p30 to p90. rcs onl+ is undetectable by sd - oct at p90 and thus is plotted as zero (). histology of retinal sections from p21, p30, p45, and p60 rcs rats was compared to locally matched sd - oct images (fig. 4). at p21 the gcl, inl, opl, onl, pr, and rpe were clearly observable in both histology and sd - oct images. at this age, os disorganization was only evident at the apical side of the rpe. by p30, os disorganization expanded and the correlation between the pr / debris layer and the hrl was evident, suggesting that both layers are comprised of is, disorganized os, and debris (fig., both modalities demonstrated that this layer is thicker in the superior retina and thins over time (fig. 4). by p45 the onl has significantly degenerated and less than half of the onl remained, which was clearly visible in the histology images. in the sd - oct images, the onl became hyperreflective, isoreflective with the opl, and was more difficult to distinguish than in the retinal sections (fig. although the onl resolution declined with disease progression, an onl+ layer was still measurable in sd - oct images at this time point (fig. when comparing the histology and sd - oct images from p21 to p60, both demonstrated an observable decrease in tr and onl thickness (fig. representative histology and sd - oct images of superior and inferior quadrants of the posterior retina from right eyes of rcs rats at p21 (a), p30 (b), p45 (c), and p60 (d). the hrl correlates with the pr / debris layer indicating a composition of is, disorganized os, and debris (arrowheads). spectral - domain oct allowed for high - resolution imaging of rcs retinal structure and quantification of retinal layers including tr, inl, onl+, rpe, and hrl. total retina and onl+ thickness degenerated over time, and the loss of the onl+ accounted primarily for tr reduction (fig. thus, the comprehensive tr and onl+ thickness measurements can be used as a baseline for future studies that assess the efficacy of potential therapies for outer retinal degeneration in the rcs rat. in addition, hrl thickness, which correlated to is, disorganized os, and debris accumulation observed in histology (fig. when comparing our sd - oct analysis to previous published anatomic measurements, we found that thickness values differed, but the rate of thickness changes was consistent. for example, both sd - oct and anatomic assessments show that half the onl thickness is decreased by approximately 7 weeks of age, that onl thinning occurs at a uniform rate in all four quadrants, and that the onl becomes absent in many areas of the posterior retina. however, manual segmentation of the onl+ resulted in thicker measurements than in previous reports, which measured distinctly around photoreceptor cell bodies (e.g., sd - oct at p17 : onl+ 90 m, tissue at p17 : onl 50 m). this difference seems reasonable for two main reasons : (1) histologic processing is known to dehydrate the tissue ; and (2) our reported onl+ measurement included opl thickness, whereas onl measurements from previous anatomic reports do not include opl thickness. hyperreflective layer thickness, acquired from sd - oct images, and an outer segment layer (osl) thickness, acquired from previous anatomic assessments, were also comparable. the osl has been quantified in order to estimate the relative volume of os debris accumulation during photoreceptor degeneration. in a previous anatomic assessment, it is reported that the osl thickness increases with age, but after maximum thickness is reached at p35, the osl decreases with progressive degeneration. these results seem comparable to the hrl identified in the rcs retina with sd - oct. hyperreflective layer thickness then decreased with progressive degeneration and was consistently thicker in the superior retina. quantification of the hrl in sd - oct images resulted in thicker values than in the quantification of the osl in retinal sections (e.g., sd - oct at p37 superior : hrl 61 m, tissue at p35 superior : osl / debris layer 48 m). two main reasons could account for this discrepancy : (1) the osl measured on retinal sections may not include the inner segment thickness ; and (2) as the hyperreflectivity increased and the olm became less distinct, it is possible that the hrl may include cells from the onl. overall, the hrl correlated with the pr / debris layer in our histology images (fig. 4), thus we suggest the hrl can be used to quantify photoreceptor inner and outer segment changes longitudinally. many histologic changes could be responsible for generating the hrl, including disorganization of os discs resulting in lamellar whorls of membranes, the expansion or swelling of apical rpe processes, or invasion of macrophages into the outer retina. our data demonstrated a correlation between preservation of onl+ thickness and increased hrl thickness in the superior retina from p30 to p37 (supplementary table s2), which supports this hypothesis. histologic analysis of retinal tissue has been considered the gold standard for characterizing retinal degeneration and assessing the efficacy of potential treatments for amd and inherited retinal dystrophies in the rcs rat. however, histologic processing precludes the ability to perform longitudinal analysis, thus increasing the number of animals required for long - term studies. in addition, artifacts from postmortem processing are unavoidable, including fixation and dehydration, which can alter layer thicknesses and mask features of in vivo disease progression. noninvasive, high - resolution sd - oct imaging offers an alternative in vivo approach to obtaining structural measurements of disease progression. spectral - domain oct imaging has been validated in multiple mouse (rho, rpe65, rd1, rd10) and rat (rho p23h) inherited retinal degeneration models. these studies have consistently reported that sd - oct is able to capture morphologic changes during disease progression and that sd - oct thickness measurements are notably higher, but in strong correlation with anatomic thickness measurements. in addition, sd - oct has been able to image well - characterized phenotypes, as in the complete absence of rod os in the rho mouse, and reveal novel in vivo findings that lead to discoveries about disease progression. for example, rpe65 mice have less distinct laminar organization, leading to the conclusion that expression of cytoskeletal elements and components of the extracellular matrix may be modified and playing a role in disease progression. in a study that characterized and revealed differences in disease progression between the rd1 and rd10 mice, retinal separations, often thought to be an artifact of histologic processing, were observed in the rd10 mice by sd - oct, confirming their in vivo existence. because of these findings, multiple studies now use retinal thickness measurements obtained from sd - oct images to demonstrate the long - term efficacy of various adeno - associated virus gene therapies for various inherited retinal degeneration mouse models (rd1, rd10, p23h rho, rs1h, rd12). despite the existing validation of sd - oct and numerous software programs available for segmentation of the retina, onl counts from h&e - stained retinal sections continue to be the main methodology used to illustrate preservation of photoreceptors after therapeutic interventions in the rcs rat. the congruency between histologic analysis and sd - oct analysis demonstrated in this study suggests that sd - oct may also be used to quantify the efficacy of various therapies longitudinally. we demonstrated that sd - oct overcomes the challenge of longitudinal anatomic assessments ; however, some limitations of this technology remain. the sd - oct analysis performed in this study was restricted to the posterior retina (up to 40 from the optic nerve head). as photoreceptor degeneration rates differ between posterior and peripheral retina in the rcs rat, this study should be used primarily as a reference for interpreting data from the posterior retina, while recognizing that rates of degeneration can differ in the peripheral retina. although sd - oct offers high - resolution imaging, manual segmentation of the onl became challenging during the course of degeneration. the onl became hyperreflective over time, slowly becoming isoreflective with the opl, olm, and hrl, which made it difficult to distinguish. to address this limitation however, since the olm became difficult to discern, this did not account for any onl that may have been masked by the hrl. although the rpe was observable in sd - oct images, the segmentation analysis showed appreciable variation between the animals. the outline of the rpe was clear in some images and less distinguished in others owing to hyperreflectance of the sclera and hrl. it is well known that the rpe is maintained in the rcs rat, but we hypothesized that due to rpe dysfunction there may be measurable changes in its thickness. our analysis suggests that if rpe thickness is changing during photoreceptor degeneration, better resolution imaging would be required to get consistent measurements of such a thin monolayer of cells. the rcs rat has been and will continue to be a fundamental model used to develop therapies for amd and inherited retinal dystrophies. we used sd - oct to quantify thickness of retinal layers in the rcs rat throughout the course of degeneration. spectral - domain oct is an important complement to histology and in many cases could be used as a surrogate when assessing the efficacy of treatments for outer retinal degeneration in the rcs rat. | purposeprospective treatments for age - related macular degeneration and inherited retinal degenerations are commonly evaluated in the royal college of surgeons (rcs) rat before translation into clinical application. historically, retinal thickness obtained through postmortem anatomic assessments has been a key outcome measure ; however, utility of this measurement is limited because it precludes the ability to perform longitudinal studies. to overcome this limitation, the present study was designed to provide a baseline longitudinal quantification of retinal thickness in the rcs rat by using spectral - domain optical coherence tomography (sd - oct).methodshorizontal and vertical linear sd - oct scans centered on the optic nerve were captured from long - evans control rats at p30, p60, p90 and from rcs rats between p17 and p90. total retina (tr), outer nuclear layer+ (onl+), inner nuclear layer (inl), and retinal pigment epithelium (rpe) thicknesses were quantified. histologic sections of rcs retina obtained from p21 to p60 were compared to sd - oct images.resultsin rcs rats, tr and onl+ thickness decreased significantly as compared to long - evans controls. changes in inl and rpe thickness were not significantly different between control and rcs retinas. from p30 to p90 a subretinal hyperreflective layer (hrl) was observed and quantified in rcs rats. after correlation with histology, the hrl was identified as disorganized outer segments and the location of accumulated debris.conclusionsretinal layer thickness can be quantified longitudinally throughout the course of retinal degeneration in the rcs rat by using sd - oct. thickness measurements obtained with sd - oct were consistent with previous anatomic thickness assessments. this study provides baseline data for future longitudinal assessment of therapeutic agents in the rcs rat. |
the hormones, glucosedependent insulinotropic polypeptide (gip) and glucagonlike peptide1 (glp1), are released from the intestine in response to nutrient ingestion and stimulate insulin secretion in a glucosedependent manner, as a result of which they have been classified as incretins. additionally, both hormones play regulatory roles in the maintenance, growth and survival of pancreatic islets, as well as acting in an integrative manner on processes involved in nutrient metabolism, including events underlying the passage of chyme through the gastrointestinal tract, and nutrient digestion, absorption and storage. the biological actions of gip and glp1 are terminated by the prolyl endopeptidase, dipeptidyl peptidase4 (dpp4). dpp4 resistant analogs of glp1 and dpp4 inhibitors are two recently introduced classes of type 2 diabetes therapeutics that mimic or potentiate, respectively, actions of the incretins. although these agents have proven to be extremely effective in improving glucose tolerance, it is unlikely that their therapeutic potential has been fully realized, and it is important to elucidate the mechanistic basis underlying incretin actions. in the present review, we focus on the current understanding of the signaling events involved in gip actions on cell function and the adipocyte, with an emphasis on studies carried out in the authors laboratory. the gip receptor (gipr) is a member of the class b g proteincoupled receptor family that has now been identified in chordates ranging from fish to mammals, although little is known about cellular signaling events in lower species. there is extensive literature on the mode of action of glp1 on the cell that has been expertly reviewed, and information that is relevant to gip action will be integrated into the current review. uptake and metabolism of glucose in the cell increases the intracellular adenosine triphosphate (atp)/adenosine diphosphate (adp) ratio, resulting in closure of atpsensitive k (katp) channels and membrane depolarization, with consequent activation of voltagedependent ca channels (vdcc), increases in intracellular ca (ica) and triggering of insulin granule exocytosis. membrane repolarization is mediated by voltagedependent k (kv) channels and casensitive k (kca) channels. both gip and glp1 have been shown to stimulate adenylyl cyclase (ac) through a stimulatory g protein (gs) coupled process resulting in increased cyclic adenosine monophosphate (camp), and this is considered to be the major signaling pathway involved in their potentiation of glucoseinduced insulin secretion. although gipstimulated insulin secretion is glucosedependent, camp production and subsequent activation of downstream signaling modules are not. as discussed further below, synergistic interaction between glucose and incretin action occurs when ca fluxes are increased through modulation of katp channels and vdcc, as well as through release from the endoplasmic reticulum (figure 1). type viii ac has been proposed to act as a coincidence detector of signals from glucose and camp, as it is activated by both cacalmodulin and gs. cacalmodulin also modulates the activity of phosphodiesterase (pde) 1c, and synergistic interactions between gs and cacalmodulin have been suggested to drive synchronous, inphase oscillations of camp and ca. signaling pathways proposed to be involved in proximal events in glucosedependent insulinotropic polypeptide (gip)mediated potentiation of glucoseinduced insulin secretion. (a) evidence has been presented supporting roles for both protein kinase a (pka) and cyclic adenosine monophosphate (camp)activated guanine nucleotide exchange factor (campgef)/exchange protein directly activated by camp (epac) in the modulation of adenosine triphosphate (atp)sensitive k (katp) channels. dissociation of campepac2 from sulfonulurea receptor 1 (sur1) binding has been proposed to activate phospholipase c (plc) through rasrelated protein 1 (rap1), resulting in phosphatidylinositol 4,5 bisphosphate (pip2) metabolism and inhibition of adenosine triphosphatesensitive channel subunit, kir6.2, membrane depolarization and activation of voltagedependent ca channels (vdcc). (b) diacylglycerolactivated pkc potentiates calciuminduced calcium release through ryanodine receptors and phosphatidylinositol trisphosphate (ip3) stimulates ca release from ip3 sensitive endoplasmic reticulum (er) ca stores. pka might act to sensitize the ca release channels (based on references 15, 20, 31, 36). ac, adenylyl cyclase ; cam, calmodulin ; gs, stimulatory g protein subunit, ip3r, inositol trisphosphate receptor ; p, phosphate ; ryr, ryanodine receptor ; serca, sarco(endo)plasmic reticulum caatpase. incretininduced increases in cell camp result in the activation of both protein kinase a (pka) and campactivated guanine nucleotide exchange factor (campgef)/exchange protein directly activated by camp (epac) (figure 1). epac2a has been identified as the major splicevariant involved in cell incretin signaling, but there are multiple pka isoforms present in cells and it is unclear as to which contribute to the modulation of insulin secretion. there is also uncertainty over the relative roles played by epac2 and pka in events leading to increases in insulin secretion, with katp channel closure, ca influx and intracellular mobilization, and processes underlying granule movement and exocytosis, all being potential targets. a number of investigators have carried out sophisticated electrophysiological and imaging studies to identify the pathways targeted by glp1 and/or gip and, as these studies have been comprehensively reviewed, only a summary of the main findings will be presented. glp1 was first suggested to modulate cell katp channel activity through a pkamediated pathway, and it was later reported that phosphorylation of the cell sulfonylurea 1 (sur1) subunit resulted in katp channel closure (figure 1a). there is now controversy over the contribution of pka to katp channel regulation, as compelling evidence points to epac2 as the major factor linking camp to katp channel closure and increasing ca influx. under nonstimulated conditions, epac2 interacts with the nucleotidebinding fold1 of sur1 (figure 1a). on binding camp, epac2 dissociates from sur1, and activates the gtpase, rasrelated protein 1 (rap1). have proposed that rap1 stimulates phospholipase c (plc), resulting in localized metabolism of plasma membrane phosphatidylinositol 4,5 bisphosphate (pip2) to phosphatidylinositol trisphosphate (ip3) and diacylglycerol (dag ; figure 1). as earlier studies showed that pip2 reduces the sensitivity of katp channels to atp, its depletion would be expected to result in potentiated atpdependent channel closure and membrane depolarization. cell stimulation by both glp1 and gip in the presence of elevated glucose has been shown to result in increased ca uptake through vdcc and nonselective ion channels, as well as stimulation of ca release from intracellular stores that, in the case of glp1, has been shown to involve epac2 activation. gip probably activates identical pathways (figure 1b), although studies on katp channeldeficient mice showed that gip actions on insulin secretion showed a greater dependency on katp channels than glp1. phospholipase c has been proposed to link epac and ica fluxes through the activation of endoplasmic reticulum (er) inositol trisphosphate (ip3) channels and protein kinase c (pkc)mediated potentiation of calciuminduced calcium release (cicr) through ryanodine receptors (figure 1b), possibly through activation of calciumcalmodulin kinase ii. in addition, pka is capable of sensitizing the intracellular ca release channels to the effects of ip3 and ca. gip also activates an islet group via caindependent phospholipase a2 (ipla2), resulting in increased arachidonic acid (aa) production from membrane lipids, and aa has been shown to increase release of ca from intracellular stores, suggesting that it might be coupled to insulin secretion. cell repolarization involves closure of vdcc, as well as opening of delayed rectifier and atype kv channels. gip and glp1 both reduce kv channel currents, prolonging cell action potentials and potentiating ca signals. kv2.1 is the major delayed rectifier channel in rodent cells, playing a dominant role in glp1, and probably gip, action. posttranslational modification of kv2.1 in response to gip and glp1 can modulate channel gating, promote inactivation and increase channel internalization through processes that involve phosphorylation by pka and pkc, and acetylation by campresponse element binding protein (creb) binding protein (cbp ; see cell prosurvival effects of gip section). as aa was also recently shown to increase the rate of inactivation of kv2.1 channels, gipactivation of ipla2 might also be linked to cell repolarization. in addition to the upstream events involved in insulin secretion, both incretins exert distal effects on secretory granule exocytosis through pka and epac dependent pathways. protein kinase a phosphorylates proteins that are components of the exocytotic machinery, including soluble nethylmaleimidesensitive fusion proteinattachment protein (snap) and mammalian uncoordinated homology 131 (munc 131). a number of models have been proposed to explain the role of epac2 in increasing the probability of granule exocytosis. proposed that epac2 interacts with sur1 associated with both the secretory granule and the plasma membrane, resulting in activation of a secretory granule chloride channel cic3, granule acidification and priming through a vtype hatpase. in a series of elegant experiments, seino. epac2a appears to be more important for regulating the readilyreleasable pool during the first phase of insulin secretion, whereas pka might be critical for second phase release. in addition to sur1 and rap1, epac2 also interacts with rasrelated in brain 3 (rab3)interactive molecule 2 (rim2), piccolo and snap25, and a camp epac2rim2 complex was shown to play a central role in secretory granule dynamics. although the mechanisms involved in complex formation are still being clarified, dissociation of camp epac2a from sur1 promotes cadependent heterodimerization of rim2 and piccolo, followed by interaction with rab3a and munc131, core components of the exocytotic apparatus. gipinduced insulin secretion was greatly impaired in rim2 knockout mice and their isolated islets, establishing its importance in the secretory pathway. additionally, phosphorylation of snapin by pka has been shown to be essential for incretinstimulated assembly of collectrin, snap25 and epac2. finally, we recently found that a selective epac agonist was capable of activating protein kinase b (pkb ; akt), emphasizing the importance of interaction between kinase pathways. it is currently unclear as to whether this interaction is related to insulin secretion or restricted to cell mitogenic and prosurvival effects of gip (see next section). cell dysfunction and reduced cell mass are major factors in the etiology of type 1 diabetes and type 2 diabetes. whereas autoimmune reactions are responsible for apoptotic loss of cells in type 1 diabetes, chronic hyperglycemia and hyperlipidemia, elevated cytokines, amyloid deposits, er stress and other factors contribute to type 2 diabetes. a number of procedures are being investigated for replenishing cell mass in diabetes patients, including the production of surrogate cells for transplantation and stimulation of residual cell proliferation. however, it appears that human cell regenerative capacity is limited to around the first three decades of life. therefore, prevention of cell loss by inhibiting apoptotic processes is an attractive alternative target. a role for incretin hormones in maintaining the normal integrity of pancreatic islets was first shown by the observation that glp1r mice have elevated levels of cell apoptosis. both gip and glp1 have been shown to exert strong prosurvival effects on cells in vitro. additionally, studies on a number of rodent models have shown that gipr or glp1r agonists exert marked antiapoptotic effects in vivo. our laboratory has focused on identifying mechanisms underlying the prosurvival actions of gip and the following overview summarizes recent findings. gip shows protective effects on cells that have been subjected to a number of apoptosisinducing stressors, including high glucose fatty acids (glucolipotoxicity), serum depletion and a low glucose environment or treatment with agents that induce genotoxic, mitochondrial or er stress. in studies on staurosporineinduced apoptosis in insulinoma1 (ins1) cells, gip was shown to exert effects at multiple levels in the apoptotic pathway, with reduced mitochondrial translocation of bcell lymphoma2 (bcl2) associated death promoter (bad) and bcl2 interacting mediator of cell deathel (bimel) and oligomerization of bcl2associated x protein (bax), release of cytochrome c and caspase 3 activation. ultimately, gip reduces the influence of mitochondriaassociated proapoptotic bcl2 family members, thus restraining the effects of stressors on the cell. although the pathways linking incretininduced production of camp and activation of pka and epac2a with the stimulation of insulin secretion have been extensively studied, the role of camp in prosurvival pathways has not been clearly defined. gipmediated antiapoptotic signaling in ins1 cells appears to be strongly dependent on camp production, as low concentrations of the adenylyl cyclase inhibitor mdl12,300a (santa cruz biotechnology, santa cruz, ca, usa) ablated the protective effects of gip on staurosporineinduced cell death. one major pathway by which gipr and glp1rmediated increases in camp production promote survival is by increasing expression of antiapoptotic genes through pka phosphorylation of creb at serine (ser)133. in the case of gip, bcl2 gene expression in ins1 cells was also found to involve pkastimulated dephosphorylation of amp activated protein kinase (ampk) and increased nuclear entry of campresponsive creb coactivator 2 (torc2). incretins activate a number of other genes involved in prosurvival pathways ; for example, expression of insulin receptor substrate 2 (irs2) is stimulated by glp1activated pka phosphorylation of creb. recently, creb was reported to be responsible for an acute phase of campdependent gene expression in cells, whereas a delayed phase involved induction of irs2/pkb pathways, activation of mammalian target of rapamycin (mtor) and increasing hypoxiainducible factor (hif) activity. this increase was shown to be associated with altered ins1 cell metabolic activity and improved cell viability. an additional effect of sustained cell stimulation by gipinduced activation of pkb is the phosphorylation and nuclear exclusion of forkhead box protein o1 (foxo1), that also promotes cell survival as a result of the requirement of nuclear foxo1 for expression of proapoptotic proteins, such as bax. it remains to be determined whether the hif pathway also modulates expression of prosurvival bcl2 family proteins. pkb phosphorylation on threonine (thr)308 and ser473 has been shown to be essential for enzyme activation in many cell types. however, in cells, gip was found to produce rapid increases in pkb activity through a nonpi3kinaseactivated pathway in the absence of detectable thr308 phosphorylation. as 8(4chlorophenylthio)2omethyl camp (8cpt camp), an epacselective agonist, mimicked the effects of gip, activation appears to be mediated by epac2, although it is currently unclear as to whether rap1 is involved. apoptosis signalregulating kinase 1 (ask1) has been shown to operate as a redox sensor that, on exposure to excessive levels of reactive oxygen species (ros), initiates the mitochondriamediated apoptotic pathway through activation of p38 mitogenactivated protein kinase (p38 mapk) and jun nterminal kinase (jnk). among the stressors shown to activate ask1 are oxidative and er stress, ca overload and receptor mediated inflammatory signals exposure of ins1 cells transfected with human ask1 to thapsigargin was found to increase phosphorylation of thr845 and reduce phosphorylation of ser83 in ask1, changes that increase its enzyme activity, resulting in phosphorylation and activation of p38 mapk and jnk, through mapk / extracellular signalregulated kinase (erk) kinase (mek) 3/6 and mek 4/7, respectively. activation of pkb with gip treatment prevented these changes in ask1 phosphorylation and downstream targets. suppressing ask1 activation with gip treatment inhibited apoptosis induced by all apoptosisinducing agents tested. as a result of the complexity of the system, the mechanism of gip action is currently only open to speculation. in nonstressed cells, ask1 forms a high molecular weight complex that has been termed the ask1 signalosome, and, in nonstressed cells when ask1 activity is inhibited, the antioxidative protein, thioredoxin (trx), is a component of the signalosome. if the oxidative state of the cell is greatly increased, the oxidized form of trx dissociates from ask1 and tumor necrosis factor (tnf) receptorassociated factor 2 (traf2) and traf6 binding activates ask1 signaling. although gipstimulated phosphorylation of ser83 by akt was clearly involved in inhibiting ask1 activity, it is also possible that the binding of thioredoxin and/or traf2/6 to ask1 were impacted on. additional gene regulatory actions of gip and glp1 involve modification of chromatin structure, thus altering the accessibility of transcription factors to target genes. a number of different posttranslational modifications of histone ntermini have been identified, and we showed that both incretin hormones modulate cell chromatin structure by increasing acetylation and phosphorylation of core h3 histones, with both elevated histone h3 acetyltransferase and reduced histone deacetylase activities contributing to the former. responses to both incretins involved activation of pka, and downstream erk1/2 (p44/42 mapk) and p38 mapk signaling modules, ultimately resulting in the activation of mitogen and stressactivated kinase1 (msk1) and creb. histone acetyltransferase (hat) inhibition resulted in significant reductions in incretinstimulated, crebactivated, bcl2 gene transcription, showing that histone h3 modification plays an important role in the regulation of apoptosisrelated proteins. both gip and glp1 also inhibit er stressinduced apoptosis through complex mechanisms that involve both reductions in the apoptotic pathway, leading to caspase activation, and altered expression or activity of a number of proteins involved in the unfolded protein response (upr), including activating transcription factor 4 (atf4), binding immunoglobin protein (bip), ccaatenhancerbinding protein homologous protein (chop) and growth arrest and deoxyribonucleic acid damageinducible protein 34 (gadd34). in recent studies, we identified an interesting link between incretinstimulated posttranslational modification of kv channels and apoptosis (figure 2). cell shrinkage (apoptotic volume decrease ; avd) occurs early in the apoptotic process, before dna fragmentation, cytochrome c release and caspase 3 activation, and it is a prerequisite for completion of programmed cell death. efflux of k is a critical component of the avd and, as activities of a number of caspases and endonucleases are suppressed at normal intracellular k concentrations, decreases in its intracellular concentration result in their activation. in different cell types, we decided to examine a possible role for the most prevalent rodent cell family member, kv2.1, in the development of apoptosis, and determine whether regulation of this process by gip and glp1 contributes to their prosurvival effects. ins1 cells, in which kv2.1 was overexpressed, showed potentiated apoptotic responses to mitochondrial and er stress, whereas gip or glp1 reduced the potentiation. in studies designed to identify their mode of action, both gip and glp1 promoted phosphorylation and acetylation of kv2.1 through pathways involving pka, and/or msk1 and hat (figure 2). we subsequently found that gip and glp1 promoted nuclear / cytoplasmic shuttling of cbp, resulting in its interaction with kv2.1 (figure 2). downregulation of cbp ablated incretininduced acetylation of kv2.1, suggesting that this hat is primarily responsible for the acetylation. as ask1 has been shown to play a key role in the proapoptotic modulation of kv channels, it is likely that the gipactivated pathway leading to phosphorylation of serine 83 in ask1 interacts with events leading to kv protein modification. proposed signaling pathways by which glucosedependent insulinotropic polypeptide (gip) regulates endocytosis of voltagedependent k (kv)2.1. binding of gip to its receptor (gipr) activates protein kinase a (pka) and, potentially, mitogen and stressactivated kinase1 (msk1), resulting in phosphorylation of kv2.1. through an unknown mechanism, cyclic adenosine monophosphateresponse element binding protein binding protein (cbp) is translocated from the nucleus to the plasma membrane, where it acetylates kv2.1. ac, adenylyl cyclase ; ac, acetylated ; creb, campresponse element binding protein ; gs, stimulatory g protein subunit ; p, phosphorylated ; torc2, transducer of regulated cyclic adenosine monophosphate response element binding protein activity. fat ingestion is a major stimulus for gip secretion in humans, dogs and rodents, and there is increasing evidence supporting a physiological role for gip in promoting fat storage. there are two pathways by which gip impacts on adipocyte metabolism : directly through interaction with gip receptors on the adipocyte and through stimulation of insulin secretion. gip infusion has been shown to promote the clearance of chylomicronassociated triglyceride (tg) in dogs, and to lower plasma tg responses to intraduodenal fat in rats. however, gip had no major effect on the rate of removal of intravenously administered tg, suggesting that gip stimulates release of tg from chylomicrons and uptake into adipose tissue. support for a role for gip in regulating adipose tissue mass came from rodent studies. mice that were treated with gipr peptide antagonists, vaccinated against gip or subjected to selective kcell ablation all showed increased resistance to highfat feedinginduced obesity, showing that gip normally promotes lipid storage. early studies on direct adipocyte actions of gip showed stimulatory effects on fatty acid (fa) synthesis from acetate in adipose tissue explants, increased uptake and incorporation of glucose into lipids, as well as enhanced free fa (ffa) incorporation into adipose tissue. adipocyte lipoprotein lipase (lpl) is responsible for the hydrolysis of tg in circulating chylomicrons, tgrich lipoproteins and very lowdensity lipoproteins, resulting in adipocyte uptake of ffa and monoacylglycerol, and the promotion of lipogenesis. gip was shown to increase lpl enzyme activity, in an insulindependent manner, in cultured 3t3l1 adipocytes, rodent adipocytes and subcutaneous human adipocytes. in view of the close correlation between human gip responses and plasma postheparin lpl levels, it has been suggested that gip acts on adipocyte storage by matching adipose tissue uptake of fa with the triglyceride load. suboptimal levels of circulating ffa result in greatly reduced cell responsiveness to subsequent glucose stimulation. in vitro studies showed that, in the absence of insulin, gip stimulates adipocyte tg hydrolysis through pka activation, and we suggested that gip primes cells during fasting by releasing adipocyte ffa into the circulation.. showed that gipstimulated increases in glycerol production were accompanied by decreased ffa in perifused adipocytes. a similar response was recently reported in humans, with gip infusion resulting in small increases in adipose tissue ffa reesterification. gip might also have longterm effects on lipid metabolism, as synthesis of pancreatic lipase and colipase were both stimulated by gip, an effect that should increase efficiency of lipid uptake. in the presence of insulin, gip signaling appears to play important roles in both the differentiation of preadipocytes and lipogenesis. a complex set of events is involved in preadipocyte to adipocyte development, including growth arrest, increased transcription factor and lipogenic enzyme expression, accumulation of lipid, and the development of sensitivity to regulatory hormones. expression of the gipr is extremely low in preadipocytes, but both messenger ribonucleic acid (mrna) and protein expression increase during differentiation of 3t3l1 cell and human preadipocytes. gip acted synergistically with insulin to increase neutral lipid accumulation during progression of 3t3l1 preadipocytes to the adipocyte phenotype. however, it was unclear as to whether synergistic effects of gip and insulin on gipr mrna levels were a result of direct effects on gene transcription or secondary to the progression of differentiation. 3t3l1 cell differentiation was associated with upregulation of nuclear levels of peroxisome proliferatoractivated receptor (ppar). treatment with the ppar receptor agonists, ly171883 and rosiglitazone, increased gipr expression in fully differentiated 3t3l1 adipocytes, whereas the antagonist, gw9662, ablated expression. acetylation of histone h3/h4 was also increased during differentiation, and both ppar and acetylated histone h3/h4 bound to a region of the gipr promoter containing the peroxisome proliferator response element (ppre). as rna interference (rnai) knockdown of ppar in differentiated 3t3l1 adipocytes greatly reduced gipr levels, ppar appears to be a critical transcription factor in regulating adipocyte receptor expression, but it is unclear as to the role played by insulin. evidence has been presented for the involvement of both ppar and ppar in the regulation of rodent cell gipr expression, and further studies are required to clarify whether there are cellselective differences in regulation. additionally, in earlier studies, it was shown that the gipr is downregulated in cells of obese rodent models of diabetes, but in studies on vancouver diabetic fatty (vdf) zucker rats, we recently found that, compared with lean controls, gipr and ppar protein levels were increased in epididymal and retroperitoneal fat pads, decreased in the perirenal fat depot and unchanged in other fat deposits (figure 3). in contrast, gipr expression in subcutaneous adipose tissue from human obese females was reported to be lower than in lean control subjects. however, these results are difficult to compare, because of the different fat depots studied. additionally, the sensitivity of gipr expression to the prevailing insulin concentration, the level of adipose tissue insulin resistance and the glycemic status of the subjects / animals could all contribute significantly to the level of gipr expression. glucosedependent insulinotropic polypeptide receptor (gipr) and peroxisome proliferatoractivated receptor (ppar) protein expression levels in adipose tissue depots from lean and obese vancouver diabetic fatty (vdf) zucker rats. tissue was collected from 18weekold rats and western blot analyses were carried out, with quantification by densitometry (n = 46 rats). keuls posthoc test. p < 0.05 vs lean control group. the pathways involved in gipstimulated lipogenesis are proving difficult to define, as a result of interactions between gip, insulin and adipokine signaling. gip stimulation of glucose uptake was shown to involve increasing plasma membrane glucose transporter4 (glut4) levels through a pkbmediated pathway. human lpl gene expression is also stimulated by gip activation of pkb, resulting in downstream reductions in lkb1 and ampk phosphorylation, and increased translocation of torc2 (campresponsive creb coactivator 2 [crtc2 ]) into the nucleus. regulation of the phosphorylation state of torc2 is complex and other members of the ampk family (saltinducible kinases [sik ] and mark2) are also capable of torc2 phosphorylation, whereas calcineurin and a campactivated pathway induce dephosphorylation. gip also enhances lpl enzyme activity in cultured 3t3l1 cells and subcutaneous human adipocytes by nontranscriptional mechanisms. in the 3t3l1 cell line, gip induces transient activation of p38 mapk and sustained activation of stressactivated protein kinase (sapk)/jnk, resulting in the release of resistin that, in turn activates pkb. somewhat surprisingly, subsequent events mimic those downstream of gip in the human adipocyte, with decreases in lkb1 and ampk phosphorylation linked to increased lpl secretion. human resistin (fizz3) shares only moderate sequence homology (53%) with the mouse peptide. additionally, fizz3/resistin is only weakly expressed in human adipocytes, and monocytes / macrophages are the major sites of fizz3/resistin production in adipose tissue. it is currently unknown whether fizz3/resistin serves a paracrine function in adipocyte regulation or whether there is an entero adipokine axis involving gip and fizz3/resistin in humans. however, fizz3/resistin has been reported to increase ffa reesterification. in the majority of in vitro studies to date, the effects of gip on adipogenesis and lipogenesis have been shown to involve synergistic actions with insulin, and there has been significant interest in gipr antagonists as potential therapies for obesity. however, adipose tissue expansion has been suggested to be an important adaptive response to increased food intake, as it protects against excess fat deposition in other sites, such as liver and muscle. gip could be an important contributor to this response and reducing its effect might not result in the anticipated benefits. additionally, studies on both transgenic mice and pigs expressing a dominantnegative gipr showed greatly reduced cell mass, with the mice becoming severely diabetic, supporting a critical role for gip signaling in cell development and proliferation. | abstractglucosedependent insulinotropic polypeptide (gip) was the first incretin to be identified. in addition to stimulating insulin secretion, gip plays regulatory roles in the maintenance, growth and survival of pancreatic islets, as well as impacting on adipocyte function. the current review focuses on the intracellular signaling pathways by which gip contributes to the regulation of cell secretion and survival, and adipocyte differentiation and lipogenesis. studies on signaling underlying the insulinotropic actions of the incretin hormones have largely been carried out with glucagonlike peptide1. they have provided evidence for contributions by both protein kinase a (pka) and exchange protein directly activated by cyclic adenosine monophosphate (epac2), and their probable role in gip signaling is discussed. recent studies have shown that inhibition of the kinase apoptosis signalregulating kinase 1 (ask1) by gip plays a key role in reducing mitochondriainduced apoptosis in cells through protein kinase b (pkb)mediated pathways, and that gipinduced posttranslational modification of voltage dependent k+ (kv) channels also contributes to its prosurvival role. through regulation of gene expression, gip tips the balance between pro and antiapoptotic members of the bcell lymphoma2 (bcl2) protein family towards cell survival. gip also plays important roles in the differentiation of preadipocytes to adipocytes, and in the regulation of lipoprotein lipase expression and lipogenesis. these events involve interactions between gip, insulin and resistin signaling pathways. (j diabetes invest, doi : 10.1111/j.20401124.2012.00196.x, 2012) |
aluminum (al) toxicity is a major constraint of crop production on acid soils. in view of the fact that 40% of world 's arable land is acidic [1, 2 ], al toxicity remains a major hurdle for increasing world food, fiber, and fuel production particularly via expansion of cultivation into acid soils. aluminum inflicts a wide range of cellular injuries in plants that ultimately result in reduced root growth, nutrient and water uptake, and productivity [1, 2 ]. plants possess some degree of tolerance to al toxicity that varies among species and genotypes [1, 36 ]. al exclusion via rhizosphere al - organic acid anion complex formation is the most widely documented physiological mechanism of al tolerance in cultivated and wild plants alike [1, 7 ]. root - exuded citrate, malate, and oxalate are the key organic acid anions involved in such mechanism. genes involved in al - induced root exudation of malate and citrate have been cloned in wheat and sorghum, and their variants are being discovered in several plant species. internal detoxification mechanisms involve the formation of al complexes with organic acids, acidic polypeptides, and/or proteins and subsequent sequestration of al in organelles away from sensitive sites in the cell [9, 10 ]. the genetic components of the internal detoxification pathways are yet to be elucidated. in soybean, al tolerance is a complex trait perhaps involving several genes and pathways [11, 12 ]. quantitative trait loci (qtl) mapping in a population derived from al tolerant pi 416937 and al sensitive young has revealed five dna markers associated with al tolerance. other reported soybean al tolerance genes include phosphoenolpyruvate carboxylase (pepc), homolog of translationally controlled tumor proteins (tctps), inosine 5-monophosphate dehydrogenases (impdhs), aluminum - induced 3 - 2 (sali3 - 2), and aluminum - induced 5 - 4a (sali 4 - 5a). ragland and soliman used gene expression as a tool to identify the above genes but the techniques used in these experiments were not sensitive enough to detect large number of genes that might be expected from the quantitative nature of soybean al tolerance trait. the objective of this study was to discover putative al tolerance genes in al - tolerant soybean line pi 416937 using dna microarrays a robust genome wide transcript profiling technology. such an approach was recently employed in wheat [15, 16 ], maize, arabidopsis, and medicago truncatula [19, 20 ] to discern the molecular basis of al tolerance in the respective species. an al - tolerant soybean plant introduction (pi 416937) highly characterized for al response [12, 21 ] was used in this experiment. seeds were surface sterilized with 20% household bleach (clorox) in water for 12 min, rinsed with distilled - deionized water several times, and were germinated in deionized water moistened standard germination paper at 25c in an incubator for 72 h. seedlings uniform in tap root length were transferred to black - painted pots filled with approximately 4 l of 800 m cacl2 background solution with 10 m al added (treated) or no al added (control) in a conviron growth chamber (16/8 h light/ dark cycle with respective temp. the ph of the culture solution was adjusted to 4.3 and maintained at that level for the entire duration of the experiment. after 2, 12, 48, or 72 h of al treatment 1 cm sections of the primary root tips of approximately 15 plants / pot were harvested, immediately flash frozen in liquid nitrogen, and stored at 70c for rna extraction. total rna was extracted from 100 mg root tissue samples using qiagen rneasy plant rna isolation kit following the manufacturer 's protocol (qiagen, inc.). the affymetrix genechip soybean genome array with over 68 000 probe sets, glycine max l. and wild soybean combined, was used for microarray analysis of the soybean genome for al tolerance. detailed procedures for rna labeling and array analysis are described in the manufacturer 's genechip expression technical manual (affymetrix). briefly, the quality of total rna was determined using the rna 6000 nano chip on agilent bioanalyzer 2100 prior to double - stranded cdna synthesis. total rna in the amount of 2 g was used for double - stranded cdna generation by linear amplification using oligo dt - t7 primer and reverse transcriptase (rt). subsequently, biotin - labeled crna was synthesized by in vitro transcription (ivt) using the enzo high yield ivt kit (enzo). quality and quantity of crna were assessed using the rna 6000 nano chip on agilent bioanalyzer 2100. arrays were hybridized overnight at 45c for 16 h in genearray hybridization oven 640 (affymetrix). the next day, arrays were washed and stained in the fluidics station 450 (affymetrix) and scanned by the high resolution genechip scanner 3000 (affymetrix). gene expression values were determined using thegenechip operating software (gcos 1.1, affymetrix). the expression levels were subjected to data query and data mining in data mining tool (dmt). statistical analysis of the data was conducted using the software packages arrayassist enterprise together with pathway assist (stratagene / agilent, santa clara, ca). the raw genechip files from genechip operating software (gcos, affymetrix, ca) were uploaded, background - subtracted, variance stabilized, and normalized with gc - rma method. the control group was used as a baseline to calculate the intensity ratio / fold changes of the treatment versus control. the p - values were obtained by an unpaired t - test assuming unequal variance. significantly upregulated and downregulated genes were annotated using protein databases accessed by blastx at national center for biotechnology information (ncbi). 24062 : f-5-tgccgaaggatcatctcaac-3, r-5-cgagggataatggttgatgg-3 ; gma.26937 : f-5-tacccaaaaggcaggcatac-3, r-5-ggccgaggtacaaacacatc-3 ; gma.4156 : f-5-tccaatgctgacaagtgctc-3, r-5-tagggacactccgtccaatc-3 ; gma.2577 : f-5-acgcctatgaacgtgaaacc-3, r-5-aacatcagcggagagcattc-3 ] from microarray experiments was conducted using the roche diagnostics light cycler 480 system with sybr green detection (roche diagnostic, corp) using beta - tubulin gene (beta - tubulin : r-5-ccatcaaacctcaaggaagc-3, f-5-tgctgtcctcttggacaatg-3) as internal control. mrna was isolated from plants grown under similar experimental conditions as in the microarray experiments. rna samples were treated with applied biosystems turbo dna - free dnase (ambion, inc.) to remove dna contamination. briefly, 2 l 10x dnase i buffer and 1 l rdnase i were added to 20 l rna sample, and the mix was incubated at 37c for 30 minutes in water bath. subsequently, 2 l resuspended dnase inactivation reagent was added and the samples mixed well and incubated at room temperature for 3 minutes. samples were then centrifuged at 10 000 g for 1.5 min (eppendorf centrifuge 5415 d) in 1.6 ml centrifuge tubes and supernatants transferred to fresh tubes. cdna was synthesized from 1 g dnase - treated rna samples using the roche diagnostics transcriptor first strand cdna synthesis kit (roche diagnostics, corp) according to manufacturer 's protocol. cdna concentration and quality was determined using nanodrop spectrophotometer brand nd-1000 (nanodrop technologies, inc.). cdna samples were diluted with nuclease - free water in varying ratios ranging from 1 : 4 to 1 : 10 depending on sample concentration. a total reaction volume of 11 l comprising 2 l cdna sample, 2 l each of the reverse and forward primers at 0.2 m concentration, and 5 l sybr mix was prepared in 96-well plates (roche diagnostics) in two biological and three technical replicates for each gene. a real - time pcr profile of preincubation at 95c for 5 min, a 45-cycle amplification at 95c for 10 second, 55c for 20 second, and 72c for 20 second, melting at 95c for 1 min, 65c for 1 min, and 95c continuous, and cooling at 40c for 30 seconds was used to amplify the samples. negative controls in which cdna sample was replaced with pcr grade water for each primer pair were included in each run. sample wells were individually assessed for data quality by evaluating amplification curves and pcr product specificity was verified by melting curve analysis. the expression level of target genes was normalized using in - run beta - tubulin gene as internal control, and transcript concentration ratios were calculated using the ct - method. the change in gene expression levels (fold change) was calculated as treatment to control ratio and compared with results from microarray. a total of 38 genes were identified as differentially expressed in the 10 m al - treated experimental plants compared to no al added controls at 2 h post al treatment (figure 1). thirty - four of them were upregulated and 4 were downregulated with a fold change ranging from 3.08 to 32.55 (table 1). at 12 and 72 h post treatment only one gene each showed significant change in expression in response to al treatment (figure 1 and table 1). the highest number of differentially expressed genes was detected at 48 h post al treatment (figures 1 and 2). the marked fold differences observed in the current research are substantially higher in comparison with results obtained by most authors but are comparable to results of [18, 24 ]. there were two genes in common between the set of genes detected at 2 h and 48 h post treatment (gma.2577, 7-fold downregulated at 2 h and 8-fold upregulated at 48 h and gma.26937, 8-fold downregulated at 2 h and 115 upregulated at 48 h). similar patterns of gene expression were observed in arabidopsis roots under al stress with few overlaps between sets of genes detected at 6 h and 48 h post al treatment. the temporal pattern of al - induced gene expression changes observed in this study diverges from results of other authors. at 12 and 72 h, the virtually no detection of al - regulated genes at 12 and 72 h post treatment seems a little odd but it is what is expressed in this soybean genotype at detection thresholds of p - value <.01 and 3-fold change in an experiment with 3 replications. gene expression is species and genotype specific [1520 ] making comparison of results across different studies difficult. the most likely explanation for the 72-hour result is that al toxicity could have already been neutralized by the 72 h, making differential gene expression unnecessary. the lack of transcriptional response at 12 h, however, is a biological puzzle, and it could represent a very unusual temporal transcriptome response of this soybean genotype to al stress. among the few reported al microarray studies, the results of kumari. in arabidopsis is the closest to ours with regard to the number of genes detected at early and late time points. they detected 127 genes at 6 h post treatment and 733 genes at 48 h post treatment using a threshold of a 2-fold change whereas we detected 38 genes at 2 h and 542 at 48 h using a 3-fold change. all of the differentially expressed genes that were functionally annotated by the genbank nonredundant protein database were grouped into five functional categories based on their putative cellular function. the functional classification showed that stress- and metabolism - related genes constitute the major fractions of al - regulated genes (figure 3). the microarray gene expression levels were validated with quantitative real - time pcr for representative genes (figure 4). in general, the microarray results were in agreement with qrt - pcr but in a few cases quantitative rt - pcr gave higher levels of expression compared to microarray. such results are obtained by a number of investigators [16, 20, 25 ]. detail discussion of factors contributing to the discrepancy between microarray and rt- pcr gene expression levels is covered in. many authors attribute the phenomenon to the high dynamic range and greater sensitivity of pcr detection. it is worth noting that the gene expression kinetics depicted in figure 1 shows the efficacy of our experimental design in capturing the full dynamic range of gene expression profiles in the soybean genotype studied. gene expression peaks at 2 and 48 h suggesting that major savings in microarray experimental expenditure could be realized by limiting sampling to these time points in future experiments. a number of transcription factors including bzip, wrky, myb, adr6, and nac were highly upregulated in the present study (tables 1 and 2). members of these families of transcription factors were previously detected under al stress in several plant species [16, 1820, 27 ]. cys2his2-type zinc finger (bzip) and auxin downregulated (adr6) factors are particularly interesting from al tolerance perspective. cys2his2-type zinc finger (bzip) protein coregulates molecular response to proton and al toxicities. it controls the expression of almt1a malate transporter protein that acts in al exclusion mechanism. in this study, cys2his2 (gma.4526, table 2) was upregulated 51-fold at 48 h post treatment suggesting that malate plays a major role in al tolerance mechanism of pi 416937 soybean. earlier physiological study by silva. showed that al stress increases exudation of both malate and citrate during the first 6 h of exposure to al in both tolerant and sensitive soybean types. but they concluded that the sustained accumulation and exudation of citrate is mainly responsible for the genotypic differences in al tolerance. in the present work, 48 h after al exposure the malate transporter regulator protein was highly expressed in contrast with the observation of silva.. we postulate that cys2his2 might regulate the expression of other al tolerance genes in addition to malate transporter. it is also possible that malate biosynthesis becomes a limiting step or malate might indeed play a major role in soybean al - tolerance contrary to earlier conclusions. adr6 transcription factors were previously reported as al tolerance genes [14, 18 ]. in the present study, the plant hormone auxin and adr6 exhibit opposite behavior in plant roots under al stress. al has been shown to inhibit auxin biosynthesis and transport genes as one possible mechanism of its toxicity. on the contrary, adr6an auxin downregulated transcription factor is induced under al stress perhaps mimicking auxin 's role of promoting root growth. these observations suggest that cys2his2 and adr6 transcription factors are important modulators of soybean molecular response to al stress. transporters, specifically malate (almts) and citrate (mate) transporters are the first al tolerance genes cloned in plants and represent the well - characterized al tolerance mechanism in a wide range of plant species [5, 8 ]. none of the family members of these two genes were detected in the present study which could be due to constitutive expression. in contrast, an abc transporter, a multidrug resistance glutathione - s - transferase - exporting atpase (gma.14080, table 2), was upregulated 27-fold at 48 h post treatment in the present study, which could detoxify xenobiotics by transporting glutathione - s - transferase conjugated toxin to the vacuole from sensitive sites in symplast. the involvement of abc transporters in al tolerance mechanism is widely documented [15, 18, 30, 31 ]. other al - induced transporters included heavy metal ion transport proteins (gma.17184 and gma.24625), lipid transport proteins (dq222982 and gma.17184), carbohydrate transport protein (gma.11888), and coatomer protein complex subunit 2-protien a polypeptide complex for membrane trafficking (gma.1654) (table 2). heavy metal transport proteins are either located in plasma membrane or subcellular membranes and detoxify heavy metals by exporting metal - ligand complexes out of the cell or by sequestration or compartmentalization of the complex in the vacuole. the internal detoxification mechanism of al involves formation of al - organic acid complexes and subsequent transport of the complex by transport proteins to leaf vacuoles in al hyperaccumulating plants that are adapted to acid soils [1, 9, 10, 32 ]. similar mechanism might operate in cultivated plants, and the heavy metal binding proteins upregulated here might function in such pathway. lipid transport proteins transport lipids to cell wall for biosynthesis of cutin layers and surface waxes as a defense mechanism against pathogen attack. lipid transport proteins loosen cell wall in a nonhydrolytic mode and enhance cell elongation, a role traditionally attributed to expansins. aluminum stress inhibits root growth by restricting cell wall extension ; hence, there should be a significance to the upregulation of lipid transport proteins under al stress. plant sugar transporters have been reported to be induced by pathogen attack and al stress [18, 35 ], as is the case in the present study (gma.11888, table 2). aluminum toxicity has been shown to elicit a wide range of stress - related proteins [19, 20, 36, 37 ]. in this study, genes known to be responsive to pathogens, oxidative stress, toxins, or al were classified under this category. several pathogenesis - related proteins including syringolide - induced protein, acidic endochitinase, pr-5, basic secretory protein, pathogenesis related protein sth-2, and proteinase inhibitors were upregulated at 48 h post al treatment (table 2). the confluence between plant molecular response to aluminum toxicity and pathogen infection likely arises from the fact that both cause oxidative stress. overexpression of peroxidase and proteinase inhibitor genes in arabidopsis did not improve al tolerance for the transformed plants relative to controls. on the other hand, overexpressing pepper basic pathogenesis - related protein 1 gene in tobacco resulted in enhanced tolerance to heavy metal cadmium and pathogen infection. other al - upregulated stress - related genes included carbohydrate oxidase, glutathione - s - transferase, and glutathione - based reductase (tables 1 and 2). carbohydrate oxidase and cell wall peroxidases have been reported to provide protection against pathogens by generating hydrogen peroxide from carbohydrate substrates in the apoplast. hydrogen peroxide has antimicrobial property and also acts as signal molecule for defense genes expression. in the case of aluminum, the activity of these enzymes is correlated with plant al sensitivity [40, 41 ]. glutathione - s - transferase and glutathione - based reductase are the key enzymes of cellular detoxification and antioxidation system. glutathione - s - transferase conjugates toxins and electrophilic compounds to reduced glutathione. the glutathione - conjugated toxin is then exported out of the cell or into the vacuole by the abc transporter proteins discussed above. the concurrent upregulation of glutathione - based reductase, glutathione - s - transferase, and abc transporter protein suggests that pi 416937 soybean may guard itself against al by extruding al out of the cell or by compartmentalization of al to the vacuole. yet there are conflicting evidences with respect to the role of the glutathione defense system in plant al tolerance. overexpression of glutathione - s - transferase in arabidopsis thaliana has been shown to enhance plant al tolerance. on the other hand, maron. found more oxidative stress genes upregulation in al sensitive cultivar of maize than in al tolerant cultivar and argue that oxidative stress genes upregulation is a symptom of al toxicity rather than a tolerance mechanism, an assertion that is supported by findings of. in addition, these genes are responsive to several biotic and abiotic stress factors and, therefore, should not be regarded as major al tolerance genes while partial role is certainly possible. the most interesting ones from al tolerance perspective are genes for biosynthesis of ascorbic acid and genes encoding cytochrome p450 and endo - xyloglucan transferases / hydrolases. all were upregulated in the present study, and the last two were previously reported to be upregulated in arabidopsis [19, 20 ] and wheat [15, 16 ] roots under al stress. oxidative stress is one aspect of al toxicity, and maintenance of cellular ascrobate homeostasis has been reported to be an essential component of plant al tolerance. cytochrome p450 may serve as monooxygenase in the biosynthetic pathways for lignin, defense compounds, hormones, pigments, fatty acids, and signaling molecules or in the detoxification pathway to catalyze the breakdown of numerous endogenous and exogenous toxic compounds. we detected two genes (gma.28852 upregulated 43-fold and gma.29655- upregulated 15-fold) which code for cytochrome p450 (table 2). gma.28852 encodes protein involved in pathways of ascorbate metabolism, coumarine and phenylpropanoid biosynthesis, and gamma hexachlorohexane degradation. members of this family of enzymes have been implicated in al tolerance [16, 1820 ]. there is a causal relationship among endoxyloglucan hydrolases, cell wall composition, and al tolerance. pectin and hemicellulose form complexes with al resulting in increased cell wall rigidity and reduced cell extension and growth [27, 43, 45 ]. endoxyloglucan hydrolases appear to relax the al - rigidified cell wall presumably by hydrolyzing the al - sugar complexes. perception of stress signal by the cell is the starting point for cascade of events leading to gene expression and change in cell metabolism in response to a stress factor. cell wall - associated receptor kinase (wak1) was the first al signaling gene discovered, but there is no evidence that demonstrate, wak1 's major role in al tolerance. microarray analyses have shown kinases, phosphates, and ef hand ca binding proteins as possible components of al signaling pathway [16, 18 ]. in the present work, a ca sensor protein (gma.35830), calcium - binding ef hand family protein (gma.7726), oxidative signal kinase (gma.8262), and a gene for growth factor phytosulfokines precursor (bk0001191) were upregulated 48 h post al treatment (table 2). the phytosulfokines growth factor is a novel al - induced gene, and it is involved in cell proliferation and growth, characteristics that confer al tolerance. we conducted a transcriptome analysis in al - tolerant soybean line pi 416937 to identify potential genetic factors underlying al tolerance trait. our results uncovered several genes which might potentially have influence on soybean al tolerance. among these, two transcription factors, cell wall metabolism enzymes and a cell proliferation gene are particularly interesting from perspective of the physiological and molecular mechanisms of plant al tolerance. the first transcription factor, cys2his2 zinc finger protein, coregulates molecular response to proton and aluminum toxicities, the major acid soil stress factors. the second transcription activator al suppresses auxin biosynthesis and transport in root system which might be one possible mechanism of al induced root growth inhibition. conversely, adr6 is triggered under al stress probably acting in a parallel pathway to auxin to restore root growth under al stress. cell wall metabolism enzymes and proteins are induced under al stress and may counteract al effects on root cell walls. it is increasingly evident that these proteins as well as cell wall pectin and hemicellulose content are important determinants of al tolerance in cereals [3, 4, 43 ]. evidence from this study also implies that cell wall remodeling enzymes and proteins may play role in soybean al tolerance. we identified a novel cell proliferation stimulating gene phytosulfokines growth factor which might reverse this effect of al. taken together ; our findings provide important insights into the molecular mechanisms of aluminum tolerance in soybean. | soybean is one of the most aluminum (al) sensitive plants. the complex inheritance of al tolerance trait has so far undermined breeding efforts to develop al - tolerant soybeans. discovering the genetic factors underlying the al tolerance mechanisms would undoubtedly accelerate the pace of such endeavor. as a first step toward this goal, we analyzed the transcriptome profile in roots of al - tolerant soybean line pi 416937 comparing al - treated and untreated control plants using dna microarrays. many genes involved in transcription activation, stress response, cell metabolism and signaling were differentially expressed. patterns of gene expression and mechanisms of al toxicity and tolerance suggest that cys2his2 and adr6 transcription activators, cell wall modifying enzymes, and phytosulfokines growth factor play role in soybean al tolerance. our data provide insights into the molecular mechanisms of soybean al tolerance and will have practical value in genetic improvement of al tolerance trait. |
data for this study were obtained from participants in the portland progression project, a prospective longitudinal study of the course and risk factors for glaucomatous progression. individuals with non - end - stage glaucoma or with ocular hypertension plus risk factors for glaucoma undergo testing with a variety of methods, including automated perimetry and sd - oct. two separate cohorts were used : a repeatability cohort, consisting of participants tested five times within a few months, and a longitudinal cohort, consisting of participants tested approximately every 6 months over several years. longitudinal data were used from the most recent eight visits at which reliable measurements (as outlined below) were acquired. inclusion criteria for both cohorts were a diagnosis of primary open - angle glaucoma and/or likelihood of developing glaucomatous damage (e.g., high - risk ocular hypertension), as determined at the discretion of each participant 's physician. exclusion criteria at entry included an inability to perform reliable visual field testing, best - corrected visual acuity worse than 20/40, or other conditions or medications that may affect the visual field. all protocols were approved and monitored by the legacy health institutional review board, and adhered to the health insurance portability and accountability act of 1996 and the tenets of the declaration of helsinki. all participants provided written informed consent once all of the risks and benefits of participation were explained to them. functional testing was performed using automated white - on - white perimetry with a humphrey field analyzer (hfa ii ; carl - zeiss meditec, dublin, ca, usa), with a size iii stimulus, sita standard algorithm, and 24 - 2 test pattern. visual field tests were excluded from analysis if they had greater than 33% false - negatives, 20% false - positives, or 33% fixation losses. for the primary analysis, mean deviation (md) was used, representing the global status of the visual field relative to age - appropriate normals, summarized across the 52 locations (excluding the blind spot) in the field. since it has been reported that the structure function relation may be nonlinear, and that functional progression also may be nonlinear when expressed in decibels, a second summary functional measure was calculated. the linear mean sensitivity (lms) was defined as the arithmetic mean of pointwise sensitivities expressed on a linear scale of 10, so that 0 db 0.001, 10 db 0.01, 20 db 0.1, and 30 db 1.0. sensitivities on this scale then may be more linearly related to axon counts and, hence, to rnflt, and also may progress more linearly over time. spectral - domain oct was performed with a spectralis oct (heidelberg engineering, heidelberg, germany). on each test date, a circle scan was performed at a radius of 6 from the center of the optic disc as placed by the operator. images were focused by the technician aiming to optimize the clarity of blood vessels within the rnfl. follow - up scans were registered in real - time to the location of the baseline reference scan for each eye. three segmentations were delineated on each circular b - scan, as shown in the example in figure 1 ; the inner limiting membrane (ilm, in green), the posterior border of the rnfl (in red), and the posterior border of the rpe / bruch 's membrane (in yellow). the instrument 's automated delineations were adjusted manually by experienced technicians when necessary to address obvious delineation errors (without reference to the functional results). the pixel corresponding to each of these three boundaries was recorded for each a - scan. in each case, this boundary pixel was assigned as belonging to the layer below the boundary ; hence pixels along the red line in figure 1 are counted not as part of the rnfl, but as part of the retinal ganglion cell layer. three structures are delineated : the ilm (green), posterior border of the rnfl (red), and the posterior border of the rpe (yellow). since overall tissue reflectance varies between scans and between individuals, due to factors including differences in the exact focal plane and the quality of preretinal optics, normalization is required to better represent alterations of the tissues ' inherent optical properties rather than the influence of these imaging artefacts. five methods for normalizing the reflectance intensity values were considered : (1) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels within the posterior vitreous (i.e., above the ilm), (2) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels coinciding with the delineated posterior border of the rpe (the yellow line on fig. 1), (3) the mean intensity of pixels within the rnfl divided by the mean intensity of a 5-pixel wide band immediately above the delineated posterior border of the rpe (designed to be entirely within the high reflectance region of rpe visible on fig. 1), (4) the mean intensity of pixels within the rnfl divided by the mean intensity of an 11-pixel wide band centered on the delineated posterior border of the rpe (designed to be similar to the method used by dwelle.), and (5) the mean intensity of pixels within the rnfl divided by the mean intensity of sub - rnfl tissue, extending from one pixel below the posterior border of the rnfl to the posterior border of the rpe (i.e., between the red and yellow lines in fig. 1 ; chosen under the assumption that using a larger portion of the cross - sectional b - scan image should provide increased stability and, hence, lower variability, especially in the face of variability caused by the precise delineation of the retinal layers). using each of these normalizations in turn, an intensity ratio was calculated for each scan in the repeatability cohort. the standard deviation of the five values for the scans obtained from each eye was calculated, and expressed as a percentage of the mean value. this coefficient of variation can be considered equivalent to the reciprocal of the signal - to - noise ratio, under the assumption that the mean value is representative of the magnitude of the signal. to determine the most repeatable measure of reflectance intensity, these were compared between normalization techniques using the signed rank test for clustered data developed by datta and satten, since data from both eyes were used. the per - eye rate of change over the most recent 8 visits in the longitudinal cohort was calculated by least - squares linear regression, for the two structural measures rnflt and reflectance intensity ratio, and for the two functional measures md and lms. models then were formed to predict the rate of functional change (by md or lms), using the concurrent rate of change of rnflt, the rate of change of the reflectance intensity ratio, and their interaction. since rates of change from both eyes were calculated, a generalized estimating equation (gee) model was used to account for correlation between the two eyes of an individual. to assess whether disease severity affected the results, secondary analyses were performed after splitting the data into two halves, according to whether the final md in the visual field series for an eye was greater or less than the median value. analyses were performed using the r language and environment for statistical computing (version 2.15.3 ; r core team, vienna, 2013, available in the public domain at http://www.r-project.org/). data for this study were obtained from participants in the portland progression project, a prospective longitudinal study of the course and risk factors for glaucomatous progression. individuals with non - end - stage glaucoma or with ocular hypertension plus risk factors for glaucoma undergo testing with a variety of methods, including automated perimetry and sd - oct. two separate cohorts were used : a repeatability cohort, consisting of participants tested five times within a few months, and a longitudinal cohort, consisting of participants tested approximately every 6 months over several years. longitudinal data were used from the most recent eight visits at which reliable measurements (as outlined below) were acquired. inclusion criteria for both cohorts were a diagnosis of primary open - angle glaucoma and/or likelihood of developing glaucomatous damage (e.g., high - risk ocular hypertension), as determined at the discretion of each participant 's physician. exclusion criteria at entry included an inability to perform reliable visual field testing, best - corrected visual acuity worse than 20/40, or other conditions or medications that may affect the visual field. all protocols were approved and monitored by the legacy health institutional review board, and adhered to the health insurance portability and accountability act of 1996 and the tenets of the declaration of helsinki. all participants provided written informed consent once all of the risks and benefits of participation were explained to them. functional testing was performed using automated white - on - white perimetry with a humphrey field analyzer (hfa ii ; carl - zeiss meditec, dublin, ca, usa), with a size iii stimulus, sita standard algorithm, and 24 - 2 test pattern. visual field tests were excluded from analysis if they had greater than 33% false - negatives, 20% false - positives, or 33% fixation losses. for the primary analysis, mean deviation (md) was used, representing the global status of the visual field relative to age - appropriate normals, summarized across the 52 locations (excluding the blind spot) in the field. since it has been reported that the structure function relation may be nonlinear, and that functional progression also may be nonlinear when expressed in decibels, a second summary functional measure was calculated. the linear mean sensitivity (lms) was defined as the arithmetic mean of pointwise sensitivities expressed on a linear scale of 10, so that 0 db 0.001, 10 db 0.01, 20 db 0.1, and 30 db 1.0. sensitivities on this scale then may be more linearly related to axon counts and, hence, to rnflt, and also may progress more linearly over time. spectral - domain oct was performed with a spectralis oct (heidelberg engineering, heidelberg, germany). on each test date, a circle scan was performed at a radius of 6 from the center of the optic disc as placed by the operator. images were focused by the technician aiming to optimize the clarity of blood vessels within the rnfl. follow - up scans were registered in real - time to the location of the baseline reference scan for each eye. three segmentations were delineated on each circular b - scan, as shown in the example in figure 1 ; the inner limiting membrane (ilm, in green), the posterior border of the rnfl (in red), and the posterior border of the rpe / bruch 's membrane (in yellow). the instrument 's automated delineations were adjusted manually by experienced technicians when necessary to address obvious delineation errors (without reference to the functional results). the pixel corresponding to each of these three boundaries was recorded for each a - scan. in each case, this boundary pixel was assigned as belonging to the layer below the boundary ; hence pixels along the red line in figure 1 are counted not as part of the rnfl, but as part of the retinal ganglion cell layer. three structures are delineated : the ilm (green), posterior border of the rnfl (red), and the posterior border of the rpe (yellow). for each pixel within the circle scan, the intensity of the raw reflectance image was extracted using custom software. since overall tissue reflectance varies between scans and between individuals, due to factors including differences in the exact focal plane and the quality of preretinal optics, normalization is required to better represent alterations of the tissues ' inherent optical properties rather than the influence of these imaging artefacts. five methods for normalizing the reflectance intensity values were considered : (1) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels within the posterior vitreous (i.e., above the ilm), (2) the mean intensity of pixels within the rnfl divided by the mean intensity of pixels coinciding with the delineated posterior border of the rpe (the yellow line on fig. 1), (3) the mean intensity of pixels within the rnfl divided by the mean intensity of a 5-pixel wide band immediately above the delineated posterior border of the rpe (designed to be entirely within the high reflectance region of rpe visible on fig. 1), (4) the mean intensity of pixels within the rnfl divided by the mean intensity of an 11-pixel wide band centered on the delineated posterior border of the rpe (designed to be similar to the method used by dwelle.), and (5) the mean intensity of pixels within the rnfl divided by the mean intensity of sub - rnfl tissue, extending from one pixel below the posterior border of the rnfl to the posterior border of the rpe (i.e., between the red and yellow lines in fig. 1 ; chosen under the assumption that using a larger portion of the cross - sectional b - scan image should provide increased stability and, hence, lower variability, especially in the face of variability caused by the precise delineation of the retinal layers). using each of these normalizations in turn, an intensity ratio was calculated for each scan in the repeatability cohort. the standard deviation of the five values for the scans obtained from each eye was calculated, and expressed as a percentage of the mean value. this coefficient of variation can be considered equivalent to the reciprocal of the signal - to - noise ratio, under the assumption that the mean value is representative of the magnitude of the signal. to determine the most repeatable measure of reflectance intensity, these were compared between normalization techniques using the signed rank test for clustered data developed by datta and satten, since data from both eyes were used. the per - eye rate of change over the most recent 8 visits in the longitudinal cohort was calculated by least - squares linear regression, for the two structural measures rnflt and reflectance intensity ratio, and for the two functional measures md and lms. models then were formed to predict the rate of functional change (by md or lms), using the concurrent rate of change of rnflt, the rate of change of the reflectance intensity ratio, and their interaction. since rates of change from both eyes were calculated, a generalized estimating equation (gee) model was used to account for correlation between the two eyes of an individual. to assess whether disease severity affected the results, secondary analyses were performed after splitting the data into two halves, according to whether the final md in the visual field series for an eye was greater or less than the median value. analyses were performed using the r language and environment for statistical computing (version 2.15.3 ; r core team, vienna, 2013, available in the public domain at http://www.r-project.org/). the repeatability cohort consisted of 53 eyes of 27 participants (one eye was excluded due to an ocular pathology unrelated to glaucoma), tested 5 times per eye within a relatively short period of time. the longitudinal cohort (restricted to eyes with at least 8 visits at which data of sufficient quality were obtained for sd - oct and perimetry) consisted of 310 eyes of 205 participants. the median rate of change of md (from linear regression) in the longitudinal cohort was 0.09 db / y, similar to a report from a clinical population of patients with glaucoma. summary of the characteristics of the two cohorts table 2 summarizes the per - eye coefficients of variation within the repeatability cohort for each of the five normalization techniques considered. using the entire region from the posterior border of the rnfl down to the posterior border of the rpe (i.e., between the red and yellow lines on fig. 1, method 5 in the list above) had significantly lower coefficients of variation (using the signed - rank test for clustered data) than normalizations based on the posterior vitreous (p = 0.011) or any of the three methods that relied upon rpe - based normalization (all p < 0.001). therefore, method 5 was used to define the reflectance intensity ratio when analyzing data from the longitudinal cohort. the 95% confidence interval for test the effect of the chosen normalization on intertest variability in the repeatability cohort is illustrated in figure 2. note that for comparison, rnflt had an average coefficient of variation in this cohort of just 1.3% (range, 0.3%4.5%). summary of the per - eye coefficients of variation (i.e., sd divided by mean) of reflectance intensity ratio, using five different normalization methods the effect of normalization on reflectance intensity measures in the repeatability cohort. for each eye, the box extends from the second lowest to the second highest measurements ; the whiskers extend to the minimum and maximum measurements for that eye. after normalization, the within - eye variability (shown by width of each box) is reduced as a proportion of the between - eye variability. therefore, it can be seen that normalization based on the average reflectance of pixels between the nerve fiber layer and rpe greatly improves test retest variability in this cohort, for which no true change should have occurred. the mean of the chosen reflectance intensity ratio across all eyes and all visits in the longitudinal cohort was 2.69 (sd, 0.99 ; range, 1.017.28). the mean per - eye rate of change of the reflectance intensity ratio in this cohort was 0.06 y (sd, 0.22 y ; range, 0.90 to + 1.13 y). the rate for a given eye was used together with the rate of change of rnflt to predict the rate of functional change. in univariate analyses, the rate of rnfl thinning was predictive of the rate of md change, with p < 0.0001 ; d / dt md = 0.062 + 0.096 d / dt rnflt (where d / dt x represents the rate of change of measure x over time). the value of d / dt md is measured in db / y, and d / dt rnflt is measured in m / y. the rate of change of the reflectance intensity ratio was not by itself significantly predictive of the rate of md change (p = 0.116). however, in a multivariable model, the interaction between the two structural rates of change improved upon predictions of the rate of functional change made using the rate of rnflt change alone : in this model, d / dt rnflt had p = 0.0005 and the interaction term had p = 0.038. as an example of the effect size : the average value of d / dt rnflt from the longitudinal cohort was 0.75 m / y. for this rate of rnfl thinning, if the reflectance intensity ratio changed at + 1.13 y (the highest value observed), then the predicted rate of functional change would be d / dt md = 0.04 db / y. by contrast, if the reflectance intensity ratio changed at 0.90 y (the lowest value observed) then the predicted rate would be d / dt md = 0.19 db / y, representing nearly a five times more rapid rate of functional deterioration. when sensitivities were transformed from db to a linear scale, results generally were similar. d / dt rnflt was predictive of d / dt lms, with p = 0.0001, but the rate of change of reflectance intensity ratio was not significantly predictive, with p = 0.256. however, in the multivariable model, where d / dt rnflt had p = 0.002 and the interaction term had p = 0.009. secondary analyses were performed after splitting the cohort into two equal parts based on the final md in each eye 's series. among those eyes with final md worse than 0.15 db (the median value in the cohort), in the same multivariable model as before, d / dt rnflt still was a significant predictor of d / dt md with p = 0.0024, but the interaction term had p = 0.650. by contrast, among eyes with final md greater than 0.15 db, d / dt rnflt had p = 0.1092, while the interaction term had p = 0.0653. clearly these results are not definitive, since they did not reach statistical significance (possibly due to the reduced sample size), but they hint that changes in reflectance intensity may be most useful at the earliest stages of the disease process. the repeatability cohort consisted of 53 eyes of 27 participants (one eye was excluded due to an ocular pathology unrelated to glaucoma), tested 5 times per eye within a relatively short period of time. the longitudinal cohort (restricted to eyes with at least 8 visits at which data of sufficient quality were obtained for sd - oct and perimetry) consisted of 310 eyes of 205 participants. the median rate of change of md (from linear regression) in the longitudinal cohort was 0.09 db / y, similar to a report from a clinical population of patients with glaucoma. table 2 summarizes the per - eye coefficients of variation within the repeatability cohort for each of the five normalization techniques considered. using the entire region from the posterior border of the rnfl down to the posterior border of the rpe (i.e., between the red and yellow lines on fig. 1, method 5 in the list above) had significantly lower coefficients of variation (using the signed - rank test for clustered data) than normalizations based on the posterior vitreous (p = 0.011) or any of the three methods that relied upon rpe - based normalization (all p < 0.001). therefore, method 5 was used to define the reflectance intensity ratio when analyzing data from the longitudinal cohort. the 95% confidence interval for test the effect of the chosen normalization on intertest variability in the repeatability cohort is illustrated in figure 2. note that for comparison, rnflt had an average coefficient of variation in this cohort of just 1.3% (range, 0.3%4.5%). summary of the per - eye coefficients of variation (i.e., sd divided by mean) of reflectance intensity ratio, using five different normalization methods the effect of normalization on reflectance intensity measures in the repeatability cohort. for each eye, the box extends from the second lowest to the second highest measurements ; the whiskers extend to the minimum and maximum measurements for that eye. after normalization, the within - eye variability (shown by width of each box) is reduced as a proportion of the between - eye variability. therefore, it can be seen that normalization based on the average reflectance of pixels between the nerve fiber layer and rpe greatly improves test retest variability in this cohort, for which no true change should have occurred. the mean of the chosen reflectance intensity ratio across all eyes and all visits in the longitudinal cohort was 2.69 (sd, 0.99 ; range, 1.017.28). the mean per - eye rate of change of the reflectance intensity ratio in this cohort was 0.06 y (sd, 0.22 y ; range, 0.90 to + 1.13 y). the rate for a given eye was used together with the rate of change of rnflt to predict the rate of functional change. in univariate analyses, the rate of rnfl thinning was predictive of the rate of md change, with p < 0.0001 ; d / dt md = 0.062 + 0.096 d / dt rnflt (where d / dt x represents the rate of change of measure x over time). the value of d / dt md is measured in db / y, and d / dt rnflt is measured in m / y. the rate of change of the reflectance intensity ratio was not by itself significantly predictive of the rate of md change (p = 0.116). however, in a multivariable model, the interaction between the two structural rates of change improved upon predictions of the rate of functional change made using the rate of rnflt change alone : in this model, d / dt rnflt had p = 0.0005 and the interaction term had p = 0.038. as an example of the effect size : the average value of d / dt rnflt from the longitudinal cohort was 0.75 m / y. for this rate of rnfl thinning, if the reflectance intensity ratio changed at + 1.13 y (the highest value observed), then the predicted rate of functional change would be d / dt md = 0.04 db / y. by contrast, if the reflectance intensity ratio changed at 0.90 y (the lowest value observed) then the predicted rate would be d / dt md = 0.19 db / y, representing nearly a five times more rapid rate of functional deterioration. when sensitivities were transformed from db to a linear scale, results generally were similar. d / dt rnflt was predictive of d / dt lms, with p = 0.0001, but the rate of change of reflectance intensity ratio was not significantly predictive, with p = 0.256. however, in the multivariable model, where d / dt rnflt had p = 0.002 and the interaction term had p = 0.009. secondary analyses were performed after splitting the cohort into two equal parts based on the final md in each eye 's series. among those eyes with final md worse than 0.15 db (the median value in the cohort), in the same multivariable model as before, d / dt rnflt still was a significant predictor of d / dt md with p = 0.0024, but the interaction term had p = 0.650. by contrast, among eyes with final md greater than 0.15 db, d / dt rnflt had p = 0.1092, while the interaction term had p = 0.0653. clearly these results are not definitive, since they did not reach statistical significance (possibly due to the reduced sample size), but they hint that changes in reflectance intensity may be most useful at the earliest stages of the disease process. retinal nerve fiber layer thickness remains an excellent structural measure for use in the diagnosis and monitoring of glaucoma. it is highly repeatable ; the average per - eye coefficient of variation in our repeatability cohort was 1.3%, which is similar to the 1.9% intravisit coefficient of variation reported previously for the cirrus oct (carl - zeiss meditec, inc.). in our longitudinal cohort, longitudinally, by contrast, the correlation between the rates of change of rnflt and md was only 0.361. in part, this is because the range of values is narrower, and so the value of the correlation coefficient is reduced. however, it also suggests that other sources of information are needed to refine predictions of longitudinal functional change. as shown here, the reflectance of the rnfl appears to provide one such source. it is possible that reflectance will prove to be even more useful in eyes with very early damage and/or ocular hypertension. importantly, reflectance information can be extracted from existing sd - oct scans without hardware modifications. it is important to note that the reflectance intensity ratio by itself was not predictive of functional change. instead, it provides a method that may be able to refine predictions made using rnflt. longitudinal changes observed in the reflectance must be considered in the context of changes observed in the rnflt, and not interpreted in isolation. it certainly is possible that the measure of reflectance intensity used here might be improved upon. most commercial oct instruments are optimized to visualize cross - sectional (b - scan) images and to measure the thickness of structures in the retina, not their reflectance. for example, improvements could involve removing the effects of vessel shadows, and/or taking into account the directionality of retinal reflectance. directionality is particularly important for cylindrical structures, such as axons and their aligned cytoskeletal components, which our normalization procedures, therefore, do not address. indeed, the test retest variability of our measure shown in figure 2 still is more than 10 times higher than the variability of rnflt measurement. improvements in the repeatability of the reflectance intensity ratio would likely improve its use for prediction of functional change. in this study, the interaction between reflectance and thickness improved prediction of the rate of md change, but this was only just significant with p = 0.038. accordingly, the standard deviation of residuals (i.e., predicted rate minus observed rate) was reduced by less than 5% by the inclusion of reflectance intensity ratio. if the test retest variability of reflectance intensity could be improved further, then we would anticipate this p value being reduced. moreover, commercial instruments may apply automatic gain controls to maintain signal strength in a more ideal range, providing benefits to imaging a broader range of eyes, but potentially confounding reflectance intensity measurements (especially those without some internal normalization). we also would note that it is possible that reflectance intensity may change before or after rnfl thinning. this study did not incorporate any potential time lag between the observed changes in the retina, since each measure was assumed to change linearly over time. furthermore, we looked solely at concurrent rates of functional change ; the aim was to predict the rate of functional loss during that same time period, rather than to predict subsequent functional loss. previous studies have used a narrow band around the rpe to normalize rnfl reflectance, but used multiple b - scans at different radial distances from the center of the optic nerve head. by contrast, in this study a single circle scan at 6 from the center of the optic nerve head was used. this is consistent with the most common current clinical approach, but means fewer pixels of information are available (1536 for this analysis) and a more limited area of rnfl is assayed by the single circular scan. using a thicker band of axial information extending all the way from the posterior border of the rnfl down to the rpe increased the number of available pixels, and so appears to give a more reliable measure of sub - rnfl reflectance when a single circle scan is used. correspondingly, the test retest variability of the reflectance intensity ratio was lower when using this thicker band. it should be noted, though, that while we tested several alternative methods for interscan normalization, these do not represent an exhaustive examination of all possibilities, and further improvement may be possible. the data used in this study consist primarily of cases of early, well - managed glaucoma. only 4 of the 53 eyes in the repeatability cohort, and 20 of 310 eyes in the longitudinal cohort, had md worse than 6 db, a value often taken to indicate moderate functional damage. approximately half of the eyes did not yet have significant functional damage when assessed by md alone. therefore, this represents an extremely important clinical scenario, where early signs of functional progression are sought. indeed, the secondary analyses hint that reflectance intensity may be more useful in preperimetric glaucoma than in eyes with established functional defects, although we can not conclude this definitively at the present time. however, our conclusions have not been tested in cases of more severe glaucoma, and so it is not yet known whether reflectance intensity remains a useful prognostic measure later in the disease process or whether it represents a very early stage of structural damage. we also would note that this study aims to predict the rate of change of md, because we sought a single continuous variable as the outcome measure to increase the statistical power of the analysis ; yet this is known not to be a particularly sensitive measure of visual field change, especially early in the disease process. future studies are needed that examine pointwise progression, and/or with much larger cohorts that would enable assessment using binary definitions of progression and stability. this study should be considered a proof of principle, rather than providing a measurement that is ready to be implemented into commercially - available instruments, given the high test however, the principle behind the measurement is something that can be qualitatively assessed already, even if quantitative assessment is not yet clinically available. figure 3 shows a magnified portion (for easier visualization) of the peripapillary rnfl b - scan shown in figure 1 ; together with the corresponding portion of a scan from the same eye 18 months later. on the date of the first (upper) scan, this participant had an average rnflt of 91.2 m and a reflectance intensity ratio averaged around the entire circle scan of 4.73. using the variability estimate from table 2, this means that the 95% confidence interval for test retest is (3.17, 6.29). by the date of the second (lower) scan, the nerve fiber layer is visibly less reflective in the lower panel. within the same time period, the md decreased from 5.0 db on the date of the first scan to 7.6 db on the date of the second scan. the results of this study suggested that clinicians should be on the lookout for similar decreases in rnfl reflectance, as they may correspond (as in this example) to worsening function. example of a section of an oct scan (top), and the same section 18 months later (bottom). in that time period, this corresponded to worsening function during that same period. in practice, light intensity and focus may vary between scans, with the result that changes in reflectance will not always be easily visible to the clinician. furthermore, to map the raw intensity information to a range compatible with most common monitors and/or clinical printouts, raw intensity values are compressed to 256 grayscale levels (8-bit range), reducing the precision available. our normalization process (which is applied to raw intensity data) may not be easy to implement qualitatively while reviewing a series of scans in a clinical setting. therefore, it is hoped that instrument manufacturers could eventually add such measures to their output, enabling more sensitive detection of changes in reflectance. in summary, we described a method to quantify the reflectance intensity of the rnfl from sd - oct scans. for a given rate of rnfl thinning, a reduction in rnfl reflectance is associated with more rapidly deteriorating function. further work will examine whether localized changes in rnfl reflectance correspond to localized functional deterioration. the causes of these reductions in reflectance intensity are not entirely clear, but they raise the intriguing possibility of dysfunctional yet surviving ganglion cell axons, which may be candidates for neuroprotection or rescue. clinically, we would recommend examination of the images produced by oct instruments, as they may reveal important changes that are not apparent from looking at rnfl thickness or other structural measures in isolation. | purposewe determined whether longitudinal changes in retinal nerve fiber layer (rnfl) reflectance provide useful prognostic information about longitudinal changes in function in glaucoma.methodsthe reflectance intensity of each pixel within spectral - domain optical coherence tomography (sd - oct) circle scans was extracted by custom software. a repeatability cohort comprising 53 eyes of 27 participants (average visual field mean deviation [md ] 1.65 db) was tested five times within a few weeks. to minimize test retest variability in their data, a reflectance intensity ratio was defined as the mean reflectance intensity of pixels within the rnfl divided by the mean between the rnfl and rpe. this was measured in a separate longitudinal cohort comprising 310 eyes of 205 participants tested eight times at 6-month intervals (average md, 0.99 db ; median rate of change, 0.09 db / y). the rate of change of this ratio, together with the rate of rnfl thinning, and their interaction, were used to predict the rate of change of md.resultsin univariate analyses, the rate of rnfl thinning was predictive of the rate of md change (p < 0.0001), but the rate of change of reflectance intensity ratio was not (p = 0.116). however, in a multivariable model, the interaction between these two rates significantly improved upon predictions of the rate of functional change made using rnfl thickness alone (p = 0.038).conclusionsfor a given rate of rnfl thinning, a reduction in the rnfl reflectance intensity ratio is associated with more rapid functional deterioration. incorporating sd - oct reflectance information may improve the structure function relation in glaucoma. |
it is characterized by unilateral or bilateral paralysis of the sixth and seventh cranial nerves, manifested clinically as external ophthalmoplegia and weakness of facial muscles. even though von graaefe described a case of congenital facial diplegia in 1880, it was paul julius mbius, a german neurologist, who drew attention to the association of congenital facial diplegia with other malformations in 1888 and 1892. only about 300 cases have been reported in the literature, and very few cases of this syndrome have been reported in the radiology literature. a 2-year - old girl was referred to our department for plain magnetic resonance imaging (mri) scan of the brain. the child 's parents noticed facial asymmetry in the child with deviation of the angle of mouth to the right side on smiling, watering and incomplete closure of the left eye since birth. the child 's birth and developmental history were normal. on physical examination, facial asymmetry, bilateral convergent squint, prominent everted upper and lower lips were noted. left facial nerve paralysis and bilateral abducent nerve paralysis mild ptosis on left side is also seen (b) picture demonstrating left - sided facial paralysis causing deviation of angle of mouth to right side on smiling mr examination was performed using a 1.5 tesla mri scanner (avanto ; siemens medical solutions, erlangen, germany). axial and sagittal t1-weighted, axial t2-weighted, fluid - attenuated inversion recovery (flair), and three - dimensional (3d) constructive interference in steady state (ciss) sequences (0.7-mm slice thickness) were evaluated. mri brain showed the following findings : absent cisternal and canalicular segments of left facial nerve [figures 2 and 3]nonvisualization of cisternal segments of bilateral abducens nerves [figures 2a and c]flattened floor of fourth ventricle with absence of bilateral facial colliculi [figure 4 ]. absent cisternal and canalicular segments of left facial nerve [figures 2 and 3 ] nonvisualization of cisternal segments of bilateral abducens nerves [figures 2a and c ] flattened floor of fourth ventricle with absence of bilateral facial colliculi [figure 4 ]. four (a d) contiguous 0.7-mm thick axial 3d ciss mr images of brain showing the absence of left facial nerve in cerebellopontine angle cistern (a and c, bent arrows). right facial nerve (a, black arrow head) and bilateral vestibulocochlear nerves (a and c, black arrows) are visualized. in addition, there is absence of bilateral abducens nerves (a, b, and d, white arrows) in prepontine cisterns, the expected position of bilateral abducens nerves (a d images are cranial to caudal sections) parasagittal ciss mr image of left internal auditory canal (iac) showing absent facial nerve in anterosuperior part of left iac (black arrow). vestibular (thick white arrow) and cochlear nerves (thin white arrow) are visualized in posterior and anteroinferior parts of left iac respectively. (anterior is to the left of image and posterior is to the right) axial t1w mr image at the level of middle cerebellar peduncles showing flattened floor of fourth (black arrow) ventricle secondary to absence of bilateral facial colliculi the left anterior inferior cerebellar artery (aica) flow void was absent, and mild prominence of the right aica was noted [figure 5 ]. axial ciss mr image showing right aica (black arrow) and absent left aica (white arrow) mbius syndrome is rare disorder, which is characterized by congenital complete or partial facial nerve paralysis with or without paralysis of other cranial nerves. the definition and criteria for diagnosis of mbius syndrome vary among authors. based on previously published reviews, kumar used the following criteria for diagnosis of this syndrome : (a) complete or partial facial nerve paralysis is an essential criterion for the diagnosis of mbius syndrome, (b) limb malformations (syndactyly, brachydactyly, absent digits, talipes) are often present, (c) bilateral or unilateral cranial nerve palsies (commonly vi, xii, also ix, x) may be seen, (d) orofacial malformations, ear deformities, and musculoskeletal deformities may also be seen. recently, verzijl., suggested facial palsy with impairment of ocular abduction as the primary criterion for mbius syndrome, with or without association of other cranial nerve paralysis, musculoskeletal defects, or orofacial malformations. mbius syndrome manifests soon after birth with inability to close the eyelids during sleep, drooling of saliva, difficulty in sucking, and mask - like facies. the hypoglossal nerve is the third most common nerve to be involved, causing paralysis and hypoplasia of the tongue. although neither the etiology nor the pathogenesis of mbius syndrome has yet been elucidated, verzijl. proposed two theories based on previous published reviews : (a) a developmental rhombomeric defect including facial cranial nerve nuclei due to a genetic cause, or (b) an interruption in the vascular supply of the brainstem secondary to an environmental, mechanical, or genetic cause resulting in ischemia in the region of facial cranial nerve nuclei. an association of absent aica with mbius syndrome is not described in the literature ; however, an association of the ischemic process resulting from an interruption of vascular supply to the brainstem in early fetal development was proposed. absent left aica with mildly prominent contralateral aica, observed in our case, may be an incidental finding and an anatomic variant. at times, either the posterior inferior cerebellar artery or the aica is absent and one artery supplies the usual territories of both arteries. however, mri is useful in demonstrating the abnormalities of the cranial nerves and orbital structures. other computed tomography (ct) and mr imaging findings include brainstem hypoplasia with straightening of floor of the fourth ventricle, indicating absence of the facial colliculus ; calcification in pons in the region of abducens nuclei ; absence of hypoglossal eminence at the medulla ; and cerebellar hypoplasia. based on history and clinicoradiological examinations (3d ciss), our case was diagnosed as mbius syndrome. craniofacial syndrome with asymmetric crying facies, congenital heart disease, and other factors may be confused with mbius syndrome. other neuromuscular disorders such as melkersson rosenthal syndrome, muscular dystrophy, congenital facial muscular atrophy, cerebral palsy, and congenital myopathies may overlap with mbius syndrome. the seventh nerve palsy in mbius syndrome primarily affects the upper face, which is differentiated from other lower motor neuron palsies affecting both upper and lower portions of the face. additionally, this can also be differentiated from supranuclear lesions, which only affect the lower half of the face. since the disease is congenital and nonprogressive, no definitive and established treatment has been described. ocular care and careful monitoring of complications that might set in, patient education, and a multidisciplinary approach by health professionals help to reduce the morbidity. we herein reported a case of mbius syndrome with absent left facial and bilateral abducens nerves. it is an irreversible condition that causes not only physical abnormalities but also social and psychological disturbances. ct and mr imaging may depict abnormalities in the brainstem, extraocular muscle hypoplasia, and other associated abnormalities. mr imaging, particularly 3d ciss sequence, is very useful in evaluating cisternal and cavernous segments of the cranial nerves. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | mbius syndrome is an extremely rare congenital disorder. we report a case of mbius syndrome in a 2-year - old girl with bilateral convergent squint and left - sided facial weakness. the characteristic magnetic resonance imaging (mri) findings of mbius syndrome, which include absent bilateral abducens nerves and absent left facial nerve, were noted. in addition, there was absence of left anterior inferior cerebellar artery (aica) and absence of bilateral facial colliculi. clinical features, etiology, and imaging findings are discussed. |
many people throughout the world live with a variety of clinical conditions, including stroke, spinal trauma, cerebral palsy, and multiple sclerosis. unfortunately, these conditions frequently present with motor deficits, which greatly reduce the quality of life for those affected. mental practice with motor imagery (mi) is currently considered a promising additional treatment to improve motor functions repetitive cognitive training exercise, during which the patient imagines performing a task or body movement without actual physical activity, has been shown to modulate the cerebral perfusion and neural activity in specific brain regions. interestingly, it has been suggested that the combination of robot - assisted training devices and brain - controlled limb assistive technology may help to induce neural plasticity, resulting in motor function improvement. despite recording noninvasively and on the same time scale as the sensorimotor control of the brain, more specifically, these signals are usually collected from multiple electrodes (or channels), which are inevitably contaminated by the noise from biological, environmental, and instrumental origins. dimensionality reduction plays a key role in many fields of data analysis. using this method, data from a high - dimensional space can be represented by vectors in a reduced, low - dimensional space in order to simplify problems without degrading performance. one of the most popular dimensionality reduction methods is principle component analysis (pca), which is theoretically guaranteed to discover the dimensionality of the subspace and produce a compact representation if the data is embedded in a linear subspace. in many real world problems, however, there is no evidence that the data is actually sampled from a linear subspace [7, 8 ]. various manifold learning techniques, including isomap, locally linear embedding (lle), and laplacian eigenmaps, have been proposed to reduce the dimensionality of fixed training sets in ways that maximally preserve certain interpoint relationships [911 ]. unfortunately, these methods do not generally provide a functional mapping between the high- and low - dimensional spaces that is valid both on and off the training data. spectral regression (sr), based on regression and spectral graph analysis, can make efficient use of both labeled and unlabeled points to discover the intrinsic discriminant structure in the data [7, 8 ]. as a result, sr has been applied to supervised, semisupervised, and unsupervised situations across different pattern recognition tasks [12, 13 ] and has shown its superiority over traditional dimensional reduction methods. empirical mode decomposition (emd) is a fully data - driven and adaptive analysis method that is widely applied within the field of biomedical signal processing [1416 ]. it decomposes a raw signal into a set of intrinsic mode functions (imfs) which represent the natural oscillatory modes contained within the original data. emd does have some limitations in processing multichannel data, since the imfs decomposed from different data channels are difficult to match in number and/or frequency [17, 18 ]. in order to resolve this problem, a noise - assisted multivariate emd (na - memd) method has been proposed recently. this method applies the dyadic filter bank property of multivariate emd to white noise and is thereby capable of reducing the mode - mixing problem significantly, achieving favorable performance in the classification of mi eeg signals. although emd and its extended versions have been widely researched and applied, there have been few studies on the selection of relevant imf levels (scales), raising the question of how to select the information - bearing imf components in an efficient way. conventional approaches make use of prior knowledge in task - related domains : relevant imfs are selected by calculating the average power spectra of the first several imfs and comparing them to the frequency distributions of the mu (812 hz) and beta rhythms (1825 hz). similarly, in the neural beta - related oscillatory activities, the informative imfs are chosen by examining the mean beta band frequency. in, the relevant modes are selected by means of partial reconstruction and measures of similarity are calculated between the probability density function of the input signal and that of each mode extracted by emd, though this is still insufficient to analyze multivariate data. recently, a novel statistical approach has been proposed to recognize the information - bearing imfs on each scale. this method uses similarity measures to compare the imfs to both the data and noise, yielding impressive results when applied to the multichannel local field potentials recorded from the cortices of monkeys during generalized flash suppressing (gfs) tasks. in this work, we propose a novel method to identify the information - bearing components from eeg data in low - dimensional space, independent of prior knowledge. the proposed method first performs na - memd on the input signal to obtain different scales of imfs. secondly, unsupervised kernel spectral regression is employed to map the decomposed imfs into a low - dimensional subspace, avoiding the eigendecomposition of dense matrices and enabling the flexible incorporation of various regularizers into the regression framework [7, 8 ]. thirdly, a gaussian mixture model (gmm) is generated, informed by the imfs from both the original signal and noise, and an optimal number of clusters and corresponding model parameters are estimated by the gmm clustering approach. finally, the information - bearing imfs from the input signal are discriminated on each scale. the gmm clustering algorithm is essentially similar to conventional clustering algorithms (e.g., k - means, performing a hard assignment of data points to clusters) except that it allows cluster parameters to be accurately estimated even when the clusters overlap substantially. compared to existing methods of identifying informative imfs, the new method has several noteworthy aspects : kernel spectral regression is employed to reduce the dimension of the decomposed imfs by constructing a nearest neighbor graph to model their intrinsic structure.the probability density function of the composite imfs is modeled by a mixture of gaussian distributions and the number of clusters which best fits the composite imfs is estimated and used to recognize the information - bearing components.the method does not depend on prior knowledge and can discriminate the informative imfs from each signal channel on each scale. kernel spectral regression is employed to reduce the dimension of the decomposed imfs by constructing a nearest neighbor graph to model their intrinsic structure. the probability density function of the composite imfs is modeled by a mixture of gaussian distributions and the number of clusters which best fits the composite imfs is estimated and used to recognize the information - bearing components. the method does not depend on prior knowledge and can discriminate the informative imfs from each signal channel on each scale. the rest of the paper is organized as follows : section 2 presents the materials and proposed signal identification method, consisting of the noise - assisted multivariate empirical mode decomposition of multichannel eeg signals, the spectral regression - based dimensionality reduction of the composite data created by combining the imfs from signal and noise channels, and gmm clustering. it then briefly introduces the common spatial patterns - based feature extraction of the reconstructed signals from the identified information - bearing imfs and support vector machine (svm) classifier. section 3 then demonstrates the experimental results, including simulation results and applications on real mi eeg datasets. finally, we provide some concluding remarks and suggestions for future work in section 4. in order to assess the proposed algorithm, the eeg data from nine subjects was obtained from two publicly available datasets. these datasets contain eeg signals recorded while subjects imagined limb movements, such as left / right hand or foot movements. they are described briefly as follows:(1)bci competition iv dataset i was provided by the berlin bci group. eeg signals were recorded using 59 electrodes from four healthy participants (a, b, f, and g) who performed two classes of mi tasks. more precisely, subjects a and f performed left hand and foot mi while subjects b and g carried out left hand and right hand mi. a total of 200 trials were available for each subject, including 100 trials for each class.(2)bci competition iii dataset iva was provided by the berlin bci group. eeg signals were recorded using 118 electrodes from five healthy subjects (aa, al, av, ay, and aw) who performed right hand and foot mi. a training set and a testing set were available for each subject, though their size differed for each subject. in total, 280 trials were available for each subject, among which 168, 224, 84, 56, and 28 trials comprised the respective training sets for subjects aa, al, av, ay, and aw, with the remaining trials belonging to their testing sets. bci competition iv dataset i was provided by the berlin bci group. eeg signals were recorded using 59 electrodes from four healthy participants (a, b, f, and g) who performed two classes of mi tasks. more precisely, subjects a and f performed left hand and foot mi while subjects b and g carried out left hand and right hand mi. a total of 200 trials were available for each subject, including 100 trials for each class. eeg signals were recorded using 118 electrodes from five healthy subjects (aa, al, av, ay, and aw) who performed right hand and foot mi. a training set and a testing set were available for each subject, though their size differed for each subject. in total, 280 trials were available for each subject, among which 168, 224, 84, 56, and 28 trials comprised the respective training sets for subjects aa, al, av, ay, and aw, with the remaining trials belonging to their testing sets. since the sensorimotor rhythms (smrs) of motor imagery are primarily linked to the central area of the brain [28, 29 ], 11 eeg channels from the experimental data were used (fc3, fc4, cz, c3, c4, c5, c6, t7, t8, ccp3, and ccp4, as recommended in). the locations of these channels are shown in figure 1. our goal is to identify the significant information - bearing imfs on each scale for multichannel data. for each set of multivariate imfs obtained by na - memd, it is key to recognize the suitable imfs bearing significant information associated with the mi eeg activities. in this section first, the na - memd algorithm is performed on the original data to obtain a set of multivariate imfs, from which the composite data is created by combining the imfs from each signal channel with those from the noise channels on each scale. secondly, the composite data is mapped into lower - dimensional subspace to extract feature vectors using unsupervised kernel spectral regression [7, 8 ]. thirdly, a gaussian mixture model is informed by exploiting the intrinsic discriminant structure of the probability distribution that generates the low - dimensional feature vectors. then, for each group of feature vectors on each scale, the maximum likelihood classification is performed to distinguish them into classes after an optimal number of clusters and corresponding model parameters are estimated by the gmm clustering approach. finally, the informative imfs from each signal channel on each scale are identified according to the clustering results. in the following sections, more details are provided for each stage of the proposed approach. for multivariate signals, the memd method is utilized by generating multidimensional envelopes, taking signal projections along different directions, and finally averaging these projections to obtain the local mean. though it is valid in processing multivariate nonstationary signals, memd still inherits a degree of mode - mixing. this has led to the recent development of the na - memd approach, which is performed by adding white noise as additional channels in the original signal. na - memd then enjoys both the benefits of the quasi - dyadic filter bank structure of memd on white noise and the additional realizations of white noise, guaranteeing the separability of the imfs that correspond to both the original signal and noise. given an n - variate input neuronal signal { s(t)}t=1 = { s1(t), s2(t),, sn(t) } with l samples per trial, memd produces j multivariate imfs:(1)st=j=1jdjt+rt, where dj(t) denotes the jth imf of s(t) and r(t) represents the n - variate residual. in practice, the sifting process for a multivariate imf can be stopped when all the projected signals fulfill a stoppage criterion. for memd sifting, a combination of emd stoppage criteria is employed as introduced in [30, 31 ]. the stoppage criterion in standard emd requires that the number of extrema and zero crossings differ at most by one for consecutive iterations in the sifting algorithm. by introducing the envelope amplitude (t) = 1/k=1|e(t) m(t)| and defining an evaluation function f(t) = |m(t)/(t)|, where denotes the total number of direction vectors in memd decomposition, e(t) represents the envelope curve along the kth (k = 1,,) set of directions given by angles k = { 1, 2,, n1 }, and m(t) is the local mean signal, another stoppage criterion is proposed. the sifting process is continued until the value of f(t) is less than or equal to some predefined threshold. similar to the given values in, = 5 and = 0.075 were chosen in this paper. spectral regression is an efficient method to reduce dimensionality from the graph embedding viewpoint [7, 8 ]. specifically, an affinity graph is first constructed to learn the responses for labeled or unlabeled data and then the ordinary regression is applied for learning the embedding function. suppose we have n data points { xi}i=1, dimensionality reduction would aim to find a lower - dimensional representation { zi}i=1, m l. given a p - nearest neighbor graph g with n vertices, where the ith vertex corresponds to a data point xi, let w be a symmetric n n matrix with wij having the weight of the edge joining vertices i and j. g and w can be defined to characterize certain statistical or geometric properties of the dataset. let v = [v1,, vn ] be the map from the graph to the real line, where t denotes a transposition. in the graph embedding approach, by introducing a linear function, vi = f(xi) = axi, we find xa = v, where x = [x1,, xn ] and a = [a1,, an ]. the optimal embedding, v, is then given by the eigenvector corresponding to the maximum eigenvalue of the generalized eigenproblem(2)xwxta=xdxtawith the eigenvalue, where d is a diagonal matrix whose entries are the column sums of w, dii = jwji. this optimization can be solved through regression by adopting the regularization technique, and its solution is then given by(3)a^=arg minai=1natxivi2+j=1laj22+j=1laj, where vi is the ith element of v, the nonnegative regularization parameter is used to control the amount of shrinkage, and some coefficients will be shrunk to exact zero if the nonnegative parameter is large enough due to the nature of the l1 penalty. when the number of features is larger than the number of samples, the sample vectors will typically be linearly independent ; thus the solutions to the optimization problem in (3) are the eigenvectors of the eigenproblem in (2) as and decrease to zero [7, 8 ]. the largest m eigenvectors of a are obtained according to the expected dimensionality of the reduced subspace in real applications. in this way, a low - dimensional representation of the sample matrix x is obtained as z = xa. similar to linear regression, by defining a nonlinear embedding function in reproducing kernel hilbert space (rkhs), that is, v = f(x) = i=1aik(x, xi) = k(x)a, where k(x, xi) is the mercer kernel of rkhs and k(x) = [k(x, x1),, k(x, xn) ], the linear spectral regression approach can be generalized to kernel spectral regression (ksr). the gaussian mixture model (gmm) is widely used as a probabilistic modeling approach to address unsupervised learning problems. based on the expectation - maximization (em) algorithm and an agglomerative clustering strategy using rissanen 's minimum description length (mdl) criterion, a gmm - based clustering approach is developed. the process begins with an initial number of clusters and a set of cluster parameters and iteratively combines the clusters until only one remains., zn ] be a set of m - dimensional samples belonging to different subclasses or clusters and let y = [y1,, yn ] be the subclass of each sample, where yi { 1,, c } denotes which gaussian distribution the sample zi belongs to and c is the number of gaussian components. (1) initialize the parameters including the initial number of clusters co and the gaussian model parameters = { { 1, 1, 1 },, { c, c, c } }, where k is the mean vector, k is the covariance matrix for the kth gaussian distribution, and k denotes the prior probability of the data point generated from the kth component, k = 1,, c. the number of initial clusters in this case should be chosen to fit the number of data types for discrimination. (2) apply an iterative em algorithm until the change in the mdl criterion (mdl(k,)) is less than a threshold, where = 0.01 (1 + m + (m + 1)m/2) log(nm):(4)mdlc,=i=1nlogk=1ckpzi yizi k,+12lognm, where pziyi(zik,) is the gaussian probability density function for the sample zi given that yi = k, log() denotes the log - transformation and is the number of continuously valued real numbers required to specify the model parameters, < 1/(2 nm). (3) record the model parameter and the value of the mdl(c,), where ifinal denotes the final iteration of the em updating process for each value of c. (4) if the number of clusters is greater than 1, apply a defined distance function to reduce the number of clusters, set c c 1, and repeat step (2). (5) choose the value c^ and the model parameters (c^,ifinal) which minimize the value of the mdl criterion. (6) based on the optimal parameters c^ and (c^,ifinal) from step (5), sample vectors are distinguished into c^ classes using the maximum likelihood classification. in this section, we introduce our algorithm for discriminating between informative and noninformative imfs. the detailed steps of our method (ksr - gmm) are described as follows. (1) generate (n + l)-channel multivariate signal consisting of the input n - channel signal and an l - channel uncorrelated gaussian white noise time - series of the same length as the input and then perform the memd decomposition on the multivariate signal, obtaining (n + l)-variate imfs denoted by (n + l) j l matrix, where j is the number of decomposition scales and (2) on the jth (j = 1,, j) scale of the resulting multivariate imfs from step (1), combine the l - channel imfs corresponding to the noise with the one - channel imfs from the original signal, giving n - groups of (l + 1)-variate composite data given by n (l + 1) l matrix. (3) at a given (jth) scale, the unsupervised ksr algorithm is performed, respectively, on the ith (i = 1,, n) group of composite data obtained in step (2), yielding n - groups of low - dimensional representation vectors denoted by n (l + 1) m matrix in the reduced subspace, where m is the number of reduced dimensions. (4) at the given scale, for each group of representation vectors extracted in step (3), the optimal number of clusters c^ is estimated by the gmm clustering approach and, based on the value of c^ and the corresponding model parameters, the representation vectors are then classified into c^ classes using the maximum likelihood classification. (5) at the given scale, the information - bearing imfs are identified according to the clustering results in step (4) : if an imf from any individual signal channel is clustered with the imfs from noise channels, then imf is rejected as noninformative. all remaining imfs the initial number of clusters is chosen to be two in the gmm clustering, since we only discriminate two kinds of data : informative and noninformative imfs. additionally, it should be noted that excessive noise levels can compromise the data - driven ability of the na - memd, though there is no technical limit on the number of the noise channels that can be added. as a rule of thumb, the variance of the noise is required to be within 210% of the variance of the input signal to produce reliable results. in the context of eeg signal processing, the common spatial patterns (csp) approach aims at finding linear spatial filters that maximize the variance of eeg signals from one class while minimizing their variance from others. mathematically, the spatial filters are the stationary points of the following optimization problem:(5)maxu ju = ute1e1tuute2e2tu = utc1uutc2us.t. u2=1,where u denotes a spatial filter, ei represents the n l data matrix from class i where n is the number of channels and l is the number of samples per channel, and ci is the estimated spatial covariance matrix from class i. using the lagrange multiplier method, the solution can be obtained as the eigenvectors of the generalized eigenvalue decomposition : c1u = c2u, where denotes the eigenvalue associated with u. the spatial filters are then the eigenvectors of c2c1, which correspond to the largest and lowest eigenvalues. with the projection matrix u = [u1,, un ], the spatially filtered signal of a trial e is given as s^=ute. for discriminating between two classes of mi tasks, the extracted feature vectors are the logarithm of the spatially filtered signal:(6)fj = logvars^ji=12mvars^i, where s^j (j=1,,2 m) denotes the m first and last rows of s^ and the symbol var() denotes the variance. the support vector machine (svm) algorithm is believed to be a state - of - the - art classification method due to its robustness to outliers and favorable generalization capability. the central idea of svm is to separate data by finding the hyperplane that produces the largest possible margin, which is the distance between nearest data points of different classes. the detailed steps of eeg processing are outlined as follows:(1)preprocess the n - channel eeg data using a 5th - order butterworth filter, obtaining filtered data with the frequency band 830 hz.(2)perform the proposed identification method on the composite signals which are acquired by combining an additional l - channel gaussian white noise with the n - channel eeg data obtained in step (1), identifying the information - bearing imfs on each scale.(3)for the n - channel eeg data, the informative imfs distinguished from step (2) are added together to construct the band - pass filtered signals.(4)process the reconstructed signals from step (3) with the csp algorithm to extract the feature vectors for different motor imagery tasks.(5)employ the svm classifier to identify the classes of eeg during different mi tasks based on the extracted feature vectors in step (4). preprocess the n - channel eeg data using a 5th - order butterworth filter, obtaining filtered data with the frequency band 830 hz. perform the proposed identification method on the composite signals which are acquired by combining an additional l - channel gaussian white noise with the n - channel eeg data obtained in step (1), identifying the information - bearing imfs on each scale. for the n - channel eeg data, the informative imfs distinguished from step (2) process the reconstructed signals from step (3) with the csp algorithm to extract the feature vectors for different motor imagery tasks. employ the svm classifier to identify the classes of eeg during different mi tasks based on the extracted feature vectors in step (4). in this section, several experiments on simulated data and real world eeg data were performed to show the effectiveness of our proposed method. the new algorithm was constructed based on the spectral regression code (http://www.cad.zju.edu.cn/home/dengcai/data/data.html) and the gmm clustering code found in the software package (https://engineering.purdue.edu/~bouman/software/cluster/). for all methods using kernel applications, a gaussian kernel function is chosen due to its validity and stability in experiments, that is, exp(xi all the methods are implemented in matlab 2013a environment on a pc with a 2.5 ghz processor and 4.0 gb ram. our proposed method is first performed on the simulated data to verify its effectiveness. unless otherwise specified, 15-channel noise data was generated using an uncorrelated gaussian white noise time - series which has the same length as that of the input signal. moreover, the variance of noise was set to be 6% of the variance of the input according to suggestions in. additionally, the number of nearest neighbors (p = 5) and the regularization parameters (= 0.001 for l2 penalty and = 0.01 for l1 penalty) were chosen by cross - validation in this simulation. in this experiment, the same simulated data was generated as in. a 3-channel synthetic signal [x(t), y(t), z(t) ] with the length n = 1000 and the sampling rate fs = 1000 hz is (7)xt = sin2f1t+sin2f2t+sin2f3t+q1t, t=1,2,,1000,where f1 = 12/fs, f2 = 26/fs, f3 = 50/fs, and q1(t), q2(t), q3(t) represent gaussian white noises. a set of 3-channel input signals with snr = 20 db was generated and an additional 15-channel white noise with snr = 6.1 db was added to the input signal to create the composite signal. our method was then performed on the composite signal and the information - bearing imfs on each scale were identified. figure 2 shows a scatter plot with class labels of sixteen samples from a two - dimensional feature vector at the first seven scales, including one sample corresponding to one signal channel and fifteen samples from noise channels. here it can be seen from figure 2 that the composite data points on the 4th, 5th, and 6th scales in x - group are all clustered into two classes, with the same being true for the 4th and 6th scales in y - group and the 4th and 5th scales in z - group, while the composite data on the remaining scales of each channel falls into one class. according to the proposed method, these imfs with two clusters are regarded as informative and the identification results are consistent with the imfs containing the true frequency components decomposed by the na - memd algorithm, as shown in figure 3. the first seven imfs are denoted as c1c7 and the residuals are represented as cres, which are the sums of the remaining scales of imfs. it can be seen that the underlying frequency components occur in the 46th imf components, which are displayed in red. (ii) to test the effect of noise with different snrs on our method, it was necessary to verify this performance since measured data often suffers from noise contamination in real applications. our method was compared with several approaches for identifying information - bearing components : (i) hu 's method, which uses the wasserstein distance to assess the similarity between the reference imfs from noise channels and the imfs from signal channels and subsequently establishes a confidence interval (e.g. 95%) for the distance by employing a monte - carlo technique, denoted as wd - ci ; and (ii) three algorithms for dimensionality reduction together with gmm clustering : pca, kernel pca (kpca), and l1-norm pca (l1pca). in order to facilitate performance comparison, two kinds of error were evaluated. these are defined as (1) type i error, which is the failure to identify true imf components bearing relevant information, and (2) type ii error, which is the improper identification of information - free imf components. first, different snrs were varied by systematically changing the variance of the white noise superimposed in the input signal, combined with separate 15-channel white noise (snr 6.1 db) as reference channels. overall, sixteen snr levels were tested with 100 trials performed at each level. in each trial, the snr of the white noise superimposed on the input signal was first changed, the relevant imfs were identified by the different algorithms, and the corresponding error rates were calculated. low rates of type i and type ii error were found at the higher snr levels for all methods. on the whole, with the exception of type i error rates in pca - based approaches, increases in snr led to decreases in error rates. when compared with other identification approaches, pca - gmm, kpca - gmm, and l1pca - gmm showed lower type i error rates but higher type ii error rates, while wd - ci yielded the lowest type ii error rate. the proposed method showed an improved type i error rate with a slightly higher type ii error rate than the wd - ci algorithm, though the overall type ii error rates of both the new method and the wd - ci algorithm remain very small, even at low snrs. these results indicate that our method is able to effectively identify the information - bearing components at low snrs and is highly resistant to white noise. next, considering that the noise contained in the signal channels is mismatched with the noise in the reference channels, the effects of red noises (1/f noise) with different snrs were tested on the proposed method. results indicate that both the new method and the wd - ci algorithm work well even when there is a mismatch between the noise contained in the data and the noise in the reference channels. this further demonstrates the robustness of our method when identifying the informative components in noisy data at low snrs. this section evaluates the performance of our proposed method on mi eeg datasets. it has already been shown that the greatest result of motor imagery is a modulation of the smrs. differential modulations in the smrs were decomposed using the na - memd method with locally orthogonal and narrowband imf bases. based on the identified information - bearing imfs, relevant imfs from the same channel were summed to get the reconstructed signal, and csp - based feature extraction and svm - based classification were performed. for each trial in the bci competition iv dataset i, we selected the eeg data from 04 s after the initiation of mi, as performed in. in contrast, the window from 0.52.5 s after initiation was used for the bci competition iii dataset iva, as in. the 11-channel eeg data was regarded as the input signal and combined with an additional 15-channel noise (snr 20 db). several parameters chosen by cross - validation in our identification algorithm are p = 5, = 0.1, and = 0. for both eeg datasets, the best model parameters were determined by fivefold cross - validation from { 2,, 2 } in svm models. according to the aforementioned steps (i) to demonstrate the identification capability of the informative imf components in eeg data using the proposed method : it is noted that, for eeg data, unlike the simulations, we do not know the ground truth of the imfs that have been identified. for all 200 trials of each subject in the bci competition iv dataset i, the average power spectra of the identified information - bearing imfs were computed and then compared to those obtained using the existing method (na - memd - pk). figure 6 shows the logarithm of average power spectra for each subject using the new method. it can be seen that the beta and mu rhythms, which are contained in the 2nd (c2) and 3rd imfs (c3), respectively, are separated clearly. moreover, the frequency bandwidths in the 1st imfs (c1) are generally broad, containing some parts of the 1530 hz frequency band. consequently, there is a trade - off in the choice of c1 ; ignoring it would sacrifice some useful information, whereas conserving it could introduce noise. to resolve this problem, the role of the first scale is decided according to the optimal classification results combined with csp - based feature extraction. for all four subjects, a paired t - test revealed no significant differences between the two approaches in the power spectra of all 200 trials at the first three imfs but found a significant difference at the 4th imf, as shown in table 1. this demonstrates the validity of the proposed approach when identifying information - bearing imfs from real eeg data. (ii) an evaluation of the classification performance of the proposed method using a fivefold cross - validation study on two mi datasets : the classification process here was repeated 100 times using the new method, the na - memd - pk algorithm, and the non - emd based approach in which raw data is directly processed by csp - based feature extraction and svm - based classification for a varying number of spatial filters (m = 1,2, 3,4). the average accuracy and standard deviation were obtained for each method and used for direct comparison. considering the size of the total data for each subject in bci competition iv dataset i, the number of eeg blocks was set at 140 for each training set and 60 for each testing set, as in. to ensure a valid comparison between the different methods, figure 7 shows the classification performances for all four subjects from the bci competition iv dataset i. the results show that the na - memd - pk approach yielded the best averaged results, with an average classification accuracy of 81.01% for all four subjects a 0.24% improvement over the csp algorithm and a 1.81% improvement over the new method. the csp method yielded the best performance among the three approaches in two subjects (a and g), whereas na - memd - pk yielded the best mean accuracy in the two remaining subjects (b and f), while our method performed slightly higher than the csp algorithm when m = 2, 3. nevertheless, a paired t - test revealed no significant difference between our method and the na - memd - pk algorithm (p = 0.195, 0.096 for m = 2, 3, resp.), no significant difference between our method and the csp approach (p = 0.074 when m = 2), and a significant difference between our method and the csp approach (p = 0.003 for m = 3). these results show that, when compared to the na - memd - pk algorithm, our method can achieve similar results without the use of prior knowledge. finally, the classification performances for the five subjects from the bci competition iii dataset iva are demonstrated. for each subject, the csp filters and classifier models were trained on the available training sets. figure 8 illustrates the classification accuracies (mean and standard deviation) obtained from these sets. the results showed that the average classification accuracy for all five subjects obtained by our method was 74.06%, yielding a 0.94% improvement over the na - memd - pk approach. a paired t - test revealed no significant difference between our method and the na - memd - pk algorithm (p = 0.225, 0.027 for m = 2, 3, resp.),and a significant difference between our method and the csp approach (p values less than 0.01). when applied to the bci competition iii data, the csp method yielded the best performance among the three approaches in two subjects (al and ay), while the proposed algorithm performed the best in subject aa when m = 1,2, 3,4. additionally, our method outperformed the na - memd - pk approach in two subjects (aa and ay), whereas the na - memd - pk algorithm performed better in two subjects (al and av) and yielded similar performance in subject aw for all four groups of spatial filters. in these experiments, the na - memd algorithm exhibited an accurate localization of the task - specific frequency bands with favorable separability for feature extraction and classification, as demonstrated in its applications to mi eeg data. for the simulations, the new method was further shown to be robust to white and colored noises with different snrs. when compared with other identification approaches (wd - ci, pca - gmm, kpca - gmm, and l1pca - gmm), the proposed method obtained relatively improved performances in terms of both type i and type ii error rates. for real eeg data, the information - bearing imfs were discriminated clearly for nine subjects during mi tasks. when compared with the na - memd - pk approach, which selects imfs based on average power spectra, the proposed method yielded similar classification performance though it did not require prior knowledge to achieve such favorable results. despite the favorable capability of the new algorithm when distinguishing the informative imfs containing task - related frequency bands and classifying mi eeg signals, it should be recognized that individual subject differences may still have a great deal of influence on the recognition ability of the algorithm. in this paper, we have shown how to discriminate the information - bearing components of motor imagery (mi) eeg independent of prior knowledge. the noise - assisted memd (na - memd) algorithm was first performed on original datasets to obtain a set of multivariate imfs, with the subsequent application of unsupervised kernel spectral regression (ksr) to generate low - dimensional feature vectors by mapping the decomposed imfs into lower - dimensional subspace. for the low - dimensional feature vectors from each signal channel, a gaussian mixture model (gmm) clustering approach was employed to estimate the optimal number of clusters and corresponding model parameters and then identify the information - bearing imfs. the common spatial pattern (csp) approach was exploited to train spatial filters to extract the task - related features from the reconstructed signals by adding the informative imfs together. a support vector machine (svm) classifier was applied to the extracted features and recognized the classes of eeg signals during different mi tasks. using these techniques, we have demonstrated that our proposed method is effective at identifying the information - bearing imf components in simulated data and mi eeg datasets and achieves excellent classification performance. in conclusion, a novel method for scale - dependent signal identification in a low - dimensional subspace has been proposed for mi task classification. although our method is independent of prior knowledge, entirely data - driven, and robust to different types of noise, several questions remain to be investigated in future work ; the spectral regression - based dimensionality reduction approach selects the nearest neighbor graph ; however this is not the only natural choice. recently there has been a great deal of interest in exploring the different ways to construct a graph to model the intrinsic geometrical and discriminant structures within eeg datasets. in addition, semisupervised clustering methods have also yielded promising results when compared with the traditional unsupervised clustering approaches. to improve the clustering performance, it will be necessary to exploit the underlying manifold structure of the data along with additional knowledge from unlabeled data. advancements such as these, in conjunction with the algorithm presented in this paper, will serve to improve the detection, classification, and evaluation of mi signals. this, in turn, can lead to improvements in eeg - based rehabilitation technologies, improving both the prediction and elicitation of motor recovery in a multitude of diseases worldwide. | motor imagery electroencephalography (eeg) has been successfully used in locomotor rehabilitation programs. while the noise - assisted multivariate empirical mode decomposition (na - memd) algorithm has been utilized to extract task - specific frequency bands from all channels in the same scale as the intrinsic mode functions (imfs), identifying and extracting the specific imfs that contain significant information remain difficult. in this paper, a novel method has been developed to identify the information - bearing components in a low - dimensional subspace without prior knowledge. our method trains a gaussian mixture model (gmm) of the composite data, which is comprised of the imfs from both the original signal and noise, by employing kernel spectral regression to reduce the dimension of the composite data. the informative imfs are then discriminated using a gmm clustering algorithm, the common spatial pattern (csp) approach is exploited to extract the task - related features from the reconstructed signals, and a support vector machine (svm) is applied to the extracted features to recognize the classes of eeg signals during different motor imagery tasks. the effectiveness of the proposed method has been verified by both computer simulations and motor imagery eeg datasets. |
honey bees (apis mellifera) contribute vital pollination services to agricultural crops and native landscapes, accounting for over $ 15 billion / year in economic value in the us. in addition, the honey bee is a model organism for epigenetic, behavioral, and host - pathogen interaction studies [210 ]. since 2006, the us and parts of europe have experienced high annual colony losses (~33% annual loss in the us) [9, 1113 ]. in the us, colony mortalities are partially attributed to colony collapse disorder (ccd) [1419 ]. these losses have stimulated greater interest in investigating honey bee biology, including the role of pathogens in colony mortalities and the role of the rna interference (rnai) mechanism in honey bee antiviral defense. pathogen incidence and abundance have been positively associated with ccd - affected colonies in the us [15, 19, 20 ] and colony losses in the us [21, 22 ], canada, and european countries, including, spain, italy [25, 26 ], belgium, and germany. in the us, israeli acute paralysis virus (iapv) was more abundant in colonies with less food stores and less developing bees / brood, and lake sinai viruses (lsv) 1 and 2 were more abundant in weak / less populated colonies from a small sample cohort ; however, this correlation was not seen in a larger sample cohort. honey bees are infected by a wide variety of pathogens (i.e., viruses, bacteria, microsporidia, and trypanosomatids) and also suffer from ectoparasitic mite (varroa destructor) infestation (reviewed in). the majority of honey bee pathogens are positive sense, single - stranded rna viruses. the short - interfering rna (sirna) pathway of rna interference (rnai) is a major antiviral immune mechanism in solitary insects (reviewed in) and is involved in honey bee antiviral defense. rnai is a post - transcriptional, sequence - specific, gene regulation mechanism conserved across several phyla, including plants, invertebrates, and mammals (reviewed in). rnai - mediated gene knockdown is a useful tool for assessing gene function in honey bees and other organisms for which additional reverse genetic tools are not available. while experimental introduction of virus sequence - specific dsrna reduced honey bee virus infections in adults and larvae [29, 3439 ], introduction of non - sequence - specific dsrna also resulted in virus reduction and altered gene expression this is consistent with global changes in honey bee gene expression, including untargeted genes (i.e., off - target effects), observed from administration of dsrna [40, 41 ]. together these results suggest that dsrna not only serves as the substrate for rnai - mediated gene regulation but also may function as a trigger of gene regulatory signal transduction cascades. future studies aimed at determining the relative contribution of rnai and other honey bee antiviral defense pathways will be important for the development of strategies that limit virus infection and for investigating immune gene function in bees. in this review, we discuss rnai as a tool for gene knockdown in honey bees, the role of the sirna pathway of rnai in honey bee antiviral defense, and additional honey bee antiviral defense pathways, including evidence of a non - sequence - specific dsrna - stimulated immune pathway in honey bees. rna silencing is a mechanism of post - transcriptional gene regulation conserved across several phyla that encompasses three distinct pathways (reviewed in [32, 42 ]), including the short - interfering rna (sirna), microrna (mirna), and piwi - interacting rna (pirna) pathways. each of these pathways is characterized by its unique biological function and involvement of distinct proteins. the sirna pathway is involved in antiviral defense in plants and invertebrates, but its function in mammalian immunology is debated (reviewed in [4345 ]). double - stranded rna is recognized and cleaved by the rnase iii enzyme, dicer (dicer-2 in drosophila [46, 47 ] and dicer - like in apis mellifera), into 21 - 22 bp short - interfering rnas (sirnas) (reviewed in) (figure 1). sirnas are short dsrnas with 5 monophosphate ends and two nucleotide overhangs at their 3 hydroxyl - termini (reviewed in). the sirnas are subsequently bound by argonaute (ago2), an endoribonuclease and catalytic component of the multiprotein rna - induced silencing complex (risc). one strand of the sirna, the passenger strand, is then released, leaving the other strand, the guide strand, to target complementary viral and transposon sequences for cleavage (reviewed in). mirnas are derived from endogenous nuclear - encoded short - hairpin rnas that are processed into shorter hairpin rnas (pre - mirna), cleaved by dicer into 21 - 22 bp segments in the cytosol and incorporated into risc (reviewed in). the mirna - containing risc then targets complementary host - encoded mrna transcripts for degradation or translational inhibition. conversely, mirnas can serve to induce transcription and translation of mrna, reduce nonsense - mediated rna decay, and improve mrna stability (reviewed in). mirnas can function in antiviral response via targeting of viral nucleic acid and host gene regulation (reviewed in). pirnas, which are larger than sirnas and mirnas (2432 nucleotides (nts)), are generated in a dicer - independent manner from single - stranded rna precursors transcribed from genomic regions (reviewed in [49, 52, 53 ]). pirnas are involved in transposon silencing, epigenome regulation, and antiviral defense (reviewed in [49, 52, 53 ]). the focus of this review is on the use of the sirna / rnai pathway in experimental gene knockdown and its role in antiviral defense in honey bees. further use of the term rnai in this review is in regard to the sirna pathway. this mechanism can also be triggered experimentally to study gene function in organisms for which the tools for facile gene knockout are not available. rnai - mediated gene knockdown has been used to study gene function in different honey bee developmental stages, including embryos [5460 ], larvae [3, 56, 6168 ], pupae [56, 69, 70 ], and fully developed adult honey bees [2, 4, 7183 ] (reviewed in). importantly, these studies have demonstrated that the rnai machinery is functional in honey bees. the variable efficacy of rnai - mediated gene knockdown observed in honey bees is likely gene-, dsrna trigger-, and tissue - specific. the most effective use of rnai - mediated gene knockdown in adult bees has been targeting of the hemolymph (insect blood) protein vitellogenin ; vitellogenin expression was reduced at the mrna (> 75%) and protein levels in several studies [2, 7274, 76, 81 ]. the efficacy of vitellogenin rnai - mediated knockdown in honey bees is likely, in part, a consequence of its involvement in a positive regulatory feedback loop in which vitellogenin and juvenile hormone mutually suppress each other [72, 85 ]. in short, rnai - mediated knockdown of vitellogenin in several studies has provided evidence for its role in aging by acting as an antioxidant and in the timing of foraging behavior [74, 76 ]. the high efficiency of rnai - mediated knockdown of honey bee vitellogenin may be partially attributed to enhanced targeting of sirnas and dsrnas to the fat body where vitellogenin is produced. similarly, intra - abdominal injection of either dsrna or sirna targeted against the glycerol-3-phosphate dehydrogenase (amgpdh) gene significantly decreased amgpdh transcripts in the fatty body (i.e., > 70% decrease), but not in the ovaries, flight muscles, or head. for comparison, a study that aimed to silence octopamine receptor expression via injection of dsrna into the antennae observed ~40% decreased expression. in addition, silencing of the hypopharyngeal amylase via dsrna injection into the abdomen resulted in ~30% decreased expression in the hypopharyngeal glands. the fat body is the site for insect humoral immunity (e.g., antimicrobial peptide production) and is involved in many metabolic processes (reviewed in [86, 88 ]). therefore, gene knockdown studies in the fat body may be useful for studying honey bee metabolic and immune pathways. the sirna / rnai is a major antiviral defense mechanism in solitary insects, including fruit flies and mosquitos (reviewed in). the piwi - interacting rna (pirna) pathway has also been linked with antiviral response in insects via the detection of pirna - sized viral rnas in persistently infected drosophila ovarian sheath cells and the discovery of ping - pong dependent pirnas in arbovirus - infected aedes spp. may have contrasting roles in insect - virus infection ; virally derived mirnas can disrupt host cell transcription and translation, but host mirnas may be used to target and disrupt viral nucleic acid (reviewed in). however, the roles of the mirna and pirna pathways in honey bee antiviral defense are not well characterized ; thus, in this review, we focus on the sirna pathway of rnai. the honey bee genome encodes the sirna / rnai machinery dicer - like, ago-2, and r2d2 [48, 91 ], and bees are readily infected by positive sense, single - stranded rna viruses (reviewed in). these single - stranded rna viruses generate double stranded rna intermediates during their replication cycle and likely have significant secondary rna structure within their genomes [92, 93 ], either of which may serve as am dicer - like substrates and trigger the honey bee sirna pathway (figure 1). the role of rnai in honey bee antiviral defense was first demonstrated when bees fed israeli acute paralysis virus (iapv) and iapv - specific dsrna had reduced iapv levels as compared to bees fed only virus. in addition, iapv - specific sirnas were detected by northern blot analysis in the iapv - specific dsrna treated bees, indicating that the dsrna and virus genomes were cleaved by dicer - like and/or ago2. iapv replication was also decreased in bees fed sirnas targeting the internal ribosomal entry site (ires) of iapv. similar results were obtained when larvae and adult bees fed dsrna targeting deformed wing virus (dwv) had reduced mortality, virus load, and deformed wing symptoms. the effect of dsrna administration on the outcome of virus infection was also examined in the eastern honey bee, apis ceranae. in turn, there has been commercial / agricultural interest in utilizing rnai - mediated antiviral treatments in honey bee colonies (reviewed in [35, 94, 95 ]). initial field studies suggested that feeding honey bees iapv - specific dsrna resulted in increased honey production and larger colony size ; however, additional research is needed to confirm the mechanism of action and further investigate additional biological effects of dsrna / sirna treatments. rnai - mediated antiviral defense in naturally infected bees, which were not fed either dsrna or sirna triggers, was documented and characterized by sequencing small rna libraries. small rna sequence data indicated that bees from ccd - affected colonies had higher amounts of 22 nt sirnas spanning the genomes of iapv, kashmir bee virus (kbv), and deformed wing virus (dwv) as compared to non - ccd colonies. most of the iapv - specific sirnas were negative sense, indicating their role as guide strands that target risc to viral genomes / mrnas. moreover, dwv virus levels were broadly proportional to the abundance of dwv - specific sirnas in both orally infected and mite - vectored infections in developing bees. transcriptome (rnaseq) sequence data also indicated the role of the rnai machinery in antiviral defense, as the expression of argonaute-2 and dicer - like were greater in bees experimentally infected with iapv as compared to mock - infected controls. intriguingly, transcriptional level regulation of the drosophila rnai genes in response to virus infection has not yet been documented [98, 99 ], suggesting that regulation of antiviral defense mechanisms in honey bees and fruit flies may differ. like many insect - infecting viruses, some honey bee viruses have likely evolved specific mechanisms to counteract rnai - mediated antiviral defense, including virus - encoded suppressors of rnai (vsr). for example, the b2 protein dimer of flock house virus binds dsrna, subsequently preventing dicer-2 cleavage of long dsrna [100, 101 ] and sirna loading into risc. dicistroviruses encode protein 1a, a vsr with differential modes of action (e.g., it binds to dicer-2 or ago2) and efficacy that varies by virus (reviewed in). based on analysis of vsr - expressing viruses (i.e., drosophila c virus and cricket paralysis virus), the presence of the highly conserved dvexnpgp motif and upstream coding sequences are indicative of the ability to express vsr proteins [102, 103 ]. sequence analysis revealed that the honey bee dicistroviruses iapv and kbv and acute bee paralysis virus (abpv) contain a dvexnpgp motif at the 5 terminus of their genomes, suggesting these honey bee - infecting viruses may encode a vsr. experimental feeding of naturally iapv - infected bees with sirnas targeting the putative iapv - encoded rnai suppressor decreased iapv loads at least three times more than treatment with sirnas targeting the iapv ires [29, 39 ]. better understanding of the importance of rnai in honey bee antiviral defense and the means by which viruses may evade the honey bee antiviral response will facilitate the manipulation of these mechanisms in the lab as well as their potential application in the field setting. the dsrna uptake mechanisms in insects and their relationship to systemic rnai and antiviral defense are not completely understood. current studies suggest that there are at least two mechanisms of dsrna uptake in insects : transmembrane channel - mediated uptake (reviewed in) and endocytosis - mediated uptake [105107 ]. sid-1 (systemic rna defective), a dsrna - transporting transmembrane protein originally identified in c. elegans [108, 109 ], has been implicated in facilitating systemic rnai in honey bees ; bees injected with dsrna had over three times greater expression of sid-1 than controls. c. elegans also encodes additional sid proteins, sid-2, sid-3, and sid-5, which have also been implicated in dsrna uptake but have not been identified in the honey bee genome [111113 ]. honey bees encode for one sid-1 ortholog with two protein isoforms (xp_006565236.1 and xp_006565237.1), which both share ~25% amino acid identity with the c. elegans sid-1 (np_504372.2). in addition, transgenic drosophila s2 cells expressing the c. elegans sid-1 protein had improved dsrna uptake. interestingly, sid-1 is not present in all insect genomes (reviewed in), including drosophila, and is not required for systemic rnai in locusts. proteins involved in phagocytosis and endocytosis may function in dsrna uptake, as the scavenger receptors sr - ci and eater and the endocytosis - associated proteins clathrin heavy chain and h+ atpase are important for dsrna uptake in drosophila s2 cells [105, 106 ]. investigating dsrna uptake and systemic rnai will be an important step towards further characterizing honey bee antiviral response. double - stranded rna treatment of honey bees has also been employed to reduce gene expression in honey bee - associated parasites including the microsporidia nosema ceranae and the ectoparasitic mite varroa destructor. honey bees inoculated with nosema spores and fed dsrna targeting nosema - specific adp / atp genes had reduced nosema spore count, and nosema had lower expression of the targeted genes. likewise, when bees were fed dsrna targeting mite sequence - specific housekeeping genes, mites had lower levels of the targeted transcripts. interestingly, long, unprocessed dsrnas were detected in bee hemolymph three days after feeding dsrna. the biological relevance of dsrna as a systemically active molecule in naturally infected honey bees is unknown, but it is remarkable that orally introduced dsrna remains stable enough to spread throughout the honey bee host and into associated parasites [117, 118 ]. it is interesting that several studies have demonstrated that dsrna / sirna feeding is an effective strategy to reduce virus loads in both larval - stage and adult bees, while achieving effective in vivo gene silencing is difficult in mammalian model systems (reviewed in). tail - vein injections of sirna in postnatal mice have been an effective strategy for gene knockdown, but overall systemic sirna delivery into mammalian systems often requires sirnas with chemical modifications such as lipophilic conjugates or nanoparticle mediated delivery (reviewed [119, 121 ]). preliminary results on the effects of rnai - mediated treatment of honey bee viruses and parasites are promising, but additional investigation is required to better understand the feasibility, effectiveness, and risk of off - target effects. additionally, it will be important to develop methods to functionally test the role of the rnai machinery via gene knockout / knockdown. genome integration of iapv also requires further examination. both genome - integrated rna viral sequences, putatively encoding for target nucleic acid or reverse - transcriptase, and rnai are involved in limiting and maintaining persistent virus infections in d. melanogaster [42, 107 ]. these and other studies will reveal the role of rnai in honey bee antiviral defense. multiple mechanisms are involved in insect immune responses, including phagocytosis, melanization, and signal transduction of the toll, imd (immune deficiency), and jak / stat (janus kinase and signal transducer and activator of transcription) innate immune response pathways which result in the production of antimicrobial peptides (amps) and other effector proteins (reviewed in [89, 90, 123 ]). there are multiple orthologous proteins utilized by both insect and mammalian immune pathways (reviewed in [124, 125 ]), including toll - like receptors (tlrs). while mammalian tlrs recognize and bind specific pathogen associated molecular patterns, the d. melanogaster toll acts downstream of pathogen recognition [90, 123 ]. honey bees encode for all the major components of the toll, imd, jnk, tor, and jak - stat pathways (except upd), amps (i.e., abaecin, hymenoptaecin, apidaecin, and defensin), and prophenoloxidases. transcriptional studies of virus - infected honey bees have implicated the jak - stat, toll, and imd pathways in antiviral defense (reviewed in). for example, bees infected with dwv had greater expression of the imd pathway member dorsal-1a, and bees fed iapv had increased expression of toll pathway members (i.e, toll-6, cactus, and the amp hymenoptaecin) and jak / stat pathway members (i.e., cbl, stat, pias, and hopscotch). nevertheless, not all infection studies investigating transcriptional responses to the same virus followed the same trends. in contrast to bees fed iapv, bees from naturally infected iapv colonies did not have differential regulation of jak / stat or imd pathways. these inconsistencies may be due to different experimental factors such as difference in virus isolate / strain utilized, infection route, age of bees, and tissue examined (reviewed in). further investigation of the role of these and other innate immune pathways in honey bee antiviral defense will lead to a better understanding of the mechanism(s) of honey bee antiviral defense and reveal unique honey bee host - virus interactions. in addition to inducing rnai, dsrna may also engage a previously uncharacterized non - sequence - specific immune pathway in honey bees (figure 1). bees coinjected with sindbis virus (sinv) and sequence - specific dsrna or non - sequence - specific dsrna had similarly decreased viral titers as compared to bees injected with virus only. likewise, adult bees treated with non - virus specific dsrna (i.e., gfp- (green fluorescent protein-) targeting) and infected with dwv had a greater rate of survival as compared to dwv - infected bees that received no dsrna - treatment. in addition, experimental introduction of nonspecific dsrna alone in honey bees perturbs honey bee gene expression [38, 41 ]. transcriptome analysis of honey bee larvae fed gfp - targeting dsrna revealed ~1,400 differentially regulated genes (degs). nine genes had sequence similarity with 21 nt regions of gfp, indicating off - target rnai. however, most degs did not share sequence similarity with dsrna - gfp and were reported to function in oxidoreductase activity, aging, cell homeostasis, morphogenesis, response to external stimulus and stress, and immune response. also, bees injected with non - sequence - specific dsrna had differential expression, including decreased expression of several apidaecin amp family members. in a recent study that examined the role of rnai - mediated antiviral defense in bumblebees (i.e., bombus terrestris), adults fed non - sequence - specific dsrna had increased survival when infected with iapv and similar virus titers as compared to bees fed dsrna targeting iapv. together these results suggest that honey bees and other members of the apidae family may have an alternative dsrna - stimulated immune pathway akin to the interferon response in mammals. mammals have dsrna recognition receptors such as toll - like receptor 3 (tlr3), protein kinase r (pkr), retinoic acid - inducible gene 1 (rig - i), and melanomadifferentiation - associated gene 5 (mda-5), that when activated, induce expression of numerous genes that contribute to an antiviral state (reviewed in). analogously, non - sequence - specific dsrna - mediated immune pathways may be important for antiviral defense in honey bees. nonspecific dsrna - triggered antiviral immunity has also been observed in other arthropods including chinese oak silk moth pupae, shrimp [130134 ], bombyx mori larvae, and sandfly cells, implicating dsrna as a viral pathogen associated molecular pattern (pamp or vamp). in addition, there is evidence that dicer-2 serves as a pathogen recognition receptor (prr) of dsrna in both d. melanogaster and culex pipiens f. molestus mosquito cells. when bound with dsrna, dicer-2 stimulates a signal - transduction cascade that results in increased expression of vago and jak - stat pathway genes (reviewed in [90, 123 ]) (figure 1). intriguingly, larvae orally infected with dwv from varroa infested colonies had significantly greater expression of the honey bee ortholog of vago as compared to control larvae from colonies with lower mite pressure. though administration of non - sequence - specific dsrna does not always improve survival or reduce viral titer in virus infected bees [34, 36 ], it is important to further examine the mechanisms involved in non - sequence - specific dsrna - mediated antiviral immunity in honey bees. since 2006, annual losses of honey bee colonies in the us have been high (i.e., averaging 33%). pathogen incidence and abundance continued investigation of honey bee host - pathogen interactions is important to better understand the role of pathogens in colony losses. honey bee virus infections result in a range of outcomes, likely caused by varying immune responses due to genetic differences [138, 139 ], coinfection with additional pathogens [20, 31, 140 ], adequate bee nutrition [141143 ], the effect of the bee microbiome [144, 145 ], and/or exposure to environmental factors including agrochemicals and weather events [146149 ]. the rnai mechanism plays a role in honey bee antiviral defense but the relative contribution of this and other immune pathways has not been fully elucidated. the efficacy of rnai - mediated treatment against honey bee viruses together with the fact that honey bee viruses encode for putative vsrs supports that rnai is an important honey bee antiviral defense mechanism. in addition, several studies implicate the involvement of innate immune pathways (i.e., jak - stat, toll, and imd) and non - sequence - specific dsrna - mediated immune responses in honey antiviral defense. honey bee gene knockout models are not yet available, so experimental induction of rnai has become an important tool for studying gene function in honey bees. although effective rnai - mediated gene knockdown has been demonstrated in the fat body, gene knockdown in other tissue types (e.g., reproductive tissue) remains a challenge. further development of honey bee cell culture systems [150153 ], and perhaps the use of the endoribonuclease crispr / cas9-mediated gene knockout system, will also facilitate future investigations of rnai and innate immune pathways in honey bees. continued investigation of honey bee host - pathogen interactions and better characterization of the honey bee immune system may result in implementation of strategies that benefit honey bee colony health and result in the discovery of additional evolutionarily conserved immune mechanisms. | honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. therefore, investigating the factors associated with high annual losses of honey bee colonies in the us is an important and active area of research. pathogen incidence and abundance correlate with colony collapse disorder- (ccd-) affected colonies in the us and colony losses in the us and in some european countries. honey bees are readily infected by single - stranded positive sense rna viruses. largely dependent on the host immune response, virus infections can either remain asymptomatic or result in deformities, paralysis, or death of adults or larvae. rna interference (rnai) is an important antiviral defense mechanism in insects, including honey bees. herein, we review the role of rnai in honey bee antiviral defense and highlight some parallels between insect and mammalian immune systems. a more thorough understanding of the role of pathogens on honey bee health and the immune mechanisms bees utilize to combat infectious agents may lead to the development of strategies that enhance honey bee health and result in the discovery of additional mechanisms of immunity in metazoans. |
schizophrenia is a severe, particularly devastating, psychiatric disorder affecting approximately 1% of the general population. even though schizophrenia is highly heritable, the research for chromosomal loci and candidate genes has not provided any consistent results. combinations of genetic, epigenetic, and environmental factors participate in the development of the disease. since these factors have not been identified, the diagnosis of schizophrenia is based on phenotypic symptoms only. therefore, the identification of susceptibility genes is likely to provide valuable insights into the etiology and pathogenesis of the disease, consequently leading to the development of more effective treatments. catechol - o - methyltransferase (comt) is an enzyme, which catalyses the o - methylation of catecholamine neurotransmitters such as dopamine, adrenaline, and noradrenaline. a reformulated hypothesis of the dopamine 's role in the disease states that hyperdopaminergic functioning in subcortical structures is associated with positive symptoms such as hallucinations and delusions, whereas hypodopaminergic functioning in prefrontal cortical regions is associated with negative and cognitive symptoms. dopamine is inactivated either by reuptake into the neurons that release dopamine into the synapse or through metabolism by monoamine oxidase or comt. in most areas of the brain, reuptake inactivation predominates, so that comt does not markedly influence dopamine levels. by contrast, in the prefrontal cortex, that mediates the cognitive functions, which are impaired in schizophrenia, dopamine levels are sensitive to comt levels. velocardiofacial syndrome (vcfs) patients with vcfs display an extremely high incidence of schizophrenia about 25% to 30%, and 22q11 deletion occurs in 2% of diagnosed schizophrenics. comt gene maps to the vcfs region of chromosome 22 [7, 8 ]. due to its involvement in the catabolic clearance of dopamine and its location in the 22q11 microdeletion area the best - studied variant is a functional single - nucleotide polymorphism, which results in a valine to methionine mutation at position 158 (commonly referred to as val158met or rs4680). the val variant has higher enzymatic comt activity and thermostability than its methionine counterpart, leading to more efficient degradation of dopamine. therefore, the val variant is related to poor performance on certain tests of working memory and to insufficient brain activation [11, 12 ]. there is weak and inconsistent evidence that the val variant may be associated with increased risk of schizophrenia. most case control studies and meta - analysis [14, 15 ] do not support association, whereas studies using a family design actually do. other variants, which have been studied for association with schizophrenia, are rs737865 and rs165599. the present study examines evidence for association of the three snps (rs737865, rs4680, and rs165599) and their haplotypes with schizophrenia in a greek population. the study sample consisted of 108 cases with verified schizophrenia and 97 healthy individuals. blood samples of schizophrenia patients were collected from hospitalized patients in the 10th clinical department of the attica 's psychiatric hospital dafni, whereas control blood samples were obtained from healthy individuals, who had free anamnesis of schizophrenia and volunteered to participate in this study. table 1 shows the distribution of cases and controls by sociodemographic characteristics. gender distribution and median age were similar in the two groups of participants. the inclusion criteria specified that all patients fulfilled the diagnostic criteria of schizophrenia according to the diagnostic and statistical manual of mental disorder (dsm - iv). all participants were of greek ethnic origin and had signed the informed - consent form. the study was approved by hospital 's ethic committee. according to the exclusion criteria, subjects who denied providing informed consent or those whose psychosis was judged secondary to substance use or a known neurological disorder, such as epilepsy, based on the consensus diagnostic procedure, were not eligible to participate. the three snps (rs737865, rs4680, and rs165599) were genotyped using pcr real - time analysis on the lightcycler 480 (roche). lightmix kit (tib - molbiol), which contained specific primers and probes for 96 pcr - rt chain reactions, and lightcycler genotyping 480 dna master of roche diagnostics, which contains tag dna polymerase, reaction buffer, mixture of dntp (with datp, dctp, dgtp, and dutp) and 15 mm mgcl2 were used. briefly 20 l of reaction master mix for comt gene was performed with h2o 10.4 l, reagent mix 1.0 l, lightcycler genotyping 480 dna master 2.0 l, mgcl2 1.6 l, dna 5.0 l (~50 ng). thermal cycling conditions for pcr were as follows : denaturation at 95c for 10 minutes, 1 cycle, followed by 45 cycles of 95c for 10 sec, 60c for 10 sec and 72c for 15 sec, followed by melting at 95c for 30 sec, 40c for 2 min and 75c for 1 sec, 1 cycle and cooling at 40c for 30 sec, 1 cycle. deviations from hardy - weinberg equilibrium (hwe) were assessed using the chi - square test at each snp locus separately in cases and controls. to assess the differences in demographic characteristics, we used chi - square for categorical data and t test for continuous data. statistical inference for single, univariate snp associations in the comt gene was derived from fisher 's exact test. the odds ratios for dominant, recessive, and additive genetic models for each individual snp were calculated. the relevant p values derived from wald test for dominant and recessive models and armitage test for trend for additive model. subsequently, all possible combinations of the three snps were constructed, and thus, differences in haplotype frequencies between schizophrenia patients and control individuals were calculated using chi - square test. a logistic regression analysis was performed to estimate the risk for schizophrenia of each one of the eight haplotypes. snp rs737865 carries the alleles t and c and the possible genotypes of that polymorphism are t / t, t / c, or c / c. snp rs4680 carries the alleles a and g, and its genotypes are a / a, g / a, or g / g. a and g are the two alleles of snp rs165599, and the possible genotypes are a / a, g / a, or g / g (table 2). to further explore the combination of genotype is from which arise the t / a / a and t / g / g haplotypes, multivariate logistic regression analysis was performed estimating the risk of schizophrenia in each combination. the sas statistical package (version 9.1, sas institute inc, cary, nc) was used to analyze the data. polymorphism rs4680 was in accordance with hardy - weinberg equilibrium (hwe), whereas borderline evidence for deviation from hwe was identified in polymorphism rs165599 among controls (x = 5.11 ; p = 0.03) and in rs737865 among cases (x = 4.15 ; p = 0.04). all the three genetic models, recessive, dominant, and additive, in the three individual snps failed to investigate any evidence for a possible genetic contribution of the snps to schizophrenia susceptibility (table 3). further, a haplotype analysis was performed by constructing all the three - site haplotypes that consisted of the alleles of snps rs4680, rs165599, and rs737865 (table 4). the most frequent haplotypes were t / a / a and t / g / g. significant differences in the frequencies were found between patients and controls regarding t / a / a and t / g / g. specifically, cases had higher frequency of t / a / a (p = 0.010) and lower frequency of t / g / g (p = 0.088) as compared to the controls. when a logistic regression analysis was performed, with schizophrenia as outcome, wild type as the baseline category, and the eight haplotypes as explanatory variables, a significant adverse effect of t / a / a was revealed. therefore, compared to the baseline expression of t / g / g, participants with t / a / a were found to be associated with 52% higher risk of schizophrenia (or = 1.52 ; 95% ci : 1.122.08 ; p = 0.008), whereas there was no evidence for association with the psychiatric illness regarding all the other haplotypes. to evaluate further the supporting evidence for the increased risk of t / a / a and for protective effect of t / g / g, a more detailed analysis was performed examining all the possible combinations of genotypes from which the t / a / a and t / g / g haplotypes were derived (tables 5 and 6). such analysis showed that after taking the most frequent expression, t / t - a / a - a / a, as baseline, participants with t / c - a / a - a / a were at increased risk for developing the disease (or = 2.13 ; 95% ci : 1.024.47 ; p = 0.045). similarly, participants with t / t - a / a - g / a were more likely to develop the disease (or = 3.20 ; 95% ci : 1.0210.05 ; p = 0.046) as compared to those with t / t - a / a - a / a. regarding t / g / g, we found a protective effect of t / t - g / g - g / g (or = 0.22 ; 95% ci : 0.090.56 ; p = 0.001) and t / t - g / a - g / g (or = 0.33 ; 95% ci : 0.120.87 ; p = 0.025) both compared to the baseline, most frequent, category t / t - g / a - g / a. in this multivariate model, two combinations, namely, t / t - g / g - g / a and t / c - g / a - g / g, were excluded given their extremely low frequencies. previously, shifman. reported a large study of comt in schizophrenia, including over 700 patients and 4000 controls. the researchers used israeli ashkenazi jewish population, a well - characterized homogeneous population, in order to reduce genetic variance, and they tested the same snps examined in our study, firstly each snp independently and further the combination of the two snps haplotype as well as the three snps haplotype. the study showed that in that specific population the association of the val / met polymorphism (rs4680) with schizophrenia was modest (p = 0.024), whereas the two other polymorphisms rs737865 and rs165599 (located in intron 1 and 3 flanking region, resp.) were highly significantly associated, as was the haplotype of all three markers g - g - g of rs737865, rs4680, and rs165599 (p = 9.6 10). two independent studies, namely the study of chen. and the study of handoko et el. have reported a significant association of comt with schizophrenia. but both of them reported different alleles of the three snps consisting the risk haplotype, than the alleles described by shifman.. specifically, chen. studied the association of comt with schizophrenia in irish families with high density of schizophrenia. they concluded that val allele might carry a small increase in disease susceptibility, but they did not confirm any association of rs737865 and rs165599. in our study none of the three snps were found to be independently associated with schizophrenia. 's hapolype analyses showed that a - g - a haplotype for snps rs737865, rs4680, and rs165599 was preferentially transmitted to the affected subjects. this finding was different from the reported g - g - g haplotype found in ashkenazi jews and different from the risk haplotype of our study. studied the above snps in a sample of australian caucasian families, containing 107 patients with schizophrenia. the haplotype analyses showed that a - a - a combination for snps rs737865, rs4680, and rs165599 was the risk haplotype for the studied population. that haplotype was in accordance with the risk haplotype found in our study in greek population. analytically, in the present study the allele frequency of comt val / met polymorphism was typical to the european population, since in europe the two alleles are very similar in frequency. furthermore, a three - marker haplotype analysis was performed, consisting of combinations of the putative functional snps. in this study, cases have higher frequency of the rs737865/rs4680/rs165599 t - a - a haplotype and lower frequency of t - g - g haplotype. the c allele at rs737865 corresponds to the g allele in the study of shifman., and the t allele, which refers to the a allele, is the wild - type allele of rs737865 polymorphism. the t - g - g haplotype, which consists of the three wild types alleles of polymorphisms rs737865, rs4680, and rs165599, is present at reasonably high frequencies in controls. furthermore, this study shows that compared to the baseline expression t - g - g, participants with t - a - a haplotype were found to be associated with 52% higher risk of schizophrenia (table 4). conclusively, among studies of european subjects, which examined the same snps described in the present study, different populations have been reported to exhibit an association between schizophrenia and the different triple snp haplotypes. as already mentioned, examples of this include the g - g - g haplotype in ashkenazi jews, the a - g - a haplotype in irish families, the a - g - g haplotype which was significantly under transmitted in australian family - affected samples, and the protective haplotype a - a - a in a case - control study of caucasian subjects. the high - risk haplotype in the ashkenazi sample was actually under transmitted to affected individuals in our sample. is the first and largest population - based association study to date to find a significant association between comt haplotype, constructed by the three snps, and schizophrenia. the ashkenazi jewish population may not be directly comparable with other european populations since this haplotype may possibly contribute risk to schizophrenia in a different pathway across multiple ethnic populations. furthermore, the phenotypic complexity of the disorder makes it difficult to achieve a great clinical homogeneity in the sample of patients. the diagnosis of the disease is based upon clinical phenomena, which may not correctly reflect the underlying biology that predisposes to illness and the absence of biological markers limits the homogeneity of diagnosis. one approach that has been suggested is to subdivide schizophrenia into components of symptom complexes and to develop targets of narrower clinical features. furthermore, all possible genotype combinations of the risk haplotype t - a - a were examined. patients with schizophrenia displayed an excess of the tc - aa - aa genotype homozygous for the two mutant alleles and the tt - aa - ga genotype, homozygous for the mutant rs4680 allele and heterozygous for rs165599. similarly, a protective effect of genotypes tt - gg - gg and tt - ga - gg was documented. in addition, bray., showed that shifman 's risk haplotype g - g - g (c - g - g) is associated with low comt mrna expression in prefrontal cortex. it is possible that the opposite haplotype a - a - a (t - a - a), which is the risk haplotype in this study confers high comt mrna expression. this speculation is compatible with the reformulated theory of hypofrontality in schizophrenia [5, 11 ]. the current study shows an association of comt gene and schizophrenia in a greek population. the haplotype analysis indicates that the t - a - a haplotype had high association with schizophrenia. | schizophrenia, a severe psychiatric condition, is characterized by disturbances of cognition, emotion, and social functioning. the disease affects almost 1% of world population. recent studies evaluating the role of catechol - o - methyltransferase enzyme (comt) polymorphisms in the pathogenesis of schizophrenia have resulted in ambiguous findings. the current study examined the association of schizophrenia with three comt polymorphisms, namely, rs737865, rs4680, and rs165599 in a greek population. there was no significant association between schizophrenia and any of the three snps examined. however, haplotype analysis showed that cases have higher frequency of the t - a - a haplotype, and participants with that haplotype were at increased risk for developing schizophrenia (or = 1.52 ; cl : 1.122.08 ; p = 0.008). furthermore, patients with schizophrenia displayed an excess of tc / aa / aa and the tt / aa / ga genotypes. similarly a protective effect of tt / gg / gg and tt / ga / gg was suggested by our results. |
the mdc (malm diet and cancer) study is a population - based, prospective cohort of 28,449 individuals examined between 1991 and 1996 (20). from this cohort, a random sample, examined between november 1991 and february 1994 (n = 6,103), was included in the mdc cardiovascular cohort, with the primary aim of studying the epidemiology of carotid artery disease (21). after exclusion of individuals lacking values from fasting plasma samples and thus missing data on prevalence of diabetes mellitus or fasting values of hdl - c, ldl - c, or gh, the cohort consisted of 4,452 persons. of these, 129 individuals had a history of cad, stroke, or chf and were excluded from further analysis. thus, 4,323 persons, age 46 to 68 years, remained to comprise the primary study cohort. all participants provided written consent, and the ethical committee at lund university, lund, sweden approved the study. at baseline (1991 to 1996), participants underwent a medical history, physical examination, and laboratory assessment. diabetes mellitus was defined as either self - report of a physician diagnosis, use of diabetes medication, or fasting venous whole blood glucose > 6.0 mmol / l (109 mg / dl). levels of hdl - c, total cholesterol, creatinine, glycated hemoglobin (hba1c), and insulin were measured according to standard procedures at the department of clinical chemistry, university hospital of malm. levels of n - terminal pro - brain natriuretic peptide (nt - probnp) were determined using the dimension rxl automated nt - probnp method (siemens diagnostics, nrnberg, germany) (22). all samples of plasma and whole blood were obtained after overnight fasting, and samples were drawn between 7:30 am and 9:00 am. gh levels were measured in stored fasting plasma samples, which were frozen immediately to 80c at the malm diet and cancer study cardiovascular cohort baseline examination. the measurement was made with a high - sensitivity chemiluminescence sandwich immunoassay similar to 1 previously described (sphingotec gmbh, borgsdorf, germany) (19). the analytical assay sensitivity (mean relative light units of 20 determinations of gh free sample + 2 sd) was 2 pg / ml gh. the functional assay sensitivity (0) according to previously described methods (28). we used multivariable risk scores with the parameters mentioned in the preceding text to estimate the risk of developing a cardiovascular event and then examined whether the addition of hs - gh improved the model and reclassified subjects, thus obtaining the nri (> 0). up - classification in the risk of individuals who developed an event or down - classification of individuals who did not develop an event improves the risk model, whereas the opposite deteriorates the model. calculation of c statistics (harrell s) (29) was used to assess model discrimination. a post - hoc analysis was made examining the case fatality of myocardial infarctions versus hs - gh. odds ratios were calculated by means of logistic regression analyses and adjusted for the risk factors included in our original model, with the addition of year of event and age at event replacing age at baseline. odds ratios for gh were expressed per 1-sd increment of the natural logarithm of hs - gh. all analyses were made with stata software version 11 (statacorp, college station, texas). women had significantly higher fasting values of hs - gh than men (table 1). individuals excluded from the study due to missing plasma samples or prevalent cardiovascular disease differed slightly in their baseline characteristics (online table 1). with the exception of age in women and systolic blood pressure and antihypertensive medication in men (online table 2), most variables were significant determinants of hs - gh in the crude regression models. in the adjusted models, age, current smoking, and hdl - c had a significant positive correlation with hs - gh, whereas ldl - c and bmi exhibited significant negative correlation in both sexes (table 2, online tables 3 and 4). prevalence of diabetes mellitus had a positive correlation with hs - gh in males and a negative correlation in females. the multiple regression models had an r value of 9.5% for males and 11.8% for females. median follow - up time ranged from 16.1 years (interquartile range : 15.4 to 16.7 years) in cad to 16.2 years (interquartile range : 15.6 to 16.7 years) in the mortality analyses. all of the cox proportional hazards models were significant when analyzing the whole cohort, with increasing hs - gh levels associated with elevated cardiovascular morbidity and mortality, independent of other cardiovascular risk factors (table 3). hrs for each sd increase of baseline hs - gh ranged from 1.11 in cad to 1.43 in cardiovascular mortality. a total of 4 of 5 male analyses were significant, with hrs ranging from 1.17 in cad to 1.44 in cardiovascular mortality, and the 5th analysis, which was chf, was nonsignificant. in females, a significant sex interaction was found in the analysis of total mortality (p = 0.02). when comparing the quartile with the highest values of hs - gh with the bottom quartile, hrs ranged from 1.33 (95% confidence interval [ci ] : 0.99 to 1.79 ; p = 0.05) in cad to 2.83 (95% ci : 1.74 to 4.61 ; p 0) was significant for the whole cohort in stroke (0.179 ; 95% ci : 0.003 to 0.293), total mortality (0.207 ; 95% ci : 0.020 to 0.383), and cardiovascular mortality (0.542 ; 95% ci : 0.205 to 0.840) (table 4, online table 8). the 2 mortality analyses were significant for males, whereas the female analyses were significant in cardiovascular mortality. improvement mostly stemmed from down - classification of nonevents in the total mortality analysis and up - classification of events in cardiovascular mortality. to evaluate the reclassification model, we tried adding creatinine and nt - probnp to the basic model before calculating the nri (> 0) for hs - gh. this attenuated the nri (> 0) in stroke and total mortality, whereas the association in cardiovascular mortality remained the same (table 4). overall, the effects on the c - statistics when adding gh to the basic model were modest. in analysis of cardiovascular mortality the c - statistics increased from 0.783 (95% ci : 0.752 to 0.814) to 0.794 (95% ci : 0.763 to 0.825) in the whole cohort, 0.751 (95% ci : 0.705 to 0.797) to 0.764 (95% ci : 0.719 to 0.809) in males, and 0.797 (95% ci : 0.751 to 0.844) to 0.807 (95% ci : 0.760 to 0.854) in females (table 5). a post - hoc analysis of the 24-h case fatality of myocardial infarctions versus hs - gh was performed to investigate the strong relationship between hs - gh and cardiovascular mortality. in a multivariate - adjusted logistic regression, the odds ratio per 1-sd increment of fasting hs - gh at baseline was 1.54 (310 events, 69 fatal ; 95% ci : 1.13 to 2.09 ; p = 0.006) in the total cohort, 1.50 (187 events, 37 fatal ; 95% ci : 1.00 to 2.23 ; p = 0.05) among males, and 1.67 (123 events, 32 fatal ; 95% ci : 0.98 to 2.85 ; p = 0.06) among females. we examined whether fasting values of hs - gh predict cardiovascular morbidity and mortality in a large population - based cohort free from cad, chf, and stroke at baseline with a longitudinal follow - up time exceeding 66,000 person - years. increasing fasting hs - gh levels were associated with higher incidence of cad, stroke, chf, all - cause mortality, and cardiovascular mortality, independent of traditional cardiovascular risk factors. adult ghd patients have an adverse cardiovascular risk factor profile (6,7) and were reported to have an increased risk of all - cause and cardiovascular mortality (24). although gh substitution ameliorates some aspects of the cardiovascular risk factor profile (6,810), there are no randomized placebo - controlled trials evaluating this therapy in relation to hard endpoints such as cardiovascular morbidity and mortality. interestingly, at the population level, we found that patients with low hs - gh levels resembled ghd patients in having an unfavorable body composition and lipoprotein profile (online table 9). however, the relationship between hs - gh and cardiovascular morbidity and mortality was the opposite of that expected from the observations in ghd patients : subjects with low hs - gh had low risk, whereas subjects with high hs - gh had high risk. although our study was performed in healthy subjects and does not prove a causal relationship between increasing gh and cardiovascular morbidity and mortality, it does cast doubts on whether gh replacement therapy in adult ghd patients is beneficial for cardiovascular health. most commonly, ghd patients are deficient for a reason, and previous cranial irradiation, surgery, and other causes may affect mortality rates independently from ghd. rather, our results, together with the only previous study of fasting gh in relation to all - cause and cardiovascular mortality in healthy subjects (15), suggest that elevated fasting gh is an independent risk factor for cardiovascular morbidity and mortality, and thus call for randomized placebo - controlled trials of gh replacement therapy to determine its effect on cardiovascular morbidity and mortality. today, gh use is not limited to individuals with ghd. for example, gh has been used as an antiaging therapy, and athletes also use it. a systematic review concluded that gh could not be recommended as antiaging therapy, and our results further highlight the impropriety and possible dangers of this practice (30). the use of gh in sports is classed as doping, and its performance - enhancing capabilities are, in many areas, doubtful (31). physical exercise results in secretion of gh ; however, regular exercise has not been shown to alter the baseline resting levels (32), which may indicate that regular physical activity does not affect the fasting value of gh. as would be expected, studies in rodents showed that mice overexpressing gh genes have a drastically shortened life span (33). what is more surprising is that mice that are either missing the gh receptor (growth hormone receptor knockout, ghrko) or ames mice (deficient in gh, prolactin, and thyroid - stimulating hormone) live longer than normal mice (34,35) while being obese compared with their siblings (36,37). although the relevance of these experimental data for humans is unclear, they suggest a link between reduced gh signaling and a phenotype of obesity and longer life span. this somewhat resembles the situation in humans with low hs - gh who have higher bmis, waist circumference, and body fat, but live longer in comparison with subjects with high hs - gh (central illustration). the outcome with the strongest independent relationship to baseline fasting hs - gh was cardiovascular mortality. in this outcome, adding hs - gh to traditional cardiovascular risk factors resulted in increments of the nri (> 0) of 0.542 in the total cohort, suggesting that addition of hs - gh improved the basic predictive model. even with the addition of creatinine and nt - probnp, the association remained significant. interestingly, the nri (> 0) in cardiovascular mortality was driven to a larger extent by up - classification of subjects with a cardiovascular death than by down - classification of healthy subjects. even though the clinical use of hs - gh for prediction would be associated with many drawbacks, these results still indicate that the predictive possibility is not negligible. in general, we found a stronger relationship between hs - gh in men than in women. our finding that women have in the order of 10-fold higher values of fasting gh than men has been previously described (38). the 24-h profiles of gh secretion in women were shown to be more irregular and to have shorter oscillatory periods and higher trough values than in men (3941). it should be kept in mind that fasting gh values are not a standard clinical test, and it is not known how these values relate to overall gh production. when examining 24-h gh profiles from previous publications, morning values in men appear quite low and stable, whereas the variation in women is larger (1618). it can thus be speculated that the more stable gh levels in the morning hours in men than in women makes a single fasting hs - gh a more accurate measure of gh secretion in men, which, in turn, could explain why associations with various outcomes are stronger in males. part of the sex discrepancy may also be attributable to power differences, as all outcomes occurred at a higher incidence rate in the male population. to date, it is unclear to what extent a fasting value of hs - gh reflects overall gh secretion. the pulsatile mode of secretion is a limitation and makes gh difficult to use in a clinical setting. it is not clear whether our findings represent a linear relationship between gh and the various outcomes, although the quartile analyses were done to evaluate this. unfortunately, splitting the material caused power difficulties and made a clear - cut answer impossible. an effort to analyze the 10th and 90th percentile versus the middle (data not shown) was also hampered by power, but did not indicate a different association. in addition, we did not measure circulating or tissue insulin - like growth factor-1, which mediates several effects of gh. despite these issues, we did observe relationships between hs - gh and gh - related phenotypes at baseline as well as associations with long - term outcomes, especially the risk of cardiovascular mortality, suggesting that fasting hs - gh does reflect a medically relevant measure. the association of higher hs - gh values with increased likelihood of a myocardial infarction being fatal may explain why our strongest findings were in the cardiovascular mortality outcome. because this was an observational study, we do not prove causation between hs - gh and our various endpoints. also, the number of nonaccepters to the initial invitation was quite large, which could have biased selection of a more healthy population. although gh secretion is known to have a strong positive correlation with estradiol levels, we have not excluded pre - menopausal or perimenopausal women from the study (42) ; this may be a potential confounder in the female analyses. however, the consequence of this would be younger women exhibiting higher gh values, which logically would weaken a positive correlation between hs - gh and the various outcomes. differences in baseline characteristics in people excluded due to missing plasma samples compared with the study cohort were minor and did not raise any suspicions of selection bias. to date, it is unclear to what extent a fasting value of hs - gh reflects overall gh secretion. the pulsatile mode of secretion is a limitation and makes gh difficult to use in a clinical setting. it is not clear whether our findings represent a linear relationship between gh and the various outcomes, although the quartile analyses were done to evaluate this. unfortunately, splitting the material caused power difficulties and made a clear - cut answer impossible. an effort to analyze the 10th and 90th percentile versus the middle (data not shown) was also hampered by power, but did not indicate a different association. in addition, we did not measure circulating or tissue insulin - like growth factor-1, which mediates several effects of gh. despite these issues, we did observe relationships between hs - gh and gh - related phenotypes at baseline as well as associations with long - term outcomes, especially the risk of cardiovascular mortality, suggesting that fasting hs - gh does reflect a medically relevant measure. the association of higher hs - gh values with increased likelihood of a myocardial infarction being fatal may explain why our strongest findings were in the cardiovascular mortality outcome. because this was an observational study, we do not prove causation between hs - gh and our various endpoints. also, the number of nonaccepters to the initial invitation was quite large, which could have biased selection of a more healthy population. although gh secretion is known to have a strong positive correlation with estradiol levels, we have not excluded pre - menopausal or perimenopausal women from the study (42) ; this may be a potential confounder in the female analyses. however, the consequence of this would be younger women exhibiting higher gh values, which logically would weaken a positive correlation between hs - gh and the various outcomes. differences in baseline characteristics in people excluded due to missing plasma samples compared with the study cohort were minor and did not raise any suspicions of selection bias. we demonstrate that higher fasting values of hs - gh are associated with cardiovascular morbidity, all - cause mortality, and, in particular, cardiovascular mortality. further research is needed to elucidate the mechanisms of this association in the general population and its potential implications in the subgroups of patients with a disturbance in gh secretion.perspectivescompetency in medical knowledge : in healthy middle - aged people, higher fasting levels of gh are associated with greater cardiovascular morbidity and mortality independent of traditional cardiovascular risk factors.competency in patient care : in healthy middle - aged individuals, measurement of fasting gh in plasma may be useful in risk stratification for cardiovascular mortality and aid clinical decision - making for primary prevention of ischemic events.translational outlook : randomized trials are needed to investigate the effect of gh replacement on cardiovascular outcomes in patients with ghd. competency in medical knowledge : in healthy middle - aged people, higher fasting levels of gh are associated with greater cardiovascular morbidity and mortality independent of traditional cardiovascular risk factors. competency in patient care : in healthy middle - aged individuals, measurement of fasting gh in plasma may be useful in risk stratification for cardiovascular mortality and aid clinical decision - making for primary prevention of ischemic events. translational outlook : randomized trials are needed to investigate the effect of gh replacement on cardiovascular outcomes in patients with ghd. | backgroundboth pathological excess and deficiency of growth hormone (gh) are associated with cardiovascular mortality.objectivesthe goal of this study was to test whether fasting levels of growth hormone measured with a high - sensitivity assay (hs - gh) predict cardiovascular morbidity and mortality at the population level.methodswe studied 4,323 participants (age 46 to 68 years ; mean age 58 years ; 59% women) of the swedish, population - based malm diet and cancer study examined in 1991 to 1994. using multivariate - adjusted cox proportional hazards models, we related baseline levels of fasting hs - gh to incidence of coronary artery disease, stroke, congestive heart failure, all - cause mortality, and cardiovascular mortality.resultsduring a median follow - up of 16.2 years, hs - gh (hazard ratio [hr]/sd increment of natural logarithm of fasting hs - gh) was independently associated with increased risk of coronary artery disease (397 events ; hr : 1.11 ; 95% confidence interval [ci ] : 1.01 to 1.23 ; p = 0.04), stroke (251 events ; hr : 1.18 ; 95% ci : 1.04 to 1.34 ; p = 0.01), congestive heart failure (107 events ; hr : 1.25 ; 95% ci : 1.03 to 1.52 ; p = 0.02), all - cause mortality (645 events ; hr : 1.17 ; 95% ci : 1.08 to 1.26 ; p 0) of 0.542 (95% ci : 0.205 to 0.840) in cardiovascular mortality.conclusionshigher values of hs - gh were associated with an increased risk of cardiovascular morbidity and mortality. |
fat redistribution in hiv - infected patients is associated with antiretroviral therapy (art), including protease inhibitors (pi) and nonnucleoside reverse transcriptase inhibitors (nnrti) [1, 2 ]. central fat accumulation or lipohypertrophy may be more common in women [3, 4 ] and has been associated with multiple metabolic abnormalities and inflammation in hiv - infected persons on art [59 ]. chronic hiv infection is also associated with persistent inflammation, and treating hiv infection improved endothelial function in treatment - nave subjects with low cardiovascular disease (cvd) risk. given the complex interactions between chronic hiv infection and art and the likelihood that traditional framingham prediction may underestimate cardiovascular risk in hiv - infected persons on art [12, 13 ], novel biomarkers are needed to assess metabolic risk in hiv infection and response to interventions. eicosanoids are endogenous products of arachidonic acid metabolism involved in oxidant stress, inflammation, and endothelial function, all of which are important in atherosclerosis and cardiovascular disease pathogenesis. biologic properties and metabolism of eicosanoids are complex and are reviewed elsewhere [1517 ]. briefly, during cellular stress, membrane phospholipids containing arachidonic acid are subjected to nonenzymatic peroxidation by free radical species to generate a variety of biologically active oxidation products, including f2-isoprostanes (f2-isops) which can cause vasoconstriction, platelet aggregation, and oxidative tissue damage. arachidonic acid can also be released from membrane phospholipids and metabolized by oxidizing enzymes during cellular stress. metabolism by cyclooxygenase (cox) enzymes yields a family of products termed prostaglandins (pgs), including pge2, which causes vasodilation or vasoconstriction and/or vascular smooth muscle proliferation ; thromboxane a2 (txa2), which causes vasoconstriction, platelet activation, and chemotaxis ; and prostacyclin (pgi2) which causes vasodilation and inhibits platelet aggregation and vascular smooth muscle proliferation. with the exception of f2-isops, parent eicosanoids are unstable in and prostaglandin metabolites, as well as f2-isops, are stable in urine and accurate indices of endogenous production [1821 ]. the primary pge2 urinary metabolite is 11--hydroxy-9,15-dioxo-2,3,4,5-tetranor - prostane-1,20-dioic acid (pge - m), while the major metabolites used to assess txa2 and pgi2 production are 11-dehydro - thromboxane b2 (txb2) and 2,3-dinor-6-keto - pgf1 (pgi - m), respectively. urinary assays for pge - m and f2-isop have low intra - individual variation over one year. in hiv - negative populations, urinary f2-isop correlates with traditional cvd risk factors and surrogate measures of cvd including brachial artery flow - mediated dilation and carotid intima media thickness [24, 25 ]. in obese children and adolescents, plasma f2-isop was positively correlated with visceral adipose tissue (vat), a marker of cvd risk. cigarette smoking is also associated with higher eicosanoids [2730 ]. in hiv - infected persons, cross - sectional studies to date have identified associations between higher f2-isop and lipoatrophy, lactic acidosis, virologic suppression on art, heavy smoking, higher body mass index (bmi) and waist circumference, elevated liver transaminases and hepatitis c virus (hcv) rna, and female sex [3135 ]. early analyses suggested higher f2-isop in persons receiving efavirenz or zidovudine, and lower levels in those on nevirapine compared with other art but consistent associations with specific art drugs or classes have not been seen. it is not yet clear why f2-isop levels are consistently higher in women (hiv - infected and uninfected) than in men. a recent study of women in haiti found higher levels of cervical cox-2 and urinary pge - m in hiv - infected women than in uninfected women and a positive correlation between systemic pge - m and both plasma hiv rna and cervical cox-2 levels. raltegravir (ral) is an hiv-1 integrase inhibitor that has not been associated with metabolic perturbations or fat redistribution during short- or long - term therapy [3739 ]. a randomized, open label study was designed to assess the effects of switching from pi- or nnrti - based art to a ral - based regimen in women with lipohypertrophy and suppressed hiv-1 rna on stable therapy. adipose tissue volumes by computerized tomography (ct), anthropometrics, and fasting metabolic parameters were performed. the objectives of these secondary analyses were to determine (a) the 24-week change in f2-isop and other urinary eicosanoid metabolites and (b) correlations between 24-week changes in f2-isop, other urinary eicosanoid metabolites, and changes in vat, the primary outcome of the parent study. we hypothesized that urinary f2-isop and other eicosanoid metabolites would decrease after 24 weeks in women switching to ral compared to those continuing a pi / nnrti and that these decreases would correlate with decreased vat. briefly, women with hiv-1 rna 94 cm or waist : hip > 0.88) were enrolled at five centers in north america from september 2008 to july 2010 and randomized 1 : 1 to switch their pi / nnrti to open label ral 400 mg twice daily (ral arm) or continue to present art for 24 weeks (pi / nnrti arm). relevant exclusion criteria included current use of metformin, thiazolidinediones, or androgen therapy, use of growth hormone or growth hormone - releasing factor in the six months prior to screening, change or initiation of lipid - lowering therapy in the three months prior to screening, and intent to significantly modify diet or exercise habits during the study. the primary endpoint of the parent trial was between - group change in percent of vat volume 24 weeks following a switch to ral versus continued pi or nnrti. all study procedures were approved by the institutional review boards of the participating institutions, and all subjects provided informed consent prior to initiation of study procedures. procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and with the helsinki declaration of the world medical association. scans were performed locally but standardized and read centrally by a blinded reader at the tufts university body composition center. waist, hip, and neck circumferences were performed according to aids clinical trials group standards at weeks 0, 12, and 24. fasting (> 8 hours) glucose, lipoprotein profile, high - sensitivity c reactive protein (hscrp), and cd4 + t cell counts were assessed at weeks 0, 12, and 24. hiv-1 rna (50 copies / ml assay sensitivity) was measured at screening and weeks 4, 8, 12, and 24. labs were performed at the individual sites in real - time and according to local standards. clean - catch urine samples were collected, and three - milliliter (ml) aliquots of urine were stored at 80c until analysis. samples were shipped overnight on dry ice to the vanderbilt university eicosanoid core laboratory where analyses were performed. urinary f2-isop, txb2, and pgi - m were measured using gas chromatography - negative ion chemical ionization mass spectrometry employing stable isotope dilution methodology, as described elsewhere [15, 18, 20, 42 ]. pge - m was measured by liquid chromatography - mass spectroscopy (lc - ms), as previously described. results for all urinary metabolites are presented as ng / mg urinary creatinine (cr). at one study site, urinary eicosanoid results from these samples were not statistically different from the other sites, and analyses with and without data from these samples were performed (data not shown). as there were no substantive changes in the results, we report results including data from all sites. baseline characteristics of the two randomization groups were compared using the mann - whitney u test for continuous variables and fisher 's exact test for categorical variables. median values and interquartile ranges (iqr) are reported for continuous variables, and percentages are reported for categorical data. comparison of median between - group 24-week change scores for eicosanoids was performed using the wilcoxon signed - rank test. the primary analysis was as - treated, excluding subjects who did not remain on the study regimen and/or did not have an observed primary endpoint. generalized linear models assessed associations between eicosanoid changes and study arm, adjusting for baseline pi use, bmi, smoking status, study site, and age (50 versus < 50 years). all statistical tests were two - sided with a nominal p level of 0.05. given the exploratory nature of these analyses, we did not adjust results for multiple testing. data analysis and management was performed using sas 9.2 (sas institute, inc., cary, nc, usa). eighteen subjects were randomized to the ral arm and 21 to continue pi / nnrti. one subject from each arm withdrew for reasons unrelated to the study intervention, leaving 37 subjects who completed the week 24 primary endpoint. complete demographic and baseline clinical characteristics of the 37 participants included in the as - treated analysis are provided in table 1. at baseline, the study groups were well balanced, with the exception of the pi / nnrti arm having a higher rate of current smoking (60% versus 24% ; p = 0.045). the median age was 43 years, bmi 32 kg / m, and 75% of subjects self - identified as black or hispanic. sixty - two percent of subjects were on a pi at entry (versus 38% nnrti), and the most commonly prescribed nrti was tenofovir (78%). baseline median (iqr) urinary f2-isop, pge - m, pgi - m, and txb2 (ng / mg cr) were 2.14 (1.493.16), 8.07 (4.4710.56), 0.10 (0.060.15), and 0.46 (0.250.73), respectively (table 1). when comparing study arms, baselines pgi - m, pge - m, and txb2 were all lower, and f2-isop was higher in the ral arm (table 1), but only pgi - m was statistically different (p = 0.005). baseline pge - m tended to be higher in current smokers (p = 0.1 ; data not shown). eicosanoid levels also tended to differ by baseline nrti, with women receiving abacavir having consistently lower levels (figure 1), including statistically significantly lower f2-isop (p = 0.05 ; figure 1(a)) and txb2 (p = 0.04 ; figure 1(d)) levels. pge - m was positively correlated with age (rho = 0.34 ; p = 0.04) and negatively correlated with body weight (rho = 0.35 ; p = 0.03). txb2 was positively correlated with vat : sat and vat : total adipose tissue and negatively correlated with sat, body weight, bmi, and hip and neck circumferences (rho = 0.37 to 0.46 ; p = 0.004 to 0.02). none of the urinary eicosanoids were correlated with fasting lipids, glucose, insulin resistance, or hscrp in this study population at baseline (data not shown). median 24-week urinary eicosanoid levels and changes from baseline are shown in figure 2 and table 2. over 24 weeks, only pgi - m in ral - treated subjects demonstrated a statistically significant within - group change (p = 0.04 ; figure 2(c)). txb2 increased in the ral arm and decreased in the pi / nnrti arm (+ 0.09 [0.04, + 0.13 ] versus 0.02 [0.20, + 0.03 ] ; figure 2(d)), but this difference was of borderline statistical significance (between - group p = 0.06). there were no other statistically significant differences between or within study arms over 24 weeks. age 50 years at baseline was associated with an increase in pge - m (median change + 3.9 versus 1.3 in subjects < 50 years of age ; p = 0.05 ; figure 3). in the pi / nnrti arm, 24-week vat change positively correlated with changes in pgi - m (rho = 0.45 ; p = 0.04) and txb2 (rho = 0.44 ; p = 0.05), with a similar trend seen for pge - m (rho = 0.41 ; p = 0.07). among persons in the ral arm, the change in pge - m correlated with an increase in hdl cholesterol (rho = 0.56 ; p = 0.02) ; this correlation was not observed in the pi / nnrti arm (rho = 0.17 ; p = 0.48). changes in eicosanoid levels over 24 weeks were not statistically different by baseline nrti (abacavir versus tenofovir ; data not shown). changes in urinary eicosanoids from baseline to 24 weeks were assessed in multivariate models adjusting for study arm (ral versus pi / nnrti), baseline bmi, age, pi use, smoking status, and study site. n = 8) was associated with 24-week pge - m increase (= 8.3 [95% ci 0.3, 16.3 ] ; p = 0.04), independent of the covariates above (table 3). no baseline factors were significantly associated with changes in other urinary eicosanoids (table 3). in these hiv - infected women with central adiposity and suppressed hiv rna on art, switching pi- or nnrti - based art to ral did not have significant effects on urinary eicosanoids over 24 weeks. overall, f2-isop, pge - m, and txb2 levels were higher and pgi - m levels were lower than published levels reported in healthy adults [15, 1820, 43, 44 ]. although formal comparisons were not performed, urinary f2-isop (lower), pge - m (higher), and txb2 (higher) levels also differed from those observed in previously studied hiv - infected women who were younger and had lower bmi. of note, several of the markers differed though not with statistical significance at baseline between the two study arms. given known effects of smoking on these biomarkers [2729 ], this difference may have been driven in part by the significantly greater number of smokers randomized to the pi / nnrti arm, and this may therefore have limited our capacity to identify differences in changes over time or due to ral switch. although baseline pge - m tended to be higher in current smokers (n = 16 ; median [iqr ] 9.9 [4.914.4 ]) than nonsmokers (n = 21 ; median [iqr ] 7.9 [4.0 - 10.0 ] ; p = 0.11), in a multivariate model, neither current smoking nor study arm was significantly associated with 24-week change in pge - m and did not attenuate the relationship between age and change in pge - m. additionally, f2-isop, which is increased in hiv - infected and uninfected smokers, tended to be higher in smokers at baseline (p = 0.06) but was not significantly higher in the pi / nnrti arm. age and bmi were lower and higher in the ral than the pi / nnrti arm, respectively (table 1), and though they were not statistically different, we did include these as covariates in adjusted models. in primary analyses, women in the switch arm had significant improvements in total and ldl cholesterol but did not have a statistically significant improvement in vat compared to women continuing an nnrti or pi. they also had a significant decrease in soluble cd14, a marker of monocyte activation. a recent analysis of extensively treatment experienced, predominantly male, subjects switching enfuvirtide to ral in france reported significant 24-week decreases in interleukin-6, d - dimer, and hscrp that were not observed in these less treatment - experienced women with central adiposity. in subjects remaining on pi / nnrti, increasing vat was marginally correlated with increasing pgi - m and pge - m over 24 weeks of follow - up, suggesting a relationship between these markers and central adiposity that was altered by a switch to ral. older age (50 years) at enrollment was associated with an increase in pge - m independent of study arm, smoking status, pi use at baseline, or other factors. this was unexpected given associations with f2-isop and txb2 in prior cross - sectional studies of hiv - infected persons [33, 34 ], and this may be due to the inclusion of both males and females in prior analyses and/or the high prevalence of obesity and relative lack of normal bmi ranges in this study population. this is the first study to prospectively assess the effects of an art switch on urinary eicosanoid metabolites. the small sample size of our study and the imbalance of smokers in the pi / nnrti arm likely limited our ability to detect differences between study arms. nonetheless, intriguing trends and preliminary associations were noted. although not routinely measured in clinical practice, eicosanoids are known markers of cardiovascular and metabolic disease risk in hiv - negative populations. in particular, f2-isop has been associated with cvd disease risk factors, hscrp, carotid intima medial thickness, coronary artery calcium, and angiographic coronary artery obstruction [23, 24, 47 ]. pge2 is a complex mediator of inflammation and vasodilation with variable effects on vascular tone depending on the tissue and prostanoid receptor. increased pge - m has also been associated with malignancies in hiv - negative populations [5053 ], and urinary pge - m correlated with plasma hiv rna and cervical cox-2 levels in a recent study of haitian women. abacavir exposure has been associated with increased cardiovascular risk in hiv - infected persons, but the association has not been consistent. although potential mechanism(s) are not clearly defined, recent studies have reported abnormal platelet reactivity with abacavir [56, 57 ]. to our knowledge, lower f2-isop in these abacavir - treated subjects and in a prior cross - sectional analysis of men and women suggests that if there is excess cardiovascular risk due to abacavir, it is independent of lipid peroxidation - related pathways ; prospective studies would be needed to determine this. due to providers ' knowledge of potential cardiovascular risk with abacavir and risk of renal toxicity with tenofovir df, it is also possible that abacavir use is simply a marker of some other unmeasured factors associated with risk for these conditions and lower urinary eicosanoid levels. in addition to the qualifications above, this analysis has other limitations that should be noted. use of aspirin or nonsteroidal anti - inflammatory drugs (nsaid) was not an exclusion criterion for the parent study, and detailed information on dosing was not collected. given the even distribution of nsaid use across study arms (table 1), we did not adjust for this variable in multivariate models and do not believe it would explain differential associations within or between groups. neither menopausal status nor sex hormone levels were ascertained as part of this study. based on data from hiv - infected women, the age distribution of our population suggests the majority of subjects were pre- or perimenopausal. the association between age and pge - m may have been due to postmenopausal changes in the older women (50 years). platelet reactivity assays were not performed, so relationships between eicosanoids and platelet function can not be determined. ct scan was used to assess sat, so we were unable to fully assess peripheral (limb) lipoatrophy. finally, this analysis may have been too small and/or of insufficient duration of follow - up to detect meaningful changes in eicosanoids. additional studies and longer - term follow - up in hiv - infected persons are needed to further elucidate the role of eicosanoids in metabolic and other aging - related comorbidities and determine their role as useful clinical biomarkers. | chronic inflammation is a hallmark of hiv infection. eicosanoids reflect inflammation, oxidant stress, and vascular health and vary by sex and metabolic parameters. raltegravir (ral) is an hiv-1 integrase inhibitor that may have limited metabolic effects. we assessed urinary f2-isoprostanes (f2-isops), prostaglandin e2 (pge - m), prostacyclin (pgi - m), and thromboxane b2 (txb2) in hiv - infected women switching to ral - containing antiretroviral therapy (art). thirty - seven women (ral = 17 ; pi / nnrti = 20) with a median age of 43 years and bmi 32 kg / m2 completed week 24. txb2 increased in the ral versus pi / nnrti arm (+ 0.09 versus 0.02 ; p = 0.06). baseline pgi - m was lower in the ral arm (p = 0.005) ; no other between - arm cross - sectional differences were observed. in the pi / nnrti arm, 24-week visceral adipose tissue change correlated with pgi - m (rho = 0.45 ; p = 0.04) and txb2 (rho = 0.44 ; p = 0.005) changes, with a trend seen for pge - m (rho = 0.41 ; p = 0.07). in an adjusted model, age 50 years (n = 8) was associated with increased pge - m (p = 0.04). in this randomized trial, a switch to ral did not significantly affect urinary eicosanoids over 24 weeks. in women continuing pi / nnrti, increased visceral adipose tissue correlated with increased pgi - m and pge - m. older age (50) was associated with increased pge - m. relationships between aging, adiposity, art, and eicosanoids during hiv - infection require further study. |
tenofovir disoproxil fumarate (tdf), the oral prodrug of tenofovir, is an acyclic nucleotide phosphonate, which is frequently used as a first - line therapy in hiv - infected patients. it can be conveniently dosed once a day and has a favorable side effect profile, making it one of the most commonly prescribed drugs in this patient population. the excretion of tdf is primarily through the kidneys. while most of this renal excretion is through filtration, 2030% of tdf is actively transported in the proximal tubule through organic anion transporter 1 (oat-1). initial studies showed tdf to be a safe drug in hiv - infected patients, with a post - marketing survey of 10 343 patients demonstrating renal side effects in only 0.5% of patients. however, after fda approval, case reports of proximal tubular dysfunction and acute kidney injury emerged [5, 6 ]. multiple observational cohorts have demonstrated the association of tdf use with a decline in estimated glomerular filtration rate (egfr) and development of chronic kidney disease (ckd) [710 ]. moreover, tdf use is associated with fanconi syndrome characterized by proximal tubular dysfunction with urinary wasting of phosphate, glucose, amino acids and bicarbonate. apoptosis of tubular cells and inhibition of mitochondrial dna replication in proximal tubular cells may be involved in the pathogenesis of tdf - induced nephropathy [12, 13 ]. risk factors for development of tdf - associated nephrotoxicity include polymorphism in genes encoding proximal tubular transporters [14, 15 ], concomitant use of protease inhibitors [1618 ], preexisting kidney disease, low body mass index, older age, advanced hiv infection, concomitant hepatitis c virus (hcv) infection and concurrent use of other nephrotoxic drugs [4, 18, 20 ]. despite the widely recognized association of tdf with proximal tubular dysfunction, the clinical course of patients with tdf - induced proximal tubular dysfunction after discontinuation of tdf remains unclear. in this study, we describe the characteristics of patients with overt proximal tubular toxicity, with documented urinary phosphate wasting attributed to tdf and their course after the discontinuation of this drug. we conducted a retrospective observational study of hiv - infected adults evaluated at the johns hopkins nephrology clinic with documented phosphate wasting in the context of tdf use. we identified 63 patients with presumed tdf toxicity that had discontinued tdf or had adjustment in the tdf dose. only 15 patients had measurements of urinary phosphate prior to drug discontinuation and were included. patients who did not have a fractional excretion of phosphate (fephos) at the time of presumed tdf toxicity were excluded. other potential causes of proximal tubule dysfunction, including multiple myeloma, amyloidosis, aminoglycoside exposure or other drug exposures were excluded in all cases demographic, clinical, laboratory and pharmacologic data were abstracted from electronic medical records at the time of the patients ' initial evaluation in the nephrology clinic. patients were classified as having diabetes mellitus or hypertension if they had a chart diagnosis of either or were on medications for the treatment of these conditions, respectively. diagnosis of hepatitis b or hcv infection was based on the presence of antibodies or detectable viral levels by pcr. fractional excretion of phosphate was calculated by the following equation : urinaryphosphate(up)plasmacreatinine(pcr)plasmaphosphate(pp)urinarycreatinine(ucr)100 phosphate wasting was defined as a fephos of > 20% among patients with normal serum phosphate levels (2.74.5 mg / dl) or > 10% among patients with hypophosphatemia (serum phosphates of 5 ml / min/1.73 m per year. we conducted a retrospective observational study of hiv - infected adults evaluated at the johns hopkins nephrology clinic with documented phosphate wasting in the context of tdf use. we identified 63 patients with presumed tdf toxicity that had discontinued tdf or had adjustment in the tdf dose. only 15 patients had measurements of urinary phosphate prior to drug discontinuation and were included. patients who did not have a fractional excretion of phosphate (fephos) at the time of presumed tdf toxicity were excluded. other potential causes of proximal tubule dysfunction, including multiple myeloma, amyloidosis, aminoglycoside exposure or other drug exposures were excluded in all cases demographic, clinical, laboratory and pharmacologic data were abstracted from electronic medical records at the time of the patients ' initial evaluation in the nephrology clinic. patients were classified as having diabetes mellitus or hypertension if they had a chart diagnosis of either or were on medications for the treatment of these conditions, respectively. diagnosis of hepatitis b or hcv infection was based on the presence of antibodies or detectable viral levels by pcr. fractional excretion of phosphate was calculated by the following equation : urinaryphosphate(up)plasmacreatinine(pcr)plasmaphosphate(pp)urinarycreatinine(ucr)100 phosphate wasting was defined as a fephos of > 20% among patients with normal serum phosphate levels (2.74.5 mg / dl) or > 10% among patients with hypophosphatemia (serum phosphates of 5 ml / min/1.73 m per year. at the time of diagnosis of tdf toxicity, the mean age of the cohort was 56 years (range 3876) with an sd of 11.3 years as shown in table 1. the mean duration of tdf therapy prior to diagnosis of tdf toxicity was 64 months (sd 21.3, range 28103). the mean serum phosphate concentration was 2.3 mg / dl (range 1.03.6 mg / dl), with 11 (73%) patients experiencing hypophosphatemia. ten patients had vitamin d level checked, and all of them had a 25-oh vitamin d level of > 15 ng / ml (mean 25-oh vitamin d level 36 ng / ml). table 1.baseline characteristics of 15 patients with tdf - induced proximal tubulopathy at the time of diagnosischaracteristicsvaluesmean age, years (range)56 (3876)male gender, n (%) 12 (80)body mass index, kg / m, mean (sd)23.2 (4.0)caucasian, n (%) 11 (73)hypertension, n (%) 9 (60)diabetes mellitus, n (%) 6 (40)hcv infection, n (%) 3 (20)current smoker, n (%) 10 (67)duration of tdf use, months mean (range)64 (28103)baseline serum creatinine, mg / dl mean (sd)(prior to tdf initiation)0.8 (0.2)baseline egfr, ml / min/1.73 m mean (sd) (prior to tdf initiation)104 (17.0)serum creatinine, mg / dl mean (sd) (at the time of tdf discontinuation)1.22 (0.3)egfr, ml / min/1.73 m mean (sd) (at the time of tdf discontinuation)69 (19.0)change in egfr from baseline/1.73 m, mean (sd)35 (18.4)serum phosphate, mg / dl mean (sd)2.3 (0.7)serum potassium, mg / dl mean (sd)3.9 (0.6)serum bicarbonate, mg / dl mean (sd)24.5 (3.2)fephos, %, mean (range)34 (2062)tmp / gfr, mean (range)1.6 (0.62.8)glycosuria, n (%) 7 (46)egfr, estimated gfr ; fephos, fractional excretion of phosphate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate. baseline characteristics of 15 patients with tdf - induced proximal tubulopathy at the time of diagnosis egfr, estimated gfr ; fephos, fractional excretion of phosphate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate. despite evidence of phosphate wasting in all the patients, only 53% had concurrent glycosuria. seven (47%) of the 15 patients had > 500 mg / g of protein on a spot urine collection. eleven (73%) patients had an egfr of > 90 ml / min/1.73 m, and the other four had a gfr of 8090 ml / min/1.73 m, with a mean serum creatinine of 0.8 mg / dl (range 0.51.0, sd 0.16). individual laboratory values of the 15 patients are shown in table 2. in assessing other features of proximal tubule dysfunction meq / l, with no bicarbonate 3.0 only one patient (# 2) had a normal tmp / gfr in the setting of a high fephos. he had concurrent glucosuria and a significant drop in his gfr on tdf therapy suggesting renal tubular dysfunction. table 2.individual characteristics of 15 patients with proximal tubular dysfunction at the time of diagnosis of tdf - induced proximal tubulopathybaseline serum creatinine (mg / dl)baseline gfr (ml / min/1.73 mmonths of tdf therapycreatinine at tdf stop (mg / dl)gfr at tdf stop (ml / min/1.73 m)change in gfr (ml / min/1.73 m)serum phosphate (mg / dl)fractional excretion of phosphatetmp / gfr (mg / dl)urine glucoseserum potassium (meq / l)serum bicarbonate (meq / l)10.897511.358392.4201.93.12020.71261031.169573.6212.8 + 3.82831.086541.374122.0211.65.02840.8121481.275462.9242.2 + 4.12351.083591.264192.4271.83.33060.8106721.552542.0301.44.22571.088701.552362.0301.44.22580.6122941.270523.1302.24.02690.8103621.077263.1332.1 + 4.428100.6115531.083322.2331.5 + 4.323110.5136561.169671.0420.6 + 3.821120.8100532.130702.2441.13.721130.9111281.185261.0450.6 + 3.322141.081501.263182.6481.44.527151.0921030.5116242.0620.8 + 3.121gfr, estimated glomerular filtration rate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate.baseline values are those recorded at the time of tdf initiation.patient had an improvement in his gfr after starting of tdf. individual characteristics of 15 patients with proximal tubular dysfunction at the time of diagnosis of tdf - induced proximal tubulopathy gfr, estimated glomerular filtration rate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate. the mean egfr for the 15 patients included at the start of tdf therapy was 104 ml / min/1.73 m (sd 17.0, range 81136) and decreased to a mean of 69 ml / min/1.73 m (sd 19.0, range 30116) with a mean egfr decline of 35 ml / min/1.73 m by the time of tdf discontinuation. only 12 of the 15 patients had multiple egfr measurements before and after discontinuation of tdf at 6-month intervals. the trend for their gfr based on their point egfr at 6-month intervals and the changes in their egfr at 6-month interval after discontinuation of tdf are shown in figure 1a. if the patient did not have an egfr measurement at the exact 6-month interval, we used the one which was closest to the 6-month mark within a 2-month interval. figure 1b depicts the mean egfr of patients in relation to discontinuation of tdf and highlights the slow decline in egfr of patients prior to tdf discontinuation. one patient (patient 15) in our group had apparent improvement in egfr after initiation of tdf. however, this may have been due to a decline in this patient 's serum creatinine as a result of poor nutrition and muscle atrophy prior to the patient 's death, which occurred within a month of tdf discontinuation, rather than an actual improvement in renal function ; his weight decreased from 76 to 62 kg in the 4 months prior to discontinuation of the tdf. 1.(a) trends of egfr values 48 months prior or 24 months after discontinuation of tdf in 12 patients. (a) trends of egfr values 48 months prior or 24 months after discontinuation of tdf in 12 patients. concomitant antiretroviral use included 11 patients on ritonavir boosted protease inhibitors (5 atazanavir), 8 patients on efavirenz and 4 on raltegravir. all patients discontinued tenofovir. new regimens included addition of the protease inhibitor darunavir with ritonavir in 2 patients (in addition to the 11 already on a boosted protease inhibitor), raltegravir in 6 and abacavir in 5. of these, six had normalization of their fephos between 8 and 60 weeks after tdf discontinuation. three others had a decrease in their fephos by the time of their last measurement between 8 and 52 weeks after drug discontinuation. all three of these had been hypophosphatemic at the time of drug discontinuation and had normalized their serum phosphate levels. one patient had an increase in fephos measured 2 months after tdf discontinuation. at that time the serum phosphate had increased from 2.0 to 2.3 mg / dl (patient 3) ; no further assessments were performed. of the eleven (73%) hypophosphatemic patients, all patients, except one, had normalization of serum phosphate levels after discontinuation of tdf. the one patient (# 3) without normalization of his serum phosphate level had an increase from 2.0 to 2.3 after 2 months without further follow - up. of the four patients who had a normal serum phosphate at the time diagnosis, three patients had an increase in their serum phosphate after discontinuation of tdf. of note, in at least three hypophosphatemic patients it took > 6 months before their serum phosphate normalized. all patients with urine glucose had resolution of glycosuria. one patient (# 10) had persistent glycosuria a year after stopping the tdf. though the fephos was not reassessed, the serum phosphate had normalized in this patient and has been in the normal range for over 2.5 years since stopping tdf. twelve (86%) of 14 with loss of kidney function on tdf had an increase in egfr after discontinuation of tdf. the egfr started to increase in these patients within 6 months and continued to improve beyond 6 months before stabilizing (figure 1a, b). one patient, who had an improvement in egfr after discontinuation of tdf, later had a decline in his egfr after 2 years as a result of biopsy - proven diabetic nephropathy. two of the 14 patients continued to experience a decline in egfr despite discontinuation of tdf. one had an underlying ckd with ongoing illicit drug use and history of non - adherence. the other had only 6 months of follow - up, and this failure to recover egfr might be explained by a residual effect from tdf use. at the time of diagnosis of tdf toxicity, the mean age of the cohort was 56 years (range 3876) with an sd of 11.3 years as shown in table 1. the mean duration of tdf therapy prior to diagnosis of tdf toxicity was 64 months (sd 21.3, range 28103). the mean serum phosphate concentration was 2.3 mg / dl (range 1.03.6 mg / dl), with 11 (73%) patients experiencing hypophosphatemia. ten patients had vitamin d level checked, and all of them had a 25-oh vitamin d level of > 15 ng / ml (mean 25-oh vitamin d level 36 ng / ml). table 1.baseline characteristics of 15 patients with tdf - induced proximal tubulopathy at the time of diagnosischaracteristicsvaluesmean age, years (range)56 (3876)male gender, n (%) 12 (80)body mass index, kg / m, mean (sd)23.2 (4.0)caucasian, n (%) 11 (73)hypertension, n (%) 9 (60)diabetes mellitus, n (%) 6 (40)hcv infection, n (%) 3 (20)current smoker, n (%) 10 (67)duration of tdf use, months mean (range)64 (28103)baseline serum creatinine, mg / dl mean (sd)(prior to tdf initiation)0.8 (0.2)baseline egfr, ml / min/1.73 m mean (sd) (prior to tdf initiation)104 (17.0)serum creatinine, mg / dl mean (sd) (at the time of tdf discontinuation)1.22 (0.3)egfr, ml / min/1.73 m mean (sd) (at the time of tdf discontinuation)69 (19.0)change in egfr from baseline/1.73 m, mean (sd)35 (18.4)serum phosphate, mg / dl mean (sd)2.3 (0.7)serum potassium, mg / dl mean (sd)3.9 (0.6)serum bicarbonate, mg / dl mean (sd)24.5 (3.2)fephos, %, mean (range)34 (2062)tmp / gfr, mean (range)1.6 (0.62.8)glycosuria, n (%) 7 (46)egfr, estimated gfr ; fephos, fractional excretion of phosphate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate. baseline characteristics of 15 patients with tdf - induced proximal tubulopathy at the time of diagnosis egfr, estimated gfr ; fephos, fractional excretion of phosphate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate. despite evidence of phosphate wasting in all the patients, only 53% had concurrent glycosuria. seven (47%) of the 15 patients had > 500 mg / g of protein on a spot urine collection. eleven (73%) patients had an egfr of > 90 ml / min/1.73 m, and the other four had a gfr of 8090 ml / min/1.73 m, with a mean serum creatinine of 0.8 mg / dl (range 0.51.0, sd 0.16). individual laboratory values of the 15 patients are shown in table 2. in assessing other features of proximal tubule dysfunction meq / l, with no bicarbonate 3.0 only one patient (# 2) had a normal tmp / gfr in the setting of a high fephos. he had concurrent glucosuria and a significant drop in his gfr on tdf therapy suggesting renal tubular dysfunction. table 2.individual characteristics of 15 patients with proximal tubular dysfunction at the time of diagnosis of tdf - induced proximal tubulopathybaseline serum creatinine (mg / dl)baseline gfr (ml / min/1.73 mmonths of tdf therapycreatinine at tdf stop (mg / dl)gfr at tdf stop (ml / min/1.73 m)change in gfr (ml / min/1.73 m)serum phosphate (mg / dl)fractional excretion of phosphatetmp / gfr (mg / dl)urine glucoseserum potassium (meq / l)serum bicarbonate (meq / l)10.897511.358392.4201.93.12020.71261031.169573.6212.8 + 3.82831.086541.374122.0211.65.02840.8121481.275462.9242.2 + 4.12351.083591.264192.4271.83.33060.8106721.552542.0301.44.22571.088701.552362.0301.44.22580.6122941.270523.1302.24.02690.8103621.077263.1332.1 + 4.428100.6115531.083322.2331.5 + 4.323110.5136561.169671.0420.6 + 3.821120.8100532.130702.2441.13.721130.9111281.185261.0450.6 + 3.322141.081501.263182.6481.44.527151.0921030.5116242.0620.8 + 3.121gfr, estimated glomerular filtration rate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate.baseline values are those recorded at the time of tdf initiation.patient had an improvement in his gfr after starting of tdf. individual characteristics of 15 patients with proximal tubular dysfunction at the time of diagnosis of tdf - induced proximal tubulopathy gfr, estimated glomerular filtration rate ; tdf, tenofovir disoproxil fumarate ; tmp, tubular maximal reabsorption of phosphate. the mean egfr for the 15 patients included at the start of tdf therapy was 104 ml / min/1.73 m (sd 17.0, range 81136) and decreased to a mean of 69 ml / min/1.73 m (sd 19.0, range 30116) with a mean egfr decline of 35 ml / min/1.73 m by the time of tdf discontinuation. only 12 of the 15 patients had multiple egfr measurements before and after discontinuation of tdf at 6-month intervals. the trend for their gfr based on their point egfr at 6-month intervals and the changes in their egfr at 6-month interval after discontinuation of tdf are shown in figure 1a. if the patient did not have an egfr measurement at the exact 6-month interval, we used the one which was closest to the 6-month mark within a 2-month interval. figure 1b depicts the mean egfr of patients in relation to discontinuation of tdf and highlights the slow decline in egfr of patients prior to tdf discontinuation. one patient (patient 15) in our group had apparent improvement in egfr after initiation of tdf. however, this may have been due to a decline in this patient 's serum creatinine as a result of poor nutrition and muscle atrophy prior to the patient 's death, which occurred within a month of tdf discontinuation, rather than an actual improvement in renal function ; his weight decreased from 76 to 62 kg in the 4 months prior to discontinuation of the tdf. 1.(a) trends of egfr values 48 months prior or 24 months after discontinuation of tdf in 12 patients. (a) trends of egfr values 48 months prior or 24 months after discontinuation of tdf in 12 patients. concomitant antiretroviral use included 11 patients on ritonavir boosted protease inhibitors (5 atazanavir), 8 patients on efavirenz and 4 on raltegravir. all patients discontinued tenofovir. new regimens included addition of the protease inhibitor darunavir with ritonavir in 2 patients (in addition to the 11 already on a boosted protease inhibitor), raltegravir in 6 and abacavir in 5. of these, six had normalization of their fephos between 8 and 60 weeks after tdf discontinuation. three others had a decrease in their fephos by the time of their last measurement between 8 and 52 weeks after drug discontinuation. all three of these had been hypophosphatemic at the time of drug discontinuation and had normalized their serum phosphate levels. one patient had an increase in fephos measured 2 months after tdf discontinuation. at that time the serum phosphate had increased from 2.0 to 2.3 mg / dl (patient 3) ; no further assessments were performed. of the eleven (73%) hypophosphatemic patients, all patients, except one, had normalization of serum phosphate levels after discontinuation of tdf. the one patient (# 3) without normalization of his serum phosphate level had an increase from 2.0 to 2.3 after 2 months without further follow - up. of the four patients who had a normal serum phosphate at the time diagnosis, three patients had an increase in their serum phosphate after discontinuation of tdf. of note, in at least three hypophosphatemic patients it took > 6 months before their serum phosphate normalized. all patients with urine glucose one patient (# 10) had persistent glycosuria a year after stopping the tdf. though the fephos was not reassessed, the serum phosphate had normalized in this patient and has been in the normal range for over 2.5 years since stopping tdf. twelve (86%) of 14 with loss of kidney function on tdf had an increase in egfr after discontinuation of tdf. the egfr started to increase in these patients within 6 months and continued to improve beyond 6 months before stabilizing (figure 1a, b). one patient, who had an improvement in egfr after discontinuation of tdf, later had a decline in his egfr after 2 years as a result of biopsy - proven diabetic nephropathy. two of the 14 patients continued to experience a decline in egfr despite discontinuation of tdf. one had an underlying ckd with ongoing illicit drug use and history of non - adherence. the other had only 6 months of follow - up, and this failure to recover egfr might be explained by a residual effect from tdf use. the potential of tdf to cause nephrotoxicity reflected by increased serum creatinine is well described in the literature [28, 29 ]. furthermore, scherzer. in a large cohort study demonstrated that each year of tdf exposure was associated with a 33% increased risk ckd. however, serum creatinine is a late marker of kidney dysfunction, and urinary phosphate wasting reflective of proximal tubular dysfunction may be a more sensitive marker for tdf - induced kidney injury. the frequency of tubulopathy reported in observational and clinical cohorts may be underestimated since most trials did not perform specific detailed analysis for the diagnosis of phosphate wasting [3133 ]. the current study demonstrates that in a series of patients with tenofovir nephrotoxicity as reflected by urine phosphate wasting the diagnosis is often late in the treatment course (average of 64 months after tdf initiation) and occurs in those who have normal kidney function at baseline. moreover, it is usually associated with hypophosphatemia though not infrequently the serum phosphate can be normal with renal phosphate wasting. this is generally not associated with other components of fanconi syndrome such as hypokalemia and metabolic acidosis. glycosuria was present in only 47 percent of patients and therefore appears to be a less sensitive marker than both serum phosphate and urine phosphate wasting. though declining egfr (increasing serum creatinine) was occurring slowly over time prior to the diagnosis of toxicity, this may not have been clinically recognized due to the very gradual absolute increase in serum creatinine over years. importantly, this study suggests that vigilance for tenofovir toxicity should remain beyond the early years of treatment. in addition, since no patient showed full recovery of egfr, earlier identification of toxicity prior to development of renal function decline is important. certainly, close monitoring of egfr over time, rather than serum creatinine alone (with clear documentation of a baseline level) might detect subtle decline and alert one earlier to the possibility of nephrotoxicity. in addition, only 5 of 15 patients had serum creatinine levels beyond the normal range of 0.51.2 mg / dl at the time of diagnosis of nephrotoxicity. this likely also explains the significant declines in egfr prior to detection and further emphasizes the need to use gfr estimation in the monitoring of kidney function. the difficulty of detecting tdf nephrotoxicity in the context of subtle changes in serum creatinine further highlights the importance of monitoring phosphate excretion, serum phosphate, urinary protein and glycosuria for tdf toxicity rather than relying on serum creatinine as a lone marker of kidney injury. earlier detection of urinary phosphate wasting or serum phosphate decline may have identified nephrotoxicity sooner. however, urine phosphate was not previously assessed in any patient, and serum phosphate levels were not monitored in the majority of the patients. current guidelines by the infectious disease society of america recommend monitoring of serum creatinine, serum phosphate, glycosuria and proteinuria twice a year in patients who are prescribed tdf and have an egfr of < 90 ml / min/1.73 m or in patients with diabetes and hypertension who have a high risk of kidney disease. despite these guidelines, only 7 (47%) of the 15 patients in this series had a serum phosphate level checked prior to their referral to the renal clinic even after years of tdf therapy. also, since not all patients developed hypophosphatemia, the diagnosis of tdf - induced tubulopathy would have been missed based on current screening guidelines in 27% of patients who had renal phosphate wasting with normal serum phosphate levels. as noted earlier, the average time for the detection of proximal tubular dysfunction after start of tdf therapy in our cohort was 64 months, a duration that is much longer than has been previously reported in other studies. this underscores the necessity of continued monitoring of side effects in patients even if they have tolerated tdf therapy for years. even after discontinuation of tdf, recovery of serum phosphate and improvement of egfr, none of the patients returned to their baseline egfr after a mean follow - up of 23 months after discontinuation of tdf. as noted earlier, the implications may be important with regard to earlier identification of tdf toxicity, as ckd is associated with a long - term increase in morbidity and mortality in hiv - infected patients [37, 38 ]. the apparent loss of kidney function, seen here and in earlier studies, despite drug discontinuation, would support monitoring potentially more sensitive markers of toxicity such as serum phosphate and fephos. it is notable that the majority of patients were on a boosted protease inhibitor at the time of development of the proximal tubular dysfunction. several studies have suggested increased toxicity of tenofovir due to the effects of protease inhibitors, particularly ritonavir, on apical membrane transporters involved in the elimination of tenofovir from the proximal tubular cell. decreased elimination has been proposed to increase intracellular concentrations of the drug and hence increase toxicity. however, since no control group is assessed, it is difficult to determine the role in protease inhibitor use in this cohort. moreover, patients who are referred to the nephrology clinic are more likely to have severe kidney disease. the retrospective nature of the study had its inherent limitations in terms of selection bias and missing data. it is also important to note that urine phosphate measurements were not obtained on a fasting urine sample, which may make measurements less reliable. finally, most of our patients had no urinary phosphate measurements at preset intervals of follow - up. tdf - induced phosphate wasting may be a more sensitive marker for kidney dysfunction than an increased serum creatinine level. moreover, tubular dysfunction may only become evident after years of tdf therapy, even in those who have normal kidney function at the time of starting tdf ; this emphasizes the importance of continued monitoring in all patients treated with tdf. | backgroundtenofovir disoproxil fumarate (tdf) may cause acute kidney injury and proximal tubular dysfunction. however, no detailed studies document urinary phosphate wasting as a marker of tdf - induced tubulopathy.methodsrecords of hiv - infected patients with presumed tdf toxicity were reviewed. we describe the characteristics and clinical course of 15 patients who had documented elevated (> 20%) fractional excretion of phosphate (fephos).resultspatients were predominantly caucasian and male (73 and 80%, respectively), with a mean age of 56 years (range 3876). of the 15 patients, 11 had a estimated glomerular filtration rate (egfr) of > 90 ml / min/1.732 at time of tdf initiation. the mean duration of tdf therapy prior to diagnosis of tdf toxicity was 64 months. mean fephos was 34% (range 2062). the mean egfr at tdf initiation was 104 ml / min/1.73 m2 [standard deviation (sd) 17.0 ] with a gradual decline to 69 ml / min/1.73 m2 (sd 19.0) by the time of tdf discontinuation. of 10 patients with repeated fephos after tdf discontinuation, 9 had improvement of their fephos. of these individuals, 6 had normalization of their fephos. estimated gfr improved in 12 patients after discontinuation of tdf, though importantly, none returned to their baseline egfr.conclusionsurinary phosphate wasting is a sensitive marker for tdf - induced proximal tubulopathy and is associated with unrecognized and permanent renal function decline. tubular dysfunction can develop after years of tdf therapy in those with normal kidney function at the time of drug initiation. this suggests that continuing vigilance be maintained in all those on tdf. |
petrous bone cholesteatomas (pbcs) are epidermoid cysts, which have developed in the petrous portion of the temporal bone and may be congenital or acquired.1 2 3 the incidence of pbcs was estimated as accounting for between 4 and 9% of all petrous bone lesions.1 2 4 a pbc gradually invades the bony labyrinth and erodes the petrous apex and the skull base around the internal auditory canal (iac) and may extend as far as the cerebellopontine angle. furthermore, these lesions may affect other vital soft tissue structures within the temporal bone such as the sigmoid sinus or the jugular vein and carotid artery.1 2 5 6 7 as regard the source and extension of cholesteatomas, sanna classified five types of pbc lesion : supralabyrinthine, infralabyrinthine, massive labyrinthine, infralabyrinthine lateral transtemporal or middle fossa approaches by microscopic surgery are usually employed for removing extensive pbcs.1 2 5 6 7 8 in recent years, the instrumentation, techniques, and knowledge relating to middle ear endoscopic surgery have greatly improved. at present, the main application of endoscopic surgery is in the surgical treatment of middle ear cholesteatoma, but the natural evolution of the technique may provide an increasing number of applications in lateral skull base surgery.9 10 11 this case report illustrates the case of a giant cholesteatoma that eroded the labyrinth and the posterior fossa dura and extended to the infralabyrinthine region, thereby affecting the body of the first cervical vertebra. few authors have dealt with the topic of a pbc, which presents this type of massive life - threatening infralabyrinthine extension, and there is apparently no literature documenting cases in which a cholesteatoma has extended to the extracranial area and cervical spine. we performed an infralabyrinthine subtotal petrosectomy by means of a combination of microscopic surgery and an adjuvant endoscopic approach to remove the cholesteatoma matrix adherent to the vertebral body and the dura of the posterior fossa. an 83-year - old caucasian male was referred to our department with otorrhea in his left ear which he had had for 3 months and a painful swelling behind the ear. he also had a left facial palsy evaluated as v grade according to the house otomicroscopic evaluation revealed total perforation of the left hand tympanic membrane, through which the middle ear cavity appeared completely occupied by cholesteatomatous soft tissue. temporal bone computed tomography revealed an extensive cholesteatoma that completely occupied the left middle ear cavity, the mastoid, and involved the petrous bones. imaging showed erosion of the tegmen tympani, the bone around the geniculate ganglion, the semicircular canals, and the cochlea too. in the posterior section, the lesion was found to extend and destroy part of the occipital bone and skull base. it extended beyond the cranial theca and went distal to the body of the first cervical vertebra (atlas) which appeared eroded (fig. 1). coronal ct ; extensive cholesteatoma eroding part of skull base bone and of the first cervical vertebra (arrow). a subsequent nuclear magnetic resonance imaging (mri) confirmed structural alteration of the petromastoid region and the presence of a 6 2 3 cm area of soft tissue which was hypointense on t1 and hyperintense on t2-weighted sequences, consistent with a diagnosis of cholesteatoma. the mri confirmed the extension of the cholesteatoma cerebrum to the first cervical vertebra (fig. 2). coronal mri, t1-weighted sequence ; presence of a hyperintense 6 2 3 cm soft tissue extended between the skull base and the first cervical vertebra. intraoperative facial nerve using nim2 was planned.12 left infratemporal and infralabyrinthine subtotal petrosectomy was performed using a combined approach microscopic and endoscopic surgery. first, the petrous bones and a massive cholesteatoma, which had occupied the lateral and posterior temporal areas on the left side and had caused widespread osteolisis of the mastoid cells, of the pyramid, and of the skull base theca, were removed through standard microscopic surgery (figs. 3 and 4). furthermore, the remaining infralabyrinthine cholesteatoma matrix, which adhered to the body of the atlas was treated by means of delicate endoscopic dissection and bipolar coagulation (fig. rigid endoscopes at 0- and 30-degree angled with an outer diameter of 4 mm (storz, germany) were used. images were recorded through a full high - definition (hd) camera (stroz, germany), which was attached to the endoscope lens, and these images were displayed on a full hd monitor. in this manner, total removal of the lesion was achieved. no postoperative cerebrospinal fluid (csf) leaks or other complications occurred. microscopic surgery ; residual cholesteatoma adherent to the transverse apophysis of the first cervical vertebra (arrowhead). microscopic view (maximum magnification) ; residual cholesteatoma adherent to the transverse apophysis of the first cervical vertebra (arrowhead) not entirely visible. adjuvant endoscopic surgery ; delicate dissection of the cholesteatoma matrix adhered to the transverse apophysis of the atlas clearly visible (round knife). pbc is an uncommon pathology but may cause severe functional damage and negatively affect the quality of life. it has a known tendency to invade the labyrinth and fallopian canal, thereby causing facial nerve paralysis and permanent hearing loss. it may also involve all the anatomical structures within the temporal bone and extend to the cerebellopontine angle or the infralabyrinthine region, which signifies a risk of damage to vital life structures (internal carotid artery, jugular vein, sigmoid sinus, dura).1 2 3 4 5 8 11 we have illustrated the case of a giant cholesteatoma which was classified as an infralabyrinthine apical cholesteatoma according to sanna that eroded the labyrinth and the posterior fossa dura. the case we have presented is a good example of a cholesteatoma which extends beyond the confines of the skull base and infralabyrinthine region. indeed, although massive intracranial involvement of the bone, nerves, and vascular system of the posterior cranial fossa by such a huge cholesteatoma is not infrequent, this case is very unusual because the lesion went beyond the theca and reached the first cervical vertebra. to our knowledge, no previous cases of a cholesteatoma of this size have been documented. traditionally, surgery performed on giant cholesteatoma of the petrous bone has contemplated lateral transtemporal or middle fossa microscopic surgery. the choice of the best surgical approach is based on the location and extent of the lesion, hearing, preoperative facial nerve paralysis, and anatomic position of the internal carotid artery and jugular bulb. it must guarantee that the cholesteatoma is visible in its entirety and ensure a sufficient exposure of the middle and posterior fossa dura, carotid artery, lateral sinus, jugular bulb, and facial nerve.2 5 6 7 8 9 10 11 13 14 15 the main problems regarding pbc removal are residual matrix, recurrence / relapse, and postoperative complications. despite the illumination and magnification offered by the microscope insufficient primary resection of the epidermal matrix, caused by a recess which was not detected by the microscope, may lead to recurrence of the disease in the patient / relapse.1 2 3 4 5 6 7 in recent years, endoscopic surgical techniques, instrumentation, and knowledge have greatly improved due to an increased use of endoscopy in middle ear and mastoid surgeries.9 10 11 marchioni recently proposed the endoscopic transcanal surgical approach for the treatment of iac and petrous bone lesions. six of the 12 patients treated had a cholesteatoma of the tympanic cavity which affected the inner ear (vestibule, geniculate ganglion, middle cranial fossa, and iac), although there was no massive infralabyrinthine extension. kanzara reported the case of a petrous apex cholesteatoma which was removed in its entirety by means of an endoscopic approach. they stated that an endoscopic permeatal approach circumvents some of the problems encountered in microscopic surgery. it provides a better operative field and excellent vision of the important structures because, unlike the microscope, it bypasses the narrowest points and provides an excellent assessment of the surrounding structures. moreover, they noted that the endoscopic approach provides more direct access to the apex. in our case, a left - sided infratemporal and infralabyrinthine subtotal petrosectomy was performed using a combined approach (microscopic and endoscopic). endoscopic petrous bone surgery offers some advantages as compared with the traditional microscopic technique, as it guarantees direct visual control of hidden areas such as the infralabyrinthine recess and its structures. in this way, the eradication of the cholesteatoma in its entirety may be possible, reducing the risk of residual persistence and recurrence (lower rate). moreover, it may lower the risk of lesions of the dura and of the other functional and vital structures of the lateral skull base during cholesteatomatous matrix removal (postoperative csf leaks).8 9 10 14 as claimed by many authors, the main limitation of the endoscopic approach is that it is a one - hand surgical procedure that does not allow adequate drilling of the temporal bone to remove extensive lesions.9 10 11 17 in conclusion, in future, endoscopic surgery will gain increasing importance in surgery of pbcs. subsequently, a complementary endoscopic approach in hidden areas should be performed to remove the cholesteatomatous matrix and reduce possible postoperative complications and relapse. | petrous bone cholesteatomas (pbcs) are epidermoid cysts, which have developed in the petrous portion of the temporal bone and may be congenital or acquired. cholesteatomas arising in this region have a tendency to invade bone and functional structures and the middle and posterior fossae reaching an extensive size. traditionally, surgery of a giant pbc contemplates lateral transtemporal or middle fossa microscopic surgery ; however, in recent years, endoscopic surgical techniques (primary or complementary endoscopic approach) are starting to receive a greater consensus for middle ear and mastoid surgeries. we report the rare case of an 83-year - old caucasian male affected by a giant cholesteatoma that eroded the labyrinth and the posterior fossa dura and extended to the infralabyrinthine region, going beyond the theca and reaching the first cervical vertebra. the giant cholesteatoma was managed through a combined approach (microscopic and, subsequently, complementary endoscopic approach). in this case report, we illustrate some advantages of this surgical choice. |
ophthalmic conditions are common in primary care practice.1 eye signs and symptoms are often the first recognized presentation of common systemic conditions, such as diabetes and hypertension. while many conditions affecting the eye can be treated, irreversible damage can occur if they are left unrecognized (eg, diabetic retinopathy, glaucoma).2 despite the prevalence of such conditions, little time is devoted to ophthalmic training in undergraduate medical education.3 a recent study revealed that canada does not have a standardized undergraduate medical education curriculum for medical schools, and that many residents of various specialties report not having confidence in managing ophthalmological cases.5 prior studies have shown that canadian medical students are not comfortable with ophthalmic clinical skills, such as the use of an ophthalmoscope.4,5 a recent study at a public medical school in california demonstrated that 26% of graduating students were not at all comfortable with performing screening eye examinations and that 57% were very interested in a skills refresher course in the future.6 this is consistent with a 1995 study which reported that, according to estimation by us primary care program directors, less than 50% of residents have adequate ophthalmic skills at the start of residency, despite 85% feeling that skills like a screening eye examination should be mastered during medical school.3 these findings support the importance of adequate undergraduate ophthalmology training.712 these studies suggest that medical students receive too little ophthalmic training and advocate increasing the exposure to ophthalmic knowledge and skills within the undergraduate medical education curriculum. the primary goal of our study was to quantify the adequacy of ophthalmic education for medical school graduates in training to be primary care physicians. our study also aimed to highlight the ophthalmic training requirements of general family practitioners, and propose necessary changes to the canadian medical school curricula accordingly. all current family medicine residents training at the university of western ontario, including those in postgraduate year (pgy) 1 and 2, were invited to participate in the study. participants were excluded if they had previously applied to or been enrolled in an ophthalmology residency program. informed consent was provided by all participants. using a paper - based questionnaire (see appendix 1), subjects were asked to provide information on the following : year of postgraduate training in family medicine residency, country of origin of medical school prior to residency, number of hours of classroom or clinical exposure to ophthalmology during undergraduate medical education, as well as percentage of rotation time involving exposure to ophthalmological conditions during family medicine residency. subjects were also asked to rate their comfort with respect to managing specific ophthalmology conditions and performing specific ophthalmic clinical skills on a likert scale from 15 (1 = not comfortable at all ; 2 = somewhat comfortable ; 3 = moderately comfortable ; 4 = comfortable ; 5 = very comfortable). the conditions and skills asked about in the questionnaire were based on the topics covered in the undergraduate medical education ophthalmology reference text published by the american academy of ophthalmology.13 descriptive statistics were generated using microsoft excel (microsoft corporation, redmond, wa) and sas software (sas institute, cary, nc). the spearman correlation test was performed to identify if there was an association between comfort level and number of hours of ophthalmology education in undergraduate medical education. descriptive statistics were generated using microsoft excel (microsoft corporation, redmond, wa) and sas software (sas institute, cary, nc). the spearman correlation test was performed to identify if there was an association between comfort level and number of hours of ophthalmology education in undergraduate medical education. of the 162 family medicine residents in pgy 1 and 2 at the university of western ontario, 54 (33.3%) volunteered to participate in the study. there was a large variation in the amount of classroom - based (range 0200 hours) and clinic - based (range 0300 hours) ophthalmology teaching during medical school. overall, it appears that international medical graduates received more ophthalmology instruction than canadian medical graduates (p < 0.05, table 2). the subjects reported that, on average, 6.3% 9.2% of their postgraduate rotations in family medicine involve exposure to eye - related cases (6.9% 10.6% for pgy 1, 5.3% 5.7% for pgy 2). eighty percent of subjects reported that they were either somewhat comfortable or not comfortable at all in dealing with ophthalmology - associated conditions. no subject reported being very comfortable with ophthalmology - associated issues (figures 1 and 2). there was no statistically significant difference in average comfort level rating from the likert scale between pgy 1 and pgy 2 (1.9 0.8 and 2.2 0.8, respectively) and between canadian medical graduates and international medical graduates (1.9 0.8 and 2.1 0.8, respectively). the spearman correlation revealed a moderately positive correlation between the hours of classroom instruction received during undergraduate medical education and overall comfort in managing ophthalmology cases (p = 0.0012). the hours of clinic instruction alone and combined classroom and clinic instruction revealed small positive correlations, albeit not statistically significant (table 3). there was a wide variation in the comfort level of family medicine residents in managing different ophthalmologic conditions (table 4), as well as in performing relevant ophthalmic clinical skills (table 5). when comparing the responses of both pgy 1 and pgy 2 in managing or coordinating care of ophthalmology - related issues, the median score differed for 22 of the 52 items. pgy 1 residents had a higher median score in 16 of all items, and pgy 2 residents higher in six items (table 4). when asked about their comfort level surrounding various ophthalmologic procedures, the median scores between pgy 1 and pgy 2 differed in 8 of 21 items. pgy 1 residents scored higher in three of all items, whereas pgy 2 residents scored higher in five items (table 5). there was a large variation in the amount of classroom - based (range 0200 hours) and clinic - based (range 0300 hours) ophthalmology teaching during medical school. overall, it appears that international medical graduates received more ophthalmology instruction than canadian medical graduates (p < 0.05, table 2). the subjects reported that, on average, 6.3% 9.2% of their postgraduate rotations in family medicine involve exposure to eye - related cases (6.9% 10.6% for pgy 1, 5.3% 5.7% for pgy 2). eighty percent of subjects reported that they were either somewhat comfortable or not comfortable at all in dealing with ophthalmology - associated conditions. no subject reported being very comfortable with ophthalmology - associated issues (figures 1 and 2). there was no statistically significant difference in average comfort level rating from the likert scale between pgy 1 and pgy 2 (1.9 0.8 and 2.2 0.8, respectively) and between canadian medical graduates and international medical graduates (1.9 0.8 and 2.1 0.8, respectively). the spearman correlation revealed a moderately positive correlation between the hours of classroom instruction received during undergraduate medical education and overall comfort in managing ophthalmology cases (p = 0.0012). the hours of clinic instruction alone and combined classroom and clinic instruction revealed small positive correlations, albeit not statistically significant (table 3). there was a wide variation in the comfort level of family medicine residents in managing different ophthalmologic conditions (table 4), as well as in performing relevant ophthalmic clinical skills (table 5). when comparing the responses of both pgy 1 and pgy 2 in managing or coordinating care of ophthalmology - related issues, the median score differed for 22 of the 52 items. pgy 1 residents had a higher median score in 16 of all items, and pgy 2 residents higher in six items (table 4). when asked about their comfort level surrounding various ophthalmologic procedures, the median scores between pgy 1 and pgy 2 differed in 8 of 21 items. pgy 1 residents scored higher in three of all items, whereas pgy 2 residents scored higher in five items (table 5). we were able to achieve a response rate of 33.3% from the entire study population, ie, 54 of 162 university of western ontario family medicine residents. there was good representation of both canadian medical graduates (n = 32) and international medical graduates (n = 22), as well as from both pgy 1 (n = 35) and pgy 2 (n = 19) years of residency. it recommends 4060 hours of ophthalmology exposure during undergraduate medical education.11 nonetheless, a recent survey by welch and eckstein revealed that medical schools in the uk did not comply with the recommended ophthalmology curriculum set out by the international council of ophthalmology.14 compared with the international council of ophthalmology task force recommendation of 4060 hours,11 our study participants received a satisfactory number of hours of both classroom - based (27.1 35.1 hours) and clinic - based ophthalmology instruction (39.8 47.1 hours). however, there was a wide variation in the hours of instruction reported, as reflected by the large standard deviations. residents received from 0200 hours of classroom instruction and 0300 hours of clinic - based instruction on ophthalmology during medical school. international medical graduates reported a higher amount of ophthalmologic instruction when compared with canadian medical graduates, although there was no significant difference in the average level of comfort in managing ophthalmic diseases between the two groups. our data suggest the need for standardizing the amount of ophthalmology instruction in undergraduate medical education. despite the amount of ophthalmology instruction in undergraduate medical education meeting the international council of ophthalmology recommendations, most (80%) of the residents in our study felt only somewhat comfortable or not comfortable at all in managing ophthalmology conditions. with the exception of dry eyes and conjunctivitis, residents felt moderately comfortable or less in managing all the specific conditions in our questionnaire. while it may be understandable that residents are not comfortable with all ocular conditions, there are certain diseases with which family medicine physicians should be familiar. for example, the medical council of canada lists strabismus, pupil abnormalities, and eye redness among its learning objectives for medical students.15 although residents felt comfortable in managing red eye, they only felt somewhat comfortable with relative afferent papillary defects, strabismus, and amblyopia. other conditions which often threaten sight in the emergency setting, such as orbital cellulitis, corneal ulcer, acute angle closure glaucoma, ocular chemical burn, and ischemic optic neuropathy secondary to giant cell arteritis, also had low median scores. all of these diseases and presenting problems are important to recognize, owing to the high risk of vision loss and diminished quality of life. comfortable with nine of them and moderately comfortable with another eight. somewhat comfortable in four specific areas, ie, tonometry, prescription of antiglaucoma medications, indications and contraindications for surgical procedures, and explaining common ophthalmological surgical procedures. emphasis should be placed on getting family medicine residents more familiar and comfortable with using a tonometer, because one of the important learning objectives of the college of family physicians of canada is to perform a focused examination and investigations to evaluate a red eye, including the measurement and evaluation of intraocular pressure.16 a similar study assessing the comfort level of canadian family medicine residents with conditions pertaining to otolaryngology / head and neck surgery also revealed low levels of comfort for important conditions and procedures.17 our results reflect the same phenomenon, ie, primary care trainees are not fully comfortable with assessing or managing some of the commonly encountered subspecialty conditions which often require acute management. when comparing the different modes of instruction (classroom versus clinic), we found that classroom - based instruction had a positive effect on raising comfort in managing ophthalmological cases. perhaps by standardizing and enriching the undergraduate medical education ophthalmology curriculum in medical schools, or by increasing the percentage of ophthalmology teaching during residency, trainees will be more confident in handling eye - related cases in the primary care setting. medical schools may consider utilizing innovative methods of education, such as computer - assisted or web - based learning, which has shown promising effectiveness in subspecialty training.18 further studies will have to be conducted to validate newer approaches to knowledge transfer in medical education. the questionnaire was administered to residents approximately 12 years following the conclusion of their undergraduate medical training. however, the responses were consistent with previous studies of undergraduate medical education ophthalmology training in other countries, which suggests validity of the results.3,4,6 our sample was limited in that it represented a selection of residents from a single postgraduate institution, although there was representation of graduates from a broad spectrum of domestic and international medical schools. it would be interesting to compare the level of comfort of residents in different family medicine residency programs. despite having received an adequate number of hours of ophthalmological instruction according to recommendations by the international council of ophthalmology, family medicine residents lack comfort in managing ophthalmic conditions. many of these conditions are common and could result in loss of vision if not treated properly, including cataracts, strabismus, pupil abnormalities, acute angle closure glaucoma, and giant cell arteritis. these specific conditions should be the focus of educational interventions, such as lectures, computer - assisted learning, continuing medical education, and refresher courses. furthermore, some residents in our study reported having received an inadequate amount of ophthalmological training during their undergraduate medical education. standardizing undergraduate ophthalmology training in canada will ensure that medical students are graduating with adequate exposure to the subspecialty.5 as primary care practitioners, family physicians play an important role in managing patients with ocular conditions. given the rising number of elderly patients, focus needs to be placed on training all physicians, including family physicians, to be aware of signs and symptoms of conditions that can lead to vision loss.19 raising family physicians comfort level with ocular conditions will help facilitate the prompt management of ocular conditions and appropriate referrals to ophthalmologists. standardizing undergraduate ophthalmology training and using effective teaching methods tailored towards primary care should help raise the comfort level of family physicians. more research is needed to evaluate various educational interventions and their effectiveness in increasing comfort in managing and referring ocular conditions. | background : this cross - sectional survey assessed the adequacy of ophthalmology teaching in undergraduate medical education and evaluated the comfort level of family medicine residents in diagnosing and managing common ophthalmic conditions.methods:postgraduate year 1 and 2 family medicine residents at the university of western ontario were recruited for this study. the main outcome measures were hours of classroom and clinic - based instruction on ophthalmology during undergraduate medical education, and the comfort level in ophthalmic clinical skills and managing various ophthalmic conditions.results:in total, 54 (33.3%) of 162 family medicine residents responded to the survey. residents reported an average of 27.1 35.1 hours and 39.8 47.1 hours of classroom and clinical ophthalmology instruction, respectively. however, most residents (80%) responded as feeling only somewhat comfortable or not at all comfortable in assessing and managing common ophthalmic conditions, including ocular emergencies, such as acute angle closure glaucoma and ocular chemical burn. a positive correlation was seen between overall comfort level and hours of classroom instruction (p < 0.05).conclusion : the number of hours of ophthalmology training received by family medicine residents during medical school meets the international council of ophthalmology task force recommendations. however, family medicine residents appear to be uncomfortable in handling treatable but potentially sight - threatening ocular conditions. standardizing the undergraduate medical education ophthalmology curriculum and increasing hours of ophthalmology training during postgraduate family medicine residency may be useful in bridging this gap in knowledge. |
a 53-year - old african man was diagnosed with unknown primary undifferentiated carcinoma with mediastinal lymph nodes and thrombosis of superior vena cava in 1993. the patient was initially treated by 8 cycles of chop (cyclophosphamide, adriamycin, vincristine, and prednisone). he relapsed in 1996 with spinal bone metastases treated by laminectomy, radiotherapy and pfl - vp16 (cddp, 5fu, leucovorin, etoposide). in 1999 he relapsed again with mediastinal lymph nodes treated by 3 cycles of navelbine and cisplatin followed by 3 cycles of carboplatin and navelbine with > 70% treatment response. biopsy at that time revealed large - cell carcinoma of bronchial or thymic origin. in 2002 newly discovered bone, pulmonary and mediastinal metastases were treated successively with taxotere / gemcitabine (6 cycles), navelbine / xeloda (6 cycles), iressa (6 months) and tarceva (8 months). in 2005, pemetrexed was maintained for only 4 cycles, then was suspended due to hematoxicity despite clinical efficiency. no maintenance treatment was used until april 2007, when new bone metastases were discovered. the patient was hence treated with pemetrexed, as it once showed its efficacy in 2005. after the sixth cycle, laboratory examination revealed serum creatinine 400 mol / l, metabolic acidosis (plasma bicarbonate 21 mmol / l), sodium 145 mmol / l, potassium 4,5 mmol / l, urea 21 mmol / l. a 24-h urine collection on the 2nd hospital day revealed a 0.55 g proteinuria without hematuria or leukocyturia. a kidney biopsy was performed showing acute tubular necrosis (atn) associated with chronic interstitial fibrosis (fig. his current renal function remained stable after 6 months follow - up of the acute renal failure (arf) episode with a stable serum creatinine level of 380 mol / l. our patient experienced severe acute kidney injury related to atn and interstitial fibrosis following sequential treatment with pemetrexed for a metastatic undifferentiated carcinoma. only few cases of arf due to pemetrexed have been reported. in a patient treated for metastatic non - small cell lung cancer, arf was associated with nephrogenic diabetes insipidus and distal renal tubular acidosis following 3 doses of pemetrexed (500 mg / m). at discharge 1 month after admission, the patient still demonstrated polyuria, hypokalemia, and metabolic acidosis despite recovery to a creatinine level of 1.7 mg / dl. in a second patient with unresectable pleural mesothelioma, pemetrexed (500 mg / m) and cisplatin (75 mg / m) for 3 cycles, then pemetrexed as a single agent induced arf appearing at the sixth cycle of pemetrexed. eighteen cases of renal failure during clinical trials of pemetrexed have also been reported. in the phase i pemetrexed maintenance therapy (pmt) study evaluating toxicity as primary end point, 8% of patients with malignant pleural mesothelioma treated with high doses (700 mg / m) of pemetrexed experienced reversible grade 1 or 2 renal failure : creatinine clearance decreased from 88 21 ml / min at the end of the induction therapy to 77 26 ml / min at the end of maintenance therapy (p < 0.05). no grade 4 toxicity was observed. in phase iii trials, all grades of renal failure and grade 4 requiring dialysis were reported in 2.4 and 0.6% of patients, respectively. in all studies, baseline creatinine clearance (estimated using the cockcroft - gault formula) indeed, pemetrexed - induced renal toxicity may potentiate an enhanced myelosuppressive response to pemetrexed. in an initial phase i study the development of severe toxicity appeared to correlate most strongly with baseline renal function. patients with an estimated creatinine clearance value less than 80 ml / min were more likely to develop severe myelosuppression (grade iv neutropenia) than those with a creatinine clearance more than 80 ml / min. this suggests that initial dosing should be based on the area under the curve as currently utilized for carboplatin rather than on body surface area and renal function. this idea was confirmed by an analysis of 10 phase ii clinical trials. in a phase i dose escalation trial including patients with various degrees of renal dysfunction, pemetrexed seems to be well tolerated at doses of 500 mg / m with vitamin supplementation in the case of creatinine clearance = 40 ml / min. the fda even recommends a creatinine clearance of greater than 45 ml / min as the threshold for administering the drug. an experimental study demonstrated that in kidney proximal tubule, the folic acid is reabsorbed via renal folate receptors and brush - border membrane vesicles. the hypothesis is that the antifolate, like the folic acid, would be less reabsorbed by urine alkalinisation and increasing urine flow rate by hydration. on the other hand, thymidine is described as an antidote for pemetrexed - related toxicity in a clinical report, though in that report the concomitant use of hemodialysis complicates the interpretation of this favorable outcome. in summary, | we report a patient with unknown primary undifferentiated carcinoma who developed acute renal failure associated with interstitial fibrosis following pemetrexed therapy. despite drug withdrawal, renal function remained altered and the patient experienced chronic renal insufficiency. pemetrexed disodium (alimta) is a multitargeted antifolate agent approved by the food and drug administration (fda) for patients diagnosed with mesothelioma and non - small cell lung cancer. this drug is almost exclusively cleared by renal excretion [1 ]. the most common side effects are hematologic dose - limiting toxicities and nonhematologic toxicities including fatigue, diarrhea, nausea, mucositis and rash. although few cases of renal failure have been published, no study has reported on the renal pathological findings in this setting. we present a case of acute tubular necrosis associated with interstitial fibrosis after pemetrexed therapy. |
acquired platelet dysfunction with eosinophilia (apde) is usually a self - limiting bleeding disorder characterized by an insidious onset of easy bruising with petechiae in an otherwise well person. hypereosinophilia is often the first clue to diagnosis, which is supported by the findings of a platelet storage pool disorder. apde has been mainly reported from thailand, malaysia, and singapore, but its true incidence has not been studied. the condition has rarely been reported elsewhere and hence travelers returning from an endemic area may present with a diagnostic challenge. an 11-year - old caucasian boy presented with recurrent cutaneous bruises and ecchymosis for five months. the child 's past health was only remarkable for occasional asthmatic attacks when he was small and he had no prior history of bleeding tendency or excessive swelling following vaccinations. fifteen months ago, the family moved from the united states and lived in central java, indonesia. five months before the consultation, he was noticed to have recurrent, unprovoked bruising of the skin with occasional epistaxis and gum bleeding. there were no symptoms of gastrointestinal, genitourinary, or intra - articular hemorrhage. he had not been noted to have worms in the stool although he had been treated with antihelminthics every 3 to 4 months. the child was otherwise well and continued to go to school as usual. on examination, there were multiple bruises from 1 to 3 cm in maximum dimension with petechiae over the limbs, the scalp, the chest and abdominal wall. laboratory investigations showed hemoglobin 12.8 g / dl, white cell count 14.810/l, eosinophils 7.410/l, platelet 22310/l, ige > 2,000 iu / ml serum biochemistries, liver transaminases, prothrombin time, partial thromboplastin time, and other immunoglobulin levels were normal. the peripheral blood film shows prominent eosinophilia with the presence of gray platelets (figure 1). the results of the platelet aggregation tests showed defective aggregation with collagen and epinephrine, consistent with a platelet storage pool disorder (table 1). figure 1photomicrograph of the blood film (100) showing an eosinophil of normal morphology (eo), a platelet of normal morphology (p), and platelets that appear pale and agranular (arrows). the latter feature is highly suggestive of thrombocytopathy and should obviate the need to measure the bleeding time. photomicrograph of the blood film (100) showing an eosinophil of normal morphology (eo), a platelet of normal morphology (p), and platelets that appear pale and agranular (arrows). the latter feature is highly suggestive of thrombocytopathy and should obviate the need to measure the bleeding time. table 1the results of the patient 's platelet aggregation tests.testresultnormal rangesadp69%64111%collage31%68117%epinephrine21%46122%ristocetin100%80115%arachidonic acid78%52110% he was empirically treated with albendazole 400 mg as single dose and a repeated dose two weeks later. the absolute eosinophil counts fell to 0.67 and 0.6310/l one and four months afterwards, respectively. acquired platelet dysfunction with eosinophilia (apde) is a unique disease that was first and almost exclusively described in the region of thailand, malaysia and singapore. the condition affects mainly patients of the pediatric age group, but adults are not spared. helminthic infestation has been associated with apde in about 50% of the cases, but how helminthes would explain the geographic occurrence is mysterious. the thrombocytopathic bleeding is evidenced by a prolonged bleeding time and positive hess 's test, degranulated platelets, and abnormal platelet aggregation tests consistent with a platelet storage pool disorder. the platelet dysfunction is believed to be secondary to eosinophilia but raised eosinophil counts are not found in all patients. reported from thailand a large cohort of 168 children diagnosed with apde. the majority of them presented with mild cutaneous bruises only, but 14 (8%) of them had severe bleeding symptoms that necessitated platelet transfusion therapy. the bleeding manifestations resolved within 6 months of diagnosis, although 12 (7%) of them had a recurrence. five of them recovered within two months after anti - helminthic treatment while the bleeding manifestations persisted for 36 months in the other patient. in particular, the presence of multiple bruises in the presence of normal platelet count and coagulation screen may be confused with accidental or non - accidental injury, while the extreme hypereosinophilia may be suggestive of hypereosinophilic syndrome. a comprehensive review by a pediatric hematologist will be essential for an accurate diagnosis without embarking on distressing medical or social investigations. as the case has illustrated, the diagnosis of apde can be reasonably reached with attention to the history, physical findings, simple laboratory tests, and examination of the platelet morphology on blood smear. sporadic reports from the united kingdom, canada, and hong kong are usually imported cases from the southeast asia (table 2). all five children presented with easy bruising, eosinophilia with normal platelet counts, but parasites were not found in any of them. some of these patients had been extensively investigated before the diagnosis of apde was made. together with this reports, the present case is illustrative of the fact that apde is not restricted to the indigenous population in the tropical countries and it may affect any child who has traveled to the southeast asia. table 2pediatric cases of acquired platelet dysfunction with eosinophilia reported from non - tropical countries.casessex/age (year)reporting countriescountries traveledparasitology / recoveryref1female/8united kingdommalaysiano parasite foundrecovered in 3 months112male/5canadamalaysiano parasite foundrecovered in 1 month123male/6canadamalaysiano parasite foundrecovered in 2 months124male/4hong kongthailandno parasite foundrecovered in 1 month135female/8hong kongnepalno parasite foundrecovered in 1 month13 | an 11-year - old american boy was staying with his family in indonesia. he presented with a 5-month history of recurrent bruises and ecchymosis. a clinical diagnosis of acquired platelet dysfunction with eosinophilia was made when his full blood counts showed hypereosinophilia (7.4109/l) with normal platelet count and gray platelets under the microscope. the diagnosis was supported by abnormal platelet aggregation tests consistent with a storage pool disorder. the bleeding symptoms and eosinophilia resolved a month later with a full course of antihelminthic therapy. hematologists should be aware of this unusual disease in travelers returning from the southeast asia. |
efforts in nanomaterials have rapidly expanded into the assembly of well - ordered two- and/or three - dimensional (2d and/or 3d) superstructures.the 3d superstructures provide possibilities to probe brand - new properties and applications due to the spatial orientation and arrangement of the nanocrystals [1 - 3 ]. different methods have been used to fabricate preferentially oriented nanowire arrays, such as, electrodeposition [4 - 6 ], vapor liquid solid (vls), thermal evaporation [9 - 11 ], lithography, aao, or mesoporous silica sba-15 -assisted methods. in these methods, sba-15 template shows a promising technique in controlling the preferential orientation without changing the nanowire morphology, and it has been explored for synthesis of ag, au, pt, ph, and si nanowires as well as binary semiconductor nanowires [19 - 22 ]. as previously reported, after removal of the silica template, the ordered mesostructure is seldom maintained, because the inorganic precursors are inclined to be absorbed on the external surface of templates and the channels are not completely filled up, which causes the framework formed inside the pores to be lacking in sufficient internal cross - linkage. recently, a single - source precursor, cadmium thioglycolate, was used to synthesize crystalline mesoporous cds nanoarrays through sba-15 silica template technique. the results demonstrated that such mesoporous semiconductor nanoarrays with high crystallinity were exactly an inverse replica of sba-15. these nanoarrays provide many opportunities for new applications as advanced materials ; however, the systematic studies of the transport, optical, and electrical properties of these nanoarrays were not reported till now. recently, some research has focused on rectification properties in the nanoscale [25 - 27 ]. the electrical properties of cds with different morphologies, such as nanoparticles, nanorods, and nanowires, were reported recently. the i v characterization of these cds systems demonstrated good ohmic contacts or highly insulating, but the rectification was not observed in these pure cds systems. the rectification can be obtained only when cds form heterostructures with others, such as, nanoparticles, polymer or si. here, the cds nanoarrays were synthesized through single - source precursor, metal alkyl xanthate. the remarkable performance on rectification within a bundle of cds nanoarrays was characterized by semiconductor characteristic measurement system (keithley 4200 scs), and the mechanism of the rectification of the cds nanoarrays was discussed. mesoporous silica sba-15 was prepared by a triblock copolymer under hydrothermal treatment at 130 c for 48 h following the general procedure reported by zhao.. for synthesis of cds nanoarrays, typically, 0.05 g sba-15 was added to a solution obtained by dissolving 0.34 g of cadmium alkyl xanthate in a certain amount of tetrahydrofuran, and then the mixture was kept stirring at room temperature until the solvent was completely vaporized. the residual powders were dried and then heated to160 c at a rate of 1 c / min and maintained at this temperature for 10 h under argon. silica composites were soaked in 2 m naoh for several hours to remove the silica template. the template - free cds products were recovered by centrifugation, washed with water, and dried at room temperature. the morphology of the samples was characterized by transmission electron microscope (tem, jem-100cx) at 100 kv. structural characterization was performed by x - ray diffraction (xrd, xpert pro mpd, with cu k radiation, = 1.54060) at 40 kv and 40 ma. scanning electron microscope (sem) measurement was carried out using a jsm-5600 lv equipped with edx (oxford isis) at 20 kv. vcurves of assembled cds nanowire arrays were measured by semiconductor characteristic system (keithley 4200-scs) at 350 nm illumination. desorption isotherms were measured on a micromeritics tristars 3000 analyzer at 196 c. before the measurements the sba-15 was synthesized at a high hydrothermal temperature of 130 c to increase the mesotunnels, which are beneficial for the production of high - quality replica materials. figure 1a exhibits the small - angle x - ray diffraction of the template - free cds nanoarrays. it indicates a highly ordered 2d hexagonal mesostructure ; at the same time, it implies that cds nanoarrays replicate well the ordered mesoporous of the sba-15 template and confirms the ordered arrangement of cds nanowires. the edx spectrum of the nanowire arrays confirms that these nanowire arrays consist of stoichiometric cds with a cd / s ratio of 1.05:1 and displays strong signals from cd to s elements without the detection of silicon element. asmall - angle diffraction pattern of template - free cds nanoarrays.beds pattern of the template - free cds nanoarrays.cwax diffraction curves of cds nanoarrays present in sba-15 pore channels and silica - free cds nanoarrays the wide - angle xrd patterns of the cds nanoarrays (fig. 1c) before and after removal of sba-15 show the (100), (002), (101), (110), (103), and (112) planes at 2 values 24.8, 26.5, 28.2, 43.7, 47.8, and 51.8, respectively, which match those of the hexagonal wurtzite structure of cds crystallite. the same xrd patterns of cds nanoarrays before and after removal of sba-15 template demonstrated that the structure of the cds nanoarrays is completely maintained during the etching. on the basis of the width of the diffraction peaks, the size of the cds nanocrystals is on the nanometer scale and the average particle size is calculated to be 7.1 nm, which is consistent with the pore diameter of the host sba-15. 2a) show typical type - ivcurves with an h1-type hysteresis loop, attributed to perfect cylindrical mesopore channels. a calcined mesoporous silica sample exhibits a high surface area of 560 m / g, the pore volume of 1.23 cm / g, and a narrow pore size distribution with a mean value of 7.3 nm. the surface area and pore size distribution of the cdr2/sba-15 nanocomposite are 28 m / g and 5.4 nm, respectively, and the adsorption volume is 0.06 cm / g, suppressed by 20 times after the one - step nanocasting process. upon the calcination at 160 c for cds@sba-15, the surface area and the pore volume decrease to 38 m / g and 0.04 cm / g, respectively, suggesting that the pores have been filled up by cdr2. desorption isotherms for the template - free cds sample exhibit a surface area of 100 m / g and a pore volume of 0.11 cm / g. a relatively narrow pore size distribution of around 3.3 nm is also exhibited, further demonstrating the high - quality mesoporous cds replicas. anitrogen sorption isotherms of the samples successively produced in the synthesis : the sba-15 hard template (), cdr2@sba-15 composites () prepared by the impregnation of cdr2into sba-15, cds@sba-15 () obtained from the cdr2@sba-15 calcined at 160 c, and cds - sba-15 replicas () etched by naoh solution.bthe corresponding pore - size distribution curves ofa figure 3demonstrates the tem images of the sba-15 template, and the cds nanoarrays after the removal of sba-15 templates (fig. 3a) templates have a uniform fiber - like morphology and well - ordered 2d mesoporous structures with uniform pore size. the high - magnification tem shows the pore size of sba-15 to be approximately 7.3 nm (fig. 3b) demonstrate that the as - prepared cds nanowire arrays have a uniform fiber - like morphology, and that the length of the cds nanowire arrays to be more than 2 m. from the insert of fig. 3b, it is obviously observed that these cds nanowire arrays are composed of uniform nanowire, and the diameter of each nanowire is about 7.3 nm, which is in reasonable agreement with the mean pore size of the silica template. these phenomena may reflect that the cds nanowire arrays are perfect duplication of the hard template and a high yield for the incorporation of cds. 3e) observed from this area can be indexed as the hexagonal wurtzite cds structure, indicating that the products consist of wurtzite cds nanocrystals, which is in agreement with the xrd data. atem images of the sba-15 sample along the direction of the hexagonal pore arrangement.btem images of the template - free cds sample. inseteis the corresponding saed pattern ofb in recent years, ac electric field assembly is widely used to construct nanodevices from nanowires and carbon nanotubes [37 - 39 ]. here, the cds nanowire arrays were ultrasonically dispersed in ethanol. after applying a droplet of the nanowire arrays suspension onto the electrodes, the pt electrodes were connected to an 8-vpp (peak - to - peak) and 10-khz ac signal for 30 s. this signal generated an alternating electrostatic force on the nanowire arrays in the solution. under the electrical polarization force, the 10-nm - thick pt electrodes were patterned on an oxidized silicon wafer with 200-nm silicon dioxide using photolithography. 4a shows the sem image of a bundle of cds nanowire arrays on pt electrodes. the electrical transport property was carried out through semiconductor characterization system (keithley 4200-scs). little current flows in reverse bias, whereas, the current increases rapidly when the bias voltage is more than 4 v. the most striking feature (fig. 4) is that both of the curves display rectifying behaviors at room temperature in air environment. under dark condition, this rectifying diode - like behavior has a threshold voltage of ~3.9 v, and no obvious breakdown current is observed even at 11 v bias. 4b) ; at the same time, the forward current is 25 na at 11 v, and their rectification ratio is about 625. under uv (= 350 nm)-illumination the turn - on voltage was dropped from 3.9 v to 3.4 v, and the breakdown current is not observed even at 11 v bias. the reverse current is 0.42 na at 11 v, the forward current is 64 na at 11 v, and their rectification ratio is about 152. the rectifying behavior is an important application of a diode [25 - 27 ]. recently, the rectifying behaviors of electric field - assembled zno nanowire have been reported. to our knowledge, such rectifying behaviors were not reported in pure cds system. in order to confirm the validity of the rectification for different metal sulfides, under dark condition, we observed the threshold voltage of ~2.0 v, the forward current of 150 na at 10 v, and the reverse current of 27 na at 10 v. for cds nanoarrays (fig. 4b), the reverse current is just 0.04 na at 11 v, and the reverse current for zns nanoarrays is much larger than that of cds nanoarrays, so the rectification ratio for zns nanoarrays is about six. but the obvious rectifying characterization was observed from the zns nanowire arrays. it is a very effective method for preparing metal sulfides with rectification through sba-15 as hard template and using a single - source precursor. thei vcurves of cds (a) and zns (b) nanoarrays at dark condition according to our previous reports on the current transport behavior of semiconductor nanowires, the nonlinear transport behavior in fig. 4 indicates that the cds nanoarrays make two schottky barrier contacts with the two pt electrodes, and a back - to - back schottky barriers structure is formed. in brief, the back - to - back schottky barrier structure is composed of two inversely connected schottky barriers, and the current in this structure is dominated by the reverse current of the reverse - biased schottky barrier. the detailed description of the properties of the back - to - back schottky barriers structure has been discussed in the previous reports of our group and other groups. in this structure, there should be no rectifying behavior if the two schottky barrier were symmetric. however, in the dielectrophoresis deposition process, the ac voltage introduces an electrostatic force on the nanoarrays, and then the nanoarrays move toward the electrodes until it gets finally deposited on them. the contacts of both ends of the nanoarrays onto the electrodes occur in a consecutive order. we consider that the side of the nanoarrays that touched the electrode first may have a firm contact with the electrode, thus forming a better contact with lower barrier height, whereas the other end that contacted later had a higher barrier, possibly leading to the formation of the schottky diode for our devices. thus, the asymmetric contacts were formed between the cds nanoarrays and pt electrodes, and resulted in the rectifying behavior. there occurs a current difference between the dark and uv illumination, because more number of electrons in the valence band are excited into the conduction band under uv illumination thereby increasing the carrier concentration of the cds nanowire arrays. as shown in fig. 4, both thei vcurves under dark and illumination conditions show linear behaviors when the bias is larger than 6 v. in this linear region, the current transport property is dominated by the nanowire itself, not by the schottky barrier. the slope of this linear region is proportional to the carrier concentration of the cds nanowire arrays. as shown in fig. 4, the slope of the current curve under illumination condition is three times higher than that of the one under dark condition. in summary, highly ordered mesoporous cds nanoarrays have been achieved by sba-15 as a template and cadmium xanthate as a single - source precursor. these materials exist as strongly nonlinear and asymmetrical, showing rectifying diode - like behavior, and provide opportunities for new applications as advanced nanodevices. the synthesis and properties of other systems, such as, mns, zns / cds composite nanoarrays, are currently in process. this study was supported by henan project for university prominent research talents (haipurt) (grant no. | highly ordered mesoporous cds nanowire arrays were synthesized by using mesoporous silica as hard template and cadmium xanthate (cdr2) as a single precursor. upon etching silica, mesoporous cds nanowire arrays were produced with a yield as high as 93 wt%. the nanowire arrays were characterized by xrd, n2adsorption, tem, and sem. the results show that the cds products replicated from the mesoporous silica sba-15 hard template possess highly ordered hexagonal mesostructure and fiber - like morphology, analogous to the mother template. the current voltage characteristics of cds nanoarrays are strongly nonlinear and asymmetrical, showing rectifying diode - like behavior. |
the utriculo - endolymphatic valve at the anteromedial wall of the utricle, was discovered by bast in 1928 in human fetuses. the utricular duct connects the utricle with the saccular space, close to the entrance of the endolymphatic duct. bast suggested that the function of the valve was closing of the utricular end of the utricular duct. bast himself proposed rotation of the valve lip at its base, where it is made - up of loose periotic tissue, as the functional mechanism. with rising intra - utricular fluid pressure a second theory proposes the opposing utricular wall as the functional part of the valve : bending of the single cell layer of the highly compliant utricular wall opposite the lip is responsible for the opening or closure of the valve [35 ]. in the early decades of the twentieth century bast reported two cases of ruptured saccules in human ears, resulting in a closed valve and an expanded utricle. bast and eyster withdrew endolymph from the cochlear duct of the guinea pig, causing a collapsed saccule and cochlear duct, while the utricle was distended and the valve closed. konishi found open valves in the guinea pig at various stages of endolymphatic hydrops, after operatively obstructing the endolymphatic duct. although there is no conclusive evidence up to now, bast s valve seems to be capable of protecting the utricle and the semicircular canal system when the evolutionary younger, cochlear part of the system ruptures. in the evolutionary chain of land living vertebrates, amphibians and reptiles have hearing organs that are small specialized regions of their vestibular systems. in birds, with a cochlear duct, a clear distinction can be made between an inferior and a superior part of the labyrinth. if the function of bast s valve in mammals is a protective one, it is expected to find a similar structure in birds between the two parts of the labyrinth. retzius made highly detailed drawings of the labyrinth of the pigeon and depicted the connection between the utricle and the saccule as a simple hole in a membrane. no utricular duct, nor a valve - like structure, is visible in fig. 1drawing of the junction between the utricle and saccule seen from the utricular side by retzius (1881) (arrow the hole between the utricle and the saccule) drawing of the junction between the utricle and saccule seen from the utricular side by retzius (1881) (arrow the hole between the utricle and the saccule) the drawing of satoh of a light microscopic slide shows a somewhat more detailed view of the same region in the pigeon. but also in this drawing little evidence for a valve - like structure between utricle and saccule can be found (fig. 2).fig. 2drawing of a light microscopical slide of the region between the utricle and saccule in pigeon by satoh (1917) (s saccule, sp posterior part of utricle, tv tegmentum vasculosum, dsu utriculo - saccular duct) drawing of a light microscopical slide of the region between the utricle and saccule in pigeon by satoh (1917) (s saccule, sp posterior part of utricle, tv tegmentum vasculosum, dsu utriculo - saccular duct) as part of a larger project to reconstruct the entire pigeon s inner ear in three dimensions, we investigated the transition region between utricle and saccule in detail using light microscopy. our objective was to obtain detailed information on a structure which resembles bast s valve to some extent. this structure consists of a duct between utricle and saccule, with an overhanging ridge and a slit - like opening on the utricular side of the duct. detailed light microscopy results and 3d - reconstructions from the light microscopy slides of the structure and its location are presented. three healthy pigeons (columbo domestica), with a weight of 400500 g, were used. animal care and use were approved by the experimental animal committee of groningen university, protocol no. 2883, in accordance with the principles of the declaration of helsinki general anesthesia was induced by intraperitoneal administration of pentobarbital (6%, overdose, hospital farmacist). after decapitation, all inner ears were fixated in an 8% neutral buffered formalin solution (ph 7.34) for at least 24 h. then the specimens were rinsed in aqua - dest. decalcification in ethylenediaminetetraacetic acid 10% solution (edta ; sigma, ed5ss, ph 7.4) took place at a temperature of 50c in a microwave oven (t / t mega microwave histoprocessor, milestone) in eight sessions of 6 h. after decalcification, the specimens were again rinsed with aqua - dest and dehydrated in a graded seven - step ethanol series (30, 50, 70, 90, 96, 100, 100%). the specimens were embedded in hpma (hydroxypropyl methacrylate, polysciences inc.) with addition of a catalyst (n, n - dimethlaniline, peg 400 ; 15:1, fluka chemie ag). as fiducial markers for 3d - reconstruction five holes (diameter 0.5 mm) were drilled in the hpma - block, perpendicular to the surface. sections of 4 m were cut and each sixth section was stained with toluidine blue and contrast - stained with basic fuchsine to facilitate reconstruction. the stored 2d images were processed with an imod (http://bio3d.colorado.edu/imod) software package for 3d - reconstruction. the input of relevant contours in each 2d - image was manually performed with a writing tablet (wacom cintiq 15x). stacking of the lm slides was executed with midas software for manual alignment and adjustment (midas is part of imod), using the drilled fiducial markers as references. corrections to be made were at most a few percent of the 2d - image cross section. if after 3d - reconstruction is larger irregularities were observed, the midas procedure was repeated for the relevant subsequent 2d - slides. figure 3a d shows subsequent (24 m distance) light - microscopy images of a duct connecting the utricular and the saccular spaces. the images show the shape of the entrance of the utricular duct at the uticular side. 3subsequent light - microscopy sections of the entrance of the utricular duct (s saccule, us utricular space, u utricle). 4 subsequent light - microscopy sections of the entrance of the utricular duct (s saccule, us utricular space, u utricle). the structures inside the rectangular box are shown in more detail in fig. 4 the valve - like structure inside the box in fig. (dense ; non - compliant) in the valve - lip surrounded by a single cell - layer and the opposing single cell - layer wall of cylindrical epithelia (hypodense ; compliant), separating the perilymphatic space from the endolymphatic space in this region, can be distinguished. furthermore, an accumulation of toluidine dyed endolymph at the entrance of the duct can be seen.fig. 4detail of fig. 3a showing a valve - like structure consisting of a ridge overhanging the entrance of the duct (single asterisk compliant cylindrical epithelia tegmentum vasculosum, double asterisks overhanging ridge consisting of rigid non - compliant connective tissue) detail of fig. 3a showing a valve - like structure consisting of a ridge overhanging the entrance of the duct (single asterisk compliant cylindrical epithelia tegmentum vasculosum, double asterisks overhanging ridge consisting of rigid non - compliant connective tissue) figures 5 and 6 show the slit - shaped opening of the utricular duct at the utricular side. the duct is drawn open on both sides to give a good impression of the flat cylindrical shape of the ductfig. 63d - reconstruction of the entrance (slit - like) of the utricular duct as seen from inside the utricule. the dashed white line follows the edge of the valve - lip overhanging the slit entrance 3d - reconstruction the utricular duct. the duct is drawn open on both sides to give a good impression of the flat cylindrical shape of the duct 3d - reconstruction of the entrance (slit - like) of the utricular duct as seen from inside the utricule. the dashed white line follows the edge of the valve - lip overhanging the slit entrance figure 6 shows a raw 3d - reconstruction of the entrance of the utricular duct as seen from inside the utricle. as far as we know this is the first description of the utricular duct and a utriculo - endolymphatic valve - like structure in the bird. 3b, the structure appears more like a hole than a duct and no clear valve - like structure is visible. 7. because we had only 2d - slides with in - between distances of 24 m available the 3d - reconstructions in figs. 5 and 6 are not very detailed, but they give a fairly good impression of the shape of the entrance of the utricular duct at the utricular side. it is filled with rigid, non - compliant connective tissue, as can be seen in fig. 4. this rigid structure, in combination with the more compliant cell - layer opposite to it, could function as a valve, like it is postulated to do in mammals.fig. 7schematic drawing by one of the authors (hpw) of the left whole labyrinth of the pigeon. the arrow marked with an asterix points toward the utricular duct and the region of the valve. bp basilar papilla, ca crista in anterior ampulla, cl crista in lateral ampulla, cp crista in posterior ampulla, es endolymphatic sac, ml macula of the lagena, mn macula neglecta, ms saccular macula, mu utricular macula schematic drawing by one of the authors (hpw) of the left whole labyrinth of the pigeon. the arrow marked with an asterix points toward the utricular duct and the region of the valve. bp basilar papilla, ca crista in anterior ampulla, cl crista in lateral ampulla, cp crista in posterior ampulla, es endolymphatic sac, ml macula of the lagena, mn macula neglecta, ms saccular macula, mu utricular macula the slit - shaped valve entrance at the urticular side (fig. 6) resembles this entrance in the guinea pig, as shown in fig. bast also referred to this opening as slit - like in the guinea pig.fig. 83d - reconstruction of the entrance (utriculo - endolymphatic duct / bast s valve) of the utricular duct in guinea pig as seen from inside the utricule (hofman. 2008) 3d - reconstruction of the entrance (utriculo - endolymphatic duct / bast s valve) of the utricular duct in guinea pig as seen from inside the utricule (hofman. 2008) shape and diameter of the utricular duct are different in the guinea pig and the pigeon. in the guinea pig the shape of the duct is that of a flat funnel with a diameter in the order of 10 m at its narrowest passage. in this way, the shape of the duct is that of a tube with an elliptical cross section with a largest width of about 200 m along its whole course. a structure comparable to the utriculo - endolymphatic (bast s) valve in mammals can be found in the pigeon. like in mammals its function is possibly the protection of superior part of the labyrinth against endolymph loss caused by a rupture of the membrane surrounding the inferior part. | the first description of the presence of a utriculo - endolymphatic valve in human fetuses was given by bast in 1928. since then this valve - like structure is called bast s valve. its exact function has not yet been established. the general opinion is that it has a protective function by having the possibility to separate the superior endolymphatic compartments of the labyrinth from the inferior compartment. phylogenetically seen birds are the first vertebrates with a cochlear duct and a distinct inferior and superior part of the labyrinth. a structure in the pigeon inner ear, resembling bast s valve in mammals, is described. |
ch is also one of the most classic diseases in newborn screening history, the incidence of which varied from 1:2000 to 1:4000 in different areas and different ethnicities. pituitary thyroid axis, which leads to a reduction of thyroxin secretion, which, in turn, causes severe damage to the brain. according to the clinical course of the disease, ch can be classified as either permanent or transient ch (pch / tch). it was reported that the incidence of pch was about 1:2000 to 1:3587 and that the incidence of tch was about 1:1580 to 1:17 000. the major etiology of pch was linked to dysgenesis, including complications such as athyreosis, atrophy, and ectopic, but the etiology of tch was not that clear. many studies suggested that the etiology of tch was multiplicity, prematurity, low birth weight, and iodine deficiency or excessive use of antithyroid drugs. in addition, an interaction between genes and the environment may also be related to the occurrence of tch. up to now, there have been very few reports regarding the incidence and etiology of pch and tch in china. guangxi province, which is located in the southwest of china, is the largest minority nationality area for the han and zhuang nations. its geography and ethnic makeup means that it is different from other areas of china. a previous study reported the incidence of ch as detected by nsp to be about 1:835 in guangxi, which is much higher than the average for the whole country. but the incidence of confirmed cases of pch and tch among patients and the reasons of the high incidence of ch in this area are still unclear and is yet to be fully reported. the objective of this study was to confirm the high incidence of ch as detected by nsp and look at the interrelated factors linked to the major types of ch in guangxi. a total of 930 612 newborns (511 555 females and 419 057 males) screened by the newborn screening program (nsp) at newborn screening center of guangxi, china, from january 2009 to december 2013, were included in this study, and the screening rate was about 93.1%. within 72 hours of birth, 3 drops of blood was taken from the heel of subjects and placed on a filter paper (s&s 903) for the measurement of thyroid stimulating hormone (tsh). patients with positive results at screening were recalled and a 90% recall rate was achieved. venous blood samples were collected from the positive patients for a confirmation test by the chemiluminescence method. at this time serum blood tsh and free thyroxin (ft4) levels after patients were confirmed to be positive for ch, it was still necessary to check the thyroid morphology by performing a thyroid ultrasound examination. for those patients where it was not possible to locate the thyroid by ultrasound, it was necessary to perform further scans using a radioisotope scanning technique. at the first diagnosis stage when recalled, patients were classified as ch, when their clinical profiles matched one of the following : (a) tsh 10 miu / ml and ft4 < 12 pmol / l and (b) the thyroid imaging examination suggested athyreosis, atrophy, or ectopic. other patients with elevated tsh continually, but normal ft4 values and normal thyroid morphology, were classified as hyperthyroxinemia (some studies also classified these patients as suffering from subclinical hypothyroidism). at the second diagnosis stage, after being followed - up for more than 2 years, patients were classified as pch, when their clinical profiles matched the following : patients who were unable to stop treatment and therefore needed constant hormone replacement therapy. those patients whose clinical profiles matched both of the following were classified as tch : (a) the thyroid imaging examination indicated the location and morphology of the thyroid to be normal and (b) the thyroid remained functional for more than 6 months after treatment was stopped and the thyroid function test would continue to be performed monthly. data were analyzed using spss 13.0, and the relationship between the multiplicity of factors and the results of the second diagnosis were analyzed with multiple logistic regression analysis. a total of 1210 patients (659 males and 551 females) were confirmed as potential ch cases among 930 612 newborns. of these the incidence of ch and hyperthyroxinemia was 60.0/100 000 (1/1665) and 69.9/100 000 (1/1430), respectively, with the total being 120.0/100 000 (1/769). in the 1210 confirmed patients, 391 patients (32.2% of the total including 331 hyperthyroxinemia patients and 60 patients with congenital hypothyroidism) were lost to follow - up, and thus 819 patients took part in the study. from december 2009 to december 2013, 273 patients were followed - up for more than 2 years, and 68 patients were diagnosed with pch, 126 patients were diagnosed with tch, and 78 patients were still under review (figure 1). based on screening of the population at the present time, the incidence of pch was 15/100 000 (1/6673), the incidence of tch was 29.5/100 000 (1/3385), and the ratio of pch to tch is approximately 1:2. distribution of permanent and transient ch in 273 patients followed - up for more than 2 years. in 194 patients (68 with pch and 126 with tch) with clear diagnosis of the disease, multiple logistic regression analysis was performed. the final diagnosis was considered as the dependent variable (pch / tch, 0/1), and the gender, gestational age, birth weight, and tsh and ft4 values at screening and diagnosis were considered as independent variables. the results indicate that tsh values at screening and diagnosis were correlated with tch. however, gender, gestational age, and birth weight were not the independent risk factors for tch (table 1). the area under the roc curve (auc) of tsh values at screening and diagnosis were 0.78 and 0.73 and the p values were 1.16 10 and 1.08 10, receptively. abbreviations : ch, congenital hypothyroidism ; auc, area under the curve ; tsh, thyroid stimulating hormone. p <.05 was considered statistical significant. to further analyze the reasons for the high incidence of ch, the tsh values of 930 612 newborns were classified following the guidelines for iodine deficiency as denoted by the world health organization. the results show that the tsh values of 7.2% newborns (67 208 out of 930 612 newborns) were above 5 miu / ml (table 2). the frequency of tsh values greater than 5 miu / l in newborns from 2009 to 2013. the nsp has been developed in guangxi since 1996, but in the early years of implementation the screening rate was lower than 10%. from 2009, when the government actively promoted the nsp in the whole province, the newborn screening laboratory of guangxi accounts for around 50% of the newborns in guangxi each year. the overall incidence of the disease was 1/769, with the incidence for ch and hyperthyroxinemia being 1/1665 and 1/1430, respectively. in the cities of beijing, shanghai, and zhejiang, the incidence of ch was reported as 1/2034, 1/2050, and 1/1618, respectively, and the incidence of the whole country was found to be approximately 1/2000. in this study, for patients with a final diagnosis made at the second diagnosis stage, the ratio of pch to tch was about 1:2, with tch being in the majority, and this was also different from other reports, and indicated that the higher incidence of ch in guangxi is mainly due to increased tch. the etiology of tch is complex, with several factors correlating to the disease including prematurity, low birth weight, intake of iodine, as well as genetic factors. based on the available data of gender, gestational age, birth weight, and tsh and ft4 values, the cause of the high incidence of ch was investigated by multivariate logistic regression analysis. the results indicate that tsh values were related to tch, and gender, gestational age, and birth weight were not independent risk factors for tch. for further analyses, the guidelines for determination of iodine deficiency as laid down by the world health organization the world health organization guidelines are based on neonatal tsh values of above 5 miu / l of whole blood and a frequency of less than 3% of the population is considered iodine deficient. frequencies of 3% to 19.9% indicate mild iodine deficiency disorders, frequencies of 20% to 39.9% and above 40% indicate moderate and severe iodine deficiency disorders, respectively. in 930 miu / l was about 7.2% and this indicated there was mild iodine deficiency in the population in guangxi. iodine is distributed all over the natural world, especially in the soil and water, but unfortunately it is not evenly distributed. the content of iodine in the soil of mountain areas tends to be the lowest with the highest levels found on the plains. the landscape of guangxi is made up of karst landforms, with the surface of the earth being rough and with a poor soil quality. both these conditions discourage the storage of iodine in the soil and therefore the water and crops inevitably possess low iodine content in comparison with other regions. our data would strongly suggest that the iodine deficiency may well be related to the high prevalence of tch in guangxi. the overall incidence of ch in guangxi was relatively high in comparison to other areas of china. among those patients, the majority had tch and this may be related to iodine deficiency in the population. future work should focus on determination of urinary iodine in the local population in order to clarify these findings. it would also be useful to explore whether the geographic and ethnic makeup of the guangxi region of china contribute to presence of tch in population by determining whether gene mutations are contributing factors. it is important to determine the etiology of the disease so that preventative strategies can be implemented to improve health care within these communities. | background. a newborn screening program (nsp) for congenital hypothyroidism (ch) was carried out in guangxi in order to understand the incidence of ch and the factors interrelated to major types of ch in this region of china. methods. during 2009 to 2013, data from 930 612 newborns attending nsp in guangxi were collected. patients were classified with either permanent ch (pch) or transient ch (tch) after 2 years of progressive study. results. a total of 1210 patients were confirmed with ch with an incidence of 1/769, including 68 pch and 126 tch cases with incidences of 1/6673 and 1/3385, respectively. the frequency of thyroid stimulating hormone values greater than 5 miu / l was 7.2%, which, based on who guidelines, suggests that the population was mildly iodine deficient. conclusions. the incidence of ch was high in guangxi. approximately two thirds of ch patients were tch, which may be due to a deficiency in iodine within the population. |
silica nanoparticles have attracted much attention for industrial and biomedical applications, such as for use as additives and in printer toners, varnishes, pharmaceutics, cosmetics, and coating materials.1,2 these wide - ranging applications stem from the properties of silica nanoparticles, which include easy synthesis, low toxicity, hydrophilicity, and the ease with which their surfaces can be modified or functionalized.35 hence, silica nanoparticles have been extensively developed for biological purposes for use as biomarkers, biosensors, dna or drug delivery, and cancer therapy.610 in this context, many studies have recently focused on biological effects of silica nanoparticles at different levels, such as on their cytotoxicities, blood compatibilities, acute and repeat dose toxicities, and biokinetics,1119 which are currently hot issues in the nanotoxicology field. however, the kinetic behaviors of silica nanoparticles at the systemic level, including their pharmacokinetics, tissue distributions, and clearances, remain unclear. in vivo biokinetic studies can be conducted by systematic and quantitative analyses of plasma, tissues, urine or feces, and other biological samples in whole animals after exposure to assess absorption, distribution, metabolism, and excretion. an understanding of the kinetic behaviors of silica nanoparticles is of importance in the context of determining absorption amounts, target organs, and residence times, which are essential for the prediction of potential adverse effects in the short- and long - term. some researchers have recently described the biodistribution and excretion kinetics of silica nanoparticles.2025 however, most of this information was obtained after intravenous injection, which introduces nanoparticles directly into the circulatory system. in practice, oral administration is important, for example, in food or water, and results in kinetic behaviors unlike those associated after intravenous injection because nanoparticles must encounter stomach acid and cross the epithelium of the gastrointestinal (gi) tract in order to reach the blood circulation. in addition, even after gi transit, nanoparticles are carried to the liver via the portal vein before entering the systemic circulation and, thus, are subject to metabolic processes that evidently reduce bioavailability. the biokinetics of silica nanoparticles at the systemic level has been less extensively explored than other inorganic nanoparticles, which appears to be due to the difficulty of detecting silica nanoparticles in biological matrices.20 the major approaches used to trace and determine their kinetics in vivo are based on the use of dye - conjugated or dye - embedded particles and subsequent fluorescence detection by microscopy or some other imaging modality.20,23,24 however, this strategy is limited with respect to the interpretation of results as dye - conjugated particles are structurally modified and, thus, molecular weights and surface charges are changed. these changes could affect biological interactions at the systemic level and could eventually modify kinetic behaviors. furthermore, the stabilities of dye - conjugated or dye - embedded particles in whole animals are also necessary considerations that complicate the interpretations of quantitative analyses. in our previous study, we devised a quantitative analytical method for measuring the amounts of silica nanoparticles in biological matrices based on a lithium borate fusion technique with a molybdenum blue spectrophotometric method.26 in the present study, we evaluated the tissue distributions and elimination kinetics of 20 nm and 100 nm silica nanoparticles after administering a single oral dose to male and female rats. in addition, the biological fates of the silica nanoparticles in target organs were determined by transmission electron microscopy (tem). colloidal silica nanoparticles (20 nm and 100 nm, dispersed in distilled water [dw ]) were purchased from e and b nanotech co, ltd. (gyeonggi - do, republic of korea) and analyzed by tem (jem-1010 ; jeol, tokyo, japan). the surface charges (zeta potentials) of nanoparticles were determined using a zeta potentiometer (zetasizer nano zs system ; malvern instruments, malvern, uk). five - week - old male and female sprague dawley rats weighing 120140 g were purchased from g - bio (seoul, republic of korea). the animals were housed in plastic lab animal cages in a ventilated room, which was maintained at 20c2c and 60%10% relative humidity under a 12-hour light / dark cycle. all animal experiments were performed in compliance with the guidelines issued by the animal and ethics review committee of seoul women s university. four groups of male and of female rats (n=6 per group) were administered a single dose of 500 or 1,000 mg / kg of nanoparticles (20 nm and 100 nm) by oral gavage. body weights, behavioral changes, and other symptoms were carefully recorded daily after treatment. for the tissue distribution study, samples of brain, heart, kidneys, liver, lungs, spleen, and testes or ovaries were collected at 1 and 6 hours, and 1, 2, 3, and 7 days post - administration after co2-induced euthanasia. to evaluate excretion profiles, urine and feces were collected at 10 hours, and 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days post - administration. silicon (si) analysis in biological samples was performed as previously described.26 briefly, a 100 mg sample was placed in a graphite crucible and 200 mg of lithium metaborate (libo2 ; sigma - aldrich, st louis, mo, usa) was added. after mixing, the crucible was heated in a furnace for 25 minutes at 1,025c, and then 2 ml of distilled deionized water (ddw), 2 ml of nitric acid (hno3 ; 70%), 2 ml scandium oxide solution (250 g / ml), and one drop of hydrogen peroxide (h2o2) were added. this mixture was then heated on a hot plate until the ash had been completely dissolved. the remaining solution was then removed by heating, and 5 ml of ddw was added. for si quantification by the molybdenum blue method, 2 to 3 drops of hydrofluoric acid (48%) were added to 1 ml of this solution, and 5 ml of ammonium molybdate solution (65 g / l) and 4 ml of hydrogen chloride (hcl ; 3.7%) were added. after adding 20 ml of ddw, the mixture was left to stand for 30 minutes, and 2 ml of oxalic acid, 2 ml of sodium sulfite solution (150 finally, 1 ml of stannous chloride solution (sncl2 50 g in 100 ml of hcl and 50 ml of ddw) was added and absorbance was measured at 820 nm (smp500 - 16509-sicx ; molecular devices llc, sunnyvale, ca, usa). quantitative analysis was carried out by external four - point - calibration with internal standard correction using spiking experiments. representative organs, such as livers and kidneys, were collected from three silica nanoparticle (1,000 mg / kg)-administered male rats 48 hours post - administration. samples were fixed using modified karnovsky s fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.05 m sodium cacodylate buffer [ph 7.2 ]), postfixed with 1:1 solution of 2% osmium tetroxide and 0.1 m sodium cacodylate for 2 hours at 4c, and then stained with uranyl acetate. blocks were sectioned using an ultramicrotome (mt - x ; boeckeler instruments, inc., tucson, az, usa) and, after coating with high purity carbon rod (ted pella, inc., redding, ca, usa), tem images were obtained using a tecnai g2 unit, equipped with an energy dispersive spectroscopy (eds) facility, at the korea basic science institute (kbsi ; gwangju branch, republic of korea). the data are presented as means standard deviations. for statistical analysis, experimental values one - way analysis of variance in sas software (tukey s test, version 11.0 ; sas institute inc., cary, nc, usa) was used to determine the significances of differences between experimental groups and controls. colloidal silica nanoparticles (20 nm and 100 nm, dispersed in distilled water [dw ]) were purchased from e and b nanotech co, ltd. (gyeonggi - do, republic of korea) and analyzed by tem (jem-1010 ; jeol, tokyo, japan). the surface charges (zeta potentials) of nanoparticles were determined using a zeta potentiometer (zetasizer nano zs system ; malvern instruments, malvern, uk). five - week - old male and female sprague dawley rats weighing 120140 g were purchased from g - bio (seoul, republic of korea). the animals were housed in plastic lab animal cages in a ventilated room, which was maintained at 20c2c and 60%10% relative humidity under a 12-hour light / dark cycle. all animal experiments were performed in compliance with the guidelines issued by the animal and ethics review committee of seoul women s university. four groups of male and of female rats (n=6 per group) were administered a single dose of 500 or 1,000 mg / kg of nanoparticles (20 nm and 100 nm) by oral gavage. body weights, behavioral changes, and other symptoms were carefully recorded daily after treatment. for the tissue distribution study, samples of brain, heart, kidneys, liver, lungs, spleen, and testes or ovaries were collected at 1 and 6 hours, and 1, 2, 3, and 7 days post - administration after co2-induced euthanasia. to evaluate excretion profiles, urine and feces were collected at 10 hours, and 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days post - administration. silicon (si) analysis in biological samples was performed as previously described.26 briefly, a 100 mg sample was placed in a graphite crucible and 200 mg of lithium metaborate (libo2 ; sigma - aldrich, st louis, mo, usa) was added. after mixing, the crucible was heated in a furnace for 25 minutes at 1,025c, and then 2 ml of distilled deionized water (ddw), 2 ml of nitric acid (hno3 ; 70%), 2 ml scandium oxide solution (250 g / ml), and one drop of hydrogen peroxide (h2o2) were added. this mixture was then heated on a hot plate until the ash had been completely dissolved. the remaining solution was then removed by heating, and 5 ml of ddw was added. for si quantification by the molybdenum blue method, 2 to 3 drops of hydrofluoric acid (48%) were added to 1 ml of this solution, and 5 ml of ammonium molybdate solution (65 g / l) and 4 ml of hydrogen chloride (hcl ; 3.7%) were added. after adding 20 ml of ddw, the mixture was left to stand for 30 minutes, and 2 ml of oxalic acid, 2 ml of sodium sulfite solution (150 finally, 1 ml of stannous chloride solution (sncl2 50 g in 100 ml of hcl and 50 ml of ddw) was added and absorbance was measured at 820 nm (smp500 - 16509-sicx ; molecular devices llc, sunnyvale, ca, usa). quantitative analysis was carried out by external four - point - calibration with internal standard correction using spiking experiments. representative organs, such as livers and kidneys, were collected from three silica nanoparticle (1,000 mg / kg)-administered male rats 48 hours post - administration. samples were fixed using modified karnovsky s fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.05 m sodium cacodylate buffer [ph 7.2 ]), postfixed with 1:1 solution of 2% osmium tetroxide and 0.1 m sodium cacodylate for 2 hours at 4c, and then stained with uranyl acetate. blocks were sectioned using an ultramicrotome (mt - x ; boeckeler instruments, inc., tucson, az, usa) and, after coating with high purity carbon rod (ted pella, inc., redding, ca, usa), tem images were obtained using a tecnai g2 unit, equipped with an energy dispersive spectroscopy (eds) facility, at the korea basic science institute (kbsi ; gwangju branch, republic of korea). the data are presented as means standard deviations. for statistical analysis, experimental values one - way analysis of variance in sas software (tukey s test, version 11.0 ; sas institute inc., cary, nc, usa) was used to determine the significances of differences between experimental groups and controls. colloidal silica nanoparticles of 20 nm and 100 nm were characterized by tem and by measuring zeta potentials (table 1). as previously reported,26 the average particle sizes of these silica nanoparticles were 153 nm and 8914 nm, respectively, and both had a spherical morphology and were negatively charged in dw at ph 7.0. survival rates, body weights, behaviors, and symptoms were carefully observed for 14 days post - administration. male and female rats that were administered the two differently sized silica nanoparticles up to 1,000 mg / kg showed no body weight loss, abnormal behaviors, or symptoms as compared with untreated controls (figure 1). the biodistribution of silica nanoparticles was examined in brain, kidneys, liver, lungs, spleen, and ovaries / testes. total si concentrations in tissues were analyzed, as described above, by determining increases in total si levels in silica - administered rats versus untreated controls. si levels were found to be significantly higher in kidneys, liver, lungs, and spleen of treated rats regardless of dose, particle size, or sex (figure 2). notably, high si concentrations were found in kidneys and livers at 6 hours to 3 days post - administration, whereas elevated si levels were detected at 6 hours to 2 days in lungs and spleens. increases in si concentrations in the gi tract (esophagus, stomach, and intestine) were not detected at 7 days post - administration. tem analysis of organs from silica - administered rats was carried out to confirm tissue distributions and to determine the biological fates of nanoparticles in target tissues. as shown in figure 3, silica nanoparticles of both 20 nm and 100 nm were observed in liver, and these had the same spherical morphology and particle sizes observed prior to administration (table 1). in particular, differently sized silica nanoparticles were localized in hepatocytes as well as in nuclei. on the other hand, irregular particle shapes were observed in kidneys, and more so in rats treated with 20 nm silica nanoparticles. tem - eds confirmed the presence of si in the particulate forms in livers and kidneys. the excretion kinetics of silica nanoparticles was evaluated by measuring increases in si levels in urine and feces. significantly, higher si concentrations in urine were detected, regardless of sex, at 12 days and 15 days after administering 20 nm silica nanoparticles at 500 or 1,000 mg / kg, respectively (figure 4). however, elimination in urine was slower after the administration of 100 nm particles, which produced elevated si levels at 13 days and 16 days at doses of 500 and 1,000 mg / kg, respectively. a similar tendency was found for fecal excretion profiles ; si concentrations were elevated at 13 days and at 14 days after the administration of 20 nm and 100 nm nanoparticles, respectively, at doses of 500 and 1,000 mg / kg, showing size - dependent elimination kinetics. it is worth noting that much higher si levels were detected in feces than in urine. colloidal silica nanoparticles of 20 nm and 100 nm were characterized by tem and by measuring zeta potentials (table 1). as previously reported,26 the average particle sizes of these silica nanoparticles were 153 nm and 8914 nm, respectively, and both had a spherical morphology and were negatively charged in dw at ph 7.0. survival rates, body weights, behaviors, and symptoms were carefully observed for 14 days post - administration. male and female rats that were administered the two differently sized silica nanoparticles up to 1,000 mg / kg showed no body weight loss, abnormal behaviors, or symptoms as compared with untreated controls (figure 1). the biodistribution of silica nanoparticles was examined in brain, kidneys, liver, lungs, spleen, and ovaries / testes. total si concentrations in tissues were analyzed, as described above, by determining increases in total si levels in silica - administered rats versus untreated controls. si levels were found to be significantly higher in kidneys, liver, lungs, and spleen of treated rats regardless of dose, particle size, or sex (figure 2). notably, high si concentrations were found in kidneys and livers at 6 hours to 3 days post - administration, whereas elevated si levels were detected at 6 hours to 2 days in lungs and spleens. increases in si concentrations in the gi tract (esophagus, stomach, and intestine) were not detected at 7 days post - administration. tem analysis of organs from silica - administered rats was carried out to confirm tissue distributions and to determine the biological fates of nanoparticles in target tissues. as shown in figure 3, silica nanoparticles of both 20 nm and 100 nm were observed in liver, and these had the same spherical morphology and particle sizes observed prior to administration (table 1). in particular, differently sized silica nanoparticles were localized in hepatocytes as well as in nuclei. on the other hand, irregular particle shapes were observed in kidneys, and more so in rats treated with 20 nm silica nanoparticles. tem - eds confirmed the presence of si in the particulate forms in livers and kidneys. the excretion kinetics of silica nanoparticles was evaluated by measuring increases in si levels in urine and feces. significantly, higher si concentrations in urine were detected, regardless of sex, at 12 days and 15 days after administering 20 nm silica nanoparticles at 500 or 1,000 mg / kg, respectively (figure 4). however, elimination in urine was slower after the administration of 100 nm particles, which produced elevated si levels at 13 days and 16 days at doses of 500 and 1,000 mg / kg, respectively. a similar tendency was found for fecal excretion profiles ; si concentrations were elevated at 13 days and at 14 days after the administration of 20 nm and 100 nm nanoparticles, respectively, at doses of 500 and 1,000 mg / kg, showing size - dependent elimination kinetics. it is worth noting that much higher si levels were detected in feces than in urine. the tissue distribution, excretion, and in vivo fates of colloidal silica nanoparticles (20 nm and 100 nm) were investigated after a single oral administration in male and female rats. particle sizes of 20 nm and 100 nm were examined, because the sizes of most of the nanoparticles developed fall in the range 1100 nm, and because these sizes varied enough to evaluate the effect of particle size on biokinetics. after a single - dose administration of silica nanoparticles, survival rates and body weight gains furthermore, no abnormal behaviors or symptoms, such as a decrease in food or water intake, diarrhea, loss of movement, or changes in pupil size or eye pigmentation, were observed during the 14 days post - administration. furthermore, the same results were obtained for different particle sizes in male and female rats. low toxicity of silica nanoparticles following oral administration was also recently reported by fu ;27 110 nm mesoporous silica nanoparticles caused no immediate toxicity, such as loss of appetite, loss of weight, death, or passive behaviors, in mice after oral administration of 5,000 mg / kg. ivanov demonstrated that intravenously injected silica nanoparticles (13 nm, 7 mg / kg) are relatively biocompatible nanomaterials when considering acute toxicity.28 it seems that silica nanoparticles do not exhibit acute toxicity at the dosages used in this study, although more extended study is needed to confirm their toxicity, for example, by biochemical analysis and histopathological examination. our previous study showed that plasma concentration - time curves for single oral doses of silica nanoparticles (20 nm and 100 nm) at 500 and 1,000 mg / kg rapidly decreased within 4 hours and 10 hours, respectively, in a dose - dependent manner, after oral administration to rats, regardless of particle size or sex.26 absorption amount was determined to be low, ranging from 6.6%9.7%, without being affected by particle size or sex.26 in our biodistribution study, significantly elevated si concentrations were detected in kidneys, liver, lungs, and spleens within 3 days, but returned to normal level after 7 days. when tissue distribution kinetics was compared, nanoparticles were found to persist longer in kidneys and liver than in lungs and spleen (3 days versus 12 days), and tissue distribution patterns were not dependent on particle size or sex. furthermore, the fact that silica nanoparticles were found in liver, lungs, and spleen also indicates that the nanoparticles were sequestered into organs by the mononuclear phagocytic system, also known as the reticuloendothelial system (res), which implies phagocytosis is involved in their uptake. increased si levels in kidneys can be closely related to the excretion pathway of the silica nanoparticles in urine. most biodistribution studies on silica nanoparticles have been conducted by administering a single intravenous injection. he reported the accumulation of spherical mesoporous silica nanoparticles (80360 nm) labeled with fluorescein isothiocyanate in liver and spleen and to a lesser extent in lungs, kidneys, and heart in rats for 5 days post - injection. he demonstrated a similar distribution pattern for 45 nm silica nanoparticles using an in vivo fluorescence imaging system. borak reported that 150 nm silica nanoparticles accumulated primarily in the lungs and kidneys, and less so in hearts and liver at 4 days post - injection.29 huang reported that mesoporous silica nanoparticles with different aspect ratios mainly accumulated in the res of liver, spleen, and lungs, and slightly in kidneys for 7 days in mice (> 80%).22 cho reported the accumulation of fluorescence dye - labeled silica nanoparticles of 50 nm, 100 nm, or 200 nm in liver, spleen, and kidneys at 24 hours post - injection, and their persistence in liver and spleen at 4 weeks in mice.24 on the other hand, malfatti investigated the long - term biodistribution of carbon-14-labeled silica nanoparticles of 33 nm in mice, and demonstrated their persistence in the res of liver, spleen, and lungs over 56 days following injection.25 taking our result and those of others into account, it appears that the liver, spleen, lungs, and kidneys are targeted by silica nanoparticles regardless of animal, silica type, or exposure routes. however, no report has demonstrated the in vivo biological fate of silica nanoparticles. in the present study, interestingly, clear intact spherical silica particles with almost the pre - administered particle size (table 1 and figure 3) were found in livers treated with 20 nm and 100 nm nanoparticles. the intracellular localization of nanoparticles was determined to be hepatocytes as well as nuclei regardless of particle size. it was reported that silica nanoparticles are internalized into cells, localized throughout cellular compartments, and subsequently penetrate into the cell nucleus.30,31 thus, the toxicity of silica nanoparticles in terms of inhibition of gene expression has to be considered. nanoparticles with an irregular (decomposed) morphology were observed in kidneys, especially when 20 nm particles were administered. the presence of si in the particulate forms was also confirmed by tem - eds. this result strongly suggests that silica nanoparticles were sequestered into liver in their intact particulate forms, but slowly decomposed or dissolved in kidneys. the excretion kinetics study showed that nanoparticles can be eliminated by urinary excretion over 2 and 5 days following the administration of 500 mg / kg and 1,000 mg / kg 20 nm particles, respectively, whereas slower excretion profiles (over 3 and 6 days) were observed for 100 nm particles. this result implies that smaller silica nanoparticles are more rapidly eliminated from the body, possibly due to their greater decomposition rates in the kidneys (figure 3). furthermore, molecules with a hydrodynamic diameter of < 6 nm can pass through the glomerular membrane,32 thus, the presence of silica nanoparticles in kidneys and urine suggests that the nanoparticles are biodegraded prior to urinary excretion. it was reported that silica nanoparticles undergo gradual biodegradation both in vitro and in vivo, resulting in the formation of silicic acid (ortho-, meta-, di-, and trisilicates) by hydrolysis.33,34 predominant excretion forms of silica nanoparticles in urine are known to be silicic acid or oligomeric silica species.35,36 thus, sodium or potassium silicic acid salts could be excreted from the organs with the urine. furthermore, it seems that absorbed nanoparticles (about 6.6%9.7%) are excreted by the urinary system. however, the majority of nanoparticles (about 75%80%) were directly excreted via feces, regardless of particle size or sex (table 3). this finding suggests that fecal and biliary excretion routes play major roles in the elimination of silica nanoparticles. in the present study, it was likely that 10%15% of administered nanoparticles remain in body tissues. several studies have demonstrated the urinary excretion of silica nanoparticles, but after intravenous injection. in one study, in vivo fluorescence imaging system in mice showed that 45 nm silica nanoparticles were eliminated via the renal route.20 in another study, the urinary excretion of mesoporous silica nanoparticles (80360 nm) was followed using a real - time in vivo imaging system,23 and, in another, 36% of 150 nm silica particles were reported to be excreted over 4 days in urine.29 however, the fecal excretion route was not examined in these studies. huang and cho investigated both the fecal and urinary excretions of silica nanoparticles in mice,22,24 but they did not calculate the amounts of silica particles excreted via urine and feces. based on our findings and previous results in the literature, both renal and fecal routes are involved in the elimination of silica nanoparticles. the effects of particle size (20 nm and 100 nm) on the tissue distribution and excretion of colloidal silica nanoparticles were investigated following a single oral administration to male and female rats. nanoparticles were found to target the kidneys, liver, lungs, and spleen regardless of particle size or sex. the primary biological fate of silica nanoparticles was found to be in particulate form in tissues. specifically, intact particulates were found in liver, but decomposed morphologies of particulates were observed in kidneys, suggesting possible nanoparticle degradation in vivo. urinary and fecal excretion kinetics were found to be size - dependent, and 20 nm nanoparticles were eliminated faster that 100 nm nanoparticles, possibly due to the more rapid decomposition of 20 nm nanoparticles. these findings will be of interest to researchers seeking to predict the potential toxicological effects of silica nanoparticles on target organs. | purposethe effects of particle size on the tissue distribution and excretion kinetics of silica nanoparticles and their biological fates were investigated following a single oral administration to male and female rats.methodssilica nanoparticles of two different sizes (20 nm and 100 nm) were orally administered to male and female rats, respectively. tissue distribution kinetics, excretion profiles, and fates in tissues were analyzed using elemental analysis and transmission electron microscopy.resultsthe differently sized silica nanoparticles mainly distributed to kidneys and liver for 3 days post - administration and, to some extent, to lungs and spleen for 2 days post - administration, regardless of particle size or sex. transmission electron microscopy and energy dispersive spectroscopy studies in tissues demonstrated almost intact particles in liver, but partially decomposed particles with an irregular morphology were found in kidneys, especially in rats that had been administered 20 nm nanoparticles. size - dependent excretion kinetics were apparent and the smaller 20 nm particles were found to be more rapidly eliminated than the larger 100 nm particles. elimination profiles showed 7%8% of silica nanoparticles were excreted via urine, but most nanoparticles were excreted via feces, regardless of particle size or sex.conclusionthe kidneys, liver, lungs, and spleen were found to be the target organs of orally - administered silica nanoparticles in rats, and this organ distribution was not affected by particle size or animal sex. in vivo, silica nanoparticles were found to retain their particulate form, although more decomposition was observed in kidneys, especially for 20 nm particles. urinary and fecal excretion pathways were determined to play roles in the elimination of silica nanoparticles, but 20 nm particles were secreted more rapidly, presumably because they are more easily decomposed. these findings will be of interest to those seeking to predict potential toxicological effects of silica nanoparticles on target organs. |
surgical aortic valve replacement (avr) still represents the gold standard among the therapeutic options in patients with severe symptomatic aortic valve stenosis. currently, patients often have a heavily calcified valve, aortic root or diffuse atherosclerosis of the aortic wall and have already undergone a previous aortic valve replacement. in order to minimize periprocedural risks and to accelerate postoperative rehabilitation, less invasive therapeutic concepts, including transcatheter aortic valve implantation (tavi) and sutureless bioprosthesis, have been developed and are increasingly used while maintaining quality and safety, especially in gray zone patients. the need for concomitant mitral valve surgery is generally viewed as a contraindication to sutureless avr because of the increased risk of interference between the two valves at the level of aorto - mitral continuity. we present the case of a 71-year - old female patient with a combination of severe stenosis of the stentless bioprosthesis and regurgitation grade ii / iv due to right coronary cusp separation. the peak transvalvular gradient was 56 mm hg and the mean gradient was 30 mm hg. concomitant severe mitral valve insufficiency grade iv / iv was present due to annulus dilatation. the ascending aorta and bicaval cannulation technique was used for initiating the cardiopulmonary bypass (cpb). a transverse aortotomy was done 1 cm distal to the sino - tubular junction, so as to leave an edge free for closure of the aortotomy after implantation of the device and to prevent closure of the aortotomy. because of a very small aortic annulus (free passage through the annulus with a 19 mm mechanical aortic valve sizer), the perceval s (sorin group, milan, italy) size small s access to the mitral valve was performed through sondergaard s groove, and mitral valve repair was performed with a semi - rigid medtronic cg future composite ring no. the selected aortic bioprosthesis perceval s was loaded and collapsed into a delivery device. to ensure correct positioning of the prosthesis, three guiding threads are temporarily positioned in the lowest part of the native leaflet insertion line for each valve sinus and the corresponding part of the bioprosthesis. once the prosthesis was completely deployed, the guiding threads were removed. to optimize the area of contact between the prosthesis and the aortic annulus, post - dilatation was carried out with a balloon catheter at a pressure of 4 atm for 30 s. the aortic cross clamp time was 74 min. the control periprocedural transesophageal echocardiogram did not indicate any paravalvular aortic regurgitation ; there was no evidence of interference between the aortic prosthesis and mitral valve ring and no evidence of mitral dysfunction. at 1-year follow - up the patient was doing well, was in nyha class 0 and showed improved symptoms in comparison with her preoperative state. transthoracic echocardiography (tte) follow - up indicated mean and peak gradients of 15 and 25 mm hg on the perceval valve and no paravalvular regurgitation. the mean mitral transvalvular gradient was 4 mm hg and mitral regurgitation grade i / iv was detected by tte. similar to conventional surgical replacement of the valve, a sutureless bioprosthesis requires valve excision (a risk reduction of paravalvular insufficiency compared with tavi) and annular decalcification, but permanent fixation sutures are not required. the perceval valve is designed for patients requiring an avr procedure, including high - risk and complex patients. with the absence of a rigid sewing ring and its elastic stent, the perceval optimizes the effective orifice area, resulting in excellent hemodynamics. the possibility to avoid placing and tying sutures may lead to shorter procedural times [2, 4 ]. in cardiac surgery, prolonged cpb and cross - clamp duration are strong independent risk factors for postoperative mortality and morbidity. the advantages of this procedure could be of benefit to patients who have no fundamental contraindications for using cardiopulmonary bypass and are undergoing complex, combined procedures or re - operations. patients with a small aortic annulus or heavy calcification of the annulus and aortic root, where positioning sutures may represent technical problems and complications, are another potential group that could benefit. it is important to accept some technical considerations that arise in the proximity of the mitral and aortic annulus at the level of aorto - mitral continuity. the cut - off point in terms of minimal aorto - mitral length for patients with a mechanical mitral prosthesis before tavi is 9 mm and is probably lower for sutureless avr, such as the perceval prosthesis [3, 6 ]. in our patient we did not measure the aorto - mitral distance. at the time of mitral valve replacement, the commissural struts could be positioned away from the aortomitral continuity to minimize the risk of interference with the intra - annular portion of the sutureless aortic prosthesis. for mitral valve repair our preferred approach is the use of a semi - rigid mitral annuloplasty ring, which offers posterior remodeling while maintaining anterior flexibility due to the presence of only textile in this part and may also minimize the risk of interference with the subannular portion of the sutureless valve in the left ventricle outflow tract. our experience demonstrates that concomitant sutureless aortic bioprosthesis implantation and mitral valve repair is feasible and safe in high - risk patients undergoing a redo operation. potential advantages include shorter aortic cross clamp times, fewer technical demands in the case of a heavily calcified and small aortic annulus, and the preservation of flexibility and movement of the mitral annulus during the cardiac cycle by using a semi - rigid annuloplasty ring. | sutureless aortic valve replacement (avr) was developed as an alternative treatment option to conventional open - heart surgery and transcatheter aortic valve implantation for gray zone patients. the need for concurrent mitral valve surgery is generally viewed as a contraindication to sutureless avr. the purpose of this brief paper is to report our experiences with sutureless valves in patients after previous cardiac procedures with degenerated aortic bioprostheses and concomitant mitral valve disease. |
over the past several decades, the entrapment of proteins in transparent, mesoporous silica has been of significant interest to scientists and engineers spanning a broad spectrum of disciplines. in more recent history, integral membrane proteins (imps) have been of particular interest for entrapment in sol gel - derived silica due to their differing functionalities that can be exploited to tailor these systems for accommodating various applications, such as biosensing, affinity chromatography, high - throughput drug screening, and bioreaction engineering. imps contain both hydrophobic and hydrophilic amino acid residues ; thus, they are either partially or completely embedded within amphiphilic lipid bilayers of cell membranes. the necessity of lipid bilayers for proper imp functionality requires an entrapment system that minimally modulates the physical and structural properties of the lipid bilayers ; direct modification of the lipid bilayer structures would adversely affect protein conformation within it. therefore, the investigation of the stability of lipid - bilayer - derived structures (i.e. nanolipoprotein particles and liposomes) entrapped within silica gel is essential to the development of viable, efficient imp - derived bioinorganic hybrid materials. during the 1990s, research groups of bright, friedman, kostic, and brennan examined the properties of various water - soluble proteins entrapped in silica gels derived from alkoxysilane precursors. their work spurred the development of optimized, biocompatible techniques for a variety of water - soluble proteins. one of the main techniques included the addition of glycerol and osmolytes, such as sugar, to alter protein hydration. however, this approach did not address the problematic presence of high concentrations of alcohol that resulted from the hydrolysis reactions of alkoxysilane precursors. the presence of alcohols is especially detrimental to lipid bilayers, as sufficiently high concentrations will lead to alcohol significantly partitioning into the bilayer, causing it to interdigitate. to address this, brennan s group further pioneered the development of biocompatible sol gel chemistries that consisted of modified alkoxysilane precursors bearing covalently attached sugar moieties and/or glycerol. depending on the specific precursor, the quantity of alcohol liberated during hydrolysis reactions was either greatly reduced or completely removed, and the additives were unable to leach from the gel. in 2002, besanger. examined the stability of 1,2-dipalmitoyl - sn - glycero-3-phosphatidylcholine (dppc) liposomes within silica gels derived from three different precursors : an unmodified alkoxysilane (tetraethyl orthosilicate or teos), an alkoxysilane with covalently attached glycerol (diglycerylsilane or dgs), and sodium silicate (ss). their work demonstrated that the use of dgs- and ss - derived gels permitted the dppc liposomes to exhibit phase transitions as they would in solution, while the use of teos - derived gels did not. also, they depicted, via confocal fluorescence imaging, that the liposomes are capable of undergoing a variety of conformational changes once trapped inside of the gel, forming aggregates or bicelles. though the dgs- and ss - derived silica gels seemed to work favorably at first, the dppc liposomes eventually lost the ability to undergo phase transitions several days later. halder. would corroborate this theory in 2004 by examining the solvation dynamics of coumarin 480 inside of liposomes entrapped in silica gel. silica interactions have elucidated that silica has a propensity for deforming liposomes, causing them to rupture and fuse to the surface. moreover, silica is actually a very common substrate for performing liposomal fusion to make supported lipid bilayers. an alternate approach for circumventing the problematic alcohol presence was presented in 2002 by ferrer. it consisted of a simple technique where rotary evaporation was used prior to incorporation of biological species. this approach was later utilized by luo. in 2005 to entrap liposomes bearing the imps bacteriorhodopsin and atp - synthase in silica gels derived from the precursor tetramethyl orthosilicate (tmos). though protein activity was observed after entrapment, the condition of the liposomal hosts was not examined. on the basis of their analysis, it is unknown whether or not the protein was functioning near its optimal activity, if the liposomes retained their structure over time, or if this approach would work well for other biologically significant imps. from these previous works, it can be seen that liposomes undergo structural changes and altered lipid dynamics upon entrapment ; thus, they are not optimal biological membrane hosts for imps inside of silica gel. in addition, the size mismatch of liposomes (100200 nm in solution) with mesoporous silica (550 nm pores) is a limiting factor in their successful implementation as biological membrane host for imps. here we look to improve upon the use of liposomes in silica gel by instead utilizing nanolipoprotein particles (nlps) as biological membrane hosts. nlps are discoidal patches of lipid bilayer associated with amphiphilic scaffold proteins that interact with the hydrophobic domain of the bilayer by wrapping around the particle periphery, making the entire structure water - soluble. nlps have an average thickness of 5 nm, with a diameter ranging from 10 to 25 nm depending on the stoichiometric ratios and types of lipids and scaffold proteins being used. this allows nlps to be more compatible with the pore size (550 nm) of mesoporous silica and bear more resemblance to water - soluble proteins the molecules for which this architecture was optimized than liposomes. therefore, here we perform entrapment of nlps using a quick, simple sol gel processing technique for tmos that includes evaporation of the majority of the methanol after the hydrolysis reactions. the lipid phase behavior of entrapped nlps in comparison to entrapped liposomes was observed using fluorescence anisotropy measurements, while the secondary structure of the scaffold protein was examined via circular dichroism spectroscopy. we found that liposomes exhibited more significant modulations in their phase behavior upon entrapment in silica gel than nlps and that modulations caused by residual methanol for both liposomes and nlps are relatively small. lipid interactions, thus strongly suggesting that there were minimal alterations in structure for nlps. our results demonstrate that nlps are more favorable for silica gel entrapment than liposomes. msp is a his - tagged membrane scaffold protein (msp1e3d1, sigma - aldrich, inc), which is comprised of residues 56243 of human apoa - i and a 22 amino acid n - terminal fusion containing the his tag, a spacer sequence, and the tev protease site. imidazole (99%), tmos (99%), 1,6-diphenyl-1,3,5-hexatriene (dph) (98%), ethanol (200 proof), sodium chloride (99%), methanol (99%), and sodium cholate (99%) were also purchased from sigma - aldrich, inc. 1,2-dipentadecanoyl - sn - glycero-3-phosphocholine (di15:0pc) was purchased in chloroform (10 mg / ml concentration) from avanti polar lipids, inc. the tris(hydroxymethyl)aminomethane (mb grade) and hydrochloric acid (12.1 n) used to prepare a tris - hcl buffer stock solution (500 mm, ph 7.5) were purchased from usb corp. and fisher scientific international, inc. all water used in these experiments was purified in a barnstead nanopure system (barnstead thermolyne, dubuque, ia) with a resistivity 17.9 mcm. for a single preparation, an appropriate aliquot of di15:0pc was removed from the 10 mg / ml chloroform stock solution, placed into a glass conical vial, dried with nitrogen, and then placed under mild vacuum for at least 4 h to fully evaporate all of the chloroform. the lipid film was then rehydrated with a reconstitution buffer (20 mm tris, 100 mm nacl, ph 7.4) to a final lipid concentration of 2 mg / ml and heated to 80 c for at least 5 min. after hydration and heating, the lipids were extruded through 100 nm pore membranes in an extruder (avestin, inc., finally, a small fraction of the resulting liposome solution was used for size determination via a particle size analyzer (brookhaven instruments corp. nlp batches were synthesized by first placing a stoichiometric excess (6 mg) of di15:0pc in chloroform solution inside of a glass conical vial. the contents were first dried using nitrogen and then placed under mild vacuum for at least 4 h. afterward, the dried lipid film was rehydrated and solubilized with a sodium cholate reconstitution buffer (40 mm sodium cholate, 20 mm tris, 100 mm nacl) and transferred to a plastic centrifuge tube, where it was allowed to further mix at room temperature (22 1 c) on a vortex mixer (fischer scientific, hampton, nh) for 30 min. next, 0.93 mg of his - tagged msp was added to the centrifuge tube and allowed to incubate at room temperature and 300 rpm for 1 h. after incubation, the nlp reaction mixture was transferred to a 10 000 mwco (molecular weight cutoff) dialysis filter (thermo scientific, rockford, il) and dialyzed against a reconstitution buffer (20 mm tris, 100 mm nacl) at 250 volume excess to remove cholate. dialysis was performed for 4 h at room temperature and 20 additional hours at 4 c. over the course of the 24 h dialysis, the buffer was exchanged three times (250 overall cholate dilution factor). nta agarose at 4 c for at least 2 h. the ratio of nlps and ni nta agarose was prepared such that the concentration of nlps was far below the maximum binding capacity of the ni after incubation, the agarose was separated via gentle centrifugation (500 rpm) and the supernatant aqueous phase was removed. centrifugation was also used during the subsequent wash and elution steps to separate the agarose from the aqueous phase. the agarose was washed four times with a wash buffer (20 mm imidazole, 20 mm tris, 100 mm nacl). the agarose was then eluted four times with an elution buffer (400 mm imidazole, 20 mm tris, 100 mm nacl). the supernatant liquid removed after each elution was concentrated using 100 000 mwco centrifugal concentrators (vivaproducts, inc. littleton, ma) and combined for a total volume of roughly 1 ml. nlps were then dialyzed again under the same conditions previously mentioned for the purpose of imidazole removal. the concentration of msp was determined using a uv vis spectrophotometer (shimadzu scientific instruments, columbia, md) to measure the concentration of msp via absorbance at 280 nm. the lipid concentration was determined by synthesizing a separate batch of nlps using di15:0pc that was laced with a trace amount of fluorescent oregon green 488 dhpe (life technologies, carlsbad, ca) and using an oregon green standard curve to measure fluorescence intensities of the resulting nlp batch with a fluorescence spectrophotometer (perkinelmer, inc., the lipid to protein ratio (4 mg:1 mg) was consistent with the expected ratio of msps to lipids (2:375). the size of the nlps was measured using a particle size analyzer (brookhaven instruments corp., the acquired stokes diameter was converted to a discoidal diameter using known thicknesses of phosphatidylcholine bilayers (see the supporting information). for a typical preparation, 5.6 ml of 0.01 m hcl in nanopure water was combined with 7.6 ml of tmos in a round - bottom flask and swirled until a uniform, cloudy phase was observed. the solution was then subjected to sonication in a bath sonicator for 10 min, followed by rotary evaporation (340 mbar reduced pressure, 50 c) for 20 min to promote rapid removal of methanol liberated during the hydrolysis reactions. the solution was then passed through a 0.45m filter, resulting in roughly 4 ml of a clear silica sol. of the 4 ml of silica sol, 1 ml was placed into a methacrylate cuvette, followed by 1 ml of a stronger reconstitution buffer (34 mm tris, 100 mm nacl) in order to neutralize the ph. afterward, 1 ml of either a 20 diluted liposome solution or 20 diluted nlp solution in regular reconstitution buffer (20 mm tris, 100 mm nacl) was added to the methacrylate cuvette. the liposome stock solution was typically 2.8 mm 15:0 pc (final concentration 56 m inside gel), while the nlp stock solution was typically 20 m msp (final concentration 400 nm inside gel). gelation typically occurred within 2 min after addition of the liposomes or nlps. ten microliters of a 100 m dph stock solution in ethanol was added to either liposome or nlp solutions in 1 ml volumes of reconstitution buffer (20 mm tris, 100 mm nacl). afterward, these samples were either diluted further (3) and used for solution anisotropy readings or added as the final 1 ml aliquot during the aforementioned entrapment / gelation process and used for gel anisotropy readings. anisotropy values (r) are determined by the difference in polarized light intensity emitted parallel and perpendicular to the excitation source normalized by the total light intensity emitted, as shown in eq 1.1 the measurements were carried out on a perkinelmer ls 55 fluorescence spectrometer equipped with a ptp-1 fluorescence peltier system (perkinelmer, inc., a wavelength of 360 nm was used for excitation, while emission intensities at 440 nm were used for determining anisotropy values. band passes of 3 and 5 nm were used on the excitation and emission monochromators, respectively. the anisotropy values were recorded at varying temperature intervals (4 c) as temperature was raised at an average rate of 0.4 c / min. anisotropy values reported are the average of at least four measurements at a given temperature. there were no fluorescence emissions at 440 nm (hence no anisotropy signals) from the silica gel, protein, or lipid ; only dph - containing samples showed fluorescence emission intensities. the anisotropy vs temperature experimental data was regressed using the method of least - squares for an empirical phase transition model shown in eq 2, where the parameters rmax, rmin, tm, and n are the maximum anisotropy, minimum anisotropy, melting temperature, and cooperativity index, respectively.2a and b are constant coefficients for the quadratic baseline. ignoring the quadratic baseline, eq 2 depicts a sigmoid function with asymptotic end behavior in the limit as t is significantly far from the phase transition region. the parameters rmax and rmin represent the vertical displacement of the plot, while tm corresponds to the inflection point (for significantly small values of b) and n corresponds to the broadness of the phase transition region. a quadratic baseline can be used in the vicinity of the phase transition region to more accurately capture the manner in which anisotropy varies with temperature, as well as any asymmetry about the inflection point. the parameters a and b were chosen to be the average of all of the individually regressed data plots. this was due to a trade - off between n and the quadratic baseline during the regression process ; a and b can influence the value of n. by fixing a and b, the change in n between different plots is almost entirely attributed to the sample, with minimal effect from other parameters. to implement this, each plot was regressed individually, while its values for a and b were recorded. once all of the values were recorded and averaged, all of the plots were reregressed with fixed values for a and b (see the supporting information). these values were the overall average from the previous regressions. in order to perform circular dichroism (cd) spectroscopy, the 20 mm tris, 100 mm nacl buffer was replaced with a 25 mm phosphate, 100 mm naf buffer due to absorbance of tris and chloride in the uv region of interest. solution and gelated measurements were performed on a jasco j-715 spectropolarimeter with a 2 cm / min scan speed (jasco, easton, md). data points were collected as averages of three scans from 260 to 190 nm at room temperature in a demountable close - ended far - uv (q) 1 mm path length quartz cuvette cell type 20c (starna cells) for the silica gel samples and in a 1 mm path length open - end quartz cuvette for the solution samples. the final concentrations of msp were 20 m in solution and 60 m in silica gel. the -helical content was determined by first converting the measured ellipticity at 222 nm to a molar ellipticity using eq 3, where []molar, mrw, 222, l, and c are the molar ellipticity (deg cm dmol), msp mean residue weight (g / mol), ellipticity (deg), cuvette path length (cm), and msp concentration (g / ml), respectively.3 once this was obtained, a common correlation shown in eq 4 was used to estimate the percentage of -helical content.4 fluorescence anisotropy of the membrane - inserting probe 1,6-diphenyl-1,3,5-hexatriene (dph) was used to investigate the phase behavior of di15:0pc carbonyl tails within liposomes and nlps. di15:0pc was used instead of other biologically prevalent pc lipids (such as dmpc or dppc) due to the compatibility of its phase transition temperature with the heating / cooling speeds of the peltier element in the experimental setup, which allowed for higher throughput of samples in a given period of time (see the supporting information). liposomes had a stokes diameter of 166.5 1.8 nm, while nlps had a stokes diameter of 11.7 2.2 nm and corresponding discoidal diameter of 14.8 4.4 nm by dynamic light scattering. we obtained fluorescence anisotropy values using eq 1 at a range of temperatures that included the main phase transition and in two environments, aqueous buffer solution and porous silica gel derived from the alkoxysilane precursor, tmos. as shown in figure 1a, the phase transition from a solid lipid phase to a liquid disordered lipid phase, observed as a decrease in anisotropy with increasing temperature, was broader for nlps than that of the liposomes in buffer. this corresponds to the disparity in the cooperativity indices (n) of the phase transition, obtained by fitting to eq 2. when lipid bilayers undergo a phase transition, the transition occurs involving multiple subunits known as cooperative units. the cooperativity index is a relative scale that is directly proportional to the size of the cooperative unit. the nlps have an average index of 0.36 0.20 and the liposomes have an average index of 1.79 0.24, both in solution. figure 1a also illustrates the shift in the midpoint of the phase transition (tm) to higher temperature for nlps in comparison to liposomes. by fitting this data to eq 2, tm values are found to be 35.1 0.1 and 37.7 0.6 c on average for the liposomes and nlps, respectively. immediately after entrapment in silica gel (day 1), phase transitions can be observed by the decrease in anisotropy with increasing temperature, as shown in figure 1b. however, the anisotropy range the overall ordinate axis difference between the maximum and minimum anisotropy values was reduced for both nlps and liposomes in comparison to solution anisotropy ranges. in addition, figure 1 and table 1 show that the tm values of both nlps and liposomes entrapped in silica gel are elevated compared to their solution values. measured anisotropy values of nlps and liposomes in (a) 20 mm tris, 100 nm nacl buffer and (b) silica gel after initial entrapment, with corresponding regression curves as temperature was increased. table 1 illustrates that the cooperativity indices for nlps and liposomes entrapped within silica gel were on the same order of magnitude, with the nlp values being slightly higher. these values were on the same order of magnitude of nlp cooperativity in solution, but were an order of magnitude lower than that of liposome cooperativity in solution. this is depicted in figure 2a, where the cooperativity of nlps and liposomes entrapped in silica gel are plotted over a 56 week period, normalized by their respective solution values. the normalized cooperativity for nlps started near unity and decayed to a constant value of approximately 0.7 over the course of 2 weeks (figure 2a). the liposomes maintained a constant normalized cooperativity between 0.1 and 0.2 over the entire 5 - 6 week period (figure 2a). in solution, cooperativity did not exhibit any significant changes over time for both nlps and liposomes as it remained unaffected over the course of 3 weeks (see tables s1 and s2, supporting information). in addition to the normalized cooperativity, the phase transition temperature (tm) of liposomes entrapped within silica gel remained relatively constant in the 4042 c region over the course of the 5 - 6 week period, as shown in figure 2b. the nlp tm was higher, as it gradually increased and leveled off in the 4650 c region, as shown in figure 2b. as with cooperativity, tm also remained relatively unaffected in solution over the course of 3 weeks for both nlps and liposomes (see tables s1 and s2, supporting information). for all samples, the observed anisotropy curves were reversible with respect to temperature in solution. however, they were not reversible in silica gel and appeared somewhat broadened (data not shown). therefore, different samples were independently aged and used to generate single sets of parameters. the plots in figure 2 are the averaged scatter plots of the regressed values for 20 silica gels entrapping nlps and 9 silica gels entrapping liposomes. regressed values for (a) cooperativity and (b) phase transition temperature of nlp and liposome samples entrapped in silica gel over the course of 56 weeks. upon synthesis of tmos - derived silica gel the methanol content in rotary evaporated silica was determined to be roughly 5 v / v%, while untreated silica had a methanol concentration of 24 v / v% (see the supporting information). due to its presence, the effect of methanol on the phase behavior of di15:0pc in nlps and liposomes was examined. as shown in figure 3, the anisotropy values of nlps in buffer solution decreased in nearly constant intervals as the concentration of methanol was increased at all temperatures. this was also observed for liposomes (see figure s3, supporting information). measured anisotropy values and corresponding regression curves of different nlp samples in 20 mm tris, 100 mm nacl at various concentrations of methanol (v / v%). moreover, the tm values for nlps and liposomes monotonically decreased and then increased with increasing methanol concentration, as shown in figure 4a, with minimum tm values of 29.1 0.1 c at 28 4 v / v% methanol and 28.3 0.1 c at 20 4 v / v% methanol, respectively. cooperativity indices, obtained by fits to eq 2, did not show corresponding minima or maxima and instead increased for nlps and decreased for liposomes with increasing methanol concentration, as shown in figure 4b as the methanol concentration was increased in solution, the size of particles in nlp samples also increased. at 0, 15, and 30 v / v%, the stokes diameter of particles in nlp solution samples was 11.7 2.2, 17.3 3.2, and 93.0 3.5 nm, respectively (see table s3, supporting information). the effect of methanol was also examined inside silica gel for nlps, where samples without rotary evaporation during the sol gel processing were compared to samples utilizing it. in figure 5 and table 1, it is shown that elimination of the rotary evaporation step, which corresponds to higher methanol concentrations, resulted in lowered anisotropy values and a reduced phase transition temperature (46.3 0.6 c for rotary evaporated samples and 40.0 0.5 c for non - rotary - evaporated samples). in addition, it was observed that the cooperativity change was small (0.33 0.06 c for rotary evaporated samples and 0.35 0.05 c for non - rotary - evaporated samples). regressed parameters for (a) phase transition temperature (tm) and (b) cooperativity (n) of nlps and liposomes in 20 mm tris, 100 mm nacl buffer in methanol measured anisotropy values of nlps in silica gel after initial entrapment with and without the use of rotary evaporation during the sol gel processing. far - uv circular dichroism spectroscopy was used to examine the secondary structures of the scaffold protein msp in lipid - free and lipid - bound conformations. figure 6 shows spectra in terms of ellipticity vs wavelength for msp alone (lipid - free) and msp assembled in nlps (lipid - bound) in both solution and gel - entrapped states. it can be seen that there were vertical shifts in spectral intensity for msp over the course of 1 week. these shifts were more prominent for samples where msp was in a lipid - free conformation (figure 6a, d) in comparison to samples where it was in a lipid - bound conformation (figure 6b, e) in both solution and silica gel, respectively. circular dichroism spectra of (a) lipid - free msp in solution and (b) lipid - bound msp (nlps) in solution and (c) corresponding solution -helix content determined from 222 nm ellipticity. circular dichroism spectra of (d) lipid - free msp in silica gel and (e) lipid - bound msp (nlps) in silica gel, with (f) corresponding gel -helix content. for all cd spectra in figure 6, two peaks characteristic of substantial -helical content the actual -helical content was estimated from the 222 nm peak using eqs 3 and 4, as other secondary structural elements have little contribution to this region of a protein spectrum. in figure 6c, the -helical content in buffer solution was shown to remain at 70% for lipid - bound msp and increase from 35% to 43% for lipid - free msp over the course of 1 week. in figure 6f, the -helical content in silica gel was shown to remain constant at 75% for lipid - bound msp and decrease from 73% to 65% for lipid - free msp over the course of 1 week. liposomes can contain tens to hundreds of thousands of lipids per structure and have cooperativity units that have been estimated to include up to 1700 lipid molecules. having fewer lipids per structure, nlps are not capable of forming cooperative units as large as those present in liposomes. in addition, a fraction of the lipids, located in a two - lipid - thick belt along the scaffold protein boundary, undergo a concomitant loss of cooperativity after nlp formation. therefore, in solution, nlps inherently have a lower cooperativity index than liposomes, as we have observed here. the presence of scaffold proteins has also been shown to elevate the phase transition temperature in solution, as observed here, for a given lipid incorporated into nlps compared to when incorporated into liposomes due to an increase in lateral pressure from the protein lipid interactions. upon entrapment in the silica gel, cooperativity of the nlp phase transition decreased slightly (93% 10% of solution value) while cooperativity of the liposomes decreased significantly (12% 3% of solution value). this leads us to believe that the liposomes are undergoing significant structural changes upon entrapment, while the nlps are not. a significant reduction in size of liposomes has been shown to result in lowered cooperativity. the rupturing of liposomes and formation of smaller lipid aggregates or bicellar structures could reasonably explain this observed behavior and has previously been observed in other works where liposomes were entrapped in silica gel. moreover, only a slight reduction in cooperativity and elevation in phase transition temperature (compared to liposomes) for nlps observed over the course of 56 weeks could indicate that they are not undergoing significant alterations in their size and structure after entrapment. upon silica gel entrapment, there was an observed elevation in phase transition temperature for nlps and liposomes, which is consistent with previous work where elevated phase transitions for entrapped liposomes were observed due to speculated excluded volume effects. this is analogous to effects observed in work with thermal unfolding of proteins in silica gel, where a higher free energy (thus higher unfolding temperature) was imposed upon entrapped, folded proteins due to decreased volume available for the unfolded form. since the lipid tails become less dense when undergoing a gel phase to liquid crystalline transition, it is reasonable to believe that the excluded volume effect can be involved. the observed decreased in anisotropy range is due either to a change in the packing and motion of the lipid tails in each phase or release of a portion of the dph into the pores of the silica gel (see the supporting information). nonetheless, a decrease in the anisotropy range does not have significant effect on the regressed values for the cooperativity index or phase transition temperature (see figure s1, supporting information). we speculate that the minor (approximately 20%) reduction in cooperativity over weeks for entrapped nlps could be related to the known phenomenon of silica gel shrinkage over time. since phospholipid headgroups are known to interact strongly with silica surfaces, changes in the geometry and size of pores could directly impact adsorbed nlps. a decrease in size of lipid structure is correlated to a decrease in cooperativity and could rationalize this observation ; however, the possibility of aggregation is not ruled out. a decrease in silica gel size corresponds to a decrease in porosity, which would enhance the excluded volume effect, explaining our observed increase in phase transition temperature over weeks for nlps. further work in analyzing different silane precursors and monolith shrinkage effects on lipid structures would be required to validate this hypothesis. overall, these results make it very plausible that the silica sol gel - entrapped nlps, unlike liposomes, maintain a structure resembling their solution counterparts for weeks at a time. due to the presence of residual methanol in the silica gels (5 v / v%), the effect of methanol on anisotropy values and phase behavior for nlps and liposomes in solution was examined. the trend in decreasing anisotropy and minimum tm values for nlps and liposomes as the methanol concentration was increased is consistent with previous fluorescence anisotropy studies involving short - chain alcohols, as well as similar studies in which lipid absorbance at 400 nm was utilized. short - chained alcohols increase the area per molecule of the lipid bilayer and induce the interdigitated phase ; thus, the dph probe is allowed to more freely rotate, lowering the anisotropy value. the minimum in the tm value is reported to correspond to the completion of the interdigitation transition. the elevation in methanol concentration required to fully interdigitate nlps could indicate that they have a slightly higher resistance to bilayer interdigitation. the presence of the scaffold proteins could perhaps prolong the bilayer structure in elevated methanol concentrations due to direct interactions with the lipid tails, making it more difficult for them to interlace. the use of tmos instead of the popular precursor teos is favorable due to its liberation of methanol, which requires exponentially higher concentrations than ethanol to significantly modulate lipid bilayer behavior. we found that in the vicinity of 5 v / v% methanol, anisotropy and tm values for nlps and liposomes were minimally changed. therefore, the more significant changes in these values for silica sol gel - entrapped nlps and liposomes were not caused by the presence of methanol. while the liposomes display a decrease in the cooperativity index, cooperativity increases for nlps with increasing methanol concentration. the interdigitated phase of bilayers tends to have a lower phase transition temperature than the gel phase ; thus, the decrease in the cooperativity that is observed is potentially the result of coexistence between the gel and interdigitated phases. this coexistence would result in the transition appearing broader, as the phase transition equation used (eq 2) only accounts for one inflection point. however, at 5 v / v% methanol, the cooperativity of the phase transition is only decreased by approximately 10% in comparison to 90% decrease observed for liposomes entrapped in silica gel. in the case of the nlps, the measured increase in aggregate size in methanol solutions would account for larger cooperative units, which would directly increase the cooperativity index. the appearance of the opposite trend, i.e., slightly decreasing cooperativity, for silica - gel - entrapped nlps illustrates that the methanol concentration is below the threshold necessary for aggregate growth. by removing most of the methanol through rotary evaporation in silica sol gel - entrapped nlps, we have avoided significant changes in anisotropy and tm caused by methanol. if we did not use rotary evaporation, the solution methanol concentration was roughly 24 v / v% with accompanying decreased anisotropy and tm values, which were consistent in magnitude with what was observed in solution experiments. however, the increase in cooperativity was minimal compared to that observed in solution. the size of the pores (550 nm) could perhaps limit the nlps from aggregating or remodeling into larger lipid structures that would have cooperative units comparable to those in solution. the presence of -helical secondary structure is essential for nlp formation and protein lipid binding. we found that the -helical content of the scaffold protein msp is significantly higher in its lipid - bound nlp - associated state (70%), versus a lipid - free state (35%43%) in solution. this magnitude of difference is consistent with previous works that examined -helical content in very similar scaffold proteins, such as msp1d1 and apolipoprotein a - i. for apolipoprotein a - i, this is due to 4 of the 10 helical regions forming a bundle in the lipid - free state, while the other 6 helical regions, along with the globular region, fold in a variety of different conformations having relatively higher random coil content. it is reasonable to believe that similar behavior is involved for msp, as it is derived directly from apolipoprotein a - i. once entrapped in silica gel interestingly, entrapped msp in its lipid - free state adopted a significantly higher -helical content than in solution, increasing to 73%. these results are consistent with previous discoveries of the biocompatible environment of silica sol gels for water - soluble proteins which are often stabilized against denaturation and aggregation in silica sol gels. the stabilization effect has been attributed to the ability of the sol gel matrix to restrict conformational flexibility and diffusional motion and to promote structural rigidity in the water environment. the confinement from the pores of the silica gel could rationalize the observed increase in -helical content for msp upon entrapment ; an estimated radius of gyration of 3 nm for randomly structured msp in solution (see the supporting information) is on the same order of magnitude as the pore size. this could potentially cause msp to adopt an alternative conformation that consists of higher -helical content. the decrease of -helical content over the course of a week (73%65%) for lipid - free msp in silica gel in comparison to the steady -helical content of 75% for lipid - bound msp could indicate that the silica gel promotes lipid association of msp, thereby maintaining the higher helical conformation of this state. however, the absence of lipids does not allow msp to maintain a constant conformation. we have demonstrated that nanolipoprotein particles (nlps) are more compatible with the nanoscale environment of the silica gel pores in comparison to liposomes. direct measurement of size of soft matter inside of mesoporous silica is difficult to obtain ; thus, we utilized biophysical characterization in the form of fluorescence anisotropy and circular dichroism spectroscopy to directly investigate lipid phase behavior and scaffold protein secondary structure, as well as indirectly correlate this behavior to aggregate size. fluorescence anisotropy, which is then used to analyze the cooperativity and temperature of the main phase transition, revealed that nlps entrapped in silica gel exhibit phase behavior with a stronger resemblance to their solution counterparts than liposomes. in particular, cooperativity indices indicate that entrapment in silica gel causes immediate large - scale changes in lipid aggregation state of liposomes toward smaller cooperative units and only minor changes for nlps over weeks of time. these large - scale changes in liposomes have been linked in the past to liposome rupture and denaturation of integral membrane proteins. by investigating these same properties for liposomes and nlps in methanol solutions, we find that the small amount of methanol remaining after evaporative removal is not sufficient to cause observed changes in these properties upon sol gel entrapment. however, if we did not remove the methanol, we found that entrapped nlps displayed shifts in anisotropy and phase transition temperature that were consistent with large fractions of methanol but that the cooperativity was relatively maintained, which we attribute to limitations in the growth of the nlps by confinement in the nanoporous environment of the silica gel. upon further investigation of conformational changes, circular dichroism revealed that the scaffold protein of the entrapped nlps maintained a consistent -helical content necessary for its structural function of belting the phospholipids. this is consistent with the known biocompatibility and structure promotion of silica gels for water - soluble proteins. future work will entail the incorporation of integral membrane proteins inside of nlps and analysis / quantification of their activity retention upon sol gel - derived entrapment via protein - specific assays. | the entrapment of nanolipoprotein particles (nlps) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. nlps are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. the nlps in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (msp), a genetically altered variant of apolipoprotein a - i. liposomes have previously been examined inside of silica sol gels and have been shown to exhibit instability. this is attributed to their size (150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer - scale pores (550 nm) of the silica gel. by contrast, the dimensional match of nlps with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. here we demonstrate that nlps are more compatible with the nanometer - scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. our results showed that the lipid phase behavior of nlps entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the msp in the nlps maintain the high degree of -helix secondary structure associated with functional protein lipid interactions after entrapment. we also examined the effects of residual methanol on lipid phase behavior and the size of nlps and found that it exerts different influences in solution and in silica gel ; unlike in free solution, silica entrapment may be inhibiting nlp size increase and/or aggregation. these findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins. |
aryl-3,4-dihydropyrimidines derivatives have recently received great attention because of their wide range of therapeutic and pharmacological properties, such as antiviral, antitumor, antibacterial and antifungal, anti - inflammatory, antihypertensive agents, and neuropeptide y (npy) antagonists. furthermore, these compounds have emerged as the integral backbones of several calcium - channel blockers. also, several alkaloids containing the dihydropyrimidine were isolated from marine sources, for example, of these are the batzelladine alkaloids, which are found to be potent hivgp-120-cd4 inhibitors [6, 7 ]. in general, the classic biginelli approach to 3,4-dihydropyrimidinones is based on the condensation of ethyl acetoacetate, aromatic aldehyde, and urea under strong acidic conditions ; this suffers, however, from low yields of products, particularly in case of substituted aromatic and aliphatic aldehydes [8, 9 ]. this problem has led to the development of multistep synthetic strategies that produce relatively higher yields, but lack the simplicity of the original one - pot - biginelli protocol. thus, the biginelli reaction has received renewed interest from researchers interested in discovering milder and more efficient procedures that are applicable to a wide range of substituents in all three components and proceed in better yields. so, the one - pot - biginelli protocol for 3,4-dihydropyrimidines synthesis was explored by varying all components and catalysts [1018 ] in protic, aprotic solvents, and solvent free conditions using either classical heating, microwave [20, 21 ], ultrasound [22, 23 ], and visible light (100 w lamp, thf) irradiations. also several improved procedures have been reported recently using not only acidic media such as lewis acids, protic acids, and ionic liquids as promoters [25, 26 ] but also nonacidic substances such as baker 's yeast, graphite, and iodine [29, 30 ]. heterogeneous solid acids are used also ; however, these are advantageous over conventional homogeneous acid catalysts as they can be easily recovered from the reaction mixture by simple filtration and can be reused after activation or without activation, thereby making the process economically viable. bakibaev and filimonov reported that piperidine as a base catalyst can promote the biginelli protocol also, to afford the corresponding 3,4-dihydropyrimidines along with hantzsch 1,4-dihydropyridines which may form in spite of urea decomposition in the reaction media, releasing ammonia. we would like to propose a new naturally and very cheap catalysts granite and quartz for the synthesis of 3,4-dihydropyrimidinones and 3,4-dihydropyrimidenthiones, using one - pot - biginelli protocol, in refluxing ethanol. it is interesting to report that the one pot reaction of a mixture of benzaldehyde, ethyl acetoacetate, and urea in the presence of granite or quartz as a catalyst in refluxing ethanol resulted in the formation of 4-phenyl-3,4-dihydropyrimidinone ia, table 1 in 64% or 68% yield according to the catalyst (scheme 1). in a similar way, urea was condensed smoothly with variety of aromatic or heterocyclic aldehydes and variety of 1,3-dicarbonyl compounds in the presence of granite or quartz in refluxing ethanol as one pot reaction to afford the corresponding 3,4-dihydropyrimidines ib q (table 1) whose composition and structures were confirmed by elemental analysis, mass, ir, and h nmr spectra of the isolated products (cf. on the other hand, carrying out of the above reaction using of 3-benzyloxybenzaldehyde, acetyl acetone, and urea in refluxing ethanol using granite as catalyst, the corresponding 5-acetyl-4-(3-(benzyloxy)phenyl)-6-methyl-3,4-dihydropyrimidin-2(1h)-one io was isolated. however, on carrying the above reaction using quartz as a catalyst, beside the proposed 3,4-dihydropyrimidinone io, another product with molecular formula (c16h16n4o2), m / z = 296 was isolated from the reaction media in 25% yield. this product can be identified as 4-amino-6-(3-(benzyloxy)phenyl)-5,6-dihydro-1,3,5-triazin-2(1h)-one iii based on the analytical and the spectral data of the isolated product, which revealed the presence of characteristic stretching vibrations due to nh, nh2, and amidic co at = 3450, 3300, and 1640 cm regions, respectively, in the ir spectrum. also, the h - nmr spectrum of the isolated product shows signals at = 5.07 (s, 2h, ch2), 5.43 (s, 1h, ch), 5.68 (s, 2h, nh2), 6.707.46 (m, 9h, ar), and 10.0 (s, 1h, nh) ppm. the c - nmr spectrum of the isolated product shows signals at = 51.2 (ch aliphatic), 62.32 (ch2 aliphatic), 165.3 (c triazine ring), 190.4 (c = o amidic), and 111160 (benzene rings). this expectation is based on the observation that the 3-benzyloxybenzaldehyde condensed with two moles of urea to give the corresponding bis - ureide ii as key intermediate which cyclized via elimination of h2o to give the extremely low yield triazine derivative iii (scheme 2). in generality of this process, various 1,3-diketones and aldehydes were reacted with thiourea in refluxing ethanol using granite or quartz as the reaction catalyst to give the corresponding 3,4-dihydropyrimidin-2(1h)-thione derivatives iv (table 2) which their structures were confirmed on the bases of the analytical and spectral data of the isolated products (cf., we noted that aromatic aldehydes carrying either electron - donating or electron - withdrawing substituents reacted well under the reaction conditions to give the corresponding products in moderate to good yields high purity in case of granite or quartz. however, the obtained yields on using quartz are higher than granite either in case of urea or thiourea. this procedure not only preserves the simplicity of the biginelli reaction but also produces good yields of the products with high purity. also, the catalyst was recovered by simple filtration and reused in subsequent reactions with consistent activity. c with methyl iodide in dry acetone in the presence of anhydrous potassium carbonate afforded the s on the other hand, heating of iva c in acetic anhydride afforded the corresponding 3-n - acetyl derivatives via c. the mass spectrum showed the molecular ion peak at m / z (%) = 378 (m, 48.03), for molecular formula c18h22n2o5s. coch3 protons at = 2.60 ppm, in addition to other singlet peaks at = 2.27, 3.67, and 3.77 ppm for methyl and two methoxy groups, respectively, and the absence of the nh proton at = 7.27. in the same time, we can use the same conditions to prepare via, b which was elucidated by correct elemental analysis and spectral data (cf. c with acetyl chloride in dmf (melting and mixed melting point) (scheme 4). in the same time, the pyrimidine derivatives viia, b can be synthesized via acetylation of the corresponding s also, it can be prepared via methylation of the n - acetyl derivatives via, b. the mass spectrum for viia showed the molecular ion peak at m / z (%) = 394 (m, 12.51), while viib illustrated the molecular ion peak at m / z (%) = 332 (m, 43.22). the h nmr revealed the presence of singlet peak at = 2.50 ppm for coch3 protons and the absence of the singlet peak at = 7.27 ppm for nh proton. methylation of va was carried out in methyl iodide in dmf in the presence of k2co3 anhydrous that yielded viiia which was confirmed by correct elemental analysis as well as spectral data. the h nmr showed the absence of singlet peak at = 7.27 ppm for nh proton and the appearance of a singlet peak at = 3.33 ppm for n heating of iva with ethylchloroacetate in ethanol and sodium acetate afforded ethyl 3-oxo-5,7-diphenyl-3,5,8,8a - tetrahydro-2h - thiazolo[3,2-a]pyrimidine-6-carboxylate xa over the unisolated intermediate ethyl 2-(2-ethoxy-2-oxoethylthio)-4,6-diphenyl-1,6 dihydropyrimidine-5-carboxylate ixa as shown in elemental analysis as well as spectral data. the mass spectrum showed the molecular ion peak at m / z (%) = 378 (m, 60.03) for molecular formula c21h18n2o3s. the h nmr revealed also the presence of one only ethyl ester group, at = 0.85 for ch3 protons (t) and 3.85 for ch2 (q), and also the absence of nh proton at = 7.27 ppm. the ir spectrum showed absorption bands at 1752, 1675, and 1589 cm for carbonyl ester, amidic carbonyl groups, and c = n, respectively. also, the isolated product xa was obtained via the reaction of iva with chloroacetyl chloride or bromoacetyl bromide in benzene and drops of triethylamine as catalyst. in the same time, compound xa can be isolated from the reaction of iva with chloro- or bromoacetic acid in acetic acid and acetic anhydride mixture in presence of anhydrous sodium acetate. similarly, compound xb was prepared from the reaction of ivb with ethylchloroacetate, chloroacetic acid, or chloroacetyl - chloride as shown in previous conditions (scheme 5). compound xa was condensed with different aromatic aldehydes in refluxing ethanolic pipredine solution to give the corresponding arylidene derivatives xia c. the h nmr showed the absence of singlet peak for ch2 protons at = 3.88 ppm and the appearance of singlet peak for = ch proton at = 7.74 ppm (scheme 5). aiming to the synthesizing of thiazolopyrimidine xii, we refluxed ivb with chloroacetone in ethanolic piperidine solution. however the corresponding 1-(5-acetyl-6-(4-methoxyphenyl)-4-methyl-1,6-dihydropyrimidin-2-ylthio)propan-2-one xiii was formed which was identified by elemental analysis as well as spectral data. the mass spectrum showed the molecular ion peak at m / z (%) = 332 (m, 5.30) for molecular formula c17h20n2o3s. the h nmr confirmed the presence of only one nh proton at = 7.11 ppm and singlet peak at = 2.46 ppm due to ch2 protons (scheme 6). on the other hand, compound va, b was reacted with thiosemicarbazide in refluxing ethanol to give the corresponding carbazide xiva, b instead of the corresponding fused pyrimidinotriazoles xv and xvi. structures xiva, b were established by elemental analysis and spectral data where the mass spectrum showed the molecular ion peak at m / z (%) = 395 (m, 24.13) for xiva and at m / z (%) = 333 (m, 12.18) for xivb (scheme 7). on the other hand, refluxing of iva, b in methyl alcohol in the presence of acetic acid and water (4 : 1 : 1) afforded 3,4-dihydropyrimidinone derivatives xviia, b. there are 5 compounds (iii, ivg, ivf, ivh, and ivc) that were tested and showed promising positive antibacterial activity. the ivh & ivc compounds showed positive antibacterial against s. aureus which are 14.5 mm and 14 mm, respectively, which are 0.25 and 0.75 mm less than the zone around streptomphenicol disc. the other three most active compounds tested are compounds ivg, ivf, and iii. the activity of these compounds against staphylococcus aureus showed positive reactions, 12.75, 12.5, and 12 mm of inhibition zones, respectively, compared to the inhibition zone of antibiotic used, as indicated in (table 3) ; this may be due to sulfur atom, two chlorine atoms, and triazine ring, respectively. all the compounds have approximately the same effect against escherichia coli bacteria as indicated by the zone of inhibition (table 3). in case of using these compounds as antimicrobial cytotoxicity, effect of these compounds in summary, we have found that quartz and granite are extremely useful and highly efficient new natural, solids for the synthesis of biologically potent aryl 3,4-dihydropyrimidines by means of three - component condensations of an aldehyde, 1,3-dicarbonyl compound, and urea or thiourea in a one - pot operation. this method is applicable to a wide range of substrates, including aromatic and heterocyclic aldehydes, and provides a variety of biologically relevant 3,4-dihydropyrimidinones and 3,4-dihydropyrimidinthiones in high yields after short reaction times. the ir spectra of samples were recorded in kbr via a shimadzu ft - ir 8101 pc infrared spectrophotometer. h nmr spectra were run at 300 mhz and recorded in cdcl3/[d6 ] dmso using tms as the internal standard. mass spectra were measured on a gcms - qp1000 ex spectrometer at 70 ev. elemental analyses were carried out at the microanalytical center of cairo university, giza, egypt. the catalyst is ground until it became fine powder. a mixture of aldehyde (1 mmol), 1,3-dicarbonyl compounds (1 mmol), urea or thiourea (1 mmol), and granite or quartz (0.5 g) in ethanol (15 ml) was heated under reflux for the required time. after completion of the reaction as monitored by t.l.c., the reaction mixture was filtered to separate the catalyst. keep the reaction mixture overnight. the solid product was filtered under suction then recrystallized from ethanol to afford pure product. i.r (kbr) : = 3300, 3100, 2950, 1700, 1630 cm. h nmr (300 mhz, cdcl3) : = 1.16 (t, j = 7.2 hz, 3h, o ch2ch3), 2.33 (s, 3h, ch3), 4.08 (q, 2h, o ch2ph), 5.37 (s, 1h, ch), 5.92 (s, 1h, nh), 6.856.94 (m, 4h, ar), 7.197.39 (m, 5h, ar), 8.31 (s, 1h, nh) ppm. mass : m / z (%) : 366 (m, 6.31), 275 (22.72), 183 (24.94), 91 (100.0). c21h22n2o4 (366) : calculated, % : c 68.84, h 6.05, n 7.65, o 17.47 ; found, % : c 68.82, h 6.10, n 7.55, o 17.46. yield quartz (65%), granite (62%). 5-acetyl-4-(3-(benzyloxy)phenyl)-6-methyl-3,4-dihydropyrimidin-2(1h)-one (ip). i.r (kbr) : = 3450, 3200, 2950, 1640, 1590 cm. h nmr (300 mhz, dmso) : = 2.08 (s, 3h, ch3), 2.27 (s, 3h, coch3), 5.05 (s, 2h, o ch2ph), 5.20 (s, 1h, ch), 6.816.92 (m, 4h, ar), 7.217.45 (m, 5h, ar), 7.81 (s, 1h, nh), 9.17 (s, 1h, nh) ppm. mass : m / z (%) : 336 (m, 1.6), 293 (1.9), 245 (33), 153 (14.6), 91 (100.0). c20h20n2o3 (336) : calculated, % : c 71.41, h 5.99, n 8.33, o 14.27 ; found, % : c 71.40, h 6.0, n 8.30, o 14.21. i.r (kbr) : = 3250, 3100, 2950, 1700, 1640 cm. h nmr (300 mhz, cdcl3) : = 1.11 (t, j = 7.2 hz, 3h, o ch2ch3), 2.40 (s, 3h, ch3), 3.87 (s, 3h, och3), 3.92 (s, 3h, och3), 4.06 (q, 2h, o ch2ch3), 5.71 (s, 1h, ch), 5.72 (s, 1h, nh), 6.73 (d, 1h, ar), 6.87 (d, 1h, ar), 6.98 (t, 1h, ar), 7.27 (s, 1h, nh) ppm. mass : m / z (%) : 320 (m, 13.7), 288 (100.0), 243 (55.2), 183 (94.9), 155 (68.9), 137 (60.1), 77 (47.3). c16h20n2o5 (320) : calculated, % : c 59.99, h 6.29, n 8.74, o 24.97 ; found, % : c 59.95, h 6.22, n 8.70, o 24.99. yield quartz (63%), granite (60%). i.r (kbr) : = 3400, 3150, 2950, 1650 cm. h nmr (300 mhz, dmso) : = 1.10 (t, j = 7.2 hz, 3h, o ch2ch3), 2.27 (s, 3h, ch3), 4.01 (q, 2h, o ch2ph), 5.15 (s, 1h, ch), 6.80 (m, 4h, ar), 7.237.42 (m, 5h, ar), 9.60 (s, 1h, nh), 10.30 (s, 1h, nh) ppm. mass : m / z (%) : 382 (m, 17.6), 199 (12.2), 91 (100.0). c21h22n2o3s (382) : calculated, % : c 65.95, h 5.80, n 7.32, o 12.55, s 8.38 ; found, % : c 65.89, h 5.60, n 7.35, o 17.46 i.r (kbr) : = 3200, 3100, 2950, 1705 cm. h nmr (300 mhz, cdcl3) : = 1.10 (t, j = 7.2 hz, 3h, o ch2ch3), 2.42 (s, 3h, ch3), 3.86 (s, 3h, och3), 3.93 (s, 3h, och3), 4.05 (q, 2h, o ch2ch3), 5.71 (s, 1h, ch), 6.68 (d, 1h, ar), 6.88 (d, 1h, ar), 6.99 (t, 1h, ar), 7.26 (s, 1h, nh), 8.10 (s, 1h, nh) ppm. mass : m / z (%) : 336 (m, 76.4), 305 (86.1), 289 (81.5), 263 (100.0), 199 (87.7), 171 (63.3), 153 (31.5), 77 (44.6). c16h20n2o4s (336) : calculated, % : c 57.12, h 5.99, n 8.33, o 19.02, s 9.53 ; found, % : c 57.13, h 5.96, n 8.35, o 19.06, s 9.51. i.r (kbr) : = 3200, 3100, 2940, 1700 cm. h nmr (300 mhz, dmso) : = 1.05 (t, j = 8.2 hz, 3h, o ch2ch3), 2.27 (s, 3h, ch3), 3.65 (s, 3h, och3), 3.72 (s, 3h, och3), 3.96 (q, 2h, o ch2ch3), 5.44 (s, 1h, ch), 6.57 (s, 1h, ar), 6.83 (d, 1h, ar), 6.94 (d, 1h, ar), 9.24 (s, 1h, nh), 10.24 (s, 1h, nh) ppm. mass : m / z (%) : 338 (m, 10.97), 279 (10.49), 256 (19.23), 166 (45.38), 149 (100.0), 105 (28.13), 69 (90.57). c16h20n2o4s (336) : calculated, % : c 57.12, h 5.99, n 8.33, o 19.02, s 9.53 ; found, % : c 57.14, h 5.96, n 8.30, o 19.04, s 9.56. i.r (kbr) : = 3150, 3000, 2900, 1750, 1640 cm. h nmr (300 mhz, cdcl3) : = 0.94 (t, j = 6.6 hz, 3h, o ch2ch3), 2.23 (s, 3h, ch3), 3.91 (q, 2h, o ch2ch3), 6.28 (s, 1h, ch), 7.057.23 (m, 3h, ar), 7.94 (s, 1h, nh), 8.32 (s, 1h, nh) ppm. mass : m / z (%) : 344 (m, 23.9), 348 (m, 7.6), 315 (34.5), 199 (100.0), 171 (36.4), 153 (16.2). c14h14cl2n2o2s (344) : calculated, % : c 48.70, h 4.09, cl 20.54, n 8.11, o 9.27, s 9.29 ; found, % : c 48.72, h 4.04, cl 20.50, n 8.13, o 9.29, s 9.30. yield quartz (55%), granite (60%). 4-amino-6-(3-(benzyloxy)phenyl)-5,6-dihydro-1,3,5-triazin-2(1h)-one (iii). = 172174c i.r (kbr) : = 3450, 3300, 1640 cm. h nmr (300 mhz, dmso) : = 5.07 (s, 2h, ch2), 5.43 (s, 1h, ch), 5.68 (s, 2h, nh2), 6.706.92 (m, 4h, ar), 6.98 (s, 1h, nh), 7.237.46 (m, 5h, ar), 10.0 (s, 1h, nh) ppm. the c - nmr (300 mhz, dmso) = 51.2 (ch aliphatic), 62.32 (ch2 aliphatic), 165.3 (c triazine ring), 190.4 (c = o amidic), 111160 (benzene rings). mass : m / z (%) : 296 (m, 54.58), 294 (82.76), 253 (57.20), 227 (63.30), 203 (100.0), 182 (31.01), 171 (55.77), 131 (99.09), 104 (29.73). c16h16n4o2 (296) : calculated, % : c 64.85, h 5.44, n 18.91, o 10.80 ; found, % : c 64.82, h 5.40, n 18.93, o 10.82. (0.005 mol) and methyl iodide (0.005 mol) was dissolved in dry acetone in the presence of pot. the formed solid was filtered off and crystallized from an appropriate solvent to give va, b, c. the formed solid was crystallized from petroleum ether / benzene 2 : 1 ; mp. = 158160c ; i.r (kbr) : = 3280, 2982, 2807, 1676, 1614, 1493 cm ; h nmr (300 mhz, cdcl3) : = 0.87 (t, j = 6.9 hz, 3h, o ch2ch3), 2.49 (s, 3h, sch3), 3.86 (q, 2h, o ch2ch3), 5.74 (s, 1h, ch), 7.27 (s, 1h, nh), 7.287.48 (m, 10h, ar) ppm ; mass : m / z (%) : 352 (m, 23.35), 337 (33.58), 323 (68.32), 275 (100.0), 77 (25.52) ; c20h20n2o2s (352) : calculated, % : c 68.16, h 5.72, n 7.95, o 9.08, s 9.10 ; found, % : c 68.13, h 5.75, n 7.97, o 9.10, s 9.12 ; yield (77%). the formed solid was crystallized from petroleum ether / ethanol 1 : 1 ; mp. = 126128c ; i.r (kbr) : = 3285, 2950, 2928, 1639, 1594 cm ; h nmr (300 mhz, dmso) : = 2.09 (s, 3h, ch3), 2.23 (s, 3h, sch3), 2.28 (s, 3h, coch3), 3.70 (s, 3h, och3), 5.54 (s, 1h, ch), 6.827.15 (d, d, 4h, ar), 9.57 (s, 1h, nh) ppm ; mass : m / z (%) : 288 (m, 29.40), 250 (22.10), 183 (19.10), 73 (67.60), 57 (100.0) ; c15h18n2o2s (290) : calculated, % : c 62.04, h 6.25, n 9.65, o 11.02, s 11.04 ; found, % : c 62.03, h 6.26, n 9.65, o 11.04, s 11.03 ; yield (86%). = 140c ; i.r (kbr) : = 3321, 2935, 2832, 1706, 1669, 1594 cm ; h nmr (300 mhz, dmso) : = 1.13 (t, j = 7.5 hz, 3h, ch3ch2o), 2.39 (s, 3h, ch3), 2.45 (s, 3h, sch3), 3.87 (s, 3h, och3), 3.93 (s, 3h, och3), 4.06 (q, 2h, ch3ch2o), 5.85 (s, 1h, ch), 6.777.02 (m, 3h, ar), 7.27 (s, 1h, nh) ppm ; mass : m / z (%) : 350 (m, 20.56), 335 (36.97), 321 (64.29), 303 (40.44), 213 (100.0), 77 (23.14) ; c17h22n2o4s (350) : calculated, % : c 58.27, h 6.33, n 7.99, o 18.26, s 9.15 ; found, % : c 58.26, h 6.33, n 7.97, o 18.27, s 9.16 ; yield (74%). method (a). a mixture of iva c (0.005 mol) and acetyl chloride (0.01 mol) was refluxed in dmf (15 ml) as a solvent containing (5 drops) of triethylamine (tea) for 1 hour and then stirred at room temperature for overnight, and then the solution was poured into ice with vigorous stirring, and then the solid product was filtered off and recrystallized from suitable solvent to afford compounds via, b, c. method (b). a solution of iva c (0.01 mol) in 15 ml of acetic anhydride the solution was then poured into 150 ml of ice - water and stirred for several hours until crystallization was complete. the precipitate was filtered and crystallized from suitable solvent to afford compounds via, b, c. the solid product was recrystallized from ethanol ; method (a) : yield (81%). = 186c ; i.r (kbr) : = 3237, 2996, 2944, 2837, 1702, 1671 cm ; h nmr (300 mhz, dmso) : = 1.17 (t, j = 6.6 hz, 3h, ch3ch2o), 2.27 (s, 3h, ch3), 2.60 (s, 3h, coch3), 3.67 (s, 3h, och3), 3.77 (s, 3h, och3), 4.08 (q, 2h, ch3ch2o), 5.50 (s, 1h, ch), 6.687.0 (m, 3h, ar), 11.6 (s, 1h, nh) ppm ; mass : m / z (%) : 378 (m, 48.03), 335 (96.81), 289 (100.0), 263 (45.57), 199 (33.62), 77 (22.44) ; c18h22n2o5s (378) : calculated, % : c 57.13, h 5.86, n 7.40, o 21.14, s 8.47 ; found, % : c 57.14, h 5.85, n 7.41, o 21.13, s 8.45. the solid product was recrystallized from benzene ; method (a) : yield (80%) ; method (b) : yield (85%). = 136c ; i.r (kbr) : = 3215, 2986, 1642, 1599, 1494 cm ; mass : m / z (%) : 380 (m, 37.39), 337 (100.0), 307 (25.7), 265 (57.72), 104 (65.03) ; c21h20n2o3s (380) : calculated, % : c 66.29, h 5.30, n 7.36, o 12.62, s 8.43 ; found, % : c 66.28, h 5.31, n 7.35, o 12.63, s 8.42. 1,1-(6-(4-methoxyphenyl)-4-methyl-2-thioxo-2,3-dihydropyrimidine-1,5(6h)-diyl)diethanone (vib). the solid product was recrystallized from ethanol ; method (a) : yield (65%) ; method (b) : yield (70%). = 130c ; i.r (kbr) : = 3243, 2962, 1698, 1609, 1509 cm. a solution of va, b (0.01 mol) in 15 ml of acetic anhydride was heated under reflux for one hour. the solution was then poured into 150 ml of ice - water and stirred for several hours until crystallization was complete. the precipitate was filtered off and washed with water then crystallized from an appropriate solvent to afford viia, b. ethyl 1-acetyl-2-(methylthio)-4,6-diphenyl-1,6-dihydropyrimidine-5-carboxylate (viia). the solid product crystallized from petroleum ether (6080) ; yield (90%) ; mp. = 102c ; i.r (kbr) : = 2978, 1697, 1601, 1533 cm ; h nmr (300 mhz, cdcl3) : 0.97 (t, j = 6.6 hz, 3h, ch3ch2o), 2.50 (s, 3h, sch3), 2.50 (s, 3h, coch3), 4.02 (q, 2h, ch3ch2o), 6.66 (s, 1h, ch), 7.277.60 (m, 10h, ar) ppm ; mass : m / z (%) : 394 (m, 12.51), 351 (100.0), 337 (10.86), 323 (28.06), 275 (84.24), 129 (18.40), 77 (29.31) ; c22h22n2o3s (394) : calculated, % : c 66.98, h 5.62, n 7.10, o 12.17, s 8.13 ; found, % : c 66.97, h 5.63, n 7.09, o 12.18, s 8.12. 1,1-(6-(4-methoxyphenyl)-4-methyl-2-(methylthio)pyrimidine-1,5(6h)-diyl)diethanone (viib). i.r (kbr) : = 2990, 1675, 1568 cm ; mass : m / z (%) : 332 (m, 43.22), 312 (39.56), 278 (51.28), 100 (100.0), 67 (50.55) ; c17h20n2o3s (332) : calculated, % : c 61.42, h 6.06, n 8.43, o 14.44, s 9.65 ; found, % : c 61.43, h 6.04, n 8.46, o 14.45, s 9.66 a mixture of va (0.005 mol) and methyl iodide (0.005 mol) was dissolved in dmf in the presence of pot. the reaction mixture was filtered on hot then the filtrate was cooled and poured onto cold water with stirring ; the formed solid was filtered off and crystallized from ethanol : benzene (3 : 1) ; mp. = 92c ; i.r (kbr) : = 3058, 2977, 2932, 1717, 1580 cm ; h nmr (300 mhz, cdcl3) : = 0.84 (t, j = 6.9 hz, 3h, o ch2ch3), 2.57 (s, 3h, sch3), 3.33 (s, 3h, nch3), 4.01 (q, 2h, o ch2ch3), 5.74 (s, 1h, ch), 7.497.64 (m, 10h, ar) ppm ; mass : m / z (%) : 366 (m, 5.21), 350 (100.0), 321 (28.48), 129 (30.96), 77 (15.87) ; c21h22n2o2s (366) : calculated, % : c 68.82, h 6.05, n 7.64, o 8.73, s 8.75 ; found, % : c 68.81, h 6.04, n 7.65, o 8.73, s 8.75 ; yield (25%). method (a). a mixture of iva, b (1 mmol) and chloroacetic acid (1 mmol) was dissolved in 40 ml of a mixture of (ac)2o / acoh (1 : 3) in the presence of 3 gm anhydrous sodium acetate that was refluxed for 4 hours. the reaction mixture was cold and poured onto cold water with stirring ; the solid formation was filtered off and crystallized from benzene / ethanol (3 : 1) to give xa, b.. a mixture of iva, b (0.005 mol) and chloroacetyl chloride with 2 drops of t.e.a was refluxed in benzene for 3 hours. then the reaction mixture was filtered off through heating then dried and crystallized from benzene / ethanol (3 : 1) to give xa, b. a mixture of iva, b (0.005 mol), ethylchloro acetate (0.005 mol), and sodium acetate trihydrate (1 gm) was refluxed in ethanol for 5 hours. the solid formation was filtered off then dried and crystallized from benzene / ethanol 3/1 to afforded xa, b. the formed solid was crystallized from benzene / ethanol (3 : 1) ; method (a) : yield (72%) ; method (b) : yield (75%) ; method (c) : yield (84%). = 1368c ; i.r (kbr) : = 2976, 2931, 2900, 1752, 1675, 1589 cm ; h nmr (300 mhz, cdcl3) : = 0.85 (t, j = 6.9 hz, 3h, o ch2ch3), 3.88 (s, 2h, ch2co), 6.19 (s, 1h, ch), 7.277.51 (m, 10h, ar) ppm ; mass : m / z (%) : 378 (m, 60.03), 350 (16.30), 301 (100.0), 273 (35.62), 129 (25.11), 77 (35.57) ; c21h18n2o3s (378) : calculated, % : c 66.65, h 4.79, n 7.40, o 12.68, s 8.47 ; found, % : c 66.65, h 4.78, n 7.41, o 12.67, s 8.47. the formed solid was crystallized from benzene and drops of ethanol ; method (a) : yield (42%). = 160162c ; i.r (kbr) : = 2983, 2936, 2876, 1756, 1655, 1612 cm ; h nmr (300 mhz, dmso) : = 2.16 (s, 3h, ch3), 2.34 (s, 3h, coch3), 3.70 (s, 3h, och3), 4.15 (s, 2h, ch2co), 5.98 (s, 1h, ch), 6.867.21 (d, d, 4h, ar) ppm ; mass : m / z (%) : 316 (m, 35.08), 301 (5.32), 273 (100.0), 245 (28.08), 230 (4.72), 181 (19.36), 115 (25.14), 77 (26.0) ; c16h18n2o3s (316) : calculated, % : c 60.36, h 5.70, n 8.80, o 15.08, s 10.07 ; found, % : c 60.35, h 5.70, n 8.81, o 15.06, s 10.08. a mixture of compound xa (1 mmol), aromatic aldehyde (1 mmol), and 2 drops of piperidine was refluxed in ethanol for 2 hours. the reaction mixture kept overnight, then the solid product was filtered off and crystallized from etoh to afford compound xi. = 1646c ; i.r (kbr) : = 3446, 1721, 1620, 1559 cm ; h nmr (300 mhz, cdcl3) : = 0.87 (t, j = 6.9 hz, 3h, o ch2ch3), 6.34 (s, 1h, ch), 7.277.54 (m, 14h, ar), 7.74 (s, 1h, = ch) ppm ; mass : m / z (%) : 500 (m, 45.80), 503 (m, 9.82), 423 (67.99), 168 (100.0), 77 (77.69) ; c28h21cln2o3s (500) : calculated, % : c 67.13, h 4.22, cl 7.08, n 5.59, o 9.58, s 6.40 ; found, % : c 67.14, h 4.24, cl 7.07, n 5.60, o 9.59, s 6.42 ; yield (88%). = 148c ; i.r (kbr) : = 34454, 1712, 1630, 1581 cm ; h nmr (300 mhz, cdcl3) : = 0.87 (t, j = 6.6 hz, 3h, o ch2ch3), 3.77 (s, 3h, och3), 3.85 (s, 3h, och3), 6.34 (s, 1h, ch), 6.84 (d, 1h ar), 6.96 (s, 1h, ar), 6.98 (d, 1h, ar), 7.357.55 (m, 10h, ar), 8.09 (s, 1h, = ch) ppm ; mass : m / z (%) : 526 (m, 42.98), 449 (100.0), 363 (27.0), 77 (5.37) ; c30h26n2o5s (526) : calculated, % : c 68.42, h 4.98, n 5.32, o 15.19, s 6.09 ; found, % : c 68.43, h 4.99, n 5.33, o 15.20, s 6.10 ; yield (85%). = 158160c ; i.r (kbr) : = 3100, 2998, 1716, 1617, 1557, 1563, 1530 cm ; h nmr (300 mhz, cdcl3) : = 0.84 (t, j = 6.9 hz, 3h, o ch2ch3), 6.31 (s, 1h, ch), 7.207.75 (m, 14h, ar), 8.31 (s, 1h, = ch) ppm ; c28h21n3o5s (511) : calculated, % : c 65.74, h 4.14, n 8.21, o 15.64, s 6.27 ; found, % : c 65.75, h 4.13, n 8.22, o 15.63, s 6.28 ; yield (92%). synthesis of 1-[5-acetyl-6-(4-methoxy - phenyl)-4-methyl-1,6-dihydro - pyrimidin-2-ylsulfanyl]-propan-2-one (xiii). a mixture of ivb (0.005 mol), chloroacetone (0.005 mol), and 2 drops of piperidine was refluxed in ethanol for 6 hours. the solid formation was filtered off then dried and crystallized from benzene / ethanol (1 : 1) ; mp. = 215c ; i.r (kbr) : = 3112, 3004, 1644, 1608, 1524 cm ; h nmr (300 mhz, dmso) : = 2.25 (s, 3h, ch3), 2.26 (s, 3h, coch3), 2.30 (s, 3h, coch3), 2.46 (s, 2h, ch2), 3.71 (s, 3h, och3), 6.44 (s, 1h, ch), 6.907.28 (d, d, 4h, ar), 7.11 (s, 1h, nh) ; mass : m / z (%) : 332 (m, 5.30), 298 (5.30), 270 (33.60), 245 (25.7), 91 (100.0) ; c17h20n2o3s (332) : calculated, % : c 61.42, h 6.06, n 8.43, o 14.44, s 9.65 ; found, % : c 61.43, h 6.05, n 8.43, o 14.42, s 9.66 ; yield (48%). a mixture of va, b (0.005 mol) and thiosemicarbazide (0.07 mol) was refluxed in ethanol (20 ml) for 7 hours in a water bath, then the reaction mixture was allowed to stand for several hours at room temperature, then the solid product was filtered off and recrystallized from ethanol to formed compounds xiva, b. ethyl 2-(2-carbamothioylhydrazinyl)-4,6-diphenyl-1,6-dihydro - pyrimidine-5-carboxylate (xiva). method (a) : yield (51%) ; method (b) : yield (46%) ; mp. = 170172c i.r (kbr) : = 3370, 3262, 3175, 1644, 1620 cm. h nmr (300 mhz, dmso) : = 0.71 (t, j = 7.5 hz, 3h, och2ch3), 3.74 (q, 2h, och2ch3), 4.50 (s, 2h, nh2), 5.26 (s, ch), 7.197.58 (m, 10h, ar), 8.65 (s, 1h, nh), 9.78 (s, 1h, nh), 10.51 (s, 1h, nh) ppm ; mass : m / z (%) : 395 (m, 24.13), 379 (21.13), 368 (49.20), 352 (76.97), 105 (86.51), 55 (100.0) ; c20h21n5o2s (395) : calculated, % : c 60.74, h 5.35, n 17.71, o 8.09, s 8.11 ; found, % : c 60.73, h 5.36, n 17.71, o 8.08. 2-(5-acetyl-6-(4-methoxyphenyl)-4-methyl-1,6-dihydropyrimidin-2-yl)hydrazinecarbothioamide (xivb). method (a) : yield (64%) ; method (b) : yield (48%) ; mp. = 92c ; i.r (kbr) : = 3284, 3145, 2049, 1604, 1510 cm ; mass : m / z (%) : 333 (m, 12.18), 274 (14.92), 233 (27.31), 215 (84.93), 178 (90.61), 136 (100.0), 76 (65.0), 51 (23.07) ; c15h19n5o2s (333) : calculated, % : c 54.04, h 5.74, n 21.01, o 9.60, s 9.62 ; found, % : c 54.05, h 5.74, n 21.0, o 9.61, s 9.62. compound iva, b (0.005 mol) was heated under reflux in (10 ml) of methanol, containing acetic acid (2.5 ml) and water (2.5 ml). after reflux for 25 hours, methanol was distilled off and the remaining solution was treated portionwise with water until precipitation was completed. after standing for several hours at room temperature, the solid product was removed by filteration to yield xviia, b which crystallized from ethanol. | synthesis of 3,4-dihydropyrimidin-2(1h)-one and 3,4-dihydropyrimidin-2(1h)-thione derivatives from aldehydes, 1,3-dicarbonyl derivatives and urea or thiourea using granite and quartz as new, natural and reusable catalysts. some of the 3,4-dihydropyrimidin-2(1h)-thione derivatives were used to prepare new heterocyclic compounds. the antimicrobial activity of selected examples of the synthesized compounds was tested and showed moderate activity. |
infections due to human immunodeficiency virus (hiv) and hepatitis b and hepatitis c viruses remain as the leading health problems. worldwide causes of viral hepatitis due to hepatitis b (hbv) and hepatitis c viruses (hcv) are also common in the geography of our country. importance of prevention from this infection is enhanced since complete eradication of hbv, which leads to life - threatening complications such as cirrhosis, hepatic insufficiency and hcc, is unlikely with current antiviral therapies. 1 hepatitis c is a global health problem and is the leading cause of cirrhosis and hepatocellular carcinoma. before 1992, when blood donors have not been screened for anti- hcv antibody, hcv, which is the most common cause of posttransfusion hepatitis, could be found anywhere in the world. it is the highest in intravenous drug users and hemophilia patients and found between 0.2% and 18% in general population according to the data from who. regions with high prevalence include far east, certain regions of africa, mediterranean countries, and eastern europe 2,3. according to the hepatitis b surface antigen (hbsag) positivity, countries with hbv prevalence > % 8 are considered high endemic regions, whereas countries with hbv prevalence between 2% and 8% are considered moderate endemic regions and countries with hbv prevalence < 2% are considered low endemic regions for hbv 4. hbv seroprevalence shows variation among geographic regions, but from west to east and south - east regions, prevalence of hbv carriers is increased to 12.5- 14.3% from 6% respectively 5. the present study aimed to investigate distribution of prevalence of hbsag, hbeag, anti - hbs antibody, anti - hbe antibody, hcv and hiv positivity among years in 8-year period, as well as the changes in population resistance against hepatitis. thus, it was aimed to attract attention to the population prevention and highlight the extent of the risk. files of the patients, who had been treated as inpatient or outpatient 992.581 due to any diagnosis between 01/01/2005 and 31/12/2012 in the clinics or policlinics of diyarbakr training and research hospital, were retrospectively reviewed using patient file database. serum samples (235.534 for hbsag, 196.727 for anti - hbs antibody, 98.497 for hbeag, 97.417 for anti - hbe antibody, 225.483 for hcv and 138.923 for hiv) of these patients, which had been processed in microbiology laboratory, were studied by chemiluminescence technique using roche e-170 (modular analytics system) device. between 2005 and 2012 ; hbsag was studied in 31.586 of 31.586 patients (13.4%), anti - hbs antibody was studied in 85,727 of 196.727 patients (43.5%), hbeag was studied in 2588 of 98.497 patients (2.6%), anti- hbe antibody was studied in 33.044 of 97.417 patients (33.9%), anti - hcv antibody was studied in 2871 of 225.483 patients (1.2%), and anti - hiv antibody was studied in 407 of 138.923 patients (0.29%) (table 1). between 2005 and 2012, prevalence of hbsag positivity according to the years was found to be 2950 (15.9%), 3629 (15.3%), 5185 (13.7%), 4662 (11.8%), 5350 (13.3%), 4937 (14.1%), 2922 (14.9%), and 1951 (% 9) respectively ; prevalence of hbeag positivity was in turn 112 (2.5%), 182 (1.8%), 521 (2.4%), 533 (% 2), 621 (3.2%), 391 (% 3.9), 214 (2.7%), and 14 (1.8%) ; prevalence of anti - hbe antibody positivity was in turn 1388 (30.4%), 4042 (42%), 7182 (33.5%), 7576 (30.1%), 6034 (32.6%), 4063 (42.5%), 2524 (32.6%), and 235 (25.2%) ; prevalence of anti - hbs antibody positivity was in turn 4456 (32.9%), 8345 (40.3%), 15162 (44.8%),12944 (42.5%), 12.497 (39.7%), 12.947 (43.9%), 8689 (51%), and 10687 (52.3%) ; prevalence of anti - hiv antibody positivity was in turn 15 (0.1%), 56 (0.7%), 120 (0.6%), 127 (0.4%), 15 (0.05%), 16 (0.06%), 52 (0.3%), and 6 (1%) ; prevalence of anti - hcv antibody positivity was in turn 181(1%), 388 (2.1%), 704 (2.2%), 559 (1.5%), 397 (1%), 349 (1.5%), 161 (1.9%), and 132 (0.7%) (table 2). prevalence of chronic hepatitis b (hbv) virus infection shows variation among regions. in the world, africa and a part of asia are high endemic regions for hbsag (8%), whereas southern europe and north and south america (except for some regions of amazon, brazil and peru) are considered low endemic regions (< 2%) 6. the most common way for transmission changes according to the endemicity of hbv infection. perinatal transmission is the primary way of transmission of hbv in high endemic regions, whereas sexual intercourse between high risk adults and shared needles among intravenous drug users are the main ways of transmission of hbv in low endemic regions 7. moreover, prevalence of hbv and hcv carriers changes according to the age, socio - economic status and occupational groups. the incidence of hepatitis b defined by the world health organization for the european union countries is 1.49 per100000 people, whereas the incidence of hcv is 8.7 per 100000 people 8. in the national studies on hbsag positivity, prevalence of hbsag positivity was found to be 2.9% in a 4-year study conducted in ankara ; 8.3% in a 8-month study and 3.27% in a 2-year study conducted in istanbul ; 10% in one - year study conducted with 10630 patients in siirt ; 15% in one - year study conducted in 5334 serum samples from malatya ; 6.6% in 2-year study conducted in 1320 serum samples from afyon ; 4.7% in 4-year study conducted in a total of 9420 women from adyaman ; and 9.6% in 9882 subjects from anlurfa 9,10,11,12,13,14,15. in diyarbakr, prevalence of hbsag positivity was 6.2% in urban area and 8.2% in rural area out of 2888 subjects. a retrospective study from kars found the prevalence of hbsag positivity to be 4.6% in 12,965 subjects, whereas it was reported to be 3.6% in 62,607 women from anlurfa, 4.22% in tunceli, 2.5% in isparta, and 5.5% in tokat 16,17,18, 19,20,21. in mersin, the prevalence of hbsag positivity was found to be 13.6% by delialiolu., whereas kandemir. found it to be 4.1% in 2800 subjects aged over 15 years, who presented to primary health care units for any reason except hepatitis 22,23. we found the prevalence of hbsag seropositivity to be 10% in a total of 276,212 serum samples in 8-year period. prevalence of hbsag positivity showed no regional difference, but was higher than that in other geographical regions of the country. contrary to the higher prevalence rates in the eastern regions of this country, there are studies suggesting a decrease in western regions. high prevalence rates reported from mersin and istanbul (8.3%, 13.6%) is conspicuous, and relevant authors attribute this to the migration from southeastern anatolian region to istanbul. another condition that should not be overlooked with regard to the high prevalence as 10% found in the present study is the fact that this study consists of all clinical forms, active, chronic and carrier, of hepatitis b. various studies investigating prevalence of hbsag in turkey have reported that south eastern anatolian region has remarkably the highest seropositivity 10. with respect to the studies conducted in other countries. in a retrospective study conducted between 2005 and 2006 in korea,, the prevalence rate was found to be 5.59% in low risk group consisted of 2540 subjects among four groups created according to the risks ; with regard to provinces, it was 4.04% in bucharest, 7.14% in craiova and 6.47% in constanta 24,25. in an 11-year surveillance study conducted in pakistan, prevalence of hbsag positivity was found to be 2.5% in 47,043 patients, whereas it was found to be 8.1% in 2,995 subjects from dhaka, bangladesh 26,27. in china, the prevalence was found 5.84% in randomly selected 8,762 subjects, whereas it was found to be 5.40% in 1352 subjects in the mianyang state of sichuan, china and 4.81% in gansu state of china 28,29,30. prevalence rates of hbsag positivity in china do not show difference among states and are between normal limits. prevalence rate found in romaine is interesting, and high percentages in bangladesh and korea is conspicuous. prevalence of hbv was found to be 11.2% in andean plateau region of latin america, which is considered as low risk region ; similar rate, however, was found (11.6%) in two high - risk groups, homeless and prostitutes, in cochabamba 31. this similarity between two different risk groups has been considered interesting by the authors ; we as well share this opinion. prevalence of hbsag positivity was found to be 1% in different regions of italy ; this rate is distributed as 0.8% for italians and as 6.4% for immigrants 32. importance of disease control and immunization against hbv infection is understood when distribution of prevalence in italy is taken into account. it should be borne in mind that, prevalence rate is decreased as living conditions are improved and power of immunization, as is in italy sample, should not be overlooked. in mersin, turkey, the prevalence was found to be 3.6% in urban area and 6.8% in rural area, showing statistically significant difference. in urban area, the prevalence rate was 2.9% in the population with high socioeconomic status, 2.8% in the population with moderate socioeconomic status and 6.7% in the population with poor socioeconomic status 23. it was observed that the prevalence of hbsag positivity is not different in various regions of the world from that in turkey. nation - wide studies reveal a prevalence rate changing between 20.6% and 52.3% for anti - hbs antibody seropositivity 10. we found the prevalence of anti - hbs antibody seropositivity to be 52.3% in our region, which explains the impact of routine hbv immunization in this region with high prevalence of hbsag positivity. comparing with nation - wide prevalence rates ; prevalence of anti - hbs antibody seropositivity was 48% in 29,227 patients from siirt, 27.2% in 1320 subjects from afyon between 2002 and 2004, 38.4% in females admitted between 2008 and 2011 in adyaman, 12.1% in tokat, 16.2% in isparta, 33% in malatya, and 46.1% in anlurfa 11,12,13,14, 15, 20,21. prevalence of anti - hbs antibody seropositivity was found 36.7% in mersin and 36.4% in ankara 9,22 (table 3). in a period of three years, prevalence of anti - hbs antibody positivity was found 29.1% in a total of 62,607 pregnant and non - pregnant women from anlurfa ; it is conspicuous that the prevalence was 44.9% in non - pregnant women but 25.0% in pregnant women 18. prevalence rate of anti - hbs antibody positivity increasing from west to east as was hbsag positivity indicates increased likelihood of hbv exposure. with regard to the studies from other countries ; prevalence of anti - hbs antibody positivity was 41.31% in 8,762 people from china 28, 28.7% in 1977 samples from gansu province, china 30, and 61.3% in mianyang 29. in italy, prevalence of anti - hbs antibody, anti - hbs+/anti - hbc+, anti - hbc antibody positivity again, another study from italy found the isolated anti - hbs antibody positivity to be 2.2% in 1991, 21.4% in 1999 and 42.9% in 2008 and reported that prevalence rates were increased by immunization 33. importance of disease control and immunization against hbv, the fact that prevalence rates are decreased as the living conditions are improved, and power of immunization, as was in italy sample, should not be overlooked. studies have proven that prevalence of anti - hbs antibody positivity increases in years. in mersin, turkey, prevalence of anti - hbs antibody positivity in urban area was 17.6% for the areas with good socioeconomic status, 22.2% for the areas with moderate socioeconomic status, and 6.0% for the areas with poor socioeconomic status ; the difference was found significantly high in favor of the regions with good and moderate socioeconomic status 23. the most important problem due to hepatitis c, one of the agents of viral hepatitis, is the chronicity rate of 85% and being usually asymptomatic. prevalence of anti - hcv antibody positivity is 1 - 2.4% 35. in this retrospective study conducted in 225,483 people between 2005 and 2012, prevalence of anti - hcv antibody positivity was found 1.2%. comparing this data with other studies from different regions of our country, we observed that, aka. found the prevalence of anti - hcv antibody positivity to be 0.52 % in 188,106 people between 2001 and 2008 in zonguldak 36, klgelier. found it to be 0.2% in 9420 females admitted between 2008 and 2011 in adyaman 15, and etinkol. found it to be 1.5% in 11,763 people in a retrospective study performed in kars between 2007 and 2008 17. found the prevalence of anti - hcv antibody positivity to be 0.62% in 29,227 patients from siirt 11 ; whereas, pehlivanolu. found it 0.65% in 37,675 patients from istanbul between 2008 and 2010 3, demirtrk. found it 2.2% in 1320 subjects from afyon between 2002 and 2004 13, and aslan. found it 2.6% in anlurfa 14 it was found to be 1.0% 20 by akam. from isparta and 2.1% by yldrm. from tokat 21. found the prevalence of anti - hcv antibody positivity to be 0.8% 18 in 62607 women from anlurfa. in mersin, the prevalence was 1.1% in general population 23. again in mersin, delialiolu. similar studies from other countries demonstrated that prevalence of hcv was 18.2% in korea between 2005 and 2006 24.. found it to be 0.39% in 9538 serum samples from 6 different regions of china ; the distribution of the prevalence rate 0.1% to 0.74% among regions was as follows ; 0.23% in beijing, 0.74% in heilongjiang, 0.26% in shandong, 0.1% in ningxia, 0.44% in gansu and 0.44% in sichuan 37. prevalence of anti - hcv antibody positivity was found to be 0.37% by zhang. in mianyang, china and 0.46% by zhonghua shi in 1977 samples from gansu province, china 29,30. it was found to be 0.58% in randomly selected 8,762 people from 6 different regions of china 28. prevalence of anti - hcv antibody in uzbekistan was 2.2% in low - risk group 38 ; whereas, it was found to be 5.4% in 2,995 people from dhaka, bengladesh 24,26, and 4.56% in romaine showing an urban distribution as 3.05% in bucharest, 3.27% in craiova and 6.37% in constanta, and high rate in constanta has been considered significant 25. again in the same region in italy, prevalence of anti - hcv antibody positivity was found 2.6% 32. prevalence of anti - hcv antibody positivity in pakistan was found 4.8% in 11-year surveillance study performed in 47,043 patients 27. high prevalence of anti - hcv antibody positivity in korea, romaine, pakistan and bangladesh, which is similar in different regions of the world, is meaningful. according to the 2011 year - end estimation of the world health organization, newly infected 2.5 million people have been detected in 2011, and a total of 1.7 million people were reported to have died of aids. according to the data from ministry of health, a mean of 500 people have been annually diagnosed with hiv within the last four years in turkey. however, this rate has been reached in the first six months of 2012 and it was reported that spread rate of the disease has reached to the highest level in the last one year. according to the data from ministry of health, number of registered hiv / aids cases in turkey is 590 in 2010, whereas the number of newly diagnosed cases increased up to 726 in 2011. however, it is conspicuous that the number of cases reached to 524 in only the first six months of 2012. according to the data in june 2012, number of registered hiv / aids cases in turkey is 5740 [the ministry of health republic of turkey ]. since the present study comprised the patients admitted to the clinics and policlinics, results of donors could not be evaluated in the discussion section of this study. in the retrospective evaluation of the prevalence of anti - hiv antibody positivity, we found it 0.1 - 1% between 2005 and 2012. these data, which was detected by elisa method, have not been confirmed by western blot method.. found anti - hiv antibody to be positive by 0.009% in only one case between 2007- 2008 in kars 17. all patients in mardin were found negative by tekin. in 2-year period 40. conducted a study in siirt in 29227 patients and found the prevalence of anti - hiv antibody seropositivity to be 0.08% 11. anti - hiv antibody positivity was not detected in any of 2629 females investigated in ankara, whereas, only one out of 1567 males showed positivity by 0.06% 9. none of the 1832 patients that underwent anti - hiv antibody testing in van showed positivity 41. although no difference is assumed between genders in terms of prevalence of viral hepatitis, it was observed to be higher in males. in the present study, of the cases with avh, 42.7% were female and 57.2% were male. in the relevant studies, yaar. found that 59% of the cases with hbsag positivity (8.3%) were male 10. in 2005, trkdoan. found chronic hepatitis b infection to be three times more prevalent in males 42. kamaz determined the prevalence of hbsag positivity to be 4.7% in males and 1.9% in females ; whereas, prevalence of anti - hbs antibody positivity was found similar (36.4%) in both genders ; prevalence of anti - hcv antibody, however, was found 0.6% in males and 0.3% in females 9. reported that 62% of the cases with positive hbsag and 54%of the cases with positive anti - hcv antibody were male 3. found the prevalence of hbsag positivity to be 66.7% in males and 33.3% in females and the prevalence of anti - hbs antibody positivity to be 58.6% in males and 41.4% in females 36. similar with the present study, in a study conducted in antakya to explore seropositivity for hepatitis a and b, prevalence of hbsag positivity was found 3.2% in 2439 preoperative cases aged between 6 months and 90 years, being 4.1% in males and 2.1% in females 43. studies from dhakka, bengladesh and pakistan reported higher prevalence rates in males versus females 26,27. in the present study, males accounted for 61% of hbsag positivity, 56% of anti - hbs antibody positivity, 45% of anti - hcv antibody positivity and 50% of anti - hiv antibody positivity. these rates suggest that middle - age - group males and females of our region are at great risk for hepatitis b virus infections. number of male patients being higher in these studies is attributed to the fact that males are more exposed to risky behaviors in terms of parenterally - transmitted hepatitis infections. a prevalence rate decreasing to 9% from 15.9% for hbsag and prevalence rate increasing to 52.3% from 32.9% for anti - hbs antibody positivity in 8-year period in our region is quite meaningful. such favorable developments in our region are of great valuable in terms of indicating to what extent could struggle against hbv is controlled by education and awareness. | distribution of hbv, hcv and hiv results of the inpatients or outpatients, who had been treated for various diagnoses in diyarbakr training and research hospital between 2005 and 2012, among years was investigated.files of the patients, who had been treated as inpatient or outpatient 992. to any diagnosis between 01/01/2005 and 31/12/2012 in the clinics or policlinics of diyarbakr 581 due training and research hospital, were retrospectively reviewed using patient file database. serum samples (235.534 for hbsag, 196.727 for anti - hbs antibody, 98.497 for hbeag, 97.417 for anti - hbe antibody, 225.483 for hcv and 138.923 for hiv) of these patients, which had been processed in microbiology laboratory, were studied by chemiluminescence technique using roche e-170 (modular analytics system) device.prevalence rates between 2005 and 2012 were as follows : 15.9%-9% for hbsag, 32.9%-52.3% for anti - hbs, 2.5%-1.8% for hbeag, 30.4%-25.2% for anti - hbe, 1%-0.7% for anti - hcv, and 0.1%-1% for anti - hiv. increase in anti - hbs prevalence is the successful outcome of routine immunization in population. this suggests that, governmental policies focused on this subject have resulted in successful outcomes and that people also take care about this.a prevalence rate decreasing to 9% from 15.9% for hbsag and prevalence rate increasing to 52.3% from 32.9% for anti - hbs antibody positivity in 8-year period in our region is quite meaningful. such favorable developments in our region are of great valuable in terms of indicating to what extent could struggle against hbv is controlled by education and awareness. |
gallstone ileus is an uncommon complication of gall stones associated with potentially serious morbidity and mortality. we reported a 60-year - old male case who presented with renal failure and pain in right hypochondriac region. he also had a history of brain infarcts along with diabetes which is an additional factor for mortality. on computed tomography of the abdomen, he was diagnosed to have cholecystocholedochal fistula including air in the gall bladder and obstruction in the distal part of the ileum. as in our case, diagnosis was challengeable because of renal failure, diabetes, septicaemia and intestinal obstruction (peritonitis). we did surgery on the basis of peritonitis which remains the only choice in such cases. gallstone ileus to refer to mechanical intestinal obstruction due to impaction of gallstones within the intestine. gallstone ileus is a rare complication of gallstones that occurs in 1 - 4% of all cases of bowel obstruction and in up to 25% of cases of non - strangulated small - bowel obstruction in patients over 65 years of age. the condition should be suspected in all patients presenting with features of small - bowel obstruction in absence of a surgical scar and external hernia, especially in elderly females. the diagnosis can always be confirmed by plain x - ray, ultrasound and computed tomography (ct) scan. a 60-year - old male presented with vomiting and abdominal pain, which had lasted for two days, in maharishi markandeshwer institute of medical sciences and research, mullana, distt - ambala, india in march, 2011. patient had been previously treated in a private hospital for intestinal obstruction using ryle 's tube aspiration, intravenous fluids, and antibiotics. he had also been kept orally nil for about 1 week, but there was no relief. there was also a history of old brain infarcts and he was a known diabetic on regular treatment. abdominal examination revealed only tenderness on the right side while the rest of the abdomen was soft and bowel sounds were present. on blood tests, elevated total leukocyte counts (15,500 /cu mm), blood urea (96 mg / dl), and creatinine (2.6 mg / dl) were observed. on contrast enhanced computed tomography (cect), there was a stone / stricture / obstruction in distal part of the ileum along with air in the gallbladder. diagnosis was made as intestinal obstruction with septicemia on basis of clinical and radiological correlations. in view of acute intestinal obstruction, laparotomy was performed and findings revealed proximal dilated loops without any collection in the peritoneum. a large stone of 4 cm in size was present one foot proximal to ileocaecal junction and was causing obstruction (figure 1). the gallstone was removed through the perforation site after milking and strictureplasty were conducted (figure 2a and 2b). on gross examination, the stone was oval in shape, hard in nature, brownish in color, and 5 cm in size (figure 3a and 3b). operative picture showing impacted stone and proximal dilated loops of the jejunum a and b. impacted stone removed from the intestine a and b. gross specimen of the stone gallstone ileus is an uncommon complication of gallstones along with mirizzi syndrome and cholecystocholedochal fistula. it accounts only for 1 - 4% of all causes of intestinal obstruction and up to 25% of cases of non - strangulated small - bowel obstruction in patients over 65 years of age. the gallstone may enter the intestine through a fistulous communication between the common bile duct and the gastrointestinal tract. however, the gallstone can impact anywhere in the gastrointestinal tract and it should be at least 2 - 2.5 cm in diameter to cause obstruction. as shown by reisner and cohen, impaction of the stone can occur at any part of the bowel including ileum (60.5%), jejunum (16.1%), stomach (14.2%), colon (4.1%), and duodenum (3.5%). it most frequently occurs in the terminal ileum and the ileocecal valve because of their narrow lumen and potentially less active peristalsis. the most common complications of gallstone disease are acute cholecystitis, acute pancreatitis, choledocholithiasis with or without cholangitis, and a gangrenous gallbladder. morbidity from gallstone ileus in the past, it was difficult to clinch the diagnosis, but the advent of ct and mri has made it easier to diagnose gallstone ileus. in 50% of cases, the classic rigler 's triad of radiography includes mechanical bowel obstruction, pneumobilia, and an ectopic gallstone within bowel lumen. however, air in the gallbladder, small bowel obstruction, and gallstone ileus imply the presence of biliary enteric fistula. the choice is between performing simple enterolithotomy with longitudinal incision on the antimesenteric border proximal to the site of obstruction, or a single - stage procedure involving enterolithotomy, cholecystectomy, and closure of the fistula. the choice of the surgical procedure is largely determined by the clinical condition of the patient. if the patient is hemodynamically stable, then single - stage procedure can be performed. however, in unstable patients with renal failure, enterolithotomy alone is considered sufficient (table 2). we thus performed enterolithotomy since our patient suffered from renal failure, diabetics, and brain infarcts. the laparoscopy - assisted techniques have been reported by sarli. who successfully treated three women with gallstone ileus. however, one should remember that laparoscopy is somehow more challenging in cases of dilated and edematous bowel and may require gentle mobilization of the bowel to prevent perforation. the final technical issues to remember are milking the stone and taking the enterotomies away from the site of impaction because it is often edematous. in addition, resection of a segment of the bowel might be necessary if the stone is firmly fixed in an edematous and inflamed segment of bowel. this entity should be kept in the back of mind as differential diagnosis of intestinal obstruction in patients with sonographic findings of inflammatory changes of gallbladder, especially elderly women. the goal of treatment in gallstone ileus is early relief of intestinal obstruction and minimization of morbidity and mortality. | background : gallstone ileus is an uncommon complication of gall stones associated with potentially serious morbidity and mortality.case report : we reported a 60-year - old male case who presented with renal failure and pain in right hypochondriac region. he also had a history of brain infarcts along with diabetes which is an additional factor for mortality. on computed tomography of the abdomen, he was diagnosed to have cholecystocholedochal fistula including air in the gall bladder and obstruction in the distal part of the ileum. computed tomography plays an important role to make the proper diagnosis and in treatment.conclusions:as in our case, diagnosis was challengeable because of renal failure, diabetes, septicaemia and intestinal obstruction (peritonitis). we did surgery on the basis of peritonitis which remains the only choice in such cases. in follow- up of 1 month patient was doing well and asymptomatic. |
spasticity is a common neurological complication following central nervous system injuries such as traumatic or acquired brain and spinal cord injury. indications for the treatment of spasticity include pain, function limitations, contracture prevention, and positioning assistance. non - pharmacological modalities include proper positioning of the affected body region, range of motion / stretching exercises, splinting, or casting. however, the use of medications can be limited by cognitive and sedative side effects. thus, the use of botulinum toxin (bont) type a and b injections has been preferred for localized spasticity. it binds presynaptic peripheral cholinergic nerve endings, acting intracellularly to cleave polypeptides involved in acetylcholine and norepinephrine exocytosis.1,2 in multiple systematic reviews and clinical trials, bont used for various muscle tone disorders has proven to reduce spasticity, increase range of motion, improve positioning, and decrease pain.36 bont is also approved for the treatment of blepharospasm, overactive bladder, chronic migraine, axillary hyperhidrosis, and strabismus.7 as with any pharmacological therapies for spasticity, onabotulinumtoxin a (botox) is also associated with adverse effects such as nausea, fatigue, bronchitis, muscular weakness, and pain.7,8 in cases of detrusor overactivity, there have been reports of hematuria associated with bont injections.9 although this may be a result of mucosal trauma from the injection itself, studies have demonstrated a greater incidence of hematuria in bont groups compared with placebo and also with higher drug concentrations.10,11 however, there are few to no reports of hematuria when it is used for spasticity in the upper limb. this case documents the unusual side effect of hematuria following bont injection for upper limb spasticity. a 29-year - old male with a history of hemophilia a presented to an outpatient specialty clinic for right upper limb spasticity secondary to traumatic brain injury. the institutional review board of university of california, irvine, orange, ca, usa approved collection of patient data with written consent. the patient reported dull pain localized to his right wrist radiating up to the shoulder (rated 68/10 on a numeric rating scale) and associated paresthesias of the second to fifth digits. the pain improved with massage, but was exacerbated with writing and long distance stick shift driving involving finger flexion with repetitive elbow and shoulder joint motions. this condition was previously treated with 140 units of bont, resulting in limited improvement of his pain. examination of his right upper limb revealed increased tone (rated 1 on the modified ashworth scale) with shoulder internal rotation and adduction, elbow flexion, and wrist flexion. he had no atrophy of the right thenar eminence, but had decreased strength of the right finger and thumb. the previous diagnosis of pain due to right upper limb spasticity was confirmed, and was further treated with a total of 500 units of onabotulinumtoxin a (100 units for right anterior deltoid, 50 units for pectoralis, 100 units for biceps, 75 units for brachioradialis, 50 units for brachialis, 75 units for extensor carpi radialis, 25 units for extensor carpi ulnaris, and 25 units for flexor digitorum superficialis). after a month, his pain decreased to 45/10 on the numeric rating scale. however, a day after leaving the clinic, the patient reported developing hematuria, which lasted 45 days and resolved upon self - infusion of factor viii as recommended by his hematologist. of note, he had not had hematuria in the past and does not require regular infusions. given his history of bladder stones, he was further evaluated in the emergency room, where the workup did not show evidence of cystoliths or urinary tract infection. bont preferentially binds and penetrates peripheral cholinergic nerve endings, acting intracellularly on metalloendoprotease to cleave polypeptides involved in acetylcholine exocytosis.1 by preventing acetylcholine release from the presynaptic membrane, bont blocks nerve impulses involved in muscle contractions, which induces chemodenervation resulting in decreased muscle tone.12 bont also causes muscle fiber atrophy and reduces the potential size of motor units, leading to weakness in muscle strength. studies have demonstrated the clinical effect of bont in patients with upper limb spasticity from traumatic brain injury and stroke resulting in improved range of motion as well as in modified ashworth scale rating and muscle tone.13,14 various side effects, including pain, fatigue, muscle weakness, bronchitis, and upper respiratory tract infections have been reported with bont injection for upper limb spasticity, but little is known of bont - associated hematuria when used for spasticity in the upper limb. this report illustrates hematuria that resolved after self - infusion of factor viii. as such, bont may play a role in the intrinsic coagulation cascade, resulting in or exacerbating spontaneous hemorrhage in those with underlying hematological deficiencies. acetylcholine is suggested to be involved in the activation of antifibrinolytic compounds.15 another study has reported similar mechanisms, implying that acetylcholine results in increased thrombin release and prothrombin gene expression.16 additionally, higher concentrations of the toxin are able to block norepinephrine exocytosis from other nerve endings.2 a report has shown norepinephrine increases thrombin and fibrin formation via 2-adrenergic receptors.17 another study demonstrated similar findings when norepinephrine was infused into patients with traumatic brain injury, resulting in a reduction in platelets possibly due to thrombus formation.18 as such, by blocking norepinephrine release, higher levels of bont may indirectly prevent clot formation, resulting in spontaneous hemorrhage. therefore, it is hypothesized that by reducing the release of acetylcholine and norepinephrine, bont causes spontaneous hemorrhage through an anticoagulation effect. furthermore, our patient had a history of factor viii deficiency, which imposes a greater risk for spontaneous bleeding. hemophilia is an inherited x - linked disorder associated with a defect of clotting factor viii or ix. spontaneous hematuria is relatively common in patients with hemophilia, and theorized to be due to tubular damage caused by immune complexes.19 this, in sequence with the negative effect of bont on coagulation, suggests that its use may convert what typically occurs as clinically insignificant microhematuria into gross hematuria by destabilizing clot formation that is used to heal the damage that occurs at baseline in patients with hemophilia. although it is difficult to explain systemic reactions caused by localized bont injection, some studies have shown subclinical effects of the toxin distant from the injection site. post - marketing surveillance reports have illustrated toxin spread in adults treated for spasticity, such as dysphagia and breathing difficulties after treatment of cervical dystonia.7 likewise, a double - blind, placebo - controlled trial reported an increased incidence of dry mouth in patients treated with bont for upper limb spasticity.20 additional findings were seen in a study that demonstrated an increased average jitter value on single fiber electromyography of the extensor digitorum after patients with craniocervical dystonia and hemifacial spasms received twice the dose of bont injections.21 a similar dose - related response was seen in our case as the patient was able to tolerate lower doses of the toxin without complication, but developed hematuria with increased doses. furthermore, dose - dependent responses have been observed in clinical accounts involving generalized weakness after upper limb injection, and detrusor weakness in patients with detrusor hyperreflexia.22,23 these studies suggest that despite localization, bont may enter the systemic circulation, resulting in generalized effects in a dose - dependent manner. clinically, it is critical to consider the potential systemic effects with localized bont injections. it can be hypothesized that bont has a negative effect on the coagulation cascade, given the effects of acetylcholine and norepinephrine on thrombin and fibrin. therefore, it is crucial that clinicians are aware of the bleeding risk with a dosage increase, especially in patients with pre - existing hematological disorders. additional studies should be undertaken to further elucidate the effect of bont in the coagulation pathway as well as to determine optimal doses and techniques that may avoid these complications. | hematuria is a documented side effect of botulinum toxin injection and has only been reported when it is used for overactive bladder. here we report a rare case of hematuria following onabotulinumtoxin a (botox) injection for upper limb spasticity in a 29-year - old male with a history of traumatic brain injury and hemophilia. hematuria resolved without further complication after self - injection of factor viii as recommended by his hematologist. botulinum toxin binds peripheral cholinergic nerve endings to prevent acetylcholine and norepinephrine exocytosis. studies have shown that both of these compounds are involved in antifibrinolytic activation, suggesting botulinum toxin may play a role in the coagulation cascade by preventing formation of fibrin. this is further supported by resolution of hematuria in our patient after self - injection of factor viii. as such, botulinum toxin injection may result in mild spontaneous hemorrhage in patients with underlying hematological deficiencies. further studies are needed to elucidate its effects in coagulation. |
ckd is a major and serious risk factor for coronary artery disease (cad). cad is extremely common in the population of uremic patients. in people with established cad, ckd predicts recurrent events as strongly as other established cardiovascular risk factors such as diabetes and elevated blood pressure. whether the cardiovascular risk in the setting of ckd is due to increased atherosclerotic burden, higher risk of plaque rupture as a result of inflammation, or other mechanisms is unknown. the incidence and severity of obstructive cad increases as glomerular filtration rate (gfr) declines. cad shows a pattern of diffuse multivessel involvement with coronary calcification ; small angiographic studies suggest that this incidence exceeds 50% in unselected ckd five - dimension patients. among patients with cad, are inversely and independently associated with kidney function, particularly at estimated gfr 50% stenosis in one or more coronary vessels were included in the studyall patients were evaluated for the evidence of ckd. serum creatinine, routine urine analysis, and spot urine for protein to creatinine ratio of all patients were measured using the standard technique in the clinical laboratory. for secondary analysis, creatinine clearance (ml / min/1.73 m of body surface area) was estimated by the cockroft gault equation. patients whose creatinine clearance is 50% stenosis in one or more coronary vessels were included in the study all patients were evaluated for the evidence of ckd. serum creatinine, routine urine analysis, and spot urine for protein to creatinine ratio of all patients were measured using the standard technique in the clinical laboratory. for secondary analysis, creatinine clearance (ml / min/1.73 m of body surface area) was estimated by the cockroft gault equation. patients whose creatinine clearance is 3 g / day. demographic variables of patients studied approximately 40% of patients had stage iii ckd during admission [figure 1 ]. approximately 55% of patients who developed aki required renal replacement therapy in the form of hemodialysis (hd). among 55%, significant number of patients requiring hd belongs to ckd group (43.8%) [figure 2 ]. contrast - induced nephropathy (cin) preventive measures in the form of hydration using normal saline or n - acetyl cysteine or sodium bicarbonate were used in 46.9% of ckd patients [table 3 ]. statistically significant number of patients developed aki in the ckd group, which did not receive any cin preventive measures (p 60 ml / min (25%) and < 15 ml / min (10.4%). recovery of renal function at the end of 3 months in ckd and non - ckd groups is shown in table 5. creatinine clearance at admission aki requiring hemodialysis aki : acute kidney injury, hd : hemodialysis, ckd : chronic kidney disease cin preventive measures and its influence on the development of aki characteristics of patients who developed aki in ckd and non - ckd group it is estimated that up to 11% of adults in the united states have ckd. recent studies have confirmed that even early ckd constitutes a significant risk factor for cardiovascular events and death, and proper management of cvd is different and more complex in patients with ckd. both the frequency of cardiovascular complications and the progression of ckd can be ameliorated in these patients by appropriate intervention. for all these reasons, we evaluated patients who got admitted with cad for the presence of ckd as part of preventive care and treatment strategies. in our study, 78% of the subjects studied were males, 22% of the subjects were females, indicating that cad is more prevalent among males than females. liu. in their study found that the prevalence of ckd among the 3513 china heart survey (chs) participants with coronary heart disease was 24.8%. mielniczuk. in their study, estimated baseline egfr for the 4181 patients with non - st or st elevation acute coronary syndromes who participated in the aggrastat - to - zocor trial. the baseline egfr in their study was 67.8 ml / min/1.73 m (range, 9.7149.2 ml / min/1.73 m) indicating that a significant number of patients had underlying ckd. in our study, 48 patients (38.4%) out of 125 developed aki (defined as an increase in serum creatinine levels 1.5 baseline). 54.2% (26 out of 48) of these aki patients required renal replacement therapy in the form of hd. requirement of hd was more in patients with underlying ckd (21 vs. 5) as compared to non - ckd patients. performed an observational study of 147,007 elderly medicare patients admitted for acute myocardial infarction (ami) as a part of the cooperative cardiovascular project. they evaluated the association between aki and all - cause mortality in the above - mentioned population. in their study, studied the incidence and clinical significance of transient versus persistent aki in 1957 patients who survived an st - elevation ami. aki developed in 46.7% of the patients, of these 3.9% (10) patients received renal replacement therapy. we followed - up 48 patients, who developed aki to find out how many had complete recovery, partial recovery, and no recovery of aki at the end of 3 months. of 14 non - ckd patients who developed aki, 8 (57.1%) patients had complete recovery and 6 (42.9%) had partial recovery of aki. of 34 ckd patients, 16 (47.1%) had complete recovery, 10 (29.4%) had partial recovery, and 8 (23.5%) had no recovery of aki. statistically significant number of patients developed no recovery of their renal function in ckd group (23.5% vs. 0) as compared to non - ckd (p < 0.001).. however, limited time that we get to do our thesis makes it difficult to include large numberslong - term follow - up was not done in our study. long - term follow - up would have given a better idea about progression of renal dysfunction and its impact on cardiovascular and renal morbidity and mortalityfinally, we did not address the influence of different therapies that were administered during the acute phase of the cad on the changes in renal function.. however, limited time that we get to do our thesis makes it difficult to include large numbers long - term follow - up was not done in our study. long - term follow - up would have given a better idea about progression of renal dysfunction and its impact on cardiovascular and renal morbidity and mortality finally, we did not address the influence of different therapies that were administered during the acute phase of the cad on the changes in renal function. in view of high prevalence of ckd (39.2%) in patients with cad, every patient undergoing cag must have renal function evaluation before cag including urine, spot urine for albumin to creatinine ratio and serum creatinine levelsconsidering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary albumin to creatinine ratio, and in patients whose creatinine clearance is < 60 ml / minaki is an important complication of cag and precautions should be taken to prevent itsignificant number of patients has worsening of renal function at the end of 3 months. we find many patients developing progressive ckd after an episode of acute coronary syndromeall patients should have their renal function assessed 3 months after the cag. in view of high prevalence of ckd (39.2%) in patients with cad, every patient undergoing cag must have renal function evaluation before cag including urine, spot urine for albumin to creatinine ratio and serum creatinine levels considering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary albumin to creatinine ratio, and in patients whose creatinine clearance is < 60 ml / min aki is an important complication of cag and precautions should be taken to prevent it significant number of patients has worsening of renal function at the end of 3 months. we find many patients developing progressive ckd after an episode of acute coronary syndrome all patients should have their renal function assessed 3 months after the cag. liu. in their study found that the prevalence of ckd among the 3513 china heart survey (chs) participants with coronary heart disease was 24.8%. estimated baseline egfr for the 4181 patients with non - st or st elevation acute coronary syndromes who participated in the aggrastat - to - zocor trial. the baseline egfr in their study was 67.8 ml / min/1.73 m (range, 9.7149.2 ml / min/1.73 m) indicating that a significant number of patients had underlying ckd. in our study, 48 patients (38.4%) out of 125 developed aki (defined as an increase in serum creatinine levels 1.5 baseline). of 48 patients who developed aki, 54.2% (26 out of 48) of these aki patients required renal replacement therapy in the form of hd. requirement of hd was more in patients with underlying ckd (21 vs. 5) as compared to non - ckd patients. performed an observational study of 147,007 elderly medicare patients admitted for acute myocardial infarction (ami) as a part of the cooperative cardiovascular project. they evaluated the association between aki and all - cause mortality in the above - mentioned population. in their study, goldberg. studied the incidence and clinical significance of transient versus persistent aki in 1957 patients who survived an st - elevation ami. aki developed in 46.7% of the patients, of these 3.9% (10) patients received renal replacement therapy. we followed - up 48 patients, who developed aki to find out how many had complete recovery, partial recovery, and no recovery of aki at the end of 3 months. of 14 non - ckd patients who developed aki, 8 (57.1%) patients had complete recovery and 6 (42.9%) had partial recovery of aki. of 34 ckd patients, 16 (47.1%) had complete recovery, 10 (29.4%) had partial recovery, and 8 (23.5%) had no recovery of aki. statistically significant number of patients developed no recovery of their renal function in ckd group (23.5% vs. 0) as compared to non - ckd (p < 0.001).. however, limited time that we get to do our thesis makes it difficult to include large numberslong - term follow - up was not done in our study. long - term follow - up would have given a better idea about progression of renal dysfunction and its impact on cardiovascular and renal morbidity and mortalityfinally, we did not address the influence of different therapies that were administered during the acute phase of the cad on the changes in renal function.. however, limited time that we get to do our thesis makes it difficult to include large numbers long - term follow - up was not done in our study. long - term follow - up would have given a better idea about progression of renal dysfunction and its impact on cardiovascular and renal morbidity and mortality finally, we did not address the influence of different therapies that were administered during the acute phase of the cad on the changes in renal function. in view of high prevalence of ckd (39.2%) in patients with cad, every patient undergoing cag must have renal function evaluation before cag including urine, spot urine for albumin to creatinine ratio and serum creatinine levelsconsidering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary albumin to creatinine ratio, and in patients whose creatinine clearance is < 60 ml / minaki is an important complication of cag and precautions should be taken to prevent itsignificant number of patients has worsening of renal function at the end of 3 months. we find many patients developing progressive ckd after an episode of acute coronary syndromeall patients should have their renal function assessed 3 months after the cag. in view of high prevalence of ckd (39.2%) in patients with cad, every patient undergoing cag must have renal function evaluation before cag including urine, spot urine for albumin to creatinine ratio and serum creatinine levels considering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary albumin to creatinine ratio, and in patients whose creatinine clearance is < 60 ml / min aki is an important complication of cag and precautions should be taken to prevent it significant number of patients has worsening of renal function at the end of 3 months. we find many patients developing progressive ckd after an episode of acute coronary syndrome all patients should have their renal function assessed 3 months after the cag. in view of high prevalence of ckd (39.2%) in patients with cad, every patient undergoing cag must have renal function evaluation before cag including urine, spot urine for albumin to creatinine ratio and serum creatinine levelsconsidering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary albumin to creatinine ratio, and in patients whose creatinine clearance is < 60 ml / minaki is an important complication of cag and precautions should be taken to prevent itsignificant number of patients has worsening of renal function at the end of 3 months. we find many patients developing progressive ckd after an episode of acute coronary syndromeall patients should have their renal function assessed 3 months after the cag. in view of high prevalence of ckd (39.2%) in patients with cad, every patient undergoing cag must have renal function evaluation before cag including urine, spot urine for albumin to creatinine ratio and serum creatinine levels considering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary albumin to creatinine ratio, and in patients whose creatinine clearance is < 60 ml / min aki is an important complication of cag and precautions should be taken to prevent it significant number of patients has worsening of renal function at the end of 3 months. we find many patients developing progressive ckd after an episode of acute coronary syndrome all patients should have their renal function assessed 3 months after the cag. | background and objective : to determine the prevalence of chronic kidney disease (ckd) and incidence of acute kidney injury (aki) in patients with coronary artery disease (cad) demonstrated on coronary angiography.materials and methods : totally, 125 patients admitted to lilavati hospital and research centre, mumbai, with cad were included in the study.results:left anterior descending artery was the major vessel involved (40%), followed by a circumflex artery (21.6%). 49 out of 125 (39.2%) were found to have underlying ckd. 69% (34) of these ckd patients developed aki. 21 out of 34 patients who developed aki required hemodialysis. only 47.1% (16 out of 34) of ckd patients had complete recovery, 29% had partial recovery, and 23% had no recovery of their renal function from aki. statistically significant number of patients in ckd group had no recovery from aki as compared to non - ckd group (23.5% vs. 0%).conclusion : our study concludes that there is a very high prevalence of ckd (39.2%) in patients with cad and aki is a very important complication (38.4%) in these patients. considering such a high prevalence of ckd, nephrology referral must be considered in patients with abnormal urinalysis, spot urinary protein to creatinine ratio and in patients whose creatinine clearance is < 60 ml / min. |
bar - retained implant overdentures (iods) are a common treatment option in implant prosthodontics, and the implants that support the prosthetic restorations exhibit high survival rates.123 it has been discussed controversially if the number of implants or a placing of extensions on bars influence the clinical outcome of iods.45678 however, the data on the prevalence of biological complications remains limited.910 it has been proven that the bone resorption for iods on prefabricated bars is lower in comparison with iods on cast bars.11 in contrast, a systematic review demonstrated similar bone loss around implants supporting iods with different attachment designs.12 this retrospective study examined the survival and success rates as well as the prevalence of peri - implantitis of iods retained by 3 different bar designs. this study is based on patient files from the departments of maxillofacial surgery and prosthodontics of the university medical center goettingen. 1/9/09). twenty - seven patients (16 female, 11 male) were treated between 1991 and 2006 with iods (n=36) that were retained by 3 different bar designs (group a = prefabricated round bars without extensions, resilient anchorage, n=7 ; group b = one - piece anterior milled bars with distal extensions, rigid anchorage, n=20 ; and group c = two bilaterally placed milled bars, with distal extensions, rigid anchorage, n=9) on 4 to 6 implants (2 - 3 per side) (n=161, mean : 4.5 per jaw) in the mandible (n=24) and/or in the maxilla (n=12). inclusion criteria for the study were in compliance with annual recall intervals, a post - operative (baseline) panoramic radiograph, and at least one follow - up panoramic radiograph after a minimum observational period of 5 years (mean : 7.3 3.6 years ; range : 5 - 19 years). furthermore, the complete documentation of all implant- and prosthesisrelated technical complications was mandatory for inclusion in the study. all radiographs were digitally analyzed (adobe photoshop cs4, san jose, ca, usa). the marginal bone levels were calculated on the basis of the radiographic linear distance from the implant shoulder to the first bone - to - implant contact (fig. 1). radiographic bone loss during the functional period was calculated by subtracting the linear distance from the implant shoulder to the marginal bone level at baseline from the distance from the implant shoulder to the marginal bone level at the last available radiograph. bone loss 3.5 mm was defined as " peri - implantitis".91013 furthermore, all prosthodontic maintenance was recorded for the survival and success analysis according to kaplan - meier. technical complication rates for each type of restoration were analyzed and compared via one - way anova and the chi - squared test (software r version 2.8, www.r-project.org). two implants (one in the maxilla after 7 years and one in the mandible after 6 years) had to be removed (7-year survival rate : 97.7%). all iods remained in function (7-year survival rate : 100%). in total, 70 of the technical complications (attachment - related : 49%, denture - related : 51%) required clinical intervention to maintain the function of the iods (overall technical complication rate : 0.37 treatments per patient per year (t / p / y)), (table 1). technical complications occurred more frequently in group a (mean : 3.5 during observational time) than in the other two groups (b : 0.8 : c : 1.0). the success rates (kaplan - meier) which represent the share of iods that were functional without any clinical intervention are presented in fig. 3. peri - implantitis was diagnosed in 20 implants (12.4%), in 12 iods (3 in the maxilla, 9 in the mandible) (a : 2 ; b : 9 ; c : 1) in 10 of 27 patients (37%). six of the 10 smokers (60%) that participated in the study, and 4 of the 17 non - smokers (23.5%) were diagnosed with peri - implantitis (fig. the implant (97.7%) and prosthetic (100%) survival rates are similar to the findings reported by other studies.123 in literature, the requirements for prosthetic maintenance of iods vary between 0.25 and 4.03 (t / p / y).12310 the overall technical complication rate of 0.37 (t / p / y) that was calculated in the present study is at the lower end of this range, it is similar to other studies that reported technical complications of bar - retained iods.12 in the present study, iods that were retained by milled bars exhibited rather lower but statistically insignificant rates of mechanical complications in comparison with iods retained by prefabricated round bars. nevertheless, this is similar to previous studies that reported lower technical complication rates for milled bars than that of prefabricated round bars.12 krennmair.6 (2012) demonstrated high implant success rates and limited prosthodontic maintenance for rigid anchoring with milled bars or telescopic attachments. in this study, bar - related complications predominantly appeared for prefabricated round bars, whereas iods retained by milled bars showed more denture - related complications. bressan.14 (2012) also demonstrated mainly bar - related complications for iods retained by round bars supported by two implants. alternatively, heschl.15 (2013) documented only limited complication rates for round bars supported by 4 implants with distal extensions. other authors controversially discussed whether the number of implants and/or the use of extensions have an impact on the complication rates of iods. ueda.8 (2011) and meijer.7 (2009) found no differences between iods supported by 2 or 4 implants. (2007 & 2012) demonstrated prevention of non - axial overloading, posterior bone resorption or denture rotation for bar - structures rigidly retained by 4 implants.567 moreover, up to now, the relevance of extensions regarding the complication rates of iods seems indistinct. placing cantilevers on bars was rated useful to offer adequate occlusal posterior support.4 however, krennmaier.5 (2007) showed that distal extensions included in their bars affected neither the degree of distal bone loss nor the implant survival rate. nevertheless, in the present study round bars on 2 implants without extensions offered higher complication rates than milled bars on 4 implants including distal extensions. in the present study, 12.4% of the implants in 37% of the patients exhibited radiographic symptoms of peri - implantitis. studies using the same diagnostic criteria have reported implant - based peri - implantitis rates between 10.1 - 11.7%, and patient - based peri - implantitis rates of 9.2 - 10.0% for mean observational periods of 5.6 - 7 years.910 the bar design might explain the comparatively high peri - implantitis rates in the present study. it may lead to a reduced accessibility for oral hygiene procedures at home.10 iods retained by milled bars exhibited significantly higher plaque index values than iods retained by telescopic crowns.6 in the present study, especially massive one - piece milled bars in the mandible showed radiological symptoms of peri - implantitis (9 out of 20, however, the rates were not statistically significant). abd el - dayem.11 (2009) demonstrated less bone resorption for iods supported by round bars compared to custom - made bars. in the present study, smoking patients exhibited a higher prevalence of peri - implantitis than non - smoking patients (60% vs. 23.5%), thus supporting data from previous studies suggesting that a smoking habit is a significant risk factor for the development of peri - implant diseases.9 within the limitations of this study, we concluded that barretained iods are an adequate treatment option for edentulous jaws. these restorations may exhibit high implant and prosthesis survival rates (> 97%) and a limited incidence of technical complications after a mean observational period of > 7 years. nevertheless, peri - implantitis was identified as a frequent and serious biological complication for this type of reconstruction. | purposethis retrospective study evaluated the outcome of implant - retained overdentures (iods) after 5 - 19 years of clinical function.materials and methodsa retrospective analysis of patient files was performed referring to 27 patients who received 36 iods with 3 different bar designs (group a = prefabricated round bars, n=7 ; group b = one - piece anterior milled bars, n=20 ; and group c = two bilaterally placed milled bars, n=9) in the mandible (n=24) and/or in the maxilla (n=12). the analysis focused on the survival and success rates (according to kaplan - meier) of the implants and prostheses. technical complication rates for each type of restoration were analyzed and compared via one - way anova and the chi - squared test. the prevalence of peri - implantitis (radiographic bone loss 3.5 mm) was evaluated by digital analysis of panoramic radiographs taken post - operative (baseline) and after 5 - 19 years of clinical function (follow - up).resultsthe mean observational time was 7.3 years. the survival rates of the prostheses and implants were 100% and 97.7%, respectively. technical complications occurred more frequently in group a (mean : 3.5 during observational time) than in the other two groups (b : 0.8 ; c : 1.0). however, this difference was not statistically significant (p=0.58). peri - implantitis was diagnosed for 12.4% of the implants in 37% of the patients.conclusionbar-retained iods are an adequate treatment option for edentulous jaws. these restorations may exhibit high implant / prosthesis survival rates (> 97%), and a limited incidence of technical complications after a mean observational period of > 7 years. nevertheless, peri - implantitis was identified as a frequent and serious biological complication for this type of reconstruction. |
interaction of plant genotype with environment is one of the central problems of plant genetics. according to modern data, environmental factors, such as, temperature, water availability, lighting conditions, mineral nutrition regulate expression of plant genes affecting diverse epigenetic processes dna methylation, histone modification, micro - rna formation (grant - downton and dickinson, 2005 ; pfluger and wagner, 2007 ; phillips., 2007 ; king., 2010 ; fisher and franklin, 2011 ; these effects in certain cases, may be stable and are maintained both in vegetative propagation and in sexual reproduction (grant - downton and dickinson, 2006 ; takeda and paszkowski, 2006 ; daxinger and whitelaw, 2010 ; d : hauser:2011 ]). nuclear and cytoplasmic (mitochondrial) genes involved in genetic control of cytoplasmic male sterility (cms) maternally heritable failure to develop fertile pollen belong to genetic systems, which are strongly sensitive to environmental factors. it is generally accepted that cms is caused be expression of specific mitochondrial genes originating from high recombination activity peculiar to mitochondrial genome (chase and gabay - laughnan, 2004 ; hanson and bentolila, 2004 ; fujii and toriyama, 2008). however, expression of these genes takes place only in hybrid combinations, when they interact with alien nuclear genomes. in fertile lines - donors of cms - inducing cytoplasms (euplasmic lines), functioning of cms - inducing genes is inhibited by nuclear fertility - restoring genes, which suppress expression of these genes at the transcriptional or post - transcriptional level. recently obtained experimental data also suggest cms to be caused by retrograde regulation from mitochondrial genome of expression of nuclear genes involved in formation of fertile pollen (fujii and toriyama, 2008 ; yang., 2008). this hypothesis seems to be true for those cms types, which originate from interaction of genetically remote nuclear and cytoplasmic genomes. such cms types were obtained, in particular, in sorghum [sorghum bicolor (l.) moench ], which presents great number of subspecies and races that can be crossed giving rise to progeny that combines nuclear and cytoplasmic genomes of genetically remote parents. these types of cms differ by mechanisms of pollen degeneration, phenotypes of sterile anthers, reaction to fertility - restorer lines, mode of action of fertility - restoring genes, types of mitochondrial, and chloroplast dnas etc. (pring., 1995). assuming significant role of retrograde signaling from chloroplasts (fernandez and strand, 2008 ; chan., 2010) and mitochondria (atkin and macherel, 2009), such genetically remote nuclear - cytoplasmic combinations may disturb cross - talk between nuclear and cytoplasmic genomes that may result in sensitivity to biotic and abiotic stresses, in particular, in drought stress response. in this connection, one may suggest that such impacts of environmental factors could down - regulate specific nuclear gene(s) involved in genetic control of pollen development. in our investigations on genetics of male fertility restoration in some types of cms - inducing cytoplasms of sorghum (9e, a4, m35 - 1a) we found an unusual inheritance pattern : the rf - genes function in the self - pollinated progenies of f1 hybrids but are not expressed or poorly expressed in backcrosses of these hybrids to parental cms - lines or in test - crosses to cms - lines with the same cytoplasm type (elkonin. similar phenomenon was also described for the a3 cytoplasm and it was explained by paramutation of the rf - genes caused by sterility - maintaining alleles (tang., 2007). in our experiments, we observed that the level of male fertility of the f1 hybrids in the 9e and m35 - 1a cytoplasms correlates with the level of plant water availability during panicle development stage (elkonin., 2005). on the basis of these data we suggested hypothesis on epigenetic regulation of fertility - restoring genes for the 9e cytoplasm (elkonin., 2006). in this paper, we report experimental results on heritable activation of fertility - restoring genes by inductive environmental conditions (plant water availability conditions) and expression of activated genes in self - pollinated progenies of fertile hybrids and in test - crosses with cms - lines in non - inductive conditions that support hypothesis on epigenetic regulation of a dominant status of fertility - restoring alleles for the the cms - lines used in this study were [9e ] tx398, [9e ] milo-10, and [9e ] volzhskoe-615 (v-615). (webster and singh, 1964). the lines [9e ] milo-10 and [9e ] v-615 were obtained by backcrossing of the line [9e ] tx398 with the lines milo-10 and volzhskoe-615. the seeds of the line [9e ] tx398 were generously provided by the late dr k. f. schertz (texas agricultural experimental station, usa). kvv-263 was obtained by self - pollination of the fertile f1 hybrids, [9e ] tx398/kvv-112 grown in greenhouse. the f2 progeny and subsequent generations were grown in field conditions. to test the hypothesis of heritable activation of fertility - restoring genes by plant water availability conditions one and the same f1, f2, and bc1 hybrids (i.e., the hybrids obtained by pollination of maternal cms - lines with the pollen of f1 hybrids) dry plot, which has a roof from translucent polycarbonate to prevent irrigation of plants by rain precipitation (the roof was put on the plot at the booting stage of plant development), and in irrigated plot, which was artificially irrigated starting from the same developmental stage (the total amount of watering was 70 l / m). in addition, sterile hybrids from f1 and f2 families grown in the dry plot and in the irrigated plot were transferred into greenhouse and were grown with regular irrigation. the progenies of fertile f1 hybrids and of fertile panicles developed on sterile plants, transferred to the greenhouse, were grown in the dry plot and in experimental field. to evaluate level of male fertility the first panicle of each plant the level of male fertility was estimated at anthesis by cytological analysis of pollen and at maturity by determining the percent seed set. on the basis of the percent seed set on the panicles the plants were classified as sterile (s) (0% seed setting or a few, no more than 1% seeds), partially sterile (ps ; 50% ; usually 90100%). for cytological analysis of pollen fertility the spikelets from different parts of the panicle were fixed in acetic alcohol (1:3), and stored in 75% alcohol. for pollen analysis the anthers from 10 to 15 spikelets the -test and exact binomial test (mcdonald, 2009) were used to determine the fit of observed ratios of sterile and fertile plants to the expected segregation ratio. the number of fertile and ps - plants in different progenies was also compared by the fisher method using f - criteria (zaitsev, 1984). results of experiments on parallel growth of hybrid populations in the dry plot and in the irrigated plot clearly demonstrate that water availability strongly affects the male fertility level of sorghum hybrids with the 9e cytoplasm (table 1). in the majority of the combinations analyzed sterile plants appeared in the f1 and bc1 generations in the dry plot ([9e ] rannee-7/kvv-263 ; [9e ] milo-10/pers-1 ; [9e ] p-614/pers-1), whereas only fertile ([9e ] milo-10/pers-1) or fertile and ps - plants were observed in the irrigated plot ([9e ] rannee-7/kvv-263 ; [9e ] p-614/pers-1). in other hybrid combinations in contrast to complete or almost complete male sterility of the dry plot. in this connection, the most significant results were obtained in the hybrid combination [9e ] milo-10/kvv-263, in which f1 plants have almost completely sterile phenotype in drought conditions. however, after artificial watering ps - plants appeared among these hybrids. in the [9e ] tx398/kvv-263, the f1 hybrids were completely fertile in the irrigated plot, while both fertile and ps - plants were found in the dry plot. remarkably, fertile line in the 9e cytoplasm, kvv-263, which was one of the sources of fertility - restoring genes was fertile both in the irrigated plot and in the dry plot. these data demonstrate that fertility - restoring genes, in homozygous state, can function in drought conditions, however, in heterozygous state, these genes need in higher level of water availability for their expression. effect of plant water availability at panicle development stage on level of male fertility of sorghum hybrids in the f, fertile (seed set > 50%) ; ps, partially sterile (50%) ; ps, partially sterile (50%) ; ps, partially sterile (50%) ; ps, partially sterile (p > 0.25 ; figure 4a). similar segregation, 3:1, was observed in this family also in the irrigated plot (= 0.545 ; 0.50 > p > 0.25). in sterile plants from the dry plot, no 188 - 8 and no 188 - 15, after their transfer to the greenhouse developed fertile panicles. in the progeny of these panicles, which were grown in field conditions, fertile and sterile individuals were noticed. after two cycles of self - pollination of fertile individuals from the progeny of 188 - 15 plant, the line no 21/10 was obtained. this line manifested complete male fertility in conditions of both irrigated plot and dry plot however, backcross hybrids of this line to maternal cms - line [9e ] milo-10 were characterized by complete male sterility. inheritance of reversion to male fertility induced in greenhouse in male - sterile plants 188 - 15 (a) and 1888 (b) from f2 family of [9e ] milo-10/perspectivnoe-1. inheritance of reversion to male fertility in the progeny of fertile panicles developed on another male - sterile plant from this family, no 188 - 8 is of special interest (figure 4b). in the self - pollinated progeny of fertile panicles (f3), segregation for plant fertile plants from this progeny were either homozygous or produced families with few sterile or ps - plants. sterile plants, being transferred to greenhouse, again developed fertile panicles, in their progeny (f4) grown in field conditions segregation was observed again. the progeny obtained from sterile plants from this family after their transfer to greenhouse (f5) was not homozygous but segregated again for male fertility ; the self - pollinated progeny of fertile plants consisted almost completely from fertile individuals, while sterile plants again reverted in the greenhouse to male fertility. it should be noted that in the self - pollinated progeny of ps - plants, as well as in the progeny of fertile plants, either only fertile, or fertile and few ps or sterile individuals were found that point on identity of genotype of fertile and ps - plants. remarkably, revertant panicles that produced fertile progeny after self - pollination, did not restore male fertility of test - cross hybrids with cms - line [9e ] tx398. principally different result was noticed in test - crosses of fertile line no 14/10 that was obtained after self - pollination of fertile panicles developed in male - sterile plant from f2 family of [9e ] tx398/persp-1 transferred from the dry plot to greenhouse (figure 5). this line was able to restore male fertility of test - cross hybrids with the cms - line [9e ] milo-10. fertility of the line 14/10 and its test - cross hybrids expressed both in the irrigated plot and in the dry plot. thus, in this case, after reversion in greenhouse conditions the line - fertility restorer was obtained on the basis of cms - plant. inheritance of reversion to male fertility induced in greenhouse in male - sterile plant from f2 family of [9e ] tx398/perspectivnoe-1. results of experiments on parallel growth of hybrid populations in the dry plot and in the irrigated plot clearly demonstrate that water availability strongly affects the male fertility level of sorghum hybrids with the 9e cytoplasm (table 1). in the majority of the combinations analyzed sterile plants appeared in the f1 and bc1 generations in the dry plot ([9e ] rannee-7/kvv-263 ; [9e ] milo-10/pers-1 ; [9e ] p-614/pers-1), whereas only fertile ([9e ] milo-10/pers-1) or fertile and ps - plants were observed in the irrigated plot ([9e ] rannee-7/kvv-263 ; [9e ] p-614/pers-1). in other hybrid combinations in contrast to complete or almost complete male sterility of the dry plot. in this connection, the most significant results were obtained in the hybrid combination [9e ] milo-10/kvv-263, in which f1 plants have almost completely sterile phenotype in drought conditions. however, after artificial watering ps - plants appeared among these hybrids. in the [9e ] tx398/kvv-263, the f1 hybrids were completely fertile in the irrigated plot, while both fertile and ps - plants were found in the dry plot. remarkably, fertile line in the 9e cytoplasm, kvv-263, which was one of the sources of fertility - restoring genes was fertile both in the irrigated plot and in the dry plot. these data demonstrate that fertility - restoring genes, in homozygous state, can function in drought conditions, however, in heterozygous state, these genes need in higher level of water availability for their expression. effect of plant water availability at panicle development stage on level of male fertility of sorghum hybrids in the f, fertile (seed set > 50%) ; ps, partially sterile (50%) ; ps, partially sterile (50%) ; ps, partially sterile (50%) ; ps, partially sterile (p > 0.25 ; figure 4a). similar segregation, 3:1, was observed in this family also in the irrigated plot (= 0.545 ; 0.50 > p > 0.25). in sterile plants from the dry plot, no 188 - 8 and no 188 - 15, after their transfer to the greenhouse developed fertile panicles. in the progeny of these panicles, which were grown in field conditions, after two cycles of self - pollination of fertile individuals from the progeny of 188 - 15 plant, the line no 21/10 was obtained. this line manifested complete male fertility in conditions of both irrigated plot and dry plot however, backcross hybrids of this line to maternal cms - line [9e ] milo-10 were characterized by complete male sterility. inheritance of reversion to male fertility induced in greenhouse in male - sterile plants 188 - 15 (a) and 1888 (b) from f2 family of [9e ] milo-10/perspectivnoe-1. inheritance of reversion to male fertility in the progeny of fertile panicles developed on another male - sterile plant from this family, no 188 - 8 is of special interest (figure 4b). in the self - pollinated progeny of fertile panicles (f3), segregation for plant fertile plants from this progeny were either homozygous or produced families with few sterile or ps - plants. sterile plants, being transferred to greenhouse, again developed fertile panicles, in their progeny (f4) grown in field conditions segregation was observed again. the progeny obtained from sterile plants from this family after their transfer to greenhouse (f5) was not homozygous but segregated again for male fertility ; the self - pollinated progeny of fertile plants consisted almost completely from fertile individuals, while sterile plants again reverted in the greenhouse to male fertility. it should be noted that in the self - pollinated progeny of ps - plants, as well as in the progeny of fertile plants, either only fertile, or fertile and few ps or sterile individuals were found that point on identity of genotype of fertile and ps - plants. remarkably, revertant panicles that produced fertile progeny after self - pollination, did not restore male fertility of test - cross hybrids with cms - line [9e ] tx398. principally different result was noticed in test - crosses of fertile line no 14/10 that was obtained after self - pollination of fertile panicles developed in male - sterile plant from f2 family of [9e ] tx398/persp-1 transferred from the dry plot to greenhouse (figure 5). this line was able to restore male fertility of test - cross hybrids with the cms - line [9e ] milo-10. fertility of the line 14/10 and its test - cross hybrids expressed both in the irrigated plot and in the dry plot. thus, in this case, after reversion in greenhouse conditions the line - fertility restorer was obtained on the basis of cms - plant. inheritance of reversion to male fertility induced in greenhouse in male - sterile plant from f2 family of [9e ] tx398/perspectivnoe-1. experimental data presented above clearly demonstrate that environmental conditions, such as water availability during panicle and pollen developmental stages, affect functioning of the fertility - restoring genes for the its influence most strongly affects male fertility of f1 and test - cross hybrid populations, in which fertility - restoring genes were in heterozygote state. in some hybrid combinations ([9e ] milo-10/kvv-263, [9e ] v-615/kvv-263), water - dependence of fertility - restoring genes is extremely strong, so that in drought conditions the f1 heterozygous plants were completely male - sterile. growing in conditions of high level of water availability or transferring into greenhouse conditions allowed obtaining male - fertile f1 hybrids in these combinations, and, on their base, fertile lines however, male fertility induced by this way, as a rule, did not transmit through the pollen in test - crosses with cms - lines in the 9e cytoplasm, with the only exception of revertant from the male - sterile plant from the f2 [9e ] tx398/persp-1 family. such absence of fertility transmission through the pollen can not be explained by cytoplasmic nature of fertility reversions because previously we have found that the reciprocal hybrids, kvv-263/tx398, obtained by pollination of emasculated panicle of fertile line kvv-263, which derived from the f1 hybrid [9e ] tx398/kvv-112 grown in greenhouse conditions, with the pollen of the line - sterility maintainer of the tx398 were male - sterile in drought conditions as well as direct hybrids, [9e ] tx398/kvv-263 (elkonin., 2005). perhaps, fertility - restoring genes are sensitive to water availability of plants during panicle development stage, and in heterozygous state produce small amount of product in drought conditions. therefore, f1 or bc1 plants in drought conditions are sterile. in wet conditions, after self - pollination and recombination, the homozygous plants for fertility - restoring genes would appear in the progeny of such heterozygotes, and these plants may be fertile in drought conditions if expression level in homozygous state is sufficient for restoration of male fertility. in other words, according to this hypothesis, water availability conditions change dominance in the nuclear loci governing fertility restoration : in drought conditions sterility - maintaining alleles are dominant, while in wet conditions fertility - restoring alleles are dominant. as follows from this hypothesis, in the f2 families grown in the irrigated plot, plants with restored male fertility (f- and ps - phenotypes) should be homozygous and heterozygous, while sterile plants (s - phenotype) should be only homozygous. in the dry plot, controversially, sterile plants should be heterozygous and homozygous, while plants with restored male fertility should be homozygous. segregation observed in the f2 progeny of ps - plant no 1 [9e ] v-615/kvv-263 (figure 2b) grown in the irrigated plot [17 (f + ps):9 s ; 3:1 = 0.167 ; 0.50 > p > 0.25 ] and in the dry plot [8 (f + ps):19 s ; 1:3 = 0.309 ; 0.75 > p > 0.50 ] confirmed this hypothesis. however, in the f2 progeny of ps - plant no 13 from the same hybrid combination no differences were observed between the irrigated plot and the dry plot (figure 2b). in both cases, the frequency of plants with restored male fertility prevailed over sterile plants ; the ratio fitted monogenic segregation 3:1 (= 0.051 ; 0.90 > p > 0.75 and = 1.301 ; 0.50 > p > 0.25 ; respectively). in addition, this hypothesis can not explain reversion to male fertility of male - sterile individuals segregating in the f2 families as recessives. therefore, more likely is another hypothesis that assumes epigenetic activation of fertility - restoring alleles by high water - availability conditions. the sensitivity of epigenetic events (such as dna methylation, histone modifications) to plant water availability conditions, is well - documented (lukens and zhan, 2007 ; chinnusamy and zhu, 2009 ; verhoeven., 2010). such a modification (dimethylation of h3 at lysine 4) associated with active gene expression was found to be high during the wet season in perennial desert plant zygophyllum dumosum boiss. (granot., 2009), or prevent methylation of fertility - restoring gene sequences that may take place in drought conditions (wang., 2011) and this methylation state is transmitted to the next sexual generations. such transgenerational inheritance of methylation state is well - known phenomenon (kakutani, 2002 ; takeda and paszkowski, 2006 ; daxinger and whitelaw, 2010 ; d : hauser:2011 ]). according to this hypothesis fertility - restoring genes are activated in wet conditions (rf / rf rf / rf ; rf / rf rf / rf, where the asterisk means activated allele) and then are transmitted to the progeny in their active state. however, taking into account complete male sterility of test - cross plants one should suppose that activated state of fertility - restoring genes inherited only under self - pollination, whereas in new genetic background (f1 hybrid genome) the epigenetic marks caused activation of fertility - restoring genes are erased and the test - cross plants become male - sterile. such inheritance is typical for epigenetically determined traits, which are caused, in particular, by changes of dna methylation pattern. data on remodeling of the parental methylation patterns in the f1 hybrids are well - documented (xiong., 1999 ; shaked., 2001 ; it should be stressed that after such activation the fertility - restoring genes become expressive not only in wet conditions but also under drought stress (figures 2a, 4a, and 5). to explain these segregation patterns, one should take into account the data from table 3. as follows from these data, both fertility - restorers, which were used in our experiments, kvv-263 and persp-1, in the wet seasons (2003, 2008) both genes functioned and segregation corresponded to 15:1 ratio, whereas in the dry seasons (2001, 2009) only one fertility - restorer gene expressed, and segregation ratio corresponded to 3:1 ratio. therefore, sterile plants which were taken from f2 families from the dry plot may have genotype rf1rf1rf2rf2 or rf1rf1rf2. after activation in the greenhouse conditions, the heterozygotes for two pairs of fertility - restoring alleles may appear (rf1rf1rf2rf2 and/or rf1rf1rf2 rf2). therefore, segregation in the progeny of such fertility revertants should correspond to digenic ratio. indeed, segregation in the progeny of fertility revertant from sterile plants 188 - 15 (f2 [9e ] m-10/persp-1 ; figure 4a) and 16 - 4 (f2 [9e ] m-10/kvv-263 ; figure 3) presumably corresponds to 9 (f + ps):7 s ratio (= 0.241 ; 0.75 > p > 0.50 ; and = 0.042 ; 0.90 > p > 0.75 ; respectively). in the self - pollinated progenies of fertile plants from these families male fertility inherited as a dominant trait, and segregation fitted to either 3:1 (families 59/11, 60/11, figure 3 : = 0.659 ; 0.50 > p > 0.25, for total data) or 9:7 (family 27/10, figure 4a : = 0.039 ; 0.90 > p > 0.75) ratios, perhaps, depending on genotype of self - pollinated plants (mono- or di - heterozygotes). ratio of fertile and male - sterile individuals in the progeny of fertility revertants from another sterile plant from this hybrid combination, 188 - 8 (figure 4b), also resembled segregation for two pairs of genes, although in this case activated fertility - restoring alleles behaved as recessives and segregation fitted to 7 (f + ps):9 s ratio (= 0.001 ; 0.99 > p > 0.95). this type of segregation means that sterility should be caused by two complementary dominant sterility - maintaining genes. in this case indeed, self - pollinated progeny of fertile plants were either fertile or contained only few sterile or ps individuals. but contrary to primary revertants (families no 120/08, 27/09, 43/10, figure 4b) the revertants from sterile individuals from the progeny of self - pollinated fertile plants produced predominantly fertile off - spring (family no 23/11, figure 4b) as it was observed in self - pollinated progenies of fertile plants. 9e cms - inducing cytoplasm of sorghum is conditioned by epigenetic changes appearing by the influence of sufficient level of water availability. this state is transmitted to self - pollination progenies but in the new genetic background in f1 or bc1 hybrid genome it might be established de novo and only under sufficient level of water availability. at present time we can only speculate about the molecular mechanisms involved in suppression of fertility - restoring genes for the recent studies on retrograde signaling from chloroplast, which are the primary targets of drought stress (chan., 2010) suggest that any molecules accumulating within these organelles under water deficit, such as reactive oxygen species, ros (suzuki., 2011), or 3-phosphoadenosine 5-phosphate, pap (estavillo., 2011), may down - regulate nuclear gene(s) involved in pollen maturation and/or anther dehiscence. in addition to chloroplasts, mitochondria play also an important role in drought response by supplying chloroplasts with atp, controlling the ros accumulation and regulating proline concentration (atkin and macherel, 2009). perhaps, withdrawal of water deficit in plants grown in the irrigated plot or in greenhouse conditions prevents accumulation of ros molecules within chloroplasts and by mechanism of retrograde signaling activate expression of nuclear genes restoring male fertility. however, such activation would not be inherited in subsequent sexual generation, unless inherent changes would occur in nuclear loci controlling pollen development, taking into account that heritable restoration of male fertility did not result from cytoplasmic mutation(s) (elkonin., 2005). therefore, to test the outlined hypothesis on activation of fertility - restoring genes by high water availability conditions it is crucially important to study the nature of molecular changes that may occur in the nuclear loci controlling fertility restoration in the future investigations, therefore, should be conducted with identification of nucleotide sequences of genes located in these loci, and investigation of epigenetic changes occurring in these loci in different plant water availability conditions. assuming that almost all of the fertility - restoring nuclear genes studied up to date encode proteins with ppr motif (fujii and toriyama, 2008), and that the ppr proteins involved in post - transcriptional regulation of gene expression in chloroplasts and mitochondria, were also found to be involved in abiotic stress response (zsigmond., 2010 ; yuan and liu 2012), one should suppose that environmentally sensitive fertility - restoring genes in our revertants also encode a protein(s) belonging to ppr family. therefore, another approach that would be helpful in investigation of hypothesis on activation of fertility - restoring genes by high water availability is to study chloroplast and mitochondria proteome in water - stressed and irrigated plants, with special attention to ppr proteins. 9e cytoplasm represent a model systems for studying regulation of plant response to drought stress. further investigation of this system as well as the molecular bases of epigenetic events in the fertility - restoring loci occurring under high water - availability conditions are needed to shed light on fundamental genetic problems, such as anterograde and retrograde signaling, gene dominance and interaction of genes with their nuclear background, and on possible application of these findings in sorghum genetic improvement. 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. | heritable changes of phenotype arising in plant ontogenesis by the influence of environmental factors belong to the most intriguing genetic phenomena. an unusual inheritance pattern was detected during examination of male fertility restoration in the cms - inducing 9e type cytoplasm of sorghum : rf - genes were functional in self - pollinated progeny of f1 hybrids yet were either not expressed or poorly expressed in backcrosses of these hybrids to cms - lines with the same cytoplasm type. in experiments on parallel growing of the same f1 hybrid combinations in the dry plot and in the irrigated plot, it was found that high level of plant water availability during panicle and pollen developmental stages significantly increased male fertility of f1 and test - cross hybrid populations, in which fertility - restoring genes were in heterozygote state, whereas in f2 populations the influences of water availability conditions cause less pronounce effects. similarly, male - sterile f1 plants, being transferred from the dry plot to greenhouse, produced male - fertile panicles. in addition, male - sterile plants from f2 families, which segregated - out as recessives, being transferred to greenhouse also produced male - fertile panicles. in the progenies of these revertants that were grown in field conditions and in the dry plot, stable inheritance of male fertility for three cycles of self - pollination was observed, and a number of stable fertile lines in the 9e cytoplasm were obtained. however, in test - crosses of these fertile lines to cms - lines with the 9e cytoplasm restoration of male fertility was not observed, except the progeny of one revertant that behaved as fertility - restorer line. these data suggest that the functional state of fertility - restoring genes for the 9e sorghum cytoplasm is epigenetically regulated trait established by the influence of environmental factors and is transmitted to sexual generations. |
in the age of health care reform, institutions are asking providers to continually improve the quality of care while reducing costs. patient satisfaction with pain management is a critically important measure of quality health care.1 while opioids are effective analgesics, their use is frequently associated with adverse effects such as dizziness, nausea, sedation, constipation, and in some cases, respiratory depression.2 guidelines suggest that opioid - sparing multimodal analgesia should be used whenever possible in order to optimize analgesic efficacy while minimizing the risk of opioid - related adverse events.2,3 regional anesthesia techniques are an effective mode of analgesia, considered safe with a low risk for complications, and are a frequently utilized component in multimodal analgesic regimens.35 transversus abdominis plane (tap) infiltration is a regional anesthesia technique that provides analgesia to the anterior abdominal wall. abdominal field blocks have been utilized for more than a century, but infiltration techniques used in tap were developed and refined within the last 15 years. tap infiltration has been demonstrated to be an effective regional anesthesia approach to controlling postsurgical pain, frequently allowing patients a faster recovery time after undergoing abdominal surgery.68 the tap is a potential anatomical space in the anterior wall that spans the entire abdomen between the internal oblique and transversus abdominis muscles (figure 1).9 the anterior rami of the lower six thoracic nerves (t7t12) and first lumbar nerve (l1) pass through the plane and provide somatic innervation to the anterior and lateral abdominal walls.6,10,11 the nerves are bound to the transversus abdominis muscle by a layer of fascia, suggesting that local anesthetics should be deposited underneath the fascial layer to ensure optimal analgesia.11 detailed anatomic studies of the tap have revealed individual variability in the extent of spinal nerve branching. in a cadaveric dissection study of innervation of the anterior abdominal wall (n=20 subjects),11 extensive branching was noted for t9l1 starting at the anterior axillary line to the midline. in addition, a large, longitudinal plexus of communicating branches was observed within the tap in the anterolateral abdominal wall lateral to the deep circumflex iliac artery. a second longitudinal plexus exists closer to the midline, running cephalocaudally with the inferior epigastric artery.11 another cadaveric study9 noted individual variation in the dimensions of the lumbar triangle of petit, usually situated just behind the highest point of the iliac crest (figure 2),6,12,13 which serves as an anatomical landmark for tap infiltration. the study also noted that the t9l1 nerves held a relatively constant course in the tap prior to the midaxillary line, but that branching after this point was commonly observed.9 tap infiltration was originally characterized as a landmark - guided abdominal field block based on the lumbar triangle of petit in 2001.12 appropriate needle placement using the landmark technique resulted in a pop or sensation of giving way, which indicated that the needle had reached the fascial plane between the internal oblique and transversus abdominis muscles.12 the technique, initially described as a field block or a regional abdominal field infiltration, was eventually referred to as a tap block.7,14 this technique was evaluated and modified through a series of cadaveric and healthy volunteer studies.9,1518 an alternative method was described in which the needle is advanced perpendicularly until the first pop is felt in between the external and internal oblique muscles, and then advanced again until a second pop is felt, indicating entry into the transversus abdominis fascial plane.6,18 as spread of the local anesthetic is critical for analgesic efficacy, an ultrasound - guided (usg) technique was a logical progression for tap infiltration. the initial usg approach involved transverse application of the ultrasound probe to the anterolateral abdominal wall where the three muscle layers are most distinct (figure 3).19,20 the needle, as well as the hypoechoic layer of local anesthetic in the fascia, was visualized in the plane.20 a series of clinical studies evaluated the extent of analgesia in multiple surgical models, including prostatectomy, abdominal surgeries (bowel resections), appendectomy, cesarean section, and abdominal hysterectomy. initial clinical results deviated from the landmark - based technique, as the spread of analgesia did not seem to be as extensive.2125 although the reason for this is not well understood, it has been suggested that the key objective in ultrasound is visualization of the spread of the injectate between the internal oblique and transversus muscles, possibly causing the operator to ignore the tactile pop that indicates optimal needle placement in the anatomical space underneath the fascial layer.8 in response to this clinical variation, the technique evolved further. multiple and single injections as well as lateral, subcostal, and posterior approaches have all been described.17,19,24,26 a report describing four different approaches to tap infiltration found that local anesthetic spread resulting from the posterior approach mirrored most closely the original landmark - guided triangle of petit approach.17 spread of local anesthetic was measured radiologically, and was found to cover t5l1 levels using these techniques.17 a four - point single - shot technique that combines both the posterior and subcostal approaches has also been described and purportedly provides wider analgesic coverage.19 in sum, there are many factors that impact analgesic outcomes in tap : in addition to method of nerve localization and needle placement, population type (pregnant vs nonpregnant, obese vs nonobese) ; surgical procedure ; practitioner s level of skill ; type, dose, and volume of local anesthetic used ; timing of injection ; and clinical assessments used all have an impacting role.8 it was determined that extent of spread was variable and nondermatomal, indicating that the specific nerves that are blocked as a result of a tap infiltration likely vary from patient to patient based on their anatomy.27 the many nuances of the technique have led to a debate in the field : is there a need for standardization of techniques or technique nomenclature ? the tap block family and regional abdominal field infiltration have both been proposed to describe the group of techniques that have evolved from the initial triangle of petit landmark technique.7,8 however, the fundamentals of the technique remain consistent across approaches : no specific nerve within the tap is targeted ; localization of the plane and the spread of local anesthetic within the plane are the key determinants of analgesia.19 multiple surgeon - assisted approaches have been described, suggesting that techniques used to access the plane itself have also broadened. intra - abdominal laparoscopic cameras operated by surgeons, rather than ultrasound, have been used to visualize the plane. for example, one report describes a technique developed to enable identification of a peritoneal bulge in the area of injection that confirms delivery of local anesthetic into the tap.28 other reports describe a surgeon - assisted intraoperative transperitoneal approach in which the blunt - tipped block needle was advanced from inside the abdominal wall through the parietal peritoneum, into the transversus abdominis muscle, and then into the tap.29,30 direct visualization of local anesthetic spread in the tap using blunt dissection of the oblique muscles has also been described.31 the goal of the surgeon - assisted approach is to conduct the same field block as traditional surgical infiltrative techniques where local anesthetic is deposited near the incision. whether local anesthetics are utilized for a peripheral nerve block or for infiltration, the objective is the same to block the sensory response. however, key technical components diverge when comparing a peripheral nerve block to an infiltration technique. as described above, tap infiltration comprises multiple approaches used to identify an anatomical plane, instead of a specific nerve or nerve plexus. for example, in a peripheral nerve block of the upper or lower extremity, usg and/or nerve stimulation are frequently used to identify the nerve that is to be blocked along with surrounding anatomy.19 the optimal needle position is near or toward that specific nerve, and spread of local anesthetic around the nerve is observed.32,33 precision and ability to maintain appropriate needle placement are of the utmost importance in peripheral nerve blocks.19 while the objective of peripheral nerve block and tap infiltration are similar in that both approaches block sensory response in order to achieve analgesia, the technical components of these two approaches are different. unlike peripheral nerve block, which involves administration of a local anesthetic in close proximity to a specific nerve or nerve plexus, tap infiltration is a field block technique in which a local anesthetic is administered into an anatomical plane. | transversus abdominis plane (tap) infiltration is a regional anesthesia technique that has been demonstrated to be effective for management of postsurgical pain after abdominal surgery. there are several different clinical variations in the approaches used for achieving analgesia via tap infiltration, and methods for identification of the tap have evolved considerably since the landmark - guided technique was first described in 2001. there are many factors that impact the analgesic outcomes following tap infiltration, and the various nuances of this technique have led to debate regarding procedural classification of tap infiltration. based on our current understanding of fascial and neuronal anatomy of the anterior abdominal wall, as well as available evidence from studies assessing local anesthetic spread and cutaneous sensory block following tap infiltration, it is clear that tap infiltration techniques are appropriately classified as field blocks. while the objective of peripheral nerve block and tap infiltration are similar in that both approaches block sensory response in order to achieve analgesia, the technical components of the two procedures are different. unlike peripheral nerve block, which involves identification or stimulation of a specific nerve or nerve plexus, followed by administration of a local anesthetic in close proximity, tap infiltration involves administration and spread of local anesthetic within an anatomical plane of the surgical site. |
we can save many teeth by endodontic procedures rather than extraction, and it has been successful and conservative mode of treatment. however, sometimes mishaps such as accidental ingestion of the endodontic instrument may occur if the treatment is carried out without application of rubber dam, especially in the child patient. such mishaps are rare but potentially very harmful to the patients and causes anxiety to the clinician. when an accidental ingestion of an endodontic instrument occurs, endodontist should have a basic knowledge about the diagnostic procedure, complications, methods of retrieving the swallowed instrument, as well as the ability to reassure the patient.1 for the endodontic instruments, the prevalence for aspiration was 0.0009/100,000 root canal treatments and the prevalence for ingestion was 0.08/100,000 root canal treatments.2 the reason for the relatively low reported incidence, when endodontic equipment is involved, may be the use of rubber dam. aspiration of foreign bodies during dental treatment is rare, however, when such accidents happen it is considered a major complication in terms of the treatment needed to avert this event, and the impact on the patient s health. the present case report describes a rare case of swallowing of a hand pro taper file used for pulpectomy in 4 years old patient and also provides the knowledge about possible complications that may arise due to accidental swallowing of such instruments and their treatment modalities depending upon the location of the instrument either in gastrointestinal tract or respiratory tract. a 4-year - old male patient reported to the department of pediatric and preventive dentistry, annasaheb chudaman patil memorial dental college, dhule with pain in his lower left back tooth. on radiographic examination, pulpectomy was planned for the tooth 74. on the same day, access opening was done with tooth 74 under local anesthesia, pulp was extirpated, working length was determined and bio - mechanical preparation was in progress when patient suddenly moved his head due to which endodontic instrument (size sx pro taper hand file, dentsply) slipped from operator s hand and patient swallowed it. after this unfortunate event, pulpectomy procedure was immediately discontinued, and measures were taken to retrieve the file. the patient was kept in the prone position with the head at lower level than the limb, and 4 - 5 thrusts were given on his back expecting the expulsion of the instrument. hence, patient was immediately shifted to the annasaheb chudaman patil memorial medical college, dhule in emergency medicine department. there a postero - anterior chest radiograph demonstrated the presence of sharp foreign object at the level l2-l3 (figure 1), just below the shadow of the diaphragm. the clinical and radiographic assessment done and opinion sought from the pediatric surgeon, who advised to keep the patient under observation as the radiographic findings were suggestive of presence of endodontic instrument in the gastrointestinal tract (stomach) and not in the respiratory tract and patient was free of any respiratory distress or coughing or sneezing. clinically, smooth breathing sound was noticed and no tenderness or pain was present on palpation of the area in which foreign body was present. chest radiograph showing protaper file at the level of l2-l3 (immediately after ingestion). high risk consent was obtained from the parents accompanying the child and patient was kept under high fibrous diet, and syrup chremaffin was prescribed for allowing the rapid transport of the instrument within the gastrointestinal tract. all the vital statistics were obtained and regularly noted every 2 h. serial radiographic examinations were performed to monitor the migration of the instrument. a repeated radiograph was taken of postero - anterior view of abdomen, 24 h after the ingestion of the instrument. this radiograph suggested the presence of the endodontic instrument in the caecum of the large intestine (figure 2). 41 h after the file ingestion, the file was found in the faeces, and confirmatory radiograph was obtained to make sure the absence of any foreign object in the gastrointestinal tract (figures 3 and 4). three weeks later, pulpectomy was completed in the department of pediatric and preventive dentistry under the rubber dam isolation. whenever a dentist loses a dental instrument, dental materials or any other foreign object inside oral cavity, he must consult a radiologist even if the patient has no complaints. according to hodges., mentally and physically handicapped children are more prone to ingestion or aspiration of dental instruments than other populations. in any event, they recommended that dental practitioners examine their instrument before use as a safeguard against any slippage, breakage.2 treatment with any endodontic device requires the use of a rubber dam. surprisingly, in a survey by whitworth. very few (< 20%) of the surveyed dentists were using rubber dam routinely while the vast majority of them (60%) have reported never to use rubber dam while performing endodontic procedures.3 there are many possible symptoms of foreign body ingestion such as chest pain, acute dysphagia, vomiting, choking, drooling, blood stained saliva. respiratory symptoms as wheezing, coughing, dyspnea may suggest that the foreign object has lodged in the upper gastrointestinal or respiratory tract. endodontic files, which are used for root canal cleaning procedure, have been reported to pass through gastrointestinal system within 3 days, but 10% require endoscopic removal and only 1% will require surgical intervention.4 patients with stomach or small - intestine foreign bodies of width < 2 cm or length < 6 cm can be discharged home with instructions on symptoms that should prompt their re - attendance.5 if a foreign object found to be lodged in respiratory tract, bronchoscopy is a must. for the sharp objects like endodontic file, when lodged in the gastrointestinal tract or respiratory tract, the management protocol is endoscopic retrieval / bronchoscopy or the careful monitoring with periodic radiographs should be undertaken. if the object fails to progress after 72 h, or the signs of bleeding, perforation, obstruction are noticed, laparotomy should be carried out immediately. rotary files are preferred over hand files for the endodontic treatment of the pediatric patient. if hand files are used, the floss should be tied to the handle of the files with the length of 18 inches or more for easy retrieval of the instrument. dentist should always work in dry environment rather than in wet conditions to minimize the chances of slippage of the instrument. for the sharp objects like endodontic file, when lodged in the gastrointestinal tract or respiratory tract, the management protocol is endoscopic retrieval / bronchoscopy or the careful monitoring with periodic radiographs should be undertaken. if the object fails to progress after 72 h, or the signs of bleeding, perforation, obstruction are noticed, laparotomy should be carried out immediately. rotary files are preferred over hand files for the endodontic treatment of the pediatric patient. if hand files are used, the floss should be tied to the handle of the files with the length of 18 inches or more for easy retrieval of the instrument. dentist should always work in dry environment rather than in wet conditions to minimize the chances of slippage of the instrument. the accidental ingestion or aspiration of the dental instrument is the potential life - threatening complication. in such cases, rubber dam isolation should be strictly applied, and dentist should be aware of handling such situation by having the knowledge of diagnosis, immediate retrieval measures and providing emergency treatment to the patient. | the majority of foreign body ingestions occur in the pediatric population, with a peak incidence between the ages of 6 months and 6 years. safety during dental treatment of children can not be overemphasized. uncooperative, mentally and physically handicapped children are more prone to ingestion or aspiration of dental instruments than other populations. in any event, it is recommended that dental practitioners examine their instrument before use, as a safeguard against any slippage, breakage. treatment with any endodontic device requires the use of proper isolation such as rubber dam. |
cementless total hip arthroplasty (tha) has been associated with favorable results at both mid- and long - term follow - ups1,2). to achieve long - term survival rates of the cementless components, as several studies have demonstrated that hydroxyapatite (ha) is a non - toxic, biocompatible, and osteoconductive material that enables strong osteointegration in a short period of time, ha coatings have long been used in tha. the abg - i (anatomical benoist giraud, howmedica, london, uk) prosthesis, designed in the 1980s as an acetabular cup and a proximal femoral stem with a surface coating of ha, has shown good clinical and radiological results by achieving stable biological fixation between the prosthetic components and the femur in studies with relatively short follow - up periods5,6,7). however, the acetabular cup revision rate is reported to be high and attributable to acetabular osteolysis caused by polyethylene wear8,9,10). although ha - coated acetabular cups have shown unfavorable outcomes, ha - coated femoral stems have been associated with more favorable outcomes11,12,13,14). despite positive results based on the femoral components, ha - coated prostheses have failed to show better clinical and radiological results than porous - coated prostheses at mid- and long - term follow - ups. the aim of the current study was to investigate the radiological results and survival rates associated with the use of ha - coated anatomical femoral stems in patients who underwent tha and were followed - up for 12 years. of the 178 hips that received tha with ha - coated hip prosthesis from april 1992 to may 1997, this study comprised 86 patients (102 hips) who underwent tha with ha - coated abg - i prosthesis and were able to undergo radiography at the final follow up. study subjects included 64 men (79 hips) and 22 women (23 hips). tha was performed on the right side in 55 hips and on the left side in 47 hips. the mean age at the time of surgery was 53.4 years (range, 30 - 75 years), and the mean duration of follow - up was 17.1 years (range, 12.1 - 21 years). the causes of tha were avascular necrosis of the femoral head in 89 cases (87%), osteoarthritis in 9 cases (8%), infectious disease in 3 cases (3%), and fracture around the hip in 1 case (1%). in the current study, abg - i was used as an anatomical femoral stem prosthesis composed of a titanium alloy (ti6al4v) and designed to achieve maximum fixation with a press - fit in the metaphyseal region. the proximal third of the femoral stem was coated with ha on a macro - relief surface to a thickness of 6010 m through a plasma spray, and the distal part had a grit - blasted surface (roughness, 2.59 m) without ha coating. all operations were performed by a single surgeon, and thas were performed in a lateral position using a posterolateral approach without involving greater trochanteric osteotomy. the interval of follow - up was 3 months for the first postoperative year, and one year thereafter. the harris hip score (hhs) was used for preoperative and final follow - up clinical evaluations. hips with a score of 90 points were defined as excellent, 80 - 89 as good, 70 - 79 as fair, and 5 mm was classified as loosening according to the method of callaghan.20). a decrease in bone mineral density caused by stress shielding was graded according to the criteria of engh.19), and the degree of stress shielding was observed during follow - up. cortical hypertrophy was defined as an increase in the diameter of the cortex measured at the point of maximum hypertrophy. revision of the femoral component was defined as the failure of survival, and the 95% confidence interval for the survival rate was calculated using a kaplan - meier survival analysis. all statistical analyses were performed using pasw statistics version 18.0 (ibm co., armonk, ny, usa), and paired t - tests and chi - square tests were conducted as appropriate. the hhs was improved from an average of 50.5 points preoperatively to an average of 84.2 points at the final follow - up. according to the hhs, 13 hips (12.7%) were graded as having excellent results, 48 (47%) were graded as good, 34 (33.3%) were graded as fair, and 7 (6.8%) were graded as having poor results. at the final follow - up, thigh pain was observed in 12 cases (11.7%) without limiting daily living activities or requiring medication. thigh pain had no statistical relationship with cortical hypertrophy, radiolucency, or pedestal formation (all p>0.05). according to radiological evaluations, fixation was graded as bony stable in 98 hips and as fibrous stable in 4 hips. in the fifth postoperative year, radiolucent lines were observed on the postoperative radiographs around the uncoated femoral stem in 69 hips (67%) and at gruen zones 3, 4, and 5. at the last follow - up, radiolucent lines were observed in only in 13 hips (12%) and were not associated with loosening. subsidence of the femoral stem occurred in 2 hips during the first postoperative year ; however, migration was less than 5 mm and these cases showed bony ingrowth fixation at the last follow - up. stress shielding of the femur was detected in 67 hips (65%), 64 of which showed first and second degree stress shielding, and 3 that showed third degree stress shielding with bone loss below the lesser trochanter according to engh's19) criteria. cortical hypertrophy was observed in 38 hips (37%), primarily in the distal area of the femoral stem. periprosthetic osteolysis was seen in 72 hips (70%) at gruen zones 1 and 7, but undetected in the distal area. postoperative complications included ipsilateral fracture of the femur in 8 hips, dislocation in 7 hips, and infection in 5 hips. of the 8 fracture cases, revision operations were conducted in 4 hips, plate fixation in 3 hips, and conservative treatment in one hip. the patient with conservative treatment had loosening during follow - up and underwent a revision operation. of the 7 cases with dislocation, 3 hips received acetabular cup revision due to recurrent dislocation, and the other 4 hips underwent 6-weeks of conservative treatment using a hip abduction orthosis. of the 5 infection cases, infection developed within the first 2 postoperative years in one patient, and at an average of 12.1 years in 4 hips. revision surgery was performed in a total of 24 hips (23.5%) due to femoral osteolysis in 14 hips, infection in 5 hips, and fracture in 5 hips. when revision was regarded as an end point indicative of failure using the kaplan - meier curve, the 17.1-year survival rate was 75% (fig. when loosening was regarded as the end point indicative of failure, the survival rate was 100%. ha - coated femoral components were first introduced for use in tha by furlong and osborn21) in 1985 and by geesink.22) in 1986. since then, a large number of authors have reported short- and mid - term follow - up results of tha with ha - coated anatomical femoral stem5,6,12). although the clinical outcomes from using ha - coated hip prostheses at mid - term follow - ups were favorable, long - term follow - up results pertaining to acetabular and femoral components showed considerable differences. the ha - coated acetabular cup was shown to have had high loosening and revision rates8,9). conversely, the ha - coated femoral stem was associated with high survival rates in both mid- and long - term follow - ups11,13). in a previous study, the significant difference in the survival rates of the acetabular cup and femoral stem in tha with ha - coated hip prosthesis was explored23). after osteolysis due to polyethylene wear had been identified as the main cause for the poor outcomes associated with the use of the acetabular cup, investigators attempted to increase its survival rate by using highly cross - linked polyethylene for the acetabular cup surfaces24,25). favorable clinical and radiological results have been suggested in studies of ha - coated hip prostheses5,6,11,12). rajaratnam.11) proposed a survival rate of 97.4% based on revision surgery in a 17-year follow - up, and a survival rate of 100% based on loosening. in the current study, the hhs was improved to an average of 84.2 points over the 17-year follow - up period, and relatively favorable results were obtained with good outcomes in 60% of subjects. the survival rates were 75% based on revision surgery, and 100% based on loosening. the common cause of revision was proximal femoral osteolysis in most cases, and revision surgery was performed concurrently with acetabular cup revision. thus, the survival rate of the femoral stem is anticipated to increase with the use of bearings generating less wear debris. however, whether the ha - coated femoral stems are associated with more favorable clinical and radiological results and longer survival rates than those of conventional porous - coated hip prostheses remains uncertain. it also remains unclear whether the ha - coated femoral stem increases survival rates remarkably. in fact, a few previous studies suggested that the ha coating had an insignificant effect on the survival rate of the femoral component26,27). ha - coated femoral components were found to have strong osteointegration with bone tissue in the early stage3,20), and the initial subsidence of the femoral stem ceased to progress28). in a study by tonino.5) on 222 patients using ha - coated femoral components, 6 femoral stems showed migrations of < 5 mm in a minimum follow - up of 2 years, but showed no further migration. in the current study, although subsidence of less than 5 mm was detected within the first postoperative year in 2 hips (1.9%), subsidence was not observed to progress any further during the remainder of the study period. bony ingrowth fixation was achieved without subsidence or loosening in 24 cases using slightly smaller prostheses incompletely fitting within the femoral metaphysis. as 98% of hips showed bony ingrowth fixation at the last follow - up, early bone formation using the ha - coated prosthesis rocci.29) reported that radiolucent lines were observed at gruen zones 3, 4, and 5 in 21% of subjects at the sixth postoperative month. according to tonino.5), radiolucent lines were observed in the uncoated distal part of the femoral stem in 12.8% of subjects. although radiolucent lines were observed at gruen zones 3, 4, and 5 without an ha coating in 69 hips (67%) in the fifth postoperative year in the current study, they were observed in only 13 hips (12%) at the last follow - up. this is thought to be attributable to radiolucent lines appearing as a response to slight motion in the uncoated distal portion of the prosthesis over - reamed during the early postoperative phase and gradually disappearing with bone formation around the distal part with a grit - blasted surface. subsidence is anticipated to be avoided by bone formation with a pedestal shape in the distal part of the femoral stem as radiolucent lines disappear when stability of a prosthesis is weakened by extensive osteolysis in the proximal part of prosthesisby reinforcing weakened stability in the proximal part caused by osteolysis. d'antonio.30) reported that cortical hypertrophy increased by 47% and that calcar resorption increased by 63% at gruen zone 5 in a 6-year follow - up study using ha - coated femoral components. additionally, canales.31) observed stress shielding in 90% of tha cases during a 10-year follow - up study that used the same femoral stem as was used in the current study. in the current study, stress shielding was observed in 67 hips (65%) postoperatively over an average follow - up of 17.1 years. stress shielding of grade 1 or 2 was seen in most cases, and grade 3 stress shielding was observed in 3 hips. cortical hypertrophy was detected in 38 hips and progressed from the distal junction to more distal regions. several other radiographic findings are thought to be attributable to the unique characteristics of abg - i femoral stems. the abg - i stems are designed to reduce the proximal femoral bone resorption due to stress shielding by facilitating osteointegration and expanding pores in the distal part with proximally ha - coated femoral prostheses. as weight loading gradually moved from the proximal to the distal direction with bone formation increasing with follow - up time around the grit - blasted distal region, cortical hypertrophy limited to gruen zones 2 and 6 expanded to gruen zones 3 and 5, and proximal bone loss occurred due to stress shielding (fig. 2). oosterbos.32) used ha - coated anatomical femoral stems and reported periprosthetic osteolysis in 8% of cases over a 10-year follow - up. moreover, bidar.13) proposed that the rate of femoral osteolysis was 65.2% at gruen zone 1 and 18.8% at gruen zone 7. in the current study, femoral osteolysis was observed in 72 hips (70.5%) and all were found at the proximal portion (gruen zones 1 and 7). of 24 hips that underwent revision of the femoral components, proximal osteolysis was the cause in 14 cases (fig. wear in acetabular components used in this study, the osteolysis rate of the proximal region was high compared to that of other prostheses with different coatings. the results of the current study support the conclusion that ha - coating of the femoral prosthesis had an insignificant influence on improving the survival rate of the femoral prosthesis and maintaining the bone quality of the femur over a long - term follow - up period after tha. a limitation of the current study was that the survival rate of the femoral prosthesis was affected by acetabular cup revision which was performed concurrently with femoral revision in all cases with osteolysis. long - term follow - up revealed good clinical outcomes in tha using ha - coated anatomical femoral stems. however, an advantage of the ha - coated femoral component was not found, possibly attributable to a relatively high revision rate due to osteolysis in this long term follow - up study. | purposeto evaluate the clinical and radiological results, as well as the survival rate, associated with total hip arthroplasty using a hydroxyapatite (ha)-coated anatomical femoral stem at a follow - up of 12 years.materials and methodsfrom april 1992 to may 1997, 86 patients (102 hips) underwent total hip arthroplasty with a ha - coated abg i (anatomical benoist giraud ; howmedica) hip prosthesis. the average age at the time of surgery was 53.4 years and the mean duration of follow - up was 17.1 years (range, 12.1 - 21.0 years). the harris hip score (hhs) and radiographic assessments of thigh pain were used to evaluate the clinical results. we observed osteointegration, cortical hypertrophy, reactive line, calcar resorption and osteolysis around the femoral stems. the survival rate of the femoral stems was evaluated by using the span of time to a revision operation for any reasons was defined as the end point.resultsthe mean hhs was 50.5 preoperatively and 84.2 at the time of last follow - up. osteolysis only around the ha - coated proximal portion of the femoral stem was observed in 72 hips, cortical hypertrophy all around the distal portion of the femoral stem was observed in 38 hips, and calcar resorption was observed in 44 hips. a reactive line was observed in 13 hips, but was unrelated to component loosening. stem revision operations were performed in 24 (23%) hips due to osteolysis (14 hips), fracture (5 hips) and infection (5 hips). the femoral stem survival rate was 75% over the mean duration of follow-up.conclusiontotal hip arthroplasty using a ha - coated anatomical femoral stem showed necessitated a high rate of revision operations due to osteolysis around the femoral stem in this long term follow - up study. |
ten humans (7 male and 3 female ; ages 5284) provided 14 postmortem eyes (8 right and 6 left) from the san diego eye bank within 48 hours of death. listed causes of death included cancer (leukemia, esophageal, and myelodysplastic syndrome), dementia, failure to thrive, liver failure, gastrointestinal bleed, myocardial infarction, and urinary tract injection. no subject was known to have a history of glaucoma. to summarize the information below, dextran experiments used 2 eyes, oct imaging used 1 eye, and trabecular bypass experiments used 11 eyes. eyes were always received as pairs, but some eyes were excluded as they were either open globes due to inadvertent trauma during procurement by the eye bank or because fluid leaks developed during experimentation such that iop was uncertain. eyes were trimmed of extraocular tissue, oriented by inferior oblique insertion location, and pinned to styrofoam. a lewicky anterior chamber (ac) maintainer (bvi visitec, alcester, uk) was inserted through a 1-mm side port (alcon, fort worth, tx, usa) into the anterior chamber. balanced salt solution (bss ; alcon) was introduced for a 1-hour preperfusion period at room temperature (rt) with a reservoir height set at 5 inches above the eye to provide a gravity - delivered pressure of 10 mm hg as previously described. 25% fluorescein (akorn, lake forest, il, usa) was diluted at rt in bss to 2.5%. louis, mo, usa) was dissolved with water into a 2% stock solution, and icg was subsequently diluted in bss to 0.4%. these concentrations were chosen because they have been described for clinical use in live humans as intraocular capsular stains for cataract surgery. in most cases, icg (n = 11) was first introduced for aqueous angiography at 10 mm hg followed by fluorescein aqueous angiography in the same eye as previously done in cows (saraswathy s,. alternatively, 3-kd fixable and fluorescent dextrans (life technologies, carlsbad, ca, usa ; diluted to 2.5 mg / ml in bss) were used (n = 2) at 10 mm hg. the eyes were placed in front of the spectralis hra+oct (heidelberg engineering, heidelberg, germany ; fluorescein capture mode : excitation wavelength = 486 nm and transmission filter set at > 500 nm ; icg capture mode : excitation wavelength = 786 nm and transmission filter set at > 800 nm) with fluorescent images taken with a 55 lens using a 25-diopter focus. confocal scanning laser ophthalmoscopic (cslo) infrared images were taken to center the eye. prior to tracer application, cslo fluorescent angiographic images using the fluorescein or icg capture mode were taken to provide a standard pretracer intensity background image, which appeared black. subsequent fluorescein or icg capture mode images were taken at various time points in various positions or face - on after tracer introduction. to prevent image signal intensity saturation over time during prolonged imaging sessions, the laser sensitivity setting on the spectralis was adjusted with each image to set the central fluorescent signal in the anterior chamber to just under signal saturation. after aqueous angiography with fluorescent dextrans for 2 minutes in human eyes (n = 2), the intracameral fluorescent dextran solution was exchanged with 4% paraformaldehyde (pfa) for 15 minutes at 10 mm hg. the entire globe was then placed in 4% pfa for an additional 15 minutes of fixation. wedges including the angle were cut from angiographically positive and negative regions, dehydrated through ethanol steps, brought through xylenes, and paraffin embedded. five - micrometer - thick sections were cut on a leitz 1512 microtome (leica biosystems, vista, ca, usa) onto superfrost plus slides (vwr, radnor, pa, usa) and air dried. slides were mounted with a 4,6-diamidino-2-phenylindole (dapi)-containing mounting medium (vector labs, burlingame, ca, usa) and viewed under a keyence bz - x700 digital imaging microscope (keyence, chicago, il, usa). all images were taken using identical settings for illumination and image capture sensitivity (keyence imaging software v.1.51). for fitc - dextrans (ex bp 470/30, dm 495, em bp 520/35) and dapi (ex bp 360/40, dm 400, em bp 460/50), appropriate filters were used, respectively. briefly, fluorescence pixel intensity was determined in a region of interest centered on the angle (photoshop cs5 v.12x32 ; adobe, san jose, ca, usa). background - adjusted intensity values were obtained by subtracting the background in each image (by sampling empty anterior chamber) from the above fluorescence pixel intensity in each angle. statistical comparison of the background - adjusted intensity values was conducted with 2-sample, equal - sized, unpaired, assumed normal variance student 's t - tests (excel 2010 ; microsoft, redmond, wa, usa). after the initial icg aqueous angiography, regions with poor angiographic signal were marked and targeted for sham (n = 5) versus trabecular bypass stents (n = 6). given the use of enucleated human eyes, the corneas were universally edematous, and despite various methods (epithelial debridement, corneal dehydration, and external transillumination), visualization of the tm for intervention through a gonioprism was not possible. therefore, an alternative approach was devised whereby a full - thickness corneal incision was made with a 1-mm side - port blade perpendicular to the proposed stent / sham site approximately 80% of the limbal white - to - white distance in that meridian. careful consideration was made not to include the wound of the 1-mm side port created for the lewicky ac maintainer. balanced salt solution was irrigated into the anterior chamber to wash out icg and improve visualization. wek - cel sponges (bvi visitec, alcester, uk) were used to remove excess fluid, and direct visualization of the angle and tm was made through a research - dedicated surgical microscope (leica, buffalo grove, il, usa). sham treatment was achieved by touching the tm at the proposed site using an 18-gauge (g) blunt fill needle (bd, franklin lakes, nj, usa). the anterior chamber was refilled with bss and the corneal wound reapproximated with cyanoacrylate glue. anterior segment oct was taken over the region of stent placement (see below). all eyes were then placed in 4% pfa overnight at 4c. the eyes were prepared for histologic sectioning after removal of the stent much as explained above with hematoxylin and eosin staining for evaluation of the angle at the stent / sham location. trabecular bypass was performed using trabecular bypass stents (provided by glaukos corporation, laguna hills, ca, usa). trabecular bypass stents (generation 1, g1) are food and drug administration (fda) approved for combined cataract and glaucoma surgery for iop lowering in cases of moderately advanced glaucoma. to facilitate placement, second - generation stents (g2w ; istent inject) 1). second - generation stents are not currently fda approved and differ from g1 stents by shape and approach to theoretically allow for easier delivery. in these experiments, the handpiece of the g2w stent was advanced into the anterior chamber through the corneal wound until the trochar tip touched the tm at the proposed injection site. depression of the injection button allowed forward delivery of the stent past the tm without the sideways motion required for g1 stents. closure of the eye and subsequent fluorescein aqueous angiography were conducted as above for sham conditions. the generation 1 (g1) stent is fda approved for moderate glaucoma combined with cataract surgery. with the snorkel - like shape, the stent is inserted into the trabecular meshwork so that one lumen is in front of and the other lumen is behind the trabecular meshwork. generation 2 (g2) stents (istent inject) are non - fda approved currently and were used for these experiments because of the mushroom shape whereby the stent is directly injected into and past the trabecular meshwork straight on, such that the mushroom head with side ports is past the trabecular meshwork while the posterior flat flange with central lumen is in communication with the anterior chamber. images provided courtesy of rob liff, glaukos corporation. to quantify changes in angiographic signal from angle intervention or sham from each individual eye, icg and fluorescein aqueous angiography intensity over time was determined as previously described. aqueous angiographic images were opened in photoshop cs5 (v.12x32) for image processing and pixel intensity measurements. both sham and experimental eyes were pooled for this purpose given the cost and precious nature of enucleated human eyes for research. briefly, (1) angiographic signal within the anterior chamber and beyond the globe horizon for each eye was cropped out to create a total ring of angiographic data. average background pixel intensity from a region of interest in the center of each eye from the pretracer image mentioned above. (3) average pixel intensities for all rings were then obtained and background adjusted by subtracting out the average background pixel intensity. (4) to control for manual adjustments to the spectralis laser sensitivity settings during image acquisition, the background - adjusted average pixel intensity from each ring was divided by the numerical value on the spectralis laser sensitivity setting to yield a normalized intensity value. quantitative assessments of the trabecular bypass experiments were then based on comparing icg (3 minutes) and fluorescein (45 seconds) images at two time points that resided on relatively linear portions of their respective curves. tracer - specific intensity over background values (ts - iob) were calculated (fig. 2). for each eye, pixel intensity was first measured using a 75 75-pixel test area that was placed over the region of interest next to the stent or sham treatment. this value was divided by another pixel intensity measure taken from a clearly signal - poor region from the same eye to determine the ratio of signal intensity of the region of interest (next to the treatment area) over background signal in each eye. the influence of the sham or trabecular bypass was then determined by dividing ts - iob (fluorescein) by ts - iob (icg). statistical comparisons between sham and stent were conducted with 2-sample, equal - sized, unpaired, assumed normal variance student 's t - tests. tracer - specific intensity over background ratio (ts - iob). to quantify the change in angiographic outflow for each eye, the perilimbal signal intensity adjacent to the area of intervention (sham or stent ; red arrows) was recorded (green box) and divided by a region of interest (red box) in a clearly signal - poor region in the exact same image to calculate a tracer - specific (indocyanine green [icg ] or fluorescein [f ]) intensity over background ratio. anterior segment oct (anterior segment module [heidelberg engineering ] on scleral mode) was concurrently conducted in one eye with icg aqueous angiography alone to determine if angiographically positive regions showed vessel anatomy compatible for aho. single line scans with a 15 scan angle (3.9-m axial and 11-m lateral resolution ; 4.5 mm) were taken with oversampling (automated real - time [art ] = 20) in angiographically positive / negative regions. anterior segment oct was also performed in one case of stent placement as mentioned above. here a volume scan was taken with a heidelberg engineering provided custom script near the area of stent placement (15 2.5 scan angle ; 128 b - scans ; axial / lateral resolution of 3.8/11 m, respectively, b - scan to b - scan distance of 11 m). the anterior segment oct images in one of the stent cases were exported as a video per manufacturer instructions on the spectralis. images were opened in photoshop and brightness / contrast adjusted to enhance visualization of particulate debris located in the anterior chamber. the presence of this debris was likely secondary to the act of the trabecular bypass / sham procedure itself. ten humans (7 male and 3 female ; ages 5284) provided 14 postmortem eyes (8 right and 6 left) from the san diego eye bank within 48 hours of death. listed causes of death included cancer (leukemia, esophageal, and myelodysplastic syndrome), dementia, failure to thrive, liver failure, gastrointestinal bleed, myocardial infarction, and urinary tract injection. no subject was known to have a history of glaucoma. to summarize the information below, dextran experiments used 2 eyes, oct imaging used 1 eye, and trabecular bypass experiments used 11 eyes. eyes were always received as pairs, but some eyes were excluded as they were either open globes due to inadvertent trauma during procurement by the eye bank or because fluid leaks developed during experimentation such that iop was uncertain. eyes were trimmed of extraocular tissue, oriented by inferior oblique insertion location, and pinned to styrofoam. a lewicky anterior chamber (ac) maintainer (bvi visitec, alcester, uk) was inserted through a 1-mm side port (alcon, fort worth, tx, usa) into the anterior chamber. balanced salt solution (bss ; alcon) was introduced for a 1-hour preperfusion period at room temperature (rt) with a reservoir height set at 5 inches above the eye to provide a gravity - delivered pressure of 10 mm hg as previously described. 25% fluorescein (akorn, lake forest, il, usa) was diluted at rt in bss to 2.5%., st. louis, mo, usa) was dissolved with water into a 2% stock solution, and icg was subsequently diluted in bss to 0.4%. these concentrations were chosen because they have been described for clinical use in live humans as intraocular capsular stains for cataract surgery. in most cases, icg (n = 11) was first introduced for aqueous angiography at 10 mm hg followed by fluorescein aqueous angiography in the same eye as previously done in cows (saraswathy s,. alternatively, 3-kd fixable and fluorescent dextrans (life technologies, carlsbad, ca, usa ; diluted to 2.5 mg / ml in bss) were used (n = 2) at 10 mm hg. the eyes were placed in front of the spectralis hra+oct (heidelberg engineering, heidelberg, germany ; fluorescein capture mode : excitation wavelength = 486 nm and transmission filter set at > 500 nm ; icg capture mode : excitation wavelength = 786 nm and transmission filter set at > 800 nm) with fluorescent images taken with a 55 lens using a 25-diopter focus. confocal scanning laser ophthalmoscopic (cslo) infrared images were taken to center the eye. prior to tracer application, cslo fluorescent angiographic images using the fluorescein or icg capture mode were taken to provide a standard pretracer intensity background image, which appeared black. subsequent fluorescein or icg capture mode images were taken at various time points in various positions or face - on after tracer introduction. to prevent image signal intensity saturation over time during prolonged imaging sessions, the laser sensitivity setting on the spectralis was adjusted with each image to set the central fluorescent signal in the anterior chamber to just under signal saturation. after aqueous angiography with fluorescent dextrans for 2 minutes in human eyes (n = 2), the intracameral fluorescent dextran solution was exchanged with 4% paraformaldehyde (pfa) for 15 minutes at 10 mm hg. the entire globe was then placed in 4% pfa for an additional 15 minutes of fixation. wedges including the angle were cut from angiographically positive and negative regions, dehydrated through ethanol steps, brought through xylenes, and paraffin embedded. five - micrometer - thick sections were cut on a leitz 1512 microtome (leica biosystems, vista, ca, usa) onto superfrost plus slides (vwr, radnor, pa, usa) and air dried. slides were mounted with a 4,6-diamidino-2-phenylindole (dapi)-containing mounting medium (vector labs, burlingame, ca, usa) and viewed under a keyence bz - x700 digital imaging microscope (keyence, chicago, il, usa). all images were taken using identical settings for illumination and image capture sensitivity (keyence imaging software v.1.51). for fitc - dextrans (ex bp 470/30, dm 495, em bp 520/35) and dapi (ex bp 360/40, dm 400, em bp 460/50), appropriate filters were used, respectively. briefly, fluorescence pixel intensity was determined in a region of interest centered on the angle (photoshop cs5 v.12x32 ; adobe, san jose, ca, usa). background - adjusted intensity values were obtained by subtracting the background in each image (by sampling empty anterior chamber) from the above fluorescence pixel intensity in each angle. statistical comparison of the background - adjusted intensity values was conducted with 2-sample, equal - sized, unpaired, assumed normal variance student 's t - tests (excel 2010 ; microsoft, redmond, wa, usa). after the initial icg aqueous angiography, regions with poor angiographic signal were marked and targeted for sham (n = 5) versus trabecular bypass stents (n = 6). given the use of enucleated human eyes, the corneas were universally edematous, and despite various methods (epithelial debridement, corneal dehydration, and external transillumination), visualization of the tm for intervention through a gonioprism was not possible. therefore, an alternative approach was devised whereby a full - thickness corneal incision was made with a 1-mm side - port blade perpendicular to the proposed stent / sham site approximately 80% of the limbal white - to - white distance in that meridian. careful consideration was made not to include the wound of the 1-mm side port created for the lewicky ac maintainer. balanced salt solution was irrigated into the anterior chamber to wash out icg and improve visualization. wek - cel sponges (bvi visitec, alcester, uk) were used to remove excess fluid, and direct visualization of the angle and tm was made through a research - dedicated surgical microscope (leica, buffalo grove, il, usa). sham treatment was achieved by touching the tm at the proposed site using an 18-gauge (g) blunt fill needle (bd, franklin lakes, nj, usa). the anterior chamber was refilled with bss and the corneal wound reapproximated with cyanoacrylate glue. anterior segment oct was taken over the region of stent placement (see below). all eyes were then placed in 4% pfa overnight at 4c. the eyes were prepared for histologic sectioning after removal of the stent much as explained above with hematoxylin and eosin staining for evaluation of the angle at the stent / sham location. trabecular bypass was performed using trabecular bypass stents (provided by glaukos corporation, laguna hills, ca, usa). trabecular bypass stents (generation 1, g1) are food and drug administration (fda) approved for combined cataract and glaucoma surgery for iop lowering in cases of moderately advanced glaucoma. to facilitate placement, second - generation stents (g2w ; istent inject) were used (fig. second - generation stents are not currently fda approved and differ from g1 stents by shape and approach to theoretically allow for easier delivery. in these experiments, the handpiece of the g2w stent was advanced into the anterior chamber through the corneal wound until the trochar tip touched the tm at the proposed injection site. depression of the injection button allowed forward delivery of the stent past the tm without the sideways motion required for g1 stents. closure of the eye and subsequent fluorescein aqueous angiography were conducted as above for sham conditions. the generation 1 (g1) stent is fda approved for moderate glaucoma combined with cataract surgery. with the snorkel - like shape, the stent is inserted into the trabecular meshwork so that one lumen is in front of and the other lumen is behind the trabecular meshwork. generation 2 (g2) stents (istent inject) are non - fda approved currently and were used for these experiments because of the mushroom shape whereby the stent is directly injected into and past the trabecular meshwork straight on, such that the mushroom head with side ports is past the trabecular meshwork while the posterior flat flange with central lumen is in communication with the anterior chamber. to quantify changes in angiographic signal from angle intervention or sham from each individual eye, icg and fluorescein aqueous angiography intensity over time was determined as previously described. aqueous angiographic images were opened in photoshop cs5 (v.12x32) for image processing and pixel intensity measurements. both sham and experimental eyes were pooled for this purpose given the cost and precious nature of enucleated human eyes for research. briefly, (1) angiographic signal within the anterior chamber and beyond the globe horizon for each eye was cropped out to create a total ring of angiographic data. (2) the background fluorescein angiography signal was then established by determining average background pixel intensity from a region of interest in the center of each eye from the pretracer image mentioned above. (3) average pixel intensities for all rings were then obtained and background adjusted by subtracting out the average background pixel intensity. (4) to control for manual adjustments to the spectralis laser sensitivity settings during image acquisition, the background - adjusted average pixel intensity from each ring was divided by the numerical value on the spectralis laser sensitivity setting to yield a normalized intensity value. quantitative assessments of the trabecular bypass experiments were then based on comparing icg (3 minutes) and fluorescein (45 seconds) images at two time points that resided on relatively linear portions of their respective curves. tracer - specific intensity over background values (ts - iob) were calculated (fig. pixel intensity was first measured using a 75 75-pixel test area that was placed over the region of interest next to the stent or sham treatment. this value was divided by another pixel intensity measure taken from a clearly signal - poor region from the same eye to determine the ratio of signal intensity of the region of interest (next to the treatment area) over background signal in each eye. the influence of the sham or trabecular bypass was then determined by dividing ts - iob (fluorescein) by ts - iob (icg). statistical comparisons between sham and stent were conducted with 2-sample, equal - sized, unpaired, assumed normal variance student 's t - tests. tracer - specific intensity over background ratio (ts - iob). to quantify the change in angiographic outflow for each eye, the perilimbal signal intensity adjacent to the area of intervention (sham or stent ; red arrows) was recorded (green box) and divided by a region of interest (red box) in a clearly signal - poor region in the exact same image to calculate a tracer - specific (indocyanine green [icg ] or fluorescein [f ]) intensity over background ratio. anterior segment oct (anterior segment module [heidelberg engineering ] on scleral mode) was concurrently conducted in one eye with icg aqueous angiography alone to determine if angiographically positive regions showed vessel anatomy compatible for aho. single line scans with a 15 scan angle (3.9-m axial and 11-m lateral resolution ; 4.5 mm) were taken with oversampling (automated real - time [art ] = 20) in angiographically positive / negative regions. anterior segment oct was also performed in one case of stent placement as mentioned above. here a volume scan was taken with a heidelberg engineering provided custom script near the area of stent placement (15 2.5 scan angle ; 128 b - scans ; axial / lateral resolution of 3.8/11 m, respectively, b - scan to b - scan distance of 11 m). the anterior segment oct images in one of the stent cases were exported as a video per manufacturer instructions on the spectralis. images were opened in photoshop and brightness / contrast adjusted to enhance visualization of particulate debris located in the anterior chamber. the presence of this debris was likely secondary to the act of the trabecular bypass / sham procedure itself. as in pigs, aqueous angiography in human enucleated model eyes demonstrated segmental angiographic signal that reflected aho as evaluated by fluorescent dextran (fig. 4). visual inspection of the initial aqueous angiography in all 14 eyes (regardless of what tracers was used first [fluorescein, icg, or fluorescent dextrans ]) showed that 11/14 eyes (78.6%) had predominately nasal angiographic signal. angiographically positive (a, d ; green lines) or diminished (a, d ; red lines) regions were identified with aqueous angiography, marked, and prepared for paraffin sectioning. in the first eye (a c), angiographically positive areas (green line in [a ] corresponds to [b ]) showed greater trapping of dextrans within outflow pathways compared to angiographically diminished (red line in [a ] corresponds to [c ]) regions. in the second eye (d f), angiographically positive areas (green line in [d ] corresponds to [e ]) also showed more trapping of dextrans in outflow pathways compared to angiographically diminished (red line in [d ] corresponds to [f ]) regions. note similar degree of nonspecific fluorescence seen in descemet 's membrane in all cases (asterisks). sc, schlemm 's canal ; tm, trabecular meshwork ; ac, anterior chamber. scale bar : 100 m. indocyanine green (icg) aqueous angiography was conducted concurrent with anterior segment oct focused on the distal angiographic signal from the right eye of an 84-year - old male (listed cause of death, myelodysplastic syndrome). (a) angiographically positive regions (yellow arrow and white arrows) demonstrated (b) intrascleral lumens compatible with aqueous humor outflow on oct. (c) in an oct b - scan taken directly below the angiographic signal depicted in (a), (d) intrascleral lumens were mostly absent. (c) this is true except one for one point on the left of the angiographic image (yellow arrow) where the oct image caught one branch of the angiographic pattern that (d) showed a single oval corresponding intrascleral lumen on oct. to look for differences in outflow in the tm and angle region, we used fixable and trappable dextrans. while unlikely imaging the tm itself in aqueous angiography, tm near - adjacent positive but not negative aqueous angiographic signal demonstrated trapping of the dextrans in aho pathways (figs. 3 ; asterisks). quantitative comparison of background - adjusted intensity values in angiographically positive compared to negative areas showed a statistically significant increase (100.76 18.52 vs. 34.19 13.21 ; background - adjusted intensity units ; average sd ; n = 12 sections for each condition ; p < 0.001 2-tailed student 's t - test). anterior segment oct supported dextran results where icg angiographically positive but not negative areas showed intrascleral lumens reminiscent of aho pathways (fig. 4). to assess whether trabecular bypass could improve regions of poor aqueous angiography signal, sequential aqueous angiography with icg followed by fluorescein was performed. areas of poor icg aqueous angiography signal were marked (figs. 5, 6 ; red arrows), and full - thickness cornea incisions were made for direct visualization of the tm for a sham treatment (fig. 5 ; n = 5) (touching the tm with a blunt - tip 18-g needle) or trabecular bypass (fig. 6 ; n = 6 [of which 5 achieved full - thickness bypass ]) with a trabecular bypass stent. corneal wounds were glued, and fluorescein aqueous angiography was performed to assess the influence of the intervention. for both sham and stent conditions (figs. 5, 6) green arrows demonstrated similar aqueous angiography patterns between icg and fluorescein. after sham, regions initially devoid of icg aqueous angiography signal continued to be so with fluorescein aqueous angiography (fig. 5). however, after trabecular bypass, regions initially devoid of icg aqueous angiography signal demonstrated qualitative increased aqueous angiography signal intensity with fluorescein (fig. sequential aqueous angiography with indocyanine green (icg) followed by fluorescein after sham intervention to the trabecular meshwork. (a d) icg aqueous angiography was first performed on the left eye of a 68-year - old male (listed cause of death, failure to thrive). this was followed by identification of a region of low signal (red arrows), full - thickness corneal incision perpendicular to this site, sham touch of the trabecular meshwork by an 18-g blunt needle, and cyanoacrylate glue closure of the wound. (e h) this procedure was followed by fluorescein aqueous angiography in the same eye demonstrating continued poor angiography signal at the sham site (red arrows). (a h) green arrows demonstrate similar angiographic patterns between icg and fluorescein outside of the sham location. j) representative images from icg and fluorescein aqueous angiography from the right eye of a 76-year - old male (listed cause of death, myocardial infarction). (k, l) representative images from icg and fluorescein aqueous angiography from the left eye of the same 76-year - old male (listed cause of death, myocardial infarction). all images were arranged such that superior is on top, and inferior is on the bottom of the image. for right eyes, nasal is on the right of the image, and temporal is on the left. sequential aqueous angiography with indocyanine green (icg) followed by fluorescein after trabecular bypass stent placement. (a d) icg aqueous angiography was first performed on the right eye of a 79-year - old male (listed cause of death, esophageal cancer). this was followed by identification of a region of low signal (red arrows and white asterisks), full - thickness corneal incision perpendicular to this site, placement of a second - generation trabecular bypass stent under direct visualization, and cyanoacrylate glue closure of the wound. (e h) this procedure was followed by fluorescein aqueous angiography in the same eye demonstrating earlier and increased angiographic signal compared to before the stent placement (red arrows and white asterisks). (a h) green arrows demonstrate similar angiographic patterns between icg and fluorescein in regions outside of stent placement. (i, j) representative images from icg and fluorescein aqueous angiography from the right eye of an 82-year - old female (listed cause of death, urinary tract infection). (k, l) representative images from icg and fluorescein aqueous angiography from the left eye of a 75-year - old female (listed cause of death, dementia). all images were arranged such that superior is on top, and inferior is on the bottom of the image. for right eyes, nasal is on the right of the image, and temporal is on the left. histologic sectioning in marked areas after sham treatment demonstrated normal - appearing tm and angle structures (fig histologic sections of marked areas after trabecular bypass demonstrated successful and full - thickness tm ablation (figs. interestingly, one case occurred in which after attempted trabecular bypass, only minimally improved fluorescent aqueous angiographic signal was seen with fluorescein (fig. histology in that case showed unsuccessful stent placement whereby the tm was only partially bypassed (fig. (a c) after sham experiments, eyes were perfusion fixed and the anterior segments prepared for paraffin sectioning and hematoxylin / eosin staining demonstrating intact trabecular meshwork. sc, schlemm 's canal ; tm, trabecular meshwork ; ac, anterior chamber. scale bar : 100 m. (a c) after trabecular bypass stent experiments, eyes were perfusion fixed and the anterior segments prepared for paraffin sectioning and hematoxylin / eosin staining. arrows (d) anterior segment optical coherence tomography in the same eye as in figures 6a through 6h and figure 8a demonstrated correct positioning of trabecular bypass stent. a) sequential aqueous angiography was performed in the right eye of a 75-year - old female (listed cause of death, dementia), first with icg showing lack of signal superior (red arrow) even after a prolonged time period. (b) subsequent fluorescein aqueous angiography barely demonstrated improved angiographic outflow signal (red arrow). (c) histologic sectioning over the stent placement site showed a cleft where the stent resided (arrowhead) with only partial tm bypass without full - thickness bypass achieved (arrow). all images were arranged such that superior is on top and inferior is on the bottom of the image. in this right eye, nasal is on the right of the image, and temporal is on the left. sc, schlemm 's canal ; tm, trabecular meshwork ; ac, anterior chamber. orange scale bar : 5 mm. given the universally edematous corneas in all eyes, a modified surgical approach was developed in the sham and stent experiments. full - thickness corneal wounds, longer than routinely used for clinical surgeries in patient care, were employed, and irrigation using bss was required to wash out fluorescent tracers to directly visualize the tm. therefore, debris was found in some cases in the anterior chamber after stent placement. in one eye (figs. h) that had stent placement in an initially icg aqueous angiography poor region with additional histology and oct - confirmed successful trabecular bypass stent placement (figs. video demonstration of these scans showed particulate matter flowing toward the trabecular bypass stent (supplementary video s1). to quantify aqueous angiographic changes in trabecular bypass experiments, experimental and sham eyes were pooled and total intensity of icg and fluorescein aqueous angiographic signal plotted over time (fig. not unlike what was shown in a previous report, there was a steady increase. (a, b) aqueous angiography with fluorescein over time demonstrated accumulated signal intensity. (c, d) aqueous angiography with indocyanine green (icg) over time also demonstrated accumulated signal intensity. total normalized pixel intensity values from 11 eyes each for (e) fluorescein and (f) icg were recorded as a function of time at 10 mm hg. orange scale bars : 5 mm. to assess for regional changes in angiographic patterns after sham or stent, ts - iob ratios were calculated for icg and fluorescein images at 3 minutes and 45 seconds, respectively (fig. these time points were chosen based on their positions at early and linear portions of each tracer 's respective curves (figs. f). after dividing fluorescein ts - iob over icg ts - iob for sham and stent conditions, a statistically significant improvement was found for the stent trabecular bypass condition (sham [n = 5 ] : 1.02 0.46 versus stent [n = 5 ] : 17.37 7.76 ; p = 0.043). 9), an intermediate value of fluorescein ts - iob over icg ts - iob (1.66) was seen. as in pigs, aqueous angiography in human enucleated model eyes demonstrated segmental angiographic signal that reflected aho as evaluated by fluorescent dextran (fig. 4). visual inspection of the initial aqueous angiography in all 14 eyes (regardless of what tracers was used first [fluorescein, icg, or fluorescent dextrans ]) showed that 11/14 eyes (78.6%) had predominately nasal angiographic signal. angiographically positive (a, d ; green lines) or diminished (a, d ; red lines) regions were identified with aqueous angiography, marked, and prepared for paraffin sectioning. in the first eye (a c), angiographically positive areas (green line in [a ] corresponds to [b ]) showed greater trapping of dextrans within outflow pathways compared to angiographically diminished (red line in [a ] corresponds to [c ]) regions. in the second eye (d f), angiographically positive areas (green line in [d ] corresponds to [e ]) also showed more trapping of dextrans in outflow pathways compared to angiographically diminished (red line in [d ] corresponds to [f ]) regions. note similar degree of nonspecific fluorescence seen in descemet 's membrane in all cases (asterisks). sc, schlemm 's canal ; tm, trabecular meshwork ; ac, anterior chamber. scale bar : 100 m. indocyanine green (icg) aqueous angiography was conducted concurrent with anterior segment oct focused on the distal angiographic signal from the right eye of an 84-year - old male (listed cause of death, myelodysplastic syndrome). (a) angiographically positive regions (yellow arrow and white arrows) demonstrated (b) intrascleral lumens compatible with aqueous humor outflow on oct. (c) in an oct b - scan taken directly below the angiographic signal depicted in (a), (d) intrascleral lumens were mostly absent. (c) this is true except one for one point on the left of the angiographic image (yellow arrow) where the oct image caught one branch of the angiographic pattern that (d) showed a single oval corresponding intrascleral lumen on oct. to look for differences in outflow in the tm and angle region, we used fixable and trappable dextrans. while unlikely imaging the tm itself in aqueous angiography, tm near - adjacent positive but not negative aqueous angiographic signal demonstrated trapping of the dextrans in aho pathways (figs. 3 ; asterisks). quantitative comparison of background - adjusted intensity values in angiographically positive compared to negative areas showed a statistically significant increase (100.76 18.52 vs. 34.19 13.21 ; background - adjusted intensity units ; average sd ; n = 12 sections for each condition ; p < 0.001 2-tailed student 's t - test). anterior segment oct supported dextran results where icg angiographically positive but not negative areas showed intrascleral lumens reminiscent of aho pathways (fig. to assess whether trabecular bypass could improve regions of poor aqueous angiography signal, sequential aqueous angiography with icg followed by fluorescein was performed. areas of poor icg aqueous angiography signal were marked (figs. 5, 6 ; red arrows), and full - thickness cornea incisions were made for direct visualization of the tm for a sham treatment (fig. 5 ; n = 5) (touching the tm with a blunt - tip 18-g needle) or trabecular bypass (fig. 6 ; n = 6 [of which 5 achieved full - thickness bypass ]) with a trabecular bypass stent. corneal wounds were glued, and fluorescein aqueous angiography was performed to assess the influence of the intervention. for both sham and stent conditions (figs. 5, 6) green arrows demonstrated similar aqueous angiography patterns between icg and fluorescein. after sham, regions initially devoid of icg aqueous angiography signal continued to be so with fluorescein aqueous angiography (fig. 5). however, after trabecular bypass, regions initially devoid of icg aqueous angiography signal demonstrated qualitative increased aqueous angiography signal intensity with fluorescein (fig. 6). sequential aqueous angiography with indocyanine green (icg) followed by fluorescein after sham intervention to the trabecular meshwork. (a d) icg aqueous angiography was first performed on the left eye of a 68-year - old male (listed cause of death, failure to thrive). this was followed by identification of a region of low signal (red arrows), full - thickness corneal incision perpendicular to this site, sham touch of the trabecular meshwork by an 18-g blunt needle, and cyanoacrylate glue closure of the wound. (e h) this procedure was followed by fluorescein aqueous angiography in the same eye demonstrating continued poor angiography signal at the sham site (red arrows). (a h) green arrows demonstrate similar angiographic patterns between icg and fluorescein outside of the sham location. (i, j) representative images from icg and fluorescein aqueous angiography from the right eye of a 76-year - old male (listed cause of death, myocardial infarction). (k, l) representative images from icg and fluorescein aqueous angiography from the left eye of the same 76-year - old male (listed cause of death, myocardial infarction). all images were arranged such that superior is on top, and inferior is on the bottom of the image. for right eyes, nasal is on the right of the image, and temporal is on the left. sequential aqueous angiography with indocyanine green (icg) followed by fluorescein after trabecular bypass stent placement. (a d) icg aqueous angiography was first performed on the right eye of a 79-year - old male (listed cause of death, esophageal cancer). this was followed by identification of a region of low signal (red arrows and white asterisks), full - thickness corneal incision perpendicular to this site, placement of a second - generation trabecular bypass stent under direct visualization, and cyanoacrylate glue closure of the wound. (e h) this procedure was followed by fluorescein aqueous angiography in the same eye demonstrating earlier and increased angiographic signal compared to before the stent placement (red arrows and white asterisks). (a h) green arrows demonstrate similar angiographic patterns between icg and fluorescein in regions outside of stent placement. (i, j) representative images from icg and fluorescein aqueous angiography from the right eye of an 82-year - old female (listed cause of death, urinary tract infection)., l) representative images from icg and fluorescein aqueous angiography from the left eye of a 75-year - old female (listed cause of death, dementia). all images were arranged such that superior is on top, and inferior is on the bottom of the image. for right eyes, nasal is on the right of the image, and temporal is on the left. histologic sectioning in marked areas after sham treatment demonstrated normal - appearing tm and angle structures (fig. histologic sections of marked areas after trabecular bypass demonstrated successful and full - thickness tm ablation (figs. interestingly, one case occurred in which after attempted trabecular bypass, only minimally improved fluorescent aqueous angiographic signal was seen with fluorescein (fig. histology in that case showed unsuccessful stent placement whereby the tm was only partially bypassed (fig. (a c) after sham experiments, eyes were perfusion fixed and the anterior segments prepared for paraffin sectioning and hematoxylin / eosin staining demonstrating intact trabecular meshwork. sc, schlemm 's canal ; tm, trabecular meshwork ; ac, anterior chamber. scale bar : 100 m. (a c) after trabecular bypass stent experiments, eyes were perfusion fixed and the anterior segments prepared for paraffin sectioning and hematoxylin / eosin staining. arrows point out successful full - thickness trabecular bypass in all cases. (d) anterior segment optical coherence tomography in the same eye as in figures 6a through 6h and figure 8a demonstrated correct positioning of trabecular bypass stent. (a) sequential aqueous angiography was performed in the right eye of a 75-year - old female (listed cause of death, dementia), first with icg showing lack of signal superior (red arrow) even after a prolonged time period. (b) subsequent fluorescein aqueous angiography barely demonstrated improved angiographic outflow signal (red arrow). (c) histologic sectioning over the stent placement site showed a cleft where the stent resided (arrowhead) with only partial tm bypass without full - thickness bypass achieved (arrow). all images were arranged such that superior is on top and inferior is on the bottom of the image. in this right eye, nasal is on the right of the image, and temporal is on the left. sc, schlemm 's canal ; tm, trabecular meshwork ; ac, anterior chamber. orange scale bar : 5 mm. given the universally edematous corneas in all eyes, a modified surgical approach was developed in the sham and stent experiments. full - thickness corneal wounds, longer than routinely used for clinical surgeries in patient care, were employed, and irrigation using bss was required to wash out fluorescent tracers to directly visualize the tm. therefore, debris was found in some cases in the anterior chamber after stent placement. in one eye (figs. h) that had stent placement in an initially icg aqueous angiography poor region with additional histology and oct - confirmed successful trabecular bypass stent placement (figs. video demonstration of these scans showed particulate matter flowing toward the trabecular bypass stent (supplementary video s1). to quantify aqueous angiographic changes in trabecular bypass experiments, experimental and sham eyes were pooled and total intensity of icg and fluorescein aqueous angiographic signal plotted over time (fig. not unlike what was shown in a previous report, there was a steady increase. (a, b) aqueous angiography with fluorescein over time demonstrated accumulated signal intensity. (c, d) aqueous angiography with indocyanine green (icg) over time also demonstrated accumulated signal intensity. total normalized pixel intensity values from 11 eyes each for (e) fluorescein and (f) icg were recorded as a function of time at 10 mm hg. graphs show mean standard error. orange scale bars : 5 mm. to assess for regional changes in angiographic patterns after sham or stent, ts - iob ratios were calculated for icg and fluorescein images at 3 minutes and 45 seconds, respectively (fig. these time points were chosen based on their positions at early and linear portions of each tracer 's respective curves (figs. f). after dividing fluorescein ts - iob over icg ts - iob for sham and stent conditions, a statistically significant improvement was found for the stent trabecular bypass condition (sham [n = 5 ] : 1.02 0.46 versus stent [n = 5 ] : 17.37 7.76 ; p = 0.043). 9), an intermediate value of fluorescein ts - iob over icg ts - iob (1.66) was seen. optical coherence tomography evaluation of distal aqueous angiography signal showed episcleral lumens compatible with aho in angiographically positive but not negative regions. with sequential aqueous angiography, the sole manipulation of trabecular bypass improved aho as measured by fluorescein aqueous angiography in areas initially devoid of icg aqueous angiography. first, the meaning of aqueous angiography in this model system is limited by the fact that the eyes are enucleated. artifacts due to diminished cell viability and episcleral venous blood clotting could affect angiographic patterns. in particular, postmortem presence of distal episcleral venous blood clots could explain segmental patterns by sectorally blocking aho. however, since tm bypass alone resulted in improved aqueous angiography signal, segmental aho as seen by aqueous angiography could not be entirely and artifacticiously due to episcleral venous clots since these clots would reside distal to the tm. lack of angiographic signal could be due to outflow pathways diving deep into the sclera such that excitation / emission wavelengths become attenuated. since tm bypass alone, which did not influence the sclera or scleral depth, resulted in improved angiographic signal, segmental aho could not be entirely due to issues of depth. additionally, depth influences interpretation of aqueous angiography results. increased depth can attenuate light transmission through sclera ; and given the location where angiographic signal appears perilimbal but also posterior to the limbus, aqueous angiography signal represents the outflow result as influenced by the entire trabecular outflow pathway without directly imaging the tm. this is in contrast to microsphere or bead methods, which have the advantage of demonstrating precise segmental tm aho but used tissue processing not compatible with live imaging. second, improving aho by ablating the tm reemphasizes to clinicians and scientists the importance of the tm. variable results of clinical tm ablation / bypass migs naturally raised the possibility of outflow obstruction in the distal outflow pathways. while distal aho is likely relevant and a source of undiscovered biology and potential disease, the results of improved angiographic aho by only trabecular bypass reinforces the role of the tm in gating aho. also, the result of diminished aho improvement with partial tm bypass is important (fig.. part of the variable clinical results from trabecular migs may come from improper surgical placement. a natural learning curve exists for all surgeries, and trabecular migs are no different. third, improving aho in regions initially devoid of aho expands on initial surgical approaches to migs. trabecular migs are typically placed in the nasal angle through a temporal clear cornea direction for several reasons. second, reports, including results here, suggest that aho is normally best nasal. however, if aho is already adequate in a particular region, it is possible that trabecular bypass to enhance aho in that region may limit further improvement due to a ceiling effect. alternatively, another approach is to place trabecular migs where aho is initially poor in an attempt to recruit these regions from a worse starting point. however, a counterargument to conducting migs in this way is that maybe the reason why aho was diminished in the first place was that the local region never had adequate outflow channels to support aho. this would be analogous to opening roads for vehicle traffic that ended up all being cul de sacs. therefore, demonstrating that regions of poor aho could be recruited for improved outflow opens the possibility of alternative surgical techniques. it is possible that better iop lowering could be achieved by placing trabecular migs in regions of initially low aho. methods such as aqueous angiography now allow ophthalmologists to test the effects of various surgical approaches such as these. additional testing needs to be done to attempt measurement of outflow facility concurrent with angiographic imaging. to do this, an aqueous humor dynamics rig will have to be situated and built around this clinical device as the center. with this, one could then test trabecular bypass in low versus high angiographic signal regions to determine if trabecular bypass in one area or the other really is better. further studies need to be conducted with aqueous angiography in live humans and animals to avoid the confounding factors related to using enucleated eyes. for example, in cases of successful trabecular bypass, the increase in fluorescein aqueous angiography was variable, with some cases rivaling native fluorescein angiographic signal in high - flow areas and in other cases to a lesser extent. this may have been due to either postmortem changes to distal outflow pathways giving a variable response to outflow recruitment for forward flow after trabecular bypass. alternatively, subtle native differences in distal outflow anatomy between different low - flow regions being accessed could have created this variability as well. additionally, while the aqueous angiography patterns and time course between fluorescein and icg were similar, they were not identical. this was likely secondary to differences in molecular properties (e.g., ph, molecular weight, protein binding). for example, the icg signal was slower, and we speculate that this could be due to lower concentration used (icg solubility is limited) or to the fact that icg was more protein bound such that icg aqueous angiography may model protein more than water movement. despite being limited in these experiments to fluorescein and icg by our commercial angiographer, given similar enough patterns and measurement of angiographic signal over specific regions of interest normalized for each dye with a ts - iob ratio, finer biochemical and molecular studies are planned that compare regions of initially greater or lesser aho to develop a better understanding of what causes more or less aho. as aho is better visualized through methods like aqueous angiography and more clinical experience with trabecular migs is gained, a better understanding of aho will arise and improved questions can be asked. ultimately, aho imaging may allow for improved glaucoma surgical results or facilitate development of novel pharmacologic or surgical treatments. | purposeto assess the ability of trabecular micro - bypass stents to improve aqueous humor outflow (aho) in regions initially devoid of aho as assessed by aqueous angiography.methodsenucleated human eyes (14 total from 7 males and 3 females [ages 5284 ]) were obtained from an eye bank within 48 hours of death. eyes were oriented by inferior oblique insertion, and aqueous angiography was performed with indocyanine green (icg ; 0.4%) or fluorescein (2.5%) at 10 mm hg. with an angiographer, infrared and fluorescent images were acquired. concurrent anterior segment optical coherence tomography (oct) was performed, and fixable fluorescent dextrans were introduced into the eye for histologic analysis of angiographically positive and negative areas. experimentally, some eyes (n = 11) first received icg aqueous angiography to determine angiographic patterns. these eyes then underwent trabecular micro - bypass sham or stent placement in regions initially devoid of angiographic signal. this was followed by fluorescein aqueous angiography to query the effects.resultsaqueous angiography in human eyes yielded high - quality images with segmental patterns. distally, angiographically positive but not negative areas demonstrated intrascleral lumens on oct images. aqueous angiography with fluorescent dextrans led to their trapping in aho pathways. trabecular bypass but not sham in regions initially devoid of icg aqueous angiography led to increased aqueous angiography as assessed by fluorescein (p = 0.043).conclusionsusing sequential aqueous angiography in an enucleated human eye model system, regions initially without angiographic flow or signal could be recruited for aho using a trabecular bypass stent. |
this work refers to the debate taking place in italy during the final decades of the nineteenth century, regarding mental health and lunatic asylums which housed an ever - increasing number of patients suffering from mental illness. overcrowding, lunatic asylums, coercive and inhuman methods, the degradation in which patients lived, medical and administrative difficulties and various deficiencies indeed called for innovative, urgent proposals. in this paper, we will mention some of the solutions proposed at the end of the nineteenth century which foresaw the establishment of new asylums and the renovation of existing ones, but also and above all the following alternatives : a) the placing of incurable, harmless chronic patients in homes and hospices ; b) family homes ; c) confinement in the home ; d) charitable institutions for poor, discharged, cured or convalescent mental patients. these new contexts would have favored life experiences and relationships in a natural family environment, from which patients had been alienated, and above all revived the sense of human dignity equally important was to have been the training of doctors and above all nurses by means of specialized schools and the re - establishment of humane relationships between doctors and patients. experiences involving solutions of this type, already existing in other european countries were also carried out in italy due to the goodwill of asylum doctors and superintendents who fostered international relationships. however, their proposals were not fully incorporated into the 1904 law on asylums and thus not put into practice. setting them aside had a negative influence on psychiatric treatment in italy and consequently fuelled the debate that 70 years later concluded with the enactment in 1978 of law 180, commonly known as the basaglia law. law 180 is the result of ideas inspired by the struggles of anti - psychiatry, new psychiatry, democratic psychiatry, etc. and by experiences in various asylums, prevalently linked to the figure and role of f. basaglia. according to dominant marxist ideology, some authors reduced mental illness to a product of society or repression or a commitment on the part of society to socialize with patients. from the viewpoint of democratic psychiatry, mental illness were denied in its existence or were completely reduced to the target of organic treatment using psychotropic drugs. in order to avoid a referendum, the hasty approval of the law does not take into account the well - constructed debate on reform, with consequences on a theoretical and practical level (see ossicini in his meticulous re - examination). indeed, if there was general consensus on closing asylums and ossicini himself had already proposed it in 1944, on the other hand, reflection on the complexity of medical, psychological, social and political responses to mental illness continued. law 180, dismantling asylums and the treatment carried out within them, shifted attention toward the surrounding territory and alternative structures, overturning the terms of the problem in respect to the past, almost as if accepting some of the alternatives in the 1904 law. we believe we can link the broadest needs underlying the law, stripped of their ideological distortions, to the spirit of the proposals not ultimately included in the 1904 law which, in the light of experience in some asylums, above all in reggio emilia, conceived of the asylum as a transitory place for acute cases ; having overcome this acute stage, other appropriate structures could take in chronically - ill mental patients and those on the road to recovery, using adequate therapeutic tools and respecting their dignity. these two outlooks differ historically, although we believe that some of the most profound arguments underlying the debate on law 180, which still persist, may be linked to unresolved problems regarding mental health at the beginning of the twentieth century. the literature on this subject is clearly extremely vast and, therefore, in this study, we have selected and closely examined only those parts focusing on this subject during the final years of the nineteenth century. with the progress in psychiatric knowledge during the last three decades of the nineteenth century in italy, as well, a debate had developed on how to treat the suffering, deviant humanity which, isolated in the course of centuries, had found hospitality in various structures which finally were transformed into asylums. the response to that distinctive expression of social deviance recognized as madness had been divided, on a theoretical level, in the attempt to scientifically interpret mental suffering in an anatomical - biological sense and on a practical level, reflecting on the value and limits of the asylum as an institution, a sophisticated therapeutic tool but at the same time segregating. it was thus made concrete with psychiatry as a science on the one hand and the asylum as a practical social solution on the other [5 - 7 ]. the development of psychiatric thinking and the interpretative paradigms of deviance phenomena went hand in hand with the debate on asylums, viewed as the answer to a problem that was considered of a prevalently social nature. it regarded the identification and classification of mental illness and at the same time the methods of treating it. in this process, the operation that attempted, on a scientific level, to transform patients into clinical cases, victims in terms of a neutral, abstract classification of mental illness, exclusively understood as physiological degeneration, was linked to reconstructing the real identity of the variegated crowd populating asylums at the end of the nineteenth century [8 - 10 ]. mental illness and the cultural and social environment in which it was manifested, responses arising from society starting from asylums and the development of psychiatry as a science thus made up the framework within which, in the last three decades of the nineteenth century, a debate went on in italy, directed towards passing legislation setting this sector in order. to put this in context, statistical research should be inserted which attempts to determine the state of asylums in italy starting in 1871. obviously, with the increase in patients, relative expenses rose exponentially, raising the question of how to solve this problem. the first regarded the establishment of new asylums or the enlarging and renovation of existing ones. others alternatively suggested, as mentioned above, solutions of various types in homes, hospices, family structures, home custody or charitable institutions. all these solutions were based on a radical change in the concept of asylum dominant at that time. it was indeed necessary to affirm the idea of the asylum as a space primarily entrusted with a therapeutic function, with treating acute mental illnesses, rather than a space to neutralize the dangerousness of patients, as it had been considered up to that time. this was a radical change in perspective, denying the binomial : madness = dangerousness, but also the equation : madness = mental illness = incurability. in conformity with the new paradigm, the logic to be followed was the one according to which mental illness is an illness that can be treated like any other, especially an acute one, thus curable in many cases. only as a last resort was the asylum to be considered the mere response to danger, the concept that had previously predominated [12, 13 ]. future psychiatric reform was thus to be inspired, on a legislative level, by the paradigm according to which : the true purpose of the asylum was to be a place, not of simple segregation and personal custody, but of efficient treatment for the mentally ill in the acute phase, carried out in a rational and above all individual way, the same way in which common illnesses were treated in hospitals ; the increasing and inevitable costs sustained by provinces for maintaining the insane were to be better employed to the advantage of the patients themselves, with modern, functional organizational reforms ; the number of patients in asylums had to respect the hygienic and technical standards of the individual institutions. to carry out this project, it was necessary to establish new asylums intended only to treat recent, acute, curable cases and create in preexisting ones separate sections for the same type of insanity, supplying them with all necessary resources. discharge from asylums all patients not belonging to these categories, to solve the problem of overcrowding and avoid a prolonged stay from becoming a factor of chronicity and incurability in mental illnesses. the patients involved in this reorganization were : idiots and imbeciles not exhibiting dangerous tendencies ; the incurable, chronically insane (of any type) but calm and identified as harmless. elderly, weak or infirm patients suffering from common chronic illnesses and permanently bedridden ; pellagrins who, after cessation of their mental disturbance, were often detained in the asylum due to risk of relapse and who, if discharged, returned to their previous alimentary habits ; alcoholics, perfectly normal if kept away from alcohol, but who easily relapsed after the slightest abuse ; epileptics without other pathological manifestations except for convulsions. for most of them, the solutions proposed regarded the establishment of special shelters, appropriate placement of the chronically insane, care in the home and charitable institutions, according to the program : of placing chronic, incurable and harmless patients in special homes and hospices, dedicating special sections to them or adapting old asylum buildings, thus reducing costs and reducing treatment to the essentials ; of encouraging and developing family charitable institutions both for the harmless chronically insane and for idiots and other patients, that is encouraging their care in the home with subsidies provided by the province under the supervision of the asylum superintendent and doctors ; of encouraging and actively developing public charity, charitable institutions for the poor, discharged, cured or convalescent mentally ill, especially pellagrins, recurrent cases, etc., offering consistent, long - term subsidies, to avoid inevitable relapses when going back to living in wretched conditions. these measures, presented for general psychiatric reform had already been put into practice in some places in tuscany, the provinces around bergamo, modena, and in particular in the reggio emilia asylum, which could boast of being a pioneer. on the other hand, they mirrored experiences abroad, as in scotland, france, germany, russia, the netherlands and switzerland, but the reference point was above all belgium, with its classical model of the gheel home. having regained the concept of the asylum as solely a place for treatment of the acute stage, attention was extended to the problem of training medical and nursing staff. therefore, a training school was necessary which, by means of theoretical lessons and practical training, would convey the special knowledge and competence required and favor the development of conscientiousness on a level with the tasks entrusted to them. schools of this type were springing up in england and in france, but the most highly qualified ones for treating the insane were in the netherlands, managed by the asylum superintendents and doctors, in particular in the meerenberg asylum, where the role of the so - called lay sisters was established. they were in charge, as supervisors, of the nursing service and treatment of patients and cooperated with doctors [15 - 17 ]. in italy, to train nurses for asylums, in 1903 a. tamburini initiated the first regular technical - professional course with the help of nurses themselves in the reggio emilia asylum. among the requisites for admission was the ability to read and write correctly, not to be addicted to alcoholic beverages and to be free from hereditary defects and mental and nervous illnesses. courses were of a theoretical - practical nature and included elementary notions of anatomy, physiology, bodily hygiene, mental functions and their principal alterations and their physical and moral causes. in addition, there were theoretical and practical rudiments on examining the patient and on assisting in care and in physical and moral treatment, notions on emergency assistance and medications and, finally, knowledge of laws and relative regulations. intervention policy was inspired by a humanitarian approach whose objective was to establish authentic human relationships and substitute the coercive treatment of the past which, no longer the rule, becomes absolutely exceptional, recognized as such solely by the mental specialist. this, was to avoid risks to health and the psyche of the patient, but above all to respect and promote his personal dignity. in the implementation of this principle, not an easy task, doctors could obtain valid assistance from nurses who had been trained professionally and humanely. we believe that the historical lines we have considered permit interpretation of the current debate on psychiatric care in another light, that is pointing out what has been done, what has been neglected but above all the fundamental principle which must guide every human and professional approach to the abyss of madness : the principle of the dignity of each individual. in this sense, we would like to conclude by recalling how these guidelines were reproposed and emphatically affirmed in the early 1900 s by the american c.w. beers, who, having been interned in an american asylum, as soon as he was discharged, published his autobiography in 1908 in the book a mind that found itself. an autobiography, thus founding the movement for mental hygiene on an international level and marking a radical turning - point in this sector. | the present work refers to the debate which took place in italy in the final years of the nineteenth century in relation to mental health and lunatic asylums, from which emerged various innovative proposals for avoiding compulsory confinement in numerous cases. some of them became part of new legislative regulations regarding asylums, but most were excluded. today, a new historical interpretation allows us to grasp a connection between law 180, dated 1978 and known as the basaglia law from the name of its promoter, and alternative proposals to asylum custody omitted from the 1904 law. |
desmoplastic fibromas are benign but locally aggressive bone tumors first described by jaffe in 1958. desmoplastic fibromas are quite rare, accounting for 0.06% of all bone tumors and 0.3% of benign bone tumors, typically in patients younger than 30 years, involving the metaphysis or diametaphysis of long bones in > 50% of cases, with no reported cases of metastases in the literature [24 ]. radiography of desmoplastic fibromas is typically lytic, expansile and well demarcated, with low signal intensity on both t1- and t2-weighted magnetic resonance imaging (mri). en bloc or wide resection of the tumors to prevent local recurrence has historically been the method of choice for treatment. some recent case reports with limited follow - up duration have advocated use of curettage with adjunctive therapy and bone grafting rather than wide resection to minimize functional loss [6, 7 ]. however, recurrence rates following curettage, excision and wide resection have been shown to be 55, 72 and 17%, respectively. the authors have obtained the guardian or patient 's informed written consent for print and electronic publication of the case report. a 10-year - old male was referred to our office after being seen by a pediatric orthopedic surgeon for a distal left forearm mass with block in pronation and supination. the patient 's past medical history is significant for a distal radius fracture 2 years prior to his aforementioned office visit. follow - up radiographs after fracture union at that time showed no evidence of disease (fig. 1). radiographs taken at the time of presentation 2 years after fracture healing revealed a 6.5 cm long lesion in the distal syndesmosis arising from the radius. the lesion resulted in pressure erosions upon the distal radius and ulna, causing a slight splaying of the bones (fig. 2). figure 1:lateral, oblique and anteroposterior (ap) views of distal radius fracture 2 years prior to presentation of desmoplastic fibroma. figure 2:ap and lateral radiographs of the left forearm showing 6.5 cm long lesion in the region of the distal syndesmosis between the radius and ulna. lateral, oblique and anteroposterior (ap) views of distal radius fracture 2 years prior to presentation of desmoplastic fibroma. ap and lateral radiographs of the left forearm showing 6.5 cm long lesion in the region of the distal syndesmosis between the radius and ulna. the differential diagnosis included primary benign and malignant bone tumors, and thus preoperative staging studies were performed. a triple - phase bone scan was administered and demonstrated increased uptake in the distal shaft of the left radius and left ulna, as well as in the distal physis of the left radius (fig. figure 3:triple - phase bone scan revealing moderately increased radiotracer uptake in the distal shaft of the left radius and left ulna, as well as in the distal physis of the left radius. triple - phase bone scan revealing moderately increased radiotracer uptake in the distal shaft of the left radius and left ulna, as well as in the distal physis of the left radius. a mri images revealed a large mass at the level of the distal radial and ulnar metadiaphyses, measuring 6 cm craniocaudad, 3.7 cm ap and 3 cm transverse (fig. figure 4:(a and b) t1 mri showing mass iso - intense to muscle. (c) t2 mri axial view with overall increased signal intensity, but with a central area of decreased signal. (c) t2 mri axial view with overall increased signal intensity, but with a central area of decreased signal. positive b - catenin nuclear immunostaining, along with histology showing fibrous tissue, supported a diagnosis of desmoplastic fibroma. upon confirmation of desmoplastic fibroma, a discussion with the patient 's mother was held, explaining the rarity of the tumor, and the likelihood that the patient would require radical resection to decrease risk of local recurrence, which would lead to a vast reduction in wrist motion. additionally, as the tumor extended to the distal radius, resection of the joint surface would be necessary. subsequently, a conversation about reconstructive options was held, including options for use of the fibula, allograft and allo - arthrodesis of the wrist joint. ultimately, the patient 's mother decided to pursue allo - arthrodesis with allograft, with plans to pursue future reconstruction with use of the fibula if this surgery was unsuccessful. upon removal of the mass dorsal plate fixation was used to stabilize the allograft, which extended from the native proximal radius to the proximal carpal row, effectively creating a single - bone forearm (fig. 5). figure 5:(a and b) post - operative radiographs of allo - arthrodesis with dorsal plate fixation. (c and d) follow - up radiographs at 7 months post - op. (a and b) post - operative radiographs of allo - arthrodesis with dorsal plate fixation. (c and d) follow - up radiographs at 7 months post - op. post - operatively, the patient began occupational therapy 1 week after surgery, completing 13 sessions over several months, with a progressive increase in strength, range of motion and coordination of the left upper extremity. at 8 weeks, the patient was able to employ pincer grasp to pick up small marbles and make a fist at 10 lbs of force, with minimal pain on palpation as well as during therapy exercises. at the request of the patient 's family at 12 months follow - up, the patient has exhibited excellent recovery, including normal range of motion at the shoulder and elbow and ability to perform all activities of daily livings despite reduced grip strength. on all follow - up imaging, the allograft has fully incorporated, with no hardware failure or loosening. although some recent literature has explored the possibility of utilizing intralesional curettage with adjuvant therapy with the intention of sparing maximum limb function, the level of invasion of the tumor and thus increased risk of recurrence in this patient made radical resection a more favorable surgical option. in the largest review of desmoplastic fibroma cases to date, recurrence rates were 38% higher following curettage than radical resection, further strengthening a decision to pursue radical resection. muramatsu reported a case of a young man with desmoplastic fibroma of the distal radius treated successfully with excision of the radius and fibular graft, free of tumor for 7 years as per last reported follow - up, with good range of motion at elbow and wrist. kesani reported a case of a 15-year - old male with recurrent desmoplastic fibroma of the radius and ulna treated successfully with distal radius allograft with radiocarpal, partial intercarpal and third metacarpal capitate arthrodesis osteosynthesis to the proximal ulna. in this particular case, the advantages of wide resection, including a substantial decrease in the rate of local recurrence, outweighed the drawbacks of the procedure, including reduced limb function. factors such as tumor size and location, as well as age and activity of the patient, must be considered in each case. previously, the senior author had a patient with a similar case which was treated with more conservative curettage several times, and ultimately required an above the elbow amputation to control extensive recurrent disease, fracture and hardware failure. this experience encouraged the author to advocate for more aggressive treatment on this patient 's behalf. this case highlights the importance of extensive discussion with parents of the child and detailed follow - up with the surgeon and physical therapists to ensure maximal limb function recovery and monitoring for recurrence of disease. | desmoplastic fibromas are rare, benign, locally aggressive bone tumors, which arise primarily in patients younger than 30 years old. historically, even with greater functional loss, en bloc or wide resection of the tumors to prevent local recurrence has been the method of choice in treatment. this article discusses the presentation of a 10-year - old male who presented with a mass in the distal forearm, after reporting difficulty in pronation and supination. the patient was ultimately treated with wide resection and allo - arthrodesis with allograft. post - operatively, the patient has exhibited excellent recovery, including normal range of motion at the shoulder and elbow, and ability to perform all activities of daily livings despite reduced grip strength compared with the contralateral side. |
hepatitis c virus (hcv) is an rna virus of the flaviviridae family that establishes persistent infection in the majority of infected patients. a potential role for nk cells in viral hepatitis was first suggested by genetic studies that described a higher odds ratio of spontaneous hcv clearance and ifn - treatment - induced hcv clearance in kir2dl3 patients who are homozygous for hla - c1 alleles as compared with patients who are homozygous or heterozygous for hla - c2 alleles. hla - c1 and hla - c2 represent two groups of hla - c alleles that differ in two amino acids in their respective hla - cw 1 domains. because the interaction between kirs on nk cells with hla molecules on target cells plays a key role in nk cell inhibition, it has been suggested that the kir2dl3/hla - c1 compound genotype results in a lower activation threshold of nk cells, thereby allowing faster nk cell activation compared with less favorable genotypes. this is supported by data in an in vitro influenza a virus infection model that demonstrate a larger hla - c regulated nk cell subset with more rapid nk cell ifn- secretion and cytotoxicity in hla - c1 than in hla - c2 homozygous patients. an increased prevalence of kir2dl3/hla - c1 homozygosity is also observed in injection drug users who remain aviremic and antibody - negative despite high - risk behavior and frequent hcv exposure. the apparent immune protection in such individuals is associated with kir2dl3 expression on nk cells and with an increased frequency of activated nk cells. at the functional level, nk cells in the blood of exposed uninfected individuals display increased ex vivo ifn production and increased in vitro cytotoxicity. these results from cross - sectional cohorts are consistent with data from a prospective study of health care workers observed after an accidental needlestick. accidental exposure to minute amounts of hcv - containing blood resulted in a transient increase the frequency of activated nk cells in the blood and their effector functions (both cytotoxicity and ifn production). the magnitude of the nk cell response correlated with that of the subsequent hcv - specific t - cell response. this likely represents an early innate response to an abortive or rapidly contained and cleared infection, because neither viremia nor hcv - specific antibodies are detected. collectively, these studies demonstrate that nk cells are sensitive biomarkers of subclinical hcv exposure. while it is possible that nk cells along with other components of the innate immune system contribute to viral containment in this setting, it is obvious that innate immune responses on their own can not clear the infection once high - level hcv viremia is established. data from prospectively studied humans and experimentally infected chimpanzees demonstrate that high - level hcv viremia persists for weeks despite induction of a large set of intrahepatic interferon - stimulated genes (isgs). this immune response is initiated in the cytoplasm and in endosomes of infected cells by the pattern recognition receptors protein kinase, retinoic acid inducible gene - i, and toll - like receptor 3 (tlr3). downstream signals, mediated by interferon regulatory factor 3 (irf3) and nuclear factor - kb, result in the transcription of the ifn gene. ifn is released from infected cells, binds to the ifn/ receptor (ifnar1 and ifnar2) on neighboring cells, and induces a diverse isg set that includes many antiviral and proinflammatory genes. however, owing to hcv s elaborate strategies to escape from ifn responses, there is no decrease in viremia, just a plateau. the onset of clinically symptomatic acute hepatitis with increased alanine aminotransferase levels occurs 8 to 10 weeks after infection. without treatment, two - thirds of the infected patients develop chronic hepatitis c, which is associated with a 23 log10 reduction in viral titer. because liver biopsies are clinically not indicated in the acute phase of hepatitis c, the intrahepatic effector responses responsible for the decrease in viremia have not been studied in patients. however, data from biopsy tissues of experimentally infected chimpanzees have clearly shown that the decrease in viremia coincides with an increase in intrahepatic ifn-mrna levels. the relative contribution of t cells and nk cells to ifn production and antiviral response is not known at this time. whereas the appearance and maintenance of hcv - specific t - cell responses in the blood, in particular cd4 t - cell proliferation and cytokine production, are the best predictors of viral clearance, nk cells are also activated and display increased cytotoxicity and ifn production. pelletier recently reported a correlation between the magnitude of t - cell response and the peripheral blood nk cell response in the acute phase of hcv infection, and kokordelis found that nk cells from patients who later cleared the infection have a greater antiviral effect in vitro than nk cells from patients who developed chronic hcv infection. this opens the interesting question of whether the increased nk cell activity in acute hcv infection is an independent event or is triggered by cd4 t - cell derived il-2. the latter would render nk cells amplifiers and even downstream effectors of the virus - specific t - cell response. they express increased levels of cd69 and hla - dr, indicating recent and more distant stimulation, respectively, and increased levels of nkp30, nkp44, nkp46, nkg2a, nkg2d, and the il-2 receptor chain cd122. increased nkg2c expression has also been reported, but has now been attributed to oligoclonal expansion of a highly differentiated nk cell subset during prior hcmv infection. nk cell activation is influenced by location, as nk cells are generally more activated in liver than in blood, even in uninfected individuals. nk cell activation is also influenced by additional factors ; for example, nkp46 levels are strongly associated with female gender and caucasian race. while exciting new information on liver - resident nk cells and nk cell memory is emerging in the general nk cell field, data on these topics are still sparse in hcv infection. this is mostly due to the limited number (about 100,000) of lymphocytes that can be isolated from subcutaneous liver biopsy tissues and the lack of liver tissue from uninfected controls. only a single study performed a side - by - side comparison of intrahepatic nk cells from surgically resected liver tissue of patients with hcv infection and uninfected controls (who underwent cholecystectomy). in that study, hcv infection was associated with an increased frequency (compared with uninfected livers) of intrahepatic nk cells that shared some phenotypic and functional features with the unique liver - resident nk cell population that has been proposed to represent the human equivalent to mouse memory nk cells in subsequent studies. this raises the interesting, but as yet unstudied question of whether hcv infection affects nk cells with memory functions in the liver. chronic hcv infection leaves a distinct signature also on peripheral blood nk cells, which has now been confirmed by several independent studies. most remarkable is a dichotomy in effector functions that is characterized by increased cytotoxicity (evidenced by increased degranulation and production of tumor necrosis factor - related apoptosis - inducing ligand [trail ]) and, upon in vitro stimulation with il-12/il-15 or il-12/il-18, decreased production of antiviral cytokines (ifn and tnf). this is unexpected because the size of the cd56 subset, which constitutes about 10% of peripheral blood nk cells in uninfected individuals, doubles in chronic hcv infection. cd56 nk cells are known to produce large amounts of ifn and trail with little perforin / granzyme - mediated cytotoxicity. these cells contain high levels of perforin and granzyme, but can also rapidly produce chemokines and cytokines. the altered subset distribution and overall decrease in the number of intrahepatic and blood nk cells should therefore not result in the selective decrease in cytokine production. what is the mechanism underlying these divergent effector functions ? increasing signaling via nkp30, nkp44, and nkp46 may directly contribute to the increase in nk cell cytotoxicity because these molecules are natural cytotoxicity receptors (ncrs) and ncr nk cells have been shown to release more perforin / granzyme containing granules and exert greater in vitro antiviral effect than ncr nk cells. increased expression of the activating receptor nkg2d and its ligands may also contribute to nk cell cytotoxicity, as may decreased expression of the inhibitory receptor nkg2a. cytokine - dependent signals are thought to play a major role in the increase in nk cell cytotoxicity and decrease in nk cell cytokine production in chronic hcv infection (figure 1). this is evidenced by increased ex vivo levels of signal transducer and activator of transcription 1 (stat1), a key molecule of ifn signaling in nk cells. because stat1 itself is an isg, increased stat1 levels are consistent with type i ifn - mediated signaling in chronic hcv infection. this is supported by increased ex vivo levels of phosphorylated stat1 and decreased levels of phosphorylated stat4 in nk cells of hcv - infected patients compared with nk cells of uninfected patients. chronic exposure to virus - induced type i ifn can explain the nk cell phenotype of increased cytotoxicity and decreased cytokine production because induction of cytotoxicity and production of ifn require differential stat1/4 signaling. based on a model initially proposed by miyagi for lymphocytic choriomeningitis virus (lcmv) infection, nk cells produce ifn in the early phase of a virus infection because of their constitutively high stat4 expression. chronic exposure to type i ifn results in increased expression of stat1, and preferential stat1 over stat4 phosphorylation, increased stat1-dependent cytotoxicity (with trail itself being an isg), and decreased stat4-dependent ifn production. the observations of increased pstat1/pstat4 ratio and increased levels of the isgs stat1 and trail in nk cells in chronic hcv infection therefore provide compelling evidence that type i ifn is produced locally in hcv infection even though it is detectable only rarely and at exceedingly low levels in the serum. candidates for type i ifn production are nonparenchymal cells, such as liver - resident macrophages (kupffer cells), plasmacytoid dendritic cells, and liver sinusoidal endothelial cells. kupffer cells were identified as a local source of ifn by immune - staining of liver sections. plasmacytoid dendritic cells were shown to respond to short - range transfer of hcv rna - containing exosomes from hepatoma cells with tlr7-induced production of type i ifn in in vitro experiments. finally, primary human liver sinusoidal endothelial cells and immortalized liver endothelial cells were shown to produce type i ifn after internalization of hcv and stimulation of tlr7 and retinoic acid - inducible gene 1 (rig - i). the dichotomy of increased cytotoxicity and decreased cytokine production by nk cells is exacerbated when hcv - infected patients undergo ifn - based therapy. this nk cell phenotype can be recapitulated when peripheral blood mononuclear cells of uninfected individuals are exposed to ifn in vitro. in addition to the local increase in type i ifn, a relative decrease in il-12, il-18, and il-2 production is thought to contribute to the suppressed cytokine production of nk cells in chronic hcv infection (see figure 1). hcv is known to activate the inflammasome in monocytes and macrophages in an infection - independent process that requires recognition of viral rna by endosomal tlr7. this can be achieved by clathrin - mediated uptake and uncoating of hcv particles and release of their rna in endosomes. it can also be achieved by cell - to - cell transfer of rna as shown in cocultures of primary human monocytes with hepatoma cells that contain replicating subgenomic hcv rna but do not release infectious hcv particles. inflammasome activation results in secretion of il-18 and il-1 in this coculture model, and both cytokines are also detected at increased levels in the blood of patients with acute hcv infection. il-18 stimulates ifn production and down - regulation of hcv replication by nk cells in cocultures with hcv - replicon cells and monocytes. interestingly, monocytes from healthy il-18 production and ifn-mediated antiviral activity of nk cells improve when monocytes from hcv - infected patients are replaced with monocytes from healthy blood donors in this system. these data show that suboptimal monocyte activation and il-18 production contribute to the defective cytokine production of nk cells in chronic hcv infection. a lack of il-2 may further contribute to the decreased cytokine production of nk cells. this scenario in chronic hcv infection clearly contrasts with acute hcv infection where serum il-18 levels peak, and where hcv - specific cd4 t - cell responses and ifn producing nk cell responses are strong and predictive of hcv clearance. in addition to monocytes, monocyte - derived dendritic cells have a reduced cytokine response in hcv infection. specifically, they produce less il-15 than those of healthy controls, which results in reduced expression of mhc class i - related chain a and b (mica / b) and in reduced nk cell stimulation via the mica / b ligand nkg2d. conversely, nk cells from hcv patients do not activate dendritic cells as much as nk cells from healthy donors. this is thought to be due to stabilization of hla - e on hcv - infected hepatocytes by an endogenously processed hcvcore peptide. what are the consequences of an nk cell phenotype that is biased toward an increase in cytotoxicity and a decrease in cytokine production ? nk cells have been shown to exert antiviral effects both via perforin / granzyme - mediated cytotoxicity and via ifn-mediated down - regulation of hcv replication in in vitro models. as initially proposed by guidotti and chisari, a cytokine - mediated antiviral effect may be more efficient than cytotoxicity in the infected liver because secreted cytokines can reach many infected hepatocytes whereas cytotoxicity requires a 1:1 interaction between cells. the altered functional phenotype of nk cells in chronic hcv infection may therefore facilitate chronic inflammation via killing of infected cells but not allow viral clearance due to impaired ifn production. because suppressed cytokine secretion is also a key characteristic of the t - cell response in chronic hcv infection, this may be a general mechanism to dampen the immune response of the host and to ameliorate disease pathogenesis. to answer the question whether the altered nk cell phenotype represents a specific mechanism of hcv persistence or a general host response to decrease immunopathology, it is interesting to study nk cells in other viral infections. hbv is a good example because it belongs to a different family of viruses and employs different mechanisms to establish persistence. most hcv infections occur in immune - competent adults whereas most hbv infections occur during the neonatal period and in early childhood when immune responses are less mature and the inflammatory response is lower than in adulthood. one of the most striking differences to hcv is the absence of detectable isg responses, even in the acute phase of hepatitis. this suggests that type i ifn the main factor that drives the increase in nk cell cytotoxicity and the decrease in ifn production in hcv infection does not play a major role in hbv infection. however, despite the differential type i ifn response, alterations in the nk cell phenotype and function in hbv infection are strikingly similar to those in hcv infection. the overall percentage of nk cells in the peripheral blood mononuclear cell population is decreased, with a relative and absolute increase of the cd56 nk cell subset. likewise, nk cells display increased expression of activating receptors such as nkp30, nkp46, and nkg2c with decreased expression of the inhibitory marker nkg2a. as in hcv infection, nk cells of chronic hbv patients have a reduced capacity to produce ifn compared with healthy controls. conversely, nk cell cytotoxicity is maintained, and the percentage of trail - expressing cd56 nk cells is increased. this altered nk cell phenotype is already apparent in pediatric patients after perinatal acquisition of hbv infection from their mothers. although nk cell phenotype and function are similar in hcv and hbv infection, the underlying causative mechanisms are different (figure 2). in hbv infection, alterations in nk cell function appear to be induced by il-10 and tgf rather than ifn. accordingly, the cytokine - induced dichotomy in nk cell effector functions increases during phases of increased disease activity with increased il-10 serum levels. some of the il-10 may be derived from regulatory b cells, which have been demonstrated in hbv infection. this nk cell phenotype can be recapitulated by in vitro exposure to il-10, which suppresses ifn production without altering cytotoxicity and il-10 production. conversely, blockade of il-10 and/or tgf restores ifn production of both cd56 and cd56 nk cells in vitro. thus, the common theme of preserved nk cell cytotoxicity and impaired ifn production is found in both chronic hbv and hcv infections. like hcv, hbv is sensitive to the antiviral effects of ifn. ifn from adoptively transferred hbv - specific cd8 t cells and from therapeutically activated nk cells has been shown to down - regulate hbv replication in transgenic mouse models. the lack of ifn production by nk cells in chronic hbv infection may therefore result in reduced viral control and inability to clear hbv, and the preserved cytotoxicity appears to contribute to liver inflammation. along this line, polymorphisms in the il-10 promoter are associated with increased il-10 production, and with increased disease severity and progression in chronic hbv infection. furthermore, liver inflammation correlates with trail expression on nk cells and trail - expressing nk cells are enriched in the liver. what could be the reason for the preserved nk cell cytotoxicity in chronic viral hepatitis ? nk cell cytotoxicity may serve a regulatory role, aimed at controlling virus - specific t - cell responses. hbv - specific t cells up - regulate trail death receptor 2 (trail - r2), which renders them susceptible to apoptosis by trail - expressing nk cells. increased trail - r2 expression is specifically observed on intrahepatic hbv - specific cd8 t cells in close contact with trail - expressing nk cells, and it correlates with the hbv titer. in contrast, the majority of t cells of other specificity do not up - regulate trail - r2, which indicates that t - cell receptor stimulation by cognate antigen rather than bystander activation is the underlying mechanism. indeed, in vitro depletion of nk cells from peripheral blood mononuclear cells of hbv - infected patients increases hbv - specific but not cmv - specific cd8 t - cell responses. a similar regulatory role of nk cells is also found in the mouse model of chronic lcmv - induced hepatitis. nk cells specifically kill lcmv - specific cd4 t cells in a perforin - dependent manner, and a reduced cd4 t - cell response is associated with a secondary reduction of the lcmv - specific cd8 t - cell response. in addition, activated cd8 t cells become targets of nkg2d nk cells because they up - regulate nkg2d ligands in lcmv infection. consequently, depletion of nk cells restores cd4 t - cell help and thereby enhances the lcmv - specific cd8 t - cell response, and blocking of the activating receptor nkg2d on nk cells directly increases the frequency of functioning, antigen - specific cd8 t cells. it has therefore been proposed that nk cells function as rheostats for virus - specific t - cell responses and that therapeutic targeting of nk cells may have differential effects based on the degree of virus - driven t - cell exhaustion. the observed down - regulation of ifn production by both nk cells and t cells in hcv and hbv infections raises the interesting question of whether their impaired function can be restored by antiviral therapy. as described in the previous sections, the suppression of nk cell ifn production is cytokine - driven that is, driven by an excess of ifn, il-10, and transforming growth factor (tgf) and by a relative lack of il-12, il-18, and il-2. in contrast, impaired cytokine production of t cells is attributed to chronic t - cell receptor stimulation by viral antigens. chronic t - cell receptor signaling induces inhibitory molecules such as programmed death 1 (pd-1), t - cell immunoglobulin domain and mucin domain-3 (tim-3), and cytotoxic t lymphocyte antigen 4 (ctla-4), and promotes an exhausted t - cell phenotype that is also observed in diseases such as hiv infection and cancer. antiviral drugs that rapidly clear hcv infection and significantly reduce hbv viremia provide an excellent clinical model to answer the question of whether the described changes in nk cell and t - cell function are reversible. ifn - based treatment regimens are not suitable to answer these questions because ifn exerts not only antiviral, but also immune - modulatory function. specifically, ifn - based therapies exacerbate the functional dichotomy of nk cells toward increased cytotoxic effector functions and reduced ifn production, suppress proliferation of t - cell and ifn production of t cells, and do not fully restore t - cell effector function even if they achieve viral clearance. in contrast, effective removal of hcv by an ifn - free regimen of direct - acting antivirals normalizes both phenotype and function of nk cells, as now shown in two independent studies on combination therapy with the hcv ns5a inhibitor daclatasvir and the ns3 protease inhibitor asu - naprevir. successful treatment of ifn nonresponders with this ifn - free regimen is associated with a rapid decrease of nk cell activation within the first 24 hours of treatment initiation and with normalization of nk cell phenotype and function by week 8 of the 24-week regimen. both nk cell cytotoxicity and ifn production reach levels that are indistinguishable from those of healthy controls. the concomitant decrease in the frequency of pstat1-expressing nk cells confirms the role of virus - induced type i ifn for the altered function of nk cell function in chronic hcv infection. this is consistent with a decrease in the expression level of the isgs rsad2, isg15, oas3, and ifit1 in the total blood leukocyte population. the effect of treatment - induced hcv clearance on the t - cell response is quite different from its effect on nk cells. spaan studied hcv - specific t - cell responses in parallel to nk cell responses in five hla - a2 patients treated with daclatasvir and asunaprevir. they used dextramers of hcv - peptide loaded hla - a2 molecules to detect hcv - specific t cells ex vivo irrespective of their function, and they report an increase in the frequency of hcv - specific t cells in the blood at the treatment time point of week 12. this is consistent with an earlier report on an increased ex vivo frequency and improved in vitro proliferation of hcv - specific cd8 t cells in 51 hcv patients who were treated with the protease inhibitor faldaprevir, the hcv ns5b polymerase inhibitor deleobuvir with or without ribavirin. cd8 t - cell populations that target the sequence of the infecting virus showed a greater improvement in in vitro proliferation than those that target sequences in which the virus had escaped, thus excluding any bias for preexisting memory t cells. notably, however, an improved ex vivo t - cell function has not yet been demonstrated in any study. in our own study of asunaprevir / daclatasvir - treated patients, we did not observe any recovery of the ifn production of hcv - specific t cells when ex vivo elispot assays with sets of overlapping hcv peptides or minimal optimal t - cell epitopes were performed (b. rehermann, unpublished results). combined, these early results suggest that successful ifn - free therapy normalizes phenotype and function of nk cells, and that it restores the proliferative capacity but not necessarily the ex vivo effector function of hcv - specific t cells. treatment of hbv infection differs from treatment of hcv infection in the sense that ifn - free hbv - specific antiviral regimens significantly decrease viremia but do not induce complete viral clearance. this is because the covalently closed circular form of the hbv dna persists in infected cells and serves as its transcriptional template. consequently, only very few patients clear hbsag with long - term nucleos(t)ide analogue treatment. consistent with suppression but not complete elimination of hbv, antiviral therapy with nucleoside analogues corrects some but not all of the abnormal nk cell parameters. the treatment - induced decrease in hbv titer is paralleled by normalization of the percentage of nk cells in peripheral blood mononuclear cells, the size of the cd56 nk cell subset, and trail expression on nk cells. consistent with low - level hbv persistence, there is no or only partial restoration of the nk cells capacity to produce ifn. as in hcv infection, restoration of t cell responses in hbv infection is mainly detectable with t cell assays that require more than a week of in vitro stimulation with viral antigens. restoration of t - cell proliferation is greater in patients who respond to long - term antiviral treatment with suppression of viremia and loss of hbsag than in those who show a decrease in viremia but remain hbsag positive. while the expanded t - cell lines also displayed detectable il-2, tnf, and ifn responses upon restimulation with their cognate antigen, t cells remain dysfunctional in ex vivo assays. furthermore, even in vitro responses are not maintained when treatment is discontinued. the t - cell responses of successfully treated patients therefore remain inferior to those of spontaneously recovered patients. collectively, these results suggest, that cytokine - induced alterations in nk cell function do normalize when the virus is cleared whereas antigen - induced alterations in t - cell function are not fully reversible. the observation that complete normalization of the nk cell phenotype and function can be achieved in hcv - infected but not in hbv - infected patients fits the observation that hcv can be completely cleared with antiviral treatment whereas hbv persists at low levels. research over the past 10 years has expanded our view on nk cells as both an antiviral and a regulatory immune cell population in viral hepatitis. impairment of ifn production is observed for both nk cells and t cells, but induced by different mechanisms. with new direct - acting antiviral treatment regimens now available as standard of care for hcv infection, research into the reversibility of these immune alterations an expansion of this new area of research may provide useful information and strategies for immunotherapy of infections such as hbv that can not be cleared with antiviral drugs alone and require an active immune response. further research in the emerging field of ilc subtypes, liver - resident nk cells, and nk - cell - mediated memory functions is important to increase our knowledge of the immune surveillance in the liver and may lead to novel strategies for immunotherapy of hepatocellular carcinoma. | natural killer (nk) cells are traditionally regarded as first - line effectors of the innate immune response, but they also have a distinct role in chronic infection. here, we review the role of nk cells against hepatitis c virus (hcv) and hepatitis b virus (hbv), two agents that cause acute and chronic hepatitis in humans. interest in nk cells was initially sparked by genetic studies that demonstrated an association between nk cell related genes and the outcome of hcv infection. viral hepatitis also provides a model to study the nk cell response to both endogenous and exogenous type i interferon (ifn). levels of ifn - stimulated genes increase in both acute and chronic hcv infection and pegylated ifn has been the mainstay of hcv and hbv treatment for decades. in chronic viral hepatitis, nk cells display decreased production of antiviral cytokines. this phenotype is found in both hcv and hbv infection but is induced by different mechanisms. potent antivirals now provide the opportunity to study the reversibility of the suppressed cytokine production of nk cells in comparison with the antigen - induced defect in ifn and tumor necrosis factor- production of virus - specific t cells. this has implications for immune reconstitution in other conditions of chronic inflammation and immune exhaustion, such as human immunodeficiency virus infection and cancer. |
the temporomandibular joints (tmj) or the craniomandibular joints are one of the important joints in the body which may be subjected to disease processes like degeneration and inflammation. the commonest of the inflammatory joint diseases is rheumatoid arthritis (ra) which is a chronic multisystem disease. the disease is characterized by a pathologic immune response that affects the synovial cells, cartilage and bone resulting in joint destruction and permanent disability. it is not specific to any particular race or region, the prevalence being 1 - 2% of the population world wide. although, it can occur at any age, the peak incidence is during the fourth and sixth decades of life. ra often affects the peripheral small joints like the fingers and toes, eventually involving other joints like knee and shoulder. studies of the incidence of tmj symptoms in ra have shown wide range of figures of involvement varying from 2 - 86%. the position and relative anatomy of tmj and its surrounding structures requires a skillful and accurate technique of imaging for a detailed study. the first reported formal study of tmj in ra was done by cadenat and blanc (1958) in fifteen patients, which revealed radiographic changes in the tmj, as well as other joints. the frequency of radiologic findings in the tmj has been reported to be between 19% and 84% in patients with ra. also, studies thus far have revealed an involvement of tmj in ra with many patients having significant changes radiographically, but less clinical signs and symptoms when compared to the peripheral joints. with this in mind, a study was undertaken to evaluate radiographically the extent of tmj involvement as compared to hand (mcp)/wrist joints in ra patients by using conventional and modern imaging techniques. also, an assessment of the tmj 's and peripheral joints on the right and left sides was done, to ascertain, if certain joints were more prone to disease processes, based on functional inequalities. this study was approved by the ethics committee of sdm college of dental sciences, karnataka, india. fifteen patients in the age group of 20 - 60 years (11 females and 4 males) were selected for this study, out of a referral pool of patients from the oral medicine, rheumatology and orthopaedics clinics. patients with a diagnosis of ra according to the american rheumatism association 's revised criteria for rheumatoid arthritis. only patients over the age of 18 patients with joint involvement other than the tmj / hand (mcp) and wrist joint were not included. patients with myofascial pain, tmj ankylosis, headache, patients with known history of previous trauma, cervical spondylosis and pregnant females were not included. detailed case history as per the questionnaire was taken. as the patients were known ra patients, the primary focus was to ascertain the onset, duration, site, type of symptoms like pain, limited / altered movement of the hands and jaw etc. clinical assessment of tmj for function and range (deviation / limitation) of movement, pain in the joint proper or while biting, audible or palpable clicking of joints were recorded. examination of the mouth was carried out at the same time as the clinical examination of the joint. examination of hand / wrist joints for soft tissue swelling, pain, limitation of movement, tenderness to palpation, difficulty in performing simple functions like writing or picking up objects and deformities was done. in all cases, laboratory tests involving erythrocyte sedimentation rate (esr - westergren 's method) and ra test using the rhelax rf slide test kit (tulip, india) was used in this study for the determination of rheumatoid factor. radiographic evaluation of the tmj was carried out using the conventional projections, transcranial and opg and advanced computed tomography (ct). transcranial views of right and left tm joints of all patients were taken using siemens vertix 100 extraoral radiographic machine with a setting of 70 - 72 mas and 74 - 82 kvp on an average. orthopantomographs were taken using the villa rotograph machine with a machine setting of 70 - 75 kvp and 60 mas. ten patients were subjected to ct scans using the toshiba 2d ct scanner model- 300 s and with a machine setting of 150 kvp and 250 mas on an average. pa views of the hand and wrist were made with the siemens vertix 100 extra oral radiographic machine with a setting of 55 kvp and 6 - 8 mas on an average. detailed analysis of the hand and wrist radiographs was performed based on larsen 's gradings [table 1 ]. for the tmj, the gradings were restricted to the condyle proper, as the glenoid fossa could not be clearly visualized in all the radiographs. since the anatomy of the tmj is different from that of the hand and foot joints, a modification of the grading system proposed by larsen. these radiographs were assessed individually by a medical and oral radiologist. during the process of deliberation the conclusion arrived at by the examiners were noted. radiographic interpretation (gradings) of hand and wrist joints from pa view modified grading system for evaluation of tmj radiographs radiographic interpretation (gradings) of tmj from transcranial, opg and ct student 's t - test was applied for the statistical analysis of the data obtained. patients with a diagnosis of ra according to the american rheumatism association 's revised criteria for rheumatoid arthritis. only patients over the age of 18 patients with joint involvement other than the tmj / hand (mcp) and wrist joint were not included. patients with myofascial pain, tmj ankylosis, headache, patients with known history of previous trauma, cervical spondylosis and pregnant females were not included. detailed case history as per the questionnaire was taken. as the patients were known ra patients, the primary focus was to ascertain the onset, duration, site, type of symptoms like pain, limited / altered movement of the hands and jaw etc. clinical assessment of tmj for function and range (deviation / limitation) of movement, pain in the joint proper or while biting, audible or palpable clicking of joints were recorded. examination of the mouth was carried out at the same time as the clinical examination of the joint. examination of hand / wrist joints for soft tissue swelling, pain, limitation of movement, tenderness to palpation, difficulty in performing simple functions like writing or picking up objects and deformities was done. in all cases, laboratory tests involving erythrocyte sedimentation rate (esr - westergren 's method) and ra test using the rhelax rf slide test kit (tulip, india) was used in this study for the determination of rheumatoid factor. radiographic evaluation of the tmj was carried out using the conventional projections, transcranial and opg and advanced computed tomography (ct). transcranial views of right and left tm joints of all patients were taken using siemens vertix 100 extraoral radiographic machine with a setting of 70 - 72 mas and 74 - 82 kvp on an average. orthopantomographs were taken using the villa rotograph machine with a machine setting of 70 - 75 kvp and 60 mas. ten patients were subjected to ct scans using the toshiba 2d ct scanner model- 300 s and with a machine setting of 150 kvp and 250 mas on an average. pa views of the hand and wrist were made with the siemens vertix 100 extra oral radiographic machine with a setting of 55 kvp and 6 - 8 mas on an average. detailed analysis of the hand and wrist radiographs was performed based on larsen 's gradings [table 1 ]. for the tmj, the gradings were restricted to the condyle proper, as the glenoid fossa could not be clearly visualized in all the radiographs. since the anatomy of the tmj is different from that of the hand and foot joints, a modification of the grading system proposed by larsen. these radiographs were assessed individually by a medical and oral radiologist. during the process of deliberation the conclusion arrived at by the examiners were noted. radiographic interpretation (gradings) of hand and wrist joints from pa view modified grading system for evaluation of tmj radiographs radiographic interpretation (gradings) of tmj from transcranial, opg and ct student 's t - test was applied for the statistical analysis of the data obtained. the study was carried out in the proposed manner and the data was statistically analyzed. of the 15 patients taken randomly, 11 (73.3%) were females and 4 (26.6%) were males. values higher than 20 mm for esr have been taken as abnormal i.e. 13 patients (87%), thereby showed abnormal values. comparison of the tmj with the hand (mcp) and wrist on the right and left side did not reveal any statistically significant results. the results were not significant when the individual joints were compared on the right and left sides [tables 4 and 5 ]. comparison of right tmj (transcranial and opg) with right hand (mcp) and wrist and comparison of left tmj (transcranial and opg) with left hand (mcp) and wrist comparison between right and left sides of hand and wrist joints and tmj it was however noticed that hand (mcp) joints were more frequently affected when compared to wrist joints and when the wrist was involved, it was bilaterally involved [figure 1 ]. no of patients with unilateral / bilateral / normal joint involvement of the hand- mcp and wrist and tmj- as visualised from transcranial (trans), opg and ct comparison of the mean gradings of the tmj and mcp and wrist joints on the right and left side indicated a higher grade on left side [figure 2 ]. rheumatoid arthritis is a chronic destructive inflammatory disease process known to commonly affect the peripheral joints. this results in joint effusion, swelling, pain, limitation in movement and deformities. the tmj like the peripheral hand and wrist joints can be a site for chronic synovial inflammation. tmj involvement in ra results in clinical symptoms like pain, swelling, crepitation, stiffness and limitation in jaw movement. radiography is a frequently used diagnostic aid to detect and assess disease severity and response to treatment. the radiographic findings ascribed to ra in the tmj are erosion and flattening of the condylar head, limitation of movement, marginal proliferations and sclerosis. in this study, the temporomandibular joints of 15 patients (30 joints) with definite ra were compared with their hand (mcp) and wrist joints to assess the degree of involvement in each case using the transcranial radiograph, opg and ct scans. of the 15 patients, 11 (73%) were females and 4 (23%) were males, in the ratio of 2.7:1. this incidence rate indicates that ra affects women approximately three times more than men and is consistent with the well documented findings of other authors. the mean age of incidence of rheumatoid arthritis in this study is 34.5 years. this finding is comparable with that of the age of patients (35) in the study conducted by mc carthy but not in agreement with that of 55 years of ogus and 56.4 years of syrjanen. this disparity could be because of the difference in sample size, the higher age group and also the racial differences in other studies. it was observed that although all the patients presented with complaints of either swelling, morning stiffness or limitation of movement of the mcp and wrist joints, the tmjs were symptomatic in five patients only (33%). tmj symptoms were less noticed by the patient who has more stiffness and pain with visible inflammatory signs in other joints. also, patients often attributed tmj symptoms to tooth related problems. in this study, other symptoms of tmj involvement like restriction in mouth opening was not observed which is consistent with previous reports. most studies indicate that tmj lesions found radiographically vary in frequency from 50% to 80% in ra. in this study, conventional methods revealed that 13 ra patients (87%) had radiographic evidence of tmj abnormalities (from slight to marked) in one or both tm joints, which is in consonance with the previous studies. however, bilateral presentation was observed in 9 (60%) and 10 (67%) patients with the transcranial and opg, respectively. symmetric involvement of both right and left joints have been reported in previous studies as well. ct scans were performed to assess whether any additional radiographic changes could be observed in the tmj and to confirm the accuracy of the findings obtained with transcranial and opg. it was found that all the patients exhibited radiographic abnormalities, with 9 (90%) patients showing bilateral presentation. the present study shows that flattening [figure 3 ] and bone erosion of the condyle was the most common radiographic manifestations. of the 15 patients i.e. 30 joints evaluated, transcranial view revealed involvement of 9 tm joints (30%) as erosions and 16 joints (44%) as flattening. this was followed by osteophytes / marginal proliferations seen in 3 joints (10%) and subcortical cyst in 1 joint (3%). opg showing flattening of the temporomandibular joint on the left side with normal right tmj in opg, 9 joints (30%) revealed bone erosions, flattening in 14 joints (46%), osteophytes in 2 joints (6%) and cyst in 1 joint (3%). the low frequency of erosions in the present study is comparable with the finding of less involvement of 24% of the joints in the study by syrjanen, 1985. ct scans of all the ten patients showed either mild or moderate involvement of the temporomandibular joint. the higher incidence of positive findings like erosions and joint space narrowing observed in this study is comparable with other studies [figures 4 and 5 ]. coronal ct showing erosive changes and decreased joint space of right and left tmj 's axial ct shows subchondral cyst in the left tmj the analysis of the data revealed possibility of a relationship between tmj abnormalities and the duration and severity of disease. it was observed that in long standing cases, there was a corresponding increase in degree of structural changes as evidenced in radiographs. in this study, 4 of the 5 patients with tmj symptoms had duration of disease exceeding 3 years. this may be attributed to the fact that as the disease progresses the chronic inflammatory process results in increased joint destruction. the most common radiographic findings are erosion and flattening of the head of the mandible and articular fossa and reduction of the joint spaces, usually noticed 5 to 10 years after the onset of symptoms. the hand (mcp) and wrist joints of all the patients exhibited radiographic abnormalities on both right and left sides [figure 6 ]. interestingly, it was noted that mcp joints were more affected than the wrist, but whenever wrist was involved, it was more likely to be bilaterally affected and showed a greater grade on radiographs. the most common findings were erosions, flattening and decreased joint space. however, one patient presented with ankylosis of the wrist joints on the left side. this patient aged 60 years had suffered from the disease for more than 10 years. pa view of the hands shows erosive changes in the mcp and wrist joints of both hands upon evaluation of the data obtained from this study, it was found that the tmj and the hand (mcp) and wrist joints of patients with rheumatoid arthritis exhibited similar radiographic abnormalities on both right and left sides., in 1991 where they found no significant differences between the changes in tmj and hand (mcp) and wrist joints. functional inequalities, if any, owing to the use of the right side over the left in the peripheral joints does not seem to have any significance with regard to the extent of disease progression. in the tmj, functional movements like chewing on the right or left side more than the other also does not cause a corresponding increase in disease activity as evidenced by radiographic findings. the results of the present study showed similar changes in the tmj and mcp and wrist joints of patients with ra. flattening and though, 87% of patients had definite radiographic changes in the tmj, only 33% of them had clinical symptoms such as pain on biting followed by tenderness to palpation. few studies have compared radiographically the bony changes in right and left tmj 's with the right and left hand (mcp) and wrist joints in rheumatoid arthritis patients. although, statistically insignificant results and sample size limits this study, it is worthwhile to note that most ra patients exhibit similar tmj bony changes along with peripheral joints. | background : a review of literature revealed that, although the involvement of temporomandibular joint (tmj) in rheumatoid arthritis (ra) patients is not uncommon, variation in presentation persist. comparative studies of bony changes in the right and left tmj with the right and left peripheral hand (metacarpophalangeal - mcp)/wrist joints have not been done, to the best of our knowledge.materials and methods : in this cross - sectional study, the temporomandibular and hand (mcp) and wrist joints of fifteen rheumatoid arthritis patients were evaluated with questionnaires, clinical and lab assessment and radiographically using conventional radiographs and computed tomography. students t - test was applied for the statistical analysis of the data obtained and a p value of 0.05 was considered as statistically significant.results:comparisons between the right tmj with right mcp / wrist joint and left tmj with left mcp / wrist joint did not reveal statistically significant results. radiographically, flattening and erosions were the common manifestations. mcp joints were more affected than the wrist, but whenever the wrist was involved, it was more likely to be bilaterally affected.conclusions:although the tmj showed osseous changes of a higher grade than the hand (mcp) and wrist joints radiographically, it was observed that patients were more aware of the peripheral joint discomfort. there were no significant differences between tmj and peripheral joints on both right and left sides. |
the number of neurons in the human brain approximates the number of stars in the galaxy. the result is an incredibly complex and sophisticated network made of roughly 100 trillion synapses. communications between neurons in the brain occur primarily through synapses formed between presynaptic and postsynaptic partners. for fast synaptic transmission, there are two types of synapses : type i synapses use glutamate as the neurotransmitter and are excitatory, whereas type ii synapses use gamma - amino butyric acid (gaba) as the major neurotransmitter and are inhibitory. while dendritic shafts are the main location for the inhibitory gabaergic synapses, dendritic spines, which are small membrane protrusions from dendritic shafts that contain glutamate receptors and postsynaptic density components, are the primary locations of excitatory synapses. a functional balance between neuronal excitation and inhibition is established during development for homeostatic control of neuronal excitability and is maintained into adulthood [14 ]. on the other hand, imbalances between neuronal excitation and inhibition have been associated with many neurological disorders including epilepsy, schizophrenia, fragile x syndrome, and autism. information can be stored in the brain by multiple synaptic mechanisms, including altered structure and chemistry of existing synapses, formation of new synapses, or elimination of old ones. such synaptic plasticity is thought to be fundamental to learning and memory in the brain. at the electrophysiological level, synaptic plasticity is reflected in processes known as long - term potentiation (ltp) and long - term depression (ltd). excitatory synapses contain ampa and nmda ionotropic glutamate receptors localized on dendritic spines, with basal synaptic transmission largely mediated by the ampa receptors. high synaptic activity opens nmda receptors, leading to long - lasting changes in postsynaptic ampa receptor number and ltp of synaptic transmission. alternatively, low levels of synaptic stimulation can activate nmda receptors to produce ltd. at the morphological level, ltp is generally associated with dendritic spine growth, whereas ltd can induce the removal of postsynaptic ampa receptors and loss of spines [1319 ]. it is thus not surprising that synaptic development, maintenance, and plasticity under normal physiological conditions are frequently associated with changes in the morphology and number of dendritic spines. in many neurodegenerative diseases, particularly those exhibiting cognitive impairments such as alzheimer 's disease (ad) and parkinson 's disease (pd), changes in dendritic spine number and morphology are also found in other disease conditions such as autism, down syndrome, drug addiction, fragile x syndrome, and schizophrenia [2024 ]. it is worth emphasizing that degeneration of synapses and dendritic spines is one of the earliest features in those neurodegenerative disease conditions, prior to subsequent loss of neurons. interventions aimed to protect the nervous system from the ravages of these disease would therefore seem more effective when the synaptic and spine pathology are prevented as early as possible. in this review article, we will summarize recent advances in our understanding of the molecular mechanisms underlying synaptic and dendritic spine pathology in neurodegenerative diseases, particularly in ad and pd. the readers are referred to some excellent previous reviews on the observation of synaptic and dendritic spine pathology in neurological disorders [2225 ]. ad is the most common neurodegenerative disease and the leading cause of dementia in the elderly. decades of intensive research have uncovered amyloid plaque and neurofibrillary tangle (nft) as the pathological hallmarks, and soluble amyloid- (a) oligomers as the leading candidate for the causative agent of ad [26, 27 ]. however, the mechanistic link between amyloid plaque and nft and the mechanism by which a oligomer may cause cognitive impairments remains poorly defined, and there is no effective treatment for this devastating disease. substantial evidences have accumulated indicating that the memory deficits in ad patients do not correlate well with amyloid plaque burden ; instead, the loss of synaptic markers is a better predictor of clinical symptoms and disease progression. together with studies using animal ad models, these studies have lent support to the hypothesis that ad could be conceptualized as a disease of synaptic failure. early structural studies of postmortem tissues showed that when compared with age - matched control brains, ad brains had reduced synapse density and number of dendritic spines in the cortex and hippocampus, principal brain areas affected by the disease, and that greater loss of dendritic spines was associated with lower mental status [29, 30 ]. these findings suggested that progressive loss of dendritic spines is directly related to the pathogenesis of ad and represents a good indicator of disease progression. studies of transgenic mouse models of ad have shown that, in the vicinity of amyloid plaques, there were dramatic spine loss and neurite dystrophy, structural changes that could lead to altered neuronal circuits and brain functions [3133 ]. further studies showed that the accumulation of soluble a might be the culprit that leads to dendritic spines loss. a is the proteolytic product of a large protein called amyloid precursor protein (app), which is cleaved by beta- and gamma - secretases to produce a and other fragments of the precursor protein. interestingly, the formation and secretion of a peptides are positively regulated by neuronal activity, and excess a peptide can in turn depress excitatory synaptic transmission onto neurons that produce a as well as nearby neurons that do not produce a. thus, activity - dependent modulation of a production may normally participate in a negative feedback regulatory loop to restrain neuronal hyperactivity, the impairment of which could contribute to ad pathogenesis. under normal conditions, a monomers could be cleared by proteolytic enzymes like neprilysin, chaperone molecule apoe, or the lysosomal and proteasomal pathways. however, under pathological conditions, soluble a levels are increased, leading to the buildup of a oligomers, which can be further sequestered into protofibrils and fibrils as seen in plaques. first, genetic studies of familial forms of ad have identified rare genetic mutations that cause ad by altering the production or metabolism of a peptides, leading to their aberrant accumulation [27, 37 ]. soluble a levels have been found to better correlate with disease progression and severity than amyloid plaques or nfts. second, a oligomers formed in vitro from synthetic peptides, purified from cultured cells expressing app, or from cortex of ad patient brains can induce synaptic dysfunction and neuritic degeneration [3841 ]. third, the reduction of soluble a levels using an immunization method in mouse ad models rescued the cognitive deficits. however, despite the overwhelming supporting evidences, the a hypothesis of ad as described above still faces challenge, since several highly publicized clinical trials targeting a had failed. the molecular mechanisms through which a might cause synaptic loss and neuronal death remain uncertain. a has been found to form pore - like structures with calcium channel activity, which could interfere with calcium signaling [43, 44 ]. a can also affect ltp and ltd by modulating glutamate receptor - dependent signaling pathways [4547 ] and trigger aberrant patterns of neural network activity. one of the earliest clues about the mechanisms of a-induced synaptic dysfunction came from studies of cultured neurons derived from tg2576 mutant app transgenic mice. among the synaptic changes observed were fewer and smaller postsynaptic compartments and fewer and enlarged active presynaptic compartments notably, the earliest observable change in synaptic components was the reduction of psd-95, which is a master regulator of the assembly and anchoring of postsynaptic density components such as glutamate receptor subunits. a was shown to be the toxic agent causing these synaptic changes since the effects were blocked by gamma - secretase inhibitor treatment and recapitulated by application of synthetic a to wild - type neurons. studies in drosophila models showed that par-1 kinase, the fly homologue of mammalian microtubule affinity regulating kinases (marks), can directly phosphorylate the fly psd-95 homologue dlg, and this phosphorylation event caused the delocalization of dlg from the postsynaptic membrane. par-1/mark kinases are known to be activated by app or a in drosophila or mammalian neurons [55, 56 ]. it would be interesting to test whether marks are critical mediators of a toxicity on mammalian synapses and dendritic spines. a significant recent advance in our understanding of the mechanisms of the synaptic toxicity of a has been the finding that a uses ltd - related signaling mechanisms to affect synaptic function and dendritic spine morphology. one of the principle mechanisms of ltd induction is the removal of ampa receptors from the postsynaptic membrane through endocytosis. overexpression of a resulted in decreased spine density and postsynaptic ampa receptor number, through signaling molecules implicated in ltd, such as p38 map kinase and calcineurin. importantly, expression of a mutant form of ampa receptor that resists ltd - driven endocytosis blocked the morphological effects and synaptic depression induced by a. this study implicated the endocytosis of ampa receptors as a major mechanism through which a causes synaptic dysfunction and subsequent degeneration, but the detailed molecular mechanisms remain unclear. recent studies using transgenic mouse models of ad have implicated the microtubule - binding protein tau as a major mediator of the toxicity of a at the postsynaptic compartment and dendritic spines. although tau abnormality has long been observed in ad, as exemplified by the formation of nfts by tau that accompany plaque pathology, and tau abnormality can cause neurodegeneration in the absence of plaque pathology as in frontotemporal dementia cases [57, 58 ], the direct involvement of tau in a-induced synaptic and dendritic spine pathology may initially appear surprising, since tau is generally considered a presynaptic protein that is primarily localized to axons. in fact, the relationship between nfts and amyloid plaques in disease pathogenesis has long been a source of considerable debate [37, 59, 60 ]. studies in mice suggested that the two lesions might be causally linked. in transgenic mouse models, intracranial injection of synthetic a, or crossing of app transgenic mice with tau transgenic mice, promoted nft pathology [6163 ], and immunization of app / psn / tau triple transgenic mice with antibodies against a reduced the levels of hyperphosphorylated tau. this was consistent with earlier studies showing that the removal of tau could relieve a-induced neurotoxicity in cultured neurons. together, these studies support the notion that the initiating event in ad is the accumulation of the toxic a peptides, and that tau abnormality is a major downstream molecular event that contributes to disease pathogenesis. previous studies have shown that a could lead to abnormal activation of a number of kinases, including cyclin - dependent kinase-5 (cdk5) [66, 67 ], fyn kinase [68, 69 ], glycogen synthase kinase-3beta (gsk3), and mark [7173 ], all of which promote tau hyperphosphorylation and could potentially affect synaptic structure and function. however, very few in vivo studies have been done to assess the roles of tau kinases or phosphatases in conferring tau toxicity and in causing ad - related memory deficit. recently studies have shown that removing endogenous tau can prevent a-induced behavioral deficits in a mouse ad model expressing human app, and block excitotoxin - induced neuronal dysfunction in both transgenic and nontransgenic mice. since current data support postsynaptic toxicity as a primary mechanism of a action in causing learning and memory deficits in ad, this study raised the possibility that tau may also act in the postsynaptic compartment. indeed, under both physiological and pathological conditions, tau was found in dendrites [75, 76 ], albeit the level of dendritic tau was much higher under disease conditions. tau was known to interact with microtubules through its microtubule - binding domain to stabilize microtubule and regulate axonal transport. it has many putative phosphorylation sites and becomes hyperphosphorylated in ad patients and transgenic animal models [57, 58 ]. apart from the notion that phosphorylation can lead to the dissociation of tau from the microtubules, other pathophysiological effects of this molecular event are unknown. a recent study has indicated that phosphorylated tau could accumulate in dendritic spines, where it may affect the synaptic trafficking and/or anchoring of glutamate receptors, thereby influencing postsynaptic function. interestingly, this effect of tau on synaptic function occurred without causing the loss of synapses or dendritic spines. this study thus revealed a critical role for tau phosphorylation in causing tau mislocalization and subsequent synaptic impairment, and it established dendritic spines as pathogenic targets of tau action. tau interacts with fyn, a protein tyrosine kinase that can phosphorylate tau and whose activity is increased in ad brain. ittner. showed that the interaction of tau with fyn leads to the targeting of fyn to dendritic spines, where fyn can phosphorylate nmda receptor subunit 2 (glur2), resulting in stabilization of the interaction between glur2 and psd-95 and enhanced excitotoxicity. tau also shows strong interaction with psd-95, providing further support for a dendritic role of tau besides its known axonal function. importantly, the toxic effects of app / a were attenuated by interfering with glur2/psd-95 interaction with a cell - permeable peptide, supporting that dendritic tau - mediated fyn recruitment and glur2/psd-95 interaction confer a toxicity at the postsynapse. thus, a tau hypothesis has been put forward based on these recent results ; a triggers the phosphorylation of tau, causing tau to dissociate from the microtubules and accumulate at the dendritic compartments. phosphorylated tau exhibits stronger interaction with fyn and thus facilitates the targeting of fyn to dendritic spines. the targeting of fyn to postsynaptic density sensitizes the nmda receptors and renders neurons more vulnerable to the toxicity of a in the postsynaptic compartment. it remains to be determined whether tau becomes hyperphosphorylated in situ in the dendritic spines as a result of altered kinase / phosphatase activities there, or that it becomes hyperphosphorylated elsewhere and is then transported to the dendritic spines. nevertheless, targeting the tau - dependent pathway, for example, by reducing tau protein level, inhibiting tau kinase activities, or increasing phosphatase activities, would represent suitable new ways of treating ad. in summary, we can consider the toxic effect of a on neuronal synapses and dendritic spines as a normal physiological process gone awry, instead of some pathological process unique to the disease process. a is continuously produced in the brain, and its production can be stimulated by neuronal activity. a can then feedback on the hyperactive neuron using a ltd - related mechanism to tune down neuronal activity, for example, by promoting ampar removal. this process normally acts as a homeostatic mechanism to restrain neuronal hyperactivation. in the disease process, however, the buildup of a tips the balance of this process toward excessive synaptic depression and ampar removal, resulting in synapse and spine loss (figure 1). the molecular mechanisms involved in a toxicity on synapses and dendritic spines are just beginning to be elucidated. we propose that a signaling cascade from a to tau and psd-95, involving tau kinases such as par-1/mark and its activating kinase lkb1, might be involved (figure 1). although much of the research on the mechanisms of a toxicity to synapses and spines has taken a approach, it is worth noting that other nonneuronal cell types in the brain play critical roles in the formation and maturation of synapses during development, and similar mechanisms may operate in the adult brain to mediate the effects of a on neuronal synapses and dendritic spines. besides providing trophic factors for neurons, glial cells have been shown to play key roles in regulating neuronal migration, axon guidance, and synapse formation. in one of the better - characterized cases, astrocytes were shown to secret signals that induce synapse formation by retinal ganglion cells (rgcs). a family of extracellular matrix proteins called thrombospondins (tsps) was identified as the synaptogenic signals coming from astrocytes. the tsp receptor from the neuronal side involved in synaptogenesis was found to be the calcium channel subunit 2-1. interestingly, the synapses formed by tsps are postsynaptically silent due to the lack of surface ampa receptors, whereas those formed by astrocyte conditioned medium are postsynaptically active, suggesting that additional factors are secreted by the astrocytes to control synaptic strength and plasticity. also, synapses are made in excess during development, and the extra synapses or weak synapses are eliminated by a process involving signals from astrocytes that induce the classical complement pathway protein c1q in neurons. in addition to secreted factors, astrocytes can regulate synapse formation using contact - mediated mechanisms. astrocytes also regulate dendritic spine morphology through a contact - mediated mechanism involving bidirectional ephrin / epha signaling. in the hippocampus, for example, astrocytes express ephrin a3, whereas neurons express the ephrin receptor epha4. perturbing ephrin / epha signaling results in defects in spine formation and maturation. one can imagine that disruption of astrocyte - neuron interaction by a could affect synapse and spine morphology through the above - mentioned mechanisms. in this respect, it is interesting to note that a recent study has shown that lentiviral - mediated delivery of ephb2 expression constructs in the dentate gyrus of happ transgenic mice reversed deficits in nmda receptor - dependent ltp and memory impairments. whether there is the other abundant glial cells in the brain are microglia. unlike the astrocytes, these cells are of mesodermal origin. the roles of microglia in disease pathogenesis in ad and other neurodegenerative diseases are very complex and controversial [87, 88 ]. this probably has to do with the diverse activities of these cells in the brain. relevant to ad pathogenesis, microglia can promote a clearance, release anti - inflammatory cytokines and neurotrophic factors on one hand, and they can also affect the activation of complement systems and elimination of synapses and spines on the other hand. thus microglia can exert neuroprotective as well as neurodegenerative effects, depending on the strength, timing, and duration of their activation. imaging studies showed that activated microglia were found in patients with mci, suggesting that neuroinflammation is an early event in the disease process. consistent with this finding, microglial activation was observed early in a tauopathy mouse model, preceding nft formation and roughly concurrent with synapse loss and impairment of synaptic function. interestingly, supplement of immunosuppressant fk506 to young mice attenuated tau pathology and increased lifespan, suggesting that microglia activation may contribute to disease. in another ad mouse model expressing the e693 mutation that causes ad by enhanced a oligomerization without fibrillization, it was found that the mice displayed age - dependent accumulation of intraneuronal a oligomers at around 8 months, when abnormal tau phosphorylation, and impairments of hippocampal synaptic plasticity and memory were observed. however, microglial activation was observed from 12 months, astrocyte activation from 18 months, and neuronal loss at 24 months. it is not known in this case whether microglial and astrocyte activation plays a neurodegenerative role or as part of a neuroprotective, compensatory response. despite the large amount of literature documenting a detrimental role for microglia and astrocyte activation in the disease process, for example, microglia are proposed to play a surveillance role by constantly monitoring and sensing synaptic health, and, in addition to the critical roles, astrocytes play in synapse formation as mentioned earlier, and these cells can also control extracellular glutamate levels, remove excess extracellular k, release gliotransmitters, store glucose and transform it into lactate as energy source of neurons, and scavenge ros to protect against oxidative damages. given these essential roles of glia to neuronal function and health, it is possible that damaging of glial cells by a may have equally harmful effect on the neurons eventually. in fact, there is evidence that glial cells can release ros upon a exposure, and glial - released cytokines may even trigger a signaling process that promotes tau hyperphosphorylation. thus, a possible role of dysfunction glial cells in ad pathogenesis should be considered, especially in the early stages of the disease process (figure 2). synapse and dendritic spine pathology have been observed in the early stages of neurodegenerative diseases before neuronal death is evident, suggesting that these cellular locations represent pathogenic sites of action by the disease - causing agents early in the disease process. at least in the case of ad, there is compelling evidence supporting a pathogenic role for the synaptic and dendritic spine abnormalities. an intriguing possibility is that, as in ad, defects in the morphology and function of synapses and dendritic spines may play a critical role in the pathogenesis of pd. in fact, alterations in synaptic plasticity as represented by ltp and ltd are observed in pd, and some familial pd - associated genes have been shown to affect synapse and dendritic spine morphology and function [95, 96 ]. it would thus be interesting to examine whether ltp- and ltd - related signaling mechanisms are involved in pink1/parkin - induced synapse and dendritic spine changes. in this respect, it would also be interesting to test the potential role of dendritic tau in mediating the synaptic effects of the fpd genes. this is particularly relevant, given the identification of tau as a susceptibility factor for pd. future studies along these directions could lead to the identification of common molecular mechanisms underlying the pathogenesis of ad, pd, and possibly other neurological disorders and offer new therapeutic strategies. | synapses are sites of cell - cell contacts that transmit electrical or chemical signals in the brain. dendritic spines are protrusions on dendritic shaft where excitatory synapses are located. synapses and dendritic spines are dynamic structures whose plasticity is thought to underlie learning and memory. no wonder neurobiologists are intensively studying mechanisms governing the structural and functional plasticity of synapses and dendritic spines in an effort to understand and eventually treat neurological disorders manifesting learning and memory deficits. one of the best - studied brain disorders that prominently feature synaptic and dendritic spine pathology is alzheimer 's disease (ad). recent studies have revealed molecular mechanisms underlying the synapse and spine pathology in ad, including a role for mislocalized tau in the postsynaptic compartment. synaptic and dendritic spine pathology is also observed in other neurodegenerative disease. it is possible that some common pathogenic mechanisms may underlie the synaptic and dendritic spine pathology in neurodegenerative diseases. |
colon cancer develops as a result of the pathologic transformation of normal colonic epithelium to adenomatous polyp, which ultimately leads to invasive cancer [13 ]. tumor induction and progression are characterized by accumulation of multiple genetic and epigenetic alterations, that confer a selective reproductive advantage to a clone, within a genetically unstable heterogeneous cell population [2, 4, 5 ]. the survival and the expansion of the neoplastic cell clone are supported by the surrounding tissue that sustains and favours these conditions [6, 7 ]. tumor - associated stroma actively fuels the colon cancer progression. among the tumor - associated cells are endothelial cells and pericytes that form the neo - vasculature. in turn, the newly formed angiogenic blood vessels supply tumor cells with nutrients and oxygen. in addition, mesenchymal cells are present in the stroma at earlier tumor stages, suggesting a co - evolution between stromal and cancer cells that leads to clonal expansion and metastasis. chronic inflammation, such as inflammatory bowel disease may predispose to malignancy and tumor progression. the chronic inflammatory microenvironment consists of immune, inflammatory and stromal cells, all of which produce cytokines, growth factors and adhesion molecules that may sustain tumor growth, its progression and spreading [8, 9 ]. the cytokines and growth factors produced by cancer cells function to create optimal growth conditions within the tumor microenvironment, while cytokines secreted by stromal cells may influence the behaviour of malignant cells. pro - inflammatory cytokines (il-6, il-1) [1012 ], growth factors (such as vegf, tgf--1, -2, -3) and their cognate receptors [1316 ] either in cancer and in stromal cells, influence not only primary gene transcription, but activate several pathways that cooperate to the aberrant clone survival, tumor expansion and metastasis [6, 10, 12 ]. in addition, alteration of microenvironmental factors induced by hypoxia, represents an hallmark of cancer progression [17, 18 ]. in this review, we discuss the role played by mirna in colon cancer initiation and progression, especially in the context of the synergism existing among hypoxic condition, expression of il-6 pro - inflammatory cytokine and up - regulation of vegfa165. we report that this cooperation could act on the neoplastic cell, also by the dysregulation of mirnas involved in colon cancer progression relevant pathways. both the over - expression and the silencing of specific mirnas recent studies have identified specific mirnas present in colorectal cancer tissues and blood that may aid the diagnosis of cancer and help to predict disease recurrence [2022 ]. such a role in oncogenesis suggested that mirnas could represent important targets for gene therapies. although little is known about the exogenous factors that interfere with the regulation of mirna expression, the microenvironment seems to play an essential role, affecting the epigenetic mechanisms that modulate mirna gene expression, including methylation, histone deacetylation, or influencing the function of proteins involved in the maturation processing of micrornas [23, 24 ]. the present review will focus on the peculiar relationship between colon cancer cells and microenvironment, that could influence mirnas dysregulation, the apoptosis escaping and the metabolic shift, leading to invasion and metastasis. micrornas (mirnas) are ~22-nt small noncoding rnas that are processed from larger (80-nt) precursor hairpins by the rnase iii enzyme dicer into mirna : mirna duplexes. one strand of these duplexes associates with the rnainduced silencing complex (risc), whereas the other is generally degraded. the mirna risc complex targets messenger rnas triggering either translational repression or mrna degradation. the total number of mirnas that are encoded in the human genome remains to be clarified. initial estimates suggested there were up to 255 human mirnas, but cloning and bioinformatic analyses have demonstrated that there are numerous nonconserved human mirnas and suggest this number may be significantly larger. thousands of mammalian messenger rnas are under selective pressure to maintain nucleotide sites matching micrornas. usually conserved targets are often highly expressed at developmental stages, before mirnas expression and their levels tend to fall as the mirna that targets them begins to accumulate. the phenomenon of the selective avoidance extended to thousand of genes enables temporal and tissue specific mirna expression pattern, playing an important role in developmental timing. it seems that the acquired differentiation and specialization is profoundly influenced by the impact of microrna on mrna repression and evolution. overall, in a well differentiated tissue mirnas are destabilizing many target messages to define tissue - specific transcript profile, but other targets could be modulated at translational level, without mrna degradation. in that regard, changes of microenvironmental factors (as seen during cancer development) could accurately modulate protein expression patterns, acting on mirna expression. a primary consequence in the transition from differentiated normal to de - differentiated neoplastic cell is the down modulation of mirnas, involved in tissue specific differentiation. in humans, aberrant expression of mirnas contributes to carcinogenesis by promoting the expression of proto - oncogenes or by inhibiting the expression of tumor suppressor genes. such oncomirs have been demonstrated in a variety of haematological and solid tumors and they contribute to cancer development and progression [2022 ]. recently, the altered mirnas expression in colon cancer has been reported (table 1). however, the role that mirnas play in the development of metastasis is more poorly defined. it is worth of note that the same mirnas could exert tumor suppressor or oncogenic effects, depending on the tumoral context and the stage of neoplastic disease. recently, arndt. have shown that the re - expression of mir-143 or mir-145 leads to tumor suppressor and oncogenic phenotypes respectively, in a metastatic crc model. in particular mir-145, reported as a tumor suppressor in a non - metastatic context, showed oncogenic effects associated with the down - regulation of the g1/s cell cycle checkpoint and neuregulin pathways in the crc metastatic setting. table 1mirnas dys - regulated in colorectal cancerhuman mirnasreferencesdown - regulatedlet-7 familymir-29[66, 68]mir-34amir-143mir-145mir-663 (targeting tgf-1)mir-31up - regulatedmir-106b-93 - 25mir-155mir-21 (epigenetic switch)[62, 63, 66]mir-181b-1 (epigenetic switch)hypermethylatedmir-124[71, 72]mir-34b[60, 71, 72]mir-137 (epigenetic silencing) mirnas dys - regulated in colorectal cancer the cancer microenvironment could contribute to mirnas dysregulation affecting the epigenetic mechanisms (such as dna methylation or histone acetylation) that regulate mirna expression or influencing the maturation process of micrornas [23, 24, 30 ]. as previously reported, dna repair failure and cell survival represent the first step in colon cancer expansion. the pro - inflammatory cytokine il-6 seems to play a role in the inactivation of dna repair mechanisms and bax - dependent apoptosis. although data on the relationship between il-6 production and tumor progression are still conflicting, recent studies suggest a pathogenetic function of the complexes formed between il-6 and its soluble receptor (sil-6r), in colon carcinoma. it seems that an increased formation of il-6-sil-6r complexes, able to interact with gp130 on the cell membrane (trans - signaling), leads to an increased expression and nuclear translocation of stat3, which can cause the induction of anti - apoptotic genes, such as the bax antagonist bcl - xl protein [10, 32, 33 ]. moreover, it has been observed that in critical conditions (hypoxia, glucose deprivation, oxidative stress), the activation of stat3, together with increased levels of hif1 (triggered by the hypoxic condition, 1% po2) influences the preferential expression of vegf - a165a isoform [17, 18, 33 ], that could lead to the inhibition of programmed cell death inducing anti - apoptotic bcl-2 protein. two different splicing isoforms of vegf165 have been identified, exerting pro- or antiangiogenic actions respectively and whose formation is strictly controlled by micro - environmental factors. the production of pro- or anti angiogenic forms of vegf depends upon splice site choice in the c - terminal. proxymal splice site selection in exon 8 generates pro - angiogenic isoforms such as vegf - a165a, and distal splice site selection results in the antagonistic anti - angiogenic isoforms the vegf - a165b. cellular choice on splice site selection, strongly influenced by microenviromental factors, depends upon the activity of rna - binding splice factors, such as asf / sf2. recent studies showed a nuclear and cytoplasmic localization of asf / sf2 splicing factor and correlated the cytoplasmic localization to the inhibition of the proangiogenic form and to a strong increase of the anti - angiogenic isoform b [34, 35 ]. in colon cancer, it seems reasonable that the cooperative interaction between il-6 and increased levels of hif1 favours the shift versus the vegf - a165 pro - angiogenic isoforms, all these factors contributing ultimately to tumor cell proliferation, apoptotic escaping and cell migration. along these lines, we found an increase of il-6 protein levels in human colon cancer tissues and the increase was positively correlated with the tumor stage. il-6, released as by the tumor itself as by tumor associated macrophages (tams), together with il-6-induced vegf - a, could influence tumor cell survival interfering with the ku - clu - bax physical interactions. in a colon cancer progression model, we had previously demonstrated that il-6 increased the expression of s - clusterin (sclu), a multifaceted protein strongly involved in cell survival, apoptosis escaping and metastasis [3639 ]. in vitro experiments have showed that the ku - clu - bax interactions are modulated by il-6, and il-6 together with vegf - a165 inhibit bax - dependent cell death increasing the production of the clusterin pro - survival form (sclu), finally shifting death into survival. in a moderately differentiated colon cancer cell line concomitantly, il-6 exposure influenced bax at protein level acting on bax - ku70-sclu physical interactions in the cytoplasm. the in vitro treatment with il-6 and vegf - a165 modulated the expression of genes involved in tumor invasion and apoptosis, as observed by microarray analysis. in particular, microarray data showed that il-6 could influence cell proliferation, not only by inducing ap1 formation, but also through the up regulation of rasp21 protein activator 1, myc and nfb. on the other hand, il-6 down - modulated the expression of genes involved in the inhibition of metastasis, such as timp1 metallopeptidase inhibitor 1 and kiss1 [40, 41 ]. these still unclear molecular interactions, underline the relevant role of the microenvironmental factors in the complicated cross talk among molecules that could effectively turn the cell fate, by a fine regulation of gene expression, also acting on mirna expression. changes of the microenvironment mediators induced by hypoxia, represent an hallmark of cancer progression. in the tumoral context, the transcriptional regulator hypoxia - inducible factor (hif1), cooperates with il-6, tgf- and vegf - a165 in the promotion of tumoral growth [10, 15, 1719 ]. the activation of hypoxia fosters neo - angiogenesis also regulating the expression of mir-210, and its target ephrin - a3, involved in tube formation and survival of endothelial cells. a group of candidate mirnas regulated by hypoxia have been recently identified : mir-16, 20let-7b, mir-17 - 5p, mir-27, mir-106, mir-107 mir-193, mir-210, mir-320 and mir-361, showing vegf as potential target [43, 73 ]. moreover, using a bioinformatic prediction program, several key genes of proliferation, metabolism, migration and apoptotic response (mir-23, mir-26, mir-27, mir-30, mir-181) were found to be potentially targets of hypoxia - regulated mirnas [57, 58 ]. in the colon cancer context, the synergism among hypoxic condition (hif-1 activation) and il-6 pro - inflammatory cytokine induces the up - regulation of vegf - a165, contributing to neoangiogenesis and metastasis formation. in colon cancer cells (caco-2), we observed that the synergism of il-6 and vegf - a165 influenced the expression of tumor suppressor mirnas, involved in epigenetic control and epithelial to mesenchymal transition (emt), which strongly correlated to the malignization of many types of cancers. in particular, the in vitro co - treatment with il-6 and vegf - a165 significantly down modulated mir-619, which targets vegf gene expression determining a positive feed - back loop, finally contributing to cancer growth and spreading. in addition, a significant decrease of mir-200 (12-folds decrease) was found, as compared to untreated colon cancer cells. the target prediction program indicates that highly conserved binding sites for the mirna200 family are present in the zeb1 and sip1 mrnas, which are repressors of ecadherin, implicated in emt transition and tumor metastasis. the same down - regulation of mir-200 was obtained in response to tgf- treatment and this effect was sufficient to induce emt, in a process requiring up - regulation of zeb1 and/or sip1. in addition, adam. demonstrated that mir-200 controls the sensitivity to the egfr therapy, which represents an actual issue in the treatment of human highly aggressive and metastatic colorectal cancer. in fact the expression of the mir-200 is sufficient to restore the egfr dependency in bladder cancer cells, targeting errfi-1 which is a novel regulator of egfr - independent growth. in the last years, great attention has been given to the development of target anti - cancer therapies, based on the knowledge of transcriptional mechanisms epigenetically regulated in tumoral tissues. the epigenetic modifications, such as dna methylation or histone acetylation, are able to affect mirnas expression causing oncomirs dysregulation, but the same microrna (epi - mirnas) may directly control the epigenetic machinery targeting its enzymatic components. evidence of the epigenetic influence of the microenvironment on tumoral cells has been observed by co - treating colon cancer cells with il-6 and vegf - a165. a strong down - modulation of the growth suppressing mir-449, which targets histone deacetylase 1 molecule (hdac-1) has been found in our system. this enzyme plays a crucial role in the epigenetic control and display high significance in oncology [48, 49 ]. aberrant hdac level and/or it has known that recruitment and activation of hdacs results in tightening of chromatin structure, thus the transcriptional machinery is prevented from accessing the dna. hence, cytokines and growth factors in the microenvironment could modulate gene transcription at epigenetic level, affecting epi - mirna (as mir-449), or influencing the activity of protein involved in the regulation of hdac activity (fig. 1). the synergistic action of il-6, and vegf - a165a promotes the acquisition of aggressiveness regulating the dna repair capacity of the cell, whose impairment is the first step of colon cancer development. the cooperative action of these soluble mediators could modulate cancer - related gene transcription (sclusterin, timp1), and affect the expression of small non coding mrna (micrornas), able to modulate the epigenetic machinery (epi - mirnas ; mir-449) and to induce the epithelial to mesenchimal transition (emt induction ; mir-200 family) cancer- stroma interactions in colon cancer progression and spreading. in colon cancer the synergistic action of il-6, and vegf - a165a promotes the acquisition of aggressiveness regulating the dna repair capacity of the cell, whose impairment is the first step of colon cancer development. the cooperative action of these soluble mediators could modulate cancer - related gene transcription (sclusterin, timp1), and affect the expression of small non coding mrna (micrornas), able to modulate the epigenetic machinery (epi - mirnas ; mir-449) and to induce the epithelial to mesenchimal transition (emt induction ; mir-200 family) the altered metabolism of tumor cells may be a potential means by which these cells evade programmed cell death, favoring survival and tumoral growth. in particular, the metabolism of fatty acids is markedly altered in the tumoral context. it has been demonstrated that the extracellular acidosis in the microenvironment of solid tumors can work in an epigenetic fashion by up - regulating the transcriptional expression of fatty acids synthase (fasn) gene, proposed as the recent reports demonstrated the existence of mirnas able to recognize and modulate the transcriptional levels of metabolic factors, relevant both in non neoplastic and in cancer cell. in particular, transfection experiments with sense and antisense sequences displayed that the increase of lipogenesis in hepg2 cells (hepatocarcinoma cell line) is directly controlled by mir-122. this mirna sequence is up - regulated by mir-370, the last therefore indirectly involved in the upregulation of lipogenic genes. interestingly the same mir-370 targets the 3utr of cpt1, down - regulating the expression of cpt1a gene and the rate of -oxidation. carnitine palmitoyl transferase 1 (cpt1) is another limiting factor in the fatty acids metabolism and it is contra - regulated by fasn substrate malonyl - coa. cpt1 resides physiologically at the mitochondrial membrane transporting long - chain fatty acids for -oxidation. recently, a cpt1a transcript splice variant, termed variant 2 (nm_001031847), was identified in colon cancer and breast cancer cells and it was undetectable in the corresponding non neoplastic cell line. this variant codifies for a protein which differs in only 11 aminoacids from cpt1a variant 1, at the c - terminus. the preferential cellular localization of the two cpt1a variants was identified, being the product of transcript variant 2 mainly localized in the nucleus of tumoral cells. the two transcript variants of cpt1a show great similarity differing only at the 3-utr level, where the recognition with mir-370 is located. a different effectiveness of mir-370 in the target recognition could be responsible for the different levels of cpt1a transcripts found in cancer cell, as compared with non neoplastic cell. in fact, mir-370 could preferentially target cpt1 isoform 1 and indirectly favour cpt1 isoform 2, localized in the nucleus of cancer cells. experiments performed on human colorectal and breast tumoral tissues and cancer cell lines (caco-2, hepg2 and mcf-7 cells) showed that the nuclear localization of cpt1a correlated to an increased hdac activity and a resulting decrease of histone acetylation. we have evidence of a different binding affinity of the two cpt1a variants for hdac1. these data are based on the dissimilar cpt1 residues at the interface with hdac1, for the two complexes. in the case of isoform 2 the interaction with hdac1 involved a region close to the c - terminus, where the differences with cpt1 isoform 1 are located. considering the great similarity of the other regions of cpt1 isoforms, this evidence suggested a higher stability of the complex cpt1 isoform2/hdac1 (unpublished data). additional data are needed to confirm if cpt1 (and in particular the nuclear isoform) could function as a novel partner of hdac-1, stabilizing hdac complexes at acetylated - histone tails of specific promoters, to silence tumor suppressor genes involved in the control of cancer cell growth and migration. experimental evidence suggest that, in tumoral context, the modulation of the metabolic factors could be driven by mirna target recognition, but also through the activation of membrane receptors signalling, such as egf / egfr family, induced by microenvironmental stimuli [51, 54 ]. the human epidermal growth factor receptor type 1 (egfr / her-1) gene encodes a membrane receptor protein in the epidermal growth factor receptor family (erbbs) and evidence suggests that egfr expression and its activation status are associated with hyper - proliferation of colon cancer cells. increased expression of egfr is independently associated with poor disease - free survival after adjustment for stage, histological grade, age, and dna mismatch repair status. epidermal growth factor and the egf receptors (known as erbb1) and erbb2 have been shown to stimulate fatty acids synthase (fasn), the major enzyme required for the synthesis of fatty acids, although the ultimate mechanisms responsible for tumour - associated fasn overexpression are not completely understood. the effects of growth factors and growth factors receptors on fasn are complex and involve activation and/or cross - talk between multiple signal - transduction pathways the egfr activation found in colorectal cancer could induce an up - regulation of fasn, determining the down - regulation of cpt1a activity in mitochondria. overall, in the neoplastic cells the gradient of microenvironmental factors (including egf / egfr activation) induces the overexpression of fasn and the resulting inhibition of fatty acids -oxidation. thus, a differential expression of cpt1a isoforms, driven by mirna target recognition, could favour the cpt1 nuclear variant that evidence suggest to be implicated in the epigenetic regulation. in conclusion, the metabolic factors altered in cancer could directly regulate the expression of genes implicated in tumor development and metastases, or modulate at epigenetic level the expression of small non coding rnas. besides, micrornas have been identified (and many others remain to be uncovered) that target metabolic factors modulating their expression. micrornas (mirnas) are ~22-nt small noncoding rnas that are processed from larger (80-nt) precursor hairpins by the rnase iii enzyme dicer into mirna : mirna duplexes. one strand of these duplexes associates with the rnainduced silencing complex (risc), whereas the other is generally degraded. the mirna risc complex targets messenger rnas triggering either translational repression or mrna degradation. the total number of mirnas that are encoded in the human genome remains to be clarified. initial estimates suggested there were up to 255 human mirnas, but cloning and bioinformatic analyses have demonstrated that there are numerous nonconserved human mirnas and suggest this number may be significantly larger. thousands of mammalian messenger rnas are under selective pressure to maintain nucleotide sites matching micrornas. usually conserved targets are often highly expressed at developmental stages, before mirnas expression and their levels tend to fall as the mirna that targets them begins to accumulate. the phenomenon of the selective avoidance extended to thousand of genes enables temporal and tissue specific mirna expression pattern, playing an important role in developmental timing. it seems that the acquired differentiation and specialization is profoundly influenced by the impact of microrna on mrna repression and evolution. overall, in a well differentiated tissue mirnas are destabilizing many target messages to define tissue - specific transcript profile, but other targets could be modulated at translational level, without mrna degradation. in that regard, changes of microenvironmental factors (as seen during cancer development) could accurately modulate protein expression patterns, acting on mirna expression. a primary consequence in the transition from differentiated normal to de - differentiated neoplastic cell is the down modulation of mirnas, involved in tissue specific differentiation. in humans, aberrant expression of mirnas contributes to carcinogenesis by promoting the expression of proto - oncogenes or by inhibiting the expression of tumor suppressor genes. such oncomirs have been demonstrated in a variety of haematological and solid tumors and they contribute to cancer development and progression [2022 ]. recently, the altered mirnas expression in colon cancer has been reported (table 1). however, the role that mirnas play in the development of metastasis is more poorly defined. it is worth of note that the same mirnas could exert tumor suppressor or oncogenic effects, depending on the tumoral context and the stage of neoplastic disease. recently, arndt. have shown that the re - expression of mir-143 or mir-145 leads to tumor suppressor and oncogenic phenotypes respectively, in a metastatic crc model. in particular mir-145, reported as a tumor suppressor in a non - metastatic context, showed oncogenic effects associated with the down - regulation of the g1/s cell cycle checkpoint and neuregulin pathways in the crc metastatic setting. table 1mirnas dys - regulated in colorectal cancerhuman mirnasreferencesdown - regulatedlet-7 familymir-29[66, 68]mir-34amir-143mir-145mir-663 (targeting tgf-1)mir-31up - regulatedmir-106b-93 - 25mir-155mir-21 (epigenetic switch)[62, 63, 66]mir-181b-1 (epigenetic switch)hypermethylatedmir-124[71, 72]mir-34b[60, 71, 72]mir-137 (epigenetic silencing) mirnas dys - regulated in colorectal cancer the cancer microenvironment could contribute to mirnas dysregulation affecting the epigenetic mechanisms (such as dna methylation or histone acetylation) that regulate mirna expression or influencing the maturation process of micrornas [23, 24, 30 ]. as previously reported, dna repair failure and cell survival represent the first step in colon cancer expansion. the pro - inflammatory cytokine il-6 seems to play a role in the inactivation of dna repair mechanisms and bax - dependent apoptosis. although data on the relationship between il-6 production and tumor progression are still conflicting, recent studies suggest a pathogenetic function of the complexes formed between il-6 and its soluble receptor (sil-6r), in colon carcinoma. it seems that an increased formation of il-6-sil-6r complexes, able to interact with gp130 on the cell membrane (trans - signaling), leads to an increased expression and nuclear translocation of stat3, which can cause the induction of anti - apoptotic genes, such as the bax antagonist bcl - xl protein [10, 32, 33 ]. moreover, it has been observed that in critical conditions (hypoxia, glucose deprivation, oxidative stress), the activation of stat3, together with increased levels of hif1 (triggered by the hypoxic condition, 1% po2) influences the preferential expression of vegf - a165a isoform [17, 18, 33 ], that could lead to the inhibition of programmed cell death inducing anti - apoptotic bcl-2 protein. two different splicing isoforms of vegf165 have been identified, exerting pro- or antiangiogenic actions respectively and whose formation is strictly controlled by micro - environmental factors. the production of pro- or anti angiogenic forms of vegf depends upon splice site choice in the c - terminal. proxymal splice site selection in exon 8 generates pro - angiogenic isoforms such as vegf - a165a, and distal splice site selection results in the antagonistic anti - angiogenic isoforms the vegf - a165b. cellular choice on splice site selection, strongly influenced by microenviromental factors, depends upon the activity of rna - binding splice factors, such as asf / sf2. recent studies showed a nuclear and cytoplasmic localization of asf / sf2 splicing factor and correlated the cytoplasmic localization to the inhibition of the proangiogenic form and to a strong increase of the anti - angiogenic isoform b [34, 35 ]. in colon cancer, it seems reasonable that the cooperative interaction between il-6 and increased levels of hif1 favours the shift versus the vegf - a165 pro - angiogenic isoforms, all these factors contributing ultimately to tumor cell proliferation, apoptotic escaping and cell migration. along these lines, we found an increase of il-6 protein levels in human colon cancer tissues and the increase was positively correlated with the tumor stage. il-6, released as by the tumor itself as by tumor associated macrophages (tams), together with il-6-induced vegf - a, could influence tumor cell survival interfering with the ku - clu - bax physical interactions. in a colon cancer progression model, we had previously demonstrated that il-6 increased the expression of s - clusterin (sclu), a multifaceted protein strongly involved in cell survival, apoptosis escaping and metastasis [3639 ]. in vitro experiments have showed that the ku - clu - bax interactions are modulated by il-6, and il-6 together with vegf - a165 inhibit bax - dependent cell death increasing the production of the clusterin pro - survival form (sclu), finally shifting death into survival. in a moderately differentiated colon cancer cell line concomitantly, il-6 exposure influenced bax at protein level acting on bax - ku70-sclu physical interactions in the cytoplasm. the in vitro treatment with il-6 and vegf - a165 modulated the expression of genes involved in tumor invasion and apoptosis, as observed by microarray analysis. in particular, microarray data showed that il-6 could influence cell proliferation, not only by inducing ap1 formation, but also through the up regulation of rasp21 protein activator 1, myc and nfb. on the other hand, il-6 down - modulated the expression of genes involved in the inhibition of metastasis, such as timp1 metallopeptidase inhibitor 1 and kiss1 [40, 41 ]. these still unclear molecular interactions, underline the relevant role of the microenvironmental factors in the complicated cross talk among molecules that could effectively turn the cell fate, by a fine regulation of gene expression, also acting on mirna expression. changes of the microenvironment mediators induced by hypoxia, represent an hallmark of cancer progression. in the tumoral context, the transcriptional regulator hypoxia - inducible factor (hif1), cooperates with il-6, tgf- and vegf - a165 in the promotion of tumoral growth [10, 15, 1719 ]. the activation of hypoxia fosters neo - angiogenesis also regulating the expression of mir-210, and its target ephrin - a3, involved in tube formation and survival of endothelial cells. a group of candidate mirnas regulated by hypoxia have been recently identified : mir-16, 20let-7b, mir-17 - 5p, mir-27, mir-106, mir-107 mir-193, mir-210, mir-320 and mir-361, showing vegf as potential target [43, 73 ]. moreover, using a bioinformatic prediction program, several key genes of proliferation, metabolism, migration and apoptotic response (mir-23, mir-26, mir-27, mir-30, mir-181) were found to be potentially targets of hypoxia - regulated mirnas [57, 58 ]. in the colon cancer context, the synergism among hypoxic condition (hif-1 activation) and il-6 pro - inflammatory cytokine induces the up - regulation of vegf - a165, contributing to neoangiogenesis and metastasis formation. in colon cancer cells (caco-2), we observed that the synergism of il-6 and vegf - a165 influenced the expression of tumor suppressor mirnas, involved in epigenetic control and epithelial to mesenchymal transition (emt), which strongly correlated to the malignization of many types of cancers. in particular, the in vitro co - treatment with il-6 and vegf - a165 significantly down modulated mir-619, which targets vegf gene expression determining a positive feed - back loop, finally contributing to cancer growth and spreading. in addition, a significant decrease of mir-200 (12-folds decrease) was found, as compared to untreated colon cancer cells. the target prediction program indicates that highly conserved binding sites for the mirna200 family are present in the zeb1 and sip1 mrnas, which are repressors of ecadherin, implicated in emt transition and tumor metastasis. the same down - regulation of mir-200 was obtained in response to tgf- treatment and this effect was sufficient to induce emt, in a process requiring up - regulation of zeb1 and/or sip1. in addition, adam. demonstrated that mir-200 controls the sensitivity to the egfr therapy, which represents an actual issue in the treatment of human highly aggressive and metastatic colorectal cancer. in fact the expression of the mir-200 is sufficient to restore the egfr dependency in bladder cancer cells, targeting errfi-1 which is a novel regulator of egfr - independent growth. in the last years, great attention has been given to the development of target anti - cancer therapies, based on the knowledge of transcriptional mechanisms epigenetically regulated in tumoral tissues. the epigenetic modifications, such as dna methylation or histone acetylation, are able to affect mirnas expression causing oncomirs dysregulation, but the same microrna (epi - mirnas) may directly control the epigenetic machinery targeting its enzymatic components. evidence of the epigenetic influence of the microenvironment on tumoral cells has been observed by co - treating colon cancer cells with il-6 and vegf - a165. a strong down - modulation of the growth suppressing mir-449, which targets histone deacetylase 1 molecule (hdac-1) has been found in our system. this enzyme plays a crucial role in the epigenetic control and display high significance in oncology [48, 49 ]. aberrant hdac level and/or it has known that recruitment and activation of hdacs results in tightening of chromatin structure, thus the transcriptional machinery is prevented from accessing the dna. hence, cytokines and growth factors in the microenvironment could modulate gene transcription at epigenetic level, affecting epi - mirna (as mir-449), or influencing the activity of protein involved in the regulation of hdac activity (fig. 1). the synergistic action of il-6, and vegf - a165a promotes the acquisition of aggressiveness regulating the dna repair capacity of the cell, whose impairment is the first step of colon cancer development. the cooperative action of these soluble mediators could modulate cancer - related gene transcription (sclusterin, timp1), and affect the expression of small non coding mrna (micrornas), able to modulate the epigenetic machinery (epi - mirnas ; mir-449) and to induce the epithelial to mesenchimal transition (emt induction ; mir-200 family) cancer- stroma interactions in colon cancer progression and spreading. in colon cancer the synergistic action of il-6, and vegf - a165a promotes the acquisition of aggressiveness regulating the dna repair capacity of the cell, whose impairment is the first step of colon cancer development. the cooperative action of these soluble mediators could modulate cancer - related gene transcription (sclusterin, timp1), and affect the expression of small non coding mrna (micrornas), able to modulate the epigenetic machinery (epi - mirnas ; mir-449) and to induce the epithelial to mesenchimal transition (emt induction ; mir-200 family) the altered metabolism of tumor cells may be a potential means by which these cells evade programmed cell death, favoring survival and tumoral growth. in particular, the metabolism of fatty acids is markedly altered in the tumoral context. it has been demonstrated that the extracellular acidosis in the microenvironment of solid tumors can work in an epigenetic fashion by up - regulating the transcriptional expression of fatty acids synthase (fasn) gene, proposed as the recent reports demonstrated the existence of mirnas able to recognize and modulate the transcriptional levels of metabolic factors, relevant both in non neoplastic and in cancer cell. in particular, transfection experiments with sense and antisense sequences displayed that the increase of lipogenesis in hepg2 cells (hepatocarcinoma cell line) is directly controlled by mir-122. this mirna sequence is up - regulated by mir-370, the last therefore indirectly involved in the upregulation of lipogenic genes. interestingly the same mir-370 targets the 3utr of cpt1, down - regulating the expression of cpt1a gene and the rate of -oxidation. carnitine palmitoyl transferase 1 (cpt1) is another limiting factor in the fatty acids metabolism and it is contra - regulated by fasn substrate malonyl - coa. cpt1 resides physiologically at the mitochondrial membrane transporting long - chain fatty acids for -oxidation. recently, a cpt1a transcript splice variant, termed variant 2 (nm_001031847), was identified in colon cancer and breast cancer cells and it was undetectable in the corresponding non neoplastic cell line. this variant codifies for a protein which differs in only 11 aminoacids from cpt1a variant 1, at the c - terminus. the preferential cellular localization of the two cpt1a variants was identified, being the product of transcript variant 2 mainly localized in the nucleus of tumoral cells. the two transcript variants of cpt1a show great similarity differing only at the 3-utr level, where the recognition with mir-370 is located. a different effectiveness of mir-370 in the target recognition could be responsible for the different levels of cpt1a transcripts found in cancer cell, as compared with non neoplastic cell. in fact, mir-370 could preferentially target cpt1 isoform 1 and indirectly favour cpt1 isoform 2, localized in the nucleus of cancer cells. experiments performed on human colorectal and breast tumoral tissues and cancer cell lines (caco-2, hepg2 and mcf-7 cells) showed that the nuclear localization of cpt1a correlated to an increased hdac activity and a resulting decrease of histone acetylation. we have evidence of a different binding affinity of the two cpt1a variants for hdac1. these data are based on the dissimilar cpt1 residues at the interface with hdac1, for the two complexes. in the case of isoform 2 the interaction with hdac1 involved a region close to the c - terminus, where the differences with cpt1 isoform 1 are located. considering the great similarity of the other regions of cpt1 isoforms, this evidence suggested a higher stability of the complex cpt1 isoform2/hdac1 (unpublished data). additional data are needed to confirm if cpt1 (and in particular the nuclear isoform) could function as a novel partner of hdac-1, stabilizing hdac complexes at acetylated - histone tails of specific promoters, to silence tumor suppressor genes involved in the control of cancer cell growth and migration. experimental evidence suggest that, in tumoral context, the modulation of the metabolic factors could be driven by mirna target recognition, but also through the activation of membrane receptors signalling, such as egf / egfr family, induced by microenvironmental stimuli [51, 54 ]. the human epidermal growth factor receptor type 1 (egfr / her-1) gene encodes a membrane receptor protein in the epidermal growth factor receptor family (erbbs) and evidence suggests that egfr expression and its activation status are associated with hyper - proliferation of colon cancer cells. increased expression of egfr is independently associated with poor disease - free survival after adjustment for stage, histological grade, age, and dna mismatch repair status. epidermal growth factor and the egf receptors (known as erbb1) and erbb2 have been shown to stimulate fatty acids synthase (fasn), the major enzyme required for the synthesis of fatty acids, although the ultimate mechanisms responsible for tumour - associated fasn overexpression are not completely understood. the effects of growth factors and growth factors receptors on fasn are complex and involve activation and/or cross - talk between multiple signal - transduction pathways the egfr activation found in colorectal cancer could induce an up - regulation of fasn, determining the down - regulation of cpt1a activity in mitochondria. overall, in the neoplastic cells the gradient of microenvironmental factors (including egf / egfr activation) induces the overexpression of fasn and the resulting inhibition of fatty acids -oxidation. thus, a differential expression of cpt1a isoforms, driven by mirna target recognition, could favour the cpt1 nuclear variant that evidence suggest to be implicated in the epigenetic regulation. in conclusion, the metabolic factors altered in cancer could directly regulate the expression of genes implicated in tumor development and metastases, or modulate at epigenetic level the expression of small non coding rnas. besides, micrornas have been identified (and many others remain to be uncovered) that target metabolic factors modulating their expression. invasion and metastasis are the deadly face of malignant tumors. considering the high rate of incidence and mortality of colorectal cancer the outcome of this non random process depends, in part, on the interaction of unique tumor cells with a compatible organ microenvironment. on the other hand, overexpression and silencing of specific mirnas it is worth of note that mirna dysregulation could play opposite role in the early stages of colon cancer, as compared to a metastatic context. as reported above, the pathway analyses may explain the observed oncogenic effects of mir-145 in metastatic crc, compared to its reported tumor suppressor effects in the non - metastatic context. the present review attempts to shed light on the fine relationship between altered gene expression profiles implicated in colon cancer progression and regulatory mirnas, induced by pro - inflammatory partners of tumor microenvironment. furthermore, in the neoplastic cells the gradient of microenvironmental factors induces the alteration of metabolic enzymes, such as fasn and cpt1a, which act on the epigenome modulating mirna expression. the other side of the coin is represented by the identification of mirnas which target the same metabolic factors, modulating their expression levels. nowadays, the identification of specific mirnas directly involved in cancer spread and metastasis may signify novel diagnostic tool in the characterization of gene targets. | the influence of the microenvironment through the various steps of cancer progression is signed by different cytokines and growth factors, that could directly affect cell proliferation and survival, either in cancer and stromal cells. in colon cancer progression, the cooperation between hypoxia, il-6 and vegf - a165 could regulate the dna repair capacity of the cell, whose impairment is the first step of colon cancer development. this cooperation redirects the activity of proteins involved in the metabolic shift and cell death, affecting the cell fate. the pathways triggered by micro environmental factors could modulate cancer - related gene transcription, affecting also small non coding mrna, micrornas. micrornas have emerged as key post - transcriptional regulators of gene expression, directly involved in human cancers. the present review will focus first on the intertwined connection between cancer microenvironment and aberrant expression of micrornas which contribute to carcinogenesis. in particular, the epigenetic mechanisms triggered by tissue microenvironment will be discussed, in view of the recent identification of mirnas able to directly or indirectly modulate the epigenetic machinery (epi - mirnas) and that are involved in the epithelial to mesenchimal transition and metastases development. |
the number of yearly deaths from melanoma continues to increase, and the overall melanoma mortality rate is one of the few cancer mortality rates not on the decline. these realities combined with increasing evidence of the lack of efficacy of the abcde criteria have necessitated ongoing efforts to enhance the earlier clinical detection of melanoma [38 ]. most approaches to melanoma diagnosis have included some predominant emphasis or combination of emphases on recognition of changing lesions, recognition of outlier (ugly - duckling) lesions, and specific melanoma characteristics, with the most utilized criteria being the abcde criteria (a for asymmetry, b for border irregularity, d for 6 mm diameter, and e for evolving lesions). many recently published strategies have rejected the diameter criterion as well as abandoned all or portions of the abcde mnemonic [3,5,912 ]. many of these proposed strategies, including the d for dark proposal i offered, have also added emphasis on recognition of darkness as a particular feature of concern in pigmented lesions [5,1012 ]. i have recently reviewed the compelling rationale for both an increased emphasis on darkness and rejection of the diameter criterion in the clinical diagnosis of melanoma. the georgia approach to melanoma diagnosis uniquely incorporates many elements of these strategies in a complementary manner to increase the sensitivity of diagnosis of early melanoma (figure 1). in their review of melanoma diagnosis, marghoob and scope discuss the concepts of a screening examination to identify lesions of possible concern and then the specific lesion assessment that follows. this distinction is important because the screening examination determines the sensitivity of melanoma recognition, and it is the screening examination that really describes how most practitioners examine patients and how patients examine each other. first, the georgia approach places increased emphasis on the screening examination by initial, distinct discussion and clarification of its function. second, the approach includes both of the two screening strategies that have been used in most melanoma educational materials, change and ugly duckling identification. third, the approach adds the easily perceived, easily communicated, highly sensitive, specific (compared to change and ugly duckling identification) screening feature of darkness. a major tenet of physical diagnosis, particular for early diagnosis, is that one sees what one looks for. a strategy based on recognition only of any lesion that changes or differs from other lesions inadequately considers this principle. the added benefit of looking specifically for dark lesions as part of a screening examination for melanoma can not be overstated ; many melanomas, particularly small melanomas, can be recognized because of, and only because of, their intensity of pigment. nonetheless, with melanoma, as with screening features for nearly every disease, no one feature has 100% sensitivity. the description of the screening examination for melanoma detection includes the instruction to examine the skin in order to detect any lesion that stands out because of being dark, different, or changing. each of the three screening features has non - redundant as well as complementary importance in the recognition of melanoma. the screening examination should usually include two looks, one for any lesion that stands out at all, which should allow detection of changed or ugly duckling lesions, and a second look to identify lesions of any size that stand out because of appearing, even focally, dark. consequently, the emphasis on darkness as a screening feature should only enhance the sensitivity of diagnosis of melanoma. though the impact of the abcde criteria on melanoma detection has been uncertain, the publication and utilization of these criteria are ubiquitous, and the criteria have many supporters. as marghoob and scope help elucidate, however, the role of the abcde criteria is not as a screening approach, as they have been utilized, but as a spot evaluation, and the criteria can also help to assess the level of concern when comparing similar lesions. thus, the criteria have value, but one that requires this more precise explanation. the meanings of the a, b, c, and e in the georgia approach are unchanged from usual use : a for asymmetry, c for color variation, and e for evolving unlike other lesions. what is critical to the utility of the abcde criteria, however, is the change of the d to signify dark and not 6 mm diameter. with this change, and without altering the familiar mnemonic, the criterion never present in the earliest melanomas is replaced by the single criterion that characterizes many early melanomas. it can now be stated more accurately that most melanomas have one or more of the abcde criteria and that the criteria are relevant to the diagnosis of early melanomas. as a criterion of an individual lesion, similar to its utilization as a screening feature, the characteristic of darkness has non - redundant value and, in addition, enhances the application of other criteria. there is increasing support for the strategy of melanoma recognition based on the concept that a melanoma will differ in appearance from one s usual moles, referred to as the ugly - duckling rule. the possible utility in this concept is reflected in the georgia approach both as a screening feature (different) and in the e for evolving description (has a mole.. unlike others on your body ?). in the third component of the georgia approach, the ugly - duckling sign is specifically defined, conveying additional emphasis on and understanding of this strategy and its application. nodular melanomas represent a minority of melanomas but contribute disproportionately to melanoma mortality, and the final portion of the georgia approach is devoted to specific education about the diagnosis of this melanoma subtype. the varied presentations of nodular melanomas, including as amelanotic lesions, are specifically discussed, as is the particular relevance of change and ugly duckling recognition to the diagnosis of these melanomas. the inclusion of the nodular melanoma pictured adds further emphasis on both the diagnosis of nodular melanomas and of the relevance of change recognition to their diagnosis. nonetheless, as nodular melanomas have metastatic potential both in a shorter time frame and when smaller in diameter than other melanomas, the previously discussed addition of dark as a screening feature and the d for dark change may have particular impact on decreasing mortality by enhancing the earlier diagnosis of this melanoma subgroup. in addition to the potential impact on the diagnosis of earlier nodular melanomas by removing any diameter consideration, many early nodular melanomas, similar to other melanoma subtypes, are characterized by their dark pigment. in their review of melanoma diagnosis, marghoob and scope discuss the concepts of a screening examination to identify lesions of possible concern and then the specific lesion assessment that follows. this distinction is important because the screening examination determines the sensitivity of melanoma recognition, and it is the screening examination that really describes how most practitioners examine patients and how patients examine each other. first, the georgia approach places increased emphasis on the screening examination by initial, distinct discussion and clarification of its function. second, the approach includes both of the two screening strategies that have been used in most melanoma educational materials, change and ugly duckling identification. third, the approach adds the easily perceived, easily communicated, highly sensitive, specific (compared to change and ugly duckling identification) screening feature of darkness. a major tenet of physical diagnosis, particular for early diagnosis, is that one sees what one looks for. a strategy based on recognition only of any lesion that changes or differs from other lesions inadequately considers this principle. the added benefit of looking specifically for dark lesions as part of a screening examination for melanoma can not be overstated ; many melanomas, particularly small melanomas, can be recognized because of, and only because of, their intensity of pigment. nonetheless, with melanoma, as with screening features for nearly every disease, no one feature has 100% sensitivity. the description of the screening examination for melanoma detection includes the instruction to examine the skin in order to detect any lesion that stands out because of being dark, different, or changing. each of the three screening features has non - redundant as well as complementary importance in the recognition of melanoma. the screening examination should usually include two looks, one for any lesion that stands out at all, which should allow detection of changed or ugly duckling lesions, and a second look to identify lesions of any size that stand out because of appearing, even focally, dark. consequently, the emphasis on darkness as a screening feature should only enhance the sensitivity of diagnosis of melanoma. though the impact of the abcde criteria on melanoma detection has been uncertain, the publication and utilization of these criteria are ubiquitous, and the criteria have many supporters. as marghoob and scope help elucidate, however, the role of the abcde criteria is not as a screening approach, as they have been utilized, but as a spot evaluation, and the criteria can also help to assess the level of concern when comparing similar lesions. thus, the criteria have value, but one that requires this more precise explanation. the meanings of the a, b, c, and e in the georgia approach are unchanged from usual use : a for asymmetry, c for color variation, and e for evolving unlike other lesions. what is critical to the utility of the abcde criteria, however, is the change of the d to signify dark and not 6 mm diameter. with this change, and without altering the familiar mnemonic, the criterion never present in the earliest melanomas is replaced by the single criterion that characterizes many early melanomas. it can now be stated more accurately that most melanomas have one or more of the abcde criteria and that the criteria are relevant to the diagnosis of early melanomas. as a criterion of an individual lesion, similar to its utilization as a screening feature, the characteristic of darkness has non - redundant value and, in addition, enhances the application of other criteria. there is increasing support for the strategy of melanoma recognition based on the concept that a melanoma will differ in appearance from one s usual moles, referred to as the ugly - duckling rule. the possible utility in this concept is reflected in the georgia approach both as a screening feature (different) and in the e for evolving description (has a mole.. unlike others on your body ?). in the third component of the georgia approach, the ugly - duckling sign is specifically defined, conveying additional emphasis on and understanding of this strategy and its application. nodular melanomas represent a minority of melanomas but contribute disproportionately to melanoma mortality, and the final portion of the georgia approach is devoted to specific education about the diagnosis of this melanoma subtype. the varied presentations of nodular melanomas, including as amelanotic lesions, are specifically discussed, as is the particular relevance of change and ugly duckling recognition to the diagnosis of these melanomas. the inclusion of the nodular melanoma pictured adds further emphasis on both the diagnosis of nodular melanomas and of the relevance of change recognition to their diagnosis. nonetheless, as nodular melanomas have metastatic potential both in a shorter time frame and when smaller in diameter than other melanomas, the previously discussed addition of dark as a screening feature and the d for dark change may have particular impact on decreasing mortality by enhancing the earlier diagnosis of this melanoma subgroup. in addition to the potential impact on the diagnosis of earlier nodular melanomas by removing any diameter consideration, many early nodular melanomas, similar to other melanoma subtypes, are characterized by their dark pigment. whatever changes occur in terms of melanoma diagnosis because of screening recommendations or technology, no strategy will reach its potential without the earliest possible clinical recognition of melanoma. the components of the georgia approach accomplish the following : first component : clarifies and emphasizes the role of a screening examination ; adds dark to both change and ugly duckling identification as screening features ; communicates the screening features simply and succinctly.second component : continues to utilize but more precisely defines the role of the abcde criteria and changes the meaning of the d to dark.third component : discusses both the ugly duckling sign as a general rule as well as specific issues relevant to the diagnosis of nodular melanomas. first component : clarifies and emphasizes the role of a screening examination ; adds dark to both change and ugly duckling identification as screening features ; communicates the screening features simply and succinctly. second component : continues to utilize but more precisely defines the role of the abcde criteria and changes the meaning of the d to dark. third component : discusses both the ugly duckling sign as a general rule as well as specific issues relevant to the diagnosis of nodular melanomas. each of these three components has non - redundant potential to enhance the diagnosis of melanoma. by unifying and integrating all of the components in a logical manner, however, the georgia approach uniquely prioritizes and maximizes the sensitivity of diagnosis of early melanomas. during this period of transition in the clinical diagnosis of melanoma, i encourage other practitioners, departments, and societies to consider and adapt the georgia approach, as well. | current clinical approaches to melanoma diagnosis have not been associated with a decrease in mortality from this cancer. the components of the new approach presented are, first, a screening examination to look for any lesion that stands out because of being dark, different, or changing ; second, when a single lesion is recognized to be of concern for any reason, that lesion is then evaluated in more detail utilizing the abcde criteria, with the d signifying dark and not 6 mm diameter in this mnemonic ; and, third, additional discussion of the ugly duckling sign and of the recognition of nodular melanomas. since the georgia society of dermatology and dermatologic surgery was the first state or national society to endorse this approach, i refer to it as the georgia approach. |
infant respiratory distress syndrome (irds) is the most common respiratory disease in preterm infants, and leads to substantial morbidity and mortality.1,2 irds is caused by lung immaturity and usually develops within minutes of birth. it is defined by tachypnea, retractions or nasal flaring, grunting respiration, and possibly central cyanosis.3 it occurs in approximately 0.3%1.2% of live - born infants.46 however, the prevalence of irds increases with decreasing gestational age.4,7 previous studies have found a prevalence of approximately 90% in premature infants born in gestational week 28.8 valid data on irds are important for clinical epidemiological research. if the coding is accurate, the danish medical registries provide excellent data to study the long - term prognosis of irds, as the registries comprise more than 30 years of medical observations.9,10 to our knowledge, no study has examined the validity of the irds diagnosis in administrative registries. we therefore conducted the present study with the objective of estimating the positive predictive value (ppv) of the irds diagnosis recorded in the population - based danish national patient registry (dnpr) according to the international classification of diseases (icd), 8th and 10th revisions, using medical records as reference standard. based on the dnpr, we identified patients diagnosed with irds from january 1, 1977 to december 31, 2008 in the northern part of denmark (corresponding to the former north jutland county). this part of denmark has approximately 500,000 inhabitants, equivalent to approximately 11% of the total danish population. the dnpr includes data on all non - psychiatric hospital admissions in the country since 1977 and outpatient clinic and emergency room visits since 1995. data include the patients civil registration number, which is a unique personal identification number assigned to all danish residents, date of admission and discharge, surgical procedure(s) performed, one primary diagnosis and up to 19 secondary diagnoses coded by the discharging physician according to the icd-8 until the end of 1993 and subsequently the icd-10. the primary diagnosis code registered is the main reason for the hospital contact. among all patients with a primary or secondary irds diagnosis, we randomly selected three irds patients for each calendar year, 96 in total, between 1977 and 2008. the irds hospital admissions were identified based on the icd-8 diagnosis code 776.19 (idiopathic respiratory distress syndrome or hyaline membrane disease) and the icd-10 diagnosis code p22.0 (idiopathic respiratory distress syndrome). based on the dnpr, we identified patients diagnosed with irds from january 1, 1977 to december 31, 2008 in the northern part of denmark (corresponding to the former north jutland county). this part of denmark has approximately 500,000 inhabitants, equivalent to approximately 11% of the total danish population. the dnpr includes data on all non - psychiatric hospital admissions in the country since 1977 and outpatient clinic and emergency room visits since 1995. data include the patients civil registration number, which is a unique personal identification number assigned to all danish residents, date of admission and discharge, surgical procedure(s) performed, one primary diagnosis and up to 19 secondary diagnoses coded by the discharging physician according to the icd-8 until the end of 1993 and subsequently the icd-10. the primary diagnosis code registered is the main reason for the hospital contact. among all patients with a primary or secondary irds diagnosis, we randomly selected three irds patients for each calendar year, 96 in total, between 1977 and 2008. the irds hospital admissions were identified based on the icd-8 diagnosis code 776.19 (idiopathic respiratory distress syndrome or hyaline membrane disease) and the icd-10 diagnosis code p22.0 (idiopathic respiratory distress syndrome). the medical records of the identified irds patients were reviewed and data were entered in epidata (epidata association, odense, denmark) by a physician (skt). where there was doubt with regard to interpretation of the medical record, another physician (cfc) we also noted gender, gestational age, treatment with continuous positive airway pressure (cpap), and whether the irds diagnosis was mentioned explicitly in the medical record. in the primary analysis, we defined irds as the presence of at least two of the four clinical symptoms (tachypnea, retractions or nasal flaring, grunting, and central cyanosis), which had to be present for more than 30 minutes. tachypnea was defined as 60 or more breaths per minute. in an additional analysis, we defined irds as two or more clinical symptoms together with a positive x - ray finding, defined as reticulogranular ground - glass appearance with air bronchograms. if no information was available on whether or not an x - ray had been taken, or if the radiologist had explicitly ruled out signs of irds, we classified the individual as not having irds. for descriptive purposes, we abstracted data on cpap treatment, but only if provided by pediatric departments and for more than 30 minutes. furthermore, we noted if the irds diagnosis was mentioned in the medical record as a confirmed diagnosis. we used the medical records as the reference standard when computing the ppv of the irds diagnosis. the ppv was defined as the proportion of patients registered with an irds diagnosis in the dnpr that was confirmed by medical record review. thus, the numerator was the number of confirmed irds cases according to the medical records, and the denominator was the selected number of patients registered with an irds diagnosis in the dnpr. the 95% confidence intervals (cis) were computed using jeffrey s method.11 we stratified the analyses by primary and secondary diagnoses, by icd-8 (19771993) and icd-10 (19942008) periods, and by gestational age (gestational week < 28, 2831, 3236, and 37). for 90 of the 96 (94%) selected patients with an irds diagnosis in the dnpr, we were able to find the corresponding medical record, of which 52 (58%) were for males and 38 (42%) were for females. gestational age was reported in the medical record for 88 irds patients, of whom 65 (74%) were preterm infants, and 23 (26%) were born at term (37 weeks of gestation or later). from the medical record we were able to confirm 73 of the 90 patients coded with an irds diagnosis. this gave us an overall ppv of 81% (95% ci 72%88%) (table 1). in the additional analysis, with irds defined as two or more clinical symptoms of irds and a confirmed x - ray (52 irds patients), the ppv was 58% (95% ci 48%68%). irds was registered in the dnpr as the primary diagnosis for 20 (22%) patients, of which 14 were confirmed by the medical record review, corresponding to a ppv of 70% (95% ci 48%86%). among 70 (78%) patients registered with irds as a secondary diagnosis, 59 were confirmed irds patients, corresponding to a ppv of 84% (95% ci 75%91%). we found a ppv of 87% (95% ci 75%94%) in the icd-8 period (19771993) and a ppv of 75% (95% ci 61%86%) in the icd-10 period (19942008). the ppv among males was 75% (95% ci 62%85%), while it was 89% (95% ci 77%96%) among females. stratified by gestational age, we found a ppv ranging from 61% (95% ci 41%79%) in infants born at 37 weeks of gestation or later, to 92% (95% ci 77%98%) in infants born between 28 and 31 weeks of gestation (table 1). the irds diagnosis was explicitly mentioned in 71 (79%) of the 90 medical records, and 82 (91%) of the patients with an irds diagnosis were treated with cpap for more than 30 minutes. in this study, we found reasonable accuracy of the coding for irds in the dnpr as confirmed by exact description of the symptoms of irds in the medical record. to our knowledge, this is the first study to examine the validity of the dnpr with regards to irds. other studies have estimated the ppv of other neonatal diagnoses in the dnpr, including the diagnoses of congenital cardiac malformations with overlapping time periods, 19942002 and 20002008.12,13 they found overall ppvs of 89% (95% ci 86%92%) and 90% (95% ci 89%91%),12,13 which is slightly higher than our ppv, probably because irds is a syndrome characterized by the co - occurrence of characteristic symptoms. we found a slightly lower ppv in the icd-10 period than in the icd-8 period. the potential decrease in ppv over time may reflect less optimal coding practices in the later period, but may also be explained by better documentation of symptoms in the medical records of the early period. we defined the irds diagnosis as the presence of a minimum of two of four clinical symptoms in the medical record. however, these symptoms may not always be described in the medical records of irds patients. if conditions such as infections or congenital heart disease were overlooked by the clinicians, we may have overestimated the ppv of the irds diagnosis. we did not include an x - ray finding of irds in our main criteria, because x - rays were not routinely performed in patients with mild irds. as expected, we found a higher ppv of the irds diagnosis among infants born preterm than among infants born at term. we only examined one region in denmark ; however, we find it reasonable to believe that the results are representative for the entire country owing to the uniform danish health care system. further, we were not able to report on sensitivity, ie, the proportion of all patients with irds actually registered in the dnpr, as we only included patients with a dnpr diagnosis of irds. however, the completeness of the dnpr has previously been estimated to be approximately 90%.12,14 further, we were not able to blind the irds diagnosis for the physician who reviewed the medical records ; however, this is unlikely to have had any major influence on our findings. the ppv of the irds diagnosis quantified in our study may be applied in sensitivity analyses in future studies, to examine the potential effect of the misclassification on study results. we found a reasonable ppv of 81% (95% ci 72%88%) of the irds diagnosis in the dnpr, when compared with symptoms described in the infants medical record. the dnpr is a useful data source for studies of irds, particularly if restricted to preterm infants. | backgroundinfant respiratory distress syndrome (irds) is the most common respiratory disease in preterm infants, and is associated with considerable morbidity and mortality. valid data on irds are important in clinical epidemiological research.objectivesthe objective of this study was to estimate the positive predictive value (ppv) of the irds diagnosis registered in the population - based danish national patient registry according to the international classification of diseases, 8th and 10th revisions.methodsbetween january 1, 1977 and december 31, 2008, we randomly selected three patients per year, 96 in total, who were registered with an irds diagnosis in the danish national patient registry and living in the northern part of denmark. data on the infants included information on the presence of predefined clinical symptoms. we defined irds as the presence of at least two of four clinical symptoms (tachypnea, retractions or nasal flaring, grunting, and central cyanosis), which had to be present for more than 30 minutes. using medical record review as the reference standard, we computed the positive predictive value of the registered irds diagnosis including 95% confidence intervals (cis).resultswe located the medical record for 90 of the 96 patients (94%), and found an overall ppv of the irds diagnosis of 81% (95% ci 72%88%). this did not vary substantially between primary and secondary diagnoses. the ppv was higher, at 89% (95% ci 80%95%), for preterm infants born before 37 weeks of gestation.conclusionthe ppv of the irds diagnosis in the danish national patient registry is reasonable when compared with symptoms described in the corresponding medical records. the danish national patient registry is a useful data source for studies of irds, particularly if restricted to preterm infants. nonetheless, the potential impact of misclassification of the irds diagnosis must be considered. |
repetitive foreign body ingestion, although a common clinical pediatric problem, is relatively rare in the adult population and occurs primarily in individuals with psychiatric conditions, such as bipolar disorder, depression, or posttraumatic stress disorder. described ingested objects include fish bones, needles, razor blades, and pins. in most cases, these objects pass spontaneously with no clinical sequelae. alternatively complications that may arise with nonsurgical modalities, however, include perforation, migration to the liver and pancreas, pancreatitis, development of gastric varices, splenic artery pseudoaneurysm, or even appearance that is indistinguishable from locally advanced pancreatic carcinoma. in these cases, a subset of psychiatric patients has a history of multiple foreign body ingestions and multiple prior surgical interventions, making successive laparoscopy and laparotomy more hazardous to the patient. we report the case of a patient with a history of multiple ingestions who had undergone multiple endoscopies and laparotomies for ingested foreign bodies and describe an innovative technique for removal of foreign bodies not amenable to endoscopic retrieval. the patient is a 54-year - old male with at least 10 previous ingestions of razor blades and nails and at least 7 prior laparotomies for removal. at admission 3 months earlier for ingestion of 2 razor blades, attempted fluoroscopic - guided endoscopic retrieval was complicated by a contained distal esophageal perforation, and only one of the 2 objects was retrieved. the remaining blade was removed after an extensive exploratory laparotomy requiring 4 hours of adhesiolysis and a large gastrotomy with fluoroscopic guidance for retrieval of the remaining razor blade. his past medical history was also notable for hiv / aids, hepatitis c, polysubstance abuse, posttraumatic stress disorder, and bipolar disorder. at the current admission, he was found to have a hemoglobin of 9.2, white blood cell count of 5.1, prothrombin time of 16.2, and inr of 1.2. abdominal films demonstrated no free air, and 2 razor blades were noted in his stomach (figure 1). an extensive discussion was held between the general surgery, gastroenterology, psychiatry, ethics, and medical legal services. because of the patient 's recent esophageal perforation following attempted endoscopic retrieval and the low likelihood of spontaneous passage of the razor blades, it was decided that the best course of action was surgical retrieval. he had numerous other well - healed incisions from prior laparotomies, which included a large midline incision, a left and right subcostal incision, a left paramedian incision, a right lower quadrant transverse incision, and a horizontal upper abdominal incision. based on the plain radiographs, we chose to make a small midline (4 cm) incision overlying his stomach through his previous midline scar. this incision was carried down to the peritoneum, which when sharply entered was found to have extensive adhesions. after a pursestring suture was placed, a 1.5-cm gastrotomy was created through which a 12-mm balloon port was placed (figures 2, 3, 4). upper gi endoscopy was then performed, which showed 2 razor blades in the cephalad portion of the stomach. using the endoscope as an intraluminal light source, insufflator, and camera, a laparoscopic grasper was placed through the balloon port to grab and atraumatically remove the 2 razor blades, located high in the fundus near the gastroesophageal junction. the patient was extubated in the or and taken to the recovery room in stable condition. he had an uncomplicated postoperative course and was tolerating a diet by postoperative day 3. on postoperative day 6, he was discharged in stable condition to a monitored living facility. a laparoscopic grasper was inserted through the balloon port, and the two razor blades were removed atraumatically. while endoscopic removal is feasible in most cases of foreign body ingestion in which spontaneous passage is not successful or possible, surgical exploration is occasionally necessary. although laparoscopic exploration and retrieval may be preferable in such circumstances, in patients with multiple prior abdominal surgeries, access to the peritoneal cavity may be difficult to obtain and may involve extensive adhesiolysis with its attendant risks of bowel injury and prolonged operative time and may be compromised by poor visualization and exposure, increasing the potential morbidity to the patient and the frustration of the surgeon. these difficulties are inherent during conventional laparotomy in this setting as well. as such, an innovative approach in these instances may minimize these risks to both the patient and surgeon. the development and application of natural orifice translumenal endoscopic surgery (notes) has generated substantial interest amongst surgeons, gastroenterologists, and the medical device industry. the feasibility of notes has been demonstrated in porcine models of the peroral transgastric endoscopic approach to the peritoneal cavity for the performance of cholecystectomy, gastrojejunostomy, splenectomy, oophorectomy and salpingectomy, and tubal ligation. more recently, notes appendectomies and laparoscopic - assisted notes cholecystectomies have been performed both via the transvaginal and transgastric route in humans. substantial technical hurdles to be overcome include optimization of gastric / visceral closure, prevention of infection, development of endoscopic suturing and anastomotic devices, multi - tasking platforms, and adequate physician training. this paradigm shift of using a natural orifice to access the peritoneal cavity as opposed to the use of abdominal wall incision(s) offers a tremendous opportunity for innovation and creative problem solving for surgeons, endoscopists, engineers, and industry. we report the first described case of foreign body removal using an approach we have termed reverse notes : rather than creating a gastrotomy endoscopically using a natural orifice, gastric access was obtained via limited, radiograph - guided laparotomy ; endoscopy provided the necessary visualization and insufflation, and conventional laparoscopic instrumentation was utilized for object retrieval. in reviewing our approach, we propose that an alternative for the positioning of the laparotomy would have been to use endoscopy to transilluminate the abdominal wall in a manner similar to percutaneous endoscopic gastrostomy. additionally, the use of carbon dioxide, rather than air insufflation, may have been preferable with regards to bowel and abdominal distention, were conversion to conventional laparotomy necessary. the endoscopic intragastric visualization we were able to obtain was superior to that of conventional gastrotomy, particularly with regards to examination of the cardia and fundus, and greatly simplified the removal of the razor blades and avoided exposure to the radiation associated with fluoroscopy. another alternative for endoscopic retrieval includes usage of a large overtube for safer retrieval of sharp objects. also, placing a peg tube at the site of the gastrotomy would provide future access to the stomach should the need arise. an obvious prerequisite to our approach is the need for endoscopic expertise either on the part of the surgeon or with the involvement of an additional endoscopist. while the flurry of research and development associated with notes is remarkable and necessary, an equally important byproduct of this intersection of endoscopy, laparoscopy, and open surgery is the opportunity for clinicians to re - think difficult clinical problems and develop innovative solutions using currently available therapies. as such, we hope that this case report provides an example of such opportunities to the practicing surgeon. | surgeons are gaining interest in natural orifice surgery because of its minimally invasive nature. the new paradigm shift of using a natural orifice, as opposed to the abdominal wall, as a conduit for entry into the abdomen has resulted in novel solutions to solving difficult surgical problems. repetitive foreign body ingestion continues to be one of those challenging dilemmas. ingested objects that can not be retrieved endoscopically must be removed by laparoscopy or laparotomy. surgical removal, however, becomes more difficult with each subsequent operation. we report a novel technique of foreign body removal that utilizes the concept of natural orifice surgery by combining both laparoscopic and endoscopic techniques. |
despite the better control of tuberculosis (tb) in the world, the proportion of extrapulmonary tb has increased in recent years. in this context, peritoneal tb (ptb) represents the most frequent form of abdominal tb. data are lacking in developed countries since most studies were done in endemic countries of tb [24 ]. the aim of the present study was to describe the clinical presentation, diagnosis, bacterial epidemiology, treatment, and outcome of ptb in two university hospitals in france over a 10-year period. all patients diagnosed with ptb in the university hospitals of lyon (croix - rousse hospital) and nancy (brabois hospital) between january 2004 and december 2014 were included in a retrospective observational study. france is a country with an incidence of 8.7 per 100,000 population (including 27% of extrapulmonary) and the two cities lyon and nancy count 1.3 million and 300,000 people, respectively. patients were identified by using the database of the administrative coding system (patients with diagnosis of ptb in the infectious disease unit) and the database of the mycobacteriological laboratory of lyon and nancy (patients with a positive culture or polymerase chain reaction [pcr ] of ascites or peritoneum). patients without culture or pcr confirmation of tb but with peritonitis consistent with ptb based on clinical, radiographic or histologic evidence were included. ascites samples were processed in biosafety level 3 facility as follows : briefly samples were centrifuged for 15 min before inoculation to solid (lwenstein - jensen) and liquid (mgit - bactec) media. pellets were heat inactivated and then used for acid fast bacilli smear test with fluorescent microscope. heat - inactivated samples were further processed for dna extraction with magna pure roche system and then used for mycobacterium tuberculosis specific in house pcr reaction. cultured strains were characterized by spoligotyping (spacer oligonucleotide typing), a pcr - based method for genotyping strains and categorized by lineage, using the spoldb4 database. all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1964, as revised in 2013. thirty - four patients were included (29 in lyon and 5 in nancy). there was male predominance, with 22 men and 12 women ; median age was 40 years. twenty - six patients (76.5%) were migrants from areas of endemic tb, mainly africa. one or more underlying disease was found in 13 patients (38.2%) : liver cirrhosis (6), diabetes mellitus (6), chronic viral hepatitis (4) or immunodepression as cancer (4), immunosuppressive therapy (3) and human immunodeficiency virus (1, who was already treated and controlled). two patients (5.9%) had history of cured tb ; history of tb exposure was reported by seven patients (20.6%). the main presentation that lead to hospitalization (29 patients : 85.3%) was a checkup of ascites or suspicion of peritoneal carcinomatosis ; two patients (5.9%) presented peritonitis. the main symptoms and signs are given in table 1. four patients (11.8%) presented acute abdominal illness requiring emergency laparotomy (two initially and two secondarily). median symptom duration before admission was 1 month.table 1symptoms and signs at the presentation (n = 34)symptoms n (%) fever and/or sweats24 (70.6%)abdominal pain24 (70.6%)ascites or abdominal swelling19 (55.9%)weight loss16 (47.1%)asthenia11 (32.4%)diarrhea4 (11.8%)anorexia3 (8.8%)abdominal mass1 (2.9%)salpingitis1 (2.9%) symptoms and signs at the presentation (n = 34) biological findings revealed biological inflammatory syndrome in all patients, with a median c - reactive protein level of 95 mg / l. lymphopenia (under 1.5 g / l) was found in 23 of the 30 patients investigated (76.7%). interferon- release assay (quantiferon - tb ; cellestis) was positive in only 16/22 patients (72.7%), and 15/24 (62.5%) had positive (at least 10 mm) tuberculin test (tubertest ; sanofi pasteur). ascitic fluid analysis found exudate in 17/21 patients (80.1%), with lymphocytic predominance in 19/21 cases (90.5%). on abdominal computed tomography (ct), ascites was found in 29/32 patients (90.6%), and was the only abnormality in 4/32 of these (13.8%). only three patients (8.9%) had no ascites on ct scan, corresponding to the dry plastic form of ptb, defined by bhargava. abdominal ultrasound was equal to ct to detect ascites (25/28, 89.3%) but less efficient in detecting peritoneal thickening (6/28, 21.4%). six patients (17.8%) had positron emission tomography, showing increased peritoneal or mesenteric and lymph node metabolic activity, which could not be differentiated from carcinomatosis. thirty - two out of 34 thoracic ct (94.1%) were abnormal with pleural effusion (12), mediastinal lymph node (9), micronodules (8), nodule (4), or excavation (1). surgery was required for diagnosis in 20 cases (58.8%), including 14 laparoscopies (41.2%) and six laparotomies (17.6%). intraoperative visual inspection was suggestive of ptb in 13 patients (65%), with typical small white peritoneal granulations or granuloma. six of the patients who did not undergo surgery (42.9%) had ultrasound - guided peritoneal biopsy. diagnostic performance of the various bacteriological and histological analyses is shown in table 2. tb pcr, performed on 13/31 ascites samples and on 8/14 peritoneal biopsies, was systematically negative on ascites and positive on only 2 peritoneal biopsies (25%). it should be noted that a large number of peritoneal biopsies were not sent to the bacteriology laboratory (8/22, 36.4%), because only malignant ascites was suspected by the surgeon. finally, histological and/or bacteriological diagnostic proof was obtained in all patients, except for three (8.8%) who did not have peritoneal biopsy but were cured by the anti - tb treatment.fig. 1abdominal computed tomography of a peritoneal tuberculosis, with peritoneal nodular thickening (white arrow) and ascitestable 2diagnostic performance of bacteriological and histological analyses in patients with peritoneal tuberculosisascitesperitoneal biopsytotal3222positive culture58.1% (18/31)71.4% (10/14)positive pcr0.0% (0/13)25.0% (2/8)total bacteriological performance58.1% (18/31)73.3% (11/15)histological performance : epithelioid giant - cell granuloma95.5% (21/22)including with caseous necrosis36.4% (8/22) pcr polymerase chain reaction abdominal computed tomography of a peritoneal tuberculosis, with peritoneal nodular thickening (white arrow) and ascites diagnostic performance of bacteriological and histological analyses in patients with peritoneal tuberculosis pcr polymerase chain reaction twenty - six infections were documented. mycobacterium bovis was isolated in 6 patients (23.1%) and m. tuberculosis in the others 20 (76.9%). all isolates were susceptible to classical anti - mycobacterial drugs, except one case of isoniazid resistance (m. bovis is naturally resistant to pyrazinamide). genetic analysis by spoligotyping, performed in 21 cases, revealed that 8 (38%) belonged to lineage t (table 3). t family lineage is characterized by the absence of spacers 3336 among the 43 spacers targeted by spoligotyping ; the prototypic shared spoligotype is sit53. there was no specific clinical characteristic for the spoligotype t.table 3spoligotyping of the 21 strains responsible for peritoneal tuberculosis spoligotyping of the 21 strains responsible for peritoneal tuberculosis sixteen patients (47.1%) showed one or more other tb location, including lung (five patients), and digestive (four patients), gynecological (four patients) and urological (two patients) tract. sputum smear examination or bronchoalveolar lavage was performed in 32 patients (94.1%) and cultures were positive for 5 patients (14.7%). extensive surgery with intestinal resection was required in two patients because of digestive perforation ; corticosteroids were prescribed for subocclusive syndrome in two patients ; and three malnourished patients required artificial nutrition. antimycobacterial therapy comprised standard regimen with daily rifampicin, isoniazid, ethambutol and pyrazinamide during 2 months followed by at least 4 months of rifampicin and isoniazid for 24 patients (70.6%) ; the others had therapy without pyrazinamide or without isoniazid and/or with quinolone because of toxicity or hepatic disorder before treatment. side - effects led to dose decrease or a change of treatment in 13 patients (38.2%). twenty - six patients (76.5%) completed treatment and were clinically and radiologically cured ; four were lost to follow - up ; one was still under treatment at the time of writing ; and three died during treatment (only one because of disseminated tb). median treatment duration was 8.5 months, and follow - up without relapse after end of treatment was 12 months. early diagnosis of ptb is a challenge as the main presentation of our cohort (85.3%) was a checkup of ascites or suspicion of peritoneal carcinomatosis. due to non - specific and insidious symptoms, there is a significant delay before diagnosis and treatment, even in developed countries. notably, most cases of ptb mimic peritoneal carcinomatosis, especially in women with elevated blood ca 125. in cirrhotic patients bacteriological performance is imperfect since culture of ascites was positive in only 58.1% and peritoneal biopsy in 73.3% of cases. histology allows to correct the diagnosis of tb in a large number of cases where bacterial culture was forgotten : it is essential to routinely do mycobacterial culture of peritoneal biopsy during an exploration of ascites or peritoneal thickening, unless there is a known intra - abdominal malignancy. laparoscopic peritoneal biopsy is the reference method for diagnosis of ptb but, as described here, ultrasound - guided biopsy may facilitate diagnosis, with a lower risk of complications. culture of ascites fluid must be systematic, even if the positivity rate is lower than for peritoneal biopsy, probably because of the low number of bacilli in the processed samples. in the present study, tb pcr seemed to be insensitive when performed on ascites, but can be useful on peritoneal biopsy. interestingly, we found that a high percentage of isolates responsible for ptb belonged to lineage t. extrapulmonary tb is usually more frequently associated with the east african - indian or central - asian lineages, whereas lineage t, like the beijing lineage, seems to be more frequently linked to pulmonary forms [13, 14 ]. the t lineage is the predominant lineage in europe, but not in africa, where most of our patients have lived before migrating in france. finally, it is not known if particular lineages are more frequently responsible for ptb and more data are required to directly incriminate the t lineage in the pathophysiology of ptb. in case of suspected ptb, especially with concordant histology, physicians should not wait for culture before initiating treatment, since treatment delay is a very important factor of poor prognosis. the present cure rate was high, and there was no relapse during follow - up. ultrasound - guided peritoneal biopsy may facilitate diagnosis. tb pcr can be useful on peritoneal biopsy. zo cavalli, florence ader, florent valour, julien saison, loc boussel, oana dumitrescu, thomas perpoint, christian chidiac, thierry may, and tristan ferry declare that they have no conflicts of interest. all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1964, as revised in 2013. this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. | introductiondiagnosis of peritoneal tuberculosis (ptb) is difficult, even in developed countries, where data are lacking. the aim of the present study was to describe the clinical presentation, diagnosis, and bacterial epidemiology of ptb in france over a 10-year period.methodsa retrospective study was conducted on ptb in two university hospitals in france, between january 2004 and december 2014.resultsamong the 34 patients, 76.5% were migrants from areas of endemic tuberculosis (tb), mainly africa. the main presentation (85.3%) was a checkup of ascites or suspicion of peritoneal carcinomatosis. on abdominal computed tomography, ascites was found in 90.6% and peritoneal thickening in 75%. surgery was required for diagnosis in 58.8% of patients. six of the patients who did not undergo surgery had ultrasound - guided peritoneal biopsy. bacteriology was positive for ascites in only 58.1% of cases, for peritoneal biopsy in 73.3%, while granuloma was found in 95.5%. tb polymerase chain reaction (pcr) was positive in 25% of peritoneal biopsy. mycobacterium bovis was isolated in 23.1% of cases and mycobacterium tuberculosis in 76.9%. isolates were fully susceptible (except m. bovis naturally resistant to pyrazinamide). many (38%) belonged to the lineage t (genetic analysis by spoligotyping). cure rate was high (76.5%), after a 69 months of anti - tuberculous therapy.conclusionin developed countries, early diagnosis of ptb is still a challenge. ultrasound - guided peritoneal biopsy may facilitate diagnosis. tb pcr can be useful on peritoneal biopsy. the lineage t was the most prevalent lineage, but more data are required to directly incriminate this lineage in the pathophysiology of ptb. |
in this study, we gathered the total sample size is 200 of mentally retarded students in north of tehran (districts 1, 2, 3) and the sample size was determined according to previous research (11 - 12) and also sample size for preliminary evaluation of reliability and validity test. data were analyzed by cronbach 's alpha coefficient for internal consistency and linear multivariate regression for construct validity (since this scale has seven items and each item measures comments and feedback of the person about one area of personal well - being, the designers (16) of this scale believe that the individual score in each area plays an important role in the distribution of overall life satisfaction scores (the first single item and separated from the other seven items). therefore, the designers have recommended linear multivariate regression analysis for validity assessment in which individual score in single item of overall life satisfaction as the dependent variable and the seven items scales as prediction variables are considered). after obtaining an authorization for the study from tehran 's exceptional education organization, we referred to sayyad shirazi girl 's exceptional school and piroozi boy 's exceptional school located in north of tehran. in these schools, a total of 200 mentally retarded students were selected of morning shift from 8 to 12 and afternoon shift from 2 to 5 and were studied in the counseling room. during this period, eight meetings with the mothers were held before research on their children and after describing the aims of the research, all mothers signed a written consent form. the test in this research was the personal well- being index- cognitive disability scale (16) that was conducted for mentally retarded students.this indicator consists of five stages. at the beginning of the test, some questions are asked about agreement responses that determine whether the student should continue the test or not. if the student succeeds, he/ she will enter the next stage which is the testing of likert scale sufficiency and the student should numerate from zero to ten.then, the student enters the total happiness scale of personal well- being index that consists of eight questions in two parts. the first part asks about life satisfaction : are you satisfied with your life ? ; and the second part consists of questions regarding the following issues : 1) life standards and conditions ; 2) health ; 3) achievements in life ; 4) relationships with others ; 5) safety and peace in life ; 6) dependence and membership among others ; 7) future safety.the student responses to each of the eight items with likert scale of zero to ten, with each number showing the following states : zero : completely dissatisfied and sad ; five : average ; ten : completely satisfied and happy ; between zero and five : completely dissatisfied and sad to average ; and between five and ten : average to completely satisfied and happy. if the student does not succeed at the likert scale stage, another three stage test will be used for him which (cube test) consists of three two cube, three- cube and five- cube stages. the fourth stage is to match with the abstract reference (step building by cubes) that consists of three stages : two steps, three steps and five steps. the fifth stage is to match with the abstract reference (images stage) in which the child shows his / her satisfaction in each item with regard to the images (two faces, three faces and five faces) (16). the construct validity of this indicator questions was 0.78 with respect to satisfaction scale ; and according to 12 studies conducted in australia, the maximum changes of the reliability was 3.1 percent of personal well- being and in australia and other world regions, cronbach 's alpha coefficient was 0.70- 0.85(16).the correlation between the questions was 30%-55% and the sum of questions correlation was at least 50% in pwi - a version (10). all the questions of personal well - being index- cognitive disability were translated and revised based on (comqol) and pwi protocol. then, the translated text was back translated, and the two forms were compared. finally, the translated text was revised and given to several professors holding a phd in psychology ; and their professional suggestions were included in the translations. to identify the face validity and initial survey, the persian version of the personal well - being index- cognitive disability scale in this study, we gathered the total sample size is 200 of mentally retarded students in north of tehran (districts 1, 2, 3) and the sample size was determined according to previous research (11 - 12) and also sample size for preliminary evaluation of reliability and validity test. data were analyzed by cronbach 's alpha coefficient for internal consistency and linear multivariate regression for construct validity (since this scale has seven items and each item measures comments and feedback of the person about one area of personal well - being, the designers (16) of this scale believe that the individual score in each area plays an important role in the distribution of overall life satisfaction scores (the first single item and separated from the other seven items). therefore, the designers have recommended linear multivariate regression analysis for validity assessment in which individual score in single item of overall life satisfaction as the dependent variable and the seven items scales as prediction variables are considered). after obtaining an authorization for the study from tehran 's exceptional education organization, we referred to sayyad shirazi girl 's exceptional school and piroozi boy 's exceptional school located in north of tehran. in these schools, a total of 200 mentally retarded students were selected of morning shift from 8 to 12 and afternoon shift from 2 to 5 and were studied in the counseling room. during this period, eight meetings with the mothers were held before research on their children and after describing the aims of the research, all mothers signed a written consent form. the test in this research was the personal well- being index- cognitive disability scale (16) that was conducted for mentally retarded students.this indicator consists of five stages. at the beginning of the test, some questions are asked about agreement responses that determine whether the student should continue the test or not. if the student succeeds, he/ she will enter the next stage which is the testing of likert scale sufficiency and the student should numerate from zero to ten.then, the student enters the total happiness scale of personal well- being index that consists of eight questions in two parts. the first part asks about life satisfaction : are you satisfied with your life ? ; and the second part consists of questions regarding the following issues : 1) life standards and conditions ; 2) health ; 3) achievements in life ; 4) relationships with others ; 5) safety and peace in life ; 6) dependence and membership among others ; 7) future safety.the student responses to each of the eight items with likert scale of zero to ten, with each number showing the following states : zero : completely dissatisfied and sad ; five : average ; ten : completely satisfied and happy ; between zero and five : completely dissatisfied and sad to average ; and between five and ten : average to completely satisfied and happy. if the student does not succeed at the likert scale stage, another three stage test will be used for him which (cube test) consists of three two cube, three- cube and five- cube stages. the fourth stage is to match with the abstract reference (step building by cubes) that consists of three stages : two steps, three steps and five steps. the fifth stage is to match with the abstract reference (images stage) in which the child shows his / her satisfaction in each item with regard to the images (two faces, three faces and five faces) (16). the construct validity of this indicator questions was 0.78 with respect to satisfaction scale ; and according to 12 studies conducted in australia, the maximum changes of the reliability was 3.1 percent of personal well- being and in australia and other world regions, cronbach 's alpha coefficient was 0.70- 0.85(16).the correlation between the questions was 30%-55% and the sum of questions correlation was at least 50% in pwi - a version (10). all the questions of personal well - being index- cognitive disability were translated and revised based on (comqol) and pwi protocol. then, the translated text was back translated, and the two forms were compared. finally, the translated text was revised and given to several professors holding a phd in psychology ; and their professional suggestions were included in the translations. to identify the face validity and initial survey, the persian version of the personal well - being index- cognitive disability scale two hundred mentally retarded students had a minimum age of 9 and a maximum age of 21 years and their mean age was 14.49 2.66. demographic characteristics of mentally retarded students cronbach 's alpha coefficient the seven items of personal well - being index- cognitive disability was between 0.56 - 0.62.besides, studying the internal consistency of these items showed that all the seven items were correlated with the total score and their scores averages were similar to each other. this indicates that the test 's questions have reliability with regard to evaluation of a common feature. cronbach 's alpha for mentally retarded students it should be mentioned that one hundred of mentally retarded students participated in test - retest reliability study. the results are presented in table 3. also, the results of coefficients test - retest using the pearson correlation between initial and retest scores were significant at 0.001 level. coefficient alpha and test - retest results of kmo and bartlett 's test showed that the sample size was sufficient for analysis. bartlett test of sphericity resulted in a chi - square value of equal to 818.974 with 406 degrees of freedom that was significant in an alpha level of 0.001. as previously mentioned, since this scale has seven items and each item measures comments and feedback of the person about one area of personal well - being, the designers of this scale believe that the individual score in each area plays an important role in the distribution of overall life satisfaction scores (the first single item and separated from the other seven items). therefore, the designers have recommended linear multivariate regression analysis for validity assessment of personal well - being index - cognitive disability (16) in which individual score in single item of overall life satisfaction as the dependent variable and the seven items scales as prediction variables are considered. and results of linear multivariate regression analysis with enter method showed that the scale items can predict 67% of distribution of overall life satisfaction. anova - linear multivariate regression by enter method for mentally retarded students besides, there was a significant relation between the questions of part one and the questions of part two, after implementation of linear multivariate regression analysis, regression coefficients were calculated for the seven items and the results showed that the among the seven items of personal well - being index- cognitive disability scale, items 2 and 7 has significant contribution to the prediction of satisfaction with life in mentally retarded students in north of tehran. cronbach 's alpha coefficient the seven items of personal well - being index- cognitive disability was between 0.56 - 0.62.besides, studying the internal consistency of these items showed that all the seven items were correlated with the total score and their scores averages were similar to each other. this indicates that the test 's questions have reliability with regard to evaluation of a common feature. cronbach 's alpha for mentally retarded students it should be mentioned that one hundred of mentally retarded students participated in test - retest reliability study. the results are presented in table 3. also, the results of coefficients test - retest using the pearson correlation between initial and retest scores were significant at 0.001 level. coefficient alpha and test - retest results of kmo and bartlett 's test showed that the sample size was sufficient for analysis. bartlett test of sphericity resulted in a chi - square value of equal to 818.974 with 406 degrees of freedom that was significant in an alpha level of 0.001. as previously mentioned, since this scale has seven items and each item measures comments and feedback of the person about one area of personal well - being, the designers of this scale believe that the individual score in each area plays an important role in the distribution of overall life satisfaction scores (the first single item and separated from the other seven items). therefore, the designers have recommended linear multivariate regression analysis for validity assessment of personal well - being index - cognitive disability (16) in which individual score in single item of overall life satisfaction as the dependent variable and the seven items scales as prediction variables are considered. and results of linear multivariate regression analysis with enter method showed that the scale items can predict 67% of distribution of overall life satisfaction. anova - linear multivariate regression by enter method for mentally retarded students besides, there was a significant relation between the questions of part one and the questions of part two, after implementation of linear multivariate regression analysis, regression coefficients were calculated for the seven items and the results showed that the among the seven items of personal well - being index- cognitive disability scale, items 2 and 7 has significant contribution to the prediction of satisfaction with life in mentally retarded students in north of tehran. this is the first study which evaluates the validity and reliability of the personal well - being index- cognitive disability on mentally retarded students in north of tehran. the results confirmed the reliability and validity for the personal well - being index- cognitive disability in mentally retarded students of exceptional schools in north districts of tehran. findings of this study are relatively harmonious with studies of lau and cummins (10), miller (12), davey (17) and davern (18). also, the results of this study is relatively harmonious with that of diener, emmons, larsen, griffin (13) and tomas (14), which reported a correlation of 0.78 between the satisfaction and life scale. lau and cummins (10) reported the eight domains form to be a single stable factor which accounts for about 50% of the variance in australia and other countries. overall, pwb is an important construct, as its low levels can lead to depression or social isolation (19, 20). in this regard, identification of factors that maintain high levels of swb such as community connectedness are important for developing strategies to prevent problems associated with low levels of swb. with regard to the extracted data from the results of this research, it can be concluded that mentally retarded students in north districts of tehran had lower emotional, mental and behavioral and, in general, improper reactions to the environmental and social desirable stimulus. therefore, using efficient and effective strategies and ability of researchers to control interfering factors have resulted in validity and reliability of this indicator in north districts of tehran. in addition, the studied children and teenagers have experienced similar mental conditions, family and economic stresses and similar environmental conditions. they accepted the facts and tried to solve their problems, engaged in group programs such as collective game, expression of events, memoirs and short stories and interacted with friends and got involved in physical activities and cognitive programs such as counting digits down, and also sports. further, they were not disappointed with undesirable attitude and response of people and society. they, with a heart full of hope, did not think about their weaknesses and disabilities and tried to see a light in the darkness and tried to be active and dynamic and to think about their capabilities. parents respect increased these children 's self- respect and created internal security and emotional immunity against cultural and social plagues. in general, it can be concluded that the personal well - being index- cognitive disability on mentally retarded students of north districts of tehran is a valid and reliable scale. therefore, we suggest that the experts of exceptional education and psychology clinics to use this index to identify the well - being of mentally retarded students. as a limited group of mentally retarded students were enrolled in this study, these findings can not be generalized. | objectivehaving a good quality of life has always been desirable for humans, and the concept of a good life and the ways of achieving it have become important over the years. personal wellbeing is the mental component of quality of life. thus, the current study was conducted to assess the reliability and validity of the personal well - being index- cognitive disability on mentally retarded students.method200 mentally retarded students in north districts of tehran (districts 1, 2 and 3) were selected by systematic random sampling. the collected data using personal well - being index- cognitive disability was analyzed by cronbach 's alpha coefficient for internal consistency and linear multivariate regression for construct validity.resultsresults confirmed the reliability and validity for the personal well - being index- cognitive disability in mentally retarded students of exceptional schools. studying the internal consistency of seven items showed that all the items were correlated with the total score and their scores averages were similar to each other. this indicates that the test 's questions have reliability with regard to evaluation of a common feature and results showed personal well - being index- cognitive disability had the most extensive coverage of construct validity.conclusionpersonal well - being index- cognitive disability scale could be applied to measure personal wellbeing in mentally retarded students. |
in india, more than 1 billion people are engaged in agricultural activities and a large quantity of pesticides is used to protect their crop against pests to get more yields. india is the largest producer of pesticides in asia and the third largest consumer of pesticides in the world. pesticide consumption has been increasing steadily in the past few years and there has been a distinct shift from organochlorine to organophosphorous (op) and carbamate pesticides. these pesticides interfere with or inhibit the activity of cholinesterase (che) enzymes in nerves and muscle tissue, which results in accumulation of the neurotransmitter acetylcholine (ach) in the nervous system. acute toxicological effects of op pesticides are a result of the inhibition of acetylcholinesterase (ache) in the nervous system, which can cause respiratory, myocardial, and neuromuscular transmission impairment. chronic effects of op exposures are not well documented ; however, several recent reports indicate that certain birth outcomes (e.g., decreased gestational age, decreased birth length) and abnormal reflex functions in infants may be associated with low level environmental exposures to op pesticides.[46 ] ache inhibition causes clinical features due to overstimulation of cholinergic synapses in the parasympathetic system, neuromuscular junction, and central nervous system. decrease in che activity by 1525%, 2535%, and 3550% is caused by low, moderate, and severe intoxication with pesticides, respectively. people are directly exposed to these pesticides through dermal contact and inhalation, and indirectly through the food chain. annually, about 3 million people worldwide are intoxicated with organophosphates ; out of this, 300,000 either die or are severely injured. a most economical blood test for the monitoring of farm workers who are exposed to op insecticide is measurement of plasma butyrylcholinesterase (bche) activity. monitoring of plasma bche has been recommended in the op - exposed population, as this could be a useful biomarker to predict and prevent health hazards of pesticides. it is recommended that the workers che level should be assessed before they start working at a pesticide applied region. in view of this, the present study was aimed to evaluate the ache and bche activities among agriculture workers occupationally exposed to pesticide. the study was conducted in the neighboring villages of chikkaballapur town, rural bangalore, south india, from december 2010 to march 2011. this study included 28 rural people who were agriculture workers, engaged in floriculture, and cultivation of cabbage, potato, and grape. a control group consisting of 13 unexposed workers, who never had any exposure to op pesticides, was taken as the reference group. a detailed history, including the personal and occupational details, was recorded through a questionnaire. a written informed consent was taken from all study subjects after explaining the importance of the study in their local language. five milliliters of venous blood was collected in dried heparinized tubes and transported in ice box to the laboratory. blood samples of voluntarily participated agriculture workers (n = 28) who have been involved mainly in pesticide spraying activities in vegetable and grape gardens were collected. a control group consisting of 13 male subjects who belonged to a similar age group and socioeconomic status and were not exposed to any kind of pesticides was selected for the study from the same localities. blood was centrifuged at 4000 rpm for 10 min at 4c to separate the plasma. che activity was determined by the method of ellman. as modified by chambers and chambers. three milliliters of 0.25 mm of 5,5-dithiobis (2-nitrobenzoic acid) (dtnb) prepared in 0.05 m phosphate buffer was pipetted out into a cuvette, in which 20 l of thoroughly mixed plasma sample and 100 l of 1 mm substrate (acetylthiocholine iodide for ache assay) were added. the sample was placed on a uv - vis spectrophotometer set at a wavelength of 410 nm. the change in absorbance with a light path of 1 cm width was recorded following time drive kinetic spectrophotometric method for 5 min to ensure that the linear phase of the reaction was measured at a time lag of 30 sec. a nonenzymatic blank was included to assess the background levels of hydrolysis of the substrate. t - test was used to compare the significance of the mean differences in che activity between exposed and control subjects. the study was conducted in the neighboring villages of chikkaballapur town, rural bangalore, south india, from december 2010 to march 2011. this study included 28 rural people who were agriculture workers, engaged in floriculture, and cultivation of cabbage, potato, and grape. a control group consisting of 13 unexposed workers, who never had any exposure to op pesticides, was taken as the reference group. a detailed history, including the personal and occupational details, was recorded through a questionnaire. a written informed consent was taken from all study subjects after explaining the importance of the study in their local language. five milliliters of venous blood was collected in dried heparinized tubes and transported in ice box to the laboratory. blood samples of voluntarily participated agriculture workers (n = 28) who have been involved mainly in pesticide spraying activities in vegetable and grape gardens were collected. a control group consisting of 13 male subjects who belonged to a similar age group and socioeconomic status and were not exposed to any kind of pesticides was selected for the study from the same localities. blood was centrifuged at 4000 rpm for 10 min at 4c to separate the plasma. che activity was determined by the method of ellman. as modified by chambers and chambers. three milliliters of 0.25 mm of 5,5-dithiobis (2-nitrobenzoic acid) (dtnb) prepared in 0.05 m phosphate buffer was pipetted out into a cuvette, in which 20 l of thoroughly mixed plasma sample and 100 l of 1 mm substrate (acetylthiocholine iodide for ache assay) were added. the sample was placed on a uv - vis spectrophotometer set at a wavelength of 410 nm. the change in absorbance with a light path of 1 cm width was recorded following time drive kinetic spectrophotometric method for 5 min to ensure that the linear phase of the reaction was measured at a time lag of 30 sec. a nonenzymatic blank was included to assess the background levels of hydrolysis of the substrate. students t - test was used to compare the significance of the mean differences in che activity between exposed and control subjects. the values of p < 0.05 were considered significant. the demographic data of lifestyle habits, type of crop cultivation, pesticides used, and frequency of application collected on both exposed and control subjects are summarized in table 1. the average age of exposed subjects and controls were 34.6 8.22 and 28.1 9.33 years, respectively. about 53.6% of study subjects were using pesticides weekly once and 21.4% were using weekly thrice for their crop protection. the majority of the workers did not use any protective equipment and a normal cloth was used to cover their face as a mask while spraying. about 6871% had complained having the symptoms of headache and eye irritation [table 1 ]. characteristics of agricultural workers (n=28) table 2 shows the list of commonly used pesticides in the study locations and world health organization (who) classification. majority of the pesticides used were in the categories of moderately hazardous to highly hazardous. pesticides used were in the chemical group of op, organochlorine, carbamates, and synthetic pyrethroids. list of commonly used pesticides in the study area ache and bche activities measured in the blood plasma of exposed and control subjects are given in table 3. ache activity among exposed subjects ranged between 1.65 and 3.54 moles / min / ml, with a mean concentration of 2.51 moles / min / ml, whereas in the control group it ranged between 2.22 and 3.51 moles / min / ml. the bche activity in agricultural workers ranged between 0.16 and 5.2 moles / min / ml, with a mean concentration of 1.66 moles / min / ml, whereas in the control group it was 2.195.06 moles / min / ml, with a mean concentration of 3.87 moles / min / ml (p < 0.05). the measured levels of ache and bche activities in exposed subjects were comparatively less than in the control subjects [figure 1 ]. cholinesterase activity (moles / min / ml) among exposed and control group subjects variation in cholinesterase activity between exposed and control groups (p<0.05, t - test) the finding suggests that the ache activity in agriculture workers was decreased (14%) when compared to that of controls due to the inhibition of ache activity by pesticides. the inhibition of ache might have resulted in the accumulation of ach at the synaptic junctions, which may lead to cytotoxicity. occupational exposures to che inhibiting pesticides used in india for agricultural pest control can impair the respiratory health of agricultural workers who work in the field. in agreement with the present study, california agricultural pesticide applicators showed considerable changes in ache inhibition and low ache activities due to exposure to pesticides during the high exposure period. their study also demonstrated that relations exist between change in che inhibition and symptoms, especially respiratory symptoms, symptoms of the cns (analysis including controls), and eye symptoms (internal analysis). studies by hillman and clarke. indicated that there was a significant decrease in activity of ache and bche found among the op pesticide sprayers as compared to the controls. reduction in plasma bche activity (56%) in the present study supports similar findings observed by various researchers. the use of che inhibiting pesticides in the agricultural activity caused the depletion of ache and bche activities among workers. from earlier reports and the present results, it can be speculated that decreased plasma che activity is due to prolonged exposures to op pesticides among the study subjects as compared to controls [figure 1 ]. this study was carried out as part of the academic dissertation and a large number of people could not be included. however, the data generated highlights the effects of pesticide exposures and it would help conduct further studies. this study was carried out as part of the academic dissertation and a large number of people could not be included. however, the data generated highlights the effects of pesticide exposures and it would help conduct further studies. the ache and bche activities measured were significantly lesser due to multiple exposures to different groups of pesticides used for agricultural activity. unscientific way of pesticide mixing, improper way of handling pesticides, and entering agriculture field immediately after pesticide application play significant roles in reducing plasma che activity. preventive measures coupled with biomonitoring of pesticide exposure using che inhibition as a marker are very much important. this will create awareness among the agriculturists, pesticide manufacturers, agriculture department, etc. | background : cholinesterase determination indicates whether the person has been under pesticide exposure is not. it is recommended that the workers cholinesterase level should be assessed for workers at a pesticide applied region. hence, cholinesterase activities in blood samples of agricultural workers exposed to vegetables and grape cultivation with age matched, unexposed workers, who never had any exposure to pesticides, were estimated.methods:the detailed occupational history and lifestyle characters were obtained by questionnaire. cholinesterase activity was determined by the method of ellman as modified by chambers and chambers.results:ache was ranging from 1.65 to 3.54moles / min / ml in exposed subjects where as it was ranged from 2.22 to 3.51moles / min / ml in control subjects. bche activity was ranging from 0.16 to 5.2moles / min / ml among exposed subjects, where as it was ranged from 2.19 to 5.06moles / min / ml in control subjects. the results showed statistically significant reduction in enzyme activities (ache 14% ; bche 56%) among exposed subjects.conclusion:it was concluded that the reduction in cholinesterase activity may lead to varieties of effects. hence it is compulsory to use protective gadgets during pesticide spray. further a continuous biomonitoring study is recommended to assess pesticide exposure. |
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